Molecular Microbiology

A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis.

Publication Date: 2012 Feb 2 PMID: 22296581
Authors: Nagorska, K. - Silhan, J. - Li, Y. - Pelicic, V. - Freemont, P. S. - Baldwin, G. S. - Tang, C. M.
Journal: Mol Microbiol

Although oxidative stress is a key aspect of innate immunity, little is known about how host-restricted pathogens successfully repair DNA damage. Base excision repair (BER) is responsible for correcting nucleobases damaged by oxidative stress, and is essential for bloodstream infection caused by the human pathogen, Neisseria meningitidis. We have characterised meningococcal BER enzymes involved in the recognition and removal of damaged nucleobases, and incision of the DNA backbone. We demonstrate that the bi-functional glycosylase/lyases Nth and MutM share several overlapping activities and functional redundancy. However MutM and other members of the GO system, which deal with 8-oxoG, a common lesion of oxidative damage, are not required for survival of N. meningitidis under oxidative stress. Instead, the nucleotide excision and mismatch repair pathways provides back-up for the GO system, while the lyase activity of Nth can substitute for the meningococcal AP endonuclease, NApe. Our genetic and biochemical evidence show that DNA repair is achieved through a robust network of enzymes that provides a flexible system of DNA repair. This network is likely to reflect successful adaptation to the human nasopharynx, and might provide a paradigm for DNA repair in other prokaryotes. (c) 2012 Blackwell Publishing Ltd.

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Expression of the RNA Recognition Motif protein RBP10 promotes a bloodstream-form transcript pattern in Trypanosoma brucei.

Publication Date: 2012 Feb 2 PMID: 22296558
Authors: Wurst, M. - Seliger, B. - Jha, B. A. - Klein, C. - Queiroz, R. - Clayton, C.
Journal: Mol Microbiol

When Trypanosoma brucei differentiates from the bloodstream form to the procyclic form, there are decreases in the levels of many mRNAs encoding proteins required for the glycolytic pathway, and the mRNA encoding the RNA Recognition Motif protein RBP10 decreases in parallel. We show that RBP10 is a cytoplasmic protein that is specific to bloodstream-form trypanosomes, where it is essential. Depletion of RBP10 caused decreases in many bloodstream-form-specific mRNAs, with increases in mRNAs associated with the early stages of differentiation. The changes were similar to, but more extensive than, those caused by glucose deprivation. Conversely, forced RBP10 expression in procyclics induced a switch towards bloodstream-form mRNA expression patterns, with concomitant growth inhibition. Forced expression of RBP10 prevented differentiation of bloodstream forms in response to cis-aconitate, but did not prevent expression of key differentiation markers in response to glucose deprivation. RBP10 was not associated with heavy polysomes, showed no detectable in vivo binding to RNA, and was not stably associated with other proteins. Tethering of RBP10 to a reporter mRNA inhibited translation, and halved the abundance of the bound mRNA. We suggest that RBP10 may prevent the expression of regulatory proteins that are specific to the procyclic form. (c) 2012 Blackwell Publishing Ltd.

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A Turning Point for Natural Products Discovery - ESF-EMBO Research Conference: Synthetic Biology of Antibiotic Production.

Publication Date: 2012 Feb 2 PMID: 22296491
Authors: Takano, E. - Bovenberg, R. - Breitling, R.
Journal: Mol Microbiol

Synthetic Biology is in a critical phase of its development: it has finally reached the point where it can move from proof-of-principle studies to real-world applications. Secondary metabolite biosynthesis, especially the discovery and production of antibiotics, is a particularly relevant target area for such applications of synthetic biology. The first international conference to explore this subject was held in Spain in October 2011. In four sessions on General Synthetic Biology, Filamentous Fungal Systems, Actinomyces Systems, and Tools and Host Structures, scientists presented the most recent technological and scientific advances, and a final-day Forward Look Plenary Discussion identified future trends in the field. (c) 2012 Blackwell Publishing Ltd.

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Aerial development in Streptomyces coelicolor requires sortase activity.

Publication Date: 2012 Feb 2 PMID: 22296345
Authors: Duong, A. - Capstick, D. S. - Di Berardo, C. - Findlay, K. C. - Hesketh, A. - Hong, H. J. - Elliot, M. A.
Journal: Mol Microbiol

Streptomyces coelicolor is a multicellular bacterium whose life cycle encompasses three differentiated states: vegetative hyphae, aerial hyphae and spores. Amongst the factors required for aerial development are the 'chaplins', a family of eight secreted proteins that coat the surface of aerial hyphae. Three chaplins (the 'long' chaplins, ChpA, B and C) possess an LAXTG-containing C-terminal sorting signal and are predicted sortase substrates. The five remaining 'short' chaplins are presumed to be associated with the cell surface through interactions with the long chaplins. We show here that two sortase enzymes, SrtE1 and SrtE2, cleave LAXTG-containing peptides at two distinct positions in vitro, and are required for cell wall anchoring of ChpC in vivo. srtE1/E2 double mutants are delayed in aerial hyphae formation, do not sporulate and fail to display all short chaplins on their aerial surfaces. Surprisingly, these mutant characteristics were not shared by a long chaplin mutant, which exhibited only modest delays in aerial development, leading us to revise the current model of chaplin-mediated aerial development. The sortase mutant phenotype, instead, appears to stem from an inability to transcribe aerial hyphae-specific genes, whose products have diverse functions. This suggests that sortase activity triggers an important, and previously unknown, developmental checkpoint. (c) 2012 Blackwell Publishing Ltd.

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Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae.

Publication Date: 2012 Feb 2 PMID: 22296329
Authors: Wholey, W. Y. - Jakob, U.
Journal: Mol Microbiol

The redox-regulated chaperone Hsp33 protects bacteria specifically against stress conditions that cause oxidative protein unfolding, such as treatment with bleach or exposure to peroxide at elevated temperatures. To gain insight into the mechanism by which expression of Hsp33 confers resistance to oxidative protein unfolding conditions, we made use of V. cholerae strain O395 lacking the Hsp33 gene hslO. We found that this strain, which is exquisitely bleach-sensitive, displays a temperature-sensitive (ts) phenotype during aerobic growth, implying that V. cholerae suffers from oxidative heat stress when cultivated at 43 degrees C. We utilized this phenotype to select for E. coli genes that rescue the ts phenotype of V. cholerae DeltahslO when overexpressed. We discovered that expression of a single protein, the elongation factor EF-Tu, was sufficient to rescue both the ts and bleach-sensitive phenotypes of V. cholerae DeltahslO. In vivo studies revealed that V. cholerae EF-Tu is highly sensitive to oxidative protein degradation in the absence of Hsp33, indicating that EF-Tu is a vital chaperone substrate of Hsp33 in V. cholerae. These results suggest an "essential client protein" model for Hsp33's chaperone action in Vibrio in which stabilization of a single oxidative stress-sensitive protein is sufficient to enhance the oxidative stress resistance of the whole organism. (c) 2012 Blackwell Publishing Ltd.

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Neck compartmentalization as the molecular basis for the different endocytic behaviour of Chs3 during budding or hyperpolarized growth in yeast cells.

Publication Date: 2012 Feb 2 PMID: 22295943
Authors: Sacristan, C. - Reyes, A. - Roncero, C.
Journal: Mol Microbiol

Yeast cells normally grow by budding, but under certain specific conditions they are also able to grow in hyperpolarized forms reminiscent of hyphal growth. During vegetative growth, the synthesis of the septum that physically separates yeast cells during cytokinesis depends on the correct assembly of the septin ring. Septins and actin patches are assembled at the neck, forming two concentric rings where the actin patch ring occupies the external-most part. This specific positioning defines a plasma membrane region at the neck from which other lateral membrane compartments are excluded. In this scenario, correct assembly of the chitin ring is dependent on the anchoring of Chs3 to the septin ring through Chs4. The anchoring of Chs3 to septins through Chs4 prevents the arrival of this protein at endocytic sites, thus reducing the endocytosis of Chs3. This allows an equilibrium to be set up between the antero- and retrograde transport of Chs3, facilitating the synthesis of the chitin ring at the neck. In contrast, hyperpolarized growth is characterized by a reduced endocytic turnover of Chs3, which in turn lead to the accumulation of Chs3 at the plasma membrane and a concomitant increase in chitin synthesis. (c) 2012 Blackwell Publishing Ltd.

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RcsB-BglJ activates the Escherichia coli leuO gene, encoding an H-NS antagonist and pleiotropic regulator of virulence determinants.

Publication Date: 2012 Feb 2 PMID: 22295907
Authors: Stratmann, T. - Pul, U. - Wurm, R. - Wagner, R. - Schnetz, K.
Journal: Mol Microbiol

The LysR-type transcription factor LeuO is involved in regulation of pathogenicity determinants and stress responses in Enterobacteriaceae, and acts as antagonist of the global repressor H-NS. Expression of the leuO gene is repressed by H-NS, and it is upregulated in stationary phase and under amino acid starvation conditions. Here, we show that the heterodimer of the FixJ/NarL-type transcription regulators RcsB and BglJ strongly activates expression of leuO and that RcsB-BglJ regulates additional loci. Activation of leuO by RcsB-BglJ is independent of the Rcs phosphorelay system. RcsB-BglJ binds to the leuO promoter region and activates one of two leuO promoters mapped in vivo. Moreover, LeuO antagonizes activation of leuO by RcsB-BglJ and acts as negative autoregulator in vivo and in vitro. Further, the H-NS paralog StpA causes repression of leuO in addition to H-NS. Together, our data suggest a complex arrangement of regulatory elements and they indicate a feedback control mechanism of leuO expression. (c) 2012 Blackwell Publishing Ltd.

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Ligand and antagonist driven regulation of the Vibrio cholerae quorum-sensing receptor CqsS.

Publication Date: 2012 Feb 2 PMID: 22295878
Authors: Wei, Y. - Ng, W. L. - Cong, J. - Bassler, B. L.
Journal: Mol Microbiol

Quorum sensing, a bacterial cell-cell communication process, controls biofilm formation and virulence factor production in Vibrio cholerae, a human pathogen that causes the disease cholera. The major V. cholerae autoinducer is (S)-3-hydroxytridecan-4-one (CAI-1). A membrane bound two-component sensor histidine kinase called CqsS detects CAI-1, and the CqsS- > LuxU- > LuxO phosphorelay cascade transduces the information encoded in CAI-1 into the cell. Because the CAI-1 ligand is known and because the signaling circuit is simple, consisting of only three proteins, this system is ideal for analyzing ligand regulation of a sensor histidine kinase. Here we reconstitute the CqsS- > LuxU- > LuxO phosphorylation cascade in vitro. We find that CAI-1 inhibits the initial auto-phosphorylation of CqsS whereas subsequent phosphotransfer steps and CqsS phosphatase activity are not CAI-1-controlled. CAI-1 binding to CqsS causes a conformational change that renders His194 in CqsS inaccessible to the CqsS catalytic domain. CqsS mutants with altered ligand detection specificities are faithfully controlled by their corresponding modified ligands in vitro. Likewise, pairing of agonists and antagonists allows in vitro assessment of their opposing activities. Our data are consistent with a two-state model for ligand control of histidine kinases. (c) 2012 Blackwell Publishing Ltd.

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A small RNA that regulates motility and biofilm formation in response to changes in nutrient availability in Escherichia coli.

Publication Date: 2012 Jan 30 PMID: 22289118
Authors: Thomason, M. K. - Fontaine, F. - De Lay, N. - Storz, G.
Journal: Mol Microbiol

In bacteria, many small regulatory RNAs (sRNAs) are induced in response to specific environmental signals or stresses and act by base-pairing with mRNA targets to affect protein translation or mRNA stability. In Escherichia coli, the gene for the sRNA IS061/IsrA, here renamed McaS, was predicted to reside in an intergenic region between abgR, encoding a transcription regulator and ydaL, encoding a small MutS-related protein. We show that McaS is a approximately 95 nt transcript whose expression increases over growth, peaking in early-to-mid stationary phase, or when glucose is limiting. McaS uses three discrete single-stranded regions to regulate mRNA targets involved in various aspects of biofilm formation. McaS represses csgD, the transcription regulator of curli biogenesis and activates flhD, the master transcription regulator of flagella synthesis leading to increased motility, a process not previously reported to be regulated by sRNAs. McaS also regulates pgaA, a porin required for the export of the polysaccharide poly beta-1,6-N-acetyl-d-glucosamine. Consequently, high levels of McaS result in increased biofilm formation while a strain lacking mcaS shows reduced biofilm formation. Based on our observations, we propose that, in response to limited nutrient availability, increasing levels of McaS modulate steps in the progression to a sessile lifestyle.

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SpyA is a membrane-bound ADP-ribosyltransferase of Streptococcus pyogenes which modifies a streptococcal peptide, SpyB.

Publication Date: 2012 Jan 30 PMID: 22288436
Authors: Korotkova, N. - Hoff, J. S. - Becker, D. M. - Quinn, J. K. - Icenogle, L. M. - Moseley, S. L.
Journal: Mol Microbiol

All sequenced genomes of Streptococcus pyogenes (Group A Streptococcus, GAS) encode a protein, SpyA, with homology to C3-like ADP-ribosyltransferase toxins. SpyA is a novel virulence factor which plays a role in pathogenesis in a mouse model of soft-tissue infection. In this study we demonstrate that SpyA is a surface-exposed membrane protein which is anchored to the streptococcal membrane by an N-terminal transmembrane sequence. We identified a small gene upstream of spyA, designated spyB, which encodes a peptide of 35 amino acids, and is co-transcribed with spyA. Expression of spyBA is strongly influenced by translational coupling: mutational inactivation of spyB translation completely abolishes translation of spyA. spyB expression increases with increasing cell density and reaches its maximum at late exponential growth phase. The SpyB N-terminus is predicted to fold into an amphipathic alpha-helix, a structural motif that targets a protein to the cytoplasmic membrane. Consistent with the prediction, we found that a SpyB fusion with peptide affinity tags is located in the streptococcal membrane. An ADP-ribosylation assay with recombinant SpyA demonstrated that SpyA modifies SpyB. Thus, our study suggests that ADP-ribosylation of SpyB may be an important function of SpyA.

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Involvement of TatD nuclease during programmed cell death in the protozoan parasite Trypanosoma brucei.

Publication Date: 2012 Jan 31 PMID: 22288397
Authors: Gannavaram, S. - Debrabant, A.
Journal: Mol Microbiol

In this report, we describe the involvement of TatD nuclease during programmed cell death (PCD) in the human protozoan parasite Trypanosoma brucei. T. brucei TatD nuclease showed intrinsic DNase activity, was localized in the cytoplasm and translocated to the nucleus when cells were treated with inducers previously demonstrated to cause PCD in T. brucei. Overexpression of TatD nuclease resulted in elevated PCD and conversely, loss of TatD expression by RNAi conferred significant resistance to the induction of PCD in T. brucei. Co-immunoprecipitation studies revealed that TatD nuclease interacts with endonucleaseG suggesting that these two nucleases could form a DNA degradation complex in the nucleus. Together, biochemical activity, RNAi and subcellular localization results demonstrate the role of TatD nuclease activity in DNA degradation during PCD in these evolutionarily ancient eukaryotic organisms. Further, in conjunction with endonucleaseG, TatD may represent a critical nuclease in a caspase-independent PCD pathway in trypanosomatid parasites since caspases have not been identified in these organisms.

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An Aspergillus nidulans bZIP response pathway hardwired for defensive secondary metabolism operates through aflR.

Publication Date: 2012 Jan 28 PMID: 22283524
Authors: Yin, W. - Amaike, S. - Wohlbach, D. J. - Gasch, A. P. - Chiang, Y. M. - Wang, C. C. - Bok, J. - Rohlfs, M. - Keller, N. P.
Journal: Mol Microbiol

The eukaryotic bZIP transcription factors are critical players in organismal response to environmental challenges. In fungi, the production of secondary metabolites (SMs) is hypothesized as one of the responses to environmental insults, e.g. attack by fungivorous insects, yet little data to support this hypothesis exists. Here we establish a mechanism of bZIP regulation of SMs through RsmA, a recently discovered YAP-like bZIP protein. RsmA greatly increases SM production by binding to two sites in the A. nidulans AflR promoter region, a C6 transcription factor known for activating production of the carcinogenic and anti-predation SM, sterigmatocystin (ST). Deletion of aflR in an overexpression rsmA (OE::rsmA) background not only eliminates ST production but also significantly reduces asperthecin synthesis. Furthermore, the fungivore, Folsomia candida, exhibited a distinct preference for feeding on wild type rather than an OE::rsmA strain. RsmA may thus have a critical function in mediating direct chemical resistance against predation. Taken together, these results suggest RsmA represents a bZIP pathway hardwired for defensive SM production. (c) 2012 Blackwell Publishing Ltd.

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A promiscuous antitoxin of bacteriophage T4 ensures successful viral replication.

Publication Date: 2012 Feb PMID: 22283468
Authors: Short, F. L. - Blower, T. R. - Salmond, G. P.
Journal: Mol Microbiol

Bacteria are constantly threatened by predation from bacteriophage parasites and, in response, have evolved an array of resistance mechanisms. These resistance mechanisms then place greater selection pressure on the infecting bacteriophages, which develop counter-strategies in a perpetual 'arms race' between virus and host. Toxin-antitoxin (TA) loci are widespread in bacteria and can confer multiple benefits, including resistance to bacteriophages. The study by Otsuka and Yonesaki, published in this issue of Molecular Microbiology, describes a new plasmid-encoded TA system, lsoAB, which confers resistance to a dmd(-) mutant of bacteriophage T4 through the activity of the LsoA toxin. Infections with wild-type T4, however, are unaffected as the Dmd protein acts as an alternative antitoxin to LsoA, thus preventing its anti-bacteriophage activity. Dmd has also been shown to negate the activity of a related toxin, RnlA. This is a striking result indicating that Dmd can act as a promiscuous antitoxin, binding and inhibiting multiple toxin partners, when antitoxin activity is generally considered to be limited to a single cognate toxin. This study is an exciting addition to both the bacteriophage resistance and TA fields, and suggests a greater role for TA system-based resistance and counter-resistance in the world's oldest predator-prey relationship.

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The Flavoprotein Cyc2p, a Mitochondrial Cytochrome C Assembly Factor, Is a NAD(P)H-Dependent Heme Reductase.

Publication Date: 2012 Jan 18 PMID: 22257001
Authors: Corvest, V. - Murrey, D. A. - Hirasawa, M. - Knaff, D. B. - Guiard, B. - Hamel, P.
Journal: Mol Microbiol

Cytochrome c assembly requires sulfhydryls at the CXXCH heme-binding site on the apoprotein and also chemical reduction of the heme co-factor. In yeast mitochondria, the cytochrome heme lyases (CCHL, CC(1) HL) and Cyc2p catalyze covalent heme attachment to apocytochromes c and c(1) . An in vivo indication that Cyc2p controls a reductive step in the heme attachment reaction is the finding that the requirement for its function can be by-passed by exogenous reductants. Although redox titrations of Cyc2p flavin (E(m ) = -290 mV) indicate that reduction of a disulfide at the CXXCH site of apocytochrome c (E(m) = -265 mV) is a thermodynamically favorable reaction, Cyc2p does not act as an apocytochrome c or c(1) CXXCH disulfide reductase in vitro. In contrast, Cyc2p is able to catalyze the NAD(P)H-dependent reduction of hemin, an indication that the protein's role may be to control the redox state of the iron in the heme attachment reaction to apocytochromes c. Using two-hybrid analysis, we show that Cyc2p interacts with CCHL and also with apocytochromes c and c(1) .We postulate that Cyc2p, possibly in a complex with CCHL, reduces the heme iron prior to heme attachment to the apoforms of cytochrome c and c(1) . (c) 2012 Blackwell Publishing Ltd.

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The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid.

Publication Date: 2012 Feb PMID: 22256861
Authors: Smith, E. J. - Corrigan, R. M. - van der Sluis, T. - Grundling, A. - Speziale, P. - Geoghegan, J. A. - Foster, T. J.
Journal: Mol Microbiol

The Sbi protein of Staphylococcus aureus comprises two IgG-binding domains similar to those of protein A and a region that triggers the activation of complement C3. Sbi is expressed on the cell surface but its C-terminal domain lacks motifs associated with wall or membrane anchoring of proteins in Gram-positive bacteria. Cell-associated Sbi fractionates with the cytoplasmic membrane and is not solubilized during protoplast formation. S. aureus expressing Sbi truncates of the C-terminal Y domain allowed identification of residues that are required for association of Sbi with the membrane. Recombinant Sbi bound to purified cytoplasmic membrane material in vitro and to purified lipoteichoic acid. This explains how Sbi partitions with the membrane in fractionation experiments yet is partially exposed on the cell surface. An LTA-defective mutant of S. aureus had reduced levels of Sbi in the cytoplasmic membrane.

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Gap1 functions as a molecular chaperone to stabilize its interactive partner Gap3 during biogenesis of serine-rich repeat bacterial adhesin.

Publication Date: 2012 Feb PMID: 22251284
Authors: Zhou, M. - Zhu, F. - Li, Y. - Zhang, H. - Wu, H.
Journal: Mol Microbiol

Serine-rich repeat glycoproteins (SRRPs) are important bacterial adhesins that are conserved in streptococci and staphylococci. Fimbriae-associated protein (Fap1) from Streptococcus parasanguinis, was the first SRRP identified; it plays an important role in bacterial biofilm formation. A gene cluster encoding glycosyltransferases and accessory secretion components is required for Fap1 biogenesis. Two glycosylation-associated proteins, Gap1 and Gap3 within the cluster, interact with each other and function in concert in Fap1 biogenesis. Here we report the new molecular events underlying contribution of the interaction to Fap1 biogenesis. The Gap1-deficient mutant rendered Gap3 unstable and degraded in vitro and in vivo. Inactivation of a gene encoding protease ClpP reversed the phenotype of the gap1 mutant, suggesting that ClpP is responsible for degradation of Gap3. Molecular chaperone GroEL was co-purified with Gap3 only when Gap1 was absent and also reacted with Gap1 monoclonal antibody, suggesting that Gap1 functions as a specific chaperone for Gap3. The N-terminal interacting domains of Gap1 mediated the Gap3 stability and Fap1 biogenesis. Gap1 homologues from Streptococcus agalactiae and Staphylococcus aureus also interacted with and stabilized corresponding Gap3 homologues, suggesting that the chaperone activity of the Gap1 homologues is common in biogenesis of SRRPs.

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Small regulatory RNAs control the multi-cellular adhesive lifestyle of Escherichia coli.

Publication Date: 2012 Jan 18 PMID: 22250746
Authors: Jorgensen, M. G. - Nielsen, J. S. - Boysen, A. - Franch, T. - Moller-Jensen, J. - Valentin-Hansen, P.
Journal: Mol Microbiol

Small regulatory RNA molecules have recently been recognized as important regulatory elements of developmental processes in both eukaryotes and bacteria. We here describe a striking example in Escherichia coli that can switch between a single-cell motile lifestyle and a multi-cellular, sessile and adhesive state that enables biofilm formation on surfaces. For this, the bacterium needs to reprogramme its gene expression, and in many E. coli and Salmonella strains the lifestyle shift relies on control cascades that inhibit flagellar expression and activate the synthesis of curli, extracellular adhesive fibres important for co-aggregation of cells and adhesion to biotic and abiotic surfaces. By combining bioinformatics, genetic and biochemical analysis we identified three small RNAs that act by an antisense mechanism to downregulate translation of CsgD, the master regulator of curli synthesis. Our demonstration that basal expression of each of the three RNA species is sufficient to downregulate CsgD synthesis and prevent curli formation indicates that all play a prominent role in the curli regulatory network. Our findings provide the first clue as to how the Rcs signalling pathway negatively regulates curli synthesis and increase the number of small regulatory RNAs that act directly on the csgD mRNA to five.

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Presence of Not5 and ubiquitinated Rps7A in polysome fractions depends upon the Not4 E3 ligase.

Publication Date: 2012 Feb PMID: 22243599
Authors: Panasenko, O. O. - Collart, M. A.
Journal: Mol Microbiol

In this study, we determine that Saccharomyces cerevisiae Not4 E3 ligase ubiquitinates Rps7A in vivo and in vitro, but not its paralogue, Rps7B. Ubiquitinated Rps7A is detectable only in 80S and polysomes, but not in free 40S fractions. A different role of the Rps7 paralogues in vivo is supported by the observation that the deletion of Rps7A but not Rps7B is sensitive to translational inhibitors and leads to an accumulation of aggregated proteins. An important accumulation of aggregated proteins that include ribosomal proteins and ribosome-associated chaperones is also observed in cells lacking Not4. A contribution of Not4 to ribosomal function extending beyond Rps7A ubiquitination is supported by the observation that the deletion of Not4 displays a synthetic slow growth phenotype when combined with the deletion of either one of the two Rps7 paralogues. Not4 is detectable in polysome fractions, as are other subunits of the Ccr4-Not complex such as Not5. The optimal presence of Not5 in polysomes is dependent upon Not4 and the deletion of Not5 leads to a dramatic reduction of polysomes. These results lead us to suggest that Not4 contributes to normal polysome levels and is important for cellular protein solubility maybe in part by ubiquitination of Rps7A.

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The product of tadZ, a new member of the parA/minD superfamily, localizes to a pole in Aggregatibacter actinomycetemcomitans.

Publication Date: 2012 Feb PMID: 22239271
Authors: Perez-Cheeks, B. A. - Planet, P. J. - Sarkar, I. N. - Clock, S. A. - Xu, Q. - Figurski, D. H.
Journal: Mol Microbiol

Aggregatibacter actinomycetemcomitans establishes a tenacious biofilm that is important for periodontal disease. The tad locus encodes the components for the secretion and biogenesis of Flp pili, which are necessary for the biofilm to form. TadZ is required, but its function has been elusive. We show that tadZ genes belong to the parA/minD superfamily of genes and that TadZ from A. actinomycetemcomitans (AaTadZ) forms a polar focus in the cell independent of any other tad locus protein. Mutations indicate that regions in AaTadZ are required for polar localization and biofilm formation. We show that AaTadZ dimerizes and that all TadZ proteins are predicted to have a Walker-like A box. However, they all lack the conserved lysine at position 6 (K6) present in the canonical Walker-like A box. When the alanine residue (A6) in the atypical Walker-like A box of AaTadZ was converted to lysine, the mutant protein remained able to dimerize and localize, but it was unable to allow the formation of a biofilm. Another essential biofilm protein, the ATPase (AaTadA), also localizes to a pole. However, its correct localization depends on the presence of AaTadZ. We suggest that the TadZ proteins mediate polar localization of the Tad secretion apparatus.

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Defying stereotypes: the elusive search for a universal model of LysR-type regulation.

Publication Date: 2012 Feb PMID: 22235937
Authors: Momany, C. - Neidle, E. L.
Journal: Mol Microbiol

LysR-type transcriptional regulators (LTTRs) compose the largest family of homologous regulators in bacteria. Considering their prevalence, it is not surprising that LTTRs control diverse metabolic functions. Arguably, the most unexpected aspect of LTTRs is the paucity of available structural information. Solubility issues are notoriously problematic, and structural studies have only recently begun to flourish. In this issue of Molecular Microbiology, Taylor et al. (2012) present the structure of AphB, a LysR-type regulator of virulence in Vibrio cholerae. This contribution adds significantly to the group of known full-length atomic LTTR structures, which remains small. Importantly, this report also describes an active-form variant. Small conformational changes in the effector-binding domain translate to global reorganization of the DNA-binding domain. Emerging from these results is a model of theme-and-variation among LTTRs rather than a unified regulatory scheme. Despite common structural folds, LTTRs exhibit differences in oligomerization, promoter recognition and communication with RNA polymerase. Such variation mirrors the diversity in sequence and function associated with members of this very large family.

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Interaction of a bacterial flagellar chaperone FlgN with FlhA is required for efficient export of its cognate substrates.

Publication Date: 2012 Feb PMID: 22233518
Authors: Minamino, T. - Kinoshita, M. - Hara, N. - Takeuchi, S. - Hida, A. - Koya, S. - Glenwright, H. - Imada, K. - Aldridge, P. D. - Namba, K.
Journal: Mol Microbiol

FlgN chaperone acts as a bodyguard to protect its cognate substrates, FlgK and FlgL, from proteolysis in the cytoplasm. Docking of the FlgN-FlgK complex with the FliI ATPase of the flagellar type III export apparatus is key to the protein export process. However, a DeltafliH-fliI flhB(P28T) mutant forms some flagella even in the absence of FliH and FliI, raising the question of how FlgN promotes the export of its cognate substrates. Here, we report that the interaction of FlgN with an integral membrane export protein, FlhA, is directly involved in efficient protein export. A DeltafliH-fliI flhB(P28T) DeltaflgN mutant caused extragenic suppressor mutations in the C-terminal domain of FlhA (FlhA(C) ). Pull-down assays using GST affinity chromatography showed an interaction between FlgN and FlhA(C) . The FlgN-FlgK complex bound to FlhA(C) and FliJ to form the FlgN-FlgK-FliJ-FlhA(C) complex. The FlgN-FlhA(C) interaction was enhanced by FlgK but not by FliJ. FlgN120 missing the last 20 residues still bound to FlgK and FliJ but not to FlhA(C) . A highly conserved Tyr-122 residue was required for the interaction with FlhA(C) . These results suggest that FlgN efficiently transfers FlgK/L subunits to FlhA(C) to promote their export.

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Polar assembly and scaffolding proteins of the virulence-associated ESX-1 secretory apparatus in mycobacteria.

Publication Date: 2012 Feb PMID: 22233444
Authors: Wirth, S. E. - Krywy, J. A. - Aldridge, B. B. - Fortune, S. M. - Fernandez-Suarez, M. - Gray, T. A. - Derbyshire, K. M.
Journal: Mol Microbiol

The ESX-1 secretion system is required for pathogenicity of Mycobacterium tuberculosis (Mtb). Despite considerable research, little is known about the structural components of ESX-1, or how these proteins are assembled into the active secretion apparatus. Here, we exploit the functionally related ESX-1 apparatus of Mycobacterium smegmatis (Ms) to show that fluorescently tagged proteins required for ESX-1 activity consistently localize to the cell pole, identified by time-lapse fluoro-microscopy as the non-septal (old) pole. Deletions in Msesx1 prevented polar localization of tagged proteins, indicating the need for specific protein-protein interactions in polar trafficking. Remarkably, expression of the Mtbesx1 locus in Msesx1 mutants restored polar localization of tagged proteins, indicating establishment of the MtbESX-1 apparatus in M. smegmatis. This observation illustrates the cross-species conservation of protein interactions governing assembly of ESX-1, as well as polar localization. Importantly, we describe novel non-esx1-encoded proteins, which affect ESX-1 activity, which colocalize with ESX-1, and which are required for ESX-1 recruitment and assembly. This analysis provides new insights into the molecular assembly of this important determinant of Mtb virulence.

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Quorum-sensing non-coding small RNAs use unique pairing regions to differentially control mRNA targets.

Publication Date: 2012 Feb PMID: 22229925
Authors: Shao, Y. - Bassler, B. L.
Journal: Mol Microbiol

Quorum sensing is a mechanism of cell-cell communication that bacteria use to control collective behaviours including bioluminescence, biofilm formation and virulence factor production. In the Vibrio harveyi and Vibrio cholerae quorum-sensing circuits, multiple non-coding small regulatory RNAs called the quorum-regulated small RNAs (Qrr sRNAs) function to establish the global quorum-sensing gene expression pattern by modulating translation of multiple mRNAs encoding quorum-sensing regulatory factors. Here we show that the Qrr sRNAs post-transcriptionally activate production of the low cell density master regulator AphA through base pairing to aphA mRNA, and this is crucial for the accumulation of appropriate levels of AphA protein at low cell density. We find that the Qrr sRNAs use unique pairing regions to discriminate between their different targets. Qrr1 is not as effective as Qrr2-5 in activating aphA because Qrr1 lacks one of two required pairing regions. However, Qrr1 is equally effective as the other Qrr sRNAs at controlling targets like luxR and luxO because it harbours all of the required pairing regions for these targets. Sequence comparisons reveal that Vibrionaceae species possessing only qrr1 do not have the aphA gene under Qrr sRNA control. Our findings suggest co-evolving relationships between particular Qrr sRNAs and particular mRNA targets.

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An unusual feature associated with LEE1 P1 promoters in enteropathogenic Escherichia coli (EPEC).

Publication Date: 2012 Feb PMID: 22229878
Authors: Jeong, J. H. - Kim, H. J. - Kim, K. H. - Shin, M. - Hong, Y. - Rhee, J. H. - Schneider, T. D. - Choy, H. E.
Journal: Mol Microbiol

Transcription start points in bacteria are influenced by the nature of the RNA polymerase.promoter interaction. For Escherichia coli RNA polymerase holoenzyme containing sigma70, it is presumed that specific sequence in one or more of the -10, extended -10 and -35 elements of the promoter guides the RNAP to select the cognate start point. Here, we investigated the promoter driving expression of the LEE1 operon in enteropathogenic E. coli and found two promoters separated by 10 bp, LEE1 P1A (+1) and LEE1 P1B (+10) using various in vitro biochemical tools. A unique feature of P1B was the presence of multiple transcription starts from five neighbouring As at the initial transcribed region. The multiple products did not arise from stuttering synthesis. Analytical software based on information theory was employed to determine promoter elements. The concentration of the NTP pool altered the preferred transcription start points, albeit the underlying mechanism is elusive. Under in vivo conditions, dominant P1B, but not P1A, was subject to regulation by IHF.

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BsrG/SR4 from Bacillus subtilis- the first temperature-dependent type I toxin-antitoxin system.

Publication Date: 2012 Feb PMID: 22229825
Authors: Jahn, N. - Preis, H. - Wiedemann, C. - Brantl, S.
Journal: Mol Microbiol

Here, we describe bsrG/SR4, a novel type I toxin-antitoxin system from the SPbeta prophage region of the Bacillus subtilis chromosome. The 294-nucleotide bsrG RNA encodes a 38-amino-acid toxin, whereas SR4 is a 180-nucleotide antisense RNA that acts as the antitoxin. Both genes overlap by 123 nucleotides. BsrG expression increases at the onset of stationary phase. The sr4 promoter is 6- to 10-fold stronger than the bsrG promoter. Deletion of sr4 stabilizes bsrG mRNA and causes cell lysis on agar plates, which is due to the BsrG peptide and not the bsrG mRNA. SR4 overexpression could compensate cell lysis caused by overexpression of bsrG. SR4 interacts with the 3' UTR of bsrG RNA, thereby promoting its degradation. RNase III cleaves the bsrG RNA/SR4 duplex at position 185 of bsrG RNA, but is not essential for the function of the toxin-antitoxin system. Endoribonuclease Y and 3'-5' exoribonuclease R participate in the degradation of both bsrG RNA and SR4, whereas PnpA processes three SR4 precursors to the mature RNA. A heat shock at 48 degrees C results in faster degradation and, therefore, significantly decreased amounts of bsrG RNA.

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Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli.

Publication Date: 2012 Jan 5 PMID: 22221182
Authors: Bateman, S. L. - Seed, P. C.
Journal: Mol Microbiol

Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor (IHF). In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNIs), primarily through de-repression of hmpA, encoding a nitric oxide-detoxifying flavohaemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX.

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N for AsN - O for StrOcture? A strand-loop-strand motif for prokaryotic O-glycosylation.

Publication Date: 2012 Jan 5 PMID: 22221153
Authors: Otzen, D.
Journal: Mol Microbiol

So far, it has not been possible to identify a general sequence motif for O-glycosylation in bacteria. In this issue, Charbonneau et al. demonstrate why O-glycosylation is mediated by a 13-residue strand-loop-strand motif which is part of a 19-residue imperfect repeat in the passenger domain of bacterial autotransporters. This motif provides a convenient 'glycosylation cassette' and raises intriguing questions about the structural regulation of the glycosylation pathway.

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The Burkholderia cenocepacia BDSF quorum sensing fatty acid is synthesized by a bifunctional crotonase homologue having both dehydratase and thioesterase activities.

Publication Date: 2012 Feb PMID: 22221091
Authors: Bi, H. - Christensen, Q. H. - Feng, Y. - Wang, H. - Cronan, J. E.
Journal: Mol Microbiol

Signal molecules of the diffusible signal factor (DSF) family have been shown recently to be involved in regulation of pathogenesis and biofilm formation in diverse Gram-negative bacteria. DSF signals are reported to be active not only on their cognate bacteria, but also on unrelated bacteria and the pathogenic yeast, Candida albicans. DSFs are monounsaturated fatty acids of medium chain length containing an unusual cis-2 double bond. Although genetic analyses had identified genes involved in DSF synthesis, the pathway of DSF synthesis was unknown. The DSF of the important human pathogen Burkholderia cenocepacia (called BDSF) is cis-2-dodecenoic acid. We report that BDSF is synthesized from a fatty acid synthetic intermediate, the acyl carrier protein (ACP) thioester of 3-hydroxydodecanoic acid. This intermediate is intercepted by protein Bcam0581 and converted to cis-2-dodecenoyl-ACP. Bcam0581 is annotated as a homologue of crotonase, the first enzyme of the fatty acid degradation pathway. We demonstrated Bcam0581to be a bifunctional protein that not only catalysed dehydration of 3-hydroxydodecanoyl-ACP to cis-2-dodecenoyl-ACP, but also cleaved the thioester bond to give the free acid. Both activities required the same set of active-site residues. Although dehydratase and thioesterase activities are known activities of the crotonase superfamily, Bcam0581 is the first protein shown to have both activities.

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The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120.

Publication Date: 2012 Feb PMID: 22220907
Authors: Higa, K. C. - Rajagopalan, R. - Risser, D. D. - Rivers, O. S. - Tom, S. K. - Videau, P. - Callahan, S. M.
Journal: Mol Microbiol

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.

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Deciphering cell wall integrity signalling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases.

Publication Date: 2012 Feb PMID: 22220813
Authors: Dichtl, K. - Helmschrott, C. - Dirr, F. - Wagener, J.
Journal: Mol Microbiol

The fungal cell wall, a conserved and highly dynamic structure, is essential for virulence and viability of fungal pathogens and is an important antifungal drug target. The cell wall integrity (CWI) signalling pathway regulates shape and biosynthesis of the cell wall. In this work we identified, localized and functionally characterized four putative CWI stress sensors of Aspergillus fumigatus, an airborne opportunistic human pathogen and the cause of invasive aspergillosis. We show that Wsc1 is specifically required for resistance to echinocandin antifungals. MidA is specifically required for elevated temperature tolerance and resistance to the cell wall perturbing agents congo red and calcofluor white. Wsc1, Wsc3 and MidA additionally have overlapping functions and are redundantly required for radial growth and conidiation. We have also analysed the roles of three Rho GTPases that have been implicated in CWI signalling in other fungi. We show that Rho1 is essential and that conditional downregulation of rho1 or deletion of rho2 or rho4 results in severely impaired CWI. Our data indicate significant functional differences between the CWI stress sensors of yeasts and moulds.

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TbPIF8, a Trypanosoma brucei protein related to the yeast Pif1 helicase, is essential for cell viability and mitochondrial genome maintenance.

Publication Date: 2012 Feb PMID: 22220754
Authors: Wang, J. - Englund, P. T. - Jensen, R. E.
Journal: Mol Microbiol

The trypanosome mitochondrial genome, kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings, including several thousand minicircles and dozens of maxicircles. The unusual complexity of kDNA would indicate that numerous proteins must be involved in its condensation, replication, segregation and gene expression. During our investigation of trypanosome mitochondrial PIF1-like helicases, we found that TbPIF8 is the smallest and most divergent. It lacks some conserved helicase domains, thus implying that unlike other mitochondrial PIF1-like helicases, this protein may have no enzymatic activity. TbPIF8 is positioned on the distal face of kDNA disk and its localization patterns vary with different kDNA replication stages. Stem-loop RNAi of TbPIF8 arrests cell growth and causes defects in kDNA segregation. RNAi of TbPIF8 causes only limited kDNA shrinkage but the networks become disorganized. Electron microcopy of thin sections of TbPIF8-depleted cells shows heterogeneous electron densities in the kinetoplast disk. Although we do not yet know its exact function, we conclude that TbPIF8 is essential for cell viability and is important for maintenance of kDNA.

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The GtaR protein negatively regulates transcription of the gtaRI operon and modulates gene transfer agent (RcGTA) expression in Rhodobacter capsulatus.

Publication Date: 2012 Feb PMID: 22211723
Authors: Leung, M. M. - Brimacombe, C. A. - Spiegelman, G. B. - Beatty, J. T.
Journal: Mol Microbiol

The gtaI gene of Rhodobacter capsulatus encodes an N-acyl-homoserine lactone (acyl-HSL) synthase. Immediately 5' of the gtaI gene is ORF rcc00328 that encodes a potential acyl-HSL receptor protein. A combination of genetic and biochemical approaches showed that rcc00328 (renamed gtaR) modulates the production of a genetic exchange element called the gene transfer agent (RcGTA), and regulates the transcription of gtaI. Although gtaI mutants exhibited decreased levels of RcGTA production, mutagenesis of gtaR did not, whereas a gtaR/gtaI double mutant produced wild-type levels of RcGTA. Because mutagenesis of gtaR suppressed the effect of the gtaI mutation, we suggest that the GtaR protein is a negative transcriptional regulator of RcGTA gene expression. We discovered that the gtaR and gtaI genes are co-transcribed, and also negatively regulated by the GtaR protein in the absence of acyl-HSL. A His-tagged GtaR protein was purified, and DNA-binding experiments revealed a binding site in the promoter region of the gtaRI operon. This GtaR protein did not bind to the RcGTA promoter region, and therefore modulation of RcGTA production appears to require at least one additional factor. We found that RcGTA production was stimulated by spent media from other species, and identified exogenous acyl-HSLs that induce RcGTA.

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Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae.

Publication Date: 2012 Feb PMID: 22211696
Authors: Fleurie, A. - Cluzel, C. - Guiral, S. - Freton, C. - Galisson, F. - Zanella-Cleon, I. - Di Guilmi, A. M. - Grangeasse, C.
Journal: Mol Microbiol

Eukaryotic-like serine/threonine-kinases are involved in the regulation of a variety of physiological processes in bacteria. In Streptococcus pneumoniae, deletion of the single serine/threonine-kinase gene stkP results in an aberrant cell morphology suggesting that StkP participates in pneumococcus cell division. To understand the function of StkP, we have engineered various pneumococcus strains expressing truncated or kinase-dead forms of StkP. We show that StkP kinase activity, but also its extracellular and cytoplasmic domains per se, are required for pneumococcus cell division. Indeed, we observe that mutant cells show round or elongated shapes with non-functional septa and a chain phenotype, delocalized sites of peptidoglycan synthesis and diffused membrane StkP localization. To gain understanding of the underlying StkP-mediated regulatory mechanism, we show that StkP specifically phosphorylates in vivo the cell division protein DivIVA on threonine 201. Pneumococcus cells expressing non-phosphorylatable DivIVA-T201A possess an elongated shape with a polar bulge and aberrant spatial organization of nascent peptidoglycan. This brings the first evidence of the importance of StkP in relationship to the phosphorylation of one of its substrates in cell division. It is concluded that StkP is a multifunctional protein that plays crucial functions in pneumococcus cell shape and division.

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Distinct roles of two ceramide synthases, CaLag1p and CaLac1p, in the morphogenesis of Candida albicans.

Publication Date: 2012 Feb PMID: 22211636
Authors: Cheon, S. A. - Bal, J. - Song, Y. - Hwang, H. M. - Kim, A. R. - Kang, W. K. - Kang, H. A. - Hannibal-Bach, H. K. - Knudsen, J. - Ejsing, C. S. - Kim, J. Y.
Journal: Mol Microbiol

Lag1p and Lac1p catalyse ceramide synthesis in Saccharomyces cerevisiae. This study shows that Lag1 family proteins are generally required for polarized growth in hemiascomycetous yeast. However, in contrast to S. cerevisiae where these proteins are functionally redundant, C. albicans Lag1p (CaLag1p) and Lac1p (CaLac1p) are functionally distinct. Lack of CaLag1p, but not CaLac1p, caused severe defects in the growth and hyphal morphogenesis of C. albicans. Deletion of CaLAG1 decreased expression of the hypha-specific HWP1 and ECE1 genes. Moreover, overexpression of CaLAG1 induced pseudohyphal growth in this organism under non-hypha-inducing conditions, suggesting that CaLag1p is necessary for relaying signals to induce hypha-specific gene expression. Analysis of ceramide and sphingolipid composition revealed that CaLag1p predominantly synthesizes ceramides with C24:0/C26:0 fatty acid moieties, which are involved in generating inositol-containing sphingolipids, whereas CaLac1p produces ceramides with C18:0 fatty acid moieties, which are precursors for glucosylsphingolipids. Thus, our study demonstrates that CaLag1p and CaLac1p have distinct substrate specificities and physiological roles in C. albicans.

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Structure of the pilus assembly protein TadZ from Eubacterium rectale: implications for polar localization.

Publication Date: 2012 Feb PMID: 22211578
Authors: Xu, Q. - Christen, B. - Chiu, H. J. - Jaroszewski, L. - Klock, H. E. - Knuth, M. W. - Miller, M. D. - Elsliger, M. A. - Deacon, A. M. - Godzik, A. - Lesley, S. A. - Figurski, D. H. - Shapiro, L. - Wilson, I. A.
Journal: Mol Microbiol

The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from Eubacterium rectale (ErTadZ), in complex with ATP and Mg(2+) , was determined to 2.1 A resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker-A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N-terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped-down 'active site'. Homology modelling of the N-terminal atypical receiver domain of CpaE indicates that it has a conserved protein-protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during an early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.

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Analysis of the role of Bacillus subtilis sigma(M) in beta-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis.

Publication Date: 2012 Feb PMID: 22211522
Authors: Luo, Y. - Helmann, J. D.
Journal: Mol Microbiol

The Bacillus subtilis extracytoplasmic function (ECF) sigma factor sigma(M) is inducible by, and confers resistance to, several cell envelope-acting antibiotics. Here, we demonstrate that sigma(M) is responsible for intrinsic beta-lactam resistance, with sigma(X) playing a secondary role. Activation of sigma(M) upregulates several cell wall biosynthetic enzymes including one, PBP1, shown here to be a target for the beta-lactam cefuroxime. However, sigma(M) still plays a major role in cefuroxime resistance even in cells lacking PBP1. To better define the role of sigma(M) in beta-lactam resistance, we characterized suppressor mutations that restore cefuroxime resistance to a sigM null mutant. The most frequent suppressors inactivated gdpP (yybT) which encodes a cyclic-di-AMP phosphodiesterase (PDE). Intriguingly, sigma(M) is a known activator of disA encoding one of three paralogous diadenylate cyclases (DAC). Overproduction of the GdpP PDE greatly sensitized cells to beta-lactam antibiotics. Conversely, genetic studies indicate that at least one DAC is required for growth with depletion leading to cell lysis. These findings support a model in which c-di-AMP is an essential signal molecule required for cell wall homeostasis. Other suppressors highlight the roles of ECF sigma factors in counteracting the deleterious effects of autolysins and reactive oxygen species in beta-lactam-treated cells.

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Genome-wide selection for increased copy number in Acinetobacter baylyi ADP1: locus and context-dependent variation in gene amplification.

Publication Date: 2012 Feb PMID: 22211470
Authors: Seaton, S. C. - Elliott, K. T. - Cuff, L. E. - Laniohan, N. S. - Patel, P. R. - Neidle, E. L.
Journal: Mol Microbiol

Renewed interest in gene amplification stems from its importance in evolution and a variety of medical problems ranging from drug resistance to cancer. However, amplified DNA segments (amplicons) are not fully characterized in any organism. Here we report a novel Acinetobacter baylyi system for genome-wide studies. Amplification mutants that consume aromatic compounds were selected under conditions requiring high-level expression from three promoters in a linked set of chromosomal genes. Tools were developed to relocate these catabolic genes to any non-essential chromosomal position, and 49 amplification mutants from five genomic contexts were characterized. Amplicon size (18-271 kb) and copy number (2-105) indicated that 30% of mutants carried more than 1 Mb of amplified DNA. Amplification features depended on genomic position. For example, amplicons from one locus were similarly sized but displayed variable copy number, whereas those from another locus were differently sized but had comparable copy number. Additionally, the importance of sequence context was highlighted in one region where amplicons differed depending on the presence of a promoter mutation in the strain from which they were selected. DNA sequences at amplicon boundaries in 19 mutants reflected illegitimate recombination. Furthermore, steady-state duplication frequencies measured under non-selective conditions (10(-4) to 10(-5) ) confirmed that spontaneous gene duplication is a major source of genetic variation.

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Spore formation in Myxococcus xanthus is tied to cytoskeleton functions and polysaccharide spore coat deposition.

Publication Date: 2012 Feb PMID: 22188356
Authors: Muller, F. D. - Schink, C. W. - Hoiczyk, E. - Cserti, E. - Higgs, P. I.
Journal: Mol Microbiol

Myxococcus xanthus is a Gram-negative bacterium that differentiates into environmentally resistant spores. Spore differentiation involves septation-independent remodelling of the rod-shaped vegetative cell into a spherical spore and deposition of a thick and compact spore coat outside of the outer membrane. Our analyses suggest that spore coat polysaccharides are exported to the cell surface by the Exo outer membrane polysaccharide export/polysaccharide co-polymerase 2a (OPX/PCP-2a) machinery. Conversion of the capsule-like polysaccharide layer into a compact spore coat layer requires the Nfs proteins which likely form a complex in the cell envelope. Mutants in either nfs, exo or two other genetic loci encoding homologues of polysaccharide synthesis enzymes fail to complete morphogenesis from rods to spherical spores and instead produce a transient state of deformed cell morphology before reversion into typical rods. We additionally provide evidence that the cell cytoskeletal protein, MreB, plays an important role in rod to spore morphogenesis and for spore outgrowth. These studies provide evidence that this novel Gram-negative differentiation process is tied to cytoskeleton functions and polysaccharide spore coat deposition.

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A kinetoplastid-specific kinesin is required for cytokinesis and for maintenance of cell morphology in Trypanosoma brucei.

Publication Date: 2012 Feb PMID: 22168367
Authors: Hu, L. - Hu, H. - Li, Z.
Journal: Mol Microbiol

Kinesins are motor-based transport proteins that play diverse roles in various cellular processes. The trypanosome genome lacks the homologues of many conserved mitotic kinesins, but encodes a number of trypanosome-specific kinesins with unknown function. Here, we report the biochemical and functional characterization of TbKIN-C, a trypanosome-specific kinesin, which was initially identified through an RNAi screen for cytokinesis genes in T. brucei. TbKIN-C possesses in vitro ATPase activity and associates with cytoskeletal tubulin microtubules in vivo. It is distributed throughout the cytoskeleton with a focal enrichment at the posterior end of the cell during early cell cycle stages. RNAi of TbKIN-C resulted in distorted cell shape with an elongated posterior filled with tyrosinated tubulin microtubules. Silencing of TbKIN-C impaired the segregation of organelles and cytoskeletal structures and led to detachment of the new flagellum and a small portion of the cytoplasm. We also show that RNAi of TbKIN-C compromised cytokinesis and abolished the trans-localization of TbCPC1, a subunit of the chromosomal passenger complex, from the central spindle to the initiation site of cytokinesis. Our results suggest an essential role of TbKIN-C in maintaining cell morphology, likely through regulating microtubule dynamics at the posterior end of the cell.

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Identification of a protein kinase A regulatory subunit from Leishmania having importance in metacyclogenesis through induction of autophagy.

Publication Date: 2012 Feb PMID: 22168343
Authors: Bhattacharya, A. - Biswas, A. - Das, P. K.
Journal: Mol Microbiol

cAMP-mediated responses act as modulators of environmental sensing and cellular differentiation of many kinetoplastidae parasites including Leishmania. Although cAMP synthesizing (adenylate cyclase) and degrading (phosphodiesterase) enzymes have been cloned and characterized from Leishmania, no cAMP-binding effector molecule has yet been identified from this parasite. In this study, a regulatory subunit of cAMP-dependent protein kinase (Ldpkar1), homologous to mammalian class I cAMP-dependent protein kinase regulatory subunit, has been identified from L. donovani. Further characterization suggested possible interaction of LdPKAR1 with PKA catalytic subunits and inhibition of PKA activity. This PKA regulatory subunit is expressed in all life cycle stages and its expression attained maximum level in stationary phase promastigotes, which are biochemically similar to the infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates LdPKAR1 expression and under starvation condition promastigotes overexpressing Ldpkar1 attained metacyclic features earlier than normal cells. Furthermore, Ldpkar1 overexpression accelerates autophagy, a starvation-induced cytological event necessary for metacyclogenesis and amastigote formation. Conditional silencing of Ldpkar1 delays the induction of autophagy in the parasite. The study, for the first time, reports the identification of a functional cAMP-binding effector molecule from Leishmania that may modulate important cytological events affecting metacyclogenesis.

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An orphan sensor kinase controls quinolone signal production via MexT in Pseudomonas aeruginosa.

Publication Date: 2012 Feb PMID: 22168309
Authors: Zaoui, C. - Overhage, J. - Lons, D. - Zimmermann, A. - Musken, M. - Bielecki, P. - Pustelny, C. - Becker, T. - Nimtz, M. - Haussler, S.
Journal: Mol Microbiol

Pseudomonas aeruginosa employs both N-acylhomoserine lactone and 2-alkyl-4(1H)-quinolone (AQ)-mediated interbacterial signalling for the orchestration of a genome-wide gene regulatory network. Despite the many advances that have been made in understanding the target genes of quorum sensing regulation, little is known on how quorum sensing systems are influenced by environmental cues. In this study, we show that AQ production is modulated by an orphan P. aeruginosa sensor kinase. Transcriptional studies of the sensor kinase (MxtR) mutant demonstrated that an induced expression of MexT, a LysR-type transcriptional regulator, largely determined the global transcriptional profile. Thereby, overexpression of the MexT-regulated MexEF-OprN efflux pump led to a delayed expression of the AQ biosynthetic genes and of AQ-dependent virulence factors. Furthermore, we demonstrated that autophosphorylation of MxtR was inhibited by ubiquinone, the central electron carrier of respiration in in vitro experiments. Our results elucidate on a mechanism by which P. aeruginosa senses environmental conditions and adapts by controlling the production of interbacterial AQ signal molecules. A regulatory function of a sensor kinase may indicate that there is a pre-emptive role of adaptation mechanisms that are turned on under distinct environmental conditions and that are important for efficient colonization and pathogenesis.

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The crystal structure of AphB, a virulence gene activator from Vibrio cholerae, reveals residues that influence its response to oxygen and pH.

Publication Date: 2012 Feb PMID: 22053934
Authors: Taylor, J. L. - De Silva, R. S. - Kovacikova, G. - Lin, W. - Taylor, R. K. - Skorupski, K. - Kull, F. J.
Journal: Mol Microbiol

Expression of the two critical virulence factors of Vibrio cholerae, toxin-coregulated pilus and cholera toxin, is initiated at the tcpPH promoter by the regulators AphA and AphB. AphA is a winged helix DNA-binding protein that enhances the ability of AphB, a LysR-type transcriptional regulator, to activate tcpPH expression. We present here the 2.2 A X-ray crystal structure of full-length AphB. As reported for other LysR-type proteins, AphB is a tetramer with two distinct subunit conformations. Unlike other family members, AphB must undergo a significant conformational change in order to bind to DNA. We have found five independent mutations in the putative ligand-binding pocket region that allow AphB to constitutively activate tcpPH expression at the non-permissive pH of 8.5 and in the presence of oxygen. These findings indicate that AphB is responsive to intracellular pH as well as to anaerobiosis and that residues in the ligand-binding pocket of the protein influence its ability to respond to both of these signals. We have solved the structure of one of the constitutive mutants, and observe conformational changes that would allow DNA binding. Taken together, these results describe a pathway of conformational changes allowing communication between the ligand and DNA binding regions of AphB.

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