Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector

Fürste, Jens P.; Pansegrau, Werner; Frank, Ronald; Blöcker, Helmut; Scholz, Peter; Bagdasarian, Michael; Lanka, Erich

doi:10.1016/0378-1119(86)90358-6

Cited by 1,006 publications

(465 citation statements)

References 37 publications

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“…The gene encoding the CzcR protein was amplified by PCR from PT5 genomic DNA (Pfu DNA polymerase, Promega) with primers czcR-F and czcR-R (see below). The 900-bp product was cloned into the SmaI site of vector pMMB66EH (23) under the tac promoter, yielding plasmid pRWT. The czcS gene from PT5 and PT1105 was amplified by PCR using primers S58 (5Ј-cggaattcgcggcgtcggctacgtcc) and S59 (5Ј-cgggatcctgcggcgagtaccggctgtggc) containing an EcoRI and a HindIII site, respectively.…”

Section: Methodsmentioning

confidence: 99%

CzcR-CzcS, a Two-component System Involved in Heavy Metal and Carbapenem Resistance in Pseudomonas aeruginosa

Perron

Caille

Rossier

et al. 2004

Journal of Biological Chemistry

291

272

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Pseudomonas aeruginosa is an environmental bacterium involved in mineralization of organic matter. It is also an opportunistic pathogen able to cause serious infections in immunocompromised hosts. As such, it is exposed to xenobiotics including solvents, heavy metals, and antimicrobials. We studied the response of P. aeruginosa upon exposure to heavy metals or antibiotics to investigate whether common regulatory mechanisms govern resistance to both types of compounds. We showed that sublethal zinc concentrations induced resistance to zinc, cadmium, and cobalt, while lethal zinc concentrations selected mutants constitutively resistant to these heavy metals. Both zinc-induced and stable zinc-resistant strains were also resistant to the carbapenem antibiotic imipenem. On the other hand, only 20% of clones selected on imipenem were also resistant to zinc. Heavy metal resistance in the mutants could be correlated by quantitative real time PCR with increased expression of the heavy metal efflux pump CzcCBA and its cognate two-component regulator genes czcR-czcS. Western blot analysis revealed reduced expression of the basic amino acid and carbapenem-specific OprD porin in all imipenem-resistant mutants. Sequencing of the czcR-czcS DNA region in eight independent zincand imipenem-resistant mutants revealed the presence of the same V194L mutation in the CzcS sensor protein. Overexpression in a susceptible wild type strain of the mutated CzsS protein, but not of the wild type form, resulted in decreased oprD and increased czcC expression. We further show that zinc is released from latex urinary catheters into urine in amounts sufficient to induce carbapenem resistance in P. aeruginosa, possibly compromising treatment of urinary tract infections by this class of antibiotics.Pseudomonas aeruginosa is a Gram-negative bacterium thriving in environments polluted with organic matter. It is also an opportunistic pathogen frequently encountered in the hospital, causing morbidity and mortality in immunocompromised and cystic fibrosis patients (1). P. aeruginosa is characterized by an intrinsically high level of resistance to xenobiotics including antimicrobial agents, solvents, and heavy metals (2), which can be accounted for by a combination of its low outer membrane permeability and the presence of multiple efflux pumps (3). These pumps belong to the resistance, nodulation, cell division (RND) 1 transporter family, present in many Gramnegative bacteria (4). To extrude substrates from the cytoplasm across the two membranes, these systems are composed of a proton antiporter located in the cytoplasmic membrane, a membrane fusion protein spanning the periplasmic space, and an outer membrane protein (5). Members of the RND family, namely the Mex pumps, have recently gained increasing interest. In particular, the constitutively expressed MexAB-OprM (6, 7) and the inducible MexXY (8) efflux pumps endow the PAO1 reference strain and other clinical isolates (9) with a natural resistance to a wide range of antimicrobial agents. Proton-dr...

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Section: Methodsmentioning

confidence: 99%

CzcR-CzcS, a Two-component System Involved in Heavy Metal and Carbapenem Resistance in Pseudomonas aeruginosa

Perron

Caille

Rossier

et al. 2004

Journal of Biological Chemistry

291

272

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“…Plasmid pLYV29 contains the 3Ј-truncated ftsA⌬27 allele (with an ochre codon spontaneously inserted instead of the Glu-394 coding triplet, thus rendering an FtsA protein lacking the last 27 residues), cloned into the vector pJF119HE (16). Plasmid pLYV32 contains the ftsA⌬23⌽36 allele (resulting from a spontaneous frameshift in the 3Ј end of the gene, rendering an FtsA protein with the last carboxy terminal 23 residues replaced by a different 36-residue sequence), cloned into pJF119EH (as pJF119HE but with the multicloning site in the opposite orientation).…”

Section: Methodsmentioning

confidence: 99%

Role of the Carboxy Terminus of Escherichia coli FtsA in Self-Interaction and Cell Division

et al. 2000

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The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of the ftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362-5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsA⌬1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsA⌬27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsA⌬27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.FtsA is an essential cell division protein of Escherichia coli that is widely conserved in bacteria. Together with ftsZ, which codes for a GTPase analog of the eukaryotic tubulin, ftsA forms one of the most frequently conserved gene pairs among the cell division genes in the eubacteria. Based on sequence homology it has been proposed that FtsA belongs to the sugar kinase/hsp70/actin superfamily (4). This superfamily comprises several proteins with a common two-domain topology and the ability to bind and hydrolyze ATP. FtsA binds to columns of ATP-agarose and can be isolated from cells either as a phosphorylated or a nonphosphorylated form (29), but so far no other biochemical function has been described for this protein.FtsA is present both in the cytoplasm and in the cytoplasmic membrane (29), where it forms a structural part of the septum (32). It has been proposed that FtsA is a component of a membrane-associated complex (septator or divisome), which would include periplasmic, transmembrane, and cytoplasmic proteins acting coordinately to perform septation (27,35). Genetic analysis suggests that FtsA may interact, directly or indirectly, with other cell division proteins, such as FtsZ, PBP3, FtsQ, and FtsN (9,10,24,33,34). The FtsZ/FtsA ratio is important for cell division, an...

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“…To purify the mature form of CelCCC from E. coli cells, another construction was carried out. The BclI-BglII fragment from pLM26, which contained the sequence coding for the mature form of CelCCC minus the first amino acid, was inserted behind an EcoRI-BclI junction linker in the multiple-cloning site of pJFll8EH expression vector [19] (Fig. 1).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Purification and characterization of endoglucanase C from Clostridium cellulolyticum

Fierobe

Bagnara-Tardif

Gaudin

et al. 1993

European Journal of Biochemistry

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An Escherichia colz clone was constructed to overproduce endoglucanase C (CelCCC) from Clostridium cellulolyticum. This construction made it easier to isolate the enzyme but, as observed in the case of endoglucanase A (CelCCA) from the same organism, the purification led to the isolation of two forms of the cellulase differing in their molecular masses, 48 kDa and 41 kDa. Nterminal sequence analysis of both purified enzymes showed that the shorter form was probably the result of partial proteolysis near the COOH-extremity. The difference in mass indicated that the shorter protein lacks the C-terminal reiterated domains (20-24-amino-acid twice-repeated sequences). These particular domains are characteristic of clostridial cellulases acting on cellulose by the mean of cellulosomal particles. Biochemical and enzymic studies were performed on each form of CelCCC, and revealed that their temperature and pH optima were identical, but their catalytic parameters were quite different. Furthermore, the differences of enzymic behavior observed between the two forms of CelCCC are almost identical to those already noted in the case of the two forms of CelCCA. The stereoselectivity of the reaction catalysed by CelCCC and CelCCA was determined using proton NMR spectroscopy ; CelCCC acts by configuration inversion, whereas CelCCA acts by configuration retention. The degradation patterns on cellodextrins (ranging from cellotriose to cellohexaose) and chromophoric cellodextrins (from p-nitrophenyl-cellobiose to p-nitrophenyl-cellopentaose) were also investigated in both forms of CelCCC and CelCCA. It emerged that the natural cellodextrins degradation patterns of CelCCC and CelCCA were very similar but the utilization of p-nitrophenyl-cellodextrins showed the existence of considerable differences between these two endoglucanases in terms of cleavage-site position and catalytic parameters. CelCCC and CelCCA were found not to act synergistically on the tested substrates.Clostridiurn cellulolyticum is a mesophilic Clostridium which is able to completely degrade crystalline cellulose. The four known cellulase sequences of this organism [l-31 exhibit reiterated domains. These particular sequences are thought to be characteristic of enzymes integrated into the cellulosome structure [4], and yet they are observed with cellulolytic systems of Clostridium thermocellum [4, 51 and Clostridium cellulovorans [6]. These organisms secrete highmolecular-mass particles named cellulosomes which are active against cellulose, and they possess an assembly factor, CbpA in C. cellulovorans [7] and CipA in C. thermocellum [8, 91. Four genes, celCCA, celCCC, coding for enzymes of the cellulolytic system of C. cellulolyticum have been completely sequenced and the four corresponding proteins are members of three different fami-Correspondence to H

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Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector

Cited by 1,006 publications

References 37 publications

CzcR-CzcS, a Two-component System Involved in Heavy Metal and Carbapenem Resistance in Pseudomonas aeruginosa

CzcR-CzcS, a Two-component System Involved in Heavy Metal and Carbapenem Resistance in Pseudomonas aeruginosa

Role of the Carboxy Terminus of Escherichia coli FtsA in Self-Interaction and Cell Division

Purification and characterization of endoglucanase C from Clostridium cellulolyticum

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