Search

The lytic and lysogenic life cycle switch of bacteriophages plays very important roles in virus-host interactions. However, the lysogeny of thermophilic bacteriophage infecting thermophile at high temperatures has not been addressed. In this study, two lysogeny-related genes encoding the CI protein and recombinase of GVE2, a thermophilic bacteriophage obtained from a deep-sea hydrothermal vent, were characterized. Temporal analyses showed that the two genes were expressed at early stages of GVE2 infection. Based on chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA), the GVE2 CI protein was bound with only one DNA fragment located at 24264-24036 bp in the GVE2 genome. This location might be the original transcription site and the lysis-lysogeny switch site, which was very different from mesophilic bacteriophages. The GVE2 CI and recombinase proteins could function only at high temperatures. Therefore our study improved our understanding of the lysogeny process of bacteriophages at high temperatures., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nonlysogenic Escherichia coli K cells exhibit a delay in lysis when infected by T4rII phage termed lysis inhibition (LIN). E. coli K cells expressing lambda rexB from either a prophage defective for rexA, or a multicopy plasmid supported T4rII infection, but prevented the establishment of LIN. In addition, E. coli null mutations in either the periplasmic "tail-specific protease" tsp, or the 10Sa RNA ssrA, completely blocked the establishment of LIN following T4 infections. The expression of rexB in the absence of rexA resulted in several cellular phenotypes, including aberrant cell surface morphology, the partial to near complete suppression of mutations of lambda S and T4t holin genes, and lysis by cells aging on plates or growing with high rexB expression at elevated temperatures. These activities of RexB were impeded in the presence of RexA.

The nucleotide sequence of a DNA fragment previously shown to contain the attachment site (attP) of Oenococcus oeni phage fOg44 (. Arch. Virol. 143, 523-536) has been determined. Sequence analysis indicated that this 6226bp EcoRI fragment harbours an integrase gene, in the vicinity of a direct repeat rich region defining attP, as well as genes encoding a muramidase-related lysin (Lys) and a holin polypeptide (Hol). Transcriptional studies suggested that lys and hol are mainly co-expressed, late in the lytic cycle, from a promotor located upstream of lys. Between the lytic cassette and the phage integration elements three additional open reading frames were found: orf217 and orf252 of unknown function and orf72, the putative product of which bears 32% identity with acidic excisionases from other Gram positive phages. We have established that the first two orfs, as well as the predicted promotor of orf72, are included in a 2143-bp DNA segment missing from the genome of the deletion mutant fOg44Delta2. Although lysogens of fOg44 and fOg44Delta2 exhibited similar properties, each phage produced two distinguishable types of lysogenic strains, differing in inducibility and immunity to other oenophages.

H-19B is a lambdoid phage that carries the genes (stx-I) encoding the two toxin subunits of a Shiga-like toxin; Escherichia coli lysogens of H-19B are converted to toxin producers. Based on the determination of a 17-kb region of the H-19B genome and functional studies, we have identified the early regulatory region and associated genes of H-19B, as well as the location of the late regulatory region and the toxin and lysis genes. A comparative analysis of the sequence of the H-19B genome reveals the presence of ORFs and genes found in analogous positions on the genomes of a number of other lambdoid phages. A cloned genomic fragment that confers immunity to an infecting H-19B phage contains an ORF of an analogous size and genomic location for a repressor gene, adjacent to a putative operator region. The lambda replication genes, O and P, are conserved in H-19B except for a 39-bp insert in the O gene creating two new O protein-binding sites in the origin of replication (ori), giving H-19B six binding sites as opposed to the four sites found in lambda. We identify ORFs and sequences involved in transcriptional regulation encoding N-like antitermination systems like those found in other lambdoid phages and nearly identical to sequences found in phage HK97. Our functional studies show that these sequences support antitermination even though they contain significant differences from those of other lambdoid phages. We also identify ORFs and sequences analogous to the Q-p'R late antiterminators-promoters found in other lambdoid phages. The Shiga-like stx-I genes are located directly downstream of the promoter, p'R, for the late genes, and upstream of the lysis genes.

We have shown that Escherichia coli pcnB mutants are lysogenized by bacteriophage lambda with lower efficiency as compared to the pcnB+ strains. Our genetic analysis revealed that expression of the lambda cII gene is decreased in the pcnB mutants. However, using various lacZ fusions we demonstrated that neither activities of pL and pR promoters nor transcription termination at tR1 were significantly impaired in the pcnB- host. On the other hand, we found that oop RNA, an antisense RNA for cII expression, is involved in this regulation. Primer protection experiments revealed that oop RNA was polyadenylated and that this polyadenylation was impaired in the pcnB mutant. We found that the oop RNA was more abundant in the pcnB mutant than in the pcnB+ strain. Furthermore, we showed that activity of the pO promoter was not stimulated in the pcnB mutant. Such findings indicated that degradation of oop RNA in the pcnB strain was slower because of inefficient polyadenylation, which could lead to more effective inhibition of cII expression by the antisense oop RNA, resulting in less efficient lysogenization of the host. The oop RNA was found previously to play a role in phage lambda development only under conditions of overproduction of this transcript. Here we demonstrate for the first time, the physiological function of oop RNA in lambda development, confirming that this short transcript plays an important role in the negative regulation of cII gene expression during lambda infection. Moreover, polyadenylation of oop RNA is one of very few known examples of specific RNA polyadenylation by PAP I in prokaryotic cells and its role in gene expression regulation.

The coordinate expression from induced lambda prophages of pLit-rexB-tImm (late immunity transcription, LIT RNA) and po-oop-t(o) (OOP RNA) has remained unexplained. The initial assigned sequence for pLit bore no relationship to po. We have identified two promoter sites for independent rexB transcription, denoted here pLit2 and pLit1, which are separated by about 330 bp. The upstream pLit1 site shares with po a common 9 bp sequence between the -10 and -35 regions, with strong homology to aspects of the SOS box or LexA operator site. This sequence is also found within OOP RNA, suggesting that OOP RNA, or another regulatory factor recognizing the common sequence, was involved in the regulation of rexB expression and hence Rex exclusion. We measured the influence of OOP synthesis from plasmids on the Rex phenotype, finding that plasmids producing OOP can suppress Rex exclusion by a lambda prophage. The possibility was suggested that low level constitutive rexB transcription occurs from pLit2. Potential binding sites were identified for DnaA, for the LexA, CI and Cro repressors and for lambda O protein in the 80 nt DNA interval upstream from and including pLit1, suggesting a complex regulatory pattern for rexB expression from this promoter.

øg1e is a temperate phage of the Lactobacillus strain G1e. The phage-host junctions attR and attL cloned from the lysogen have a 24-bp common (core) sequence implicated in recombination. DNA sequencing analysis of a 5.2-kbp SacI fragment of the øg1e phage genome (42.5 kbp) revealed two possible open reading frames (ORF), xis and int, and the phage attachment (recombination) site (attP), whose 24-bp sequence is identical to the core sequence detected in attR and attL. The deduced int product (Int) is a basic protein of 391 amino acids with an estimated pI of 9.70, and significantly resembles other presumed integrases encoded by the Lactobacillus and Lactococcus phages including øadh and øLC3, as well as the Escherichia coli phages such as lambda. The predicted øg1e xis protein (Xis) is small and very acidic (66 amino acids; pI 4.55), and shows a resemblance (32% overall identity) with a putative excisionase encoded by the Staphylococcus phage ø11. The øg1e Int with a deduced molecular mass of 45.5 kDa was overproduced in E. coli cells, and electrophoretically analyzed.

Lysogens of Shigella flexneri harbouring the temperate bacteriophage, Sf6, have been previously shown to undergo a serotype conversion due to O-acetylation of the O-antigen of the lipopolysaccharide. A partial physical map of the phage genome has been constructed. Analysis of the phage DNA suggests that the phage packages by a headful mechanism and that the mature DNA molecules are terminally redundant. Cloning of the PstI fragments of Sf6 enabled the region encoding the serotype conversion to be localized, showing that this was clearly phage-encoded. The gene was further localized by mutagenesis with Tn5 and the nucleotide sequence of the entire 2693-bp PstI fragment was determined. Two major open reading frames (ORFs) were found capable of encoding proteins of 44.1 and 37.2 kDa. The latter corresponds to the O-antigen acetylase and its gene has been designated oac. The oac gene is capable of converting Sh. flexneri serotypes X, Y, 1a and 4a to 3a, 3b, 1b and 4b, respectively. The Oac protein bears a high degree of homology to the NodX protein of Rhizobium leguminosarum suggesting that it, too, may be a sugar acetylase. The second ORF immediately upstream from oac corresponds to the bacteriophage Sf6 integrase responsible for chromosomal integration and is highly homologous to the integrases of Escherichia coli bacteriophages P4 and phi 80, but less closely related to those of P1, P2, P22, 186 and lambda.