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1.  Genomic Avenue to Avian Colisepticemia 
mBio  2015;6(1):e01681-14.
ABSTRACT
Here we present an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78 that represent the major cause of avian colisepticemia, an invasive infection caused by avian pathogenic Escherichia coli (APEC) strains. It is associated with high mortality and morbidity, resulting in significant economic consequences for the poultry industry. To understand the genetic basis of the virulence of avian septicemic E. coli, we sequenced the entire genome of a clinical isolate of serotype O78—O78:H19 ST88 isolate 789 (O78-9)—and compared it with three publicly available APEC O78 sequences and one complete genome of APEC serotype O1 strain. Although there was a large variability in genome content between the APEC strains, several genes were conserved, which are potentially critical for colisepticemia. Some of these genes are present in multiple copies per genome or code for gene products with overlapping function, signifying their importance. A systematic deletion of each of these virulence-related genes identified three systems that are conserved in all septicemic strains examined and are critical for serum survival, a prerequisite for septicemia. These are the plasmid-encoded protein, the defective ETT2 (E. coli type 3 secretion system 2) type 3 secretion system ETT2sepsis, and iron uptake systems. Strain O78-9 is the only APEC O78 strain that also carried the regulon coding for yersiniabactin, the iron binding system of the Yersinia high-pathogenicity island. Interestingly, this system is the only one that cannot be complemented by other iron uptake systems under iron limitation and in serum.
IMPORTANCE
Avian colisepticemia is a severe systemic disease of birds causing high morbidity and mortality and resulting in severe economic losses. The bacteria associated with avian colisepticemia are highly antibiotic resistant, making antibiotic treatment ineffective, and there is no effective vaccine due to the multitude of serotypes involved. To understand the disease and work out strategies to combat it, we performed an extensive genomic and genetic analysis of Escherichia coli strains of serotype O78, the major cause of the disease. We identified several potential virulence factors, conserved in all the colisepticemic strains examined, and determined their contribution to growth in serum, an absolute requirement for septicemia. These findings raise the possibility that specific vaccines or drugs can be developed against these critical virulence factors to help combat this economically important disease.
doi:10.1128/mBio.01681-14
PMCID: PMC4313913  PMID: 25587010
2.  Complete Genome Sequence of the Solvent Producer Clostridium saccharobutylicum NCP262 (DSM 13864) 
Genome Announcements  2013;1(6):e00997-13.
Clostridium saccharobutylicum was employed for the production of acetone and butanol in South Africa until the 1970s. The genome comprises a single replicon (5,107,814 bp) harboring all the genes necessary for solvent production and the degradation of various organic compounds, such as fructose, cellobiose, sucrose, and mannose.
doi:10.1128/genomeA.00997-13
PMCID: PMC3869335  PMID: 24285650
3.  First Insights into the Genome of the Gram-Negative, Endospore-Forming Organism Sporomusa ovata Strain H1 DSM 2662 
Genome Announcements  2013;1(5):e00734-13.
The genome of Sporomusa ovata strain H1 DSM 2662, an anaerobic, Gram-negative endospore-forming bacterium, was sequenced. S. ovata uses N-methyl compounds, primary alcohols, fatty acids, and H2 and CO2 as energy and carbon sources to produce acetate. The genome harbors one chromosome, which encodes proteins typical for sporulation.
doi:10.1128/genomeA.00734-13
PMCID: PMC3772150  PMID: 24029766
4.  Genome-guided analysis of physiological and morphological traits of the fermentative acetate oxidizer Thermacetogenium phaeum 
BMC Genomics  2012;13:723.
Background
Thermacetogenium phaeum is a thermophilic strictly anaerobic bacterium oxidizing acetate to CO2 in syntrophic association with a methanogenic partner. It can also grow in pure culture, e.g., by fermentation of methanol to acetate. The key enzymes of homoacetate fermentation (Wood-Ljungdahl pathway) are used both in acetate oxidation and acetate formation. The obvious reversibility of this pathway in this organism is of specific interest since syntrophic acetate oxidation operates close to the energetic limitations of microbial life.
Results
The genome of Th. phaeum is organized on a single circular chromosome and has a total size of 2,939,057 bp. It comprises 3.215 open reading frames of which 75% could be assigned to a gene function. The G+C content is 53.88 mol%. Many CRISPR sequences were found, indicating heavy phage attack in the past. A complete gene set for a phage was found in the genome, and indications of phage action could also be observed in culture. The genome contained all genes required for CO2 reduction through the Wood-Ljungdahl pathway, including two formyl tetrahydrofolate ligases, three carbon monoxide dehydrogenases, one formate hydrogenlyase complex, three further formate dehydrogenases, and three further hydrogenases. The bacterium contains a menaquinone MQ-7. No indications of cytochromes or Rnf complexes could be found in the genome.
Conclusions
The information obtained from the genome sequence indicates that Th. phaeum differs basically from the three homoacetogenic bacteria sequenced so far, i.e., the sodium ion-dependent Acetobacterium woodii, the ethanol-producing Clostridium ljungdahlii, and the cytochrome-containing Moorella thermoacetica. The specific enzyme outfit of Th. phaeum obviously allows ATP formation both in acetate formation and acetate oxidation.
doi:10.1186/1471-2164-13-723
PMCID: PMC3551663  PMID: 23259483
5.  Genome Sequence of Staphylococcus aureus VC40, a Vancomycin- and Daptomycin-Resistant Strain, To Study the Genetics of Development of Resistance to Currently Applied Last-Resort Antibiotics 
Journal of Bacteriology  2012;194(8):2107-2108.
The increasing emergence of multidrug-resistant Staphylococcus aureus is a problem of global importance. Here, we report the genome of S. aureus VC40, which is resistant to the last-resort antibiotics vancomycin and daptomycin. Its genome sequence will allow insights into the mechanisms that convey full resistance to these compounds.
doi:10.1128/JB.06631-11
PMCID: PMC3318483  PMID: 22461548
6.  An Ancient Pathway Combining Carbon Dioxide Fixation with the Generation and Utilization of a Sodium Ion Gradient for ATP Synthesis 
PLoS ONE  2012;7(3):e33439.
Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes.
doi:10.1371/journal.pone.0033439
PMCID: PMC3315566  PMID: 22479398
7.  Complete Genome Sequences of the Chemolithoautotrophic Oligotropha carboxidovorans Strains OM4 and OM5 
Journal of Bacteriology  2011;193(18):5043.
We report on genome sequencing of Oligotropha carboxidovorans strain OM4 and resequencing of strain OM5. The genomes of both are composed of one chromosome and two plasmids. The presence of two plasmids in the OM5 genome is inconsistent with the previously published sequence, for which only one plasmid was described (D. Paul, S. Bridges, S. Burgess, Y. Dandass, and M. Lawrence, BMC Genomics 11:511, 2010).
doi:10.1128/JB.05619-11
PMCID: PMC3165685  PMID: 21742883
8.  Sequence of the hyperplastic genome of the naturally competent Thermus scotoductus SA-01 
BMC Genomics  2011;12:577.
Background
Many strains of Thermus have been isolated from hot environments around the world. Thermus scotoductus SA-01 was isolated from fissure water collected 3.2 km below surface in a South African gold mine. The isolate is capable of dissimilatory iron reduction, growth with oxygen and nitrate as terminal electron acceptors and the ability to reduce a variety of metal ions, including gold, chromate and uranium, was demonstrated. The genomes from two different Thermus thermophilus strains have been completed. This paper represents the completed genome from a second Thermus species - T. scotoductus.
Results
The genome of Thermus scotoductus SA-01 consists of a chromosome of 2,346,803 bp and a small plasmid which, together are about 11% larger than the Thermus thermophilus genomes. The T. thermophilus megaplasmid genes are part of the T. scotoductus chromosome and extensive rearrangement, deletion of nonessential genes and acquisition of gene islands have occurred, leading to a loss of synteny between the chromosomes of T. scotoductus and T. thermophilus. At least nine large inserts of which seven were identified as alien, were found, the most remarkable being a denitrification cluster and two operons relating to the metabolism of phenolics which appear to have been acquired from Meiothermus ruber. The majority of acquired genes are from closely related species of the Deinococcus-Thermus group, and many of the remaining genes are from microorganisms with a thermophilic or hyperthermophilic lifestyle. The natural competence of Thermus scotoductus was confirmed experimentally as expected as most of the proteins of the natural transformation system of Thermus thermophilus are present. Analysis of the metabolic capabilities revealed an extensive energy metabolism with many aerobic and anaerobic respiratory options. An abundance of sensor histidine kinases, response regulators and transporters for a wide variety of compounds are indicative of an oligotrophic lifestyle.
Conclusions
The genome of Thermus scotoductus SA-01 shows remarkable plasticity with the loss, acquisition and rearrangement of large portions of its genome compared to Thermus thermophilus. Its ability to naturally take up foreign DNA has helped it adapt rapidly to a subsurface lifestyle in the presence of a dense and diverse population which acted as source of nutrients. The genome of Thermus scotoductus illustrates how rapid adaptation can be achieved by a highly dynamic and plastic genome.
doi:10.1186/1471-2164-12-577
PMCID: PMC3235269  PMID: 22115438
9.  Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-Aggregative-Haemorrhagic Escherichia coli (EAHEC) 
Archives of Microbiology  2011;193(12):883-891.
The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic Escherichiacoli (EAHEC).
Electronic supplementary material
The online version of this article (doi:10.1007/s00203-011-0725-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00203-011-0725-6
PMCID: PMC3219860  PMID: 21713444
EHEC outbreak; EAHEC; Genome sequencing; Pathotype; Genome evolution
10.  Comparative Genomics and Transcriptomics of Propionibacterium acnes 
PLoS ONE  2011;6(6):e21581.
The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that is occasionally associated with inflammatory diseases. Recent work has indicated that evolutionary distinct lineages of P. acnes play etiologic roles in disease while others are associated with maintenance of skin homeostasis. To shed light on the molecular basis for differential strain properties, we carried out genomic and transcriptomic analysis of distinct P. acnes strains. We sequenced the genome of the P. acnes strain 266, a type I-1a strain. Comparative genome analysis of strain 266 and four other P. acnes strains revealed that overall genome plasticity is relatively low; however, a number of island-like genomic regions, encoding a variety of putative virulence-associated and fitness traits differ between phylotypes, as judged from PCR analysis of a collection of P. acnes strains. Comparative transcriptome analysis of strains KPA171202 (type I-2) and 266 during exponential growth revealed inter-strain differences in gene expression of transport systems and metabolic pathways. In addition, transcript levels of genes encoding possible virulence factors such as dermatan-sulphate adhesin, polyunsaturated fatty acid isomerase, iron acquisition protein HtaA and lipase GehA were upregulated in strain 266. We investigated differential gene expression during exponential and stationary growth phases. Genes encoding components of the energy-conserving respiratory chain as well as secreted and virulence-associated factors were transcribed during the exponential phase, while the stationary growth phase was characterized by upregulation of genes involved in stress responses and amino acid metabolism. Our data highlight the genomic basis for strain diversity and identify, for the first time, the actively transcribed part of the genome, underlining the important role growth status plays in the inflammation-inducing activity of P. acnes. We argue that the disease-causing potential of different P. acnes strains is not only determined by the phylotype-specific genome content but also by variable gene expression.
doi:10.1371/journal.pone.0021581
PMCID: PMC3124536  PMID: 21738717
11.  Complete Genome Sequence of Methanothermobacter marburgensis, a Methanoarchaeon Model Organism▿  
Journal of Bacteriology  2010;192(21):5850-5851.
The circular genome sequence of the chemolithoautotrophic euryarchaeon Methanothermobacter marburgensis, with 1,639,135 bp, was determined and compared with that of Methanothermobacter thermautotrophicus. The genomes of the two model methanogens differ substantially in protein coding sequences, in insertion sequence (IS)-like elements, and in clustered regularly interspaced short palindromic repeats (CRISPR) loci.
doi:10.1128/JB.00844-10
PMCID: PMC2953689  PMID: 20802048
12.  More Than 200 Genes Required for Methane Formation from H2 and CO2 and Energy Conservation Are Present in Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus 
Archaea  2011;2011:973848.
The hydrogenotrophic methanogens Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus can easily be mass cultured. They have therefore been used almost exclusively to study the biochemistry of methanogenesis from H2 and CO2, and the genomes of these two model organisms have been sequenced. The close relationship of the two organisms is reflected in their genomic architecture and coding potential. Within the 1,607 protein coding sequences (CDS) in common, we identified approximately 200 CDS required for the synthesis of the enzymes, coenzymes, and prosthetic groups involved in CO2 reduction to methane and in coupling this process with the phosphorylation of ADP. Approximately 20 additional genes, such as those for the biosynthesis of F430 and methanofuran and for the posttranslational modifications of the two methyl-coenzyme M reductases, remain to be identified.
doi:10.1155/2011/973848
PMCID: PMC3087415  PMID: 21559116
13.  Host Imprints on Bacterial Genomes—Rapid, Divergent Evolution in Individual Patients 
PLoS Pathogens  2010;6(8):e1001078.
Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strain's evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization.
Author Summary
Bacterial virulence results from the interaction between bacteria and their hosts. This interaction provides selection pressure for bacterial adaptation towards increased fitness or virulence. Basic mechanisms involved in bacterial adaptation at the genetic level are point mutations and recombination. As bacterial genome plasticity is higher in vivo than in vitro, host-pathogen interaction may facilitate bacterial adaptation. Comparative genomics has so far been almost entirely focused on genomic changes upon prolonged bacterial growth in vitro. To achieve a better comprehension of bacterial genome plasticity and the capacity to adapt in response to their host, we studied bacterial genome evolution in vivo. We analyzed the impact of individual hosts on genome-wide bacterial adaptation under controlled conditions, by administration of asymptomatic bacteriuria E. coli isolate 83972 to several hosts. Interestingly, the different hosts appeared to personalize their microflora. Adaptation at the genomic level included point mutations in several metabolic and virulence-related genes, often affecting pleiotropic regulators, but re-isolates from each patient showed a distinct pattern of genetic alterations in addition to random changes. Our results provide new insights into bacterial traits under selection during E. coli in vivo growth, further explaining the mechanisms of bacterial adaptation to specific host environments.
doi:10.1371/journal.ppat.1001078
PMCID: PMC2928814  PMID: 20865122
14.  The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids 
PLoS ONE  2010;5(7):e10986.
Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as “B. cereus variety (var.) anthracis”.
doi:10.1371/journal.pone.0010986
PMCID: PMC2901330  PMID: 20634886
15.  Rhizobium sp. Strain NGR234 Possesses a Remarkable Number of Secretion Systems▿ †  
Applied and Environmental Microbiology  2009;75(12):4035-4045.
Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.
doi:10.1128/AEM.00515-09
PMCID: PMC2698369  PMID: 19376903
16.  Genome sequence of Desulfobacterium autotrophicum HRM2, a marine sulfate reducer oxidizing organic carbon completely to carbon dioxide 
Environmental Microbiology  2009;11(5):1038-1055.
Sulfate-reducing bacteria (SRB) belonging to the metabolically versatile Desulfobacteriaceae are abundant in marine sediments and contribute to the global carbon cycle by complete oxidation of organic compounds. Desulfobacterium autotrophicum HRM2 is the first member of this ecophysiologically important group with a now available genome sequence. With 5.6 megabasepairs (Mbp) the genome of Db. autotrophicum HRM2 is about 2 Mbp larger than the sequenced genomes of other sulfate reducers (SRB). A high number of genome plasticity elements (> 100 transposon-related genes), several regions of GC discontinuity and a high number of repetitive elements (132 paralogous genes Mbp−1) point to a different genome evolution when comparing with Desulfovibrio spp. The metabolic versatility of Db. autotrophicum HRM2 is reflected in the presence of genes for the degradation of a variety of organic compounds including long-chain fatty acids and for the Wood–Ljungdahl pathway, which enables the organism to completely oxidize acetyl-CoA to CO2 but also to grow chemolithoautotrophically. The presence of more than 250 proteins of the sensory/regulatory protein families should enable Db. autotrophicum HRM2 to efficiently adapt to changing environmental conditions. Genes encoding periplasmic or cytoplasmic hydrogenases and formate dehydrogenases have been detected as well as genes for the transmembrane TpII-c3, Hme and Rnf complexes. Genes for subunits A, B, C and D as well as for the proposed novel subunits L and F of the heterodisulfide reductases are present. This enzyme is involved in energy conservation in methanoarchaea and it is speculated that it exhibits a similar function in the process of dissimilatory sulfate reduction in Db. autotrophicum HRM2.
doi:10.1111/j.1462-2920.2008.01825.x
PMCID: PMC2702500  PMID: 19187283
17.  A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli 
Clostridial collagenases are foe and friend: on the one hand, these enzymes enable host infiltration and colonization by pathogenic clostridia, and on the other hand, they are valuable biotechnological tools due to their capacity to degrade various types of collagen and gelatine. However, the demand for high-grade preparations exceeds supply due to their pathogenic origin and the intricate purification of homogeneous isoforms. We present the establishment of an Escherichia coli expression system for a variety of constructs of collagenase G (ColG) and H (ColH) from Clostridium histolyticum and collagenase T (ColT) from Clostridium tetani, mimicking the isoforms in vivo. Based on a setup of five different expression strains and two expression vectors, 12 different constructs were expressed, and a flexible purification platform was established, consisting of various orthogonal chromatography steps adaptable to the individual needs of the respective variant. This fast, cost-effective, and easy-to-establish platform enabled us to obtain at least 10 mg of highly pure mono-isoformic protein per liter of culture, ideally suited for numerous sophisticated downstream applications. This production and purification platform paves the way for systematic screenings of recombinant collagenases to enlighten the biochemical function and to identify key residues and motifs in collagenolysis.
doi:10.1007/s00253-009-1953-4
PMCID: PMC3085789  PMID: 19333597
Clostridial collagenases; Expression; Purification; Platform
18.  Re-Citrate Synthase from Clostridium kluyveri Is Phylogenetically Related to Homocitrate Synthase and Isopropylmalate Synthase Rather Than to Si-Citrate Synthase† ▿ 
Journal of Bacteriology  2007;189(11):4299-4304.
The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity.
doi:10.1128/JB.00198-07
PMCID: PMC1913417  PMID: 17400742
19.  Metagenomics of the Deep Mediterranean, a Warm Bathypelagic Habitat 
PLoS ONE  2007;2(9):e914.
Background
Metagenomics is emerging as a powerful method to study the function and physiology of the unexplored microbial biosphere, and is causing us to re-evaluate basic precepts of microbial ecology and evolution. Most marine metagenomic analyses have been nearly exclusively devoted to photic waters.
Methodology/Principal Findings
We constructed a metagenomic fosmid library from 3,000 m-deep Mediterranean plankton, which is much warmer (∼14°C) than waters of similar depth in open oceans (∼2°C). We analyzed the library both by phylogenetic screening based on 16S rRNA gene amplification from clone pools and by sequencing both insert extremities of ca. 5,000 fosmids. Genome recruitment strategies showed that the majority of high scoring pairs corresponded to genomes from Rhizobiales within the Alphaproteobacteria, Cenarchaeum symbiosum, Planctomycetes, Acidobacteria, Chloroflexi and Gammaproteobacteria. We have found a community structure similar to that found in the aphotic zone of the Pacific. However, the similarities were significantly higher to the mesopelagic (500–700 m deep) in the Pacific than to the single 4000 m deep sample studied at this location. Metabolic genes were mostly related to catabolism, transport and degradation of complex organic molecules, in agreement with a prevalent heterotrophic lifestyle for deep-sea microbes. However, we observed a high percentage of genes encoding dehydrogenases and, among them, cox genes, suggesting that aerobic carbon monoxide oxidation may be important in the deep ocean as an additional energy source.
Conclusions/Significance
The comparison of metagenomic libraries from the deep Mediterranean and the Pacific ALOHA water column showed that bathypelagic Mediterranean communities resemble more mesopelagic communities in the Pacific, and suggests that, in the absence of light, temperature is a major stratifying factor in the oceanic water column, overriding pressure at least over 4000 m deep. Several chemolithotrophic metabolic pathways could supplement organic matter degradation in this most depleted habitat.
doi:10.1371/journal.pone.0000914
PMCID: PMC1976395  PMID: 17878949
20.  Complete Nucleotide Sequence of the 113-Kilobase Linear Catabolic Plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a and Transcriptional Analysis of Genes Involved in Quinaldine Degradation▿ †  
Journal of Bacteriology  2007;189(10):3855-3867.
The nucleotide sequence of the linear catabolic plasmid pAL1 from the 2-methylquinoline (quinaldine)-degrading strain Arthrobacter nitroguajacolicus Rü61a comprises 112,992 bp. A total of 103 open reading frames (ORFs) were identified on pAL1, 49 of which had no annotatable function. The ORFs were assigned to the following functional groups: (i) catabolism of quinaldine and anthranilate, (ii) conjugation, and (iii) plasmid maintenance and DNA replication and repair. The genes for conversion of quinaldine to anthranilate are organized in two operons that include ORFs presumed to code for proteins involved in assembly of the quinaldine-4-oxidase holoenzyme, namely, a MobA-like putative molybdopterin cytosine dinucleotide synthase and an XdhC-like protein that could be required for insertion of the molybdenum cofactor. Genes possibly coding for enzymes involved in anthranilate degradation via 2-aminobenzoyl coenzyme A form another operon. These operons were expressed when cells were grown on quinaldine or on aromatic compounds downstream in the catabolic pathway. Single-stranded 3′ overhangs of putative replication intermediates of pAL1 were predicted to form elaborate secondary structures due to palindromic and superpalindromic terminal sequences; however, the two telomeres appear to form different structures. Sequence analysis of ORFs 101 to 103 suggested that pAL1 codes for one or two putative terminal proteins, presumed to be covalently bound to the 5′ termini, and a multidomain telomere-associated protein (Tap) comprising 1,707 amino acids. Even if the putative proteins encoded by ORFs 101 to 103 share motifs with the Tap and terminal proteins involved in telomere patching of Streptomyces linear replicons, their overall sequences and domain structures differ significantly.
doi:10.1128/JB.00089-07
PMCID: PMC1913324  PMID: 17337569
21.  Structural and Functional Characterization of Three Polyketide Synthase Gene Clusters in Bacillus amyloliquefaciens FZB 42 
Journal of Bacteriology  2006;188(11):4024-4036.
Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp0), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide.
doi:10.1128/JB.00052-06
PMCID: PMC1482889  PMID: 16707694
22.  The Genome Sequence of Methanosphaera stadtmanae Reveals Why This Human Intestinal Archaeon Is Restricted to Methanol and H2 for Methane Formation and ATP Synthesis†  
Journal of Bacteriology  2006;188(2):642-658.
Methanosphaera stadtmanae has the most restricted energy metabolism of all methanogenic archaea. This human intestinal inhabitant can generate methane only by reduction of methanol with H2 and is dependent on acetate as a carbon source. We report here the genome sequence of M. stadtmanae, which was found to be composed of 1,767,403 bp with an average G+C content of 28% and to harbor only 1,534 protein-encoding sequences (CDS). The genome lacks 37 CDS present in the genomes of all other methanogens. Among these are the CDS for synthesis of molybdopterin and for synthesis of the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex, which explains why M. stadtmanae cannot reduce CO2 to methane or oxidize methanol to CO2 and why this archaeon is dependent on acetate for biosynthesis of cell components. Four sets of mtaABC genes coding for methanol:coenzyme M methyltransferases were found in the genome of M. stadtmanae. These genes exhibit homology to mta genes previously identified in Methanosarcina species. The M. stadtmanae genome also contains at least 323 CDS not present in the genomes of all other archaea. Seventy-three of these CDS exhibit high levels of homology to CDS in genomes of bacteria and eukaryotes. These 73 CDS include 12 CDS which are unusually long (>2,400 bp) with conspicuous repetitive sequence elements, 13 CDS which exhibit sequence similarity on the protein level to CDS encoding enzymes involved in the biosynthesis of cell surface antigens in bacteria, and 5 CDS which exhibit sequence similarity to the subunits of bacterial type I and III restriction-modification systems.
doi:10.1128/JB.188.2.642-658.2006
PMCID: PMC1347301  PMID: 16385054
23.  The Pathogenicity Island-Associated K15 Capsule Determinant Exhibits a Novel Genetic Structure and Correlates with Virulence in Uropathogenic Escherichia coli Strain 536  
Infection and Immunity  2004;72(10):5993-6001.
The K15 capsule determinant of uropathogenic Escherichia coli strain 536 (O6:K15:H31) is part of a novel 79.6-kb pathogenicity island (PAI) designated PAI V536 that is absent from the genome of nonpathogenic E. coli K-12 strain MG1655. PAI V536 shows typical characteristics of a composite PAI that is associated with the pheV tRNA gene and contains the pix fimbriae determinant as well as genes coding for a putative phosphoglycerate transport system, an autotransporter protein, and hypothetical open reading frames. A gene cluster coding for a putative general secretion pathway system, together with a kpsK15 determinant, is localized downstream of a truncated pheV gene (′pheV) also present in this chromosomal region. The distribution of genes present on PAI V536 was studied by PCR in different pathogenic and nonpathogenic E. coli isolates of various sources. Analysis of the 20-kb kps locus revealed a so far unknown genetic organization. Generally, the kpsK15 gene cluster resembles that of group 2 and 3 capsules, where two conserved regions (regions 1 and 3) are located up- or downstream of a highly variable serotype-specific region (region 2). Interestingly, recombination of a group 2 and 3 determinant may have been involved in the evolution of the K15 capsule-encoding gene cluster. Expression of the K15 capsule is important for virulence in a murine model of ascending urinary tract infection but not for serum resistance of E. coli strain 536.
doi:10.1128/IAI.72.10.5993-6001.2004
PMCID: PMC517556  PMID: 15385503
24.  Analysis of the Genome Structure of the Nonpathogenic Probiotic Escherichia coli Strain Nissle 1917 
Journal of Bacteriology  2004;186(16):5432-5441.
Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases. To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been analyzed by three approaches: (i) sequence context screening of tRNA genes as a potential indication of chromosomal integration of horizontally acquired DNA, (ii) sequence analysis of 280 kb of genomic islands (GEIs) coding for important fitness factors, and (iii) comparison of Nissle 1917 genome content with that of other E. coli strains by DNA-DNA hybridization. PCR-based screening of 324 nonpathogenic and pathogenic E. coli isolates of different origins revealed that some chromosomal regions are frequently detectable in nonpathogenic E. coli and also among extraintestinal and intestinal pathogenic strains. Many known fitness factor determinants of strain Nissle 1917 are localized on four GEIs which have been partially sequenced and analyzed. Comparison of these data with the available knowledge of the genome structure of E. coli K-12 strain MG1655 and of uropathogenic E. coli O6 strains CFT073 and 536 revealed structural similarities on the genomic level, especially between the E. coli O6 strains. The lack of defined virulence factors (i.e., alpha-hemolysin, P-fimbrial adhesins, and the semirough lipopolysaccharide phenotype) combined with the expression of fitness factors such as microcins, different iron uptake systems, adhesins, and proteases, which may support its survival and successful colonization of the human gut, most likely contributes to the probiotic character of E. coli strain Nissle 1917.
doi:10.1128/JB.186.16.5432-5441.2004
PMCID: PMC490877  PMID: 15292145
25.  Complete Nucleotide Sequence and Genetic Organization of the 210-Kilobase Linear Plasmid of Rhodococcus erythropolis BD2 
Journal of Bacteriology  2003;185(17):5269-5274.
The complete nucleotide sequence of the linear plasmid pBD2 from Rhodococcus erythropolis BD2 comprises 210,205 bp. Sequence analyses of pBD2 revealed 212 putative open reading frames (ORFs), 97 of which had an annotatable function. These ORFs could be assigned to six functional groups: plasmid replication and maintenance, transport and metalloresistance, catabolism, transposition, regulation, and protein modification. Many of the transposon-related sequences were found to flank the isopropylbenzene pathway genes. This finding together with the significant sequence similarities of the ipb genes to genes of the linear plasmid-encoded biphenyl pathway in other rhodococci suggests that the ipb genes were acquired via transposition events and subsequently distributed among the rhodococci via horizontal transfer.
doi:10.1128/JB.185.17.5269-5274.2003
PMCID: PMC180976  PMID: 12923100

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