Analysis of transcription from the trfA promoter of broad host range plasmid RK2 in Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa (original) (raw)
Related papers
Fems Microbiology Letters, 1990
The broad-host-range plasmid, pRW2, is a derivative of pRK 2501 carrying the Escherichia coli lac operon without a promoter, downstream of a polylinker sequence. We have cloned a number of DNA fragments carrying promoters into this plasmid and measured promoter activity in both E. coli and Pseudomonas aeruginosa. Promoters carrying consensus −10 and −35 sequences were active in both backgrounds and the depencence of activity on the nucleotide sequence of the 35 region was the same in both cases. We also measured the activity of two promoters at which transcription in E. coli was totally dependent on the E. coli activators CRP and FNR: both promoters were found to be ative in P. aeruginosa.
1998
The xylR and xylS genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive 70 -dependent tandem promoters that drive expression of xylR, while expression of the xylS gene is driven from Ps2, a constitutive 70 -dependent promoter, and by the regulatable 54 class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions ؊2 to ؊30 (؊2/؊30 region) and the ؊137/؊156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a 54 -deficient Pseudomonas putida background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing 54 modulates expression from the downstream Ps2 70dependent promoter. In a P. putida IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the 54 -dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing 54 at the upstream promoter may modulate expression from the Ps2 promoter.
Genetics, 1992
A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.
Escherichia coli and Pseudomonas putida RNA polymerases display identical contacts with promoters
Molecular and General Genetics, 1984
Methylation protection experiments with four promoters (P1 and P2 of the pBR322 plasmid, lacUV5 and lambda P0) have shown that the RNA polymerases from Escherichia coli and Pseudomonas putida, while differing in the primary structure of the subunits involved in DNA binding, display identical patterns of DNA contacts. Nor do these enzymes differ in covalent cross-linking patterns with a partially apurinized promoter. We conclude that the two RNA polymerases have very similar structures of DNA binding centers. The evolutionary conservation of this structure may account for the fact that diverse RNA polymerases often recognize and efficiently use promoters of distant bacterial species.
The EMBO journal, 1984
Expression of the meta-cleavage pathway operon of TOL plasmid pWW0 of Pseudomonas putida is positively regulated by the xylS gene product. We have sequenced the promoter region of this operon and localized the transcription initiation sites. Two overlapping promoters, designated Pm1 and Pm2, are responsible for the positively regulated expression of the meta-pathway operon. Mutants of P. putida were isolated that expressed the meta-cleavage pathway operon constitutively. Several plasmid-located mutations that led to constitutivity were characterized by sequencing and the transcription initiation sites on mutant plasmids localized. This resulted in the identification of newly created promoters whose functioning did not require the xylS product. Comparison of the promoter sequences obtained suggests a tentative consensus sequence for promoters of P. putida which is significantly different from that of E. coli.
Journal of Molecular Biology, 1990
The transcription of the gEtX gene encoding the glutamyl-tRNA synthetase and of the adjacent vallJ and ala W tRNA operons of Escherichia coli K-12 has been studied. The ala W operon containing two tRNA& g enes, is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand. The valU operon, containing three tRNAp$ and one tRNALyS nun (the wild-type allele of supN) genes, is adjacent to gltX and is transcribed from the opposite strand. Its only promoter is upstream from the gltX promoters. The gltX gene transcript is monocistronic and its transcription initiates at three promoters, Pl, P2 and P3. The transcripts from one or more of these promoters are processed by RNase E to generate two major species of gEtX mRNA, which are stable and whose relative abundance varies with growth conditions. The stability of g&X mRNA decreases in an RNase E-strain and its level increases with growth rate about three times more than that of the glutamyl-tRNA synthetase. The 5' region of these mRNAs can adopt a stable secondary structure (close to the ribosome binding site) that is similar to the anticodon and part of the dihydroU stems and loops of tRNAG1", and which might be involved in translational regulation of GluRS synthesis. The gZtX and vaEU promoters share the same AT-rich and bent upstream region, whose position coincides with the position of the upstream activating sequences of tRNA and rRNA promoters to which they are similar. This suggests that gltX and valU share transcriptional regulatory mechanisms.
Syst Appl Microbiol, 2005
The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5 0-16S-23S-5S-3 0 structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three À10 and À35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.
Journal of Bacteriology, 2000
We have previously shown that both ends of the Tn3 family transposon Tn4652 contain integration host factor (IHF) binding sites and that IHF positively regulates expression of the Tn4652 transposase gene tnpA in Pseudomonas putida (R. Hõrak, and M. Kivisaar, J. Bacteriol. 180:2822-2829, 1998). Tn4652 can activate silent genes by creating fusion promoters during the transposition. The promoters are created as fusions between the ؊35 hexamer provided by the terminal inverted repeats of Tn4652 and the ؊10 hexamers in the target DNA. Two fusion promoters, PRA1 and PLA1, that contain sequences of the right and left termini of Tn4652, respectively, were chosen for the study of mechanisms of transcription activation. Gel mobility shift analysis using crude extracts from P. putida cells allowed us to detect specific binding of P. putida IHF to the ends of the transposon Tn4652. We found that the rate of transcription from the fusion promoter PRA1 is enhanced by IHF. Notably, the positive effect of IHF on transcription from the promoter PRA1 appeared only when cells of P. putida reached the stationary growth phase. We speculate that the intracellular concentration of IHF might be critical for the in vivo effect of IHF on transcription from the fusion promoters in P. putida. In the case of PLA1, the mechanism of transcription modulation by IHF is different than that observed for PRA1. Our results demonstrate that transcription of neighboring genes from outwardly directed promoters at the ends of a mobile DNA element could be influenced by the same factors that control transposition of the element.
In-vivo-generated fusion promoters in Pseudomonas putida
Gene, 1993
carrying the promoterless pheBA operon was cloned into Pseudomonas putidu PaW85, and phenolutilizing colonies were isolated on minimal plates containing phenol as the only carbon and energy source. In these clones, chromosomally located Tn4652 was transposed upstream from the coding sequence of pheA (encoding phenol monooxygenase). Sequence analysis together with mapping of the transcription start point of the pheBA operon in the recombinant plasmids revealed that fusions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated functional promoters under selective pressure in P. putida ceils. These promoter sequences show similarity to the Escherichia coli RNA polymerase 07' promoter consensus sequence. In three of the six fusion promoters studied, the generation combined two distinct events: transposition of Tn46.52 into DNA containing potential -10 sequences and point mutations in these sequences. These mutations made the -10 sequences more like the 07' promoter consensus sequences.
Plasmid, 1991
Two small plasmid RK2 derivatives, pSV6 and pSV16, were constructed and used for the isolation and characterization of trfA mutants temperature-sensitive (ts) for replication in Escherichia coli. Four of the mutants were examined for their ability to initiate replication from the RK2 replication origin in E. coli when present in cis with respect to the origin and in trans when present on a multicopy pBR322 replicon. Each of the mutant trfA genes exhibited temperaturesensitivity in supporting replication from the RK2 origin when present in cis, and the lowest nonpermissive temperature varied depending on the mutant. When the mutant trfA genes were present on the multicopy replicon (in trans), three of the four mutant genes could support replication of the RK2-oriV plasmid pSV 16 at all temperatures tested. However, with the exception of one of the mutants, the activity was reduced when compared to wild-type. The increased activity in trans possibly is the result of the increased cellular level of the TrfA protein when compared with the in cis situation where the mutant trfA gene is at a much lower copy-number. Two of the mutants also were tested in cis for temperature sensitivity in Pseudomonas aeruginosa. One of the mutants did not exhibit temperature sensitivity under the conditions employed. The second mutant showed some temuerature sensitivity but the nonpermissive temperature pattern was different than that found in E. co/i.