Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12

Lupski, James R.; Ruiz, A.; Godson, G. N.

doi:10.1007/bf00341439

Cited by 99 publications

(58 citation statements)

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“…Units are millimoles of galactose phosphate produced per minute per milliliter of culture (optical density at 600 nm of 1) per plasmid copy (25). Plasmid copy number was determined by ␤-lactamase assay (17). Each assay was performed at least three times.…”

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confidence: 99%

hlyM, a transcriptional silencer downstream of the promoter in the hly operon of Escherichia coli

et al. 1995

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Transcription of the hly operon of transmissible plasmids in Escherichia coli is subject to a tight regulation which also involves various chromosomal genes, such as hha. We have identified a 200-bp region within the hlyC gene, designated hlyM, which modulates hemolysin expression. The deletion of hlyM increased the activity of hly::galK fusion 20-fold. hlyM does not contain any internal promoter, nor is it capable of acting in trans. Our data suggest that the chromosomal Hha protein interacts with hlyM in order to silence the hly promoter. In addition, hlyR, a positive activator of hemolysin expression, seems to suppress the modulatory effect dictated by the Hha protein on the hlyM region.Synthesis and secretion of hemolysin are determined by the hly operon located either on the chromosome or on conjugative plasmids (10). Transcription of the hly operon produces a major 4-kb hlyCA transcript and a minor 8-kb hlyCABD transcript (31). The two transcripts initiate at the same promoter, located upstream of the hlyC gene (12, 31). The minor transcript is thought to be produced by readthrough, or antitermination, at a Rho-independent transcriptional terminator located between hlyA and hlyB (15). Similar patterns of expression have been reported for the leukotoxins of Pasteurella (27) and Actinobacillus (26) species.In the case of the plasmid hly operons, a 600-bp sequence (hlyR) located 1.5 kb upstream of hlyC is known to increase hemolysin expression (29). The primary mechanism by which hlyR exerts its action has been proposed to be antitermination at the hlyA-hlyB transcriptional terminator (15,16).Hemolysin expression is a rather complex process in which chromosomal genes are involved as well. Three of them, tolC (30), hha (9, 21), and more recently sfrB (1), have been identified. The hha gene was identified during analysis of hemolysin expression in cells harboring the hemolytic recombinant plasmid pANN202-312, which contains the cluster hlyCABD from

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confidence: 99%

hlyM, a transcriptional silencer downstream of the promoter in the hly operon of Escherichia coli

et al. 1995

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“…The promoter strength was expressed by the ratio of CAT activity to /?-lactamase activity (Lupski, 1984) and was also assessed by the level of chloramphenicol resistance of the host (Table 1). a 10-fold phenicol with P3.…”

Section: T T C T G T T T C T a T C A G C T G T C C C T C C T G T T C mentioning

confidence: 99%

An IS1-like element is responsible for high-level synthesis of extended-spectrum -lactamase TEM-6 in Enterobacteriaceae

Goussard

Sougakoff

Mabilat

et al. 1991

Journal of General Microbiology

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Resistance of Escherichiu coli strain HB251 to the newer p-lactam antibiotics, in particular ceftazidime and aztreonam, results from production of the extended-spectrum p-lactamase TEM-6. The corresponding structural gene, bfuT-6, and its promoter region were amplified by the polymerase chain reaction. Analysis of the sequence of the amplification product showed that bluT-6 differed by two nucleotide substitutions from bluT-1, the gene encoding TEM-1 penicillinase in plasmid pBR322. The mutations led to the substitution of a lysine for a glutamic acid at position 102 and of a histidine for an arginine at position 162 of the unprocessed TEM-1 protein. The presence of a 116 bp DNA insert upstream from bluT-6 resulted in the creation of hybrid promoter P6 in which the -10 region was that of TEM-1 promoter P3 whereas the -35 canonical sequence TTGACA was provided by the right end of the insert. P6 was found to be 10 times more active than P3 and to confer higher levels of antibiotic resistance upon the host. Analysis of the sequence of the insert indicated that the 116 bp fragment is related to insertion sequence IS1 but differs from it by three internal deletions that removed regions encoding the transposase. The distribution of the ISl-like element in clinical isolates of Enterobucferiaceae was studied by the polymerase chain reaction and by DNA-DNA hybridization. The element appeared to be widespread and was detected in strains producing TEM-6 or other TEM variants.

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“…The 3-galactosidase activities of cultures of cells harboring the various PAB-lacZ fusion plasmids were assayed by a modification of the method of Miller (18) as described previously (9). The relative copy numbers of the PAB-lacZ fusion plasmids that contained the bla gene were estimated from gene dosage by assaying their P-lactamase activities as described by Lupski et al (14), using cephaloridine (Sigma) as the colorigenic reagent. Protein concentrations were determined with Bio-Rad protein assay kits (Bio-Rad Laboratories) by following the instructions supplied with the kits.…”

Section: Methodsmentioning

confidence: 99%

Autoregulation of the stability operon of IncFII plasmid NR1

et al. 1992

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The stb locus of IncFII plasmid NR1, which mediates stable inheritance of the plasmid, is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The two tandem genes, stbA and stbB, are transcribed as an operon from promoter PAB. Using PAB-lacZ gene fusions, it was found that the stb operon is autoregulated. A low-copy-number stb+ plasmid introduced into the same cell with the PA.acZ fusion plasmid repressed (-galactosidase activity about 5-fold, whereas a high-copy-number stb+ plasmid repressed 13-galactosidase about 15-fold. IncFII plasmid NR1, like most low-copy-number plasmids that have been examined (1, 4, 23), encodes functions that ensure its stable inheritance in the population of cells (17,27). The stb (stability) locus of NR1 (17, 27), which may be equivalent to the parA locus of closely related IncFII plasmid Ri (11), is thought to participate in the partitioning of plasmid molecules to daughter cells during cell division, which is essential for stable plasmid inheritance. Mutations that inactivate the stb function result in plasmid instability, so that plasmid-free cells are segregated at a rate consistent with random distribution of plasmid copies at cell division (17,25).Within the 95-kb genome of the self-transmissible antibiotic resistance plasmid NR1 (30), stb is located approximately at coordinates 24.5 to 26.1 kb and is contained within a 1.7-kb region bounded by NaeI and TaqI restriction sites ( Fig. 1) (27). The three essential elements of the stb locus are a cis-acting DNA site and two tandem genes, stbA and stbB. These genes encode trans-acting stability proteins of 36,000 and 13,000 Da, respectively (27), and are transcribed together from promoter PAB (19). The cis-acting DNA site is located at or near PAB and can stabilize a plasmid if StbA and StbB are provided in trans (20,27). Therefore, the stb locus of NR1 is basically similar to the stabilizing loci of plasmids F (sop) and P1 prophage (par), which also are composed of two genes that encode essential trans-acting proteins and a cis-acting site (1-3, 6, 12, 15, 21, 22, 24). A primary difference among these plasmid-stabilizing loci is that for F and P1, the cis-acting partition sites lie downstream from the two genes, whereas for the stb locus of NR1, the site is upstream from the genes and may overlap promoter PAB. This fundamental difference may signal differences in the ways the loci function and in how they are regulated.Several lines of indirect evidence suggest that the stb operon is autoregulated. Mutants BglII fragment that contains stbB and the 3' half of stbA ( Fig. 1) produce an overabundance of truncated StbA protein (11,27) and have a high rate of transcription of the remaining stb sequences (19). Other mutants that have a transposon insertion in stbA, which prevents transcription of the region downstream from the insertion including stbB, produce an overabundance of mRNA that hybridizes to probes from the upstream region of the operon (19). Also, a...

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Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12

Cited by 99 publications

References 50 publications

hlyM, a transcriptional silencer downstream of the promoter in the hly operon of Escherichia coli

hlyM, a transcriptional silencer downstream of the promoter in the hly operon of Escherichia coli

An IS1-like element is responsible for high-level synthesis of extended-spectrum -lactamase TEM-6 in Enterobacteriaceae

Autoregulation of the stability operon of IncFII plasmid NR1

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