Environmental Microbiology

Sensing of environmental signals: classification of chemoreceptors according to the size of their ligand binding regions.

Publication Date: 2010 Aug 25 PMID: 20738376
Authors: Lacal, J. - Garcia-Fontana, C. - Munoz-Martinez, F. - Ramos, J. L. - Krell, T.
Journal: Environ Microbiol

Summary Central to the different forms of taxis are methyl-accepting chemotaxis proteins (MCPs). The increasing number of genome sequences reveals that MCPs differ enormously in sequence, topology and genomic abundance. This work is a one-by-one bioinformatic analysis of the almost-totality of MCP genes available and a classification of motile bacteria according to their lifestyle. On average, motile archaea have 6.7 MCP genes per genome whereas motile bacteria have more than twice as much. We show that the number of MCPs per genome depends on bacterial lifestyle and metabolic diversity, but weakly on genome size. Signal perception at an MCP occurs at the N-terminal ligand binding region (LBR). Here we show that around 88% of MCPs possess an LBR that remains un-annotated in SMART. MCPs can be classified into two clusters according to the size of the LBR. Cluster I receptors have an LBR between 120 and 210 amino acids whereas cluster II receptors have larger LBRs of 220-299 amino acids. There is evidence that suggests that some cluster II LBRs are composed of two cluster I LBRs. Further evidence indicates that other cluster II LBRs might harbour novel sensor domains. Cluster II receptors are dominant in archaea whereas cluster I receptors are prevalent in bacteria. MCPs can be classified into six different receptor topologies and this work contains a first estimation of the relative abundance of different receptor topologies in bacteria and archaea. Topologies involving extracytoplasmic sensing are prevalent in bacteria whereas topologies with cytosolic signal recognition are abundant in archaea.

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Evolutionary and functional diversity of the Pseudomonas type IVa pilin island.

Publication Date: 2010 Aug 25 PMID: 20738375
Authors: Giltner, C. L. - Rana, N. - Lunardo, M. N. - Hussain, A. Q. - Burrows, L. L.
Journal: Environ Microbiol

Summary In Pseudomonas aeruginosa, most proteins involved in type IVa pilus (T4aP) biogenesis are highly conserved except for the major pilin PilA and the minor pilins involved in pilus assembly. Here we show that each of the five major pilin alleles is associated with a specific set of minor pilins, and unrelated strains with the same major pilin type have identical minor pilin genes. The sequences of the minor pilin genes of strains with group III and V pilins are identical, suggesting that these groups diverged recently through further evolution of the major pilin cluster. Both gene clusters are localized on a single 'pilin island' containing putative tRNA recombinational hotspots, and a similar organization of pilin genes was identified in other Pseudomonas species. To address the biological significance of group-specific differences, cross-complementation studies using group II (PAO1) and group III (PA14) minor pilins were performed. Heterologous minor pilins complemented twitching motility to various extents except in the case of PilX, which was non-functional in non-native backgrounds. A recombinant PA14 strain expressing the PAO1 minor pilins regained motility only upon co-introduction of the PA14 pilX gene. Comparison of PilX and PilQ secretin sequences from group II, III and V genomes revealed discrete regions of sequence that co-varied between groups. Our data suggest that changes in PilX sequence have led to compensatory changes in the PilQ secretin monomer such that heterologous PilX proteins are no longer able to promote opening of the secretin to allow pili to appear on the cell surface.

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A comparative molecular analysis of water-filled limestone sinkholes in north-eastern Mexico.

Publication Date: 2010 Aug 25 PMID: 20738374
Authors: Sahl, J. W. - Gary, M. O. - Harris, J. K. - Spear, J. R.
Journal: Environ Microbiol

Summary Sistema Zacaton in north-eastern Mexico is host to several deep, water-filled, anoxic, karstic sinkholes (cenotes). These cenotes were explored, mapped, and geochemically and microbiologically sampled by the autonomous underwater vehicle deep phreatic thermal explorer (DEPTHX). The community structure of the filterable fraction of the water column and extensive microbial mats that coat the cenote walls was investigated by comparative analysis of small-subunit (SSU) 16S rRNA gene sequences. Full-length Sanger gene sequence analysis revealed novel microbial diversity that included three putative bacterial candidate phyla and three additional groups that showed high intra-clade distance with poorly characterized bacterial candidate phyla. Limited functional gene sequence analysis in these anoxic environments identified genes associated with methanogenesis, sulfate reduction and anaerobic ammonium oxidation. A directed, barcoded amplicon, multiplex pyrosequencing approach was employed to compare approximately 100 000 bacterial SSU gene sequences from water column and wall microbial mat samples from five cenotes in Sistema Zacaton. A new, high-resolution sequence distribution profile (SDP) method identified changes in specific phylogenetic types (phylotypes) in microbial mats at varied depths; Mantel tests showed a correlation of the genetic distances between mat communities in two cenotes and the geographic location of each cenote. Community structure profiles from the water column of three neighbouring cenotes showed distinct variation; statistically significant differences in the concentration of geochemical constituents suggest that the variation observed in microbial communities between neighbouring cenotes are due to geochemical variation.

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Diversity of virus-host systems in hypersaline Lake Retba, Senegal.

Publication Date: 2010 Aug 25 PMID: 20738373
Authors: Sime-Ngando, T. - Lucas, S. - Robin, A. - Tucker, K. P. - Colombet, J. - Bettarel, Y. - Desmond, E. - Gribaldo, S. - Forterre, P. - Breitbart, M. - Prangishvili, D.
Journal: Environ Microbiol

Summary Remarkable morphological diversity of virus-like particles was observed by transmission electron microscopy in a hypersaline water sample from Lake Retba, Senegal. The majority of particles morphologically resembled hyperthermophilic archaeal DNA viruses isolated from extreme geothermal environments. Some hypersaline viral morphotypes have not been previously observed in nature, and less than 1% of observed particles had a head-and-tail morphology, which is typical for bacterial DNA viruses. Culture-independent analysis of the microbial diversity in the sample suggested the dominance of extremely halophilic archaea. Few of the 16S sequences corresponded to known archeal genera (Haloquadratum, Halorubrum and Natronomonas), whereas the majority represented novel archaeal clades. Three sequences corresponded to a new basal lineage of the haloarchaea. Bacteria belonged to four major phyla, consistent with the known diversity in saline environments. Metagenomic sequencing of DNA from the purified virus-like particles revealed very few similarities to the NCBI non-redundant database at either the nucleotide or amino acid level. Some of the identifiable virus sequences were most similar to previously described haloarchaeal viruses, but no sequence similarities were found to archaeal viruses from extreme geothermal environments. A large proportion of the sequences had similarity to previously sequenced viral metagenomes from solar salterns.

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Comparative proteomics and activity of a green sulfur bacterium through the water column of Lake Cadagno, Switzerland.

Publication Date: 2010 Aug 20 PMID: 20731699
Authors: Habicht, K. S. - Miller, M. - Cox, R. P. - Frigaard, N. U. - Tonolla, M. - Peduzzi, S. - Falkenby, L. G. - Andersen, J. S.
Journal: Environ Microbiol

Summary Primary production in the meromictic Lake Cadagno, Switzerland, is dominated by anoxygenic photosynthesis. The green sulfur bacterium Chlorobium clathratiforme is the dominant phototrophic organism in the lake, comprising more than half of the bacterial population, and its biomass increases 3.8-fold over the summer. Cells from four positions in the water column were used for comparative analysis of the Chl. clathratiforme proteome in order to investigate changes in protein composition in response to the chemical and physical gradient in their environment, with special focus on how the bacteria survive in the dark. Although metagenomic data are not available for Lake Cadagno, proteome analysis was possible based on the completely sequenced genome of an isolated strain of Chl. clathratiforme. Using LC-MS/MS we identified 1321 Chl. clathratiforme proteins in Lake Cadagno and quantitatively compared 621 of these in the four samples. Our results showed that compared with cells obtained from the photic zone, cells collected from the dark part of the water column had the same expression level of key enzymes involved in carbon metabolism and photosynthetic light harvesting. However, most proteins participating in nitrogen and sulfur metabolism were twofold less abundant in the dark. From the proteome analysis we were able to show that Chl. clathratiforme in the photic zone contains enzymes for fixation of N(2) and the complete oxidation of sulfide to sulfate while these processes are probably not active in the dark. Instead we propose that Chl. clathratiforme cells in the dark part of the water column obtain energy for maintenance from the fermentation of polyglucose. Based on the observed protein compositions we have constructed possible pathways for C, N and S metabolism in Chl. clathratiforme.

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Solenicola setigera is the first characterized member of the abundant and cosmopolitan uncultured marine stramenopile group MAST-3.

Publication Date: 2010 Aug 16 PMID: 20722698
Authors: Gomez, F. - Moreira, D. - Benzerara, K. - Lopez-Garcia, P.
Journal: Environ Microbiol

Summary Culture-independent molecular methods based on the amplification, cloning and sequencing of small-subunit (SSU) rRNA genes are a powerful tool to study the diversity of prokaryotic and eukaryotic microorganisms for which morphological features are not conspicuous. In recent years, molecular data from environmental surveys have revealed several clades of protists lacking cultured and/or described members. Among them are various clades of marine stramenopiles (heterokonts), which are thought to play an essential ecological role as grazers, being abundant and distributed in oceans worldwide. In this work, we show that Solenicola setigera, a distinctive widespread colonial marine protist, is a member of the environmental clade MArine STramenopile 3 (MAST-3). Solenicola is generally considered as a parasite or an epiphyte of the diatom Leptocylindrus mediterraneus. So far, the ultrastructural, morphological and ecological data available were insufficient to elucidate its phylogenetic position, even at the division or class level. We determined SSU rRNA gene sequences of S. setigera specimens sampled from different locations and seasons in the type locality, the Gulf of Lions, France. They were closely related, though not identical, which, together with morphological differences under electron microscopy, suggest the occurrence of several species. Solenicola sequences were well nested within the MAST-3 clade in phylogenetic trees. Since Solenicola is the first identified member of this abundant marine clade, we propose the name Solenicolida for the MAST-3 phylogenetic group.

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Heterogenic expression of genes encoding secreted proteins at the periphery of Aspergillus niger colonies.

Publication Date: 2010 Aug 16 PMID: 20722697
Authors: Vinck, A. - de Bekker, C. - Ossin, A. - Ohm, R. A. - de Vries, R. P. - Wosten, H. A.
Journal: Environ Microbiol

Summary Colonization of a substrate by fungi starts with the invasion of exploring hyphae. These hyphae secrete enzymes that degrade the organic material into small molecules that can be taken up by the fungus to serve as nutrients. We previously showed that only part of the exploring hyphae of Aspergillus niger highly express the glucoamylase gene glaA. This was an unexpected finding since all exploring hyphae are exposed to the same environmental conditions. Using GFP as a reporter, we here demonstrate that the acid amylase gene aamA, the alpha-glucuronidase gene aguA, and the feruloyl esterase gene faeA of A. niger are also subject to heterogenic expression within the exploring mycelium. Coexpression studies using GFP and dTomato as reporters showed that hyphae that highly express one of these genes also highly express the other genes encoding secreted proteins. Moreover, these hyphae also highly express the amylolytic regulatory gene amyR, and the glyceraldehyde-3-phosphate dehydrogenase gene gpdA. In situ hybridization demonstrated that the high expressers are characterized by a high 18S rRNA content. Taken together, it is concluded that two subpopulations of hyphae can be distinguished within the exploring mycelium of A. niger. The experimental data indicate that these subpopulations differ in their transcriptional and translational activity.

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Host migration impacts on the phylogeography of Lyme Borreliosis spirochaete species in Europe.

Publication Date: 2010 Aug 16 PMID: 20722696
Authors: Vollmer, S. A. - Bormane, A. - Dinnis, R. E. - Seelig, F. - Dobson, A. D. - Aanensen, D. M. - James, M. C. - Donaghy, M. - Randolph, S. E. - Feil, E. J. - Kurtenbach, K. - Margos, G.
Journal: Environ Microbiol

Summary The geographic patterns of transmission opportunities of vector-borne zoonoses are determined by a complex interplay between the migration patterns of the host and the vector. Here we examine the impact of host migration on the spread of a tick-borne zoonotic disease, using Lyme Borreliosis (LB) spirochaetal species in Europe. We demonstrate that the migration of the LB species is dependent on and limited by the migration of their respective hosts. We note that populations of Borrelia spp. associated with birds (Borrelia garinii and B. valaisiana) show limited geographic structuring between countries compared with those associated with small mammals (Borrelia afzelii), and we argue that this can be explained by higher rates of migration in avian hosts. We also show the presence of B. afzelii strains in England and, through the use of the multi-locus sequence analysis scheme, reveal that the strains are highly structured. This pattern in English sites is very different from that observed at the continental sites, and we propose that these may be recent introductions.

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The plant pathogen Pseudomonas fuscovaginae contains two conserved quorum sensing systems involved in virulence and negatively regulated by RsaL and the novel regulator RsaM.

Publication Date: 2010 Aug 3 PMID: 20701623
Authors: Mattiuzzo, M. - Bertani, I. - Ferluga, S. - Cabrio, L. - Bigirimana, J. - Guarnaccia, C. - Pongor, S. - Maraite, H. - Venturi, V.
Journal: Environ Microbiol

Pseudomonas fuscovaginae is a Gram-negative fluorescent pseudomonad pathogenic towards several plant species. Despite its importance as a plant pathogen, no molecular studies of virulence have thus far been reported. In this study we show that P. fuscovaginae possesses two conserved N-acyl homoserine lactone (AHL) quorum sensing (QS) systems which we designated PfsI/R and PfvI/R. The PfsI/R system is homologous to the BviI/R system of Burkholderia vietnamiensis and produces and responds to C10-HSL and C12-HSL whereas PfvI/R is homologous to the LasI/R system of Pseudomonas aeruginosa and produces several long-chain 3-oxo-HSLs and responds to 3-oxo-C10-HSL and 3-oxo-C12-HSL and at high AHL concentrations can also respond to structurally different long-chain AHLs. Both systems were found to be negatively regulated by a repressor protein which was encoded by a gene located intergenically between the AHL synthase and LuxR-family response regulator. The pfsI/R system was regulated by a novel repressor designated RsaM while the pfvI/R system was regulated by both the RsaL repressor and by RsaM. The two systems are not transcriptionally hierarchically organized but share a common AHL response and both are required for plant virulence. Pseudomonas fuscovaginae has therefore a unique complex regulatory network composed of at least two different repressors which directly regulate the AHL QS systems and pathogenicity.

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