Genetic Requirements of Phage λ Red-Mediated Gene Replacement in
            <i>Escherichia coli</i>
            K-12

Poteete, Anthony R.; Fenton, Anita C.

doi:10.1128/jb.182.8.2336-2340.2000

Cited by 30 publications

(30 citation statements)

References 35 publications

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“…Escherichia coli BW25113 [⌬(araD-araB)567 ⌬lacZ4787(::rrnB-3) lacIp-4000(lacI q ) Ϫ rph-1 ⌬(rhaD-rhaB)568 hsdR514] (4) and BW27784 (BW25113 ⌬araFGH ⌬araEp::P CP18 -araE) (11) were obtained from the E. coli stock center. Strain TP608 (AB1157 ⌬(recC-ptr-recB-recD)::P tac -gam-red-pae-cI822 sulA6209::tet lexA71::Tn5) was kindly provided by A. R. Poteete (20). sulA6209::tet, followed by lexA71::Tn5, was transduced into strain BW27784 by serial P1 transduction using a P1vir lysate grown with strain TP608, as previously described (18).…”

Section: Methodsmentioning

confidence: 99%

Vibrio cholerae LexA Coordinates CTX Prophage Gene Expression

Kimsey

Waldor

2009

J Bacteriol

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The filamentous bacteriophage CTX⌽ transmits the cholera toxin genes by infecting and lysogenizing its host, Vibrio cholerae. CTX⌽ genes required for virion production initiate transcription from the strong P A promoter, which is dually repressed in lysogens by the phage-encoded repressor RstR and the host-encoded SOS repressor LexA. Here we identify the neighboring divergent rstR promoter, P R , and show that RstR both positively and negatively autoregulates its own expression from this promoter. LexA is absolutely required for RstR-mediated activation of P R transcription. RstR autoactivation occurs when RstR is bound to an operator site centered 60 bp upstream of the start of transcription, and the coactivator LexA is bound to a 16-bp SOS box centered at position ؊23.5, within the P R spacer region. Our results indicate that LexA, when bound to its single site in the CTX⌽ prophage, both represses transcription from P A and coactivates transcription from the divergent P R . We propose that LexA coordinates P A and P R prophage transcription in a gene regulatory circuit. This circuit is predicted to display transient switch behavior upon induction of CTX⌽ lysogens.The horizontal transfer of CTX⌽, the lysogenic filamentous phage that carries the genes encoding cholera toxin, contributes to the emergence of new toxigenic strains of Vibrio cholerae, the cause of epidemic cholera (6, 31). For stable CTX⌽ transfer to occur, the infecting single-stranded DNA CTX⌽ genome must integrate site-specifically into the V. cholerae genome and establish a lysogenic program that involves the transcriptional repression of CTX⌽ genes involved in bacteriophage replication and morphogenesis (17). The CTX⌽ prophage expresses a 14-kDa repressor, RstR, that represses the expression of CTX⌽ structural genes and provides immunity to secondary CTX⌽ infection (12, 32). The CTX⌽ prophage can be induced with DNA-damaging agents such as mitomycin C and UV radiation, a process that is mediated by a second repressor, the host SOS repressor LexA (25). Prophage induction does not kill the host bacterium and results in only a modest increase in CTX⌽ titers, unlike the massive phage growth and host lysis that occurs upon induction of phage lambda.The CTX⌽ replication and morphogenesis genes initiate transcription from the strong P A promoter located in ig-2, an intergenic region separating the rstA replication gene from the divergently transcribed rstR gene encoding the CTX⌽ repressor (12) (Fig. 1B). RstR strongly represses P A transcription in CTX⌽ lysogens by binding to a set of three operator sites that surround P A (13). The host SOS repressor LexA also represses P A by binding to a single SOS box centered Ϫ48.5 bp from the start of P A transcription (25) (Fig. 1B). LexA, together with RecA, regulates a network of more than 30 host genes that monitors and responds to DNA damage (7). Upon DNA damage or replication fork arrest, RecA, together with singlestranded DNA and ATP, stimulates the autoproteolysis of LexA (14), leading to the transient d...

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Section: Methodsmentioning

confidence: 99%

Vibrio cholerae LexA Coordinates CTX Prophage Gene Expression

Kimsey

Waldor

2009

J Bacteriol

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“…This hyper-rec state of a bacterium expressing some of the phage recombination genes (gam, bet, and exo) partially models the conditions which prevail in a phage-infected cell. It has been found that expression in a red ϩ cell of genes from the nin region of the chromosome, including rap, does not further stimulate recombination but makes recombination partially independent of recF, recO, recR, and ruvC (12). In this study we show that the rap gene accounts for some of this activity, complementing the recombination defect of a ruvC mutant.…”

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confidence: 56%

“…Starting with an E. coli strain bearing a (recC-ptr-recB-recD)⌬:: P tac -gam-betexo-pae-cI substitution as well as deletions of recG and sulA, two isogenic sets of rap ϩ and mutant rap strains with mutations in various other recombination genes were constructed. The reason for employing a background lacking recG is that deletion of recG elevates the frequency of Red-mediated recombination; deletion of sulA was found to enhance the viability of recBCD⌬::red strains lacking recF, recO, or recR function (12). The results of crosses in these strains are summarized in Table 2.…”

Section: Strainsmentioning

confidence: 97%

Recombination-Promoting Activity of the Bacteriophage λ Rap Protein in Escherichia coli K-12

2002

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The rap gene of bacteriophage was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recG⌬ red ؉ rap ؉ strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicolresistant bacterial progeny. The results of these experiments indicated that the rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.

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“…1) as a sole sulfur source, the metA and metB genes were expressed to ϳ100-fold greater levels ( Table 2). To determine if the repression of the metA and metB genes was mediated by the MetJ protein, a metJ::ble mutation-whereby the entirety of the metJ coding sequence is replaced with a cassette expressing a protein conferring zeomycin resistance-was constructed by directed gene knockout (24). As expected, the MetA::LacZ and MetB::LacZ fusion proteins were expressed to high levels in the presence of methionine in a metJ::ble mutant background (Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…Following amplification of the appropriate antibiotic resistance gene by PCR with these tailed primers, allelic replacement was performed by electroporation of the fragment into a host cell bearing plasmid pTP223 (24), which expresses the red recombinase system. Replacement of the chromosomal gene with the antibiotic resistance gene was confirmed by (i) amplification of the target locus via PCR and determination of the nucleotide sequences of the join points between native chromosomal DNA and the introduced antibiotic resistance genes and (ii) linkage analysis of the antibiotic resistance gene to known neighboring loci via P1 cotransduction assays.…”

Section: Methodsmentioning

confidence: 99%

Two Transsulfurylation Pathways in Klebsiella pneumoniae

Seiflein

Lawrence

2006

J Bacteriol

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In most bacteria, inorganic sulfur is assimilated into cysteine, which provides sulfur for methionine biosynthesis via transsulfurylation. Here, cysteine is transferred to the terminal carbon of homoserine via its sulfhydryl group to form cystathionine, which is cleaved to yield homocysteine. In the enteric bacteria Escherichia coli and Salmonella enterica, these reactions are catalyzed by irreversible cystathionine-␥-synthase and cystathionine-␤-lyase enzymes. Alternatively, yeast and some bacteria assimilate sulfur into homocysteine, which serves as a sulfhydryl group donor in the synthesis of cysteine by reverse transsulfurylation with a cystathionine-␤-synthase and cystathionine-␥-lyase. Herein we report that the related enteric bacterium Klebsiella pneumoniae encodes genes for both transsulfurylation pathways; genetic and biochemical analyses show that they are coordinately regulated to prevent futile cycling. Klebsiella uses reverse transsulfurylation to recycle methionine to cysteine during periods of sulfate starvation. This methionine-to-cysteine (mtc) transsulfurylation pathway is activated by cysteine starvation via the CysB protein, by adenosyl-phosphosulfate starvation via the Cbl protein, and by methionine excess via the MetJ protein. While mtc mutants cannot use methionine as a sulfur source on solid medium, they will utilize methionine in liquid medium via a sulfide intermediate, suggesting that an additional nontranssulfurylation methionine-to-cysteine recycling pathway(s) operates under these conditions.Bacterial physiology is a complex network of interwoven biochemical pathways involving numerous shared intermediates, disparately regulated enzymes, and keystone metabolites with important roles as substrates, products, regulatory effectors, or a combination of all three. When genomes gain and lose genes-as they do with startling frequency (20)-the addition of new enzymes and loss of others alter pools of metabolites, overall energy demands, drains on carbon and nitrogen pools, and the balance between transport and de novo biosynthesis. While comparative genomics provides a record of changes in the gene inventory, insight into how the cell responds to such changes is achieved by comparative genetic analyses, where metabolic differences among related organisms are correlated with changes both in their genetic and genomic makeup and in the deployment of their regulatory apparatuses.Here we examine differences in sulfur amino acid metabolism among three well-studied enteric bacteria, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. E. coli and Salmonella serve as model systems for the physiology of sulfur assimilation. In these organisms, sulfate is reduced to sulfide and assimilated into acyl-activated serine to form cysteine (14); cysteine then serves as the sulfur group donor, either directly or indirectly, for the synthesis of all other sulfur-bearing molecules in the cell, including methionine. In methionine biosynthesis, cysteine is combined with acyl-activated homoserine to ...

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Genetic Requirements of Phage λ Red-Mediated Gene Replacement in Escherichia coli K-12

Cited by 30 publications

References 35 publications

Vibrio cholerae LexA Coordinates CTX Prophage Gene Expression

Vibrio cholerae LexA Coordinates CTX Prophage Gene Expression

Recombination-Promoting Activity of the Bacteriophage λ Rap Protein in Escherichia coli K-12

Two Transsulfurylation Pathways in Klebsiella pneumoniae

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