Identifying the suite of genes central to swimming in the biocontrol bacterium Pseudomonas protegens Pf-5.

Swimming motility is a key bacterial trait, important to success in many niches. Biocontrol bacteria, such as Pseudomonas protegens Pf-5, are increasingly used in agriculture to control crop diseases, where motility is important for colonization of the plant rhizosphere. Swimming motility typically involves a suite of flagella and chemotaxis genes, but the specific gene set employed for both regulation and biogenesis can differ substantially between organisms. Here we used transposon-directed insertion site sequencing (TraDIS), a genome-wide approach, to identify 249 genes involved in P. protegens Pf-5 swimming motility. In addition to the expected flagella and chemotaxis, we also identified a suite of additional genes important for swimming, including genes related to peptidoglycan turnover, O-antigen biosynthesis, cell division, signal transduction, c-di-GMP turnover and phosphate transport, and 27 conserved hypothetical proteins. Gene knockout mutants and TraDIS data suggest that defects in the Pst phosphate transport system lead to enhanced swimming motility. Overall, this study expands our knowledge of pseudomonad motility and highlights the utility of a TraDIS-based approach for analysing the functions of thousands of genes. This work sets a foundation for understanding how swimming motility may be related to the inconsistency in biocontrol bacteria performance in the field.

QTL analysis of natural Saccharomyces cerevisiae isolates reveals unique alleles involved in lignocellulosic inhibitor tolerance.

Decoding the genetic basis of lignocellulosic inhibitor tolerance in Saccharomyces cerevisiae is crucial for rational engineering of bioethanol strains with enhanced robustness. The genetic diversity of natural strains present an invaluable resource for the exploration of complex traits of industrial importance from a pan-genomic perspective to complement the limited range of specialised, tolerant industrial strains. Natural S. cerevisiae isolates have lately garnered interest as a promising toolbox for engineering novel, genetically encoded tolerance phenotypes into commercial strains. To this end, we investigated the genetic basis for lignocellulosic inhibitor tolerance of natural S. cerevisiae isolates. A total of 12 quantitative trait loci underpinning tolerance were identified by next-generation sequencing linked bulk-segregant analysis of superior interbred pools. Our findings corroborate the current perspective of lignocellulosic inhibitor tolerance as a multigenic, complex trait. Apart from a core set of genetic variants required for inhibitor tolerance, an additional genetic background-specific response was observed. Functional analyses of the identified genetic loci revealed the uncharacterised ORF, YGL176C and the bud-site selection XRN1/BUD13 as potentially beneficial alleles contributing to tolerance to a complex lignocellulosic inhibitor mixture. We present evidence for the consideration of both regulatory and coding sequence variants for strain improvement.

Microbial communities are sensitive indicators for freshwater sediment copper contamination.

Anthropogenic activities, such as mining and agriculture, have resulted in many freshwater systems having elevated concentrations of copper. Despite the prevalence of this contamination, and the vital ecological function of prokaryotes, just three studies have investigated prokaryote community responses to copper concentration in freshwater sediments. To address this, the current study investigated these communities in outdoor mesocosms spiked with varying copper concentrations. We profiled the prokaryotic communities at the taxonomic level, using next-generation high-throughput sequencing techniques, as well as their function, using baiting with leaf analogues, and Biolog Ecoplates for community-level physiological profiling. Sediments containing just 46 mg kg-1 of copper, had distinctly different microbial communities compared with controls, as determined by both DNA and RNA 16S ribosomal RNA gene (rRNA) profiling. In addition to this, sediment communities displayed a greatly reduced utilisation of carbon substrates under elevated copper, while the communities recruited onto leaf analogues were also disparate from those of control ponds. Given the vital role of prokaryotes in ecosystem processes, including carbon cycling, these changes are potentially of great ecological relevance, and are seen to occur well below the 'low risk' sediment quality guideline values (SQGV) used by regulatory bodies internationally.

Genetic identification of a high-affinity Ni transporter and the transcriptional response to Ni deprivation in Synechococcus sp. strain WH8102.

One biological need for Ni in marine cyanobacteria stems from the utilization of the Ni metalloenzyme urease for the assimilation of urea as a nitrogen source. In many of the same cyanobacteria, including Synechococcus sp. strain WH8102, an additional and obligate nutrient requirement for Ni results from usage of a Ni superoxide dismutase (Ni-SOD), which is encoded by sodN. To better understand the effects of Ni deprivation on WH8102, parallel microarray-based analysis of gene expression and gene knockout experiments were conducted. The global transcriptional response to Ni deprivation depends upon the nitrogen source provided for growth; fewer than 1% of differentially expressed genes for Ni deprivation on ammonium or urea were concordantly expressed. Surprisingly, genes for putative Ni transporters, including one colocalized on the genome with sodN, sodT, were not induced despite an increase in Ni transport. Knockouts of the putative Ni transporter gene sodT appeared to be lethal in WH8102, so the genes for sodT and sodN in WH8102 were interrupted with the gene for Fe-SOD, sodB, and its promoter from Synechococcus sp. strain WH7803. The sodT::sodB exconjugants were unable to grow at low Ni concentrations, confirming that SodT is a Ni transporter. The sodN::sodB exconjugants displayed higher growth rates at low Ni concentrations than did the wild type, presumably due to a relaxed competition between urease and Ni-SOD for Ni. Both sodT::sodB and sodN::sodB lines exhibited an impaired ability to grow at low Fe concentrations. We propose a posttranslational allosteric SodT regulation involving the binding of Ni to a histidine-rich intracellular protein loop.

Statistical analysis of microarray data with replicated spots: a case study with synechococcus WH8102.

Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in part to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition.

Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity.

The extent to which cultured strains represent the genetic diversity of a population of microorganisms is poorly understood. Because they do not require culturing, metagenomic approaches have the potential to reveal the genetic diversity of the microbes actually present in an environment. From coastal California seawater, a complex and diverse environment, the marine cyanobacteria of the genus Synechococcus were enriched by flow cytometry-based sorting and the population metagenome was analysed with 454 sequencing technology. The sequence data were compared with model Synechococcus genomes, including those of two coastal strains, one isolated from the same and one from a very similar environment. The natural population metagenome had high sequence identity to most genes from the coastal model strains but diverged greatly from these genomes in multiple regions of atypical trinucleotide content that encoded diverse functions. These results can be explained by extensive horizontal gene transfer presumably with large differences in horizontally transferred genetic material between different strains. Some assembled contigs showed the presence of novel open reading frames not found in the model genomes, but these could not yet be unambiguously assigned to a Synechococcus clade. At least three distinct mobile DNA elements (plasmids) not found in model strain genomes were detected in the assembled contigs, suggesting for the first time their likely importance in marine cyanobacterial populations and possible role in horizontal gene transfer.

Genome of Geobacter sulfurreducens: metal reduction in subsurface environments.

The complete genome sequence of Geobacter sulfurreducens, a delta-proteobacterium, reveals unsuspected capabilities, including evidence of aerobic metabolism, one-carbon and complex carbon metabolism, motility, and chemotactic behavior. These characteristics, coupled with the possession of many two-component sensors and many c-type cytochromes, reveal an ability to create alternative, redundant, electron transport networks and offer insights into the process of metal ion reduction in subsurface environments. As well as playing roles in the global cycling of metals and carbon, this organism clearly has the potential for use in bioremediation of radioactive metals and in the generation of electricity.

Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis.

The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.

Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440.

Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

Phylogeny of multidrug transporters.

We currently recognize five large ubiquitous superfamilies and one small eukaryotic-specific family in which cellular multidrug efflux pumps occur. One, the ABC superfamily, includes members that use ATP hydrolysis to drive drug efflux, but the MFS, RND, MATE and DMT superfamilies include members that are secondary carriers, functioning by drug:H(+)or drug:Na(+)antiport mechanisms. The small MET family seems to be restricted to endosomal membranes of eukaryotes, and only a single such system has been functionally characterized. In this review article, these families of drug transporters are discussed and evaluated from phylogenetic standpoints.

Comparative genomics of microbial drug efflux systems.

The complete genome sequences of 36 microorganisms have now been published and this wealth of genome data has enabled the development of comparative genomic and functional genomic approaches to investigate the biology of these organisms. Comparative genomic analyses of membrane transport systems have revealed that transporter substrate specificities correlate with an organism's lifestyle. The types and numbers of predicted drug efflux systems vary dramatically amongst sequenced organisms. Microarray and gene knockout studies to date have suggested that predicted drug efflux genes often appear to be a) non-essential and b) expressed at detectable levels under standard laboratory growth conditions.

Complete genome sequence of Caulobacter crescentus.

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

TIGRFAMs: a protein family resource for the functional identification of proteins.

TIGRFAMs is a collection of protein families featuring curated multiple sequence alignments, hidden Markov models and associated information designed to support the automated functional identification of proteins by sequence homology. We introduce the term 'equivalog' to describe members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families where possible, and otherwise into protein families with other hierarchically defined homology types. TIGRFAMs currently contains over 800 protein families, available for searching or downloading at www.tigr.org/TIGRFAMs. Classification by equivalog family, where achievable, complements classification by orthology, superfamily, domain or motif. It provides the information best suited for automatic assignment of specific functions to proteins from large-scale genome sequencing projects.

Whole genome analyses of transporters in spirochetes: Borrelia burgdorferi and Treponema pallidum.

The completely sequenced genomes of two spirochetes, Borrelia burgdorferi(Bbu) and Treponema pallidum (Tpa) were analyzed for the distribution of transporter types. Both organisms exhibited fewer proteins with >7 alpha-helical transmembrane spanners (TMSs), and fewer identified transport systems per megabase pair of DNA than most other prokaryotes analyzed. Each organism exhibits one recognizable ion channel protein of the MscS family. Tpa has twice as many primary carriers as Bbu but lacks PTS permeases that are plentiful in Bbu. Tpa is the only prokaryote so far sequenced which has two F-type ATPases. Large families of secondary nutrient uptake carriers (MFS and APC) that are prevalent in other organisms are essentially lacking in Spirochetes. The largest Spirochete secondary carrier families consist of efflux systems. While both Bbu and Tpa exhibit an unusual degree of transporter diversity, major differences in specificity exist between these two organisms.

Status of genome projects for nonpathogenic bacteria and archaea.

Since the first microbial genome was sequenced in 1995, 30 others have been completed and an additional 99 are known to be in progress. Although the early emphasis of microbial genomics was on human pathogens for obvious reasons, a significant number of sequencing projects have focused on nonpathogenic organisms, beginning with the release of the complete genome sequence of the archaeon Methanococcus jannaschii in 1996. The past 18 months have seen the completion of the genomes of several unusual organisms, including Thermotoga maritima, whose genome reveals extensive potential lateral transfer with archaea; Deinococcus radiodurans, the most radiation-resistant microorganism known; and Aeropyrum pernix, the first Crenarchaeota to be completely sequenced. Although the functional characterization of genomic data is still in its initial stages, it is likely that microbial genomics will have a significant impact on environmental, food, and industrial biotechnology as well as on genomic medicine.