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PubMed1982

DNA methyltransferase-dependent transcription of the phage Mu mom gene.

The phage Mu mom gene controls an unusual DNA modification. Expression of the mom function requires an active host (dam+) DNA adenine methylase [S-adenosyl-L-methionine:DNA (6-aminopurine)-methyltransferase]; in dam- hosts, Mu development is normal except that the viral DNA does not undergo the mom modification. The present communication compares transcription of the mom gene in dam+ versus dam- cells. 32P-labeled probes were prepared by nick-translation of a purified mom gene-containing restriction fragment and of virion DNA, respectively. These probes were hybridized with various RNAs blotted onto nitrocellulose filters (after fractionation by agarose gel electrophoresis). The salient findings are: (i) mom-specific RNA was readily detected in dam+ lysogenic cells, but only after induction of the Mu prophage; (ii) the level of mom RNA was decreased at least to 1/20th in induced dam- Mu lysogens; and (iii) little difference, if any, was observed between dam+ and dam- cells with respect to total Mu transcripts produced after prophage induction. These results are in accord with the known pattern of mom gene expression and Mu development. They show that the host (dam+) DNA adenine methylase activity is required for transcription of the mom gene. This represents a unique example where a DNA methylase exerts a positive regulatory role in mRNA transcription; alternative mechanisms for this process will be discussed.

Coliphages↗
PubMed1982

Escherichia coli single-strand DNA binding protein from wild type and lexC113 mutant affects in vitro proteolytic cleavage of phage lambda repressor.

In Escherichia coli, the single-strand DNA-binding protein (SSB) is required for DNA replication. A mutation of the ssb gene, lexC113, imparts to the cells UV sensitivity and inability to induce lambda prophage and to amplify recA protein, indicating participation of SSB in DNA repair and viral induction processes. We report the effect of purified SSB, isolated from wild-type and lexC113 strains, on the recA-mediated proteolysis of lambda repressor in vitro. (i) These proteins abolished the inhibition produced by excess single-strand DNA and (ii) in the presence of the binding proteins, the apparent stoichiometry--1 monomer of recA to 6 nucleotides of single-strand DNA [Craig, N. L. & Roberts, J. W. (1980) Nature (London) 283, 26-30] was no longer observed. (iii) At the optimal concentration--1 protein monomer to 8 nucleotides--they increased the rate and extent of repressor cleavage at all single-strand DNA concentrations, including that observed at the apparent optimal DNA concentration. (iv) At binding protein/nucleotide ratios greater than or equal to 1:3, SSB from lexC113 inhibited repressor cleavage while that from wild type did not. (v) These results are consistent with the notion that SSB is probably involved in the induction of prophages in vivo.

Bacterial Proteins↗
PubMed1984

The rnh gene is essential for growth of Escherichia coli.

We have determined that a functional gene coding for ribonuclease H seems to be essential for cell growth in Escherichia coli. A strain was made with two copies of the rnh gene by lysogenizing an E. coli strain with a lambda phage bearing a copy of the rnh gene. Inactivation of one of the two copies of the rnh gene was accomplished by transformation with a linear DNA molecule that had the gene for chloramphenicol acetyltransferase inserted near the middle of the rnh gene. In recombinants that had an inactive gene replacing the normal chromosomal rnh gene, the lambda rnh prophage supplies an intact functional copy of the rnh gene. Curing the cells of the lambda rnh prophage left the cell with an inactive rnh gene and resulted in cell death. An intact functional rnh gene provided on a plasmid permits normal curing, and cured survivors were readily obtained. The technique described is probably generally applicable for assessing the requirement for other E. coli genes.

Bacteriophage lambda↗
PubMed1985

Interaction of the lambda site-specific recombination protein Xis with attachment site DNA.

Nuclease protection experiments show that Xis protein of bacteriophage lambda specifically binds attachment (att) site DNA. The region of Xis binding, present in both the phage att site and the right prophage att site, extends from position -102 to position -62 in the P arm. The sequence of this region, the positions of purines protected by Xis against methylation, and the binding of Xis to a resected att site indicate the presence of two binding sites. The postulated recognition elements, contained in 13-base-pair direct repeats separated by 7 base pairs, are situated on the same face of the DNA helix. Protection experiments performed with DNase I suggest that the DNA wraps around (or along the surface of) the bound Xis protein. The Xis binding data presented here establishes that Xis, like the other two proteins involved in lambda site-specific recombination, interacts specifically with att DNA. This rules out that class of models in which the profound effects of Xis on the directionality of site-specific recombination are mediated solely through protein-protein interactions or modification of another protein. In addition, nuclease protection experiments with pairwise combinations of the proteins show that Xis and integration host factor (IHF), or Xis and Int, can bind simultaneously to either the phage or right prophage att sites, and the DNA sequences protected are the sum of those protected with each protein alone. It is therefore unlikely that the effect of Xis on the direction of recombination is exerted by directly blocking the binding of Int or IHF to one or more of their respective binding sites.

Attachment Sites, Microbiological↗
PubMed1994

Translation elongation factor Tu cleaved by a phage-exclusion system.

Bacteriophage T4 multiples poorly in Escherichia coli strains carrying the defective prophage, e14; the e14 prophage contains the lit gene for late inhibitor of T4 in E. coli. The exclusion is caused by the interaction of the e14-encoded protein, Lit, with a short RNA or polypeptide sequence encoded by gol from within the major head protein gene of T4. The interaction between Lit and the gol product causes a severe inhibition of all translation and prevents the transcription of genes downstream of the gol site in the same transcription unit. However, it does not inhibit most transcription, nor does it inhibit replication or affect intracellular levels of ATP. Here we show that the interaction of gol with Lit causes the cleavage of translation elongation factor Tu (EF-Tu) in a region highly conserved from bacteria to humans. The depletion of EF-Tu is at least partly responsible for the inhibition of translation and the phage exclusion. The only other phage-exclusion system to be understood in any detail also attacks a highly conserved cellular component, suggesting that phage-exclusion systems may yield important reagents for studying cellular processes.

Amino Acid Sequence↗
PubMed1997

5-Methylcytosine is not a mutation hot spot in nondividing Escherichia coli.

Spontaneous deamination of 5-methylcytosine (5meC) causes hot spots of CxG --> TxA mutations in Escherichia coli and in human cells. In E. coli, the resulting TxG mispairs can be corrected to CxG by very short patch (VSP) repair, which requires the product of gene vsr. Mutation hot spots in genes of replicating vsr+ bacteria are attributable to low Vsr activity. To determine the rate of deamination of 5meC and the efficiency of VSP repair in nondividing bacteria, we used kanamycin-sensitive (KanS) lysogens containing a lambda kan- prophage. Deamination of a 5meC in the kan- gene resulted in mutation to kanamycin resistance (KanR). Lysogens containing a single lambda kan- prophage per bacterial genome were grown in synthetic medium with limiting amino acids and stored at 15 degrees C or 37 degrees C. In the absence of VSP repair, KanR mutants accumulated at the rate of approximately 1.3 x 10(-7) per bacterium per day at 37 degrees C. This is similar to the 5meC --> T mutation rate reported for DNA in solution. In vsr+ bacteria, the KanR accumulation rate was 3 x 10(-9) per bacterium per day, which is not significantly higher than the rate observed when the target cytosine was unmethylated. The increase in KanR mutants was barely detectable in vsr+ cultures stored at 15 degrees C for 4 months. It is likely that mutation hot spots at 5meC in rapidly dividing cells are attributable to insufficient time for TxG correction in the interval between deamination of 5meC and subsequent DNA replication. DNA synthesis occurred in bacteria starved for amino acids and this synthesis was not highly mutagenic.

5-Methylcytosine↗
PubMed1998

In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family.

The genome of the broad host range Streptomyces temperate phage, phiC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in phiC31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.

Amino Acid Sequence↗
PubMed2000

Genetic analysis of a bacterial genetic exchange element: the gene transfer agent of Rhodobacter capsulatus.

An unusual system of genetic exchange exists in the purple nonsulfur bacterium Rhodobacter capsulatus. DNA transmission is mediated by a small bacteriophage-like particle called the gene transfer agent (GTA) that transfers random 4.5-kb segments of the producing cell's genome to recipient cells, where allelic replacement occurs. This paper presents the results of gene cloning, analysis, and mutagenesis experiments that show that GTA resembles a defective prophage related to bacteriophages from diverse genera of bacteria, which has been adopted by R. capsulatus for genetic exchange. A pair of cellular proteins, CckA and CtrA, appear to constitute part of a sensor kinase/response regulator signaling pathway that is required for expression of GTA structural genes. This signaling pathway controls growth-phase-dependent regulation of GTA gene messages, yielding maximal gene expression in the stationary phase. We suggest that GTA is an ancient prophage remnant that has evolved in concert with the bacterial genome, resulting in a genetic exchange process controlled by the bacterial cell.

Amino Acid Sequence↗
PubMed2000

Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice.

Shiga toxin-producing Escherichia coli (STEC) cause significant disease; treatment is supportive and antibiotic use is controversial. Ciprofloxacin but not fosfomycin causes Shiga toxin-encoding bacteriophage induction and enhanced Shiga toxin (Stx) production from E. coli O157:H7 in vitro. The potential clinical relevance of this was examined in mice colonized with E. coli O157:H7 and given either ciprofloxacin or fosfomycin. Both antibiotics caused a reduction in fecal STEC. However, animals treated with ciprofloxacin had a marked increase in free fecal Stx, associated with death in two-thirds of the mice, whereas fosfomycin did not. Experiments that used a kanamycin-marked Stx2 prophage demonstrated that ciprofloxacin, but not fosfomycin, caused enhanced intraintestinal transfer of Stx2 prophage from one E. coli to another. These observations suggest that treatment of human STEC infection with bacteriophage-inducing antibiotics, such as fluoroquinolones, may have significant adverse clinical consequences and that fluoroquinolone antibiotics may enhance the movement of virulence factors in vivo.

Animals↗
PubMed1999

Sequence analysis of Stx2-converting phage VT2-Sa shows a great divergence in early regulation and replication regions.

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.

Amino Acid Sequence↗
PubMed2001

Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12.

Escherichia coli O157:H7 is a major food-borne infectious pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Here we report the complete chromosome sequence of an O157:H7 strain isolated from the Sakai outbreak, and the results of genomic comparison with a benign laboratory strain, K-12 MG1655. The chromosome is 5.5 Mb in size, 859 Kb larger than that of K-12. We identified a 4.1-Mb sequence highly conserved between the two strains, which may represent the fundamental backbone of the E. coli chromosome. The remaining 1.4-Mb sequence comprises of O157:H7-specific sequences, most of which are horizontally transferred foreign DNAs. The predominant roles of bacteriophages in the emergence of O157:H7 is evident by the presence of 24 prophages and prophage-like elements that occupy more than half of the O157:H7-specific sequences. The O157:H7 chromosome encodes 1632 proteins and 20 tRNAs that are not present in K-12. Among these, at least 131 proteins are assumed to have virulence-related functions. Genome-wide codon usage analysis suggested that the O157:H7-specific tRNAs are involved in the efficient expression of the strain-specific genes. A complete set of the genes specific to O157:H7 presented here sheds new insight into the pathogenicity and the physiology of O157:H7, and will open a way to fully understand the molecular mechanisms underlying the O157:H7 infection.

Bacterial Proteins↗
PubMed2002

A satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer.

CTXphi is a filamentous bacteriophage whose genome encodes cholera toxin, the principal virulence factor of Vibrio cholerae. We have found that the CTXphi-related element RS1 is a satellite phage whose transmission depends upon proteins produced from a CTX prophage (its helper phage). However, unlike other satellite phages and satellite animal viruses, RS1 can aid the CTX prophage as well as exploit it, due to the RS1-encoded protein RstC. RstC, whose function previously was unknown, is an antirepressor that counteracts the activity of the phage repressor RstR. RstC promotes transcription of genes required for phage production and thereby promotes transmission of both RS1 and CTXphi. Antirepression by RstC also induces expression of the cholera toxin genes, ctxAB, and thus may contribute to the virulence of V.cholerae. In vitro, RstC binds directly to RstR, producing unusual, insoluble aggregates containing both proteins. In vivo, RstC and RstR are both found at the cell pole, where they again appear to form stable complexes. The sequestration/inactivation process induced by RstC resembles those induced by mutant polyglutamine-containing proteins implicated in human neurodegenerative disorders.

Bacterial Proteins↗
PubMed1993

Lethal transposition of Mud phages in Rec- strains of Salmonella typhimurium.

Under several circumstances, the frequency with which Mud prophages form lysogens is apparently reduced in rec strains of Salmonella typhimurium. Lysogen formation by a MudI genome (37 kb) injected by a Mu virion is unaffected by a host rec mutation. However when the same MudI phage is injected by a phage P22 virion, lysogeny is reduced in a recA or recB mutant host. A host rec mutation reduces the lysogenization of mini-Mu phages injected by either Mu or P22 virions. When lysogen frequency is reduced by a host rec mutation, the surviving lysogens show an increased probability of carrying a deletion adjacent to the Mud insertion site. We propose that the rec effects seen are due to a failure of conservative Mu transposition. Replicative Mud transposition from a linear fragment causes a break in the host chromosome with a Mu prophage at both broken ends. These breaks are lethal unless repaired; repair can be achieved by Rec functions acting on the repeated Mu sequences or by secondary transposition events. In a normal Mu infection, the initial transposition from the injected fragment is conservative and does not break the chromosome. To account for the conditions under which rec effects are seen, we propose that conservative transposition of Mu depends on a protein that must be injected with the DNA. This protein can be injected by Mu but not by P22 virions. Injection or function of the protein may depend on its association with a particular Mu DNA sequence that is present and properly positioned in Mu capsids containing full-sized Mu or MudI genomes; this sequence may be lacking or abnormally positioned in the mini-Mud phages tested.

Bacteriophage mu↗
PubMed1972

Isolation of plaque-forming, galactose-transducing strains of phage lambda.

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.-A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.

Centrifugation, Density Gradient↗
PubMed1974

Isolation of P22 specialized transducing phage followong F'-episome fusion.

A P22 specialized transducing phage has been constructed which carries the structural gene for aspartate transcarbamylase (ATCase). This gene (pyrB) was first brought close to the P22 attachment site by fusing an F' pyrB(+) episome to an F' prolac episome which carries a P22 prophage attachment site. A prophage was added to these fused F' episomes and the lysogen was UV-induced. The specialized transducing phage was isolated from the resulting lysate. The phage also carries argI, the structure gene for ornithine transcarbamylase.

Aspartate Carbamoyltransferase↗
PubMed2025

Short-term virus-host interactions and functional dynamics in recently deglaciated Antarctic tundra soils.

Long-term chronosequence studies have shown that, as glaciers retreat, newly exposed soils become colonized through primary succession. To determine the key drivers of this process and their vulnerability to climate change, the short-term responses of these pioneering microbial communities also need to be elucidated. Here, we investigated how the taxonomic and functional structure of microbial communities, including viruses, changed over a 7-year period in an Antarctic glacier forefield. Using metagenomics and metatranscriptomics we assessed the influence of both abiotic and biotic factors on these communities. Our results revealed a highly heterogeneous bacteria-dominated microbial community, with Pseudomonas as the most abundant genus, followed by Lysobacter, Devosia, Cellulomonas, and Brevundimonas. This community exhibited the capacity for aerobic anoxygenic phototrophy, carbon and nitrogen fixation, and sulfur cycling, processes vital for survival in nutrient-poor environments. 52 high-quality metagenome-assembled genomes (MAGs) were recovered, representing both transient and cosmopolitan taxa, some of which were able to rapidly respond to environmental changes. A diverse and highly dynamic collection of lytic and temperate viruses was identified across all samples, with high clonal viral genomes typically detected in only one of the eight samples analyzed. Metatranscriptomic analyses confirmed the activity of lytic viruses, while prophage genomes featured much lower expression levels. Prophages appeared to influence host fitness through the expression of genes encoding membrane transporters. Additionally, the abundance of genes linked to antimicrobial compound synthesis and resistance, along with antiphage defense systems, highlights the importance of biotic interactions in driving microbial community succession and shaping short-term responses to environmental fluctuations.

Antarctica↗
PubMed1995

Bacterial ribonuclease: mutagenic effect in microbial test-systems.

Pure enzyme samples of ribonuclease from Bacillus intermedius 7P (known commercially as 'binase') were investigated for genotoxicity in four microbial tests: the Ames plate incorporation method, AraR-assay; the prophage induction test; and the DNA-repair test. The weak mutagenic effect of binase at high concentrations (0.1 mg/plate, 1 mg/plate) was established by induction of forward AraR-mutations and histidine-reverse mutations (both frameshift mutations and base pair substitution). Metabolic activation with rat or chicken liver, human placenta or plant (from tulip bulbs) microsomal fractions in vitro was seen to abolish the binase mutagenicity. Bacillus intermedius 7P ribonuclease appears to possess DNA damaging activity in uvrA- and polA- mutants, but not in the recA-deficient Escherichia coli strain, and exhibits an induction of recA-dependent mutagenesis detected by the 8-fold increase of the prophage-induction level in lysogenic Bacillus subtilis culture and by the 5-fold increase of this level in the Streptomyces lavendulae 3 lysogenic strain. The importance of the roles of both of enzyme catalytic activity and native structure is emphasized. A proposed mechanism for exogenous ribonuclease action is discussed. Bacillus intermedius 7P ribonuclease probably does not act as a direct genotoxic agent interacting with DNA, but could provoke nucleotide imbalance through its catalytic action on membrane-associated RNAs, which results in alteration of DNA replication and, as a consequence, in recA-dependent mutagenesis.

Arabinose↗
PubMed1999

Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis.

Glycosyl hydrolase (GH) genes from Escherichia coli and Bacillus subtilis were used to search for cases of horizontal gene transfer. Such an event was inferred by G + C content, codon usage analysis, and a phylogenetic congruency test. The codon usage analysis used is a procedure based on a distance derived from a Pearson linear correlation coefficient determined from a pairwise codon usage comparison. The distances are then used to generate a distance-based tree with which we can define clusters and rapidly compare codon usage. Three genes (yagH from E. coli and xynA and xynB from B. subtilis) were determined to have arrived by horizontal gene transfer and were located in E. coli CP4-6 prophage, and B. subtilis prophages 6 and 5, respectively. In this study, we demonstrate that with codon usage analysis, the proposed horizontally transferred genes can be distinguished from highly expressed genes.

Bacillus subtilis↗