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PubMed2005

Plasmid partition system of the P1par family from the pWR100 virulence plasmid of Shigella flexneri.

P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.

Amino Acid Sequence↗
PubMed2005

Comparative analysis of differentially expressed genes in Shewanella oneidensis MR-1 following exposure to UVC, UVB, and UVA radiation.

We previously reported that Shewanella oneidensis MR-1 is highly sensitive to UVC (254 nm), UVB (290 to 320 nm), and UVA (320 to 400 nm). Here we delineated the cellular response of MR-1 to UV radiation damage by analyzing the transcriptional profile during a 1-h recovering period after UVC, UVB, and UVA exposure at a dose that yields about a 20% survival rate. Although the SOS response was observed with all three treatments, the induction was more robust in response to short-wavelength UV radiation (UVB and UVC). Similarly, more prophage-related genes were induced by short-wavelength UV radiation. MR-1 showed an active detoxification mechanism in response to UVA, which included the induction of antioxidant enzymes and iron-sequestering proteins to scavenge reactive oxygen species. In addition, a great number of genes encoding multidrug and heavy metal efflux pumps were induced following UVA irradiation. Our data suggested that activation of prophages appears the major lethal factor in MR-1 following UVC or UVB irradiation, whereas oxidative damage contributes greatly to the high UVA sensitivity in MR-1.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2005

Type III restriction is alleviated by bacteriophage (RecE) homologous recombination function but enhanced by bacterial (RecBCD) function.

Previous works have demonstrated that DNA breaks generated by restriction enzymes stimulate, and are repaired by, homologous recombination with an intact, homologous DNA region through the function of lambdoid bacteriophages lambda and Rac. In the present work, we examined the effect of bacteriophage functions, expressed in bacterial cells, on restriction of an infecting tester phage in a simple plaque formation assay. The efficiency of plaque formation on an Escherichia coli host carrying EcoRI, a type II restriction system, is not increased by the presence of Rac prophage-presumably because, under the single-infection conditions of the plaque assay, a broken phage DNA cannot find a homologue with which to recombine. To our surprise, however, we found that the efficiency of plaque formation in the presence of a type III restriction system, EcoP1 or EcoP15, is increased by the bacteriophage-mediated homologous recombination functions recE and recT of Rac prophage. This type III restriction alleviation does not depend on lar on Rac, unlike type I restriction alleviation. On the other hand, bacterial RecBCD-homologous recombination function enhances type III restriction. These results led us to hypothesize that the action of type III restriction enzymes takes place on replicated or replicating DNA in vivo and leaves daughter DNAs with breaks at nonallelic sites, that bacteriophage-mediated homologous recombination reconstitutes an intact DNA from them, and that RecBCD exonuclease blocks this repair by degradation from the restriction breaks.

Bacteriophage lambda↗
PubMed2005

GIL16, a new gram-positive tectiviral phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements.

One of the most notable characteristics of Tectiviridae resides in their double-layer coats: the double-stranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and phiNS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating from Bacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated from B. thuringiensis serovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by the Bacillus cereus reference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to the Tectiviridae.

Bacillus cereus↗
PubMed2006

Transcriptome analysis applied to survival of Shewanella oneidensis MR-1 exposed to ionizing radiation.

The ionizing radiation (IR) dose that yields 20% survival (D20) of Shewanella oneidensis MR-1 is lower by factors of 20 and 200 than those for Escherichia coli and Deinococcus radiodurans, respectively. Transcriptome analysis was used to identify the genes of MR-1 responding to 40 Gy (D20). We observed the induction of 170 genes and repression of 87 genes in MR-1 during a 1-h recovery period after irradiation. The genomic response of MR-1 to IR is very similar to its response to UV radiation (254 nm), which included induction of systems involved in DNA repair and prophage synthesis and the absence of differential regulation of tricarboxylic acid cycle activity, which occurs in IR-irradiated D. radiodurans. Furthermore, strong induction of genes encoding antioxidant enzymes in MR-1 was observed. DNA damage may not be the principal cause of high sensitivity to IR, considering that MR-1 carries genes encoding a complex set of DNA repair systems and 40 Gy IR induces less than one double-strand break in its genome. Instead, a combination of oxidative stress, protein damage, and prophage-mediated cell lysis during irradiation and recovery might underlie this organism's great sensitivity to IR.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2005

Switches in bacteriophage lambda development.

The lysis-lysogeny decision of bacteriophage lambda (lambda) is a paradigm for developmental genetic networks. There are three key features, which characterize the network. First, after infection of the host bacterium, a decision between lytic or lysogenic development is made that is dependent upon environmental signals and the number of infecting phages per cell. Second, the lysogenic prophage state is very stable. Third, the prophage enters lytic development in response to DNA-damaging agents. The CI and Cro regulators define the lysogenic and lytic states, respectively, as a bistable genetic switch. Whereas CI maintains a stable lysogenic state, recent studies indicate that Cro sets the lytic course not by directly blocking CI expression but indirectly by lowering levels of CII which activates cI transcription. We discuss how a relatively simple phage like lambda employs a complex genetic network in decision-making processes, providing a challenge for theoretical modeling.

Bacteriolysis↗
PubMed2003

Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: induction of chromosomal mutations but not gene mutations.

Arsenic is a prevalent human carcinogen whose mutagenicity has not been characterized fully. Exposure to either form of inorganic arsenic, As(III) or As(V), can result in the formation of at least four organic metabolites: monomethylarsonic acid, monomethylarsonous acid (MMA(III)), dimethylarsinic acid, and dimethylarsinous acid (DMA(III)). The methylated trivalent species, as well as some of the other species, have not been evaluated previously for the induction of chromosome aberrations, sister chromatid exchanges (SCE), or toxicity in cultured human peripheral blood lymphocytes; for mutagenicity in L5178Y/Tk(+/-) mouse lymphoma cells or in the Salmonella reversion assay; or for prophage-induction in Escherichia coli. Here we evaluated the arsenicals in these assays and found that MMA(III) and DMA(III) were the most potent clastogens of the six arsenicals in human lymphocytes and the most potent mutagens of the six arsenicals at the Tk(+/-) locus in mouse lymphoma cells. The dimethylated arsenicals were also spindle poisons, suggesting that they may be ultimate forms of arsenic that induce aneuploidy. Although the arsenicals were potent clastogens, none were potent SCE inducers, similar to clastogens that act via reactive oxygen species. None of the six arsenicals were gene mutagens in Salmonella TA98, TA100, or TA104; and neither MMA(III) nor DMA(III) induced prophage. Our results show that both methylated As(V) compounds were less cytotoxic and genotoxic than As(V), whereas both methylated As(III) compounds were more cytotoxic and genotoxic than As(III). Our data support the view that MMA(III) and DMA(III) are candidate ultimate genotoxic forms of arsenic and that they are clastogens and not gene mutagens. We suggest that the clastogenicity of the other arsenicals is due to their metabolism by cells to MMA(III) or DMA(III).

Arsenicals↗
PubMed1980

I. Bacterial mutagenicity of particulates from Houston air.

This study was designed to examine suspended air particulates from the Houston atmosphere, Airborne particulates were collected using either a hi-vol sampler (one stage from 0.01 to 25 micrometer) or an Anderson Cascade Impactor, the five stages of which roughly resemble the human respiratory tract. After organic extraction, the Ames assay was used to determine the mutagenic content of extracts, and the ability to induce prophage was assessed. Also DNA-repair-deficient cells were employed to see if the extracts caused DNA damage and what portion of the premutational lesions was repaired in normal cells. Results indicate that extracts of particulates from Houston air cause a significant number of mutations in bacteria and that the highest frequency of reversions is associated with the smallest particulates. An excision repair system is operative in bacteria which is able to assuage damage done to DNA by these extracts. The extracts did not cause prophage induction.

Air Pollutants↗
PubMed1983

IS2 insertion is a major cause of spontaneous mutagenesis of the bacteriophage P1: non-random distribution of target sites.

Insertion mutations arising spontaneously in the P1 prophage and affecting vegetative phage reproduction were screened for the presence of insertion sequence 2 (IS2). Filter hybridization identified 28 out of 44 independent insertions as IS2. Their target specificity is not random. A region that amounts to < 2% of the phage genome had trapped 15 of the 28 IS2 elements. However, precise mapping of nine mutants in this hot spot segment revealed no preferred insertion site. Rather, the nine IS2 are distributed over the whole target segment and IS2 are found in both orientations. Sequence data indicate that at least two sequence variants of IS2 participated in mutagenesis of the phage genome. The detectable transposition of IS2 from the host chromosome to the prophage occurs with a frequency of 3 x 10(-5) per cell per generation under the particular experimental conditions. It is concluded that IS2, a natural resident of Escherichia coli K12 strains, is an important agent for spontaneous mutagenesis and exerts this action non-randomly along the genome.

Bacteriophage P1↗
PubMed2001

A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.

Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective lambda prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era.

Animals↗
PubMed1999

Mechanism of DNA gyrase-mediated illegitimate recombination: characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype.

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we isolated temperature-sensitive gyrA mutants that confer spontaneous illegitimate recombination and spontaneous induction of lambda prophage at higher frequencies than that in the wild-type. After reconstruction of single mutations by targeted mutagenesis, we confirmed that two single mutations, gyrAL492P and gyrAL488P, and a double mutation, gyrAI203V+gyrAI205V, show the same properties as those described above. With respect to the phenotypes of hyper-recombination and higher induction of lambda prophage, these mutations were dominant over the wild-type. Analysis of recombination junctions of lambdabio transducing phages formed spontaneously in these mutants showed that the parental E. coli bio and lambda recombination sites have a homologous sequence of only 0. 7 base-pair on average, indicating that homology is not required for this illegitimate recombination. Analysis of nucleotide sequences of mutant gyrA genes revealed that the gyrAL492P and gyrAL488P mutations contain amino acid substitutions of Leu492-->Pro and Leu488-->Pro, respectively, which correspond to the alpha18 helix in the breakage-reunion domain of DNA gyrase A subunit. The gyrAI203V and gyrAI205V mutations contain Ile203-->Val and Ile205-->Val, respectively, which correspond to the alpha10' helix, also in the breakage-reunion domain of DNA gyrase A subunit. Biochemical analysis indicated that the GyrA63 protein that contains the L492P mutation has an apparently normal supercoiling activity, but it also produces a small amount of linear DNA in the absence of DNA gyrase inhibitor during the supercoiling reaction, suggesting that the mutant DNA gyrase may have a defect at the step of religation or a defect in the subunit interaction. These results suggest that the recombination is induced by defects of religation and/or dimer formation in the mutant DNA gyrases, implying that two alpha helices, alpha10' and alpha18, of DNA gyrase A subunit have crucial roles in subunit interaction and/or resealing of DNA.

Alleles↗
PubMed2000

Genomic sequence and analysis of the atypical temperate bacteriophage N15.

N15 is a temperate bacteriophage that forms stable lysogens in Escherichia coli. While its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the prophage form replicates autonomously as a linear DNA molecule with closed hairpin telomeres. Here, we describe the genomic architecture of N15, and its global pattern of gene expression, which reveal that N15 contains several plasmid-derived genes that are expressed in N15 lysogens. The tel site, at which processing occurs to form the prophage ends is close to the center of the genome in a similar location to that occupied by the attachment site, attP, in lambda and its relatives and defines the boundary between the left and right arms. The left arm contains a long cluster of structural genes that are closely related to those of the lambda-like phages, but also includes homologs of umuD', which encodes a DNA polymerase accessory protein, and the plasmid partition genes, sopA and sopB. The right arm likewise contains a mixture of apparently phage- and plasmid-derived genes including genes encoding plasmid replication functions, a phage repressor, a transcription antitermination system, as well as phage host cell lysis genes and two putative DNA methylases. The unique structure of the N15 genome suggests that the large global population of bacteriophages may exhibit a much greater diversity of genomic architectures than was previously recognized.

Bacteriolysis↗
PubMed2001

The plasmid status of satellite bacteriophage P4.

P4 is a natural phasmid (phage-plasmid) that exploits different modes of propagation in its host Escherichia coli. Extracellularly, P4 is a virion, with a tailed icosahedral head, which encapsidates the 11.6-kb-long double-stranded DNA genome. After infection of the E. coli host, P4 DNA can integrate into the bacterial chromosome and be maintained in a repressed state (lysogeny). Alternatively, P4 can replicate as a free DNA molecule; this leads to either the lytic cycle or the plasmid state, depending on the presence or absence of the genome of a helper phage P2 in the E. coli host. As a phage, P4 is thus a satellite of P2 phage, depending on the helper genes for all the morphogenetic functions, whereas for all its episomal functions (integration and immunity, multicopy plasmid replication) P4 is completely autonomous from the helper. Replication of P4 DNA depends on its alpha protein, a multifunctional polypeptide that exhibits primase and helicase activity and binds specifically the P4 origin. Replication starts from a unique point, ori1, and proceeds bidirectionally in a straight theta-type mode. P4 negatively regulates the plasmid copy number at several levels. An unusual mechanism of copy number control is based on protein-protein interaction: the P4-encoded Cnr protein interacts with the alpha gene product, inhibiting its replication potential. Furthermore, expression of the replication genes cnr and alpha is regulated in a complex way that involves modulation of promoter activity by positive and negative factors and multiple mechanisms of transcription elongation-termination control. Thus, the relatively small P4 genome encodes mostly regulatory functions, required for its propagation both as an episomal element and as a temperate satellite phage. Plasmids that, like P4, propagate horizontally via a specific transduction mechanism have also been found in the Archaea. The presence of P4-like prophages or cryptic prophages often associated with accessory bacterial functions attests to the contribution of satellite phages to bacterial evolution.

Bacteriophage T4↗
PubMed1996

Defining the SOS operon of coliphage 186.

We have sequenced the LexA-controlled operon of coliphage 186 that carries the tum gene, whose product is necessary for UV induction of the 186 prophage. The operon consists of orf95 and orf97, and we have identified orf95 as the tum gene. The major translation products from orf95 result from internal initiations and modulate Tum activity. Tum is the product of the full-length Orf95 protein. The second gene of the operon, orf97, is of unknown function but, while it has little effect on prophage induction, its presence in the cell totally blocks infection of that cell by 186.

Amino Acid Sequence↗
PubMed2000

Homologous recombination between a lactococcal bacteriophage and the chromosome of its host strain.

Genetic exchanges constitute a significant means by which bacteriophages acquire novel characteristics. Phages of Lactococcus lactis occupy a particular niche, the dairy factory environment, where their populations are subjected to constant changes. Little is known about the mechanisms of evolution that lead to the genetic diversity of lactococcal phages. In this study, we described two DNA exchanges involving the lytic phage ul36, a member of the P335 species, and its L. lactis host. They occurred by homologous recombination with phage-related sequences present in the host chromosome. Both mutants generated by these recombination events are insensitive to the phage resistance mechanism AbiK and one has a reduced burst size as well as a new origin of replication. We propose that this type of DNA exchange with prophages or remnants of prophages occurs frequently within the P335 species as supported by DNA-DNA comparisons between P335-like phages.

Bacterial Proteins↗
PubMed1982

Low level and high level DNA rearrangements in Escherichia coli.

It can be argued that all organisms exhibit two levels of DNA rearrangements. At a low level they may occur sporadically in cells, perhaps largely because of spontaneous activity of transposable genetic elements. A high level may be induced in special circumstances if functions that cause rearrangements are hyperactive. As an example of low level genetic rearrangements, we have studied the occurrence of spontaneous polar mutations in the early regions of prophage Mu. We isolated 49 independent prophage mutants, which are defective in replication ad expression of late genes; 44 were in the B region and 5 were in the A region. In the B region, 68% were IS1 insertions, 9% were IS5 insertions and 9% were IS2 insertions; 14% showed no insertion. In the A region, all 5 were IS5 insertions. Thus most spontaneous polar mutations in Escherichia coli appear to the insertions. IS1 is the most common insertion; however, certain DNA rearrangements are exemplified by DNA fusion and DNA dissociation that occur when replication-transposition functions of Mu are induced.

Bacteriophage mu↗
PubMed1975

Recovery of phage lambda from ultraviolet damage.

Recovery of phage lambda from ultraviolet damage can occur, in the dark, through three types of repair processes as defined by microbiological tests: (1) host-cell reactivation, (2) prophage reactivation, and (3) UV reactivation. This paper reviews the properties of the three repair processes, analyzes their dependence on the functioning of bacterial and phase genes, and discusses their relationship. Progress in the understanding of the molecular mechanisms underlying the three repair processes has been relatively slow, particularly for UV reactivation. It has been shown that host-cell reactivation is due to pyrimidine dimer excision and that prophage reactivation is due to genetic recombination (prereplicative). We provide evidence showing that neither of these mechanisms accounts for UV reactivation of phage lambda. Furthermore, UV reactivation differs from the other repair processes in that it is inducible and error-prone. Whether UV-damaged bacterial DNA is subject to a similar repair process is still an open question.

Cell Survival↗