P1 lysogens of Escherichia coli carry the prophage as a stable low copy number plasmid. The frequency with which viable cells cured of prophage are produced is about 10(-5) per cell per generation. Here we show that a significant part of this remarkable stability can be attributed to a plasmid-encoded mechanism that causes death of cells that have lost P1. In other words, the lysogenic cells appear to be addicted to the presence of the prophage. The plasmid withdrawal response depends on a gene named doc (death on curing), encoding a 126 amino acid protein. Expression of doc is not SOS-inducing and killing by Doc is recA-independent. In cells that retain P1 the killing is prevented by the product of a gene named phd (prevent host death), encoding a 73 amino acid protein. The genes phd and doc have been cloned and expressed from a 0.7 kb segment of P1 DNA. The two genes constitute an operon and the synthesis of Doc appears to be translationally coupled to that of Phd. Homologs of the P1 addiction genes are found elsewhere, but phd and doc are unrelated to previously described genes of other plasmids that also cause an apparent increase in plasmid stability by post-segregational killing.
The number of copies of the genes leuB, nifH, nifD, and nifK per cell of Azotobacter vinelandii has been determined to be about 80. A beta-lactamase gene was integrated into the A. vinelandii chromosome by single-point crossover. Subsequently, we have been able to detect nearly 80 copies of this beta-lactamase gene per cell of A. vinelandii when cultured for a large number of generations in the presence of ampicillin. The multiple copies of the beta-lactamase gene do not seem to be present on a single chromosome, as evident from the fragment obtained by digestion of cellular DNA with the appropriate restriction endonuclease. The kinetics of renaturation of DNA of A. vinelandii is suggestive of complexity similar to that of Escherichia coli. The DNA content of A. vinelandii, however, is 40 times that of E. coli. All these indicate the presence of multiple chromosomes, possibly as many as 80, in A. vinelandii.
The leucine-responsive regulatory protein Lrp regulates the expression of a number of operons in Escherichia coli, including the ilvIH operon. Earlier in vitro experiments showed purified Lrp binding to two regions of DNA proximal to the ilvIH promoter, an upstream region (؊260 to ؊190) and a downstream region (؊150 to ؊40). The effect of mutations in these regions on ilvIH promoter expression in vivo led to the proposal that activation of transcription required Lrp binding to downstream sites 3, 4, 5, and 6. Binding of Lrp to upstream sites 1 and 2 seemed to enhance promoter expression but was not absolutely required (Q. Wang and J. M. Calvo, J. Mol. Biol. 229:306-318, 1993). Here we present data that require a reevaluation of the above conclusion. Constructs having either a deletion of DNA or a 100-bp substitution of DNA upstream of position ؊160 showed no ilvIH promoter activity in vivo. These results unambiguously establish that DNA at or upstream of position ؊160 is required for ilvIH promoter expression. Together with previous results, we conclude that Lrp bound at downstream sites is necessary but not sufficient for promoter activation. In addition, insertion of 4, 6, 8, or 10 bp between the upstream and downstream regions also resulted in a very strong reduction of in vivo promoter expression, even though the binding of Lrp in vitro was not greatly affected by these mutations. Closer inspection showed that the affinity of Lrp for the upstream region of all of these constructs was about the same but that Lrp bound to the downstream region of the wild-type construct with a higher degree of cooperativity than in the case of the others. These mutations may have reduced promoter activity in vivo by eliminating a binding site for some transcription factor other than Lrp. Alternatively, the small-addition mutations may have affected the geometry of these complexes, preventing either an interaction between Lrps bound at upstream and downstream sites (which might be necessary for promoter expression) or preventing the positioning of Lrp bound at upstream sites for productive interaction with the promoter.
The DNA-binding protein MetR belongs to the LysR family of transcriptional activators and is required for expression of the metE and metH promoters in Escherichia coli. However, it is not known if this activation is mediated by a direct interaction of MetR with RNA polymerase. In a search for RNA polymerase mutants defective in MetR-mediated activation of the metE gene, we isolated a mutation in the ␣ subunit of RNA polymerase that decreases metE expression independently of the MetR protein. The mutation does not affect expression from the metH promoter, suggesting that the ␣ subunit of RNA polymerase interacts differently at these two promoters. The mutation was mapped to codon 261 of the rpoA gene, resulting in a change from a glutamic acid residue to a lysine residue. Growth of the mutant is severely impaired in minimal medium even when supplemented with methionine and related amino acids, indicating a pleiotropic effect on gene expression. This rpoA mutation may identify either a site of contact with an as yet unidentified activator protein for metE expression or a site of involvement by the ␣ subunit in sequence-specific recognition of the metE promoter.
SummaryOctopine-type Ti plasmids such as pTi15955, pTiA6 and pTiR10 direct the catabolism of at least eight compounds called opines that are released from crown gall tumours. Four of these compounds are denoted mannityl opines, each of which possesses a D-mannityl substituent on the nitrogen atom of either glutamate or glutamine. We have analysed a 20 kb region of the Ti plasmid pTi15955 that is required for the catabolism of two such opines, mannopinic acid and agropinic acid. A total of 12 genes in four operons were identified by DNA sequence analysis. Transposons Tn5lacZ and MudK were used to mutagenize these genes and to create aga-lacZ and moa-lacZ translational fusions. The expression of all fusions was induced by agropinic acid and by mannopinic acid. One of these four operons encodes an agropinic acid permease, whereas a second one encodes a mannopinic acid permease. A third operon contains three genes encoding probable catabolic enzymes, two of which (AgaF and AgaG) are thought to convert agropinic acid to mannopinic acid, while the third (AgaE) probably converts mannopinic acid to mannose and glutamate. AgaE resembles a bacterial amino acid deaminase, whereas AgaF and AgaG resemble two bacterial proteins that together catabolize substituted hydantoins, whose chemical structure resembles that of agropinic acid. The remaining operon encoded the MoaR protein, a negative regulator of itself and of the other three operons.
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 ci immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 cl.100 prophage, whose replication at 32°C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 cl.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.P1 prophage replicates in Escherichia coli as a stable, low-copy-number, 90-kilobase (kb) plasmid. (See reference 47 for all but selected references to primary sources concerning P1.) A 4-kb segment of P1 DNA, the plasmid replicon, suffices to support stable plasmid maintenance at the low copy number. This segment consists of a 1.5-kb module, responsible for controlled replication from an origin known as oriR, and an adjacent 2.5-kb module, responsible for partitioning of the daughter plasmids at cell division.Replication from oriR requires the oriR-specific initiator, the product of the adjacent repA gene. A regulatory element, incA, is situated immediately downstream of r-epA and consists of a set of sites that, like oriR itself, bind initiator and interfere with oriR function. Also required for replication from oriR is the bacterial dnaA gene product, for which presumptive binding sites in oriR are present. P1 lytic replication initiates at an origin known as oriL (14,22,38). The lytic replicon appears not to require dnaA function (23). It does depend on the integrity of an open reading frame (repL) within which oriL is probably embedded (22,38). Control of the lytic replicon is exerted by the ci immunity repressor acting at an operator (designated Op53) to repress an operon that includes repL and an additional gene, not essential for replication, upstream of it (22,38). Repression by ci is antagonized by ant, normally controlled in P1 lysogens by the secondary immunity gene c4. The products of ci and c4 do not inhibit replication from the plasmid replicon (5), nor does the control element of the plasmid replicon (incA) inhibit the lytic replicon (unpublished results).We have examined the growth of P1 lysogens when either or both replicons are inhibited. Replication from oriR was inhibited in one of three ways: (i) mutational inactivation of
A mutation in the rpoA gene (which encodes the ␣ subunit of RNA polymerase) that changed the glutamic acid codon at position 261 to a lysine codon decreased the level of expression of a metE-lacZ fusion 10-fold; this decrease was independent of the MetR-mediated activation of metE-lacZ. Glutamine and alanine substitutions at this position are also metE-lacZ down mutations, suggesting that the glutamic acid residue at position 261 is essential for metE expression. In vitro transcription assays with RNA polymerase carrying the lysine residue at codon 261 indicated that the decreased level of metE-lacZ expression was not due to a failure of the mutant polymerase to respond to any other trans-acting factors, and a deletion analysis using a metE-lacZ gene fusion suggested that there is no specific cis-acting sequence upstream of the ؊35 region of the metE promoter that interacts with the ␣ subunit. Our data indicate that the glutamic acid at position 261 in the ␣ subunit of RNA polymerase influences the intrinsic ability of the enzyme to transcribe the metE core promoter.The efficiency with which transcription is initiated by RNA polymerase depends on the intrinsic strength of the promoter as well as on the influence of trans-acting regulatory factors. Positive control during transcription initiation is a common mechanism for the regulation of gene expression (23,28). Although a large number of trans-acting regulatory proteins and their binding sites on DNA have been identified (25, 28), relatively little is known about their mechanisms of action. The involvement of direct protein-protein contacts between RNA polymerase and transcription factors has been proposed to explain transcriptional activation (27, 29), though we have limited knowledge of the mechanism involved or the sites of contact, especially in RNA polymerase. Recently, several groups have reported that the sites of interaction of a number of activators with RNA polymerase are localized in its ␣ subunit, which is encoded by the rpoA gene, specifically in the carboxy-terminal region (13-16, 32, 34, 43). Studies indicate that the amino-terminal two-thirds of the ␣ subunit is sufficient for the formation of active enzyme molecules (9,11,12). More recently there have been reports that the ␣ subunit may also make contacts with DNA to activate transcription (1, 30). An AT-rich sequence located upstream of the Ϫ35 region of the Escherichia coli rRNA promoter rrnB P1 stimulates transcription in the absence of any accessory proteins, and mutations in the carboxy-terminal region of the ␣ subunit prevent this stimulation (30).The DNA-binding protein MetR belongs to the LysR family of bacterial activator proteins (10, 31) and is required for the activation of a number of methionine biosynthetic genes in E. coli and Salmonella typhimurium (4,8,21,22,39). For two of the genes, metE and metH, the MetR binding sites required for activation were defined genetically and biochemically and lie just upstream of the RNA polymerase binding site (3,22,38,42). It is possible that...
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