Construction of improved plasmid vectors for promoter characterization in Pseudomonas aeruginosa and other Gram-negative bacteria

Rist, M

doi:10.1016/s0378-1097(98)00481-9

Cited by 16 publications

(24 citation statements)

References 6 publications

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“…This is consistent with previous observations that the lac promoter is expressed weakly in pseudomonads (Matthysse et al, 1996;Rist and Kertesz, 1998).…”

Section: Discussionsupporting

confidence: 93%

Adherence and Infectivity of Green Fluorescent Protein-Labeled Pseudomonas plecoglossicida to Ayu Plecoglossus altivelis.

Sukenda

Wakabayashi

2001

Fish Pathol.

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ABSTRACT--We report green fluorescent protein (GFP) as a useful reporter molecule to track the occurrence of Pseudomonas plecoglossicida in ayu Plecoglossue altivelis. The gfp gene was placed in a shuttle vector pME4510 and then three kinds of GFP vectors, pSKL01, pSKT03 and pSKN04 were constructed, in which lac, tac, and neophosphotransferase II (npt2) promoters drove the expression of GFP, respectively. A promoterless GFP vector was designated as pSKP02. P. plecoglossicida carrying pSKT03 gave the highest expression of GFP. In addition, pSKT03 was relatively stable under non-selective pressure and there was no significant changes in the in vitro growth and pathogenicity of the cells carrying this plasmid.The cells of fluorescent P. plecoglossicida attaching to the body surface of ayu were easily detected under a fluorescence microscope.It was revealed that they adhered predominantly to the microscopic injuries in the skin and fins.

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“…This is consistent with previous observations that the lac promoter is expressed weakly in pseudomonads (Matthysse et al, 1996;Rist and Kertesz, 1998).…”

Section: Discussionsupporting

confidence: 93%

Adherence and Infectivity of Green Fluorescent Protein-Labeled Pseudomonas plecoglossicida to Ayu Plecoglossus altivelis.

Sukenda

Wakabayashi

2001

Fish Pathol.

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“…2) was cloned into pBBR1MCS-5 (Kovach et al, 1995), to produce pBBRsfnR. Since the lac promoter is expressed constitutively in Pseudomonas species (Rist & Kertesz, 1998), pBBRsfnR should express sfnR in Dfi74J. pBBRsfnR was introduced into Dfi74J by electrotransformation.…”

Section: Complementation Of Dfi74j With Sfnrmentioning

confidence: 99%

A CysB-regulated and σ54-dependent regulator, SfnR, is essential for dimethyl sulfone metabolism of Pseudomonas putida strain DS1

et al. 2003

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Pseudomonas putida strain DS1 utilizes dimethyl sulfide (DMS) as a sulfur source, and desulfurizes it via dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO 2 ) and methanesulfonate (MSA). Its Tn5 mutant, Dfi74J, no longer utilized DMS, DMSO and DMSO 2 , but could oxidize DMS to DMSO 2 , suggesting that the conversion of DMSO 2 to MSA was interrupted in the mutant. Sequencing of the Tn5 flanking region of Dfi74J demonstrated that a gene, sfnR (designated for dimethyl sulfone utilization), encoding a transcriptional regulator containing an ATP-dependent s 54 -association domain and a DNA-binding domain, was disrupted. sfnR is part of an operon with two other genes, sfnE and sfnC, located immediately upstream of sfnR and in the same orientation. The genes encode NADH-dependent FMN reductase (SfnE) and FMNH 2 -dependent monooxygenase (SfnC). Complementation of Dfi74J with an sfnR-expressing plasmid led to restoration of its growth on DMS, DMSO and DMSO 2 . An rpoN-defective mutant of strain DS1, which lacks the s 54 factor, grew on MSA, but not on DMS, DMSO and DMSO 2 , indicating that SfnR controls expression of gene(s) involved in DMSO 2 metabolism by interaction with s 54 -RNA polymerase. Northern hybridization and a reporter gene assay with an sfn-lacZ transcriptional fusion elucidated that expression of the sfnECR operon was induced under sulfate limitation and was dependent on a LysR-type transcriptional regulator, CysB. This is believed to be the first report that a s 54 -dependent transcriptional regulator induced under sulfate limitation is involved in sulfur assimilation.

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“…In the case of pCAR1, it is possible that the Par proteins supplied from pBBRparW, -parA, or -parB were not present in the correct amounts and ratios, since each of the par genes in the above plasmids was located downstream of the lac promoter, which is known to be expressed constitutively in Pseudomonas spp. (46). Like the oriV region of pCAR1, the 3.3-kb par operon of pCAR1 was conserved in other (putative) IncP-7 plasmids, i.e., pND6-1, pWW53, and pL6.5.…”

Section: Resultsmentioning

confidence: 94%

Characterization of the Replication, Maintenance, and Transfer Features of the IncP-7 Plasmid pCAR1, Which Carries Genes Involved in Carbazole and Dioxin Degradation

Shintani

Yano

Habe

et al. 2006

Appl Environ Microbiol

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Isolated from Pseudomonas resinovorans CA10, pCAR1 is a 199-kb plasmid that carries genes involved in the degradation of carbazole and dioxin. The nucleotide sequence of pCAR1 has been determined previously. In this study, we characterized pCAR1 in terms of its replication, maintenance, and conjugation. By constructing miniplasmids of pCAR1 and testing their establishment in Pseudomonas putida DS1, we show that pCAR1 replication is due to the repA gene and its upstream DNA region. The repA gene and putative oriV region could be separated in P. putida DS1, and the oriV region was determined to be located within the 345-bp region between the repA and parW genes. Incompatibility testing using the minireplicon of pCAR1 and IncP plasmids indicated that pCAR1 belongs to the IncP-7 group. Monitoring of the maintenance properties of serial miniplasmids in nonselective medium, and mutation and complementation analyses of the parWABC genes, showed that the stability of pCAR1 is attributable to the products of the parWAB genes. In mating assays, the transfer of pCAR1 from CA10 was detected in a CA10 derivative that was cured of pCAR1 (CA10dm4) and in P. putida KT2440 at frequencies of 3 ؋ 10 ؊1 and 3 ؋ 10 ؊3 per donor strain, respectively. This is the first report of the characterization of this completely sequenced IncP-7 plasmid.In the past 30 years, many plasmids carrying genes for enzymes that degrade organic xenobiotics have been isolated (reviewed in references 9, 41, 42, and 69). The most detailed analyses have been conducted on the toluene/xylene-degradative plasmid pWW0 (20,37,70,71) and the 2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4 (11,12,65). These two plasmids belong to incompatibility (Inc) groups P-9 and P-1␤, respectively, which contain many other degradative plasmids, such as NAH7 (IncP-9) (13), pADP-1 (IncP-1␤) (10, 36), pDTG1 (IncP-9) (9, 54), pUO1 (IncP-1␤) (30, 59), and SAL1 (IncP-9) (7). These are mobile genetic elements that spread various degradative genes in the environment (26,60).Plasmid pCAR1 is involved in the degradation of carbazole (CAR), which is a xenobiotic azarene compound composed of a dibenzopyrrole ring. pCAR1 was isolated from the CARdegrading bacterium Pseudomonas resinovorans CA10 (40, 43). The CAR-catabolic enzymes encoded by pCAR1 can also degrade dibenzo-p-dioxin and dibenzofurans, which are the parental compounds of dioxins (21,39,50). Previously, we reported the following characteristics of pCAR1: (i) this 199.035-kb plasmid carries the 72.8-kb class II transposon Tn4676, which contains the CAR degradation (car) operon (35, 58); (ii) the backbone of pCAR1 (the region that roughly corresponds to the outside of Tn4676) shows mosaicity, i.e., it is composed of putative genes for replication that are homologous to those of other Pseudomonas sp. plasmids, as well as genes for transfer, which are similar to those of plasmids of Enterobacteriaceae (35); (iii) the putative repA gene of pCAR1 does not show similarity to the rep genes of IncP-1, IncP-9, or other replicons w...

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Construction of improved plasmid vectors for promoter characterization in Pseudomonas aeruginosa and other Gram-negative bacteria

Cited by 16 publications

References 6 publications

Adherence and Infectivity of Green Fluorescent Protein-Labeled Pseudomonas plecoglossicida to Ayu Plecoglossus altivelis.

Adherence and Infectivity of Green Fluorescent Protein-Labeled Pseudomonas plecoglossicida to Ayu Plecoglossus altivelis.

A CysB-regulated and σ54-dependent regulator, SfnR, is essential for dimethyl sulfone metabolism of Pseudomonas putida strain DS1

Characterization of the Replication, Maintenance, and Transfer Features of the IncP-7 Plasmid pCAR1, Which Carries Genes Involved in Carbazole and Dioxin Degradation

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