M13 and pUC vectors with new unique restriction sites for cloning

Beneš, Vladimír; Hostomský, Zdeněk; Arnold, Luboš; Pačes, Václav

doi:10.1016/0378-1119(93)90360-f

Cited by 44 publications

(19 citation statements)

References 6 publications

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“…The crcZ promoter of P. aeruginosa contains conserved Ϫ24/Ϫ12 boxes (TGGCACGN 4 CTGCT) that are typical of 54 promoters (consensus, TGGCACGN 4 TTGC T / A , where the most highly conserved nucleotides are in boldface and N indicates any nucleotide [3]) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa

et al. 2011

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In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein.Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and 54 RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.Pseudomonas aeruginosa, like other fluorescent pseudomonads, is a metabolically versatile bacterium; it utilizes more than 100 different organic substrates for growth (37). This versatility requires a complex regulatory network that ensures the cellular carbon-nitrogen balance and determines the order in which growth substrates are degraded. In general, substrates that yield high energy and promote fast growth are degraded preferentially. For instance, intermediates of the tricarboxylic acid (TCA) cycle such as succinate prevent glucose degradation in P. aeruginosa (21). As a result, growth on a mixture of succinate and glucose is biphasic (diauxic) because the bacterium utilizes first succinate and then glucose (39). The underlying mechanism of carbon catabolite repression involves the CbrA/ CbrB two-component system (16,30), the small RNA (sRNA) CrcZ (36), and the RNA-binding protein Crc (6,22,25,26,33) as key regulatory elements. They are conserved in fluorescent pseudomonads (38; Pseudomonas Genome Database).

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Section: Resultsmentioning

confidence: 99%

Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa

et al. 2011

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“…Digestion of both plasmids with EcoRI produced 11 bands for pSOG1 and 10 bands for pSOG2, ranging from 0?3 to 7?2 kbp. All of these fragments were cloned in the EcoRI site of pUC28 (Benes et al, 1993). Fragments obtained by digestion with BamHI, HindIII, PstI and XbaI in the size range 0?8-4?5 kb were also cloned in the corresponding sites of pUC28 to obtain an overlapping clone library for pSOG1 and pSOG2.…”

Section: Methodsmentioning

confidence: 99%

Two novel conjugative plasmids from a single strain of Sulfolobus

et al. 2006

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Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4. The plasmids were separated from each other and transferred into Sulfolobus solfataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number and is stably maintained (pSOG2). Plasmid pSOG2 is the first Sulfolobus CP found to have these characteristics. The genomes of both pSOG plasmids have been sequenced and were compared to each other and the available Sulfolobus CPs. Interestingly, apart from a very well-conserved core, 70 % of the pSOG1 and pSOG2 genomes is largely different and composed of a mixture of genes that often resemble counterparts in previously described Sulfolobus CPs. However, about 20 % of the predicted genes do not have known homologues, not even in other CPs. Unlike pSOG1, pSOG2 does not contain a gene for the highly conserved PlrA protein nor for obvious homologues of partitioning proteins. Unlike pNOB8 and pKEF9, both pSOG plasmids lack the so-called clustered regularly interspaced short palindrome repeats (CRISPRs). The sites of recombination between the two genomes can be explained by the presence of recombination motifs previously identified in other Sulfolobus CPs. Like other Sulfolobus CPs, the pSOG plasmids possess a gene encoding an integrase of the tyrosine recombinase family. This integrase probably mediates plasmid site-specific integration into the host chromosome at the highly conserved tRNA Glu loci.

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“…Additionally, all positive-reacting cosmids were hybridized in the same way with the insert of pMec23 to screen for the tfdB gene. Suitable fragments were then subcloned in pUC vectors (5), yielding the plasmids listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Genetic Analysis of Phenoxyalkanoic Acid Degradation in Sphingomonas herbicidovorans MH

Müller

Byrde

Werlen

et al. 2004

Appl Environ Microbiol

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Phenoxyalkanoic acid degradation is well studied in Beta-and Gammaproteobacteria, but the genetic background has not been elucidated so far in Alphaproteobacteria. We report the isolation of several genes involved in dichlor-and mecoprop degradation from the alphaproteobacterium Sphingomonas herbicidovorans MH and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-D). Two genes for ␣-ketoglutarate-dependent dioxygenases, sdpA MH and rdpA MH , were found, both of which were adjacent to sequences with potential insertion elements. Furthermore, a gene for a dichlorophenol hydroxylase (tfdB), a putative regulatory gene (cadR), two genes for dichlorocatechol 1,2-dioxygenases (dccA I/II ), two for dienelactone hydrolases (dccD I/II ), part of a gene for maleylacetate reductase (dccE), and one gene for a potential phenoxyalkanoic acid permease were isolated. In contrast to other 2,4-D degraders, the sdp, rdp, and dcc genes were scattered over the genome and their expression was not tightly regulated. No coherent pattern was derived on the possible origin of the sdp, rdp, and dcc pathway genes. rdpA MH was 99% identical to rdpA MC1 , an (R)-dichlorprop/␣-ketoglutarate dioxygenase from Delftia acidovorans MC1, which is evidence for a recent gene exchange between Alpha-and Betaproteobacteria. Conversely, DccA I and DccA II did not group within the known chlorocatechol 1,2-dioxygenases, but formed a separate branch in clustering analysis. This suggests a different reservoir and reduced transfer for the genes of the modified ortho-cleavage pathway in Alphaproteobacteria compared with the ones in Beta-and Gammaproteobacteria.

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M13 and pUC vectors with new unique restriction sites for cloning

Cited by 44 publications

References 6 publications

Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa

Promoter Recognition and Activation by the Global Response Regulator CbrB in Pseudomonas aeruginosa

Two novel conjugative plasmids from a single strain of Sulfolobus

Genetic Analysis of Phenoxyalkanoic Acid Degradation in Sphingomonas herbicidovorans MH

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