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PubMed1974

Int-constitutive mutants of bacteriophage lambda.

The constitutive production of small amounts of trpB enzyme in an Escherichia coli strain carrying lambdacI857 prophage within the trpC gene has been examined in derivatives of this strain from which portions of the prophage have been deleted. Enzyme production requires a site (p(I)) within the prophage close to the left prophage end. Selection for mutants of this lysogen that grow on low concentrations of indole yielded two types of mutations within the prophage: (a) v2-type, in which all phage genes controlled by the major leftward operator are derepressed; and (b) int-c type, in which the only phage gene derepressed is int. The int-c mutations lie in the same part of the prophage as p(I). All int-c mutants appear deficient in xis gene function, even when derepressed.

Chromosome Mapping↗

PubMed1994

Central location of the Mu strong gyrase binding site is obligatory for optimal rates of replicative transposition.

The bacteriophage Mu genome contains a strong DNA gyrase binding site (SGS) near its center, and disruption of the SGS by deletion or by insertion results in long delays in replication following induction of the appropriate lysogen. To determine if the central location of the SGS is obligatory for its function in Mu replication, we pursued two lines of investigation. First, fragments of Mu DNA containing the SGS were inserted into various locations in a Mu prophage lacking the central SGS. Replication following induction was restored in all of the lysogens constructed, but the observed rate of replication for different prophages decreased with increasing distance between the new location of the SGS and the center of the genome. We also deleted different lengths of DNA from within the right half of a wild-type prophage, retaining the SGS and displacing it from a central location. Replication rates of the deleted prophages were reduced, with larger deletions resulting in larger reductions. Pairing deletions in the right half of the prophage with a deletion in the left half resulted in substantially higher rates of replication than observed with the right half deletions alone. We conclude that the SGS must be located centrally between the Mu termini for optimal rates of Mu replication. These results are discussed in terms of a model that proposes that the SGS is involved in organizing the topology of supercoiled prophage DNA to assist in synapsis of the Mu termini.

Bacteriophage mu↗

PubMed1977

Isolation and characterization of nondefective transducing lambda bacteriophages carrying fla genes of Escherichia coli K-12.

In Escherichia coli K-12, 11 fla genes and a hag gene are located between his and uvrC, making two clusters at map positions 42.5 and 43.0 min. Nondefective transducing lambda phages for these genes were isolated. Low-frequency-transducing donors were constructed starting from lysogens of lambda cI857 in which the prophage is integrated at a secondary attachment site at 44 min on the E. coli map. Two strategies were used to delete the region between the prophage and the fla genes. Deletion mutants of the supD locus between fla and the prophage were isolated by selecting for loss of Su1+, an allele of supD. A strain with a deletion starting within the prophage and ending at a position close to the fla genes was isolated from heat-resistant derivatives of the lysogen. A lysogen of lambda b2 was then constructed in which the prophage had integrated at the site of the defective prophage by means of recombination with residual lambda deoxyribonucleic acid. From low-frequency-transducing lysate of the donor strains thus constructed, either directly or in combination with a procedure that extends the loci transduced, various lambda pfla's were isolated. lambda pflaL1 carries all nine fla genes at 43 min, and lambda pflaH14 carries hag and two fla genes at 42.5 min.

Bacteriophage lambda↗

PubMed2025

CRISPRi-mediated repression of three cI repressors induces the expression of three related Neisseria gonorrhoeae bacteriophages.

The Neisseria gonorrhoeae FA1090 isolate encodes nine prophage islands (Ngoɸ1-9). Ngoɸ1-3 contain genes consistent with a Siphoviridae-dsDNA bacteriophage (phage). Saturating transposon-sequencing screens using two different N. gonorrhoeae isolates predicted that multiple prophage genes were essential, including three putative transcriptional repressors: ngo0479 (present in Ngoɸ1), ngo1116 (present in Ngoɸ2), and ngo1630 (present in Ngoɸ3). All three genes display homology to the Lambda phage cI, a regulator important for maintaining the lysogenic state and inhibiting lytic induction, but these proteins are not close paralogs. Using a Neisseria lactamica-derived Type I-C CRISPR-interference system, we show that these cI orthologs are essential, as the knockdown of each gene results in bacterial death. We determined that the repression of the three cI orthologs resulted in the significant induction of phage gene expression. Finally, we detected Siphoviridae-like phage particles released from N. gonorrhoeae following repression of ngo0479, ngo1116, or ngo1630. We hypothesize that these cI orthologs are critical for preventing phage lytic infection and cell death and allow N. gonorrhoeae to benefit from the carriage and expression of prophage genes.IMPORTANCEBacteriophage, or phage, are bacteria-infecting viruses and are the most abundant natural entities in the world. Here, we report that Neisseria gonorrhoeae's three most complete double-stranded DNA prophage islands each encode essential and related transcriptional repressors. CRISPRi-mediated repression of these transcriptional repressors leads to a significant increase in prophage gene expression and phage induction. This study marks an important initial step in studying the interaction between N. gonorrhoeae and its resident phage.

Neisseria gonorrhoeae↗

PubMed2005

Discrimination within phenotypically closely related definitive types of Salmonella enterica serovar typhimurium by the multiple amplification of phage locus typing technique.

Multilocus sequence typing (MLST) is a relatively new high-resolution typing system employed for epidemiological studies of bacteria, including Salmonella. Discrimination based on MLST of housekeeping genes may be problematical, due to the high identity of gene sequences of closely related Salmonella species. The presence of genomic sequences derived from stable temperate phages in Salmonella offers an alternative for MLST of Salmonella. We have used MLST of prophage loci in Salmonella enterica serovar Typhimurium to discriminate closely related isolates of serovar Typhimurium. We have compared these results to MLST of five housekeeping genes, as well as pulsed-field gel electrophoresis (PFGE). The presence or absence of prophage loci in the 73 serovar Typhimurium isolates tested, as well as allelic variation as detected by sequencing, provided greater discrimination between isolates than either MLST of housekeeping genes or PFGE. Amplification of prophage loci alone separated serovar Typhimurium isolates into 27 groups comprising multiple isolates or individual strains. Sequencing of isolates found within the clusters separated isolates even further. By contrast, PFGE could only divide the 73 isolates into five distinct groups. MLST using housekeeping genes did not provide any significant separation of isolates in comparison to amplification or MLST of prophage loci. The results demonstrate that the amplification and sequencing of prophage loci provides a high-resolution, objective method for the discrimination of closely related isolates of serovar Typhimurium. It is proposed that multiple amplification of phage locus typing may provide sufficient discrimination for epidemiological purposes without recourse to MLST.

Bacterial Proteins↗

PubMed1978

The activity of ant product of the Salmonella phage P22 against the closely related but heteroimmune phage L.

P22 mutants defective in the early gene 24 are complemented by phage L in mixed infection. P22 12- and P22 23- mutants are not complemented by phage L. Gene function 24 of an L prophage is turned on by a superinfecting P22 24- mutant and complements the missing function of the defective P22 phage. Since this transactivation of prophage gene 24 depends on a functional gene ant in the superinfecting P22 mutant, it indicates derepression for leftward directed gene expression in prophage L. On the contrary neither the rightward directed expression of gene 12 nor of gene 23 in prophage L. can be turned on by superinfecting P22 24- 12- or P22 24- 23- mutants (and also not by P22 12- and P22 23-) to a degree sufficient for complementation of simultaneously superinfecting L virB 12- or L virB 23- mutants. The failure to detect release of repression for rightward directed gene expression of prophage L corresponds to the earlier observation (Prell, 1975) that P22 superinfecting L lysogens cannot release replication inhibition for simultaneously infecting phage L. The results are discussed with respect to the mechanism underlying the different action of P22 antirepressor in L and in P22 lysogens.

DNA Replication↗

PubMed1975

Regulation of gene expression in Salmonella phage P22. II. Regulation of expression of late functions.

Transactivation experiments were performed involving the genetically related Salmonella phages P22, L and Px1 in order to find out if more than one positively acting regulatory product is engaged in the expression of vegetative gene functions of each of these phages. The results obtained with Px1- and L-lysogenic cells superinfected with P22 suggest the following conclusions: 1. The expression of the early genes 12 and 23 and of the late gene 19 (lysozyme synthesis) is positively regulated by two different regulatory products, since P22 transactivates in prophage Px1 both early and late genes (Prell, 1973), in prophage L only late genes. 2. The transactivation by P22 of the lysozyme gene of prophage L takes place in the presence of L repressor. This conclusion is suggested, since the superinfecting P22 does not derepress early gene expression (see 1.), and is confirmed by demonstration of replication inhibition for L phage in L lysogenic cells doubly superinfected with L and P22 phages (Thomas-Bertani-experiment). 3. The late gene regulatory protein seems to be synthesized by gene 23, as transactivation experiments with both L- and Px1 prophages suggest. 4. The expression of gene 23 itself is turned on by an early regulatory product. The gene which codes for it is still unidentified. However its product seems to by highly specific, since it is active on Px1- but not on L-prophage.

Bacteriophage P22↗

PubMed1986

Reconstitution of an operon from overlapping fragments: use of the lambda SV2 integrative cloning system.

We have used the lambda SV2 system [Howard and Gottesman. In Gluzman (Ed.), Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 211-216; in Inouye, M. (Ed.) Experimental Manipulations of Gene Expression. Academic Press, New York, 1983, pp. 137-153] to reconstitute the Salmonella typhimurium his operon from overlapping fragments. lambda SV2 can be propagated as an autonomously replicating plasmid or as a prophage integrated in the Escherichia coli chromosome at the lambda attachment site; our reconstitution was accomplished in the integrated state. We first inserted a portion of the his operon into lambda SV2 and integrated the resulting plasmid by site-specific recombination into the E. coli chromosome. This was achieved by brief induction of a resident prophage. The lysogen was then transformed with DNA from a lambda SV2 clone carrying the remainder of the his operon on an overlapping DNA fragment. The second plasmid was forced to integrate into the first by homologous recombination. When this recombination occurs at the his overlap, a lysogen carrying two lambda SV2 prophages is produced. One prophage carries the entire his operon and the other carries the his overlap region. The latter is removed by site-specific recombination, permitting further contiguous sequences to be sequentially added to the remaining prophage. This method should be applicable for the reconstitution and maintenance of large genes or gene clusters in the E. coli genome.

Alleles↗

PubMed2005

Phage regulatory circuits and virulence gene expression.

In many pathogenic bacteria, genes that encode virulence factors are located in the genomes of prophages. Clearly bacteriophages are important vectors for disseminating virulence genes, but, in addition, do phage regulatory circuits contribute to expression of these genes? Phages of the lambda family that have genes encoding Shiga toxin are found in certain pathogenic Escherichia coli (known as Shiga toxin producing E. coli) and the filamentous phage CTXphi, that carries genes encoding cholera toxin (CTX), is found in Vibrio cholerae. Both the lambda and CTXphi phages have repressor systems that maintain their respective prophages in a quiescent state, and in both types of prophages this repressed state is abolished when the host cell SOS response is activated. In the lambda type of prophages, only binding of the phage-encoded repressor is involved in repression and this repressor ultimately controls Shiga toxin production and/or release. In the CTXphi prophage, binding of LexA, the bacterial regulator of SOS, in addition to binding of the repressor is involved in repression; the repressor has only limited control over CTX production and has no influence on its release.

Bacteriophages↗

PubMed2001

Evidence for a rolling-circle mechanism of phage DNA synthesis from both replicative and integrated forms of CTXphi.

The genes encoding cholera toxin, the principal virulence factor of Vibrio cholerae, are part of the circular single-stranded DNA genome of CTXphi. In toxigenic V. cholerae strains, the CTXphi genome is typically found in integrated arrays of tandemly arranged CTX prophages. Infected cells that lack a chromosomal integration site harbour the CTXphi genome as a plasmid (pCTX). We studied the replication of pCTX and found several indications that this plasmid replicates via a rolling-circle (RC) mechanism. The initiation and termination sites for pCTX plus-strand DNA synthesis were mapped to a 22 bp sequence that contains inverted repeats and a nonanucleotide motif found in the plus-strand origins of several RC replicons. Furthermore, similar to other RC replicons, replication of plasmids containing duplicated pCTX origins resulted in the deletion of sequences between the two origins and the formation of a single chimeric origin. Our previous work revealed that CTX prophage arrays give rise to hybrid CTX virions that contain sequences derived from two adjacent prophages. We now report that the boundaries between the sequences contributed to virions by the upstream and the downstream prophages in an array correspond to the site at which synthesis of plus-strand pCTX DNA is initiated and terminated. These data support the model that plus-strand CTXphi DNA is generated from chromosomal prophages via a novel process analogous to RC replication.

Bacteriophages↗

PubMed2000

An efficient recombination system for chromosome engineering in Escherichia coli.

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

Bacteriophage lambda↗

PubMed2006

Complexity of the genomic diversity in enterohemorrhagic Escherichia coli O157 revealed by the combinational use of the O157 Sakai OligoDNA microarray and the Whole Genome PCR scanning.

Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic uremic syndrome, is one of the leading worldwide public health threats. Genome sequencing of two O157 strains have revealed that the chromosome is comprised of a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences. Most of the large O157-specific sequences are prophages and prophage-like elements, which have carried many virulence genes into the O157 genome. This suggests that bacteriophages have played the key roles in the emergence of O157. The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the other hand, revealed a high level of genomic diversity in O157. Variation of prophages has also been suggested as a major factor generating such diversity. In this study, we analyzed the gene content of O157 strains, by an oligoDNA microarray, using the same set of strains as examined by the WGPScanning method. Although most of the strains were typical O157 : H7, they differed remarkably in gene composition, particularly in those on prophages, and we identified more than 400 'variably absent or present' genes which included virulence-related genes. This confirms the role of prophages in generating the genomic diversity, and raises a possibility that some level of variation in potential virulence is present among O157 strains. Fine comparison of the two datasets obtained by microarray and WGPScanning provided much further details on the O157 genome diversity than illustrated by each method alone, indicating the usefulness of this combinational approach in the genomic comparison of closely related strains.

Combinatorial Chemistry Techniques↗

PubMed1997

A spontaneous Escherichia coli K12 mutant which inhibits the excision-reintegration process of Mu gem2ts.

Escherichia coli K12 strains lysogenic for Mu gem2ts with the prophage inserted in a target gene (i.e., lacZ::Mu gem2ts lysogenic strains) revert to Lac+ by prophage precise excision with a relatively high frequency (about 1 x 10(-6)). The revertants obtained are still lysogens with the prophage inserted elsewhere in the bacterial chromosome. We have observed that, with the time of storage in stabs, bacterial cultures lysogenic for Mu gem2ts lose the ability to excise the prophage. The mutation responsible for this effect was co-transducible with the gyrB gene. After the removal of the prophage by P1 vir transduction from these strains, one randomly chosen clone, R3538, was further analyzed. It shows an increment of DNA supercoiling of plasmid pAT153, used as a reporter, and a reduced beta-galactosidase activity. On the other hand, R3538 is totally permissive to both lytic and lysogenic cycles of bacteriophage Mu.

Bacterial Proteins↗

PubMed1973

Deletion mapping and orientation of the histidine operon of Escherichia coli on a transducing bacteriophage.

The defective prophage phi80ilambdac(I)857dhis has been mapped through both marker rescue and deletion analysis. Deletions have been isolated which put residual his genes close to trp genes. Analysis of these deletions shows that the histidine operon on the prophage is oriented clockwise as on the bacterial chromosome, thus opposite to the orientation of the trp operon. The presence of the his promoter-operator region is inferred by the ability of the prophage-carrying strain to derepress sequentially under conditions in which the histidine concentration is limiting. In addition to his, the gnd gene is also present on the prophage and is located between his and trp operons. The bacterial genes are inserted in the right arm of the prophage and substitute for all of the late function genes, except for the first three. These data indicate that the "sense" strand for transcription of the his operon in vivo must be the "R" strand.

Chromosome Mapping↗

PubMed1989

Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage.

Alleles↗

PubMed1999

The Vibrio cholerae O139 Calcutta bacteriophage CTXphi is infectious and encodes a novel repressor.

CTXphi is a lysogenic, filamentous bacteriophage. Its genome includes the genes encoding cholera toxin (ctxAB), one of the principal virulence factors of Vibrio cholerae; consequently, nonpathogenic strains of V. cholerae can be converted into toxigenic strains by CTXphi infection. O139 Calcutta strains of V. cholerae, which were linked to cholera outbreaks in Calcutta, India, in 1996, are novel pathogenic strains that carry two distinct CTX prophages integrated in tandem: CTX(ET), the prophage previously characterized within El Tor strains, and a new CTX Calcutta prophage (CTX(calc)). We found that the CTX(calc) prophage gives rise to infectious virions; thus, CTX(ET)phi is no longer the only known vector for transmission of ctxAB. The most functionally significant differences between the nucleotide sequences of CTX(calc)phi and CTX(ET)phi are located within the phages' repressor genes (rstR(calc) and rstR(ET), respectively) and their RstR operators. RstR(calc) is a novel, allele-specific repressor that regulates replication of CTX(calc)phi by inhibiting the activity of the rstA(calc) promoter. RstR(calc) has no inhibitory effect upon the classical and El Tor rstA promoters, which are instead regulated by their cognate RstRs. Consequently, production of RstR(calc) renders a CTX(calc) lysogen immune to superinfection by CTX(calc)phi but susceptible (heteroimmune) to infection by CTX(ET)phi. Analysis of the prophage arrays generated by sequentially integrated CTX phages revealed that pathogenic V. cholerae O139 Calcutta probably arose via infection of an O139 CTX(ET)phi lysogen by CTX(calc)phi.

Bacterial Proteins↗

PubMed1978

Genetic characterization of Mu-like bacteriophage D108.

Infection of Escherichia coli by bacteriophage D108 was shown to result in the generation of apparently random chromosomal mutations. Approximately 1% of the cells lysogenized by D108, as with Mu, acquired new auxotrophic mutations. D108-induced mutations were nonreverting and were most probably the result of insertion of the D108 genome into regions of genetic function. D108 and Mu shared many similar properties but were heteroimmune and had different host ranges. Lytic infections of Mu lysogens with D108 and D108 lysogens with Mu resulted in 100-fold increases in release of phage with prophage markers over those due to spontaneous induction. Phenotypic mixing was common, with most phage carrying the prophage immunity being packaged in particles with the host range of the superinfecting phage. A fraction of the superinfecting phage genomes were, however, packaged in particles with the prophage-specified host range. Although 10% of the prophage progeny were D108-Mu genetic hybrids, superinfecting phage-induced release of the prophage with reciprocal phenotypic mixing occurred in recA hosts, in which the frequency of D108-Mu genetic hybrids was reduced 100-fold.

Coliphages↗

PubMed1979

Heat-sensitive lambda repressors retain partial activity during bacteriophage induction.

At 43 degrees C, lambda cIts prophages are "induced" and enter the lytic cycle. Lac- lysogens containing heat-inducible lambda N- prophages were superinfected with a lambda trp/lac N+cI- phage containing a lacZ+ gene whose expression is controlled by the lambda cI product (repressor). Lysogens were then heated, and the synthesis of beta-galactosidase and release of progeny phage were measured. In lambda N- cIts2 or lambda N-cIts16 lysogens superinfected with lambda trp/lac N+, beta-galactosidase appeared earlier and was synthesized more rapidly than in superinfected lysogens containing lambda N- cIts857 prophage. Even at 45 degrees C, the cI857 repressor retained some activity. Lysogens containing other N-cIts mutant prophages producing renaturable repressors were also only partially derepressed at 43 degrees C. Partial derepression of lambda "early" transcription is sufficient for induction of lambda N+ prophages.

Bacteriophage lambda↗