Stimulation of bacterial cytokinesis by bacteriophage predation

Bacteriophage (phage) are ubiquitous in the water column and in the sediments of most natural water. Because of their colloidal nature, they can either aggregate into clumps large enough to settle into the sediment or departing upon the physiochemical conditions or disassociate and reenter the water column. About 80% of the bacterial strains isolated from New River sediment have a virulent phage that can be isolated with them.Liquid cultures of a strain of Pseudomonas aeruginosa isolated from the New River along with its phage were set up. One was infected with the virulent phage and another kept as a control. Daily counts were made of bacterial numbers. After 10 days the control culture was infected and counted for 3 more days.Both cultures divided exponentially at first. The infected culture continued to divide at about half the initial rate. The uninfected culture nearly ceased division, but when phage were added it quickly began to divide.The virulent phage infection clearly stimulated host division. The effect was to establish itself as an endemic infection which did not outpace its host's division rate. Further, the enhanced division rate may act to increase the host's share of available nutrients and benefit its competitive position in the system.     

Cryo-EM structure of a bacteriophage T4 gp24 bypass mutant: the evolution of pentameric vertex proteins in icosahedral viruses

Many large viral capsids require special pentameric proteins at their fivefold vertices. Nevertheless, deletion of the special vertex protein gene product 24 (gp24) in bacteriophage T4 can be compensated by mutations in the homologous major capsid protein gp23. The structure of such a mutant virus, determined by cryo-electron microscopy to 26 angstroms, shows that the gp24 pentamers are replaced by mutant major capsid protein (gp23) pentamers at the vertices, thus re-creating a viral capsid prior to the evolution of specialized major capsid proteins and vertex proteins. The mutant gp23* pentamer is structurally similar to the wild-type gp24* pentamer but the insertion domain is slightly more distant from the gp23* pentamer center. There are additional SOC molecules around the gp23* pentamers in the mutant virus that were not present around the gp24* pentamers in the wild-type virus.     

Rule-based computing system for microbial interactions and communications: evolution in virtual bacterial populations

We have developed a novel rule-based computing system of microbial interactions and communications, referred to as COSMIC-Rules, for simulating evolutionary processes within populations of virtual bacteria. The model incorporates three levels: the bacterial genome, the bacterial cell and an environment inhabited by such cells. The virtual environment in COSMIC-Rules can contain multiple substances, whose relative toxicity or nutrient status is specified by the genome of the bacterium. Each substance may be distributed uniformly or in a user-defined manner. The organisms in COSMIC-Rules possess individually-defined physical locations, size, cell division status and genomes. Genes and/or gene systems are represented by abstractions that may summate sometimes complex phenotypes. Central to COSMIC-Rules is a simplified representation of bacterial species, each containing a functional genome including, where desired, extrachromosomal elements such as plasmids and/or bacteriophages. A widely applicable computer representation of biological recognition systems based on bit string matching is essential to the model. This representation permits, for example, the modelling of protein-protein interactions, receptor-ligand interactions and DNA-DNA transactions. COSMIC-Rules is intended to inform studies on bacterial adaptation and evolution, and to predict behaviour of populations of pathogenic bacteria and their viruses. The framework is constructed for parallel execution across a large number of machines and efficiently utilises a 64 processor development cluster. It will run on any Grid system and has successfully tested simulations with millions of bacteria, of multiple species and utilising multiple substrates. The model may be used for large-scale simulations where a genealogical record for individual organisms is required.     

Ecology and evolution of phages encoding anti-CRISPR proteins

CRISPR-Cas are prokaryotic defence systems that provide protection against invasion by mobile genetic elements (MGE), including bacteriophages. MGE can overcome CRISPR-Cas defences by encoding anti-CRISPR (Acr) proteins. These proteins are produced in the early stages of the infection and inhibit the CRISPR-Cas machinery to allow phage replication. While research on Acr has mainly focused on their discovery, structure and mode of action, and their applications in biotechnology, the impact of Acr on the ecology of MGE as well as on the coevolution with their bacterial hosts only begins to be unravelled. In this review, we summarise our current understanding on the distribution of anti-CRISPR genes in MGE, the ecology of phages encoding Acr, and their coevolution with bacterial defence mechanisms. We highlight the need to use more diverse and complex experimental models to better understand the impact of anti-CRISPR in MGE-host interactions.     

Complex systems, evolution, and animal models

In this paper, we respond to arguments made concerning our position regarding animal models (Shelley, 2010) by briefly examining the fact that animals (human and nonhuman) are complex systems that have different evolutionary trajectories. This historical fact has implications for using animals as predictive models for human response to drugs and disease.     

Optimality models of phage life history and parallels in disease evolution

Optimality models constitute one of the simplest approaches to understanding phenotypic evolution. Yet they have shortcomings that are not easily evaluated in most organisms. Most importantly, the genetic basis of phenotype evolution is almost never understood, and phenotypic selection experiments are rarely possible. Both limitations can be overcome with bacteriophages. However, phages have such elementary life histories that few phenotypes seem appropriate for optimality approaches. Here we develop optimality models of two phage life history traits, lysis time and host range. The lysis time models show that the optimum is less sensitive to differences in host density than suggested by earlier analytical work. Host range evolution is approached from the perspective of whether the virus should avoid particular hosts, and the results match optimal foraging theory: there is an optimal "diet" in which host types are either strictly included or excluded, depending on their infection qualities. Experimental tests of both models are feasible, and phages provide concrete illustrations of many ways that optimality models can guide understanding and explanation. Phage genetic systems already support the perspective that lysis time and host range can evolve readily and evolve without greatly affecting other traits, one of the main tenets of optimality theory. The models can be extended to more general properties of infection, such as the evolution of virulence and tissue tropism.     

Organization and evolution of the mitochondrial genome of yeast

Summary The mitochondrial genome of yeast (S. cerevisiae orS. carlsbergensis) appears to be formed by 60–70 genetic units, each one of which is formed by (1) a GC-rich sequence, possibly having a regulatory role; (2) a gene, and (3) an AT-rich spacer, which probably is not transcribed. Recombination in this genome appears to underlie a number of important phenomena. The organization of the mitochondrial genome of yeast and these recombinational events are discussed in relationship with the organization and evolution of the nuclear genome of eukaryotes.     

A simulation of T4 bacteriophage assembly and operation

A model is presented for the self-assembly and operation of a bacteriophage comparable with the T4 bacteriophage that infects Escherichia coli. The model treats protein molecules as simple units obeying the principle free energy minimization, and exhibiting the properties of quasi-equivalence and conformational switching. A computer program incorporating the model has been developed. The results of simulation using this program are presented.     

Rule-based simulation of temperate bacteriophage infection: Restriction–modification as a limiter to infection in bacterial populations

An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria.     

Isolation and characterization of bacteriophage PM3 from Aeromonas hydrophila the bacterial receptor for which is the monopolar flagellum

PM3 is an Aeromonas-specific bacteriophage which was isolated and characterized on A. hydrophila strain TF7. Spontaneous mutants resistant to PM3 were non-motile having lost their characteristic monopolar flagellum. In addition, purified flagella inactivated PM3. PM3 is the first filamentous bacteriophage isolated on Aeromonas, the adsorption site for which is the monopolar flagellum.     

Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

The crystal structure of the bifunctional primase-helicase of bacteriophage T7

Within minutes after infecting Escherichia coli, bacteriophage T7 synthesizes many copies of its genomic DNA. The lynchpin of the T7 replication system is a bifunctional primase-helicase that unwinds duplex DNA at the replication fork while initiating the synthesis of Okazaki fragments on the lagging strand. We have determined a 3.45 A crystal structure of the T7 primase-helicase that shows an articulated arrangement of the primase and helicase sites. The crystallized primase-helicase is a heptamer with a crown-like shape, reflecting an intimate packing of helicase domains into a ring that is topped with loosely arrayed primase domains. This heptameric isoform can accommodate double-stranded DNA in its central channel, which nicely explains its recently described DNA remodeling activity. The double-jointed structure of the primase-helicase permits a free range of motion for the primase and helicase domains that suggests how the continuous unwinding of DNA at the replication fork can be periodically coupled to Okazaki fragment synthesis.     

Core-binding specificity of bacteriophage integrases

The site-specific recombination systems of bacteriophages λ and HK022 share the same mechanism and their integrase proteins show strong homology. Nevertheless the integrase protein of each phage can only catalyze recombination between its own att sites. Previous work has shown that the specificity determinants in the att sites are located within the sequences that bind the integrase to the core of att. DNA fragments that carry attL and attR sites of each phage were challenged with each of the two integrases and the DNA-protein complexes were examined by the gel- retardation technique. The results show that each integrase can form higher-order DNA-protein complexes only with its cognate att sites, suggesting that differences in the mode of binding to the core are responsible for the specificity difference between the two integrases. Received: 16 November 1999 / Accepted: 26 January 2000     

细菌重组系统及其应用研究进展

利用重组酶和辅助蛋白共同作用于DNA片段上,使不同基因重新组合以完成基因重组的现象在细菌中广泛存在,基因重组对于细菌的遗传多样性、进化等具有重要意义。目前,细菌基因重组主要分为同源重组、位点特异性重组和转座重组3种类型。本文主要对细菌重组系统重组酶的种类、作用机制及其在细菌遗传操作中的应用策略进行阐述。     

Major coat proteins of bacteriophage Pf3 and M13 as model systems for Sec-independent protein transport

Abstract: The membrane insertion of bacteriophage coat proteins occurs independent of the Sec-translocase of Escherichia coli . Detailed study of the Pf3 and M13 coat proteins has elucidated two fundamental mechanisms of how proteins invade the membrane, most likely by direct interaction with the lipid bilayer. The Sec-independent translocation of amino-terminal regions across the inner membrane is limited to a short length and a small number of charged residues. Protein regions that contain several charged residues are efficiently translocated across the membrane when these regions are flanked by two adjacent hydrophobic segments interacting synergistically. The relevance of these findings for the membrane insertion mechanism of multispanning membrane proteins is discussed.     

Holins: form and function in bacteriophage lysis

Abstract: During the lytic cycle of most bacteriophages, a phage-encoded peptidoglycan-degrading activity is elaborated. At least four entirely distinct types of enzymes fulfill this role and are given the generic name 'endolysin'. Endolysins characterized to date are synthesized without a signal sequence and thus accumulate fully folded and active in the cytosol during the vegetative phase. Small membrane proteins are required in order for endolysins to gain access to the peptidoglycan. Because the available data suggest that the membrane lesion formed by these proteins is stable and non-specific, these proteins have been given the designation 'holins' ('hole'-formers). Analysis of the primary sequence suggests a simple membrane topology with two or more membrane-spanning helical domains and a highly charged, hydrophilic C-terminus. Comparison of the sequences of holins from phages of Gram-negative hosts suggests there are at least two major holin groups. Putative holin genes have also been found in bacteriophages of Gram-positive bacteria. Altogether, in phages of Eubacteria, 11 or more unrelated gene families which share the functional and structural characteristics of holins have been identified. Genetic and physiological analysis suggest that holins are primarily regulated at the level of function. Holin function is modulated in some cases by a second protein encoded by the holin gene. The primary regulation of holin function, however, appears to be intrinsic to the holin structure itself, since a missense allele of the S holin gene of phage λ has been found which abolishes the normal delay that allows the vegetative phase to generate a useful number of progeny.     

Organization and evolution of the glandular kallikrein locus in Mus musculus

The gene of tissue kallikrein and closely related genes constitute the glandular kallikrein (GK) gene family. The number of members varies between species, ranging from three human to 25 murine. Recently, the gene family was extended with 12 new members, KLK4-KLK15, that were identified adjacent to the classical GK genes on human chromosome 19. In this report, the structure and phylogeny of the mouse GK gene locus are described. A comparison of the human and murine loci shows that the locations of the tissue kallikrein gene and KLK4-KLK15 are conserved. The region between the tissue kallikrein gene and KLK15, devoid of genes in human, is expanded and contains 23 classical GK genes in mouse. Downstream of KLK15, where the genes encoding PSA and hK2 are located in human, mouse carries the pseudogene PsimGK25. Phylogenetic analyses show that classical GK genes emerged after the separation of the primate and rodent lineages, forming a subgroup within the newly extended GK family.     

A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.     

High-throughput analysis of growth differences among phage strains

Although methods such as spectrophotometry are useful for identifying growth differences among bacterial strains, it is currently difficult to similarly determine whether bacteriophage strains differ in growth using high throughput methods. Here we use automated spectrophotometry to develop an in vitro method for indirectly distinguishing fitness (growth) differences among virus strains, based on direct measures of their infected bacterial hosts. We used computer simulations of a mathematical model for phage growth to predict which features of bacterial growth curves were best associated with differences in growth among phage strains. We then tested these predictions using the in vitro method to confirm which of the inferred viral growth traits best reflected known fitness differences among genotypes of the RNA phage phi-6, when infecting a Pseudomonas syringae host. Results showed that the inferred phage trait of time-to-extinction (time required to drive bacterial density below detectable optical density) reliably correlated with genotype rankings based on absolute fitness (phage titer per ml). These data suggested that the high-throughput analysis was valuable for identifying growth differences among virus strains, and that the method may be especially useful for high throughput analyses of fitness differences among phage strains cultured and/or evolved in liquid (unstructured) environments.     

Dynamics of protein evolution

Protein sequences of the SWISS-PROT data bank were analysed by fractal techniques and harmonic analysis. In both cases, the results show the presence of self-affinity, a kind of self-similarity, in the sequences. Self-similarity is a sign of fractality and fractality is a consequence of a chaotic dynamical process. The evolution of the protein sequences is modelled as a dynamical system. The abundance of the fractal form in biology and creation of fractal forms as a result of “chaos” is already established. It may be noted that the word “chaos” here implies that most predictable processes can also become unpredictable under certain conditions, and that the most unpredictable processes are not as unpredictable as they are expected to be. In evolutionary dynamics, this allows scope for mutations and variations in otherwise predictable situations, potentially leading to increased diversity. Part of this work was presented at the National Symposium on Evolution of Life.