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PubMed1971

Heat induction of prophage phi 105 in Bacillus subtilis: replication of the bacterial and bacteriophage genomes.

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.

Bacillus subtilis↗

PubMed1984

[Mutagenic action of alkylating agents on prophage lambda].

The lethal and mutagenic effects of 7 alkylating agents: N-nitroso-N-methylurea (NMU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), nitrogen mustard (HN2), mitomycin C (MC), bifunctional acridine mustard (AM)--and of cyanate (KNCO) on heat inducible lambda cI857 prophage were studied. After treatment of lysogenic cells with mutagens, prophage was heat-induced either immediately or after 90 min incubation in nutrient broth and c mutants forming clear plaques at 32 degrees C were scored. NMU (0.02 M) when immediately induced with heat, induces c mutants very efficiently (maximal yield 10%) not only in the wild-type cells but also in repair-deficient mutants recA13, lexA102, uvrA6 umuC36, recF143, xthA9, polA1, uvrD3 and uvrD502. These data show that NMU-induced mutations are fixed as replication errors due to mispairing modified bases. After delayed heat induction, the prophage survival enhances and the frequency of c mutations declines considerably in host cells of all repair genotypes tested. Carbamoylation is not involved in the mutagenic action of NMU, because KNCO (0.02 M) has a very slight lethal effect and does not induce mutations. MNNG (100 micrograms/ml) and EMS (0.1 M) also induce mutations by replicative mechanism, because maximal yield of c mutations does not depend on RecA+ and is about 15 and 2%, respectively. MMS is a mutagen of the repair type, since its mutagenic action is suppressed by recA mutation of the host. NH2 only inactivates prophage, but does not induce mutations. MC (50 micrograms/ml) and AM (150 micrograms/ml) induce mutations rather inefficiently (the maximal yield 0.1 and 0.3%, respectively) both in recA+ and recA- hosts. The mutagenic action of these agents is probably due to intercalation.

Alkylating Agents↗

PubMed1975

[The influence of several prophages on the genetic behavior of Flexner dysentery bacteria].

In conjugation experiments of Escherichia coli K-12 Hfr strains and converted clones of Shigella flexneri var. y(-:3,4) that had acquired the capacity to synthesize type of antigens IV or V it is confirmed that the locus linked to lac-pro region in Sh. flexneri chromosome called Tp locus is a site of an attachment of prophages responsible for certain type specific antigens. Lac+ hybrids of the clone converted by phage IV lost the type specific antigen IV with the frequency comparable with the loss of the aforementioned antigen by wild strains of Sh. flexneri ser 4 in similar experiments carried out previously (90,8% and 97% respectively). Lac+ hybrids of the clones converted by phage V did not lose the type antigen that corresponded to the behaviour of wild strains of serotype 5b (V: 7,8) with double lysogenicity. It was shown that the clones converted by the aforementioned phage V had acquired an immunity not only to phage V, but to phage 7,8 as well. An independent segregation of immunity to phages V and 7,8 in lac+ hybrids of the converted clone was observed. It indicates that donor strain NTCC 595/52 carries two prophages, V and "defect" 7,8 (not expressing factor 7,8). A maintenance of antigen V observed in lac+ hybrids of the converted clone in this case confirms previous suggestions that an attachment of prophage 7,8 creates a state of some inhomology in this region disturbing the recombination process. The converting phage V isolated from the strain NTCC 595/52 is probably recombinant. An analysis of the hybrid classes allows to suggest the following approximate order of markers of the prophage on the chromosome of the converted clone: aV--imm V--imm 7,8--lac.

Bacteriophages↗

PubMed1993

Induction of prophage lambda by nitrofurantoin and its modulation by butylated hydroxytoluene, sodium arsenite and alpha tocopherol.

Nitrofurantoin induced prophage-lambda in E. coli K12 strain GY5027(lambda) in a dose dependent manner, the maximum induction being 10-fold the spontaneous induction level and the maximum efficiency of induction 74%. The lever extract used as a metabolizing mixture enhanced the induction level significantly. Chloramphenicol at a concentration of 20 micrograms/ml inhibited the prophage induction by nitrofurantoin, indicating that the induction required concomitant protein synthesis. Butylated hydroxytoluene(BHT) and sodium arsenite enhanced the nitrofurantoin induced prophage-lambda induction in E. coli GY 5027(lambda) cells in a dose dependent manner. The maximum modulations in induction level (I/Io) were achieved with 100 micrograms/ml BHT and 250 micrograms/ml sodium arsenite corresponding to a nitrofurantoin concentration of 15 micrograms/ml and were found significant on statistical analysis. alpha-tocopherol, however, did not produce any effect on the prophage-lambda induction by nitrofurantoin.

Arsenic↗

PubMed2005

Effect of glutathione on UV induction of prophage lambda.

The effect of glutathione (GSH) on the ultraviolet (UV) induction of lambda prophage was investigated in lysogenic Escherichia coli. The data showed that extracellular GSH could inhibit the UV induction of lambda prophage. The inhibitory rates were concentration dependent, and the maximal rate obtained was 94% with 3.0 M GSH. The effect was also measured in three different lambda lysogens: a wild-type strain (wt), an isogenic GSH-deficient strain, and an isogenic strain producing increased amounts of GSH. The result showed that when subjected to UV irradiation (254 nm, 60 J m(-2)), GSH-deficient strain was approximately fivefold more sensitive to be lysed than wt, whereas the strain with higher intracellular GSH levels was only 28% susceptible to be lysed. With electron spin resonance and spin trapping techniques, we observed that free radical signals occurred in the suspensions of UV irradiated lysogenic cells and the intensity of signals was influenced by GSH levels. These results indicate that GSH can significantly inhibit the UV induction of lambda prophage, and that this effect is correlated to its capacity to scavenge free radicals generated after UV irradiation.

Bacteriophage lambda↗

PubMed2006

Role of the lytic repressor in prophage induction of phage lambda as analyzed by a module-replacement approach.

Using a module exchange approach, we have tested a long-standing model for the role of Cro repressor in lambda prophage induction. This epigenetic switch from lysogeny to the lytic state occurs on activation of the host SOS system, which leads to specific cleavage of CI repressor. It has been proposed that Cro repressor, which operates during lytic growth and which we shall term the lytic repressor, is crucial to prophage induction. In this view, Cro binds to the O(R)3 operator, thereby repressing the cI gene and making the switch irreversible. Here we tested this model by replacing lambda Cro with a dimeric form of Lac repressor and adding several lac operators. This approach allowed us to regulate the function of the lytic repressor at will and to prevent it from repressing cI, because lac repressor could not repress P(RM) in our constructs. Repression of cI by the lytic repressor was not required for prophage induction to occur. However, our evidence suggests that this binding can make induction more efficient, particularly at intermediate levels of DNA damage that otherwise cause induction of only a fraction of the population. These results indicate that this strategy of module exchange will have broad applications for analysis of gene regulatory circuits.

Bacterial Proteins↗

PubMed2006

Hypervariable prophage WO sequences describe an unexpected high number of Wolbachia variants in the mosquito Culex pipiens.

Wolbachia are maternally inherited endosymbiotic bacteria that infect many arthropod species and may induce cytoplasmic incompatibility (CI) resulting in abortive embryonic development. Among all the described host species, mosquitoes of the Culex pipiens complex display the highest variability of CI crossing types. Paradoxically, searches for polymorphism in Wolbachia infecting strains and field populations hitherto failed or produced very few markers. Here, we show that an abundant source of the long-sought polymorphism lies in WO prophage sequences present in multiple copies dispersed in the genome of Wolbachia infecting C. pipiens (wPip). We identified up to 66 different Wolbachia variants in C. pipiens strains and field populations and no occurrence of superinfection was observed. At least 49 different Wolbachia occurred in Southern Europe C. pipiens populations, and up to 10 different Wolbachia were even detected in a single population. This is in sharp contrast with North African and Cretan samples, which exhibited only six variants. The WO polymorphism appeared stable over time, and was exclusively transferred maternally. Interestingly, we found that the CI pattern previously described correlates with the variability of Gp15, a prophage protein similar to a bacterial virulence protein. WO prophage sequences thus represent variable markers that now open routes for approaching the molecular basis of CI, the host effects, the structure and dynamics of Wolbachia populations.

Animals↗

PubMed2005

Organization of the CTX prophage in environmental isolates of Vibrio mimicus.

The organization of the CTX prophage in environmental strains of Vibrio mimicus was investigated. Sixteen hundred non-sucrose fermenting vibrios were examined for ctx gene by hybridization. Out of 1,600 isolates, 6 V. mimicus isolates contained ctxA gene. The organization of CTX prophage was determined by RFLP using ctxA probe. The CTX prophage integrated at a single site in V. mimicus genome which was present as a single copy flanked by at least a single RS element. Ribotype pattern revealed that a particular clone of V. mimicus acquired the CTXPhi in the aquatic environment. This study demonstrated that V. mimicus could act as a reservoir of CTXPhi in the aquatic environment.

DNA, Viral↗

PubMed2002

Phenotypes of lexA mutations in Salmonella enterica: evidence for a lethal lexA null phenotype due to the Fels-2 prophage.

The LexA protein of Escherichia coli represses the damage-inducible SOS regulon, which includes genes for repair of DNA. Surprisingly, lexA null mutations in Salmonella enterica are lethal even with a sulA mutation, which corrects lexA lethality in E. coli. Nine suppressors of lethality isolated in a sulA mutant of S. enterica had lost the Fels-2 prophage, and seven of these (which grew better) had also lost the Gifsy-1 and Gifsy-2 prophages. All three phage genomes included a homologue of the tum gene of coliphage 186, which encodes a LexA-repressed cI antirepressor. The tum homologue of Fels-2 was responsible for lexA lethality and had a LexA-repressed promoter. This basis of lexA lethality was unexpected because the four prophages of S. enterica LT2 are not strongly UV inducible and do not sensitize strains to UV killing. In S. enterica, lexA(Ind(-)) mutants have the same phenotypes as their E. coli counterparts. Although lexA null mutants express their error-prone DinB polymerase constitutively, they are not mutators in either S. enterica or E. coli.

Bacterial Proteins↗

PubMed2003

Integration and distribution of Lactobacillus johnsonii prophages.

In Lactobacillus johnsonii strain NCC533, two prophages were integrated into tRNA genes and one was disrupted by integration. In a survey, the prophages were restricted to strains sharing an essentially identical restriction pattern. Microarray analysis showed that the prophage DNA represents about 50% of the NCC533 strain-specific DNA.

Animals↗

PubMed2003

Prophage contribution to bacterial population dynamics.

Cocultures of Salmonella strains carrying or lacking specific prophages undergo swift composition changes as a result of phage-mediated killing of sensitive bacteria and lysogenic conversion of survivors. Thus, spontaneous prophage induction in a few lysogenic cells enhances the competitive fitness of the lysogen population as a whole, setting a selection regime that forces maintenance and spread of viral DNA. This is likely to account for the profusion of prophage sequences in bacterial genomes and may contribute to the evolutionary success of certain phylogenetic lineages.

Biological Evolution↗

PubMed2004

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis.

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.

Animals↗

PubMed2005

Complete nucleotide sequence of the LE1 prophage from the spirochete Leptospira biflexa and characterization of its replication and partition functions.

The first and, to date, only extrachromosomal circular replicon identified in the spirochete Leptospira is the LE1 prophage from Leptospira biflexa. The 74-kb LE1 genome has a GC content of 36%, which is similar to the GC content of Leptospira spp. Most of the 79 predicted open reading frames (ORFs) showed no similarities to known ORFs. However 21 ORFs appeared to be organized in clusters that could code for head and tail structural proteins and immunity repressor proteins. In addition, the pattern of gene expression showed that several LE1 genes are expressed specifically either in LE1 prophage or in L. biflexa late after infection. Since the LE1 prophage replicates autonomously as a circular replicon in L. biflexa, we were able to engineer an L. biflexa-Escherichia coli shuttle vector from a 5.3-kb DNA fragment of LE1 (Saint Girons et al., J. Bacteriol. 182:5700-5705, 2000), opening this genus to genetic manipulation. In this study, base compositional asymmetry confirms the location of the LE1 replication region and suggests that LE1 replicates via a bidirectional Theta-like replication mechanism from this unique origin. By subcloning experiments, the replication region can be narrowed down to a 1-kb region. This minimal replication region consists of a rep encoding a protein of 180 amino acids. Upstream from rep, putative partitioning genes, called parA and parB, were found to be similar to the par loci in Borrelia plasmids. A significant increase of plasmid stability in L. biflexa can be seen only when both parA and parB are present. These results enable the construction of new shuttle vectors for studying the genetics of Leptospira spp. This study will also contribute to a better knowledge of phages unrelated to lambdoid phages.

Amino Acid Sequence↗

PubMed2005

Identification of genes of VSH-1, a prophage-like gene transfer agent of Brachyspira hyodysenteriae.

VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. Purified VSH-1 virions are noninfectious, contain random 7.5-kb fragments of the bacterial genome, and mediate generalized transduction of B. hyodysenteriae cells. In order to identify and sequence genes of this novel gene transfer agent (GTA), proteins associated either with VSH-1 capsids or with tails were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of 11 proteins were determined. Degenerate PCR primers were designed from the amino acid sequences and used to amplify several VSH-1 genes from B. hyodysenteriae strain B204 DNA. A lambda clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence overlapping DNA inserts containing additional VSH-1 genes. VSH-1 genes spanned 16.3 kb of the B. hyodysenteriae chromosome and were flanked by bacterial genes. VSH-1 identified genes and unidentified, intervening open reading frames were consecutively organized in head (seven genes), tail (seven genes), and lysis (four genes) clusters in the same transcriptional direction. Putative lysis genes encoding endolysin (Lys) and holin proteins were identified from sequence and structural similarities of their translated protein products with GenBank bacteriophage proteins. Recombinant Lys protein hydrolyzed peptidoglycan purified from B. hyodysenteriae cells. The identified VSH-1 genes exceed the DNA capacity of VSH-1 virions and do not encode traditional bacteriophage early functions involved in DNA replication. These genome properties explain the noninfectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, GTAs of other bacterial species, such as the GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this GTA and should facilitate investigations of VSH-1-like prophages from other Brachyspira species.

Cloning, Molecular↗

PubMedDate not supplied

[Inductive effect of hypoxanthine-xanthine oxidase system on lambda prophage].

Hypoxanthine-xanthine oxidase (HX-XO) system is a classical system that can generate superoxide anions. The inductive effect of the HX-XO system for lambda prophage has been investigated in this study. The results showed that the system can induce lambda prophage from lysogenic state to lytic growth. The inductive effect was directly proportional to the concentration of HX and XO and inversely related to the time of preliminary incubation of HX with XO. The cell density of the lysogenic bacteria also greatly affected the inductive effect. The maximal PFU number of 2.9 x 10(4) PFU/ml was recorded at 0.86 mM HX, 1.6 x 10(-2) U/ml XO, and a cell density of 10(8) cells/ml. The inductive effect of the HX-XO system was inhibited in the suspensions by glutathione, superoxide dismutase, and catalase. The results provide evidence that free radicals are the primary factors in the induction of lambda prophage in lysogenic bacteria.

Bacteriophage lambda↗

PubMed1978

Control of lambda repressor prophage and establishment transcription by the product of gene tof.

Control of expression of the bacteriophage lambda (lambda) repressor was studied by measuring repressor transcription in noninduced and derepressed lambda lysogens. Three distinct modes of leftward transcription were observed from cI and the adjacent genes associated with the control of repressor synthesis: The prophage or maintenance mode Prm-cI-rex-ti repressor transcript occurs from repressed lysogens; the oop (Po-oop-to) transcript, and the lit (lit-ti) RNA, from the distal half of gene rex, both occur from induced tof+ prophage; the repressor establishment mode of transcription is observed throughout the rex-cI-tof-y-cII-oop interval between Po and ti from induced tof- prophage. The overall level of establishment mRNA synthesis is partially template dependent. However, the actual initiation step for repressor establishment transcription requires the participation of the lambda cIII, cII products, and also either requires the activity of Escherichia coli replication proteins, or is triggered by a replication initiation event. The cII cIII products do not positively stimulate de novo initiation of establishment transcription, but rather act after an initial replication-dependent step. Initiation of the establishment mode of repressor transcription is totally inhibited by more than 125-fold, in an all or none fashion, by the lambda antirepressor (Tof), the product of gene tof (cro). Since Tof only reduces the in vivo rightward transcription of cII from Pr by about 2-fold, we suggest that Tof inhibits repressor establishment transcription by either uncoupling the replication and cII-cIII dependent events, or by inhibiting the activity rather than the expression of the cIII, cII products. Our results do not fully support either of the present hypotheses that establishment transcription is initiated from the hypothetical Pre promoter in the y-interval, or arises through antitermination of the oop RNA. Since the initiation and control of the establishment mode of repressor transcription parallels the control of lit RNA synthesis, we propose a common mechanism underlies the initiation of these transcripts.

Coliphages↗

PubMed1984

Prophage inactivation in recB-proficient Escherichia coli K12 (lambda) lysogens after ultraviolet irradiation.

If ultraviolet irradiated, lambda-lysogenic Escherichia coli K12 bacteria are incubated for 4 to 6 h at 30 degrees C, lambda prophage becomes inactive in the non-surviving cells. This was demonstrated by the use of lambda cIts857ind1 prophage which can be induced by heat but not by ultraviolet light. An analysis with various bacterial mutants showed that RecBC recombination enzyme is required in conjunction with RecA protein for prophage inactivation.

Bacteriophage lambda↗

PubMed1986

Physical mapping of LP51 and LP52 prophages of lysogenic strains of Bacillus licheniformis.

The physical maps of the LP51 and LP52 prophages in lysogenic strains of Bacillus licheniformis were constructed on the basis of data obtained by hybridization of phage DNA probes with Southern blots of restricted DNA of the lysogens. The data were compatible with the Campbell model for chromosomal integration; the attP site was mapped at 58.7-61.8 map units of the genomes of both phages. Identification of prophage-host DNA junction fragments indicated the presence of a unique attB site on the bacterial chromosome; the set of junction fragments in the strain B. licheniformis ATCC 10716 was identical to that of ATCC 11946, but different from ATCC 8187. Both the LP51 and LP52 phages used the same integration sites. Upon reinfection with either phage, the cured strains UM12 and UM18 (i.e. 10716 and 11946 cured of LP52 or LP51, respectively) turned out to be integration deficient. In surface cultures the reinfected bacteria could be maintained in the lysogenic state without, however, integrating the phage genome; when these bacteria were passaged in submerged cultures, several modes of anomalous integration were observed, and the phage segregated into a variety of forms, discernible by virulence and plaque morphology. In liquid cultures of UM12(LP51) or UM12(LP52) lytic forms finally predominated, while most lysogenized UM18 were converted into defective lysogens which contained a defective prophage in a stably integrated form.

Bacillus↗