Bacterial nomenclature in the era of genomics.

The remarkable success of taxonomic discovery, powered by culturomics, genomics and metagenomics, creates a pressing need for new bacterial names while holding a mirror up to the slow pace of change in bacterial nomenclature. Here, I take a fresh look at bacterial nomenclature, exploring how we might create a system fit for the age of genomics, playing to the strengths of current practice while minimizing difficulties. Adoption of linguistic pragmatism-obeying the rules while treating recommendations as merely optional-will make it easier to create names derived from descriptions, from people or places or even arbitrarily. Simpler protologues and a relaxed approach to recommendations will also remove much of the need for expert linguistic quality control. Automated computer-based approaches will allow names to be created en masse before they are needed while also relieving microbiologists of the need for competence in Latin. The result will be a system that is accessible, inclusive and digital, while also fully capable of naming the unnamed millions of bacteria.

Draft Genome Sequences of 11 Clinical Isolates of Acinetobacter baumannii.

The development of multidrug-resistantAcinetobacter baumanniiis of serious concern in the hospital setting. Here, we report draft genome sequences of 11A. baumanniiisolates that were isolated from a single patient over a 65-day period, during which time the isolates exhibited increased antimicrobial resistance.

Draft Genome Sequence of the Pandoraea apista LMG 16407 Type Strain.

Pandoraea species, in particular Pandoraea apista, are opportunistic, multidrug-resistant pathogens in persons with cystic fibrosis (CF). To aid in understanding the role of P. apista in CF lung disease, we used Illumina MiSeq and nanopore MinION technology to sequence the whole genome of the P. apista LMG 16407(T).

Diagnostic metagenomics: potential applications to bacterial, viral and parasitic infections.

The term 'shotgun metagenomics' is applied to the direct sequencing of DNA extracted from a sample without culture or target-specific amplification or capture. In diagnostic metagenomics, this approach is applied to clinical samples in the hope of detecting and characterizing pathogens. Here, I provide a conceptual overview, before reviewing several recent promising proof-of-principle applications of metagenomics in virus discovery, analysis of outbreaks and detection of pathogens in contemporary and historical samples. I also evaluate future prospects for diagnostic metagenomics in the light of relentless improvements in sequencing technologies.

Epidemiological investigation of Pseudomonas aeruginosa isolates from a six-year-long hospital outbreak using high-throughput whole genome sequencing.

Although previous bacterial typing methods have been informative about potential relatedness of isolates collected during outbreaks, next-generation sequencing has emerged as a powerful tool to not only look at similarity between isolates, but also put differences into biological context. In this study, we have investigated the whole genome sequence of five Pseudomonas aeruginosa isolates collected during a persistent six-year outbreak at Nottingham University Hospitals National Health Service (NHS) Trust ­ City Campus, United Kingdom. Sequencing, using both Roche 454 and Illumina, reveals that most of these isolates are closely related. Some regions of difference are noted between this cluster of isolates and previously published genome sequences. These include regions containing prophages and prophage remnants such as the serotype-converting bacteriophage D3 and the cytotoxin-converting phage phi CTX. Additionally, single nucleotide polymorphisms (SNPs) between the genomic sequence data reveal key single base differences that have accumulated during the course of this outbreak, giving insight into the evolution of the outbreak strain. Differentiating SNPs were found within a wide variety of genes, including lasR, nrdG, tadZ, and algB. These have been generated at a rate estimated to be one SNP every four to five months. In conclusion, we demonstrate that the single base resolution of whole genome sequencing is a powerful tool in analysis of outbreak isolates that can not only show strain similarity, but also evolution over time and potential adaptation through gene sequence changes.

High-throughput whole-genome sequencing to dissect the epidemiology of Acinetobacter baumannii isolates from a hospital outbreak.

Shared care of military and civilian patients has resulted in transmission of multidrug-resistant Acinetobacter baumannii (MDR-Aci) from military casualties to civilians. Current typing technologies have been useful in revealing relationships between isolates of A. baumannii but they are unable to resolve differences between closely related isolates from small-scale outbreaks, where chains of transmission are often unclear. In a recent hospital outbreak in Birmingham, six patients were colonised with MDR-Aci isolates indistinguishable using standard techniques. We used whole-genome sequencing to identify single nucleotide polymorphisms in these isolates, allowing us to discriminate between alternative epidemiological hypotheses in this setting.

Genome sequence of the emerging pathogen Helicobacter canadensis.

We determined the genome sequence of the type strain of Helicobacter canadensis, an emerging human pathogen with diverse animal reservoirs. Potential virulence determinants carried by the genome include systems for N-linked glycosylation and capsular export. A protein-based phylogenetic analysis places H. canadensis close to Wolinella succinogenes.

Bacterial flagella and Type III secretion: case studies in the evolution of complexity.

Bacterial flagella at first sight appear uniquely sophisticated in structure, so much so that they have even been considered 'irreducibly complex' by the intelligent design movement. However, a more detailed analysis reveals that these remarkable pieces of molecular machinery are the product of processes that are fully compatible with Darwinian evolution. In this chapter we present evidence for such processes, based on a review of experimental studies, molecular phylogeny and microbial genomics. Several processes have played important roles in flagellar evolution: self-assembly of simple repeating subunits, gene duplication with subsequent divergence, recruitment of elements from other systems ('molecular bricolage'), and recombination. We also discuss additional tentative new assignments of homology (FliG with MgtE, FliO with YscJ). In conclusion, rather than providing evidence of intelligent design, flagellar and non-flagellar Type III secretion systems instead provide excellent case studies in the evolution of complex systems from simpler components.

An abundance of bacterial ADP-ribosyltransferases--implications for the origin of exotoxins and their human homologues.

ADP-ribosylation is a post-translational modification that can be seen in many contexts, including as the primary mechanism of action of many important bacterial exotoxins. By data-mining complete and incomplete bacterial genome sequences, we have discovered >20 novel putative ADP-ribosyltransferases, including several new potential toxins.

Coiled-coil domain of enteropathogenic Escherichia coli type III secreted protein EspD is involved in EspA filament-mediated cell attachment and hemolysis.

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic Escherichia coli the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic E. coli mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.

An embarrassment of sortases - a richness of substrates?

A range of surface proteins is anchored to the cell walls of Gram-positive pathogens such as Staphylococcus aureus by the transpeptidase sortase. Until now, sortase-like proteins and their substrates appeared to be limited mainly to such pathogens. However, by searching for sortase homologues among complete and incomplete genome sequences, we have found them to be present in almost all Gram-positives, a single Gram-negative bacterium and an archaean. There is usually more than one sortase-like protein encoded in each Gram-positive genome, and the genes encoding the sortase-like proteins are often clustered with genes encoding their likely substrates.

Tricorn-like proteases in bacteria.

The tricorn protease is an archaeal protease that forms massive proteasome-like capsids with a hollow chamber. beta-Propeller and PDZ domains are thought to play a role in substrate selection. By analysis of predicted proteins from novel bacterial genome sequences, we have identified four new bacterial tricorn-like proteases, complete with similar beta-propeller, PDZ and catalytic domains. We propose various hypotheses as to the function of these domains that can now be tested in the laboratory.

The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences.

Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium-properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain-Barré syndrome. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.

The coiled-coil domain of EspA is essential for the assembly of the type III secretion translocon on the surface of enteropathogenic Escherichia coli.

Enteropathogenic E. coli (EPEC) utilize a type III secretion system to deliver virulence-associated effector proteins to the host cell. Four proteins, EspA, EspB, EspD, and Tir, which are integral to the formation of characteristic "attaching and effacing" (A/E) intestinal lesions, are known to be exported via the EPEC type III secretion system. Recent work demonstrated that EspA is a major component of a filamentous structure, elaborated on the surface of EPEC, which is required for translocation of EspB and Tir. The carboxyl terminus of EspA is predicted to comprise an alpha-helical region, which demonstrates heptad periodicity whereby positions a and d in the heptad repeat unit abcdefg are occupied by hydrophobic residues, indicating a propensity for coiled-coil interactions. Here we demonstrate multimeric EspA isoforms in EPEC culture supernatants and EspA:EspA interaction on solid phase. Non-conservative amino acid substitution of specific EspA heptad residues generated EPEC mutants defective in filament assembly but which retained the ability to induce A/E lesions; additional mutation totally abolished EspA filament assembly and A/E lesion formation. These results demonstrate a similarity to flagellar biosynthesis and indicate that the coiled-coil domain of EspA is required for assembly of the EspA filament-associated type III secretion translocon.

beta-propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection.

Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain beta-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed beta-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the beta-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.

Microbial genomes.

Microbial genome sequencing is driven by the need to understand and control pathogens and to exploit extremophiles and their enzymes in bioremediation and industry. It is hard for the traditional bacteriologist to grasp the scale and pace of the venture. Around two dozen microbial genomes have now been completed and, within a decade, genomes from every significant species of bacterial pathogen of humans, animals and plants will have been sequenced. Indeed, we will often have more than one sequence from a species or genus--for example, we already have sequences from two strains of Helicobacter pylori, from two strains of Mycobacterium tuberculosis and from three species of Pyrococcus. However, genome sequencing risks becoming expensive molecular stamp-collecting without the tools to mine the data and fuel hypothesis-driven laboratory-based research. Bioinformatics, twinned with the new experimental approaches forming functional genomics', provides some of the needed tools. Nonetheless, there will be an increasing need for us to explore the detailed implications of genomic findings. Microbial genome sequencing thus represents not a threat, but an exciting opportunity for molecular microbiologists.