Parallel Evolution of Host-Attachment Proteins in Phage PP01 Populations Adapting to Escherichia coli O157:H7.

The emergence of antibiotic resistance has sparked interest in phage therapy, which uses virulent phages as antibacterial agents. Bacteriophage PP01 has been studied for potential bio-control of Escherichia coli O157:H7, its natural host, but in the laboratory, PP01 can be inefficient at killing this bacterium. Thus, the goal of this study was to improve the therapeutic potential of PP01 through short-term experimental evolution. Four replicate populations of PP01 were serially passaged 21 times on non-evolving E. coli O157:H7 with the prediction that the evolved phage populations would adsorb faster and more efficiently kill the host bacteria. Dead-cell adsorption assays and in vitro killing assays confirmed that evolved viruses improved their adsorption ability on E. coli O157:H7, and adapted to kill host bacteria faster than the wildtype ancestor. Sequencing of candidate tail-fiber genes revealed that the phage populations evolved in parallel; the lineages shared two point mutations in gp38 that encodes a host recognition protein, and surprisingly shared a ~600 bp deletion in gp37 that encodes the distal tail fibers. In contrast, no mutations were observed in the gp12 gene encoding PP01’s short tail fibers. We discuss the functional role of the observed mutations, including the possible adaptive role of the evolved deletions. This study demonstrates how experimental evolution can be used to select for viral traits that improve phage attack of an important bacterial pathogen, and that the molecular targets of selection include loci contributing to cell attachment and phage virulence.

Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa.

Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections.

Complete Draft Genome Sequence of Escherichia coli JF733.

ITALIC! Escherichia coliJF733 is a strain with a long history in research on membrane proteins and processes. However, tracing back the strain development raises some questions concerning the correct genotype of JF733. Here, we present the complete draft genome of ITALIC! E. coliJF733 in order to resolve any remaining uncertainties.

The U.S. Culture Collection Network Lays the Foundation for Progress in Preservation of Valuable Microbial Resources.

The U.S. Culture Collection Network was formed in 2012 by a group of culture collection scientists and stakeholders in order to continue the progress established previously through efforts of an ad hoc group. The network is supported by a Research Coordination Network grant from the U.S. National Science Foundation (NSF) and has the goals of promoting interaction among collections, encouraging the adoption of best practices, and protecting endangered or orphaned collections. After prior meetings to discuss best practices, shared data, and synergy with genome programs, the network held a meeting at the U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado in October 2015 specifically to discuss collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. The meeting allowed collection curators who had already backed up their resources at the USDA NCGRP to visit the site, and brought collection owners, managers, and stakeholders together. Eight formal collections have established off-site backups with the USDA-ARS, ensuring that key material will be preserved for future research. All of the collections with backup at the NCGRP are public distributing collections including U.S. NSF-supported genetic stock centers, USDA-ARS collections, and university-supported collections. Facing the retirement of several pioneering researchers, the community discussed the value of preserving personal research collections and agreed that a mechanism to preserve these valuable collections was essential to any future national culture collection system. Additional input from curators of plant and animal collections emphasized that collections of every kind face similar challenges in developing long-range plans for sustainability.

The United States Culture Collection Network (USCCN): Enhancing Microbial Genomics Research through Living Microbe Culture Collections.

The mission of the United States Culture Collection Network (USCCN; http://usccn.org) is "to facilitate the safe and responsible utilization of microbial resources for research, education, industry, medicine, and agriculture for the betterment of human kind." Microbial culture collections are a key component of life science research, biotechnology, and emerging global biobased economies. Representatives and users of several microbial culture collections from the United States and Europe gathered at the University of California, Davis, to discuss how collections of microorganisms can better serve users and stakeholders and to showcase existing resources available in public culture collections.

Antibiotic resistance correlates with transmission in plasmid evolution.

Conjugative (horizontally transmissible) plasmids are autonomous replicators, whose "self-interests" do not necessarily overlap with those of their hosts. This situation causes plasmids and bacteria to sometimes experience differing selection pressures. Escherichia coli plasmid pB15 contains genes for resistance to several antibiotics, including tetracycline. When plasmid-bearing cells were experimentally evolved in the laboratory, changes in resistance level in the unselected tetracycline marker coincided with changes in plasmid rates of vertical versus horizontal transmission. Here, we used minimum inhibitory assays that measure resistance levels as quantitative traits to determine phenotypic correlations among plasmid characters and to estimate divergence among plasmid lineages. Results suggested that plasmid-level evolution led to formation of two phenotypically dissimilar groups: virulent (highly infectious) and avirulent (weakly infectious) plasmids. In contrast, measures of carbon-source utilization, and fitness assays relative to a common competitor revealed that bacterial hosts generally converged in phenotypic performance, despite divergence among their associated plasmids. Preliminary sequence analyses suggested that divergence in plasmid conjugation was due to altered configurations of a shufflon region (a site-specific recombination system), where genetic rearrangements affect conjugative ability. Furthermore, we proposed that correlated resistance and transmission in pB15 derivatives were caused by a tetracycline-resistance transposon inserted into a transfer operon, allowing transcription from its promoter to simultaneously affect both plasmid resistance and transmission.

Detecting key structural features within highly recombined genes.

Many microorganisms exhibit high levels of intragenic recombination following horizontal gene transfer events. Furthermore, many microbial genes are subject to strong diversifying selection as part of the pathogenic process. A multiple sequence alignment is an essential starting point for many of the tools that provide fundamental insights on gene structure and evolution, such as phylogenetics; however, an accurate alignment is not always possible to attain. In this study, a new analytic approach was developed in order to better quantify the genetic organization of highly diversified genes whose alleles do not align. This BLAST-based method, denoted BLAST Miner, employs an iterative process that places short segments of highly similar sequence into discrete datasets that are designated "modules." The relative positions of modules along the length of the genes, and their frequency of occurrence, are used to identify sequence duplications, insertions, and rearrangements. Partial alleles of sof from Streptococcus pyogenes, encoding a surface protein under host immune selection, were analyzed for module content. High-frequency Modules 6 and 13 were identified and examined in depth. Nucleotide sequences corresponding to both modules contain numerous duplications and inverted repeats, whereby many codons form palindromic pairs. Combined with evidence for a strong codon usage bias, data suggest that Module 6 and 13 sequences are under selection to preserve their nucleic acid secondary structure. The concentration of overlapping tandem and inverted repeats within a small region of DNA is highly suggestive of a mechanistic role for Module 6 and 13 sequences in promoting aberrant recombination. Analysis of pbp2X alleles from Streptococcus pneumoniae, encoding cell wall enzymes that confer antibiotic resistance, supports the broad applicability of this tool in deciphering the genetic organization of highly recombined genes. BLAST Miner shares with phylogenetics the important predictive quality that leads to the generation of testable hypotheses based on sequence data.

Evolution of transcription regulatory genes is linked to niche specialization in the bacterial pathogen Streptococcus pyogenes.

Streptococcus pyogenes is a highly prevalent bacterial pathogen, most often giving rise to superficial infections at the throat or skin of its human host. Three genotype-defined subpopulations of strains exhibiting strong tropisms for either the throat or skin (specialists) or having no obvious tissue site preference (generalists) are recognized. Since the microenvironments at the throat and skin are distinct, the signal transduction pathways leading to the control of gene expression may also differ for throat versus skin strains of S. pyogenes. Two loci (mga and rofA/nra) encoding global regulators of virulence gene expression are positioned 300 kb apart on the genome; each contains alleles forming two major sequence clusters of approximately 25 to 30% divergence that are under balancing selection. Strong linkage disequilibrium is observed between sequence clusters of the transcription regulatory loci and the subpopulations of throat and skin specialists, against a background of high recombination rates among housekeeping genes. A taxonomically distinct commensal species (Streptococcus dysgalactiae subspecies equisimilus) shares highly homologous rof alleles. The findings provide strong support for a mechanism underlying niche specialization that involves orthologous replacement of regulatory genes following interspecies horizontal transfer, although the directionality of gene exchange remains unknown.

Chimeric nature of two plasmids of Hafnia alvei encoding the bacteriocins alveicins A and B.

The complete nucleotide sequences of two bacteriocin-encoding plasmids isolated from Hafnia alvei (pAlvA and pAlvB) were determined. Both plasmids resemble ColE1-type replicons and carry mobilization genes, as well as colicin-like bacteriocin operons. These bacteriocins appear to be chimeras consisting of translocation domains from Tol-dependent colicins, unique binding domains, and killing and immunity domains similar to those of the pore-forming colicin Ia. Just as is found for colicin Ia, these H. alvei bacteriocins (alveicins) lack lysis genes. The alveicins are unusually small at 408 and 358 amino acids for alveicin A and B, respectively, which would make alveicin B the smallest pore-forming bacteriocin yet discovered. The pattern of nucleotide substitution in the alveicins suggests that the dominant forces in the evolution of their killing domains and immunity genes are neutral mutation and random genetic drift rather than diversifying selection, which has been implicated in the evolution of other colicins. Five of six bacteriocinogenic isolates of H. alvei were found to carry plasmids identical to pAlvA. Comparisons of the levels of nucleotide divergence in five housekeeping genes to the levels of divergence in their respective plasmids led us to conclude that pAlvA is transferring laterally through the H. alvei population relatively rapidly.

The Ecology and Evolution of Microbial Defense Systems in Escherichia coli.

Microbes produce an extraordinary array of microbial defense systems. These include broad-spectrum classical antibiotics critical to human health concerns; metabolic by-products, such as the lactic acids produced by lactobacilli; lytic agents, such as lysozymes found in many foods; and numerous types of protein exotoxins and bacteriocins. The abundance and diversity of this biological arsenal are clear. Lactic acid production is a defining trait of lactic acid bacteria. Bacteriocins are found in almost every bacterial species examined to date, and within a species, tens or even hundreds of different kinds of bacteriocins are produced. Halobacteria universally produce their own version of bacteriocins, the halocins. Streptomycetes commonly produce broad-spectrum antibiotics. It is clear that microbes invest considerable energy in the production and elaboration of antimicrobial mechanisms. What is less clear is how such diversity arose and what roles these biological weapons play in microbial communities. One family of microbial defense systems, the bacteriocins, has served as a model for exploring evolutionary and ecological questions. In this review, current knowledge of how the extraordinary range of bacteriocin diversity arose and is maintained in one species of bacteria, Escherichia coli, is assessed and the role these toxins play in mediating microbial dynamics is discussed.

Bacteriocin diversity: ecological and evolutionary perspectives.

The bacteriocin family is the most abundant and diverse group of bacterial defense systems. Bacteriocins range from the well-studied narrow spectrum, high molecular weight colicins produced by Escherichia coli and the short polypeptide lantibiotics of lactic acid bacteria to the relatively unknown halocins produced almost universally by the haolobacteria. The abundance and diversity of this potent arsenal of weapons is clear. Less clear is their evolutionary origins and the role they play in mediating microbial interactions. The goal of this review is to explore what we know about the evolution and ecology of the best-characterized family of bacteriocins, the colicins. We summarize current knowledge of how such extraordinary protein diversity arose and is maintained in microbial populations and what role these toxins play in mediating microbial population-level and community-level dynamics.

Bacteriocins: evolution, ecology, and application.

Microbes produce an extraordinary array of microbial defense systems. These include classical antibiotics, metabolic by-products, lytic agents, numerous types of protein exotoxins, and bacteriocins. The abundance and diversity of this potent arsenal of weapons are clear. Less clear are their evolutionary origins and the role they play in mediating microbial interactions. The goal of this review is to explore what we know about the evolution and ecology of the most abundant and diverse family of microbial defense systems: the bacteriocins. We summarize current knowledge of how such extraordinary protein diversity arose and is maintained in microbial populations and what role these toxins play in mediating microbial population-level and community-level dynamics. In the latter half of this review we focus on the potential role bacteriocins may play in addressing human health concerns and the current role they serve in food preservation.