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Regional replication of the bacterial chromosome induced by derepression of prophage lambda. IV. Escape synthesis of gal operon in phage 82.

Derepression of prophage lambda in E. coli strain K12 results in constitutive synthesis of the enzymes directed by the nearby bacterial operon, gal (escape synthesis). Phage 82 fails to cause escape synthesis despite that it lysogenizes the strain K12 at the site identical to that of lambda on the host chromosome. The reason for the observed difference between 82 and lambda is studied in the light of the recent finding that escape synthesis in lambda-lysogen is closely associated to phage-promoted replication of bacterial chromosome contiguous to the prophage including gal operon (escape replication). Excision-defective mutants from 82, 82int or 82xis, do initiate escape synthesis, suggest that the prophage 82 is normally excised too quickly after induction to allow sufficient escape replication. In support of this, much more DNA hybridizable to bacterial DNA contained in lambdagal accumulates after induction if 82int than after induction of 82. Studies with various hybrid phages between 82 and lambda have suggested: 1. The occurrence of gal escape synthesis depends on the nature of the region between b2 and N in the lambda map. 2. Regions of the 82 genome on both sides of the attachment site contribute independently to prevent gal escape synthesis. Implications of these results are discussed with regard to the factors involved in the prophage excision.

Chromosomes, Bacterial↗

The requirement of ArcA and Fnr for peak expression of the cyd operon in Escherichia coli under microaerobic conditions.

Transcriptional regulation of cyd, encoding the cytochrome d complex for O2 scavenging, was studied in Escherichia coli by monitoring phi(cyd-lac) expression under atmospheres containing 0-21% O2. Peak expression of this operon occurred under microaerobic conditions. Mutations in arcA (a trans-acting regulatory gene involved in aerobic respiration) greatly lowered phi(cyd-lac) expression under all conditions. Mutations in fnr (a trans-acting regulatory gene involved in anaerobic respiration) shifted the pattern of phi(cyd-lac) expression, lowering the microaerobic and aerobic expression. In an arcA-fnr double mutant, phi(cyd-lac) expression became insignificant, irrespective of the availability of O2. It thus appears that the expression of the phi(cyd-lac) operon is under dual control by the two pleiotropic activators, ArcA and Fnr, which interact to give the peak microaerobic expression.

Aerobiosis↗

TdcA, a transcriptional activator of the tdcABC operon of Escherichia coli, is a member of the LysR family of proteins.

The tdcB and tdcC genes of the tdcABC operon of Escherichia coli encode threonine dehydratase and a threonine-serine permease, respectively. These proteins are involved in transport and metabolism of threonine and serine during anaerobic growth. In this study, we functionally characterized tdcA, which encodes a 35 kDa polypeptide consisting of 312 amino acid residues. Non-polar and partially polar mutations introduced into tdcA drastically reduced the expression of the genes down-stream from tdcA. Complementation studies using single-copy chromosomal integrants of a tdcB-lacZ fusion harboring an in-frame deletion of tdcA with chromosomal or plasmid-borne tdcA+ in trans showed complete restoration of tdc operon expression in vivo. The amino acid sequence at the amino-terminal end of TdcA revealed a significant homology to the helix-turn-helix motifs of typical DNA binding proteins. Sequence alignment of TdcA with LysR also showed considerable sequence similarity throughout their entire lengths. Our results suggest that TdcA is related to the LysR family of proteins by common ancestry and, based on its functional role in tdc expression, belongs to the LysR family of transcriptional activators.

Amino Acid Sequence↗

Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli.

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind-) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations oc in the umuDC and umuD'C operons and also used the oc98-recA mutation. The oc1-umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutagenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.

Bacterial Proteins↗

Organization and nucleotide sequence of ten ribosomal protein genes from the region equivalent to the spectinomycin operon in the archaebacterium Halobacterium marismortui.

The nucleotide sequence has been determined of a 4700 bp region from a ribosomal protein gene cluster of Halobacterium marismortui (Haloarcula marismortui), which is equivalent to part of the spectinomycin operon of Escherichia coli. The genes were localized on the recombinant lambda EMBL3 clone PP*7, which also contains several other ribosomal protein genes from the DNA region in H. marismortui equivalent to the linked S10/spc operon. The genes analysed encode ten ribosomal proteins, namely HmaL5, HmaS14, HmaS8, HmaL6, HL5, HL24, HmaL18, HmaS5, HmaL30 and HmaL15. The gene organization of the archaebacterial cluster is similar to that in eubacteria but has two additional genes, namely those encoding HL5 and HL24, which were identified as extra proteins that are apparently not present in E. coli. These correspond to the gene products of orfd and orfe in Methanococcus vannielii and also have eukaryotic counterparts.

Amino Acid Sequence↗

Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon.

The structural genes for NRZ, the second nitrate reductase of Escherichia coli, have been sequenced. They are organized in a transcription unit, narZYWV, encoding four subunits, NarZ, NarY, NarW and NarV. The transcription unit is homologous (73% identity) to the narGHJI operon which encodes the genes for NRA, the better characterized nitrate reductase of this organism. The level of homology between the corresponding polypeptides ranges from 69% for the NarW/NarJ pair to 86% for the NarV/NarI pair. The NarZ polypeptide contains the five conserved regions present in all other known molybdoproteins of E. coli and their relative order is the same. The NarY polypeptide, which contains the same four cysteine clusters in the same order as NarH, is probably an electron transfer unit of the complex. Upstream of narZ, an open reading frame, ORFA, is present which could encode a product which has homology (73% identity) with the COOH-terminal end of NarK. The ORFA-narZ intergenic region, however, is about 80 nucleotides long and does not contain the cis-acting elements, NarL and Fnr boxes, nor the terC4 terminator sequence present in the 500 nucleotide narK-narG intergenic region. This might explain why the narZYWV and the narGHJI operons are regulated differently. Our results tend to support the hypothesis that a DNA fragment larger than that encompassing the narGHJI genes has been duplicated.

Amino Acid Sequence↗

Characterization of three genes in the dam-containing operon of Escherichia coli.

The dam-containing operon in Escherichia coli is located at 74 min on the chromosomal map and contains the genes aroK, aroB, a gene called urf74.3, dam and trpS. We have determined the nucleotide sequence between the dam and trpS genes and show that it encodes two proteins with molecular weights of 24 and 27 kDa. Furthermore, we characterize the three genes urf74.3, 24kDa, 27kDa and the proteins they encode. The predicted amino acid sequences of the 24 and 27 kDa proteins are similar to those of the CbbE and CbbZ proteins, respectively, of the Alcaligenes eutrophus cbb operon, which encodes enzymes involved in the Calvin cycle. In separate experiments, we have shown that the 24 kDa protein has d-ribulose-5-phosphate epimerase activity (similar to CbbE), and we call the gene rpe. Similarly, the 27 kDa protein has 2-phosphoglycolate phosphatase activity (similar to CbbZ), and we name the gene gph. The Urf74.3 protein, with a predicted molecular weight of 46 kDa, migrated as a 70 kDa product under denaturing conditions. Overexpression of Urf74.3 induced cell filamentation, indicating that Urf74.3 directly or indirectly interferes with cell division. We present evidence for translational coupling between aroB and urf74.3 and also between rpe and gph. Proteins encoded in the dam superoperon appear to be largely unrelated: Dam, and perhaps Urf74.3, are involved in cell cycle regulation, AroK, AroB, and TrpS function in aromatic amino acid biosynthesis, whereas Rpe and Gph are involved in carbohydrate metabolism.

Amino Acid Sequence↗

Regulation of the rpsU-dnaG-rpoD macromolecular synthesis operon and the initiation of DNA replication in Escherichia coli K-12.

We have fused the E. coli dnaG 5' regulatory region to the TcR structural gene in the promoter probe plasmids pPV33 and pKK175-6 to demonstrate that a promoter activity is located on a 250 bp SacII-HindIII restriction fragment and that a transcription terminator, previously reported by nucleotide sequence (Smiley et al. 1982) to immediately precede the dnaG gene, acts as such in vivo. We have determined the complete nucleotide sequence of this controlling region and report: 1) tandem promoters on the same SacII-HindIII restriction fragment which promotes tet expression in the gene fusion experiments, 2) an open reading frame between these promoters and the dnaG gene which is the rpsU (ribosomal protein S21) gene, 3) a sequence homologous to the lambda nut site, 4) a possible LexA protein binding site on one of the dnaG promoters. This places the order on the E. coli genetic map at 66 min in the clockwise direction as rpsU-dnaG-rpoD which are all contained in a single macromolecular synthesis operon. We postulate a model for regulation of the initiation of DNA replication based on antitermination of the rpsU-dnaG-rpoD operon.

Bacteriophage lambda↗

Anomalous expression of the E. coli lac operon in Proteus mirabilis. I. Effects of L8 and L8 UV5.

The lac operon shows anomalous expression in Proteus mirabilis: the maximal induced level is 10% or less of that in E. coli, while repression reduces this by a factor of only 2-5. We have sought to determine whether this effect relates in any way to CRP-mediated activation of expression, by comparing expression in P. mirabilis of lac operons (introduced for technical reasons on IncP1 plasmids) either regulatorily wild-type or bearing L8 or L8UV5. Derivatives of RP1 bearing L8UV5 were obtained by homogenotisation of pGC9114 (RP1::Tn951) in a L8UV5 background; while derivatives of RP4 bearing lac+, L8 or L8UV5 were obtained by Mu-mediated translocation of chromosomal regions bearing these alleles, following partial heat-induction of Mucts62 on pGM14 (RP4::Mucts62) in the appropriate hosts. These plasmids could be readily transferred to, and stably maintained in, the P. mirabilis strains employed. It was found that L8 reduced the maximal level of beta-galactosidase activity, and L8UV5 restored this activity to around wild-type, in P. mirabilis quantitatively very much as in E. coli. Nevertheless, the low maximal level of expression and high basal level characteristic of the former host were unchanged. The simplest explanation of these results is that P. mirabilis contains a protein that mimics the E. coli CRP protein in interacting with the appropriate DNA binding site and thereby stimulating transcription; and that the anomalous regulation of lac in this host is unconnected with the CRP system.

Escherichia coli↗

The regulatory region of the uxuAB operon in Escherichia coli K12.

The uxuAB operon is composed of two genes coding for enzymes involved in hexuronate degradation. This operon is negatively controlled by the uxuR and exuR regulatory gene products. Starting from uxu hybrid plasmids, a 300 bp Sau3A restriction fragment was isolated and shown to contain the entire uxu regulatory region using in vitro gene fusions that brought lac gene expression under the control of the transcriptional and translational signals of the uxuA gene. The nucleotide sequence of this fragment was established. The start of the uxu mRNA was localized by S1 mapping experiments (the presence of a CAP binding site upstream of the promoter was shown by DNAse I footprinting and in vitro titration). The N-terminal amino acid sequence of the purified uxuA-lacZ gene product allowed the mapping of the uxuA initiation codon at 115 bp from the start of transcription.

Base Sequence↗

Involvement of the nusB gene products in transcription of Escherichia coli tryptophan operon in vitro.

Synthesis of trp mRNA in vitro directed by plasmid DNA carrying the entire trp operon was studied using crude protein extracts (S-100) from Escherichia coli strains carrying the nusA or nusB mutation or both. It was found that the levels of trp mRNA transcribed from the promoter-distal genes (trpCBA) relative to that from the promoter-proximal genes (trpED) was markedly lower with extracts from the nus- mutants than that from the nus+ strain. Kinetic experiments suggest that termination of RNA transcripts at intragenic transcriptional barriers is prevented by the nus gene products from allowing efficient expression of the operon.

Bacterial Proteins↗

Mutation probe of gene structure in E. coli: suppressor mutations in the seven-tRNA operon.

Cells defective in uracil-DNA glycosylase (ung::Tn10) were used in two ways to reveal differences in select point mutations (GC to AT transitions) within the seven-tRNA operon of E. coli. The mutations were indicated as de novo or converted glutamine tRNA suppressor mutations in the genes glnU and/or glnV: the kinetics of photoenzymatic monomerization of pyrimidine dimers quantitated by ung-dependent UV mutagenesis indicated more rapid repair of dimers at sites for converted suppressor mutation than of dimers at sites for de novo suppressor mutation, and spontaneous deamination of cytosine was considerably more frequent at sites for converted suppressor mutation than at sites for de novo suppressor mutation. To explain these results we suggest the physical structure of the DNA in vivo is different at different sites in the seven-tRNA operon. The non-transcribed strand including specifically the anticodon region of the site for converted suppressor mutation may frequently be looped out in a single strand so that a T = C dimer is more accessible to DNA photolyase or a free cytosine residue of non-irradiated DNA is in an aqueous environment conducive to deamination. In addition, we analysed the spontaneous de novo suppressor mutation data to determine an estimate for the in vivo rate of cytosine deamination in double strand DNA of 3.2 X 10(-13)/sec.

DNA Glycosylases↗

Autoregulation of the ccd operon in the F plasmid.

Mini-F sequences, including the promoter and portions of the ccd region, were inserted upstream of lacZ in promoterless lacZ vectors, and beta-galactosidase specific activities were measured. The results showed that the H (ccdA), G (ccdB) and D genes, together with a promoter, comprise an operon. Ccd operon expression was shown to be regulated at the level of transcription by the G gene product, probably in concert with the H gene product. Thus expression is autoregulated. Expression of the D gene was largely dependent on the ccd promoter, although low levels of transcription from another promoter within the ccd coding region were detected.

Escherichia coli↗

Attenuation in the threonine operon: effects of amino acids present in the presumed leader peptide in addition to threonine and isoleucine.

We have studied in vivo the contribution of amino acids corresponding to codons of the leader sequence other than the so-called "regulatory codons" (for threonine and isoleucine) in the expression of the threonine operon. In the presence of threonine and isoleucine, addition of each amino acid encoded in the proximal part of the leader sequence resulted in a significant decrease of the expression of the operon, over and above that decrease observed in the sole presence of threonine and isoleucine. These effects were cumulative. No such effect was found with the amino acids encoded by the distal part of the leader sequence. These findings are discussed in the light of the current model of attenuation.

Amino Acid Sequence↗

Studies on the involvement of the UGA readthrough process in the mechanism of attenuation of the tryptophan operon of Escherichia coli.

We asked if UGA suppression by charged tRNATrp, a process called UGA readthrough, is involved in the mechanism of attenuation of the tryptophan (trp) operon in Escherichia coli. For this purpose we used two mutations: strA(LD1) which causes restriction of UGA readthrough, and revA which partially overcomes the restriction of UGA readthrough caused by strA(LD1)(Engelberg-Kulka et al. 1982). trp attenuation was monitored by the regulation of the synthesis of the trp operon enzyme anthranilate synthetase (ASase) in trpR strains. We showed that the strA(LD1) mutation causes a significant increase in the level of synthesis of ASase in the presence of an excess of tryptophan, while the revA mutation reverses this effect, indicating that transcription termination at the trp attenuator site is relieved by restriction of UGA readthrough. Based on our results and the sequence data of the trp leader RNA of E. coli (Oxender et al. 1979), we offer a model for the involvement of the UGA readthrough process in trp attenuation. We suggest that the UGA readthrough process permits trp attenuation to respond to slight changes in the cellular concentration of charged tRNATrp.

Anthranilate Synthase↗

Shadow promoters in the F plasmid transfer operon.

The in vivo transcription start site for PYZ, the promoter of the F plasmid transfer operon, has been located. When transcription start sites for PYZ are deleted, two additional shadow promoters, upstream of PYZ, are activated in vivo. The shadow promoters correspond to those previously identified by in vitro run-off transcription experiments. The activated shadow promoters are stimulated by TraJ protein, a positive regulator of the transfer operon.

Base Sequence↗

A DNA sequence containing the control regions of the malEFG and malK-lamB operons in Escherichia coli K12.

The malB region in E. coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes. We have established the DNA sequence of a 600 base pair segment which contains the sites necessary for initiation of transcription of the malB operons and the translation start sites for malE and malK. We have also determined the position of a 147 base pair deletion, malB delta 100, in this sequence. The control region contains two short segments (about 35 base pairs) showing unusual distributions of bases: the GC content (85%) is higher than average and most of the purines (85%) are on one strand. We discuss the possible relevance of this and other salient features of the DNA sequence to the mechanisms by which the expression of the malB region is positively regulated.

Base Composition↗

In vitro synthesis of enzymes of the tryptophan operon of Escherichia coli. Evidence for positive control of transcription.

A protein fraction, called At (= anti termination) factor, has been isolated from extracts of E. coli and partially purified. The At factor stimulates the synthesis in vitro of anthranilate synthetase, an enzyme encoded by two genes of the tryptophan (trp) operon, but has no effect on the synthesis of T7 RNA polymerase and other T7- and T4 coded proteins. The At factor stimulates the synthesis of trp mRNA; it has no effect on the translation of trp mRNA. We conclude that in vitro transcription of the trp operon is positively controlled.

Anthranilate Synthase↗