O-antigens of Gram-negative bacteria modulate the interactions of bacterial cells with diverse external factors, including the components of the immune system and bacteriophages. Some phages need to ...acquire specific adhesins to overcome the O-antigen layer. For other phages, O-antigen is required for phage infection. In this case, interaction of phage receptor binding proteins coupled with enzymatic degradation or modification of the O-antigen is followed by phage infection. Identification of the strategies used by newly isolated phages may be of importance in their consideration for various applications. Here we describe an approach based on screening for host LPS alterations caused by selection by bacteriophages. We describe an optimized LPS profiling procedure that is simple, rapid and suitable for mass screening of mutants. We demonstrate that the phage infection strategies identified using a set of engineered E. coli 4 s mutants with impaired or altered LPS synthesis are in good agreement with the results of simpler tests based on LPS profiling of phage-resistant spontaneous mutants.
The T5 family of viruses are tailed bacteriophages characterized by a long non-contractile tail. The bacteriophage DT57C is closely related to the paradigmal T5 phage, though it recognizes a ...different receptor (BtuB) and features highly divergent lateral tail fibers (LTF). Considerable portions of T5-like phages remain structurally uncharacterized. Here, we present the structure of DT57C determined by cryo-EM, and an atomic model of the virus, which was further explored using all-atom molecular dynamics simulations. The structure revealed a unique way of LTF attachment assisted by a dodecameric collar protein LtfC, and an unusual composition of the phage neck constructed of three protein rings. The tape measure protein (TMP) is organized within the tail tube in a three-stranded parallel α-helical coiled coil which makes direct contact with the genomic DNA. The presence of the C-terminal fragment of the TMP that remains within the tail tip suggests that the tail tip complex returns to its original state after DNA ejection. Our results provide a complete atomic structure of a T5-like phage, provide insights into the process of DNA ejection as well as a structural basis for the design of engineered phages and future mechanistic studies.
The imbalance of the renin-angiotensin system is currently considered as a potentially important factor of the pathogenesis of COVID-19 disease. It has been shown previously in the murine model, that ...the expression of angiotensin-converting enzyme 2 (ACE2) on the cell surface is downregulated in response to the infection by SARS-CoV virus or recombinant spike protein (S protein) alone. In the case of natural infection, circulation of the S protein in a soluble form is unlikely. However, in SARS-CoV-2, a large fraction of S protein trimers is pre-processed during virion morphogenesis due to the presence of furin protease cleavage site between the S1 and S2 subunits. Therefore, S protein transition into the fusion conformation may be accompanied by the separation of the S1 subunits carrying the receptor-binding domains from the membrane-bound S2 subunits. The fate of the S1 particles shed due to the spontaneous “firing” of some S protein trimers exposed on the virions and on the surface of infected cells has been never investigated. We hypothesize that the soluble S1 subunits of the SARS-CoV-2 S protein shed from the infected cells and from the virions
in vivo
may bind to the ACE2 and downregulate cell surface expression of this protein. The decrease in the ACE2 activity on the background of constant or increased ACE activity in the lungs may lead to the prevalence of angiotensin II effects over those of angiotensin (1-7), thus promoting thrombosis, inflammation, and pulmonary damage. This hypothesis also suggests the association between less pronounced shedding of the S1 particles reported for the S protein carrying the D614G mutation (vs. the wild type D614 protein), and lack of increased severity of the COVID-19 infection caused by the mutant (D614G) SARS-CoV-2 strain, despite its higher infectivity and higher
in vivo
viral load.
The contemporary understanding of complex interactions in natural microbial communities and the numerous mechanisms of bacterial communication challenge the classical concept of bacteria as ...unicellular organisms. Microbial populations, especially those in densely populated habitats, appear to behave cooperatively, coordinating their reactions in response to different stimuli and behaving as a quasi-tissue. The reaction of such systems to viral infection is likely to go beyond each cell or species tackling the phage attack independently. Bacteriophage infection of a fraction of the microbial community may also exert an influence on the physiological state and/or phenotypic features of those cells that have not yet had direct contact with the virus or are even intrinsically unable to become infected by the particular virus. These effects may be mediated by sensing the chemical signals released by lysing or by infected cells as well as by more indirect mechanisms.
is an important industrial microorganism and a widely used model object for research in the field of lactic acid bacteria (LAB) biology. The development of new
and related LAB strains with improved ...properties, including phage-resistant strains for dairy fermentation, LAB-based vaccines or strains with altered genotypes for research purposes, are hindered by the lack of genome-editing tools that allow for the easy and straightforward incorporation of a significant amount of the novel genetic material, such as large genes or operons, into the chromosomes of these bacteria. We recently employed a suggested system based on the CRISPR-Cas-associated transposon for the editing of the
genome. After the in-depth redesign of the system, we were able to achieve the stable incorporation of the fragments that were sized up to 10 kbp into the
beta-galactosidase gene. The efficiency of editing under the optimized conditions were 2 × 10
and 4 × 10
for 1 kbp and 10 kbp, respectively, which are sufficient for fast and easy modifications if a positive selection marker can be used.
Adaptive immunity systems found in different organisms fall into two major types. Prokaryotes possess CRISPR-Cas systems that recognize former invaders using memorized (captured) pieces of their DNA ...as pathogen signatures. Mammals possess a vast repertoire of antibodies and T-cell receptor variants generated in advance. In this second type of adaptive immunity, a pathogen presentation to the immune system specifically activates the cells that express matching antibodies or receptors. These cells proliferate to fight the infection and form the immune memory. The principle of preemptive production of diverse defense proteins for future use can hypothetically take place in microbes too. We propose a hypothesis that prokaryotes employ diversity-generating retroelements to prepare defense proteins against yet-unknown invaders. In this study, we test this hypothesis with the methods of bioinformatics and identify several candidate defense systems based on diversity-generating retroelements.
The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ₀) synthesis and DNA metabolism ...genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2′-deoxy-preQ₀ and 2′-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ∼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction–modification role for the cluster in Enterobacteriaceae. Another preQ₀ derivative, 2′-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism.
The power of most of the enterobacterial O antigen types to provide robust protection against direct recognition of the cell surface by bacteriophage receptor-recognition proteins (RBP) has been ...recently recognized. The bacteriophages infecting O antigen producing strains of E. coli employ various strategies to tackle this nonspecific protection. T-even related phages, including RB49-like viruses, often have wide host ranges, being considered good candidates for use in phage therapy. However, the mechanisms by which these phages overcome the O antigen barrier remain unknown. We demonstrate here that RB49 and related phages Cognac49 and Whisky49 directly use certain types of O antigen as their primary receptors recognized by the virus long tail fibers (LTF) RBP gp38, so the O antigen becomes an attractant instead of an obstacle. Simultaneously to recognize multiple O antigen types, LTFs of each of these phages can bind to additional receptors, such as OmpA protein, enabling them to infect some rough strains of E. coli. We speculate that the mechanical force of the deployment of the short tail fibers (STF) triggered by the LTF binding to the O antigen or underneath of it, allows the receptor binding domains of STF to break through the O polysaccharide layer.
Summary
Bacteriophages recognize and bind to their hosts with the help of receptor‐binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great ...variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C – tailspikes gp63.1 and gp66 – are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N‐terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1‐gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1‐gp66 interaction is widespread among all three families of tailed bacteriophages.
N4‐like phage G7C recognizes its E. coli 4s host cell with the help of gp63.1 and gp66 tailspike proteins. Gp66 is attached to the phage particle directly whereas gp63.1 binds to gp66. Gp63.1 is an esterase that deacetylates the E. coli 4s O‐antigen while leaving its polysaccharide backbone intact. This minor modification of the O‐antigen is absolutely essential for G7C infection.
The complete genomes of the new
Erwinia amylovora
bacteriophages Loshitsa2 and Micant are 43,092 bp and 43,028 bp long, respectively, encode 51 putative proteins, and have two tRNA genes. Comparative ...analysis with representatives of the class
Caudoviricetes
suggests that bacteriophages Loshitsa2 and Micant are related to LIMElight bacteriophage belonging to the family
Autographiviridae
and could be proposed to be members of a novel subfamily.
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