SEDS proteins are a widespread family of bacterial cell wall polymerases.

Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis, this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development.

Analysis of a phase-variable restriction modification system of the human gut symbiont Bacteroides fragilis.

The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum.

Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli.

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Thermosipho africanus Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

Coupling mutagenesis and parallel deep sequencing to probe essential residues in a genome or gene.

The sequence of a protein determines its function by influencing its folding, structure, and activity. Similarly, the most conserved residues of orthologous and paralogous proteins likely define those most important. The detection of important or essential residues is not always apparent via sequence alignments because these are limited by the depth of any given gene's phylogeny, as well as specificities that relate to each protein's unique biological origin. Thus, there is a need for robust and comprehensive ways of evaluating the importance of specific amino acid residues of proteins of known or unknown function. Here we describe an approach called Mut-seq, which allows the identification of virtually all of the essential residues present in a whole genome through the application of limited chemical mutagenesis, selection for function, and deep parallel genomic sequencing. Here we have applied this method to T7 bacteriophage and T7-like virus JSF7 of Vibrio cholerae.

Genomic science in understanding cholera outbreaks and evolution of Vibrio cholerae as a human pathogen.

Modern genomic and bioinformatic approaches have been applied to interrogate the V. cholerae genome, the role of genomic elements in cholera disease, and the origin, relatedness, and dissemination of epidemic strains. A universal attribute of choleragenic strains includes a repertoire of pathogenicity islands and virulence genes, namely the CTXϕ prophage and Toxin Co-regulated Pilus (TCP) in addition to other virulent genetic elements including those referred to as Seventh Pandemic Islands. During the last decade, the advent of Next Generation Sequencing (NGS) has provided highly resolved and often complete genomic sequences of epidemic isolates in addition to both clinical and environmental strains isolated from geographically unconnected regions. Genomic comparisons of these strains, as was completed during and following the Haitian outbreak in 2010, reveals that most epidemic strains appear closely related, regardless of region of origin. Non-O1 clinical or environmental strains may also possess some virulence islands, but phylogenic analysis of the core genome suggests they are more diverse and distantly related than those isolated during epidemics. Like Haiti, genomic studies that examine both the Vibrio core and pan-genome in addition to Single Nucleotide Polymorphisms (SNPs) conclude that a number of epidemics are caused by strains that closely resemble those in Asia, and often appear to originate there and then spread globally. The accumulation of SNPs in the epidemic strains over time can then be applied to better understand the evolution of the V. cholerae genome as an etiological agent.

The origin of the Haitian cholera outbreak strain.

BACKGROUND: Although cholera has been present in Latin America since 1991, it had not been epidemic in Haiti for at least 100 years. Recently, however, there has been a severe outbreak of cholera in Haiti. METHODS: We used third-generation single-molecule real-time DNA sequencing to determine the genome sequences of 2 clinical Vibrio cholerae isolates from the current outbreak in Haiti, 1 strain that caused cholera in Latin America in 1991, and 2 strains isolated in South Asia in 2002 and 2008. Using primary sequence data, we compared the genomes of these 5 strains and a set of previously obtained partial genomic sequences of 23 diverse strains of V. cholerae to assess the likely origin of the cholera outbreak in Haiti. RESULTS: Both single-nucleotide variations and the presence and structure of hypervariable chromosomal elements indicate that there is a close relationship between the Haitian isolates and variant V. cholerae El Tor O1 strains isolated in Bangladesh in 2002 and 2008. In contrast, analysis of genomic variation of the Haitian isolates reveals a more distant relationship with circulating South American isolates. CONCLUSIONS: The Haitian epidemic is probably the result of the introduction, through human activity, of a V. cholerae strain from a distant geographic source. (Funded by the National Institute of Allergy and Infectious Diseases and the Howard Hughes Medical Institute.).

Vibrio parahaemolyticus and its specific bacteriophages as an indicator in cockles (Anadara granosa) for the risk of V. parahaemolyticus infection in Southern Thailand.

Correlation between the numbers of Vibrio parahaemolyticus and its specific bacteriophages in cockles was investigated from June 2009 to May 2010 in Hat Yai, Songkhla, Thailand. Cockles obtained monthly from a local market were sampled to determine the numbers of V. parahaemolyticus and bacteriophages that could form plaques on ten strains of pandemic and nonpandemic V. parahaemolyticus. In addition, V. parahaemolyticus isolates from clinical samples from Hat Yai hospital over the same period were investigated. All 139 cockles sampled were positive for V. parahaemolyticus. However, only 76 of them were positive for bacteriophages. During the testing period, the number of bacteriophages was not significantly correlated with the incidence of V. parahaemolyticus-infected patients, but the numbers of V. parahaemolyticus isolates from the cockle samples were closely related to the number of infected patients. The bacteriophages isolated from V. parahaemolyticus also infected Vibrio alginolyticus and Vibrio mimicus, suggesting that the broad host range of phages may be a factor of providing the possibility of their participation in the processes of genetic exchange between V. parahaemolyticus and closely related Vibrio spp. In conclusion, this study indicated that the number of V. parahaemolyticus in cockles may be a useful tool for predicting the relative risk of infection by V. parahaemolyticus in this area of Thailand.

Covariance predicts conserved protein residue interactions important for the emergence and continued evolution of SARS-CoV-2 as a human pathogen.

SARS-CoV-2 is one of three recognized coronaviruses (CoVs) that have caused epidemics or pandemics in the 21st century and that likely emerged from animal reservoirs. Differences in nucleotide and protein sequence composition within related ß-coronaviruses are often used to better understand CoV evolution, host adaptation, and their emergence as human pathogens. Here we report the comprehensive analysis of amino acid residue changes that have occurred in lineage B ß-coronaviruses that show covariance with each other. This analysis revealed patterns of covariance within conserved viral proteins that potentially define conserved interactions within and between core proteins encoded by SARS-CoV-2 related ß-coronaviruses. We identified not only individual pairs but also networks of amino acid residues that exhibited statistically high frequencies of covariance with each other using an independent pair model followed by a tandem model approach. Using 149 different CoV genomes that vary in their relatedness, we identified networks of unique combinations of alleles that can be incrementally traced genome by genome within different phylogenic lineages. Remarkably, covariant residues and their respective regions most abundantly represented are implicated in the emergence of SARS-CoV-2 and are also enriched in dominant SARS-CoV-2 variants.

Covariance predicts conserved protein residue interactions important to the emergence and continued evolution of SARS-CoV-2 as a human pathogen.

SARS-CoV-2 is one of three recognized coronaviruses (CoVs) that have caused epidemics or pandemics in the 21st century and that likely emerged from animal reservoirs. Differences in nucleotide and protein sequence composition within related ß-coronaviruses are often used to better understand CoV evolution, host adaptation, and their emergence as human pathogens. Here we report the comprehensive analysis of amino acid residue changes that have occurred in lineage B ß-coronaviruses that show covariance with each other. This analysis revealed patterns of covariance within conserved viral proteins that potentially define conserved interactions within and between core proteins encoded by SARS-CoV-2 related ß-coranaviruses. We identified not only individual pairs but also networks of amino acid residues that exhibited statistically high frequencies of covariance with each other using an independent pair model followed by a tandem model approach. Using 149 different CoV genomes that vary in their relatedness, we identified networks of unique combinations of alleles that can be incrementally traced genome by genome within different phylogenic lineages. Remarkably, covariant residues and their respective regions most abundantly represented are implicated in the emergence of SARS-CoV-2 are also enriched in dominant SARS-CoV-2 variants.

Protein covariance networks reveal interactions important to the emergence of SARS coronaviruses as human pathogens.

SARS-CoV-2 is one of three recognized coronaviruses (CoVs) that have caused epidemics or pandemics in the 21 st century and that have likely emerged from animal reservoirs based on genomic similarities to bat and other animal viruses. Here we report the analysis of conserved interactions between amino acid residues in proteins encoded by SARS-CoV-related viruses. We identified pairs and networks of residue variants that exhibited statistically high frequencies of covariance with each other. While these interactions are likely key to both protein structure and other protein-protein interactions, we have also found that they can be used to provide a new computational approach (CoVariance-based Phylogeny Analysis) for understanding viral evolution and adaptation. Our data provide evidence that the evolutionary processes that converted a bat virus into human pathogen occurred through recombination with other viruses in combination with new adaptive mutations important for entry into human cells.

Defective lytic transglycosylase disrupts cell morphogenesis by hindering cell wall de-O-acetylation in Neisseria meningitidis.

Lytic transglycosylases (LT) are enzymes involved in peptidoglycan (PG) remodeling. However, their contribution to cell-wall-modifying complexes and their potential as antimicrobial drug targets remains unclear. Here, we determined a high-resolution structure of the LT, an outer membrane lipoprotein from Neisseria species with a disordered active site helix (alpha helix 30). We show that deletion of the conserved alpha-helix 30 interferes with the integrity of the cell wall, disrupts cell division, cell separation, and impairs the fitness of the human pathogen Neisseria meningitidis during infection. Additionally, deletion of alpha-helix 30 results in hyperacetylated PG, suggesting this LtgA variant affects the function of the PG de-O-acetylase (Ape 1). Our study revealed that Ape 1 requires LtgA for optimal function, demonstrating that LTs can modulate the activity of their protein-binding partner. We show that targeting specific domains in LTs can be lethal, which opens the possibility that LTs are useful drug-targets.

Bacteria are surrounded by a tough yet flexible wall that protects the cell and serves as an anchor for several of the cell's structures. This cell wall contains a large mesh-like molecule called peptidoglycan made of many repeated building blocks. When a bacterial cell divides in two, it needs to make more of this material. Making peptidoglycan involves two different sets of enzymes working together: "polymerases" are the enzymes that link the individual building blocks to peptidoglycan, one after the other; while "lytic transglycosylases" are enzymes that modify the peptidoglycan to create space for the addition of new building blocks and for assemblies of proteins that must span the cell wall. Lytic transglycosylases are known to assemble with other proteins and enzymes to form the cell's peptidoglycan-modifying machinery, but it was not clear exactly what purpose they serve within these "enzyme complexes". It was also unclear whether these enzymes would be good targets for new antibiotics. To help answer these questions, Williams et al. looked at a lytic transglycoslyase called LtgA. This enzyme is originally from Neisseria meningitidis, a bacterium that can cause meningitis and life-threatening sepsis in humans. Williams et al. discovered that part of the enzyme's active site ­ the region of an enzyme where the chemical reaction takes ­ can switch from an ordered helix to a disordered, flexible loop. Bacteria were then genetically engineered to make a version of the enzyme that lacked this helix. These bacteria had weaker cell walls and were deformed; they were also less able to grow and divide, both in the laboratory and in a mouse model of infection. Further analysis showed that the deletion of the helix from the enzyme resulted in the peptidoglycan being modified much more than normal, which could likely explain their reduced virulence. Williams et al. also found that deleting the helix from LtgA interfered with the activity of a protein that interacts with this enzyme, called Ape1, which also contributed to the fragility of the cell wall. This shows that lytic transglycosylases assembled into enzyme complexes can alter the activities of other proteins in the complex. Together these findings show that researchers could target one enzyme in a complex in bacteria, and disrupt the activity of other proteins in that complex. This highlights the possibility of considering enzyme complexes as useful targets for new drugs, which is important considering the current problem of antibiotic resistance.

The Drosophila Immune Deficiency Pathway Modulates Enteroendocrine Function and Host Metabolism.

Enteroendocrine cells (EEs) are interspersed between enterocytes and stem cells in the Drosophila intestinal epithelium. Like enterocytes, EEs express components of the immune deficiency (IMD) innate immune pathway, which activates transcription of genes encoding antimicrobial peptides. The discovery of large lipid droplets in intestines of IMD pathway mutants prompted us to investigate the role of the IMD pathway in the host metabolic response to its intestinal microbiota. Here we provide evidence that the short-chain fatty acid acetate is a microbial metabolic signal that activates signaling through the enteroendocrine IMD pathway in a PGRP-LC-dependent manner. This, in turn, increases transcription of the gene encoding the endocrine peptide Tachykinin (Tk), which is essential for timely larval development and optimal lipid metabolism and insulin signaling. Our findings suggest innate immune pathways not only provide the first line of defense against infection but also afford the intestinal microbiota control over host development and metabolism.

The acetate switch of an intestinal pathogen disrupts host insulin signaling and lipid metabolism.

Vibrio cholerae is lethal to the model host Drosophila melanogaster through mechanisms not solely attributable to cholera toxin. To examine additional virulence determinants, we performed a genetic screen in V. cholerae-infected Drosophila and identified the two-component system CrbRS. CrbRS controls transcriptional activation of acetyl-CoA synthase-1 (ACS-1) and thus regulates the acetate switch, in which bacteria transition from excretion to assimilation of environmental acetate. The resultant loss of intestinal acetate leads to deactivation of host insulin signaling and lipid accumulation in enterocytes, resulting in host lethality. These metabolic effects are not observed upon infection with ΔcrbS or Δacs1 V. cholerae mutants. Additionally, uninfected flies lacking intestinal commensals, which supply short chain fatty acids (SCFAs) such as acetate, also exhibit altered insulin signaling and intestinal steatosis, which is reversed upon acetate supplementation. Thus, acetate consumption by V. cholerae alters host metabolism, and dietary acetate supplementation may ameliorate some sequelae of cholera.

A hybrid approach for the automated finishing of bacterial genomes.

Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.

RNA-Seq-based monitoring of infection-linked changes in Vibrio cholerae gene expression.

Pathogens adapt to the host environment by altering their patterns of gene expression. Microarray-based and genetic techniques used to characterize bacterial gene expression during infection are limited in their ability to comprehensively and simultaneously monitor genome-wide transcription. We used massively parallel cDNA sequencing (RNA-seq) techniques to quantitatively catalog the transcriptome of the cholera pathogen, Vibrio cholerae, derived from two animal models of infection. Transcripts elevated in infected rabbits and mice relative to laboratory media derive from the major known V. cholerae virulence factors and also from genes and small RNAs not previously linked to virulence. The RNA-seq data was coupled with metabolite analysis of cecal fluid from infected rabbits to yield insights into the host environment encountered by the pathogen and the mechanisms controlling pathogen gene expression. RNA-seq-based transcriptome analysis of pathogens during infection produces a robust, sensitive, and accessible data set for evaluation of regulatory responses driving pathogenesis.