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1.  Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation 
BMC Genomics  2013;14:693.
Background
Lyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. “bavariensis” (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. “finlandensis” (1).
Results
Robust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases.
Conclusions
Intra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues.
doi:10.1186/1471-2164-14-693
PMCID: PMC3833655  PMID: 24112474
Borrelia burgdorferi; Lyme borreliosis; Pan-genome; Single-nucleotide polymorphisms; Phylogenetic tree; Genome evolution simulation
2.  Integrated Metagenomics/Metaproteomics Reveals Human Host-Microbiota Signatures of Crohn's Disease 
PLoS ONE  2012;7(11):e49138.
Crohn's disease (CD) is an inflammatory bowel disease of complex etiology, although dysbiosis of the gut microbiota has been implicated in chronic immune-mediated inflammation associated with CD. Here we combined shotgun metagenomic and metaproteomic approaches to identify potential functional signatures of CD in stool samples from six twin pairs that were either healthy, or that had CD in the ileum (ICD) or colon (CCD). Integration of these omics approaches revealed several genes, proteins, and pathways that primarily differentiated ICD from healthy subjects, including depletion of many proteins in ICD. In addition, the ICD phenotype was associated with alterations in bacterial carbohydrate metabolism, bacterial-host interactions, as well as human host-secreted enzymes. This eco-systems biology approach underscores the link between the gut microbiota and functional alterations in the pathophysiology of Crohn's disease and aids in identification of novel diagnostic targets and disease specific biomarkers.
doi:10.1371/journal.pone.0049138
PMCID: PMC3509130  PMID: 23209564
3.  THE IMPORTANCE OF A MULTI-FACETED APPROACH TO CHARACTERIZING THE MICROBIAL FLORA OF CHRONIC WOUNDS 
Chronic wounds contain complex polymicrobial communities of sessile organisms that have been underappreciated because of limitations of standard culture techniques. The aim of this work is to combine recently developed next-generation investigative techniques to comprehensively describe the microbial characteristics of chronic wounds. Tissue samples were obtained from 15 patients with chronic wounds presenting to the Johns Hopkins Wound Center. Standard bacteriological cultures demonstrated an average of 3 common bacterial species in wound samples. By contrast, high-throughput pyrosequencing revealed increased bacterial diversity with an average of 17 genera in each wound. Data from microbial community profiling of chronic wounds was compared to published sequenced analyses of bacteria from normal skin. Increased proportions of anaerobes, Gram-negative rods and Gram-positive cocci were found in chronic wounds. In addition, chronic wounds had significantly lower populations of Propionibacterium compared to normal skin. Using epifluorescence microscopy, wound bacteria were visualized in highly organized thick confluent biofilms or as scattered individual bacterial cells. Fluorescent in-situ hybridization allowed for the visualization of Staphylococcus aureus cells in a wound sample. Quorum sensing molecules were measured by bioassay to evaluate signaling patterns amongst bacteria in the wounds. A range of autoinducer-2 activities were detected in the wound samples. Collectively, these data provide new insights into the identity, organization, and behavior of bacteria in chronic wounds. Such information may provide important clues to effective future strategies in wound healing.
doi:10.1111/j.1524-475X.2011.00720.x
PMCID: PMC3227014  PMID: 22092791
Chronic Wound; Microbiome; Biofilm; Microbiology; Pyrosequencing; Epifluorescence Microscopy; Fluorescent In-Situ Hybridization; Quorum Sensing
4.  Whole-Genome Sequences of Borrelia bissettii, Borrelia valaisiana, and Borrelia spielmanii 
Journal of Bacteriology  2012;194(2):545-546.
It has been known for decades that human Lyme disease is caused by the three spirochete species Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii. Recently, Borrelia valaisiana, Borrelia spielmanii, and Borrelia bissettii have been associated with Lyme disease. We report the complete genome sequences of B. valaisiana VS116, B. spielmanii A14S, and B. bissettii DN127.
doi:10.1128/JB.06263-11
PMCID: PMC3256645  PMID: 22207749
5.  Whole-Genome Sequences of Two Borrelia afzelii and Two Borrelia garinii Lyme Disease Agent Isolates 
Journal of Bacteriology  2011;193(24):6995-6996.
Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B. burgdorferi, and B. bavariensis sp. nov. Whole-genome sequencing is an excellent tool for investigating and understanding the influence of bacterial diversity on the pathogenesis and etiology of Lyme disease. We report here the whole-genome sequences of four isolates from two of the Borrelia species that cause human Lyme disease, B. afzelii isolates ACA-1 and PKo and B. garinii isolates PBr and Far04.
doi:10.1128/JB.05951-11
PMCID: PMC3232841  PMID: 22123755
6.  Comparative Genomics of Vancomycin-Resistant Staphylococcus aureus Strains and Their Positions within the Clade Most Commonly Associated with Methicillin-Resistant S. aureus Hospital-Acquired Infection in the United States  
mBio  2012;3(3):e00112-12.
ABSTRACT
Methicillin-resistant Staphylococcus aureus (MRSA) strains are leading causes of hospital-acquired infections in the United States, and clonal cluster 5 (CC5) is the predominant lineage responsible for these infections. Since 2002, there have been 12 cases of vancomycin-resistant S. aureus (VRSA) infection in the United States—all CC5 strains. To understand this genetic background and what distinguishes it from other lineages, we generated and analyzed high-quality draft genome sequences for all available VRSA strains. Sequence comparisons show unambiguously that each strain independently acquired Tn1546 and that all VRSA strains last shared a common ancestor over 50 years ago, well before the occurrence of vancomycin resistance in this species. In contrast to existing hypotheses on what predisposes this lineage to acquire Tn1546, the barrier posed by restriction systems appears to be intact in most VRSA strains. However, VRSA (and other CC5) strains were found to possess a constellation of traits that appears to be optimized for proliferation in precisely the types of polymicrobic infection where transfer could occur. They lack a bacteriocin operon that would be predicted to limit the occurrence of non-CC5 strains in mixed infection and harbor a cluster of unique superantigens and lipoproteins to confound host immunity. A frameshift in dprA, which in other microbes influences uptake of foreign DNA, may also make this lineage conducive to foreign DNA acquisition.
IMPORTANCE
Invasive methicillin-resistant Staphylococcus aureus (MRSA) infection now ranks among the leading causes of death in the United States. Vancomycin is a key last-line bactericidal drug for treating these infections. However, since 2002, vancomycin resistance has entered this species. Of the now 12 cases of vancomycin-resistant S. aureus (VRSA), each was believed to represent a new acquisition of the vancomycin-resistant transposon Tn1546 from enterococcal donors. All acquisitions of Tn1546 so far have occurred in MRSA strains of the clonal cluster 5 genetic background, the most common hospital lineage causing hospital-acquired MRSA infection. To understand the nature of these strains, we determined and examined the nucleotide sequences of the genomes of all available VRSA. Genome comparison identified candidate features that position strains of this lineage well for acquiring resistance to antibiotics in mixed infection.
doi:10.1128/mBio.00112-12
PMCID: PMC3372964  PMID: 22617140
7.  Genome Stability of Lyme Disease Spirochetes: Comparative Genomics of Borrelia burgdorferi Plasmids 
PLoS ONE  2012;7(3):e33280.
Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33–40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ∼900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.
doi:10.1371/journal.pone.0033280
PMCID: PMC3303823  PMID: 22432010
8.  Whole Genome Sequence of an Unusual Borrelia burgdorferi Sensu Lato Isolate▿  
Journal of Bacteriology  2011;193(6):1489-1490.
Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named “Borrelia finlandensis.”
doi:10.1128/JB.01521-10
PMCID: PMC3067611  PMID: 21217002
9.  Whole-Genome Sequences of Thirteen Isolates of Borrelia burgdorferi▿ †  
Journal of Bacteriology  2010;193(4):1018-1020.
Borrelia burgdorferi is a causative agent of Lyme disease in North America and Eurasia. The first complete genome sequence of B. burgdorferi strain 31, available for more than a decade, has assisted research on the pathogenesis of Lyme disease. Because a single genome sequence is not sufficient to understand the relationship between genotypic and geographic variation and disease phenotype, we determined the whole-genome sequences of 13 additional B. burgdorferi isolates that span the range of natural variation. These sequences should allow improved understanding of pathogenesis and provide a foundation for novel detection, diagnosis, and prevention strategies.
doi:10.1128/JB.01158-10
PMCID: PMC3028687  PMID: 20935092
10.  Bacterial diversity in the oral cavity of ten healthy individuals 
The ISME journal  2010;4(8):962-974.
The composition of the oral microbiota from 10 individuals with healthy oral tissues was determined using culture-independent techniques. From each individual, 26 specimens, each from different oral sites at a single point in time, were collected and pooled. An eleventh pool was constructed using portions of the subgingival specimens from all 10 individuals. The 16S rRNA gene was amplified using broad-range bacterial primers, and clone libraries from the individual and subgingival pools were constructed. From a total of 11 368 high-quality, non-chimeric, near full-length sequences, 247 species-level phylotypes (using a 99% sequence identity threshold) and 9 bacteria phyla were identified. At least 15 bacterial genera were conserved among all 10 individuals, with significant interindividual differences at the species and strain level. Comparisons of these oral bacterial sequences to near full-length sequences found previously in the large intestines and feces of other healthy individuals suggest that the mouth and intestinal tract harbor distinct sets of bacteria. Co-occurrence analysis demonstrated significant segregation of taxa when community membership was examined at the level of genus, but not at the level of species, suggesting that ecologically-significant, competitive interactions are more apparent at a broader taxonomic level than species. This study is one of the more comprehensive, high-resolution analyses of bacterial diversity within the healthy human mouth to date, and highlights the value of tools from macroecology for enhancing our understanding of bacterial ecology in human health.
doi:10.1038/ismej.2010.30
PMCID: PMC2941673  PMID: 20336157
oral microbiota; ribosomal RNA sequences; human microbial ecology
11.  Fast, Adaptive Evolution at a Bacterial Host-Resistance Locus: the PFam54 Gene Array in Borrelia burgdorferi 
Gene  2009;445(1-2):26-37.
Microbial pathogens have evolved sophisticated mechanisms for evasion of host innate and adaptive immunities. PFam54 is the largest paralogous gene family in the genomes of Borrelia burgdorferi, the Lyme disease bacterium. One member of PFam54, the complement-regulator acquiring surface proteins 1 (BbCRASP-1), is able to abort the alternative pathway of complement activation via binding human complement regulator factor H (FH). The gene coding for BbCRASP-1 exists in a tandem array of PFam54 genes in the B. burgdorferi genome, a result apparently of repeated gene duplications. To help elucidate the functions of the large number of PFam54 genes, we performed phylogenomic and structural analyses of the PFam54 gene array from ten B. burgdorferi genomes. Analyses based on gene tree, genome synteny, and structural models revealed rapid adaptive evolution of this array through gene duplication, gene loss, and functional diversification. Individual PFam54 genes, however, do not show high intra-population sequence polymorphisms as genes providing evasion from adaptive immunity generally do. PFam54 members able to bind human FH are not monophyletic, suggesting that human FH affinity, however strong, is an incidental rather than main function of these PFam54 proteins. The large number of PFam54 genes existing in any single B. burgdorferi genome may target different innate-immunity proteins of a single host species or the same immune protein of a variety of host species. Genetic variability of the PFam54 gene array suggests that universally present PFam54 lineages such as BBA64, BBA65, BBA66, and BBA73 may be better candidates for the development of broad-spectrum vaccines or drugs than strain-restricted lineages such as BbCRASP-1.
doi:10.1016/j.gene.2009.05.017
PMCID: PMC2743244  PMID: 19505540
Borrelia burgdorferi; Complement-Regulator Acquiring Surface Protein-1; Lyme disease; Factor H; immune evasion; cspA
12.  Hydrogenomics of the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus▿ †  
Applied and Environmental Microbiology  2008;74(21):6720-6729.
Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO2, and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium.
doi:10.1128/AEM.00968-08
PMCID: PMC2576683  PMID: 18776029
13.  The Pangenome Structure of Escherichia coli: Comparative Genomic Analysis of E. coli Commensal and Pathogenic Isolates▿ †  
Journal of Bacteriology  2008;190(20):6881-6893.
Whole-genome sequencing has been skewed toward bacterial pathogens as a consequence of the prioritization of medical and veterinary diseases. However, it is becoming clear that in order to accurately measure genetic variation within and between pathogenic groups, multiple isolates, as well as commensal species, must be sequenced. This study examined the pangenomic content of Escherichia coli. Six distinct E. coli pathovars can be distinguished using molecular or phenotypic markers, but only two of the six pathovars have been subjected to any genome sequencing previously. Thus, this report provides a seminal description of the genomic contents and unique features of three unsequenced pathovars, enterotoxigenic E. coli, enteropathogenic E. coli, and enteroaggregative E. coli. We also determined the first genome sequence of a human commensal E. coli isolate, E. coli HS, which will undoubtedly provide a new baseline from which workers can examine the evolution of pathogenic E. coli. Comparison of 17 E. coli genomes, 8 of which are new, resulted in identification of ∼2,200 genes conserved in all isolates. We were also able to identify genes that were isolate and pathovar specific. Fewer pathovar-specific genes were identified than anticipated, suggesting that each isolate may have independently developed virulence capabilities. Pangenome calculations indicate that E. coli genomic diversity represents an open pangenome model containing a reservoir of more than 13,000 genes, many of which may be uncharacterized but important virulence factors. This comparative study of the species E. coli, while descriptive, should provide the basis for future functional work on this important group of pathogens.
doi:10.1128/JB.00619-08
PMCID: PMC2566221  PMID: 18676672
14.  Extensive Genome Rearrangements and Multiple Horizontal Gene Transfers in a Population of Pyrococcus Isolates from Vulcano Island, Italy▿ †  
Applied and Environmental Microbiology  2008;74(20):6447-6451.
The extent of chromosome rearrangements in Pyrococcus isolates from marine hydrothermal vents in Vulcano Island, Italy, was evaluated by high-throughput genomic methods. The results illustrate the dynamic nature of the genomes of the genus Pyrococcus and raise the possibility of a connection between rapidly changing environmental conditions and adaptive genomic properties.
doi:10.1128/AEM.01024-08
PMCID: PMC2570278  PMID: 18723649
15.  Insights into Plant Cell Wall Degradation from the Genome Sequence of the Soil Bacterium Cellvibrio japonicus▿ †  
Journal of Bacteriology  2008;190(15):5455-5463.
The plant cell wall, which consists of a highly complex array of interconnecting polysaccharides, is the most abundant source of organic carbon in the biosphere. Microorganisms that degrade the plant cell wall synthesize an extensive portfolio of hydrolytic enzymes that display highly complex molecular architectures. To unravel the intricate repertoire of plant cell wall-degrading enzymes synthesized by the saprophytic soil bacterium Cellvibrio japonicus, we sequenced and analyzed its genome, which predicts that the bacterium contains the complete repertoire of enzymes required to degrade plant cell wall and storage polysaccharides. Approximately one-third of these putative proteins (57) are predicted to contain carbohydrate binding modules derived from 13 of the 49 known families. Sequence analysis reveals approximately 130 predicted glycoside hydrolases that target the major structural and storage plant polysaccharides. In common with that of the colonic prokaryote Bacteroides thetaiotaomicron, the genome of C. japonicus is predicted to encode a large number of GH43 enzymes, suggesting that the extensive arabinose decorations appended to pectins and xylans may represent a major nutrient source, not just for intestinal bacteria but also for microorganisms that occupy terrestrial ecosystems. The results presented here predict that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases. Most importantly, the genome of C. japonicus is remarkably similar to that of the gram-negative marine bacterium, Saccharophagus degradans 2-40T. Approximately 50% of the predicted C. japonicus plant-degradative apparatus appears to be shared with S. degradans, consistent with the utilization of plant-derived complex carbohydrates as a major substrate by both organisms.
doi:10.1128/JB.01701-07
PMCID: PMC2493263  PMID: 18556790
16.  Serial Analysis of rRNA Genes and the Unexpected Dominance of Rare Members of Microbial Communities▿  
Applied and Environmental Microbiology  2007;73(14):4532-4542.
The accurate description of a microbial community is an important first step in understanding the roles of its components in ecosystem function. A method for surveying microbial communities termed serial analysis of rRNA genes (SARD) is described here. Through a series of molecular cloning steps, short DNA sequence tags are recovered from the fifth variable (V5) region of the prokaryotic 16S rRNA genes from microbial communities. These tags are ligated to form concatemers comprised of 20 to 40 tags which are cloned and identified by DNA sequencing. Four agricultural soil samples were profiled with SARD to assess the method's utility. A total of 37,008 SARD tags comprising 3,127 unique sequences were identified. A comparison of duplicate profiles from one soil genomic DNA preparation revealed that the method was highly reproducible. The large numbers of singleton tags, together with nonparametric richness estimates, indicated that a significant amount of sequence tag diversity remained undetected with this level of sampling. The abundance classes of the observed tags were scale-free and conformed to a power law distribution. Numerically, the majority of the total tags observed belonged to abundance classes that were each present at less than 1% of the community. Over 99% of the unique tags individually made up less than 1% of the community. Therefore, from either a numerical or diversity standpoint, taxa with low abundance comprised a significant proportion of the microbial communities examined and could potentially make a large contribution to ecosystem function. SARD may provide a means to explore the ecological roles of these rare members of microbial communities in qualitative and quantitative terms.
doi:10.1128/AEM.02956-06
PMCID: PMC1932806  PMID: 17526780
17.  Secrets of Soil Survival Revealed by the Genome Sequence of Arthrobacter aurescens TC1 
PLoS Genetics  2006;2(12):e214.
Arthrobacter sp. strains are among the most frequently isolated, indigenous, aerobic bacterial genera found in soils. Member of the genus are metabolically and ecologically diverse and have the ability to survive in environmentally harsh conditions for extended periods of time. The genome of Arthrobacter aurescens strain TC1, which was originally isolated from soil at an atrazine spill site, is composed of a single 4,597,686 basepair (bp) circular chromosome and two circular plasmids, pTC1 and pTC2, which are 408,237 bp and 300,725 bp, respectively. Over 66% of the 4,702 open reading frames (ORFs) present in the TC1 genome could be assigned a putative function, and 13.2% (623 genes) appear to be unique to this bacterium, suggesting niche specialization. The genome of TC1 is most similar to that of Tropheryma, Leifsonia, Streptomyces, and Corynebacterium glutamicum, and analyses suggest that A. aurescens TC1 has expanded its metabolic abilities by relying on the duplication of catabolic genes and by funneling metabolic intermediates generated by plasmid-borne genes to chromosomally encoded pathways. The data presented here suggest that Arthrobacter's environmental prevalence may be due to its ability to survive under stressful conditions induced by starvation, ionizing radiation, oxygen radicals, and toxic chemicals.
Synopsis
Soil systems contain the greatest diversity of microorganisms on earth, with 5,000–10,000 species of microorganism per gram of soil. Arthrobacter sp. strains have a primitive life cycle and are among the most frequently isolated, indigenous soil bacteria, found in common and deep subsurface soils, arctic ice, and environments contaminated with industrial chemicals and radioactive materials. To better understand how these bacteria survive in environmentally harsh conditions, the authors used a structural genomics approach to identify genes involved in soil survival of Arthrobacter aurescens strain TC1, a bacterium originally isolated for its ability to degrade the herbicide atrazine. They found that the genome of this bacterium comprises a single circular chromosome and two plasmids that encode for a large number proteins involved in stress responses due to starvation, desiccation, oxygen radicals, and toxic chemicals. A. aurescens' metabolic versatility is in part due to the presence of duplicated catabolic genes and its ability to funnel plasmid-derived intermediates into chromosomally encoded pathways. Arthrobacter's array of genes that allow for survival in stressful conditions and its ability to produce a temperature-tolerant “cyst”-like resting cell render this soil microorganism able to survive and prosper in a variety of environmental conditions.
doi:10.1371/journal.pgen.0020214
PMCID: PMC1713258  PMID: 17194220
18.  Secrets of Soil Survival Revealed by the Genome Sequence of Arthrobacter aurescens TC1 
PLoS Genetics  2006;2(12):e214.
Arthrobacter sp. strains are among the most frequently isolated, indigenous, aerobic bacterial genera found in soils. Member of the genus are metabolically and ecologically diverse and have the ability to survive in environmentally harsh conditions for extended periods of time. The genome of Arthrobacter aurescens strain TC1, which was originally isolated from soil at an atrazine spill site, is composed of a single 4,597,686 basepair (bp) circular chromosome and two circular plasmids, pTC1 and pTC2, which are 408,237 bp and 300,725 bp, respectively. Over 66% of the 4,702 open reading frames (ORFs) present in the TC1 genome could be assigned a putative function, and 13.2% (623 genes) appear to be unique to this bacterium, suggesting niche specialization. The genome of TC1 is most similar to that of Tropheryma, Leifsonia, Streptomyces, and Corynebacterium glutamicum, and analyses suggest that A. aurescens TC1 has expanded its metabolic abilities by relying on the duplication of catabolic genes and by funneling metabolic intermediates generated by plasmid-borne genes to chromosomally encoded pathways. The data presented here suggest that Arthrobacter's environmental prevalence may be due to its ability to survive under stressful conditions induced by starvation, ionizing radiation, oxygen radicals, and toxic chemicals.
Synopsis
Soil systems contain the greatest diversity of microorganisms on earth, with 5,000–10,000 species of microorganism per gram of soil. Arthrobacter sp. strains have a primitive life cycle and are among the most frequently isolated, indigenous soil bacteria, found in common and deep subsurface soils, arctic ice, and environments contaminated with industrial chemicals and radioactive materials. To better understand how these bacteria survive in environmentally harsh conditions, the authors used a structural genomics approach to identify genes involved in soil survival of Arthrobacter aurescens strain TC1, a bacterium originally isolated for its ability to degrade the herbicide atrazine. They found that the genome of this bacterium comprises a single circular chromosome and two plasmids that encode for a large number proteins involved in stress responses due to starvation, desiccation, oxygen radicals, and toxic chemicals. A. aurescens' metabolic versatility is in part due to the presence of duplicated catabolic genes and its ability to funnel plasmid-derived intermediates into chromosomally encoded pathways. Arthrobacter's array of genes that allow for survival in stressful conditions and its ability to produce a temperature-tolerant “cyst”-like resting cell render this soil microorganism able to survive and prosper in a variety of environmental conditions.
doi:10.1371/journal.pgen.0020214
PMCID: PMC1713258  PMID: 17194220
19.  Chromosome Evolution in the Thermotogales: Large-Scale Inversions and Strain Diversification of CRISPR Sequences 
Journal of Bacteriology  2006;188(7):2364-2374.
In the present study, the chromosomes of two members of the Thermotogales were compared. A whole-genome alignment of Thermotoga maritima MSB8 and Thermotoga neapolitana NS-E has revealed numerous large-scale DNA rearrangements, most of which are associated with CRISPR DNA repeats and/or tRNA genes. These DNA rearrangements do not include the putative origin of DNA replication but move within the same replichore, i.e., the same replicating half of the chromosome (delimited by the replication origin and terminus). Based on cumulative GC skew analysis, both the T. maritima and T. neapolitana lineages contain one or two major inverted DNA segments. Also, based on PCR amplification and sequence analysis of the DNA joints that are associated with the major rearrangements, the overall chromosome architecture was found to be conserved at most DNA joints for other strains of T. neapolitana. Taken together, the results from this analysis suggest that the observed chromosomal rearrangements in the Thermotogales likely occurred by successive inversions after their divergence from a common ancestor and before strain diversification. Finally, sequence analysis shows that size polymorphisms in the DNA joints associated with CRISPRs can be explained by expansion and possibly contraction of the DNA repeat and spacer unit, providing a tool for discerning the relatedness of strains from different geographic locations.
doi:10.1128/JB.188.7.2364-2374.2006
PMCID: PMC1428405  PMID: 16547022
20.  A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes 
PLoS Computational Biology  2005;1(6):e60.
Clustered regularly interspaced short palindromic repeats (CRISPRs) are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21–37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas) protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer “immunity” against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.
Synopsis
The family of clustered regularly interspaced short palindromic repeats (CRISPRs) describes a class of DNA repeats found in nearly half of all bacterial and archaeal genomes. These DNA repeat regions have a remarkably regular structure: unique sequences of constant size, called spacers, sit between each pair of repeats. The DNA repeats do not encode proteins, but appear to be transcribed and processed into small RNAs that may have any number of functions, including resistance to any phage (i.e., virus of bacteria) whose sequence matches a spacer; spacers change rapidly as microbial strains evolve. This work describes 41 new CRISPR-associated (cas) gene families, which are always found near these repeats, in addition to the four previously known. It shows that CRISPR systems belong to different classes, with different repeat patterns, sets of genes, and species ranges. Most of these seem to come and go rather rapidly from their host genomes. These possibly beneficial mobile genetic elements may play an important role in driving prokaryotic evolution.
doi:10.1371/journal.pcbi.0010060
PMCID: PMC1282333  PMID: 16292354
21.  A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes 
PLoS Computational Biology  2005;1(6):e60.
Clustered regularly interspaced short palindromic repeats (CRISPRs) are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21–37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas) protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer “immunity” against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.
Synopsis
The family of clustered regularly interspaced short palindromic repeats (CRISPRs) describes a class of DNA repeats found in nearly half of all bacterial and archaeal genomes. These DNA repeat regions have a remarkably regular structure: unique sequences of constant size, called spacers, sit between each pair of repeats. The DNA repeats do not encode proteins, but appear to be transcribed and processed into small RNAs that may have any number of functions, including resistance to any phage (i.e., virus of bacteria) whose sequence matches a spacer; spacers change rapidly as microbial strains evolve. This work describes 41 new CRISPR-associated (cas) gene families, which are always found near these repeats, in addition to the four previously known. It shows that CRISPR systems belong to different classes, with different repeat patterns, sets of genes, and species ranges. Most of these seem to come and go rather rapidly from their host genomes. These possibly beneficial mobile genetic elements may play an important role in driving prokaryotic evolution.
doi:10.1371/journal.pcbi.0010060
PMCID: PMC1282333  PMID: 16292354
22.  Microbial metagenomics 
Genome Biology  2005;6(10):347.
A report on the 105th Annual Meeting of the American Society for Microbiology, Atlanta, USA, 5-9 June 2005.
A report on the 105th Annual Meeting of the American Society for Microbiology, Atlanta, USA, 5-9 June 2005.
doi:10.1186/gb-2005-6-10-347
PMCID: PMC1257460  PMID: 16207365
23.  The first decade of microbial genomics: what have we learned and where are we going next? 
Genome Biology  2005;6(9):341.
A report on the International Conference on Microbial Genomics, Halifax, Canada, 13-16 April 2005.
A report on the International Conference on Microbial Genomics, Halifax, Canada, 13-16 April 2005.
doi:10.1186/gb-2005-6-9-341
PMCID: PMC1242203  PMID: 16168094
24.  Gene Transfer and Genome Plasticity in Thermotoga maritima, a Model Hyperthermophilic Species†  
Journal of Bacteriology  2005;187(14):4935-4944.
The genome sequence of the hyperthermophilic bacterium Thermotoga maritima MSB8 presents evidence for lateral gene transfer events between bacterial and archaeal species. To estimate the extent of genomic diversity across the order Thermotogales, a comparative genomic hybridization study was initiated to compare nine Thermotoga strains to the sequenced T. maritima MSB8. Many differences could be associated with substrate utilization patterns, which are most likely a reflection of the environmental niche that these individual species occupy. A detailed analysis of some of the predicted variable regions demonstrates many examples of the deletion/insertion of complete cassettes of genes and of gene rearrangements and insertions of DNA within genes, with the C or N terminus being retained. Although the mechanism for gene transfer in this lineage remains to be elucidated, this analysis suggests possible associations with repetitive elements and highlights the possible benefits of rampant genetic exchange to these species.
doi:10.1128/JB.187.14.4935-4944.2005
PMCID: PMC1169497  PMID: 15995209
25.  Insights on Evolution of Virulence and Resistance from the Complete Genome Analysis of an Early Methicillin-Resistant Staphylococcus aureus Strain and a Biofilm-Producing Methicillin-Resistant Staphylococcus epidermidis Strain†  
Journal of Bacteriology  2005;187(7):2426-2438.
Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the ∼2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the ∼2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis.
doi:10.1128/JB.187.7.2426-2438.2005
PMCID: PMC1065214  PMID: 15774886

Results 1-25 (31)