Environmental Microbiology

Quorum sensing-controlled Evr regulates a conserved cryptic pigment biosynthetic cluster and a novel phenomycin-like locus in the plant pathogen, Pectobacterium carotovorum.

Publication Date: 2010 Feb 26 PMID: 20192973
Authors: Williamson, N. R. - Commander, P. M. - Salmond, G. P.
Journal: Environ Microbiol

Summary Pectobacterium carotovorum SCRI193 is a phytopathogenic Gram-negative bacterium. In this study, we have identified a novel cryptic pigment biosynthetic locus in P. carotovorum SCRI193 which we have called the Pectobacterium orange pigment (pop) cluster. The pop cluster is flanked by two tRNA genes and contains genes that encode non-ribosomal peptide synthases and polyketide synthase and produces a negatively charged polar orange pigment. Orange pigment production is activated when an adjacent transcriptional activator sharing sequence similarity with the Erwinia virulence regulator (Evr) is overexpressed. Evr was shown to positively activate its own transcription and that of the pigment biosynthetic genes and an unlinked locus encoding a phenomycin homologue. In addition, the expression of Evr and orange pigment production was shown to be regulated by N-(3-oxohexanoyl)-HSL (OHHL) quorum sensing and have a virulence phenotype in potato. Finally, by comparative genomics and Southern blotting we demonstrate that this pigment biosynthetic cluster is present in multiple P. carotovorum spp., Pectobacterium brasiliensis 1692 and a truncated version of the cluster is present in Pectobacterium atrosepticum. The conserved nature of this cluster in P. carotovorum and P. brasiliensis suggests that the pop cluster has an important function in these broad-host-range soft rotting bacteria, which is no longer required in the narrow-host-range P. atrosepticum SCRI1043.

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Temperature controls on aquatic bacterial production and community dynamics in arctic lakes and streams.

Publication Date: 2010 Feb 25 PMID: 20192972
Authors: Adams, H. E. - Crump, B. C. - Kling, G. W.
Journal: Environ Microbiol

Summary The impact of temperature on bacterial activity and community composition was investigated in arctic lakes and streams in northern Alaska. Aquatic bacterial communities incubated at different temperatures had different rates of production, as measured by (14)C-leucine uptake, indicating that populations within the communities had different temperature optima. Samples from Toolik Lake inlet and outlet were collected at water temperatures of 14.2 degrees C and 15.9 degrees C, respectively, and subsamples incubated at temperatures ranging from 6 degrees C to 20 degrees C. After 5 days, productivity rates varied from 0.5 to approximately 13.7 microg C l(-1) day(-1) and two distinct activity optima appeared at 12 degrees C and 20 degrees C. At these optima, activity was 2- to 11-fold higher than at other incubation temperatures. The presence of two temperature optima indicates psychrophilic and psychrotolerant bacteria dominate under different conditions. Community fingerprinting via denaturant gradient gel electrophoresis (DGGE) of 16S rRNA genes showed strong shifts in the composition of communities driven more by temperature than by differences in dissolved organic matter source; e.g. four and seven unique operational taxonomic units (OTUs) were found only at 2 degrees C and 25 degrees C, respectively, and not found at other incubation temperatures after 5 days. The impact of temperature on bacteria is complex, influencing both bacterial productivity and community composition. Path analysis of measurements of 24 streams and lakes sampled across a catchment 12 times in 4 years indicates variable timing and strength of correlation between temperature and bacterial production, possibly due to bacterial community differences between sites. As indicated by both field and laboratory experiments, shifts in dominant community members can occur on ecologically relevant time scales (days), and have important implications for understanding the relationship of bacterial diversity and function.

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Microbial diversity and biogeochemistry of the Guaymas Basin deep-sea hydrothermal plume.

Publication Date: 2010 Feb 25 PMID: 20192971
Authors: Dick, G. J. - Tebo, B. M.
Journal: Environ Microbiol

Summary Hydrothermal plumes are hot spots of microbial biogeochemistry in the deep ocean, yet little is known about the diversity or ecology of microorganisms inhabiting plumes. Recent biogeochemical evidence shows that Mn(II) oxidation in the Guaymas Basin (GB) hydrothermal plume is microbially mediated and suggests that the plume microbial community is distinct from deep-sea communities. Here we use a molecular approach to compare microbial diversity in the GB plume and in background deep seawater communities, and cultivation to identify Mn(II)-oxidizing bacteria from plumes and sediments. Despite dramatic differences in Mn(II) oxidation rates between plumes and background seawater, microbial diversity and membership were remarkably similar. All bacterial clone libraries were dominated by Gammaproteobacteria and archaeal clone libraries were dominated by Crenarchaeota. Two lineages, both phylogenetically related to methanotrophs and/or methylotrophs, were consistently over-represented in the plume. Eight Mn(II)-oxidizing bacteria were isolated, but none of these or previously identified Mn(II) oxidizers were abundant in clone libraries. Taken together with Mn(II) oxidation rates measured in laboratory cultures and in the field, these results suggest that Mn(II) oxidation in the GB hydrothermal plume is mediated by genome-level dynamics (gene content and/or expression) of microorganisms that are indigenous and abundant in the deep sea but have yet to be unidentified as Mn(II) oxidizers.

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Persistence of bacterial and archaeal communities in sea ice through an Arctic winter.

Publication Date: 2010 Feb 25 PMID: 20192970
Authors: Collins, R. E. - Rocap, G. - Deming, J. W.
Journal: Environ Microbiol

Summary The structure of bacterial communities in first-year spring and summer sea ice differs from that in source seawaters, suggesting selection during ice formation in autumn or taxon-specific mortality in the ice during winter. We tested these hypotheses by weekly sampling (January-March 2004) of first-year winter sea ice (Franklin Bay, Western Arctic) that experienced temperatures from -9 degrees C to -26 degrees C, generating community fingerprints and clone libraries for Bacteria and Archaea. Despite severe conditions and significant decreases in microbial abundance, no significant changes in richness or community structure were detected in the ice. Communities of Bacteria and Archaea in the ice, as in under-ice seawater, were dominated by SAR11 clade Alphaproteobacteria and Marine Group I Crenarchaeota, neither of which is known from later season sea ice. The bacterial ice library contained clones of Gammaproteobacteria from oligotrophic seawater clades (e.g. OM60, OM182) but no clones from gammaproteobacterial genera commonly detected in later season sea ice by similar methods (e.g. Colwellia, Psychrobacter). The only common sea ice bacterial genus detected in winter ice was Polaribacter. Overall, selection during ice formation and mortality during winter appear to play minor roles in the process of microbial succession that leads to distinctive spring and summer sea ice communities.

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Aqueous films limit bacterial cell motility and colony expansion on partially saturated rough surfaces.

Publication Date: 2010 Feb 25 PMID: 20192969
Authors: Wang, G. - Or, D.
Journal: Environ Microbiol

Summary Bacterial motility is a key mechanism for survival in a patchy environment and is important for ecosystem biodiversity maintenance. Quantitative description of bacterial motility in soils is hindered by inherent heterogeneity, pore-space complexity and dynamics of microhydrological conditions. Unsaturated conditions result in fragmented aquatic habitats often too small to support full bacterial immersion thereby forcing strong interactions with mineral and air interfaces that significantly restrict motility. A new hybrid model was developed to study hydration effects on bacterial motility. Simulation results using literature parameter values illustrate sensitivity of colony expansion rates to hydration conditions and are in general agreement with measured values. Under matric potentials greater than -0.5 kPa (wet), bacterial colonies grew fast at colony expansion rates exceeding 421 +/- 94 microm h(-1); rates dropped significantly to 31 +/- 10 microm h(-1) at -2 kPa; as expected, no significant colony expansion was observed at -5 kPa because of the dominance of capillary pinning forces in the submicrometric water film. Quantification of hydration-related constraints on bacterial motion provides insights into optimal conditions for bacterial dispersion and spatial ranges of resource accessibility important for bioremediation and biogeochemical cycles. Results define surprisingly narrow range of hydration conditions where motility confers ecological advantage on natural surfaces.

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DNA sequence-based analysis of the Pseudomonas species.

Publication Date: 2010 Feb 25 PMID: 20192968
Authors: Mulet, M. - Lalucat, J. - Garcia-Valdes, E.
Journal: Environ Microbiol

Summary Partial sequences of four core 'housekeeping' genes (16S rRNA, gyrB, rpoB and rpoD) of the type strains of 107 Pseudomonas species were analysed in order to obtain a comprehensive view regarding the phylogenetic relationships within the Pseudomonas genus. Gene trees allowed the discrimination of two lineages or intrageneric groups (IG), called IG P. aeruginosa and IG P. fluorescens. The first IG P. aeruginosa, was divided into three main groups, represented by the species P. aeruginosa, P. stutzeri and P. oleovorans. The second IG was divided into six groups, represented by the species P. fluorescens, P. syringae, P. lutea, P. putida, P. anguilliseptica and P. straminea. The P. fluorescens group was the most complex and included nine subgroups, represented by the species P. fluorescens, P. gessardi, P. fragi, P. mandelii, P. jesseni, P. koreensis, P. corrugata, P. chlororaphis and P. asplenii. Pseudomonas rhizospherae was affiliated with the P. fluorescens IG in the phylogenetic analysis but was independent of any group. Some species were located on phylogenetic branches that were distant from defined clusters, such as those represented by the P. oryzihabitans group and the type strains P. pachastrellae, P. pertucinogena and P. luteola. Additionally, 17 strains of P. aeruginosa, 'P. entomophila', P. fluorescens, P. putida, P. syringae and P. stutzeri, for which genome sequences have been determined, have been included to compare the results obtained in the analysis of four housekeeping genes with those obtained from whole genome analyses.

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Biosynthesis of uronamide sugars in Pseudomonas aeruginosa O6 and Escherichia coli O121 O antigens.

Publication Date: 2010 Feb 25 PMID: 20192967
Authors: King, J. D. - Vinogradov, E. - Tran, V. - Lam, J. S.
Journal: Environ Microbiol

Summary The major component of the outer leaflet of the outer membrane of Gram-negative bacteria is lipopolysaccharide (LPS). The outermost domain of LPS is a polysaccharide called O antigen. Pseudomonas aeruginosa establishes biofilms on wet surfaces in a wide range of habitats and mutations in O-antigen biosynthesis genes affect bacterial adhesion and the structure of these biofilms. The P. aeruginosa O6 O antigen contains a 2-acetamido-2-deoxy-d-galacturonamide (d-GalNAcAN) residue. O-antigen biosynthesis in this serotype requires the wbpS gene, which encodes a protein with conserved domains of the glutamine-dependent amidotransferase family. Replacement of conserved amino acids in the N-terminal glutaminase conserved domain of WbpS inhibited O-antigen biosynthesis under restricted-ammonia conditions, but not in rich media; suggesting that this domain functions to provide ammonia for O-antigen biosynthesis under restricted-ammonia conditions, by hydrolysis of glutamine. Escherichia coli O121 also produces a d-GalNAcAN-containing O antigen, and possesses a homologue of wbpS called wbqG. An E. coli O121 wbqG mutant was cross-complemented by providing wbpS in trans, and vice versa, showing that these two genes are functionally interchangeable. The E. coli O121 wbqG mutant O antigen contains 2-acetamido-2-deoxy-d-galacturonate (d-GalNAcA), instead of d-GalNAcAN, demonstrating that wbqG is specifically required for biosynthesis of the carboxamide in this sugar.

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Thermophilic anaerobes in Arctic marine sediments induced to mineralize complex organic matter at high temperature.

Publication Date: 2010 Feb 18 PMID: 20192966
Authors: Hubert, C. - Arnosti, C. - Bruchert, V. - Loy, A. - Vandieken, V. - Jorgensen, B. B.
Journal: Environ Microbiol

Summary Marine sediments harbour diverse populations of dormant thermophilic bacterial spores that become active in sediment incubation experiments at much higher than in situ temperature. This response was investigated in the presence of natural complex organic matter in sediments of two Arctic fjords, as well as with the addition of freeze-dried Spirulina or individual high-molecular-weight polysaccharides. During 50 degrees C incubation experiments, Arctic thermophiles catalysed extensive mineralization of the organic matter via extracellular enzymatic hydrolysis, fermentation and sulfate reduction. This high temperature-induced food chain mirrors sediment microbial processes occurring at cold in situ temperatures (near 0 degrees C), yet it is catalysed by a completely different set of microorganisms. Using sulfate reduction rates (SRR) as a proxy for organic matter mineralization showed that differences in organic matter reactivity determined the extent of the thermophilic response. Fjord sediments with higher in situ SRR also supported higher SRR at 50 degrees C. Amendment with Spirulina significantly increased volatile fatty acids production and SRR relative to unamended sediment in 50 degrees C incubations. Spirulina amendment also revealed temporally distinct sulfate reduction phases, consistent with 16S rRNA clone library detection of multiple thermophilic Desulfotomaculum spp. enriched at 50 degrees C. Incubations with four different fluorescently labelled polysaccharides at 4 degrees C and 50 degrees C showed that the thermophilic population in Arctic sediments produce a different suite of polymer-hydrolysing enzymes than those used in situ by the cold-adapted microbial community. Over time, dormant marine microorganisms like these are buried in marine sediments and might eventually encounter warmer conditions that favour their activation. Distinct enzymatic capacities for organic polymer degradation could allow specific heterotrophic populations like these to play a role in sustaining microbial metabolism in the deep, warm, marine biosphere.

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Time-resolved genetic responses of Lactococcus lactis to a dairy environment.

Publication Date: 2010 Feb 18 PMID: 20192965
Authors: Bachmann, H. - de Wilt, L. - Kleerebezem, M. - van Hylckama Vlieg, J. E.
Journal: Environ Microbiol

Summary Lactococcus lactis is one of main bacterial species found in mixed dairy starter cultures for the production of semi-hard cheese. Despite the appreciation that mixed cultures are essential for the eventual properties of the manufactured cheese the vast majority of studies on L. lactis were carried out in laboratory media with a pure culture. In this study we applied an advanced recombinant in vivo expression technology (R-IVET) assay in combination with a high-throughput cheese-manufacturing protocol for the identification and subsequent validation of promoter sequences specifically induced during the manufacturing and ripening of cheese. The system allowed gene expression measurements in an undisturbed product environment without the use of antibiotics and in combination with a mixed strain starter culture. The utilization of bacterial luciferase as reporter enabled the real-time monitoring of gene expression in cheese for up to 200 h after the cheese-manufacturing process was initiated. The results revealed a number of genes that were clearly induced in cheese such as cysD, bcaP, dppA, hisC, gltA, rpsE, purL, amtB as well as a number of hypothetical genes, pseudogenes and notably genetic elements located on the non-coding strand of annotated open reading frames. Furthermore genes that are likely to be involved in interactions with bacteria used in the mixed strain starter culture were identified.

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The effects of submerged aquatic vegetation on the persistence of environmental populations of Enterococcus spp.

Publication Date: 2010 Feb 18 PMID: 20192964
Authors: Badgley, B. D. - Thomas, F. I. - Harwood, V. J.
Journal: Environ Microbiol

Summary Enterococcus spp. are utilized worldwide as faecal indicator bacteria, but certain strains exhibit extended survival in environmental habitats and the factors influencing their persistence are poorly understood. We used flowing freshwater mesocosms to explore the effect of submerged aquatic vegetation (SAV) on the persistence of natural enterococci populations from a subtropical lake. The highest mean densities of culturable enterococci over 2 weeks occurred in SAV [8.6 x 10(2) colony-forming units (cfu) per 100 g wet weight], followed by sediments (1.3 x 10(2) cfu per 100 g) and water (18 cfu per 100 ml). However, due to relative differences in the total mass of each substrate in the entire system (water > sediments > SAV), SAV-associated enterococci represented only a minor proportion of the total population. Vegetated mesocosms harboured significantly higher mean cfu per mesocosm and cfu densities in sediments compared with their unvegetated counterparts, suggesting that SAV indirectly facilitates persistence in aquatic habitats. Populations were dominated (> 96%) by a single Enterococcus casseliflavus strain according to BOX-PCR genotyping, which did not change over the 10-month study and strongly suggests bacterial replication in the lake. The presence of such strains in the environment may represent highly competitive, naturalized and reproducing indicator bacteria populations that are not directly related to pollution events.

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Identification of the trehalose biosynthetic loci of Pseudomonas syringae and their contribution to fitness in the phyllosphere.

Publication Date: 2010 Feb 18 PMID: 20192963
Authors: Freeman, B. C. - Chen, C. - Beattie, G. A.
Journal: Environ Microbiol

Summary Surprisingly little is known of the trehalose biosynthetic pathways in pseudomonads, despite the importance of trehalose to protecting cells from environmental stresses such as low water availability. The genome of the foliar pathogen Pseudomonas syringae pv. tomato strain DC3000 contains genes for two trehalose biosynthetic pathways, TreS and TreYZ, and lacks genes for the more common OtsAB pathway. Deletion of either the treS (PSPTO_2760-2762) or treY/treZ (PSPTO_3125-3134) locus eliminated trehalose accumulation and reduced bacterial growth under hyperosmotic conditions. In evaluating the role of trehalose in P. syringae fitness on leaves, we found that a double deletion mutant lacking these loci exhibited poorer survival than the wild type on tomato leaves over a 2-week period in a growth chamber. Similarly, this mutant exhibited reduced survival on leaves of susceptible and resistant cultivars of the host plant tomato and of the non-host plant soybean over a 10-day period in field plots. Thus, the trehalose biosynthetic loci in P. syringae, which are highly conserved among pseudomonads, contributed to DC3000 fitness on leaves, supporting a role for trehalose in P. syringae survival and population maintenance in the phyllosphere.

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Population ecology of nitrifying Archaea and Bacteria in the Southern California Bight.

Publication Date: 2010 Feb 18 PMID: 20192962
Authors: Beman, J. M. - Sachdeva, R. - Fuhrman, J. A.
Journal: Environ Microbiol

Summary Marine Crenarchaeota are among the most abundant microbial groups in the ocean, and although relatively little is currently known about their biogeochemical roles in marine ecosystems, recognition that Crenarchaeota posses ammonia monooxygenase (amoA) genes and may act as ammonia-oxidizing archaea (AOA) offers another means of probing the ecology of these microorganisms. Here we use a time series approach combining quantification of archaeal and bacterial ammonia oxidizers with bacterial community fingerprints and biogeochemistry, to explore the population and community ecology of nitrification. At multiple depths (150, 500 and 890 m) in the Southern California Bight sampled monthly from 2003 to 2006, AOA were enumerated via quantitative PCR of archaeal amoA and marine group 1 Crenarchaeota 16S rRNA genes. Based on amoA genes, AOA were highly variable in time - a consistent feature of marine Crenarchaeota- however, average values were similar at different depths and ranged from 2.20 to 2.76 x 10(4)amoA copies ml(-1). Archaeal amoA genes were correlated with Crenarchaeota 16S rRNA genes (r(2) = 0.79) and the slope of this relationship was 1.02, demonstrating that the majority of marine group 1 Crenarchaeota present over the dates and depths sampled possessed amoA. Two AOA clades were specifically quantified and compared with betaproteobacterial ammonia-oxidizing bacteria (beta-AOB) amoA genes at 150 m; these AOA groups were found to strongly co-vary in time (r(2) = 0.70, P < 0.001) whereas AOA : beta-AOB ratios ranged from 13 to 5630. Increases in the AOA : beta-AOB ratio correlated with the accumulation of nitrite (r(2) = 0.87, P < 0.001), and may be indicative of differences in substrate affinities and activities leading to periodic decoupling between ammonia and nitrite oxidation. These data capture a dynamic nitrogen cycle in which multiple microbial groups appear to be active participants.

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The Pseudomonas aeruginosa patatin-like protein PlpD is the archetype of a novel Type V secretion system.

Publication Date: 2010 Feb 23 PMID: 20192961
Authors: Salacha, R. - Kovacic, F. - Brochier-Armanet, C. - Wilhelm, S. - Tommassen, J. - Filloux, A. - Voulhoux, R. - Bleves, S.
Journal: Environ Microbiol

Summary We discovered a novel secreted protein by Pseudomonas aeruginosa, PlpD, as a member of the bacterial lipolytic enzyme family of patatin-like proteins (PLPs). PlpD is synthesized as a single molecule consisting of a secreted domain fused to a transporter domain. The N-terminus of PlpD includes a classical signal peptide followed by the four PLP conserved blocks that account for its lipase activity. The C-terminus consists of a POTRA (polypeptide transport-associated) motif preceding a putative 16-stranded beta-barrel similar to those of TpsB transporters of Type Vb secretion system. We showed that the C-terminus remains inserted into the outer membrane while the patatin moiety is secreted. The association between a TpsB component and a passenger protein is a unique hybrid organization that we propose to classify as Type Vd. More than 200 PlpD orthologues exist among pathogenic and environmental bacteria, which suggests that bacteria secrete numerous PLPs using this newly defined mechanism.

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Relationship between cystic fibrosis respiratory tract bacterial communities and age, genotype, antibiotics and Pseudomonas aeruginosa.

Publication Date: 2010 Feb 23 PMID: 20192960
Authors: Klepac-Ceraj, V. - Lemon, K. P. - Martin, T. R. - Allgaier, M. - Kembel, S. W. - Knapp, A. A. - Lory, S. - Brodie, E. L. - Lynch, S. V. - Bohannan, B. J. - Green, J. L. - Maurer, B. A. - Kolter, R.
Journal: Environ Microbiol

Summary Polymicrobial bronchopulmonary infections in cystic fibrosis (CF) cause progressive lung damage and death. Although the arrival of Pseudomonas aeruginosa often heralds a more rapid rate of pulmonary decline, there is significant inter-individual variation in the rate of decline, the causes of which remain poorly understood. By coupling culture-independent methods with ecological analyses, we discovered correlations between bacterial community profiles and clinical disease markers in respiratory tracts of 45 children with CF. Bacterial community complexity was inversely correlated with patient age, presence of P. aeruginosa and antibiotic exposure, and was related to CF genotype. Strikingly, bacterial communities lacking P. aeruginosa were much more similar to each other than were those containing P. aeruginosa, regardless of antibiotic exposure. This suggests that community composition might be a better predictor of disease progression than the presence of P. aeruginosa alone and deserves further study.

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Trade-off between antibiotic resistance and biological fitness in Acinetobacter sp. strain DR1.

Publication Date: 2010 Feb 23 PMID: 20192959
Authors: Kang, Y. S. - Park, W.
Journal: Environ Microbiol

Summary Rifampicin, a bactericidal antibiotic drug, is routinely used to make an environmental recipient selective in laboratory-conjugation experiments. We noticed, inadvertently, that the rifampicin-resistant Acinetobacter sp. strain DR1, a recently discovered hexadecane-degrading environmental isolate, exhibited a substantial loss of quorum sensing signalling. The domesticated ampicillin-resistant strain, DR1, evidenced more dramatic phenotypic changes than were observed in the rifampicin-resistant cells: a complete loss of quorum sensing, a loss in swimming and swarming motilities, poor fimbrial expression, increased rigidity in membrane fatty acid composition and reduced hexadecane degradation capability. Interestingly, the motility of strain DR1 grown adjacent to a streptomycin-producing Streptomyces griceus was permanently abrogated, where this change was heritable and other phenotypic changes could not be detected. In this study, we have reported for the first time that the in situ acquisition of antibiotic resistance may reduce biological fitness, including losses in the production of quorum sensing signals, motility and substrate utilization, and each antibiotic is associated with different degrees of phenotypic and genetic alterations. Our data also suggested that the domestication of environmental isolates should be approached with caution, as there are phenotypic variations in antibiotic-resistant cells that might not be noticeable unless all possible phenotypic assays are conducted.

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Phylogenetic diversity and metagenomics of candidate division OP3.

Publication Date: 2010 Feb 11 PMID: 20158507
Authors: Glockner, J. - Kube, M. - Shrestha, P. M. - Weber, M. - Glockner, F. O. - Reinhardt, R. - Liesack, W.
Journal: Environ Microbiol

Summary Except for environmental 16S rRNA gene sequences, no information is available for members of the candidate division OP3. These bacteria appear to thrive in anoxic environments, such as marine sediments, hypersaline deep sea, freshwater lakes, aquifers, flooded paddy soils and methanogenic bioreactors. The 16S rRNA phylogeny suggests that OP3 belongs to the Planctomycetes/Verrucomicrobia/Chlamydiae (PVC) superphylum. Metagenomic fosmid libraries were constructed from flooded paddy soil and screened for 16S rRNA gene-containing fragments affiliated with the PVC superphylum. The screening of 63 000 clones resulted in 23 assay-positive fosmids, of which three clones were affiliated with OP3. The 16S rRNA gene sequence divergence between the fragments OP3/1, OP3/2 and OP3/3 ranges from 18% to 25%, indicating that they belong to different OP3 subdivisions. The 23S rRNA phylogeny confirmed the membership of OP3 in the PVC superphylum. Sequencing the OP3 fragments resulted in a total of 105 kb of genomic information and 90 ORFs, of which 47 could be assigned a putative function and 11 were conserved hypothetical. Using BLASTP searches, a high proportion of ORFs had best matches to homologues from Deltaproteobacteria, rather than to those of members of the PVC superphylum. On the fragment OP3/3, a cluster of nine ORFs was predicted to encode the bacterial NADH dehydrogenase I. Given the high proportion of homologues present in deltaproteobacteria and anoxic conditions in the natural environment of OP3 bacteria, the detection of NADH dehydrogenase I may suggest an anaerobic respiration mode. Oligonucleotide frequencies calculated for OP3/1, OP3/2 and OP/3 show high intraphylum correlations. This novel sequence information could therefore be used to identify OP3-related fragments in large metagenomic data sets using marker gene-independent procedures in the future. In addition to the OP3 fragments, a single metagenomic fragment affiliated with the candidate division BRC1 was obtained and analysed.

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Identification of conditionally essential genes for growth of Pseudomonas putida KT2440 on minimal medium through the screening of a genome-wide mutant library.

Publication Date: 2010 Feb 11 PMID: 20158506
Authors: Molina-Henares, M. A. - de la Torre, J. - Garcia-Salamanca, A. - Molina-Henares, A. J. - Herrera, M. C. - Ramos, J. L. - Duque, E.
Journal: Environ Microbiol

Summary In silico models for Pseudomonas putida KT2440 metabolism predict 68 genes to be essential for growth on minimal medium. In this study a genome-wide collection of single-gene P. putida KT2440 knockouts was generated by mini-Tn5 transposon mutagenesis and used to identify genes essential for growth in minimal medium with glucose. Our screening of the knockout library allowed us to rescue mutants for 48 different knockouts that were conditionally essential for growth on minimal medium. The in vivo screening showed that 24 of these mutants had a insertion in genes proposed to be conditionally essential based on in silico models, whereas another 24 newly implicated conditionally essential genes have been found. For 10 of the in silico proposed conditionally essential genes not found in the screening, knockout mutants were available at the Pseudomonas Reference Culture Collection. These mutants were tested for conditional growth on minimal medium, but none of them was shown to be essential, suggesting that the in silico proposal was inaccurate. Among the set of identified conditionally essential genes were a number of genes involved in the biosynthesis of certain amino acids and vitamins. Auxotrophs for all amino acids predicted by the in silico models were found and, in addition, we also found auxotrophs for proline, serine, threonine and methionine, as well as auxotrophs for biotin, nicotinate and vitamin B12 that were not predicted in silico. Metabolic tests were performed to validate the mutants' phenotypes. Auxotrophies for l-Arg, l-Leu, l-Pro and l-Cys were bypassed by external addition of the corresponding d-amino acids, suggesting the existence of number of d- to l-amino acid racemases encoded by the KT2440 genome. Therefore, the in vivo high-throughput analysis presented here provides relevant insights into the metabolic cross-road of biosynthetic pathways in this microorganism, as well as valuable information for the fine tuning of current in silico metabolic models.

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