Closely Related Vibrio alginolyticus Strains Encode an Identical Repertoire of Caudovirales-Like Regions and Filamentous Phages

Many filamentous vibriophages encode virulence genes that lead to the emergence of pathogenic bacteria. Most genomes of filamentous vibriophages characterized up until today were isolated from human pathogens. Despite genome-based predictions that environmental Vibrios also contain filamentous phages that contribute to bacterial virulence, empirical evidence is scarce. This study aimed to characterize the bacteriophages of a marine pathogen, Vibrio alginolyticus (Kiel-alginolyticus ecotype) and to determine their role in bacterial virulence. To do so, we sequenced the phage-containing supernatant of eight different V. alginolyticus strains, characterized the phages therein and performed infection experiments on juvenile pipefish to assess their contribution to bacterial virulence. We were able to identify two actively replicating filamentous phages. Unique to this study was that all eight bacteria of the Kiel-alginolyticus ecotype have identical bacteriophages, supporting our previously established theory of a clonal expansion of the Kiel-alginolyticus ecotype. We further found that in one of the two filamentous phages, two phage-morphogenesis proteins (Zot and Ace) share high sequence similarity with putative toxins encoded on the Vibrio cholerae phage CTXΦ. The coverage of this filamentous phage correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phage contributes to bacterial virulence.

HK97 gp74 Possesses an α-Helical Insertion in the ββα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities

ABSTRACT The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ββα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two β-strands of the ββα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ββα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle. IMPORTANCE This study demonstrates that residues outside the canonical ββα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.

Involvement of the Major Capsid Protein and Two Early-Expressed Phage Genes in the Activity of the Lactococcal Abortive Infection Mechanism AbiT

ABSTRACTThe dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB)Lactococcus lactisto produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives:e14(bIL170 [e14bIL170]),orf41(P008 [orf41P008]), andorf6(p2 [orf6p2] and P008 [orf6P008]). The genese14bIL170andorf41P008are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function.orf6is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression inL. lactisof ORF6p2and ORF5p2, a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.

Genetic Background and Mobility of Variants of the GenenleAin Attaching and Effacing Escherichia coli

ABSTRACTIn this study, we characterized the genetic background of variousnleAvariants in 106 Shiga toxin-producingEscherichia coli(STEC) and enteropathogenicEscherichia coli(EPEC) strains. The flanking regions of eightnleAvariants were analyzed by DNA sequencing and compared with the corresponding regions of five previously described NleA-encoding prophages. The analyzednleAvariants were all located downstream of the DNA region responsible for phage morphogenesis. In particular, the type III effector genesavrA,ospB,nleH, andnleGand IS elements were detected in the neighborhood ofnleA. The structure of the eight analyzed regions flankingnleAprimarily resembled the corresponding region of the NleA4795-encoding prophage BP-4795. Using PCR, the gene order flanking 13nleAvariants in strains of different serogroups was compared to the respective regions in reference strains. The analyses showed that strains which harbor prophages with conserved flanking regions of a particularnleAvariant predominantly occurred, and IS elements were additionally detected in these regions. We were able to mobilizenleAby transduction in 20% of strains determined, which comprised in particular EPEC strains harboring annleAvariant, the gene encoding the protein known as “EspI-like.” Plaque hybridization was used to identify phages that harbor the genesstxandnleA. However, only two strains harbored variantnleA4795in the genome of an Stx1 prophage.

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

ABSTRACT The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (ΦAb-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the ΦAb-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of ΦAb-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.

Dual Regulatory Control of a Particle Maturation Function of Bacteriophage P1

ABSTRACT A unique arrangement of promoter elements was found upstream of the bacteriophage P1 particle maturation gene (mat). A P1-specific late-promoter sequence with conserved elements located at positions −22 and −10 was expected from the function of the gene in phage morphogenesis. In addition to a late-promoter sequence, a −35 element and an operator sequence for the major repressor protein, C1, were found. The −35 and −10 elements constituted an activeEscherichia coli ς70 consensus promoter, which was converted into a P1-regulated early promoter by the superimposition of a C1 operator. This combination of early- and late-promoter elements regulates and fine-tunes the expression of the particle maturation gene. During lysogenic growth the gene is turned off by P1 immunity functions. Upon induction of lytic growth, the expression of mat starts simultaneously with the expression of other C1-regulated P1 early functions. However, while most of the latter functions are downregulated during late stages of lytic growth the expression of mat continues throughout the entire lytic growth cycle of bacteriophage P1. Thus, the maturation function has a head start on the structural components of the phage particle.

Bacteriophage T4 gp2 Interferes with Cell Viability and with Bacteriophage Lambda Red Recombination

ABSTRACT The T4 head protein, gp2, promotes head-tail joining during phage morphogenesis and is also incorporated into the phage head. It protects the injected DNA from degradation by exonuclease V during the subsequent infection. In this study, we show that recombinant gp2, a very basic protein, rapidly kills the cells in which it is expressed. To further illustrate the protectiveness of gp2 for DNA termini, we compare the effect of gp2 expression on Red-mediated and Int-mediated recombination. Red-mediated recombination is nonspecific and requires the transient formation of double-stranded DNA termini. Int-mediated recombination, on the other hand, is site specific and does not require chromosomal termini. Red-mediated recombination is inhibited to a much greater extent than is Int-mediated recombination. We conclude from the results of these physiological and genetic experiments that T4 gp2 expression, like Mu Gam expression, kills bacteria by binding to double-stranded DNA termini, the most likely mode for its protection of entering phage DNA from exonuclease V.