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PubMed1998

Sequence and DNA structural determinants of N4 virion RNA polymerase-promoter recognition.

Coliphage N4-coded, virion-encapsidated RNA polymerase (vRNAP) is able to bind to and transcribe promoter-containing double-stranded DNAs when the template is supercoiled and Escherichia coli single-stranded DNA-binding protein (Eco SSB) is present. We report that vRNAP-promoter recognition and activity on these templates require specific sequences and a hairpin structure on the template strand. Hairpin extrusion, induced by Mg(II) and physiological superhelical density, is essential to provide the correct DNA structure for polymerase recognition, as mutant promoters that do not form hairpins show reduced in vitro activity. Therefore, a supercoil-induced DNA structural transition regulates N4 vRNAP transcription. Eco SSB activates transcription at physiological superhelical densities by stabilizing the template-strand hairpin. Specific sequences at the promoters are conserved to provide proper contacts for vRNAP, to support hairpin extrusion, or both. We propose a model for in vivo utilization of the vRNAP promoters, and discuss the roles of DNA supercoiling and Eco SSB in promoter activation.

Base Sequence↗
PubMed2004

Effect of oxygen on survival of faecal pollution indicators in drinking water.

AIMS: The aim of this study was to determine the effect of oxygen on the survival of faecal pollution indicators including Escherichia coli in nondisinfected drinking water. METHODS AND RESULTS: Aerobic and anaerobic drinking water microcosms were inoculated with E. coli ATCC 25922 or raw sewage. Survival of E. coli was monitored by membrane filtration combined with cultivation on standard media, and by in situ hybridization with 16S rRNA-targeted fluorescent oligonucleotide probes. Anaerobic conditions significantly increased the survival of E. coli in drinking water compared with aerobic conditions. Escherichia coli ATCC 25922 showed a biphasic decrease in survival under aerobic conditions with an initial first-order decay rate of -0.11 day(-1) followed by a more rapid rate of -0.35 day(-1). In contrast, the first-order decay rate under anaerobic conditions was only -0.02 day(-1). After 35 days, <0.01% of the initial E. coli ATCC 25922 population remained detectable in aerobic microcosms compared with 48% in anaerobic microcosms. A poor survival was observed under aerobic conditions regardless of whether E. coli ATCC 25922 or sewage-derived E. coli was examined, and regardless of the detection method used (CFU or fluorescent in situ hybridization). Aerobic conditions in drinking water also appeared to decrease the survival of faecal enterococci, somatic coliphages and coliforms other than E. coli. CONCLUSIONS: The results indicate that oxygen is a major regulator of the survival of E. coli in nondisinfected drinking water. The results also suggest that faecal pollution indicators other than E. coli may persist longer in drinking water under anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of oxygen should be considered when evaluating the survival potential of enteric pathogens in oligotrophic environments.

Aerobiosis↗
PubMed2004

Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments.

AIMS: To compare the suitability of various bacterial and viral indicators to assess the removal of faecal micro-organisms by primary and secondary wastewater treatment processes. METHODS AND RESULTS: The numbers of several bacterial indicators [faecal coliforms (FC), enterococci (ENT) and sulphite-reducing clostridia (SRC)] and bacteriophages (somatic coliphages, F-specific RNA phages and bacteriophages infecting Bacteroides fragilis strain RYC2056) were determined in incoming raw sewage and effluents from various primary and secondary wastewater treatment processes in several geographical areas. Reductions in the numbers of indicators were calculated as log10 reductions. Processes based on removal and mild disinfection, showed no significant differences in the elimination of any of the indicators tested or between geographical areas. In contrast, treatment processes that include strong microbial inactivation, such as lime-aided flocculation and lagooning, showed significant differences between the log10 reductions of the various micro-organisms studied, FC showing the highest reduction and spores of SRC and phages infecting B. fragilis the lowest. CONCLUSIONS: The microbial elimination performance of treatment processes based principally on removal and mild disinfection can be evaluated with a single indicator. In contrast, processes with additional disinfecting capabilities require more than one indicator for accurate evaluation of the treatment; bacteriophages are good candidates for use as second indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages provide additional information for the evaluation of microbial elimination in some treatment plants. The easy, fast and cheap methods available for phage determination are feasible both in industrialized and developing countries.

Bacteria↗
PubMed1994

Neither absence nor excess of lambda O initiator-digesting ClpXP protease affects lambda plasmid or phage replication in Escherichia coli.

Owing to rapid proteolysis of the coliphage lambda-coded initiator protein, lambda O, this protein is considered to carry a rate-limiting step in lambda DNA replication. The discovery of ClpXP protease responsible for lambda O protein turnover allowed an opportunity to verify this hypothesis. However, neither absence nor excess of this protease significantly affected the transformation efficiency and copy number of lambda plasmid, or the kinetics of the lambda phage growth. These results are also incompatible with the hypothesis that the stabilization of lambda O plays a role in the switch from early (circle-to-circle) to late (rolling-circle) lambda phage DNA replication. Transcriptional activation of ori lambda, probably assisted by the Escherichia coli DnaA function, remains as the possible rate-limiting step in lambda DNA replication.

ATP-Dependent Proteases↗
PubMed1994

Identification of an HP1 phage protein required for site-specific excision.

Transposon insertion mutagenesis and transformation were used to locate genes responsible for excision in the temperature phage HP1 of Haemophilus influenzae. A 6.5 kb segment of DNA near the left end of the phage genome was sequenced, and 11 new open reading frames were identified. Two face-to-face overlapping promoter sequences organized these open reading frames into two operons transcribed in opposite directions. Interruption of the first open reading frame in the rightward operon created lysogens unable to produce phages. Provision of the uninterrupted open reading frame in trans restored phage production. The gene identified by this procedure, cox, was cloned and the protein product was expressed at high levels in Escherichia coli. The Cox protein is a 79-residue basic protein with a predicted strong helix-turn-helix DNA-binding motif. Extracts induced to express high levels of Cox contained a 9 kDa protein. These extracts inhibited integrative recombination and were required for excisive recombination mediated by HP1 integrase. The HP1 cox gene location is similar to that of the homologous excisive and regulatory genes from coliphages P2 and 186. These phages appear to share a distinctive organization of recombination proteins and transcriptional domains differing markedly from phage lambda and its relatives.

Amino Acid Sequence↗
PubMed1995

Transcription antitermination: the lambda paradigm updated.

Coliphage lambda employs systems of transcription termination and antitermination to regulate gene expression. Early gene expression is regulated by the phage-encoded N protein working with a series of Escherichia coli proteins, Nus, at RNA sites, NUT, to modify RNA polymerase to a termination-resistant form. Expression of lambda late genes is regulated by the phage-encoded Q antitermination protein. Q, which appears to use only one host factor, acts at a DNA site, qut, to modify RNA polymerase to a termination-resistant form. This review focuses on recent studies which show that: (i) N can mediate antitermination in vitro, independent of Nus proteins. (ii) Early genes in another lambdoid phage HK022 are also regulated by antitermination, where only an RNA signal appears necessary and sufficient to create a termination-resistant RNA polymerase. (iii) A part of the qut signal appears to be read from the non-template DNA strand. (iv) A host-encoded inhibitor of N antitermination appears to act through the NUT site as well as with the alpha subunit of RNA polymerase, and is antagonized by NusB protein.

Bacteriophage lambda↗
PubMed1995

A role for bacteriophages in the evolution and transfer of bacterial virulence determinants.

A virulence-associated region in the genome of Dichelobacter nodosus has been shown to contain an integrase gene which is highly related to the integrases of Shigella flexneri phage Sf6 and coliphages P4 and phi R73, together with open reading frames (vapB, C and D) related to genes borne on plasmids in Neisseria gonorrhoeae, Escherichia coli, Actinobacillus actinomycetemcomitans and Treponema denticola. Similar to P4 and phi R73, the vap region is bracketed by putative bacteriophage att sites and is adjacent to a tRNA gene, which suggests that the vap region has been derived by the integration of a bacteriophage, or a plasmid carrying a bacteriophage-related integrase gene. Many similarities in genes and genes clusters encoding virulence determinants have been found in distantly related bacteria. These genes are often located on plasmids in one organism but on the chromosome in others, implying that transmission of the genes has been followed by integration. Thus, the events which have generated the vap regions of D. nodosus may represent a common mechanism for transfer of virulence determinants. A number of genes involved in the virulence of bacterial pathogens are found on integrated bacteriophages, and we suggest that others will prove to be associated with tRNA genes and/or integrase genes derived from bacteriophages. The use of tRNA genes as integration sites for many bacteriophages and plasmids may favour intergeneric transmission, as tRNA genes are highly conserved.

Attachment Sites, Microbiological↗
PubMed2004

Bacteriophage T4 endonuclease II: concerted single-strand nicks yield double-strand cleavage.

In vivo, endonuclease II (EndoII) of coliphage T4 cleaves sites with conserved sequence elements (CSEs) to both the left and the right of the cleaved bonds, 16 bp altogether with some variability tolerated. In vitro, however, single-strand nicks in the lower strand predominate at sites containing only the left-side CSE that determines the precise position of lower strand nicks. Upper strand nick positions vary both in vivo and in vitro. A 24 bp substrate was nicked with the same precision as in longer substrates, showing that the conserved sequence suffices for precise nicking by EndoII. Using DNA ligase in vitro, we found that EndoII nicked both strands simultaneously at an in vivo-favoured site but not at an in vitro-favoured site. This indicates that the right-side CSE at in vivo-favoured sites is important for simultaneous nicking of both strands, generating double-strand cleavage. Separate analysis of the two strands following in vitro digestion at two in vitro-favoured sites showed that EndoII nicked the lower strand about 1.5-fold faster than the upper strand. In addition, the upper and lower strands were nicked independently of each other, seldom resulting in double-strand cleavage. Thus, cleavage by EndoII is the fortuitous outcome of two separate nicking events.

Bacteriophage T4↗
PubMed2005

CTXphi and Vibrio cholerae: exploring a newly recognized type of phage-host cell relationship.

The genes encoding cholera toxin, one of the principal virulence factors of the diarrhoeal pathogen Vibrio cholerae, are part of the genome of CTXphi, a filamentous bacteriophage. Thus, CTXphi has played a critical role in the evolution of the pathogenicity of V. cholerae. Unlike the well-studied F pilus-specific filamentous coliphages, CTXphi integrates site-specifically into its host chromosome and forms stable lysogens. Here we focus on the CTXphi life cycle and, in particular, on recent studies of the mechanism of CTXphi integration and the factors that govern lysogeny. These and other processes illustrate the remarkable dependence of CTXphi on host-encoded factors.

Inovirus↗
PubMed1979

The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1.

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromosuccinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to a molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Q beta, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1. Q beta and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.

Amino Acid Sequence↗
PubMed1982

Ribitol dehydrogenase messenger RNA from an enzyme superproducer strain of Klebsiella aerogenes. Purification, cell-free translation and studies in vitro and in vivo.

1. Ribitol dehydrogenase messenger RNA, from a strain of Klebsiella aerogenes that had been evolved to superproduce this enzyme, has been purified in a single step by labelling extracted polysomes with rabbit anti(ribitol dehydrogenase) and immunoprecipitating with sheep anti-(rabbit IgG). 2. The extracted mRNA is stable in a protein synthesis system in vitro and directs synthesis 35-40-times more efficiently than RNA from coliphages MS2 or Q beta, to give ribitol dehydrogenase as sole major product. 3. Its size distribution shows a major band of 1500 nucleotides plus fragments 400-1400 nucleotides, with only traces of size 2400-3000 nucleotides. Only the latter could encode both proteins of the operon: ribitol dehydrogenase and D-ribulokinase. 4. Ribitol dehydrogenase mRNA represents 24% of total mRNA in cells harvested just after a 'switch' point' in mid-exponential phase. About half of the polysomes containing this mRNA are unattached to DNA, whereas only 3% of other mRNAs are unattached to DNA. 5. This mRNA is not outstandingly stable in vivo, though there are indications that it may be more stable than average. Hence the high level of synthesis of ribitol dehydrogenase (up to 30% of total protein in an extract) seems to be due to very efficient transcription and translation from multiple copies of a constitutive rbtD gene.

DNA-Directed RNA Polymerases↗
PubMed1984

A new method for detection of small modifications in genomic DNA, applied to the human delta-beta globin gene cluster.

Cloned DNA fragments were subcloned in filamentous coliphages fd 103 or M 13; the recombinant single-stranded DNAs were then used to form hybrids with genomic DNA as well as with complementary recombinant single-stranded DNA. Hybrids were submitted to S1-nuclease treatment alone or in combination with restriction enzyme digestions. This method was used to analyze the delta-beta globin gene cluster from the total genomic DNA of a beta 0-thalassemic patient. A modification located approximately 530 base pairs upstream from the cap site of the beta-globin gene was detected in only one thalassemic chromosome of this patient. Sequence analysis have shown that the patient was homozygous for a single nucleoside change (dC----dT) which remains undetected by our hybridization method, leading to a codon 39 nonsense mutation; they have demonstrated too that he was heterozygous for the modification mentioned and detected by S1-nuclease, which corresponds to an additional sequence d(T-A-T-A) in a 52 alternating purine-pyrimidine run, leading to a complex change from d[(A-T)7(T)7] to d[(A-T)11(T)3].

Base Sequence↗
PubMed2006

Transcription of a 'photosynthetic' T4-type phage during infection of a marine cyanobacterium.

The transcription of S-PM2 phage following infection of Synechococcus sp. WH7803, a marine cyanobacterium, was analysed by quantitative real-time PCR. Unlike the distantly related coliphage T4, there were only two (early and late) instead of three (early, middle and late) classes of transcripts during the developmental cycle of the phage. This difference is consistent with the absence from the S-PM2 genome of T4-like middle mode promoter sequences and the transcription factors associated with their recognition. Phage S-PM2 carries the 'photosynthetic' genes psbA and psbD that encode homologues of the host photosystem II proteins D1 and D2. Transcripts of the phage psbA gene appeared soon after infection and remained at high levels until lysis. Throughout the course of infection, the photosynthetic capacity of the cells remained constant. A considerable transient increase in the abundance of the host psbA transcripts occurred shortly after infection, suggesting that the host responds to the trauma of phage infection in a similar way as it does to a variety of other environmental stresses. The very substantial transcription of the phage psbA gene during the latter phase of phage infection suggests that S-PM2 has acquired this cellular gene to ensure that D1 levels and thus photosynthesis are fully maintained until the infected cell finally lyses. Unexpectedly, transcripts of a phage-encoded S-layer protein gene were among the earliest and most abundant detected, suggesting that this partial homologue of a host protein plays an important role in the S-PM2 infection process.

Bacteriophages↗
PubMed2000

Peptide mimics elicit antibody responses against the outer-membrane lipooligosaccharide of group B neisseria meningitidis.

As an alternative approach towards the development of a meningococcal vaccine, the potential of peptide mimics of lipooligosaccharide (LOS) to elicit cross-reactive immune responses against LOS was investigated. The heptapeptides SMYGSYN and APARQLP were identified by enrichment from a coliphage display library with a LOS-specific monoclonal antibody. Mice immunised with these peptides conjugated to diphtheria toxoid elicited a total IgG response to LOS with geometric mean titres 2-4 times higher compared with non-immunised controls. There was an increase in LOS-specific IgG1 immunoglobulin, whereas specific IgG2a and IgG3 decreased slightly in response to immunisation. The data demonstrated that peptide mimics can elicit immune responses against meningococcal LOS.

Animals↗
PubMed2002

Amino acid alterations in Gp38 of host range mutants of PP01 and evidence for their infection of an ompC null mutant of Escherichia coli O157:H7.

The previously isolated T-even type coliphage PP01, specifically infective to Escherichia coli O157:H7, uses the outer membrane protein OmpC as a receptor. The characterization of a spontaneous PP01-resistant strain indicated that it had lost ompC due to the deletion of a 14-kbp region upstream of and partially including ompC. Two host range mutants, able to infect an ompC null mutant, were isolated. Sequencing of gene 38, which codes for the receptor recognition protein Gp38, indicated three mutations in one mutant and two in the other. Both mutant proteins had a Gly208Arg, a Gly161Arg or Gly101His replacement, respectively, and the one mutant phage in addition a Trp189Arg replacement. These alterations suggest that the host range was mediated by a more positively charged Gp38.

Bacteriophages↗
PubMed1997

Microorganisms as tracers in groundwater injection and recovery experiments: a review.

Modern day injection and recovery techniques designed to examine the transport behavior of microorganisms in groundwater have evolved from experiments conducted in the late 1800s, in which bacteria that form red or yellow pigments were used to trace flow paths through karst and fractured-rock aquifers. A number of subsequent groundwater hydrology studies employed bacteriophage that can be injected into aquifers at very high concentrations (e.g., 10(13) phage ml-1) and monitored through many log units of dilution to follow groundwater flow paths for great distances, particularly in karst terrain. Starting in the 1930s, microbial indicators of fecal contamination (particularly coliform bacteria and their coliphages) were employed as tracers to determine potential migration of pathogens in groundwater. Several injection and recovery experiments performed in the 1990s employed indigenous groundwater microorganisms (both cultured and uncultured) that are better able to survive under in situ conditions. Better methods for labeling native bacteria (e.g. by stable isotope labeling or inserting genetic markers, such as the ability to cause ice nucleation) are being developed that will not compromise the organisms' viability during the experimental time course.

Enterobacteriaceae↗
PubMed2006

Imaging RNA polymerase-amelogenin gene complexes with single molecule resolution using atomic force microscopy.

The AMELX gene encoding the enamel matrix protein, amelogenin, is located within (and in the opposite orientation to) the first intron of the ARHGAP6 gene, which encodes a GTPase-activating protein. The orientation of these two genes with respect to each other raises the possibility that they may undergo simultaneous convergent transcription during amelogenesis. The aim of this study was to use atomic force microscopy (AFM) to study a transcriptionally active amelogenin DNA template and to investigate the binding of RNA polymerase to convergently aligned promoters. Images of RNA polymerases stalled on DNA templates were obtained following incubation of the template with RNA polymerases and ribonucleotide triphosphates. A linear DNA template incorporating an intact rat amelogenin cDNA flanked by convergently aligned coliphage T7 and T3 promoters was constructed and shown to be transcriptionally active in vitro. Atomic force microscopy images of transcription complexes revealed globular structures, corresponding to single RNA polymerase molecules bound at specific locations on the DNA templates. These results indicate that AFM allows the visualization of individual RNA polymerases on DNA templates, offering a realistic approach to investigating the concept of convergent transcription of nested genes, which may lead to an understanding of whether the simultaneous expression of AMELX and ARHGAP6 is possible during the formation of tooth enamel.

Amelogenesis↗
PubMed1991

Functional importance of sequence in the stem-loop of a transcription terminator.

Intrinsic transcription terminators of prokaryotes are distinguished by a common RNA motif: a stem-loop structure high in guanine and cytosine content, followed by multiple uridine residues. Models explaining intrinsic terminators postulate that the stem-loop sequence is necessary only to form structure. In the tR2 terminator of coliphage lambda, single-nucleotide changes reducing potential RNA stem stability eliminated tR2 activity, and a compensatory change that restored the stem structure restored terminator activity. However, multiple changes in the stem sequence that should have either maintained or increased stability reduced terminator activity. These results suggest that the ability of the stem-loop structure to signal transcription termination depends on sequence specificity and secondary structure.

Bacteriophage lambda↗