Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria

Keen, N. T.; Tamaki, Stanley; Kobayashi, Donald Y.; Trollinger, David

doi:10.1016/0378-1119(88)90117-5

Cited by 1,438 publications

(618 citation statements)

References 16 publications

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“…Plasmid pT7/ T3-18U or 19U (Bethesda Research Laboratories) was used for cloning as well as expression of single-stranded DNA in E. coli. Plasmid pRK415, a broad-hostrange plasmid, was used for transferring genes from E. coli to R. sphaeroides (17). Plasmids pSUP202 and pJP5603 were used as suicide vectors (25,30).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3

et al. 1997

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A gene cluster which includes genes required for the expression of nitric oxide reductase in Rhodobacter sphaeroides 2.4.3 has been isolated and characterized. Sequence analysis indicates that the two proximal genes in the cluster are the Nor structural genes. These two genes and four distal genes apparently constitute an operon. Mutational analysis indicates that the two structural genes, norC and norB, and the genes immediately downstream, norQ and norD, are required for expression of an active Nor complex. The remaining two genes, nnrT and nnrU, are required for expression of both Nir and Nor. The products of norCBQD have significant identity with products from other denitrifiers, whereas the predicted nnrT and nnrU gene products have no similarity with products corresponding to other sequences in the database. Mutational analysis and functional complementation studies indicate that the nnrT and nnrU genes can be expressed from an internal promoter. Deletion analysis of the regulatory region upstream of norC indicated that a sequence motif which has identity to a motif in the gene encoding nitrite reductase in strain 2.4.3 is critical for nor operon expression. Regulatory studies demonstrated that the first four genes, norCBQD, are expressed only when the oxygen concentration is low and nitrate is present but that the two distal genes, nnrTU, are expressed constitutively.Denitrification is the reduction of nitrate (NO 3 Ϫ ) to gaseous intermediates, principally nitrogen gas. Nitric oxide (NO), an obligatory intermediate during denitrification, is generated from the one-electron reduction of nitrite (NO 2 Ϫ ) (41). NO reduction is coupled to energy generation (18,29). NO is also a well-known cytotoxic compound, so its production during denitrification has the potential of causing significant cell damage. To mitigate the toxicity of NO, its steady-state concentration during denitrification is maintained at low-nanomolar levels (11). The protein responsible for NO reduction, NO reductase (Nor), catalyzes the reaction 2NO ϩ 2H ϩ 3N 2 O ϩ H 2 O. Nitrous oxide (N 2 O) is an inert, nontoxic intermediate that is frequently the terminal product of denitrification (42). Nor has been purified and shown to be a heterodimeric membrane protein (9,14,16). Metal analysis has shown that it contains only iron in stoichiometric amounts. Recently, the Nor structural genes have been characterized, and sequence analysis revealed that Nor was related to the cytochrome c oxidase superfamily (36). In particular, Nor is most closely related to the heme b-containing oxidases, which are expressed under conditions of low oxygen concentration. It has been suggested that Nor was the original member of this family and that the other members arose by modifying the Nor structure (26).The genetic organization of the region of the chromosome encoding the nor structural genes varies among denitrifiers. In Pseudomonas stutzeri, the two structural genes form a distinct transcriptional unit (43). In Pseudomonas aeruginosa, the two structural genes ...

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Section: Methodsmentioning

confidence: 99%

Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3

et al. 1997

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“…Cloning and sequencing of the pssA gene Cosmid pC7 was purified using the Jetstar kit (Genomed, Bad Oeynhausen, Germany), digested with Pst I and the resulting fragments were cloned into the broad host range vector pRK415 (Keen et al, 1988). Subfragments of the resulting plasmid pPC7 were obtained after digestion with EcoRI, which were subsequently ligated into pUC19.…”

Section: Construction and Screening Of A Cosmid Librarymentioning

confidence: 99%

Characterization of an exported protease from Shiga toxin‐producing Escherichia coli

Djafari

Ebel

Deibel

et al. 1997

Molecular Microbiology

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SummaryThe gene for a novel, high molecular weight protein secreted by Shiga toxin-producing Escherichia coli (STEC) has been cloned, sequenced and characterized with respect to its activity. This gene, designated pssA, is localized on the large plasmid that also harbours the STEC haemolysin operon. Sequencing of a region comprising 10 630 nt revealed that the sequences flanking the pssA gene are composed of several remnants of different insertion elements. The PssA protein is produced as a 142 kDa precursor molecule that, after N-and C-terminal processing, is released into the culture supernatant as a mature polypeptide of approximately 104 kDa. The primary sequence of PssA is highly related to a family of autonomously transported putative virulence factors from different Gram-negative pathogens, which includes the Tsh protein of an avian-pathogenic E. coli strain, the SepA protein from Shigella flexneri and the EspC protein from enteropathogenic E. coli. A common motif present in all four proteins is reminiscent of the catalytic centre of certain serine proteases. PssA (protease secreted by STEC) indeed shows serine protease activity in a casein-based assay and is moreover cytotoxic for Vero cells. This activity of PssA and probably of other proteins of the Tsh family may be of functional importance during infection of the mucosal cell layer by the bacterial pathogen.

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“…Complementation plasmid pED5 expressing the R. capsulatus nuoH and nuoI genes was constructed by cloning the 2385 bp BamHI-EcoRI restriction fragment containing these genes into pRK415 (Keen et al, 1988; Table 1). The 5Ј extremity of this fragment is located 17 bp upstream of the translation start of nuoH and thus contains the putative ribosome binding site for this gene.…”

Section: Plasmid Complementationmentioning

confidence: 99%

Distal genes of the nuo operon of Rhodobacter capsulatus equivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH–ubiquinone oxidoreductase

Dupuis

Darrouzet

Duborjal

et al. 1998

Molecular Microbiology

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SummarySeven out of the 13 proteins encoded by the mitochondrial genome of mammals (peptides ND1 to ND6 plus ND4L) are subunits of the respiratory NADH-ubiquinone oxidoreductase (complex I). The function of these ND subunits is still poorly understood. We have used the NADH-ubiquinone oxidoreductase of Rhodobacter capsulatus as a model for the study of the function of these proteins. In this bacterium, the 14 genes encoding the NADH-ubiquinone oxidoreductase are clustered in the nuo operon. We report here on the biochemical and spectroscopic characterization of mutants individually disrupted in five nuo genes, equivalent to mitochondrial genes nd1, nd2, nd5, nd6 and nd4L. Disruption of any of these genes in R. capsulatus leads to the suppression of NADH dehydrogenase activity at the level of the bacterial membranes and to the disappearance of complex I-associated iron-sulphur clusters. Individual NUO subunits can still be immunodetected in the membranes of these mutants, but they do not form a functional subcomplex. In contrast to these observations, disruption of two ORFs (orf6 and orf7 ), also present in the distal part of the nuo operon, does not suppress NADH dehydrogenase activity or complex I-associated EPR signals, thus demonstrating that these ORFs are not essential for the biosynthesis of complex I.

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Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria

Cited by 1,438 publications

References 16 publications

Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3

Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3

Characterization of an exported protease from Shiga toxin‐producing Escherichia coli

Distal genes of the nuo operon of Rhodobacter capsulatus equivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH–ubiquinone oxidoreductase

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