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.

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.

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.

Membrane transport proteins: implications of sequence comparisons.

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.

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.

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.

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.

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.

Major facilitator superfamily.

The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane alpha-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living 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.

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.

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.

Phylogenetic characterization of novel transport protein families revealed by genome analyses.

As a result of recent genome sequencing projects as well as detailed biochemical, molecular genetic and physiological experimentation on representative transport proteins, we have come to realize that all organisms possess an extensive but limited array of transport protein types that allow the uptake of nutrients and excretion of toxic substances. These proteins fall into phylogenetic families that presumably reflect their evolutionary histories. Some of these families are restricted to a single phylogenetic group of organisms and may have arisen recently in evolutionary time while others are found ubiquitously and may be ancient. In this study we conduct systematic phylogenetic analyses of 26 families of transport systems that either had not been characterized previously or were in need of updating. Among the families analyzed are some that are bacterial-specific, others that are eukaryotic-specific, and others that are ubiquitous. They can function by either a channel-type or a carrier-type mechanism, and in the latter case, they are frequently energized by coupling solute transport to the flux of an ion down its electrochemical gradient. We tabulate the currently sequenced members of the 26 families analyzed, describe the properties of these families, and present partial multiple alignments, signature sequences and phylogenetic trees for them all.

Microbial genome analyses: global comparisons of transport capabilities based on phylogenies, bioenergetics and substrate specificities.

We have conducted genome sequence analyses of seven prokaryotic microorganisms for which completely sequenced genomes are available (Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Mycoplasma genitalium, Synechocystis PCC6803 and Methanococcus jannaschii). We report the distribution of encoded known and putative polytopic cytoplasmic membrane transport proteins within these genomes. Transport systems for each organism were classified according to (1) putative membrane topology, (2) protein family, (3) bioenergetics, and (4) substrate specificities. The overall transport capabilities of each organism were thereby estimated. Probable function was assigned to greater than 90% of the putative transport proteins identified. The results show the following: (1) Numbers of transport systems in eubacteria are approximately proportional to genome size and correspond to 9.7 to 10.8% of the total encoded genes except for H. pylori (5.4%), Synechocystis (4.7%) and M. jannaschii (3.5%) which exhibit substantially lower proportions. (2) The distribution of topological types is similar in all seven organisms. (3) Transport systems belonging to 67 families were identified within the genomes of these organisms, and about half of these families are also found in eukaryotes. (4) 12% of these families are found exclusively in Gram-negative bacteria, but none is found exclusively in Gram-positive bacteria, cyanobacteria or archaea. (5) Two superfamilies, the ATP-binding cassette (ABC) and major facilitator (MF) superfamilies account for nearly 50% of all transporters in each organism, but the relative representation of these two transporter types varies over a tenfold range, depending on the organism. (6) Secondary, pmf-dependent carriers are 1.5 to threefold more prevalent than primary ATP-dependent carriers in E. coli, H. influenzae, H. pylori and B. subtilis while primary carriers are about twofold more prevalent in M. genitalium and Synechocystis. M. jannaschii exhibits a slight preference for secondary carriers. (7) Bioenergetics of transport generally correlate with the primary forms of energy generated via available metabolic pathways but ecological niche and substrate availability may also be determining factors. (8) All organisms display a similar range of transport specificities with quantitative differences presumably reflective of disparate ecological niches. (9) M. jannaschii and Synechocystis have a two to threefold increased proportion of transporters for inorganic ions with a concomitant decrease in transporters for organic compounds. (10) 6 to 18% of all transporters in these bacteria probably function as drug export systems showing that these systems are prevalent in non-pathogenic as well as pathogenic organisms. (11) All seven prokaryotes examined encode proteins homologous to known channel proteins, but none of the channel types identified occurs in all of these organisms. (12) The phosphoenolpyruvate:sugar phosphotransferase system is prevalent in the large genome organisms, E. coli and B. subtilis, and is present in the small genome organisms, H. influenzae and M. genitalium, but is totally lacking in H. pylori, Synechocystis and M. jannaschii. Details of the information summarized in this article are available on our web sites, and this information will be periodically updated and corrected as new sequence and biochemical data become available.

Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes.

Here, we present a comprehensive analysis of solute transport systems encoded within the completely sequenced genomes of 18 prokaryotic organisms. These organisms include four Gram-positive bacteria, seven Gram-negative bacteria, two spirochetes, one cyanobacterium and four archaea. Membrane proteins are analyzed in terms of putative membrane topology, and the recognized transport systems are classified into 76 families, including four families of channel proteins, four families of primary carriers, 54 families of secondary carriers, six families of group translocators, and eight unclassified families. These families are analyzed in terms of the paralogous and orthologous relationships of their protein members, the substrate specificities of their constituent transporters and their distributions in each of the 18 organisms studied. The families vary from large superfamilies with hundreds of represented members, to small families with only one or a few members. The mode of transport generally correlates with the primary mechanism of energy generation, and the numbers of secondary transporters relative to primary transporters are roughly proportional to the total numbers of primary H(+) and Na(+) pumps in the cell. The phosphotransferase system is less prevalent in the analyzed bacteria than previously thought (only six of 14 bacteria transport sugars via this system) and is completely lacking in archaea and eukaryotes. Escherichia coli is shown to be exceptionally broad in its transport capabilities and therefore, at a membrane transport level, does not appear representative of the bacteria thus far sequenced. Archaea and spirochetes exhibit fewer proteins with multiple transmembrane segments and fewer net transporters than most bacteria. These results provide insight into the relevance of transport to the overall physiology of prokaryotes.

Topology, structure and evolution of two families of proteins involved in antibiotic and antiseptic resistance in eukaryotes and prokaryotes--an analysis.

Analysis of deduced amino acid sequences has demonstrated that the sequences of eukaryotic and prokaryotic proteins mediating resistance to antibiotics and antiseptics are highly related. Hydropathy analysis and alignment of conserved motifs revealed that these proteins can be divided into two separate families with either 12 or 14 transmembrane segments (TMS). Conserved motifs have been identified which are either characteristic for each family or conserved in both families. The conservation of these motifs suggested that they may be essential for the function of these proteins. Phylogenetic and structural analysis revealed that the two families may have evolved from a common ancestor with six TMS.

Analysis of a transfer region from the staphylococcal conjugative plasmid pSK41.

The nucleotide sequence of a 14.4-kb region (tra) associated with DNA transfer of the staphylococcal conjugative plasmid, pSK41, has been determined. Analysis of the sequence revealed the presence of 15 genes potentially involved in the conjugative process. Polypeptide products likely to correspond to ten of these genes have been identified, of which one was found to be a lipoprotein. Comparison of the deduced tra products to the protein databases revealed several interesting similarities, one of which suggests an evolutionary link between this Gram+ bacterial conjugation system and DNA transfer systems of Gram- bacteria, such as Escherichia coli and Agrobacterium tumefaciens. The nt sequence also provided an insight into the transcriptional organisation and regulation of the region.

Characterisation of sin, a potential recombinase-encoding gene from Staphylococcus aureus.

The staphylococcal beta-lactamase (Bla) transposon Tn4002 has previously been reported to have a high level of insertional specificity for a 1.8-kb region on the plasmid pSK1. Nucleotide sequences of this region and of a related region on plasmid pI9789 were determined. Sequence analysis revealed that these two plasmids contain sin, a gene whose deduced product shows similarity to a family of DNA recombinases. Southern hybridisation analysis indicated that sin is located on alpha-, beta- and gamma-families of Bla plasmids and on pSK1 family plasmids. A region of dyad symmetry located upstream from sin on pSK1 and pI9789 was identified as the site of insertions of Tn552 and Tn4002 in separate isolates.

Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae.

We present the complete inventory of currently recognized and putative transporters encoded within the genome of Saccharomyces cerevisiae. These 258 transporters are classified into 42 families according to phylogenetic and substrate specificity criteria. Twelve of these yeast families are found only in eukaryotic organisms, and four are so far unique to yeast. Putative yeast-specific families transport heavy metals, arsenite and calcium. The phylogenetic analyses reported allow classification of 139 functionally uncharacterized yeast transporters into families of known functions. The relative proportions of yeast transporters specific for different classes of substrates differ only slightly from those reported for Escherichia coli. However, the ratio of secondary transporters (uniporters, cation symporters and antiporters) to primary ATP-driven transporters is much higher for yeast than for bacteria.

Paralogous genes encoding transport proteins in microbial genomes.

The largest superfamilies of prokaryotic genes encode transport proteins, but many transporters are encoded by orphan genes or those that comprise very small families. We have analyzed eighteen completely sequenced prokaryotic genomes for paralogous transport systems and have thereby identified 76 permease families. In this short review, we present and discuss the paralogues in some of these families and interpret the most prominent results, particularly those relevant to the largest permease superfamilies.