Molecular Microbiology

The Bacteroides fragilis transcriptome response to oxygen and H(2)O(2): the role of OxyR and its effect on survival and virulence.

Publication Date: 2008 Jun PMID: 18476922
Authors: Sund, C. J. - Rocha, E. R. - Tzianabos, A. O. - Wells, W. G. - Gee, J. M. - Reott, M. A. - O'Rourke, D. P. - Smith, C. J.
Journal: Mol Microbiol

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Stochastic assembly of chemoreceptor clusters in Escherichia coli.

Publication Date: 2008 Jun PMID: 18476921
Authors: Thiem, S. - Sourjik, V.
Journal: Mol Microbiol

Chemoreceptors and cytoplasmic chemotaxis proteins in Escherichia coli form clusters that play a key role in signal processing. These clusters localize at cell poles and at specific positions along the cell body which correspond to future division sites, but the details of cluster formation and the mechanism of cluster distribution remain unclear. Here, we used fluorescence microscopy to investigate how the numbers and sizes of receptor clusters depend on the expression level of chemotaxis proteins and on the cell length. We show that the average cluster number saturates at high levels of protein expression at approximately 3.7 clusters per cell, well below the number of available positioning sites. Correspondingly, distances between clusters in filamentous cells saturate at an average of 1 mum but, even at saturating expression levels, individual cluster numbers and distances show a broad distribution around the mean. Our data imply a stochastic mode of cluster assembly, where a defined average interval between clusters along the cell body arises from competition between nucleation of new clusters and growth of existing clusters. Upon subsequent anchorage to defined lateral sites, clusters grow with rates that inversely depend on their size, and become polar upon several rounds of cell division.

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Sweet to the extreme: protein glycosylation in Archaea.

Publication Date: 2008 Jun PMID: 18476920
Authors: Yurist-Doutsch, S. - Chaban, B. - VanDyke, D. J. - Jarrell, K. F. - Eichler, J.
Journal: Mol Microbiol

Post-translational modifications account for much of the biological diversity generated at the proteome level. Of these, glycosylation is the most prevalent. Long thought to be unique to Eukarya, it is now clear that both Bacteria and Archaea are also capable of N-glycosylation, namely the covalent linkage of oligosaccharides to select target asparagine residues. However, while the eukaryal and bacterial N-glycosylation pathways are relatively well defined, little is known of the parallel process in Archaea. Of late, however, major advances have been made in describing the process of archaeal N-glycosylation. Such efforts have shown, as is often the case in archaeal biology, that protein N-glycosylation in Archaea combines particular aspects of the eukaryal and bacterial pathways along with traits unique to this life form. For instance, while the oligosaccharides of archaeal glycoproteins include nucleotide-activated sugars formed by bacterial pathways, the lipid carrier on which such oligosaccharides are assembled is the same as used in eukaryal N-glycosylation. By contrast, transfer of assembled oligosaccharides to their protein targets shows Archaea-specific properties. Finally, addressing N-glycosylation from an archaeal perspective is providing new general insight into this event, as exemplified by the solution of the first crystal structure of an oligosaccharide transferase from an archaeal source.

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Functional interplay between the Yersinia pseudotuberculosis YpsRI and YtbRI quorum sensing systems modulates swimming motility by controlling expression of flhDC and fliA.

Publication Date: 2008 May 5 PMID: 18466299
Authors: Atkinson, S. - Chang, C. Y. - Patrick, H. L. - Buckley, C. M. - Wang, Y. - Sockett, R. E. - Camara, M. - Williams, P.
Journal: Mol Microbiol

Quorum sensing (QS) in Yersinia pseudotuberculosis involves two pairs of LuxRI orthologues (YpsRI and YtbRI) and multiple N-acylhomoserine lactones (AHLs). In a ypsI/ytbI mutant, AHL synthesis was abolished, unaffected in a ypsR/ytbR double mutant and substantially reduced in a ypsI/ytbR mutant indicating that neither YpsR nor YtbR is essential for AHL synthesis. To determine the inter-relationship between YpsRI and YtbRI we constructed chromosomal lux-promoter fusions to ypsR, ypsI, ytbR and ytbI and examined their expression in each of the QS mutant backgrounds. The YpsRI system negatively autoregulates itself but positively regulates the expression of the ytbRI system whereas the ytbRI system is positively autoregulated but only at the level of ytbI expression. YtbRI does not control expression of ypsR or ypsI. This hierarchical QS system controls swimming motility via regulation of flhDC and fliA. The AHLs synthesised via YtbI play a dual role, activating flhDC, in conjunction with YpsR but repressing fliA in conjunction with YtbR and YpsR. In liquid and plate assays, the early onset of motility observed in ypsR and ypsI mutants was abolished in ytbI, ytbRypsI/ytbI, ypsR/ytbR mutants indicating that QS regulates motility both positively (via YtbRI) and negatively (via YpsRI).

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Targeted deletion of SAP1 abolishes the expression of infectivity factors necessary for successful malaria parasite liver infection.

Publication Date: 2008 May 5 PMID: 18466298
Authors: Aly, A. S. - Mikolajczak, S. A. - Rivera, H. S. - Camargo, N. - Jacobs-Lorena, V. - Labaied, M. - Coppens, I. - Kappe, S. H.
Journal: Mol Microbiol

Malaria parasite sporozoites prepare for transmission to a mammalian host by upregulation of UIS (Upregulated in Infectious Sporozoites) genes. A number of UIS gene products are essential for the establishment of the intrahepatocytic niche. However, the factors that regulate the expression of genes involved in gain of infectivity for the liver are unknown. Herein, we show that a conserved Plasmodium sporozoite low complexity asparagine-rich protein, SAP1 (Sporozoite Asparagine-rich Protein1), has an essential role in malaria parasite liver infection. Targeted deletion of SAP1 in the rodent malaria parasite P. yoelii generated mutant parasites that traverse and invade hepatocytes normally but cannot initiate liver stage development in vitro and in vivo. Moreover, immunizations with Pysap1(-) sporozoites confer long lasting sterile protection against wildtype sporozoite infection. Strikingly, lack of SAP1 abolished expression of essential UIS genes including UIS3, UIS4 and P52 but not the constitutively expressed genes encoding, among others, sporozoite proteins CSP and TRAP. SAP1 localization to the cell interior but not the nucleus of sporozoites suggests its involvement in a posttranscriptional mechanism of gene expression control. These findings demonstrate that SAP1 is essential for liver infection possibly by functioning as a selective regulator controlling the expression of infectivity-associated parasite effector genes.

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Yeast alpha-tubulin suppressor Ats1/Kti13 relates to the Elongator complex and interacts with Elongator partner protein Kti11.

Publication Date: 2008 May 5 PMID: 18466297
Authors: Zabel, R. - Bar, C. - Mehlgarten, C. - Schaffrath, R.
Journal: Mol Microbiol

The alpha-tubulin suppressor 1 (ATS1) gene and the killer toxin insensitive 13 (KTI13) locus from Saccharomyces cerevisiae are allelic. The Ats1/Kti13 gene product interacts with the cell polarity factor Nap1 and promotes growth inhibition of S. cerevisiae by zymocin, a tRNAse toxin complex from Kluyveromyces lactis. Kti13 removal causes zymocin resistance, a trait that is typical of defects in the Elongator complex. Here, we show that Kti13 copurifies with the Elongator partner protein Kti11 and that the Kti11 interaction, not the Nap1 partnership, requires the C-terminus of Kti13. Moreover, Kti13 functionally relates to roles of the Elongator complex in tRNA wobble uridine modification, tRNA suppression of nonsense (SUP4) and missense (SOE1) mutations and tRNA restriction by zymocin. Also, inactivation of Kti13 or Elongator rescues the thermosensitive growth defect of secretory mutants (sec2-59(ts), sec12-4(ts)) suggesting that Kti13 and Elongator affect secretion processes that depend on the GTP exchange factors Sec2 and Sec12, respectively. Distinct from tandem deletions in KTI13 and Elongator genes, a kti13Deltakti11Delta double deletion induces synthetic sickness or lethality. In sum, our data suggest that Kti13 and Kti11 support Elongator functions and that they both share Elongator-independent role(s) that are important for cell viability.

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Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harboring drug tolerant bacteria.

Publication Date: 2008 May 5 PMID: 18466296
Authors: Ojha, A. K. - Baughn, A. D. - Sambandan, D. - Hsu, T. - Trivelli, X. - Guerardel, Y. - Alahari, A. - Kremer, L. - Jacobs, W. R. Jr - Hatfull, G. F.
Journal: Mol Microbiol

Successful treatment of human Tuberculosis (TB) requires 6 to 9 months therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably due to reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters is not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms - organized communities of surface attached cells - but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, that contain an extracellular matrix rich in free mycolic acids, and harbor an important drug-tolerant population that persist despite exposure to high levels of antibiotics.

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The Schizosaccharomyces pombe endo-1,3-beta-glucanase Eng1 contains a novel carbohydrate binding module required for septum localization.

Publication Date: 2008 May 5 PMID: 18466295
Authors: Martin-Cuadrado, A. B. - Del Dedo, J. E. - de Medina-Redondo, M. - Fontaine, T. - Del Rey, F. - Latge, J. P. - de Aldana, C. R.
Journal: Mol Microbiol

Cell separation in Schizosaccharomyces pombe is achieved through the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ring-like structure that surrounds the septum. Correct localization of these hydrolases requires the presence of both the septins and the exocyst. In this work, we show that the glucanase Eng1 contains a region at the C-terminus that acts as a carbohydrate-binding module (CBM) and that it is not present in other members of glycoside hydrolases family 81 (GH81). In vitro, the purified CBM has affinity for beta-1,3 glucan chains with a minimum degree of polymerization of 30 glucose units. Deletion of the CBM results in a protein that is largely defective in complementing the separation defect of eng1Delta mutants. This defect is due to a reduction in the catalytic activity against insoluble substrates and to a defect in targeting of Eng1 to the septum, since the truncated protein localizes to the lateral cell wall of the cell. Thus, the targeting of Eng1 to the primary septum requires not only trans-factors (septins and the exocyst complex) but also a cis-element localized to the C-terminus of the protein.

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An endocytic mechanism for haemoglobin-iron acquisition in Candida albicans.

Publication Date: 2008 May 5 PMID: 18466294
Authors: Weissman, Z. - Shemer, R. - Conibear, E. - Kornitzer, D.
Journal: Mol Microbiol

The fungal pathogen Candida albicans is able to utilize haemin and haemoglobin as iron sources. Haem-iron utilization is facilitated by Rbt5, an extracellular, GPI-anchored, haemin- and haemoglobin-binding protein. Here, we show that Rbt5 and its close homolog Rbt51 are short-lived plasma membrane proteins, degradation of which depends on vacuolar activity. Rbt5 facilitates the rapid endocytosis of haemoglobin into the C. albicans vacuole. We relied on recapitulation of the Rbt51-dependent haem-iron utilization in S. cerevisiae to identify mutants defective in haemoglobin utilization. Homologs of representative mutants in S. cerevisiae were deleted in C. albicans and tested for haemoglobin iron utilization and haemoglobin uptake. These mutants define a novel endocytosis-mediated haemoglobin utilization mechanism that depends on acidification of the lumen of the late secretory pathway, on a Type I myosin, and on the activity of the ESCRT pathway.

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Suppression of a cold-sensitive mutation in ribosomal protein S5 reveals a role for RimJ in ribosome biogenesis.

Publication Date: 2008 May 6 PMID: 18466225
Authors: Roy-Chaudhuri, B. - Kirthi, N. - Kelley, T. - Culver, G. M.
Journal: Mol Microbiol

A specific mutation of Escherichia coli ribosomal protein S5, in which glycine is changed to aspartate at position 28 [S5(G28D)], results in cold sensitivity and defects in ribosome biogenesis and translational fidelity. In an attempt to understand the roles of S5 in these essential cellular functions, we selected extragenic suppressors and identified rimJ as a high-copy suppressor of the cold-sensitive phenotype associated with the S5(G28D) mutation. Our studies indicate that RimJ overexpression suppresses the growth defects, anomalous ribosome profiles and mRNA misreading exhibited by the S5(G28D) mutant strain. Although previously characterized as the N-acetyltransferase of S5, our data indicate that RimJ, when devoid of acetyltransferase activity, can suppress S5(G28D) defects thus indicating that the suppression activity of RimJ is not dependent on its acetyltransferase activity. Additionally, RimJ appears to associate with pre-30S subunits indicating that it acts on the ribonucleoprotein particle. These findings suggest that RimJ has evolved dual functionality; it functions in r-protein acetylation and as a ribosome assembly factor in E. coli.

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Large-Scale Transposon Mutagenesis of Mycoplasma pulmonis.

Publication Date: 2008 Apr 28 PMID: 18452587
Authors: French, C. T. - Lao, P. - Loraine, A. E. - Matthews, B. T. - Yu, H. - Dybvig, K.
Journal: Mol Microbiol

To obtain mutants for the study of the basic biology and pathogenic mechanisms of mycoplasmas, the insertion site of transposon Tn4001T was determined for 1,700 members of a library of Mycoplasma pulmonis mutants. After evaluating several criteria for gene disruption, we concluded that 321 of the 782 protein coding regions were inactivated. The dispensable and essential genes of M. pulmonis were compared to those reported for Mycoplasma genitalium and Bacillus subtilis. Perhaps the most surprising result of the current study was that unlike other bacteria, ribosomal proteins S18 and L28 were dispensable. Carbohydrate transport and the susceptibility of selected mutants to UV irradiation were examined to assess whether active transposition of Tn4001T within the genome would confound phenotypic analysis. In contrast to earlier reports suggesting that mycoplasmas were limited in their DNA repair machinery, mutations in recA, uvrA, uvrB and uvrC resulted in a DNA-repair deficient phenotype. A mutant with a defect in transport of N-acetylglucosamine was identified.

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Essential protective role attributed to the surface lipoproteins of Borrelia burgdorferi against innate defences.

Publication Date: 2008 May 6 PMID: 18452586
Authors: Xu, Q. - McShan, K. - Liang, F. T.
Journal: Mol Microbiol

To initiate infection, a microbial pathogen must be able to evade innate immunity. Here we show that the Lyme disease spirochete Borrelia burgdorferi depends on its surface lipoproteins for protection against innate defences. The deficiency for OspC, an abundantly expressed surface lipoprotein during early infection, led to quick clearance of B. burgdorferi after inoculation into the skin of SCID mice. Increasing expression of any of the four randomly chosen surface lipoproteins, OspA, OspE, VlsE or DbpA, fully protected the ospC mutant from elimination from the skin tissue of SCID mice; moreover, increased OspA, OspE or VlsE expression allowed the mutant to cause disseminated infection and restored the ability to effectively colonize both joint and skin tissues, albeit the dissemination process was much slower than that of the mutant restored with OspC expression. When the ospC mutant was modified to express OspA under control of the ospC regulatory elements, it registered only a slight increase in the 50% infectious dose than the control in SCID mice but a dramatic increase in immunocompetent mice. Taken together, the study demonstrated that the surface lipoproteins provide B. burgdorferi with an essential protective function against host innate elimination.

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Identification of a novel alpha(1-->6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan- and lipoarabinomannan-deficient mutant.

Publication Date: 2008 May 7 PMID: 18452585
Authors: Mishra, A. K. - Alderwick, L. J. - Rittmann, D. - Wang, C. - Bhatt, A. - Jacobs Jr, W. R. - Takayama, K. - Eggeling, L. - Besra, G. S.
Journal: Mol Microbiol

Mycobacterium tuberculosis and Corynebacterium glutamicum share a similar cell wall structure and orthologous enzymes involved in cell wall assembly. Herein, we have studied C. glutamicum NCgl1505, the orthologue of putative glycosyltransferases Rv1459c from M. tuberculosis and MSMEG3120 from Mycobacterium smegmatis. Deletion of NCgl1505 resulted in the absence of lipomannan (Cg-LM-A), lipoarabinomannan (Cg-LAM) and a multi-mannosylated polymer (Cg-LM-B) based on a 1,2-di-O-C(16)/C(18:1)-(alpha-D-glucopyranosyluronic acid)-(1-->3)-glycerol (GlcAGroAc(2)) anchor, while syntheses of triacylated-phosphatidyl-myo-inositol dimannoside (Ac(1)PIM(2)) and Man(1)GlcAGroAc(2) were still abundant in whole cells. Cell-free incubation of C. glutamicum membranes with GDP-[(14)C]Man established that C. glutamicum synthesized a novel alpha(1-->6)-linked linear form of Cg-LM-A and Cg-LM-B from Ac(1)PIM(2) and Man(1)GlcAGroAc(2) respectively. Furthermore, deletion of NCgl1505 also led to the absence of in vitro synthesized linear Cg-LM-A and Cg-LM-B, demonstrating that NCgl1505 was involved in core alpha(1-->6) mannan biosynthesis of Cg-LM-A and Cg-LM-B, extending Ac(1)PI[(14)C]M(2) and [(14)C]Man(1)GlcAGroAc(2) primers respectively. Use of the acceptor alpha-D-Manp-(1-->6)-alpha-D-Manp-O-C(8) in an in vitro cell-free assay confirmed NCgl1505 as an alpha(1-->6) mannopyranosyltransferase, now termed MptB. While Rv1459c and MSMEG3120 demonstrated similar in vitroalpha(1-->6) mannopyranosyltransferase activity, deletion of the Rv1459c homologue in M. smegmatis did not result in loss of mycobacterial LM/LAM, indicating a functional redundancy for this enzyme in mycobacteria.

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Disruption of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene causes aberrant gametogenesis.

Publication Date: 2008 Apr 28 PMID: 18452584
Authors: Taylor, C. J. - McRobert, L. - Baker, D. A.
Journal: Mol Microbiol

Phosphodiesterase (PDE) and guanylyl cyclase (GC) enzymes are key components of the cGMP signalling pathway and are encoded in the genome of Plasmodium falciparum. Here we investigate the role of specific GC and PDE isoforms in gamete formation - a process that is essential for malaria transmission and occurs in the Anopheles mosquito midgut following feeding on an infected individual. Details of the intracellular signalling events controlling development of the male and female gametes from their precursors (gametocytes) remain sparse in P. falciparum. Previous work involving the addition of pharmacological agents to gametocytes implicated cGMP in exflagellation - the emergence of highly motile, flagellated male gametes from the host red blood cell. In this study we show that decreased GC activity in parasites having undergone disruption of the PfGCbeta gene had no significant effect on gametogenesis. By contrast, decreased cGMP-PDE activity during gametocyte development due to disruption of the PDEdelta gene, led to a severely reduced ability to undergo gametogenesis. This suggests that the concentration of cGMP must be maintained below a threshold in the developing gametocyte to allow subsequent differentiation to proceed normally. The data indicate that PfPDEdelta plays a crucial role in regulating cGMP levels during sexual development.

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The Novel Cladosporium fulvum Lysin Motif Effector Ecp6 Is A Virulence Factor With Orthologs In Other Fungal Species.

Publication Date: 2008 Apr 28 PMID: 18452583
Authors: Bolton, M. D. - van Esse, H. P. - Vossen, J. H. - de Jonge, R. - Stergiopoulos, I. - Stulemeijer, I. J. - van den Berg, G. C. - Borras-Hidalgo, O. - Dekker, H. L. - de Koster, C. G. - de Wit, P. J. - Joosten, M. H. - Thomma, B. P.
Journal: Mol Microbiol

During tomato leaf colonization, the biotrophic fungus Cladosporium fulvum secretes several effector proteins into the apoplast. Eight effectors have previously been characterized and show no significant homology to each other or to other fungal genes. To discover novel C. fulvum effectors that might play a role in virulence, we utilized two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to visualize proteins secreted during C. fulvum-tomato interactions. Three novel C. fulvum proteins were identified; CfPhiA, Ecp6, and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides. Ecp6 contains lysin motifs (LysM domains) that are recognized as carbohydrate-binding modules. Ecp7 encodes a small, cysteine-rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNAi-mediated gene silencing we demonstrate that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologs in other organisms, conserved Ecp6 orthologs were identified in various fungal species. Homology based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding.

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Regulation of bacterial gene expression by the NTP substrates of transcription initiation.

Publication Date: 2008 Apr 28 PMID: 18452582
Authors: Turnbough, C. L. Jr
Journal: Mol Microbiol

Many mechanisms of gene regulation in bacteria do not employ repressor or activator proteins. One class of these mechanisms includes those in which the key regulatory element is the control of transcription initiation by the availability of NTP substrates. In this commentary, several distinct examples of initiating NTP-mediated gene regulation are discussed, including a mechanism reported by Krasnyet al. in this issue of Molecular Microbiology. These researchers show that during the stringent response induced by amino acid starvation of Bacillus subtilis, increases in the intracellular level of ATP permit up-regulation of promoters with +1A start sites, while concurrent decreases in the intracellular level of GTP cause down-regulation of promoters with +1G start sites. This regulation is restricted to stringently-controlled promoters.

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YscU cleavage and the assembly of Yersinia type III secretion machine complexes.

Publication Date: 2008 Apr 29 PMID: 18452514
Authors: Riordan, K. E. - Schneewind, O.
Journal: Mol Microbiol

YscU, a component of the Yersinia type III secretion machine, promotes auto-cleavage at asparagine 263 (N263). Mutants with an alanine substitution at yscU codon 263 displayed secretion defects for some substrates (LcrV, YopB and YopD); however, transport of effector proteins into host cells (YopE, YopH, YopM) continued to occur. Two yscU mutations were isolated that, unlike N263A, completely abolished type III secretion; YscU(G127D) promoted auto-cleavage at N263, whereas YscU(G270N) did not. When fused to glutathione S-transferase (Gst), the YscU C-terminal cytoplasmic domain promoted auto-cleavage and Gst-YscU(C) also exerted a dominant-negative phenotype by blocking type III secretion. Gst-YscU(C/N263A) caused a similar blockade and Gst-YscU(C/G270N) reduced secretion. Gst-YscU(C) and Gst-YscU(C/N263A) bound YscL, the regulator of the ATPase YscN, whereas Gst-YscU(C/G270N) did not. When isolated from Yersinia, Gst-YscU(C) and Gst-YscU(C/N263A) associated with YscK-YscL-YscQ; however, Gst-YscU(C/G270N) interacted predominantly with the machine component YscO, but not with YscK-YscL-YscQ. A model is proposed whereby YscU auto-cleavage promotes interaction with YscL and recruitment of ATPase complexes that initiate type III secretion.

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PbSR is synthesized in macrogametocytes and involved in formation of the malaria crystalloids.

Publication Date: 2008 Apr 29 PMID: 18452513
Authors: Carter, V. - Shimizu, S. - Arai, M. - Dessens, J. T.
Journal: Mol Microbiol

Crystalloids are transient organelles that form in developing malaria ookinetes and disappear after ookinete-to-oocyst transition. Their origins and functions remain poorly understood. The Plasmodium berghei scavenger receptor-like protein PbSR is essential for mosquito-to-host transmission of the parasite: PbSR knockout parasites produce normal numbers of oocysts that fail to form sporozoites, pointing to a role for PbSR in the oocyst during sporogony. Here, using fluorescent protein tagging and targeted gene disruption, we show that PbSR is synthesized in macrogametocytes, gets targeted to the crystalloids of developing ookinetes and is involved in crystalloid formation. While oocyst sporulation rates of PbSR knockout parasites are highly reduced in parasite-infected mosquitoes, sporulation rates in vitro are not adversely affected, supporting the view that mosquito factors could be involved in the PbSR loss-of-function phenotype. These findings are the first to identify a parasite protein involved with the crystalloid organelle, and suggest a novel protein-trafficking mechanism to deliver PbSR to the oocysts.

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Localization and nucleotide specificity of Blastocystis succinyl-CoA synthetase.

Publication Date: 2008 Apr 29 PMID: 18452512
Authors: Hamblin, K. - Standley, D. M. - Rogers, M. B. - Stechmann, A. - Roger, A. J. - Maytum, R. - van der Giezen, M.
Journal: Mol Microbiol

The anaerobic lifestyle of the intestinal parasite Blastocystis raises questions about the biochemistry and function of its mitochondria-like organelles. We have characterized the Blastocystis succinyl-CoA synthetase (SCS), a tricarboxylic acid cycle enzyme that conserves energy by substrate-level phosphorylation. We show that SCS localizes to the enigmatic Blastocystis organelles, indicating that these organelles might play a similar role in energy metabolism as classic mitochondria. Although analysis of residues inside the nucleotide-binding site suggests that Blastocystis SCS is GTP-specific, we demonstrate that it is ATP-specific. Homology modelling, followed by flexible docking and molecular dynamics simulations, indicates that while both ATP and GTP fit into the Blastocystis SCS active site, GTP is destabilized by electrostatic dipole interactions with Lys 42 and Lys 110, the side-chains of which lie outside the nucleotide-binding cavity. It has been proposed that residues in direct contact with the substrate determine nucleotide specificity in SCS. However, our results indicate that, in Blastocystis, an electrostatic gatekeeper controls which ligands can enter the binding site.

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Scavenger receptor gp340 aggregates group A streptococci by binding pili.

Publication Date: 2008 Apr 29 PMID: 18452511
Authors: Edwards, A. M. - Manetti, A. G. - Falugi, F. - Zingaretti, C. - Capo, S. - Buccato, S. - Bensi, G. - Telford, J. L. - Margarit, I. - Grandi, G.
Journal: Mol Microbiol

Group A streptococci (GAS) are the most frequent cause of bacterial pharyngitis. The first obstacle to GAS colonization of the pharynx is saliva. As well as forming a physical barrier, saliva contains components of innate and acquired immunity. Previous work has shown that saliva induces bacterial aggregation, which may serve as a clearance mechanism. As the aggregation of some oral streptococci in saliva is mediated by long proteinaceous appendages, we hypothesized that pili of GAS might behave similarly. Wild-type GAS M1 strain SF370 aggregated in saliva, while pilus-defective mutants did not. Similarly, heterologous expression of diverse GAS pili on the surface of Lactococcus lactis induced aggregation in saliva, while control strains were unaffected. Further studies revealed that aggregating bacteria bound salivary component gp340. Purified gp340 aggregated wild-type GAS and L. lactis expressing GAS pili, but not control strains. GAS pilus-defective mutants were abrogated in gp340 binding and aggregation. Furthermore, gp340-mediated aggregation reduced bacterial adhesion to human epithelial cells, suggesting a role in host defence.

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Selective repression by Fis and H-NS at the Escherichia coli dps promoter.

Publication Date: 2008 Apr 28 PMID: 18452510
Authors: Grainger, D. C. - Goldberg, M. D. - Lee, D. J. - Busby, S. J.
Journal: Mol Microbiol

Dps is a nucleoid-associated protein that plays a major role in condensation of the Escherichia coli chromosome in stationary phase. Here we show that two other nucleoid-associated proteins, Fis and H-NS, can bind at the dps gene promoter and downregulate its activity. Both Fis and H-NS selectively repress the dps promoter, preventing transcription initiation by RNA polymerase containing sigma(70), the housekeeping sigma factor, but not by RNA polymerase containing sigma(38), the stationary-phase sigma factor. Fis represses by trapping RNA polymerase containing sigma(70) at the promoter. In contrast, H-NS functions by displacing RNA polymerase containing sigma(70), but not RNA polymerase containing sigma(38). Dps levels are known to be very low in exponentially growing cells and rise sharply as cells enter stationary phase. Conversely, Fis levels are high in growing cells but fall to nearly zero in stationary-phase cells. Our data suggest a simple model to explain how the Dps-dependent super-compaction of the folded chromosome is triggered as cell growth ceases.

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Staphylococcus aureus primase has higher initiation specificity, interacts with single-stranded DNA stronger, but is less stimulated by its helicase than Escherichia coli primase.

Publication Date: 2008 Apr 28 PMID: 18452509
Authors: Koepsell, S. A. - Larson, M. A. - Frey, C. A. - Hinrichs, S. H. - Griep, M. A.
Journal: Mol Microbiol

The study of primases from model organisms such as Escherichia coli, phage T7 and phage T4 has demonstrated the essential nature of primase function, which is to generate de novo RNA polymers to prime DNA polymerase. However, little is known about the function of primases from other eubacteria. Their overall low primary sequence homology may result in functional differences. To help understand which primase functions were conserved, primase and its replication partner helicase from the pathogenic Gram-positive bacteria Staphylococcus aureus were compared in detail with that of E. coli primase and helicase. The conserved properties were to primer initiation and elongation and included slow kinetics, low fidelity and poor sugar specificity. The significant differences included S. aureus primase having sixfold higher kinetic affinity for its template than E. coli primase under equivalent conditions. This naturally higher activity was balanced by its fourfold lower stimulation by its replication fork helicase compared with E. coli primase. The most significant difference between the two primases was that S. aureus helicase stimulation did not broaden the S. aureus primase initiation specificity, which has important biological implications.

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Cell architecture comes to phage biology.

Publication Date: 2008 Jun PMID: 18435709
Authors: Hendrix, R.
Journal: Mol Microbiol

Nothing is more iconic of the early days of molecular biology than the image of a bacteriophage infecting an Escherichia coli cell. It is perhaps surprising therefore that more than 50 years later, it is still possible to learn something entirely new and unexpected about how phage infection works, as we see in the paper by Edgar et al. of this issue of Molecular Microbiology. The results give fundamental new insight into the way these viruses infect their hosts and promise to open new windows on the virus-host interactions that have shaped the evolution of both.

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Biosynthesis of the Escherichia coli K1 group 2 polysialic acid capsule occurs within a protected cytoplasmic compartment.

Publication Date: 2008 Jun PMID: 18435708
Authors: Steenbergen, S. M. - Vimr, E. R.
Journal: Mol Microbiol

Capsular polysaccharides are important virulence determinants in a wide range of invasive infectious diseases. Although capsule synthesis has been extensively investigated, understanding polysaccharide export from the cytoplasm to the external environment has been more difficult. Here we present the results of a novel protection assay indicating that synthesis and export of the Escherichia coli K1 group 2 capsular polysialic acid (K1 antigen) occur within a protected subcellular compartment designated the sialisome. In addition to the polymerase encoded by neuS, localization and complementation analyses indicated that the sialisome includes the accessory membrane protein NeuE. The requirement for NeuE was suppressed by overproducing NeuS, suggesting that NeuE functions by stabilizing the polymerase or facilitating its assembly in the sialisome. Although an interaction between NeuE and NeuS could not be demonstrated with a bacterial two-hybrid system that reconstitutes an intracellular cell-signalling pathway, interactions between NeuS and KpsC as well as other sialisome components were detected. The combined results provide direct evidence for specific protein-protein interactions in the synthesis and export of group 2 capsular polysaccharides under in vivo conditions. The approaches developed here will facilitate further dissection of the sialisome, suggesting similar methodology for understanding the biosynthesis of other group 2 capsules.

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Evolution of a new enzyme activity from the same motif fold.

Publication Date: 2008 Apr 21 PMID: 18433454
Authors: Leiman, P. G. - Molineux, I. J.
Journal: Mol Microbiol

The host cell recognition protein of the Escherichia coli bacteriophage HK620 is a large homotrimeric tailspike that cleaves the O18A1 type O antigen. The crystal structure of HK620 tailspike determined in the apo and substrate-bound form is reported by Barbirz et al. in this issue of Molecular Microbiology. Lacking detectable sequence similarity, the fold and overall organization of the HK620 tailspike are similar to those of the tailspikes of the related phages P22 and Sf6. The substrate-binding site is intra-subunit in P22 and HK620 tailspikes, but inter-subunit in Sf6, demonstrating how phages can adapt the same protein fold to recognize different substrates.

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Systematic functional analysis of calcium signaling proteins in the genome of the rice blast fungus, Magnaporthe oryzae, using a high-throughput RNA silencing system.

Publication Date: 2008 Apr 21 PMID: 18433453
Authors: Quoc, N. B. - Kadotani, N. - Kasahara, S. - Tosa, Y. - Mayama, S. - Nakayashiki, H.
Journal: Mol Microbiol

We developed an RNA silencing vector, pSilent-Dual1 (pSD1), with a convergent dual Pol II promoter system that provides a high-throughput platform for functional genomics research in filamentous fungi. In the pSD1 system, the target gene was designed to be transcribed as a chimeric RNA with enhanced green fluorescent protein (eGFP) RNA. This enabled us to efficiently screen the resulting transformants using GFP fluorescence as an indicator of gene silencing. A model study with the eGFP gene showed that pSD1-based vectors induced gene silencing via the RNAi pathway with slightly lower efficiency than did hairpin eGFP RNA-expressing vectors. To demonstrate the applicability of the pSD1 system for elucidating gene function in the rice blast fungus Magnaporthe oryzae, 37 calcium signaling-related genes that include almost all known calcium signaling proteins in the genome were targeted for gene silencing by the vector. Phenotypic analyses of the silenced transformants showed that at least 26, 35, and 15 of the 37 genes examined were involved in hyphal growth, sporulation and pathogenicity, respectively, in M. oryzae. These included several novel findings such as that Pmc1-, Spf1- and Neo1-like Ca(2+) pumps, calreticulin, and calpactin heavy chain were essential for fungal pathogenicity.

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An amphiphilic region in the cytoplasmic domain of KdpD is recognized by the signal recognition particle and targeted to the Escherichia coli membrane.

Publication Date: 2008 Apr 29 PMID: 18433452
Authors: Maier, K. S. - Hubich, S. - Liebhart, H. - Krauss, S. - Kuhn, A. - Facey, S. J.
Journal: Mol Microbiol

The sensor protein KdpD of Escherichia coli is composed of a large N-terminal hydrophilic region (aa 1-400), four transmembrane regions (aa 401-498) and a large hydrophilic region (aa 499-894) at the C-terminus. KdpD requires the signal recognition particle (SRP) for its targeting to the membrane. Deletions within KdpD show that the first 50 residues are required for SRP-driven membrane insertion. A fusion protein of the green fluorescent protein (GFP) with KdpD is found localized at the membrane only when SRP is present. The membrane targeting of GFP was not observed when the first 50 KdpD residues were deleted. A truncated mutant of KdpD containing only the first 25 amino acids fused to GFP lost its ability to specifically interact with SRP, whereas a specific interaction between SRP and the first 48 amino acids of KdpD fused to GFP was confirmed by pull-down experiments. Conclusively, a small amphiphilic region of 27 residues within the amino-terminal domain of KdpD (aa 22-48) is recognized by SRP and targets the protein to the membrane. This shows that membrane proteins with a large N-terminal region in the cytoplasm can be membrane-targeted early on to allow co-translational membrane insertion of their distant transmembrane regions.

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Plasmodium falciparum antigenic variation. Mapping mosaic var gene sequences onto a network of shared, highly polymorphic sequence blocks.

Publication Date: 2008 Apr 28 PMID: 18433451
Authors: Bull, P. C. - Buckee, C. O. - Kyes, S. - Kortok, M. M. - Thathy, V. - Guyah, B. - Stoute, J. A. - Newbold, C. I. - Marsh, K.
Journal: Mol Microbiol

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, encoded by an extremely diverse gene family called var. Understanding of the genetic organization of var genes is hampered by sequence mosaicism that results from a long history of non-homologous recombination. Here we have used software designed to analyse social networks to visualize the relationships between large collections of short var sequences tags sampled from clinical parasite isolates. In this approach, two sequences are connected if they share one or more highly polymorphic sequence blocks. The results show that the majority of analysed sequences including several var-like sequences from the chimpanzee parasite Plasmodium reichenowi can be either directly or indirectly linked together in a single unbroken network. However, the network is highly structured and contains putative subgroups of recombining sequences. The major subgroup contains the previously described group A var genes, previously proposed to be genetically distinct. Another subgroup contains sequences found to be associated with rosetting, a parasite virulence phenotype. The mosaic structure of the sequences and their division into subgroups may reflect the conflicting problems of maximizing antigenic diversity and minimizing epitope sharing between variants while maintaining their host cell binding functions.

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The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements.

Publication Date: 2008 Apr 28 PMID: 18433450
Authors: Behnke, M. S. - Radke, J. B. - Smith, A. T. - Sullivan, W. J. Jr - White, M. W.
Journal: Mol Microbiol

Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis-elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii, we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis-acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6-8 bp resolution and these minimal cis-elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis-elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a 'poised' chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.

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The identity of the transcription +1 position is crucial for changes in gene expression in response to amino acid starvation in Bacillus subtilis.

Publication Date: 2008 Apr 29 PMID: 18433449
Authors: Krasny, L. - Tiserova, H. - Jonak, J. - Rejman, D. - Sanderova, H.
Journal: Mol Microbiol

We identify here a pattern in the transcription start sites (+1A or +1G) of sigma(A)-dependent promoters of genes that are up-/downregulated in response to amino acid starvation (stringent response) in Bacillus subtilis. Upregulated promoters initiate mostly with ATP and downregulated promoters with GTP. These promoters appear to be sensitive to changes in initiating nucleoside triphosphate concentrations. During the stringent response in B. subtilis, when ATP and GTP levels change reciprocally, the identity of the +1 position (A or G) of these promoters is a factor important in their regulation. Mutations that change the identity of position +1 (A for G and vice versa) change the response of the promoter to amino acid starvation.

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Silencing the crowd: high-throughput functional genomics in Magnaporthe oryzae.

Publication Date: 2008 May 6 PMID: 18433448
Authors: Caracuel-Rios, Z. - Talbot, N. J.
Journal: Mol Microbiol

A new high-throughput RNA-silencing system has been developed for use in the rice blast fungus Magnaporthe oryzae, allowing rapid generation of transformants in which individual genes have been silenced. Development of this system will allow large-scale functional analysis of genes in the fungus to define the cellular processes required for plant infection and disease symptoms. Functional analysis of 37 genes predicted to be involved in calcium signalling was carried out by RNA silencing to validate the new strategy and has provided new insight into the role of calcium-mediated signal transduction in plant pathogenic fungi.

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Ancestral roles of eukaryotic frataxin: mitochondrial frataxin function and heterologous expression of hydrogenosomal Trichomonas homologues in trypanosomes.

Publication Date: 2008 May 6 PMID: 18433447
Authors: Long, S. - Jirku, M. - Mach, J. - Ginger, M. L. - Sutak, R. - Richardson, D. - Tachezy, J. - Lukes, J.
Journal: Mol Microbiol

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe-S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin-depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe-S cluster biogenesis and protection from reactive oxygen species.

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Co-regulation of yeast purine and phosphate pathways in response to adenylic nucleotide variations.

Publication Date: 2008 May 6 PMID: 18433446
Authors: Gauthier, S. - Coulpier, F. - Jourdren, L. - Merle, M. - Beck, S. - Konrad, M. - Daignan-Fornier, B. - Pinson, B.
Journal: Mol Microbiol

Adenylate kinase (Adk1p) is a pivotal enzyme in both energetic and adenylic nucleotide metabolisms. In this paper, using a transcriptomic analysis, we show that the lack of Adk1p strongly induced expression of the PHO and ADE genes involved in phosphate utilization and AMP de novo biosynthesis respectively. Isolation and characterization of adk1 point mutants affecting PHO5 expression revealed that all these mutations also severely affected Adk1p catalytic activity, as well as PHO84 and ADE1 transcription. Furthermore, overexpression of distantly related enzymes such as human adenylate kinase or yeast UMP kinase was sufficient to restore regulation. These results demonstrate that adenylate kinase catalytic activity is critical for proper regulation of the PHO and ADE pathways. We also establish that adk1 deletion and purine limitation have similar effects on both adenylic nucleotide pool and PHO84 or ADE17 expression. Finally, we show that, in the adk1 mutant, upregulation of ADE1 depends on synthesis of the previously described effector(s) (S)AICAR ((N-succinyl)-5-aminoimidazol-4-carboxamide ribotide), while upregulation of PHO84 necessitates the Spl2p positive regulator. This work reveals that adenylic nucleotide availability is a key signal used by yeast to co-ordinate phosphate utilization and purine synthesis.

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Amino-acid dependent growth of Campylobacter jejuni : Key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate.

Publication Date: 2008 Apr 21 PMID: 18433445
Authors: Guccione, E. - Del Rocio Leon-Kempis, M. - Pearson, B. M. - Hitchin, E. - Mulholland, F. - van Diemen, P. M. - Stevens, M. P. - Kelly, D. J.
Journal: Mol Microbiol

Amino-acids are key carbon and energy sources for the asaccharolytic food-borne human pathogen Campylobacter jejuni. During microaerobic growth in amino-acid rich complex media, aspartate, glutamate, proline and serine are the only amino-acids significantly utilised by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase (aspA) mutant (unable to utilise any amino-acid except serine) and a Cj0762c (aspB) mutant lacking aspartate:glutamate aminotransferase (unable to utilise glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be up-regulated by oxygen-limitation, and aspartate enhanced oxygen-limited growth of C. jejuni in an aspA dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81-176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.

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Fine-tuned growth phase control of dps, encoding a DNA protection protein, by FIS and H-NS.

Publication Date: 2008 May 6 PMID: 18433444
Authors: Schnetz, K.
Journal: Mol Microbiol

Dps, a ferritin-like DNA-binding protein, protects bacterial DNA against oxidative stress and hyper-compacts the nucleoid into a crystalline-like structure. In Escherichia coli, transcription of dps from a single promoter is directed by sigmaS-RNA polymerase in stationary phase and by sigma70-RNA polymerase in fast-dividing cells exposed to oxidative stress. In this issue of Molecular Microbiology, Grainger and co-workers demonstrate that the nucleoid-associated proteins, FIS and H-NS, control sigma factor selection at the dps promoter by a novel mechanism. The finding suggests a simple mechanism of growth phase control of Dps production and has implications for the response to oxidative stress by pathogenic enterobacteria.

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Identification and characterization of KpsS, a novel polysaccharide sulphotransferase in Mesorhizobium loti.

Publication Date: 2008 Jun PMID: 18430142
Authors: Townsend, G. E. 2nd - Keating, D. H.
Journal: Mol Microbiol

Plants enter into symbiotic relationships with bacteria that allow survival in nutrient-limiting environments. The bacterium Mesorhizobium loti enters into a symbiosis with the legume host, Lotus japonicus, which results in the formation of novel plant structures called root nodules. The bacteria colonize the nodules, and are internalized into the cytoplasm of the plant cells, where they reduce molecular dinitrogen for the plant. Symbiosis between M. loti and L. japonicus requires bacterial synthesis of secreted and cell-surface polysaccharides. We previously reported the identification of an unusual sulphate-modified form of capsular polysaccharide (KPS) in M. loti. To better understand the physiological function of sulphated KPS, we isolated the sulphotransferase responsible for KPS sulphation from M. loti extracts, determined its amino acid sequence and identified the corresponding M. loti open reading frame, mll7563 (which we have named kpsS). We demonstrated that partially purified KpsS functions as a fucosyl sulphotransferase in vitro. Furthermore, mutants deficient for this gene exhibit a lack of KPS sulphation and a decreased rate of nodule formation on L. japonicus. Interestingly, the kpsS gene product shares no significant amino acid similarity with previously identified sulphotransferases, but exhibited sequence identity to open reading frames of unknown function in diverse bacteria that interact with eukaryotes.

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A Csr-type regulatory system, including small non-coding RNAs, regulates the global virulence regulator RovA of Yersinia pseudotuberculosis through RovM.

Publication Date: 2008 Jun PMID: 18430141
Authors: Heroven, A. K. - Bohme, K. - Rohde, M. - Dersch, P.
Journal: Mol Microbiol

The MarR-type regulator RovA controls expression of virulence genes of Yersinia pseudotuberculosis in response to environmental signals. Using a genetic strategy to discover components that influence rovA expression, we identified new regulatory factors with homology to components of the carbon storage regulator system (Csr). We showed that overexpression of a CsrB- or a CsrC-type RNA activates rovA, whereas a CsrA-like protein represses RovA synthesis. We further demonstrate that influence of the Csr system on rovA is indirect and occurs through control of the LysR regulator RovM, which inhibits rovA transcription. The CsrA protein had also a major influence on the motility of Yersinia, which was independent of RovM. The CsrB and CsrC RNAs are differentially expressed in Yersinia. CsrC is highly induced in complex but not in minimal media, indicating that medium-dependent rovM expression is mediated through CsrC. CsrB synthesis is generally very low. However, overexpression of the response regulator UvrY was found to activate CsrB production, which in turn represses CsrC synthesis independent of the growth medium. In summary, the post-transcriptional Csr-type components were shown to be key regulators in the co-ordinated environmental control of physiological processes and virulence factors, which are crucial for the initiation of Yersinia infections.

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Trypanosoma brucei BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination.

Publication Date: 2008 Jun PMID: 18430140
Authors: Hartley, C. L. - McCulloch, R.
Journal: Mol Microbiol

Antigenic variation in Trypanosoma brucei has selected for the evolution of a massive archive of silent Variant Surface Glycoprotein (VSG) genes, which are activated by recombination into specialized expression sites. Such VSG switching can occur at rates substantially higher than background mutation and is dependent on homologous recombination, a core DNA repair reaction. A key regulator of homologous recombination is BRCA2, a protein that binds RAD51, the enzyme responsible for DNA strand exchange. Here, we show that T. brucei BRCA2 has undergone a recent, striking expansion in the number of BRC repeats, a sequence element that mediates interaction with RAD51. T. brucei BRCA2 mutants are shown to be significantly impaired in antigenic variation and display genome instability. By generating BRCA2 variants with reduced BRC repeat numbers, we show that the BRC expansion is crucial in determining the efficiency of T. brucei homologous recombination and RAD51 localization. Remarkably, however, this appears not to be a major determinant of the activation of at least some VSG genes.

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Lipid spirals in Bacillus subtilis and their role in cell division.

Publication Date: 2008 Jun PMID: 18430139
Authors: Barak, I. - Muchova, K. - Wilkinson, A. J. - O'Toole, P. J. - Pavlendova, N.
Journal: Mol Microbiol

The fluid mosaic model of membrane structure has been revised in recent years as it has become evident that domains of different lipid composition are present in eukaryotic and prokaryotic cells. Using membrane binding fluorescent dyes, we demonstrate the presence of lipid spirals extending along the long axis of cells of the rod-shaped bacterium Bacillus subtilis. These spiral structures are absent from cells in which the synthesis of phosphatidylglycerol is disrupted, suggesting an enrichment in anionic phospholipids. Green fluorescent protein fusions of the cell division protein MinD also form spiral structures and these were shown by fluorescence resonance energy transfer to be coincident with the lipid spirals. These data indicate a higher level of membrane lipid organization than previously observed and a primary role for lipid spirals in determining the site of cell division in bacterial cells.

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The type III secretion system tip complex and translocon.

Publication Date: 2008 Jun PMID: 18430138
Authors: Mueller, C. A. - Broz, P. - Cornelis, G. R.
Journal: Mol Microbiol

The type III secretion machinery of Gram-negative bacteria, also known as the injectisome or needle complex, is composed of a basal body spanning both bacterial membranes and the periplasm, and an external needle protruding from the bacterial surface. A set of three proteins, two hydrophobic and one hydrophilic, are required to allow translocation of proteins from the bacterium to the host cell cytoplasm. These proteins are involved in the formation of a translocation pore, the translocon, in the host cell membrane. Exciting progress has recently been made on the interaction between the translocators and the injectisome needle and the assembly of the translocon in the host cell membrane. As expected, the two hydrophobic translocators insert into the target cell membrane. Unexpectedly, the third, hydrophilic translocator, forms a complex on the distal end of the injectisome needle, the tip complex, and serves as an assembly platform for the two hydrophobic translocators.

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Mini-III, a fourth class of RNase III catalyses maturation of the Bacillus subtilis 23S ribosomal RNA.

Publication Date: 2008 Jun PMID: 18430137
Authors: Olmedo, G. - Guzman, P.
Journal: Mol Microbiol

Ribonuclease III (RNase III) type of enzymes are double-stranded RNA (dsRNA)-specific endoribonucleases that have important roles in RNA maturation and mRNA decay. They are involved in processing precursors of ribosomal RNA (rRNA) in bacteria as well as precursors of short interfering RNAs (siRNAs) and microRNAs (miRNAs) in eukaryotes. RNase III proteins have been grouped in three major classes according to their domain organization. In this issue of Molecular Microbiology, Redko et al. identified a novel class of bacterial RNase III, named Mini-III, consisting only of the RNase III catalytic domain and functioning in the maturation of the 23S rRNA in Bacillus subtilis. Its absence from proteobacteria reveals that this step is mechanistically different from the corresponding step in Escherichia coli. The fact that Mini-III orthologues are present in unicellular photosynthetic eukaryotes and in plants opens new opportunities for functional studies of this type of RNases.

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Fold and function of polypeptide transport-associated domains responsible for delivering unfolded proteins to membranes.

Publication Date: 2008 Jun PMID: 18430136
Authors: Knowles, T. J. - Jeeves, M. - Bobat, S. - Dancea, F. - McClelland, D. - Palmer, T. - Overduin, M. - Henderson, I. R.
Journal: Mol Microbiol

Membranes of Gram-negative bacteria, mitochondria and chloroplasts receive and fold beta-barrel transmembrane proteins through the action of polypeptide transport-associated (POTRA) domains. In Escherichia coli, folding substrates are inserted into the outer membrane by the essential protein YaeT, a prototypic Omp85 protein. Here, the articulation between tandem POTRA domains in solution is defined by nuclear magnetic resonance (NMR) spectroscopy, indicating an unprecedented juxtaposition. The novel solution orientations of all five POTRA domains are revealed by small-angle X-ray scattering of the entire 46 kDa periplasmic region. NMR titration studies show that strands from YaeT's canonical folding substrate, PhoE, bind non-specifically along alternating sides of its mixed beta sheets, thus providing an ideal platform for helping to fold nascent outer-membrane proteins. Together, this provides the first structural model of how multiple POTRA domains recruit substrates from the periplasmic solution into the outer membrane.

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The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli.

Publication Date: 2008 Jun PMID: 18430135
Authors: Traxler, M. F. - Summers, S. M. - Nguyen, H. T. - Zacharia, V. M. - Hightower, G. A. - Smith, J. T. - Conway, T.
Journal: Mol Microbiol

The stringent response to amino acid starvation, whereby stable RNA synthesis is curtailed in favour of transcription of amino acid biosynthetic genes, is controlled by the alarmone ppGpp. To elucidate the extent of gene expression effected by ppGpp, we designed an experimental system based on starvation for isoleucine, which could be applied to both wild-type Escherichia coli and the multiauxotrophic relA spoT mutant (ppGpp(0)). We used microarrays to profile the response to amino acid starvation in both strains. The wild-type response included induction of the general stress response, downregulation of genes involved in production of macromolecular structures and comprehensive restructuring of metabolic gene expression, but not induction of amino acid biosynthesis genes en masse. This restructuring of metabolism was confirmed using kinetic Biolog assays. These responses were profoundly altered in the ppGpp(0) strain. Furthermore, upon isoleucine starvation, the ppGpp(0) strain exhibited a larger cell size and continued growth, ultimately producing 50% more biomass than the wild-type, despite producing a similar amount of protein. This mutant phenotype correlated with aberrant gene expression in diverse processes, including DNA replication, cell division, and fatty acid and membrane biosynthesis. We present a model that expands and functionally integrates the ppGpp-mediated stringent response to include control of virtually all macromolecular synthesis and intermediary metabolism.

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The receiver domain of FrzE, a CheA-CheY fusion protein, regulates the CheA histidine kinase activity and downstream signalling to the A- and S-motility systems of Myxococcus xanthus.

Publication Date: 2008 Jun PMID: 18430134
Authors: Inclan, Y. F. - Laurent, S. - Zusman, D. R.
Journal: Mol Microbiol

The Frz chemosensory system is a two-component signal transduction pathway that controls cell reversals and directional movements for the two motility systems in Myxococcus xanthus. To trigger cell reversals, FrzE, a hybrid CheA-CheY fusion protein, autophosphorylates the kinase domain at His-49, and phosphoryl groups are transferred to aspartate residues (Asp-52 and Asp-220) in the two receiver domains of FrzZ, a dual CheY-like protein that serves as the pathway output. The role of the receiver domain of FrzE was unknown. In this paper, we characterize the FrzE protein in vitro and show that the receiver domain of FrzE negatively regulates the autophosphorylation activity of the kinase domain of FrzE. Unexpectedly, it does not appear to play a direct role in phospho-relay as in most other histidine kinase receiver domain hybrid systems. The regulatory role of the FrzE receiver domain suggests that it may interact with or be phosphorylated by an unknown protein. We also show the dynamics of motility system-specific marker proteins in FrzE mutants as cells move forward and reverse. Our studies indicate that the two motility systems are functionally co-ordinated and that any system-specific branching of the pathway most likely occurs downstream of FrzE.

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A novel regulatory protein governing biofilm formation in Bacillus subtilis.

Publication Date: 2008 Jun PMID: 18430133
Authors: Chu, F. - Kearns, D. B. - McLoon, A. - Chai, Y. - Kolter, R. - Losick, R.
Journal: Mol Microbiol

Production of an extracellular matrix is a hallmark of biofilm formation. In the spore-forming bacterium Bacillus subtilis, the matrix consists of an exopolysaccharide, which is specified by the epsA-O operon, and a secreted protein TasA, which is encoded by the yqxM-sipW-tasA operon. Past and present evidence establish that the epsA-O and yqxM-sipW-tasA operons are controlled by the repressor proteins SinR and AbrB. Here, we report the identification of a novel regulatory protein Slr that promotes transcription of the yqxM-sipW-tasA operon but is not needed for expression of the epsA-O operon. We further show that the gene for Slr is itself under the negative control of SinR and AbrB. These findings reveal that matrix production is governed by an intricate network involving the interplay of negatively and positively acting regulatory proteins.

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Loss of tafazzin in yeast leads to increased oxidative stress during respiratory growth.

Publication Date: 2008 May PMID: 18430085
Authors: Chen, S. - He, Q. - Greenberg, M. L.
Journal: Mol Microbiol

The tafazzin (TAZ) gene is highly conserved from yeast to humans, and the yeast taz1 null mutant shows alterations in cardiolipin (CL) metabolism, mitochondrial dysfunction and stabilization of supercomplexes similar to those found in Barth syndrome, a human disorder resulting from loss of tafazzin. We have previously shown that the yeast tafazzin mutant taz1Delta, which cannot remodel CL, is ethanol-sensitive at elevated temperature. In the current report, we show that in response to ethanol, CL mutants taz1Delta as well as crd1Delta, which cannot synthesize CL, exhibited increased protein carbonylation, an indicator of reactive oxygen species (ROS). The increase in ROS is most likely not due to defective oxidant defence systems, as the CL mutants do not display sensitivity to paraquat, menadione or hydrogen peroxide (H2O2). Ethanol sensitivity and increased protein carbonylation in the taz1Delta mutant but not in crd1Delta can be rescued by supplementation with oleic acid, suggesting that oleoyl-CL and/or oleoyl-monolyso-CL enables growth of taz1Delta in ethanol by decreasing oxidative stress. Our findings of increased oxidative stress in the taz1Delta mutant during respiratory growth may have important implications for understanding the pathogenesis of Barth syndrome.

MeSH Categories: Acyltransferases/*genetics, Cardiolipins/genetics/metabolism, Ethanol/metabolism/pharmacology, *Heat, Hydrogen Peroxide/pharmacology, Oleic Acid/metabolism/pharmacology, Oxidative Stress/*genetics, Paraquat/pharmacology, Protein Carbonylation/genetics, Reactive Oxygen Species/metabolism, Saccharomyces cerevisiae/genetics/*growth & development/metabolism, Saccharomyces cerevisiae Proteins/*genetics, Vitamin K 3/pharmacology

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A novel autotransporter of uropathogenic Proteus mirabilis is both a cytotoxin and an agglutinin.

Publication Date: 2008 May PMID: 18430084
Authors: Alamuri, P. - Mobley, H. L.
Journal: Mol Microbiol

One of the six predicted Proteus mirabilis autotransporters (ATs), ORF c2341, is predicted to contain a serine protease motif and was earlier identified as an immunogenic outer membrane protein in P. mirabilis. The 3.2 kb gene encodes a 117 kDa protein with a 58-amino-acid-long signal peptide, a 75-kDa-long N-terminal passenger domain and a 30-kDa-long C-terminal translocator. Affinity-purified 110 kDa AT exhibited chymotrypsin-like activity and hydrolysed N-Suc-Ala-Ala-Pro-Phe-pNa and N-Suc-Ala-Ala-Pro-Leu-pNa with a K(M) of 22 muM and 31 muM, respectively, under optimal pH of 8.5-9.0 in a Ca(2+)-dependent manner. Activity was inhibited by subtilase-specific inhibitors leupeptin and chymostatin. Both the cell-associated and purified form elicited cytopathic effects on cultured kidney and bladder epithelial cells. Substrate hydrolysis as well as cytotoxicity was associated with the passenger domain and was compromised upon mutation of any of the catalytic residues (Ser366, His147 and Asp533). At alkaline pH and optimal cell density, the AT also promoted autoaggregation of P. mirabilis and this function was independent of its protease activity. Cytotoxicity, autoaggregation and virulence were significantly reduced in an isogenic pta mutant of P. mirabilis. Proteus toxic agglutinin (Pta) represents a novel autotransported cytotoxin with no bacterial homologues that works optimally in the alkalinized urinary tract, a characteristic of urease-mediated urea hydrolysis during P. mirabilis infection.

MeSH Categories: Agglutinins/chemistry/genetics/*metabolism, Amino Acid Motifs, Amino Acid Sequence, Animals, Bacterial Outer Membrane Proteins/chemistry/genetics/metabolism, Bacterial Toxins/chemistry/genetics/*metabolism, Disease Models, Animal, Humans, Mice, Mice, Inbred CBA, Molecular Sequence Data, Protein Sorting Signals, Protein Structure, Tertiary, Proteus Infections/microbiology, Proteus mirabilis/genetics/*metabolism/*pathogenicity, Substrate Specificity, Subtilisins/chemistry/genetics/*metabolism, Urinary Bladder/microbiology, Urinary Tract Infections/microbiology, Virulence/genetics

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The LAMP-like protein p67 plays an essential role in the lysosome of African trypanosomes.

Publication Date: 2008 May PMID: 18430083
Authors: Peck, R. F. - Shiflett, A. M. - Schwartz, K. J. - McCann, A. - Hajduk, S. L. - Bangs, J. D.
Journal: Mol Microbiol

RNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific approximately 90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (approximately 20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four- to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH--chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.

MeSH Categories: Amino Acid Sequence, Animals, Endocytosis, Gene Order, Genome, Protozoan, Humans, Hydrogen-Ion Concentration, Lipoproteins, HDL/immunology, Lysosome-Associated Membrane Glycoproteins/genetics/*metabolism, Lysosomes/*metabolism/ultrastructure, Molecular Sequence Data, Peptide Hydrolases/metabolism, Protozoan Proteins/genetics/*metabolism, RNA Interference, Trypanosoma brucei brucei/genetics/*metabolism/ultrastructure, Trypanosomiasis, African/*parasitology

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Mycothiol regulates and is regulated by a thiol-specific antisigma factor RsrA and sigma(R) in Streptomyces coelicolor.

Publication Date: 2008 May PMID: 18430082
Authors: Park, J. H. - Roe, J. H.
Journal: Mol Microbiol

Mycothiol (MSH) is a small thiol molecule with a cysteine-ligated disaccharide structure found in actinomycetes that include streptomycetes and mycobacteria. In Streptomyces coelicolor, a model organism for antibiotic production and differentiation, the amount of MSH is under the control of a sigma factor sigma(R), which is regulated by an antisigma factor RsrA with a thiol-disulphide redox switch. We found that the first gene (mshA) in the biosynthetic pathway for MSH and the gene for amidase (mca) that participates in detoxifying mycothiol-reactive drugs are under direct control of sigma(R). The sigma(R) target genes are induced not only by a thiol oxidant diamide, but also by alkylating agents that cause a rapid decrease in MSH. Expression of the sigma(R) regulon was also elevated in MSH-deficient mutants, suggesting that a decrease in the level of MSH is a natural intracellular trigger for sigma(R) activation. We found that MSH was capable of reducing RsrA to bind sigma(R), whereas glutathione was not. These results support a proposal that the RsrA-sigma(R) system senses the intracellular level of reduced MSH, and that MSH serves as a natural modulator of the transcription system for its own replenishment in addition to being a redox buffer and drug detoxifier.

MeSH Categories: Alkylating Agents/pharmacology, Amidohydrolases/genetics/metabolism, Anti-Bacterial Agents/pharmacology, Bacterial Proteins/*metabolism, Cysteine/*genetics/*metabolism, Drug Resistance, Bacterial/genetics, *Gene Expression Regulation, Bacterial, Glycopeptides/*genetics/*metabolism, Inositol/*genetics/*metabolism, Mutation, *Regulon, Sigma Factor/*metabolism, Streptomyces coelicolor/drug effects/*genetics/metabolism, Transcription Factors/*metabolism

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PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum.

Publication Date: 2008 May PMID: 18430081
Authors: Hillmann, F. - Fischer, R. J. - Saint-Prix, F. - Girbal, L. - Bahl, H.
Journal: Mol Microbiol

Clostridia belong to those bacteria which are considered as obligate anaerobe, e.g. oxygen is harmful or lethal to these bacteria. Nevertheless, it is known that they can survive limited exposure to air, and often eliminate oxygen or reactive derivatives via NAD(P)H-dependent reduction. This system does apparently contribute to survival after oxidative stress, but is insufficient to establish long-term tolerance of aerobic conditions. Here we show that manipulation of the regulatory mechanism of this defence mechanism can trigger aerotolerance in the obligate anaerobe Clostridium acetobutylicum. Deletion of a peroxide repressor (PerR)-homologous protein resulted in prolonged aerotolerance, limited growth under aerobic conditions and rapid consumption of oxygen from an aerobic environment. The mutant strain also revealed higher resistance to H2O2 and activities of NADH-dependent scavenging of H2O2 and organic peroxides in cell-free extracts increased by at least one order of magnitude. Several genes encoding the putative enzymes were upregulated and identified as members of the clostridial PerR regulon, including the heat shock protein Hsp21, a reverse rubrerythrin which was massively produced and became the most abundant protein in the absence of PerR. This multifunctional protein is proposed to play the crucial role in the oxidative stress defence.

MeSH Categories: Aerobiosis/genetics, Anaerobiosis/genetics, Bacterial Proteins/genetics/*metabolism, Clostridium acetobutylicum/drug effects/*genetics, Gene Deletion, *Gene Expression Regulation, Bacterial, Genes, Bacterial, Hydrogen Peroxide/pharmacology, Oxidative Stress/*genetics, Oxygen/metabolism, Reactive Oxygen Species/metabolism, *Regulon, Repressor Proteins/genetics/*metabolism, Transcription Factors/genetics/*metabolism

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LysM, a widely distributed protein motif for binding to (peptido)glycans.

Publication Date: 2008 May PMID: 18430080
Authors: Buist, G. - Steen, A. - Kok, J. - Kuipers, O. P.
Journal: Mol Microbiol

Bacteria retain certain proteins at their cell envelopes by attaching them in a non-covalent manner to peptidoglycan, using specific protein domains, such as the prominent LysM (Lysin Motif) domain. More than 4000 (Pfam PF01476) proteins of both prokaryotes and eukaryotes have been found to contain one or more Lysin Motifs. Notably, this collection contains not only truly secreted proteins, but also (outer-)membrane proteins, lipoproteins or proteins bound to the cell wall in a (non-)covalent manner. The motif typically ranges in length from 44 to 65 amino acid residues and binds to various types of peptidoglycan and chitin, most likely recognizing the N-acetylglucosamine moiety. Most bacterial LysM-containing proteins are peptidoglycan hydrolases with various cleavage specificities. Binding of certain LysM proteins to cells of Gram-positive bacteria has been shown to occur at specific sites, as binding elsewhere is hindered by the presence of other cell wall components such as lipoteichoic acids. Interestingly, LysM domains of certain plant kinases enable the plant to recognize its symbiotic bacteria or sense and induce resistance against fungi. This interaction is triggered by chitin-like compounds that are secreted by the symbiotic bacteria or released from fungi, demonstrating an important sensing function of LysMs.

MeSH Categories: Acetylglucosamine/metabolism, *Amino Acid Motifs, Animals, Bacterial Proteins/chemistry/metabolism, Consensus Sequence, Evolution, Molecular, Peptidoglycan/*metabolism, Protein Structure, Tertiary, Proteins/metabolism, Repetitive Sequences, Amino Acid, Substrate Specificity

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The 'core' and 'accessory' regulons of Pseudomonas-specific extracytoplasmic sigma factors.

Publication Date: 2008 May PMID: 18430079
Authors: Cornelis, P.
Journal: Mol Microbiol

Pyoverdine is a fluorescent, high-affinity peptide siderophore produced by different Pseudomonas species. The genes for pyoverdine biosynthesis depend on PvdS, an extracytoplasmic sigma factor. In this issue of Molecular Microbiology, Swingle et al. demonstrate that in the plant pathogen Pseudomonas syringae PvdS not only regulates the production of pyoverdine (core regulon), but also controls expression of other genes likely to be involved in the adaptation to the environment (accessory regulon). This accessory regulon is variable, as different sets of genes seem to be recruited according to the Pseudomonas species and its specific ecological niche.

MeSH Categories: Adaptation, Physiological/genetics, Cytoplasm/metabolism, *Gene Expression Regulation, Bacterial, Oligopeptides/biosynthesis/*genetics, Pseudomonas syringae/genetics/metabolism/*physiology, *Regulon, Sigma Factor/*metabolism

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Regulation of mycothiol metabolism by sigma(R) and the thiol redox sensor anti-sigma factor RsrA.

Publication Date: 2008 May PMID: 18430078
Authors: Newton, G. L. - Fahey, R. C.
Journal: Mol Microbiol

Mycothiol (MSH) is the major thiol in Actinobacteria and plays a role analogous to that of glutathione. The biosynthetic pathway has been established in mycobacteria and is initiated by the glycosyltransferase MshA. A key mycothiol-dependent detoxification pathway utilizes the amidase (Mca) to cleave mycothiol S-conjugates to produce GlcN-Ins and a mercapturic acid excreted from the cell. How expression of mycothiol genes is regulated in mycobacteria has been unclear so the report in this issue by Park and Roe showing that in Streptomyces coelicolor the redox controlled anti-sigma factor RsrA that binds the regulator sigma(R) controls key elements of mycothiol metabolism is a major advance. Conditions that deplete thiols are shown to induce directly expression of sigR, rsrA, mshA and mca, as well as the thioredoxin reductase-thioredoxin system, generating an autoregulatory cycle that persists until the thiol-depleting condition is alleviated. Evidence for indirect induction of mshB-D to support mycothiol biosynthesis is also presented. It was shown in vitro that mycothiol, like reduced thioredoxin and dithiothreitol, can reduce oxidized RsrA to activate its binding to sigma(R). These studies establish for the first time how mycothiol metabolism is regulated to cope with stress from thiol reactive toxins.

MeSH Categories: Actinobacteria/genetics/*metabolism, Bacterial Proteins/*metabolism, Cysteine/*genetics/*metabolism, *Gene Expression Regulation, Bacterial, Glycopeptides/*genetics/*metabolism, Inositol/*genetics/*metabolism, Sigma Factor/*metabolism, Streptomyces coelicolor/genetics/metabolism, Sulfhydryl Compounds/metabolism, Transcription Factors/*metabolism

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The essentiality and involvement of Streptococcus intermedius histone-like DNA-binding protein in bacterial viability and normal growth.

Publication Date: 2008 Jun PMID: 18410499
Authors: Liu, D. - Yumoto, H. - Murakami, K. - Hirota, K. - Ono, T. - Nagamune, H. - Kayama, S. - Matsuo, T. - Miyake, Y.
Journal: Mol Microbiol

Streptococcus intermedius histone-like DNA-binding protein (Si-HLP) is a homodimeric protein and, conserved with Escherichia coli HU, a well-documented nucleoid-associated protein (NAP). In E. coli, HU plays important roles as both structural and regulatory factors, but it is not essential for E. coli viability. Streptococcal HLP has been found to bind host cells and induce cytokine production, but its physiological role remains poorly defined. In the present study, using gene insertion knockout and tetracycline-regulated antisense RNA expression techniques, we determined whether Si-HLP is essential for bacterial viability and normal growth in S. intermedius. The Si-HLP-downregulated S. intermedius strain showed alterations in its morphology and surface properties. Downregulation of Si-HLP led to an expanded nucleoid to fill the intracellular space. Transcription levels of several genes, including virulence-associated factors, were found to be activated or repressed in the antisense Si-hlp RNA-expressing strain by real-time PCR and reverse-transcription PCR. Collectively, these data suggest that Si-HLP serves as an essential NAP governing the nucleoid architecture and controlling the gene transcription profile in S. intermedius.

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The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes.

Publication Date: 2008 Jun PMID: 18410498
Authors: Spycher, C. - Rug, M. - Pachlatko, E. - Hanssen, E. - Ferguson, D. - Cowman, A. F. - Tilley, L. - Beck, H. P.
Journal: Mol Microbiol

During the intra-erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein-trafficking system beyond its confines. Membrane-bound structures called Maurer's clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane-associated histidine-rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurer's cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurer's cleft-resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurer's clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1-mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.

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DNA dynamics vary according to macrodomain topography in the E. coli chromosome.

Publication Date: 2008 Apr 28 PMID: 18410497
Authors: Espeli, O. - Mercier, R. - Boccard, F.
Journal: Mol Microbiol

The organization of the Escherichia coli chromosome has been defined genetically as consisting of four insulated macrodomains and two less constrained regions. Here we have examined the movement of chromosomal loci by tracking fluorescent markers in time-lapse microscopy during a complete cell cycle. Analysing the positioning, the segregation pattern and the motility of markers allowed us to show that the dynamic behaviour of loci belonging to various macrodomains and less constrained regions is radically different. In macrodomains constraints on mobility are apparent whereas in non-structured regions, markers exhibited a greater motility that may explain their ability to interact with flanking macrodomains. Following replication, duplicated markers belonging to macrodomains show a colocalization step and this landmark is not apparent in non-structured regions. Chromosome segregation occurs in three steps: first, the origin-proximal half of the chromosome consisting of the Ori macrodomain and the two non-structured region segregates concomitantly in a short period of time. Second, the Right and Left macrodomains segregate progressively following the genetic map. Third, the Ter macrodomain is rapidly segregated before division, after a significant period of colocalization. Macrodomain territories defined as cellular spaces occupied by the different macrodomains can be identified.

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Protein N-glycosylation determines functionality of the Saccharomyces cerevisiae cell wall integrity sensor Mid2p.

Publication Date: 2008 May 5 PMID: 18410496
Authors: Hutzler, F. - Gerstl, R. - Lommel, M. - Strahl, S.
Journal: Mol Microbiol

The fungal cell wall is a highly dynamic structure that is essential to maintain cell shape and stability. Hence in yeasts and fungi cell wall integrity is tightly controlled. The Saccharomyces cerevisiae plasma membrane protein Mid2p is a putative mechanosensor that responds to cell wall stresses and morphological changes during pheromone induction. The extracellular domain of Mid2p, which is crucial to sensing, is highly O- and N-glycosylated. We showed that O-mannosylation is determining stability of Mid2p. If and how N-glycosylation is linked to Mid2p function was unknown. Here we demonstrate that Mid2p contains a single high mannose N-linked glycan at position Asn-35. The N-glycan is located close to the N-terminus and is exposed from the plasma membrane towards the cell wall through a highly O-mannosylated domain that is predicted to adopt a rod-like conformation. In contrast to O-mannosylation, lack of the N-linked glycan affects neither, stability of Mid2p nor distribution at the plasma membrane during vegetative and sexual growth. However, non-N-glycosylated Mid2p fails to perceive cell wall challenges. Our data further demonstrate that both the extent of the N-linked glycan and its distance from the plasma membrane affect Mid2p function, suggesting the N-glycan to be directly involved in Mid2p sensing.

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The Ustilago maydis CysHis-type zinc finger transcription factor Mzr1 regulates fungal gene expression during the biotrophic growth stage.

Publication Date: 2008 Apr 18 PMID: 18410495
Authors: Zheng, Y. - Kief, J. - Auffarth, K. - Farfsing, J. W. - Mahlert, M. - Nieto, F. - Basse, C. W.
Journal: Mol Microbiol

The smut fungus Ustilago maydis establishes a biotrophic relationship with its host plant maize to progress through sexual development. Here, we report the identification and characterization of the Cys(2)His(2)-type zinc finger protein Mzr1 that functions as a transcriptional activator during host colonization. Expression of the U. maydis mig2 cluster genes is tightly linked to this phase. Upon conditional overexpression, Mzr1 confers induction of a subset of mig2 genes during vegetative growth and this requires the same promoter elements that confer inducible expression in planta. Furthermore, expression of the mig2-4 and mig2-5 genes during biotrophic growth is strongly reduced in cells deleted in mzr1. DNA-array analysis led to the identification of additional Mzr1-induced genes. Some of these genes show a mig2-like plant-specific expression pattern and Mzr1 is responsible for their high-level expression during pathogenesis. Mzr1 function requires the b-dependently regulated Cys(2)His(2)-type cell cycle regulator Biz1, indicating that two stage-specific regulators mediate gene expression during host colonization. In spite of a role as transcriptional activator during biotrophic growth, mzr1 is not essential for pathogenesis; however, conditional overexpression interfered with proliferation during vegetative growth and mating ability, caused a cell separation defect, and triggered filamentous growth. We discuss the implications of these findings.

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The role of laccase in prostaglandin production by Cryptococcus neoformans.

Publication Date: 2008 May 7 PMID: 18410494
Authors: Erb-Downward, J. R. - Noggle, R. M. - Williamson, P. R. - Huffnagle, G. B.
Journal: Mol Microbiol

Recently, it has been demonstrated that the opportunistic fungal pathogen Cryptococcus neoformans can synthesize authentic immunomodulatory prostaglandins. The mechanism by which this takes place is unclear as there is no cyclooxygenase homologue in the cryptococcal genome. In this study, we show that cryptococcal production of both PGE(2) and PGF(2alpha) can be chemically inhibited by caffeic acid, resveratrol and nordihydroguaiaretic acid. These polyphenolic molecules are frequently used as inhibitors of lipoxygenase enzymes; however, blast searches of the cryptococcal genome were unable to identify any homologues of mammalian, plant or fungal lipoxygenases. Next we investigated cryptococcal laccase, an enzyme known to bind polyphenols, and found that either antibody depletion or genetic deletion of the primary cryptococcal laccase (lac1Delta) resulted in a loss of cryptococcal prostaglandin production. To determine how laccase is involved, we tested recombinant laccase activity on the prostaglandin precursors, arachidonic acid (AA), PGG(2) and PGH(2). Using mass spectroscopy we determined that recombinant Lac1 does not modify AA or PGH(2), but does have a marked activity toward PGG(2) converting it to PGE(2) and 15-keto-PGE(2). These data demonstrate a critical role for laccase in cryptococcal prostaglandin production, and provides insight into a new and unique fungal prostaglandin pathway.

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Plasmodium falciparum Sec24 marks transitional ER that exports a model cargo via a diacidic motif.

Publication Date: 2008 Apr 29 PMID: 18410493
Authors: Lee, M. C. - Moura, P. A. - Miller, E. A. - Fidock, D. A.
Journal: Mol Microbiol

Exit from the endoplasmic reticulum (ER) often occurs at distinct sites of vesicle formation known as transitional ER (tER) that are enriched for COPII vesicle coat proteins. We have characterized the organization of ER export in the malaria parasite, Plasmodium falciparum, by examining the localization of two components of the COPII machinery, PfSec12 and PfSec24a. PfSec12 was found throughout the ER, whereas the COPII cargo adaptor, PfSec24a, was concentrated at distinct foci that likely correspond to tER sites. These foci were closely apposed to cis-Golgi sites marked by PfGRASP-GFP, and upon treatment with brefeldin A they accumulated a model cargo protein via a process dependent on the presence of an intact diacidic export motif. Our data suggest that the cargo-binding function of PfSec24a is conserved and that accumulation of cargo in discrete tER sites depends upon positive sorting signals. Furthermore, the number and position of tER sites with respect to the cis-Golgi suggests a co-ordinated biogenesis of these domains.

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The beta-1,3-glucanosyltransferase gas4p is essential for ascospore wall maturation and spore viability in Schizosaccharomyces pombe.

Publication Date: 2008 Jun PMID: 18410286
Authors: de Medina-Redondo, M. - Arnaiz-Pita, Y. - Fontaine, T. - Del Rey, F. - Latge, J. P. - Vazquez de Aldana, C. R.
Journal: Mol Microbiol

Meiosis is the developmental programme by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. The formation of the Schizosaccharomyces pombe ascospore wall requires the co-ordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. During sporogenesis, the beta-1,3-glucan synthase bgs2p synthesizes linear beta-1,3-glucans, which remain unorganized and alkali-soluble until covalent linkages are set up between beta-1,3-glucans and other cell wall components. Several proteins belonging to the glycoside hydrolase family 72 (GH72) with beta-1,3-glucanosyltransferase activity have been described in other organisms, such as the Saccharomyces cerevisiae Gas1p or the Aspergillus fumigatus Gel1p. Here we describe the characterization of gas4(+), a new gene that encodes a protein of the GH72 family. Deletion of this gene does not lead to any apparent defect during vegetative growth, but homozygous gas4Delta diploids show a sporulation defect. Although meiosis occurs normally, ascospores are unable to mature or to germinate. The expression of gas4(+) is strongly induced during sporulation and a yellow fluorescent protein (YFP)-gas4p fusion protein localizes to the ascospore periphery during sporulation. We conclude that gas4p is required for ascospore maturation in S. pombe.

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Systematic deletion of Salmonella small RNA genes identifies CyaR, a conserved CRP-dependent riboregulator of OmpX synthesis.

Publication Date: 2008 May PMID: 18399940
Authors: Papenfort, K. - Pfeiffer, V. - Lucchini, S. - Sonawane, A. - Hinton, J. C. - Vogel, J.
Journal: Mol Microbiol

Post-transcriptional repression of porin synthesis has emerged as a major function of Hfq-dependent, small non-coding RNAs (sRNAs). Many enterobacteria express OmpX-like porins, a family of outer membrane proteins whose physiological roles and structural properties have been studied intensively. While regulatory sRNAs have been identified for most major and many minor porins of Salmonella and Escherichia coli, a post-transcriptional regulator of OmpX levels has never been found. Here, we have taken a 'reverse target search' approach by systematic inactivation of Salmonella sRNA genes, and screening 35 sRNA deletion strains for effects on OmpX synthesis. We have identified the Hfq-dependent CyaR (formerly RyeE) sRNA as an ompX repressor. Global transcriptomic profiling following induction of CyaR expression suggests that ompX mRNA is the primary target of this sRNA under standard growth conditions. The results of phylogenetic and mutational analyses suggest that a conserved RNA hairpin of CyaR, featuring a C-rich apical loop, acts to sequester the Shine-Dalgarno sequence of ompX mRNA and to inhibit translational initiation. We have also discovered that cyaR expression is tightly controlled by the cyclic AMP receptor protein, CRP. This represents a new link between porin repression and nutrient availability that is likely to be widely conserved among enterobacteria.

MeSH Categories: 5' Untranslated Regions/genetics/metabolism, Bacterial Outer Membrane Proteins/biosynthesis/*genetics, Base Sequence, Cyclic AMP Receptor Protein/genetics/*metabolism, Gene Deletion, *Gene Expression Regulation, Bacterial, MicroRNAs/genetics/*metabolism, Molecular Sequence Data, Porins/biosynthesis/*genetics, RNA, Bacterial/genetics/*metabolism, Salmonella typhimurium/*genetics/metabolism, Transcription, Genetic

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Polarized growth in fungi--interplay between the cytoskeleton, positional markers and membrane domains.

Publication Date: 2008 May PMID: 18399939
Authors: Fischer, R. - Zekert, N. - Takeshita, N.
Journal: Mol Microbiol

One kind of the most extremely polarized cells in nature are the indefinitely growing hyphae of filamentous fungi. A continuous flow of secretion vesicles from the hyphal cell body to the growing hyphal tip is essential for cell wall and membrane extension. Because microtubules (MT) and actin, together with their corresponding motor proteins, are involved in the process, the arrangement of the cytoskeleton is a crucial step to establish and maintain polarity. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, actin-mediated vesicle transportation is sufficient for polar cell extension, but in S. pombe, MTs are in addition required for the establishment of polarity. The MT cytoskeleton delivers the so-called cell-end marker proteins to the cell pole, which in turn polarize the actin cytoskeleton. Latest results suggest that this scenario may principally be conserved from S. pombe to filamentous fungi. In addition, in filamentous fungi, MTs could provide the tracks for long-distance vesicle movement. In this review, we will compare the interaction of the MT and the actin cytoskeleton and their relation to the cortex between yeasts and filamentous fungi. In addition, we will discuss the role of sterol-rich membrane domains in combination with cell-end marker proteins for polarity establishment.

MeSH Categories: Actins/*metabolism, Aspergillus nidulans/*growth & development/metabolism, *Cell Polarity, Fungal Proteins/*metabolism, Membrane Microdomains/metabolism, Microtubules/*metabolism, Saccharomyces cerevisiae/*growth & development/metabolism, Schizosaccharomyces/*growth & development/metabolism

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