Cloning and nucleotide sequence of the Pseudomonas aeruginosa nfxB gene, conferring resistance to new quinolones

The gene nfxB is one of the genes which affect the cell membrane permeability of quinolones in Pseudomonas aeruginosa PAO. Both wild-type nfxB and a mutant nfxB (nfx13E) were cloned and the DNA sequences were determined. The wild-type gene was dominant in PAO strains. The nfxB mutation was a point mutation (cytosine----guanine) which generates an amino acid exchange (arginine----glycine) in the putative nfxB product. The amino acid sequence of the wild-type NfxB protein revealed that it has a helix-turn-helix motif which may be responsible for the ability to bind in a sequence-specific manner to DNA. This finding indicated that the NfxB protein may regulate the expression of genes that are associated with cell permeability of drugs in P. aeruginosa. The position of the amino acid substitution between the NfxB protein and the Nfx13E protein was located within a possible DNA-binding domain, suggesting that the mutant protein (Nfx13E) may have lost DNA binding ability and regulator activity.     

Cloning and expression in Escherichia coli of the structural gene coding for the monomeric protein of the S layer of Thermus thermophilus HB8.

The gene coding for the 100 kDa monomeric protein (P100) of the S layer of Thermus thermophilus HB8 has been cloned in the Escherichia coli plasmid vector pUC9. Recombinant plasmids were selected by colony screening with anti-P100 rabbit antiserum. The gene, named slpA (for surface layer protein A), was identified in a bacterial clone harboring a hybrid plasmid, pMF4, with a 5.8-kbp insert. This plasmid consistently expressed a protein specifically recognized by anti-P100 antiserum. Expression was apparently independent of Plac, indicating that the promoter for P100 is functional in E. coli. Most E. coli strains transformed with plasmids containing the 5.8-kbp insert cloned in pMF4 expressed two proteins with apparent masses of 52 and 50 kDa, which were strongly recognized by anti-P100 antiserum in Western immunoblots. The 52-kDa fragment could be overproduced, and the sequence of the N-terminal undecapeptide, determined by microsequencing, indicated that it could correspond to the N-terminal domain of P100. Expression of slpA in lon mutants of E. coli led to accumulation of a protein indistinguishable from native P100, indicating that the complete gene was cloned and that the product of lon, protease La, was involved in proteolytic degradation of P100 synthesized in E. coli.     

Mutator effects in Escherichia coli caused by the expression of specific foreign genes

Certain genes from Lactococcus lactis and Pseudomonas aeruginosa, including the nfxB gene, generate a mutator phenotype in Escherichia coli. The results of this study, together with those of a previous study, support conservation of regulatory sequences in E. coli and P. aeruginosa and suggest that some efflux pumps prevent mutagenicity by exporting mutagenic products of metabolism.     

Identification of RegA protein from Pseudomonas aeruginosa using anti-RegA antibody

The product of Pseudomonas aeruginosa regA gene acts as a positive regulator of exotoxin A expression. The protein, RegA, was overproduced in E. coli transformed with an expression vector containing the regA gene. The overproduced RegA accumulated in E. coli in the form of inclusion bodies. The latter were isolated and served as a source of antigen for raising polyclonal antibodies. The antibodies reacted specifically with a P. aeruginosa protein whose molecular weight corresponded to that predicted for RegA from its known DNA sequence, and whose response to modulating factors matched that expected for RegA. The immunodetectable RegA was localized in the membrane fraction of P. aeruginosa strain PA103.     

Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.     

Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene.

A 5.9-kb DNA fragment was cloned from Pseudomonas aeruginosa PA103 by its ability to functionally complement a fur mutation in Escherichia coli. A fur null mutant E. coli strain that contains multiple copies of the 5.9-kb DNA fragment produces a 15-kDa protein which cross-reacts with a polyclonal anti-E. coli Fur serum. Sequencing of a subclone of the 5.9-kb DNA fragment identified an open reading frame predicted to encode a protein 53% identical to E. coli Fur and 49% identical to Vibrio cholerae Fur and Yersinia pestis Fur. While there is extensive homology among these Fur proteins, Fur from P. aeruginosa differs markedly at its carboxy terminus from all of the other Fur proteins. It has been proposed that this region is a metal-binding domain in E. coli Fur. A positive selection procedure involving the isolation of manganese-resistant mutants was used to isolate mutants of strain PA103 that produce altered Fur proteins. These manganese-resistant Fur mutants constitutively produce siderophores and exotoxin A when grown in concentrations of iron that normally repress their production. A multicopy plasmid carrying the P. aeruginosa fur gene restores manganese susceptibility and wild-type regulation of exotoxin A and siderophore production in these Fur mutants.     

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.     

Nucleotide sequences and expression in Escherichia coli of the in-phase overlapping Pseudomonas aeruginosa plcR genes.

The translation products of chromosomal DNAs of Pseudomonas aeruginosa encoding phospholipase C (heat-labile hemolysin) have been examined in T7 promoter plasmid vectors and expressed in Escherichia coli cells. A plasmid carrying a 4.7-kilobase (kb) DNA fragment was found to encode the 80-kilodalton (kDa) phospholipase C as well as two more proteins with an apparent molecular mass of 26 and 19 kDa. Expression directed by this DNA fragment with various deletions suggested that the coding region for the two smaller proteins was contained in a 1-kb DNA region. Moreover, the size of both proteins was reduced by the same amount by an internal BglII-BglII DNA deletion, suggesting that they were translated from overlapping genes. Similar results were obtained with another independently cloned 6.1-kb Pseudomonas DNA, which in addition coded for a 31-kDa protein of opposite orientation. The nucleotide sequence of the 1-kb region above revealed an open reading frame with a signal sequence typical of secretory proteins and a potential in-phase internal translation initiation site. Pulse-chase and localization studies in E. coli showed that the 26-kDa protein was a precursor of a secreted periplasmic 23-kDa protein (PlcR1) while the 19-kDa protein (PlcR2) was mostly cytoplasmic. These results indicate the expression of Pseudomonas in-phase overlapping genes in E. coli.     

Molecular cloning and sequence of the thdF gene, which is involved in thiophene and furan oxidation by Escherichia coli.

Our previous work resulted in the isolation of mutant strains of Escherichia coli K-12 which were able to oxidize furans and thiophenes as a result of mutations in several novel genes. Some of the genes involved in thiophene oxidation were cloned into the multicopy vector pUC19. The plasmid pKA10 carries a 3.8-kb chromosomal fragment which encodes a previously undiscovered gene involved in thiophene oxidation. Three proteins with approximate molecular sizes of 48, 30, and 26 kDa were overproduced by cells carrying pKA10. Maxicell experiments and DNA sequence analysis indicated that the 48- and 26-kDa proteins are encoded by pKA10, whereas the 30-kDa protein is apparently chromosomally derived. A cassette specifying kanamycin resistance was inserted into various sites on pKA10. An insertion which abolished the 48-kDa protein also abolished thiophene oxidation. Chromosomal integration of pKA10::Kan allowed us to locate the chromosomal insert of pKA10 at 84 min on the E. coli genetic map by transduction. Since no previously identified genes involved in thiophene metabolism are located in this region, we designated the gene for the 48-kDa protein as thdF. Sequencing of the 3.8-kb insert revealed an overlap of several hundred bases with the regulatory and structural regions of the tnaA gene, which is also located at 84 min. The 26-kDa protein is probably truncated tnaA protein. An open reading frame corresponding to the 48-kDa thdF protein was located next to the tnaA gene, which encodes tryptophanase, but was transcribed in the opposite sense.     

Cloning of the protein D2 gene of Pseudomonas aeruginosa and its functional expression in the imipenem-resistant host

Protein D2 forms the water-filled pore across the outer membrane of Pseudomonas aeruginosa and allows the penetration of imipenem. We cloned the protein D2 gene by the antibody screening technique. When the imipenem-resistant mutant lacking protein D2 harbored the plasmid with the cloned D2 gene, the mutant overproduced protein D2 in the outer membrane. These transformants exhibited fully-restored imipenem susceptibility. The results prove unequivocally that protein D2 forms the imipenem-permeable pore in the P. aeruginosa outer membrane.     

The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins.

The product of the secY (prlA) gene (the SecY protein) involved in protein export in Escherichia coli was overproduced and localized in the cytoplasmic (inner) membrane. Because of its strong interaction with a non-ionic detergent (NP40), it partitioned into the detergent layer during electroblotting through a NP40-containing gel (detergent blotting), and it formed a horizontal streak in the O'Farrell two-dimensional gel electrophoretic system. Consequently, we developed an alternative two-dimensional gel procedure, which proved useful for analysis of integral membrane proteins, especially in combination with detergent blotting. SDS-gel electrophoresis was carried out successively through gels of lower (first dimension) and higher (second dimension) sieving effects. Many membrane proteins, unlike soluble proteins, formed spots off and above the diagonal line, and all of these spots partitioned exclusively into the detergent layer. A characteristic pattern of integral membrane proteins of E. coli was thus obtained and the spot of the SecY protein in the cytoplasmic membrane was identified even when it was not overproduced. These results show that the gene secY specifies an integral membrane component of the protein export machinery.     

The identification of a new family of sugar efflux pumps in Escherichia coli

Using a functional cloning strategy with an Escherichia coli genomic plasmid library, we have identified a new family of sugar efflux proteins with three highly homologous members in the E. coli genome. In addition, two open reading frames, one present in Yersinia pestis and the other in Deinococcus radiodurans, appear to encode closely related proteins. An in vitro transport assay using inside-out membrane vesicles prepared from overproducing strains was used to demonstrate that members of this new family can efflux [14C]-lactose. As sugar efflux phenomena have been reported previously in several bacterial species including E. coli, the identification of a new family of sugar efflux proteins may help to reveal the physiological role of sugar efflux in metabolism. It is proposed that the E. coli members of this family, whose functions were previously unknown, be given the gene family designation SET for sugar efflux transporter.     

Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane

A DNA/membrane complex extracted from a miniplasmid derivative of the broad host range plasmid RK2 cultured in Escherichia coli capable of synthesizing new plasmid supercoiled DNA in vitro was treated with antibodies that were made against or reacted with the dnaA and dnaK host-encoded proteins, respectively. Anti-dnaA protein antibody inhibited total plasmid DNA synthesis significantly and the synthesis of supercoil plasmid DNA almost completely. In contrast, anti-dnaK protein antibody and nonimmune serum had little or no effect on total plasmid DNA synthesis. Both proteins were found to be present in the inner but not outer membrane fraction of E. coli. A variety of miniplasmid-encoded proteins which had previously been found in the DNA/membrane complex have also been localized to the inner but not outer membrane fraction. These include an essential initiation protein of 32 kDa (and an overlapping protein of 43 kDa coded for by the same gene), as well as a 30-kDa protein that may be linked to incompatibility functions. Various extraction methods were used to distinguish between the associated and the integral nature of the plasmid-encoded proteins. The results demonstrated that the essential replication proteins (32 and 43 kDa) as well as the 30-kDa protein was tightly bound to the inner membrane. Computer analysis of the amino acid sequence of the 32 (and 43)-kDa protein revealed a hydrophobic region that is only half that normally required to span the membrane. Other interactions are discussed with respect to attaching this protein to the membrane.     

Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3' end in mRNA stability

The major outer membrane protein (MOMP)-encoding gene (omp1) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant omp1 is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous omp1 promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3' end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed omp1 gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.     

Overexpression, refolding, and purification of the histidine-tagged outer membrane efflux protein OprM of Pseudomonas aeruginosa

This paper describes the overproduction and purification of the C-terminus polyhistidine-tagged outer membrane protein OprM, which is a part of the MexA-MexB-OprM active efflux system of Pseudomonas aeruginosa. Renaturation of the protein from inclusion bodies of Escherichia coli was achieved using guanidine-HCl as denaturing agent and n-octylpolyoxyethylene (C8POE) and n-octyltetraoxyethylene (C8E4) as nonionic detergents. The refolded protein was purified by ion-exchange and nickel-affinity chromatography. The final yield was 6 mg of pure histidine-tagged OprM per liter of E. coli culture. Renaturation was monitored by the effects of heating prior to SDS-PAGE, using a typical and exclusive property of outer membrane proteins. Immunoblotting revealed that the recombinant protein is addressed to the outer membrane of E. coli, after maturation by excision of its N-terminal signal sequence. Complementation of an oprM deletion mutant with the plasmid encoded histidine-tagged OprM protein restored antibiotic susceptibilities to wild-type levels, demonstrating functionality of recombinant OprM.     

SecA protein is directly involved in protein secretion in Escherichia coli

A high-expression plasmid for the secA gene was constructed. The SecA protein was then overproduced in E. coli and purified. The purified SecA stimulated the in vitro translocation of a model secretory protein into inverted membrane vesicles pretreated with 4 M urea. Membrane vesicles from a secAts mutant exhibited lower translocation activity, which was enhanced by SecA. These results indicate that SecA is directly involved in protein secretion across the cytoplasmic membrane.     

Identification of a novel membrane-associated gene product that suppresses toxicity of a TrfA peptide from plasmid RK2 and its relationship to the DnaA host initiation protein

The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli. The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form. The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E. coli. Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added. Recently, the Dp was independently shown to help prevent overinitiation in E. coli and was termed Hda (S. Kato and T. Katayama, EMBO J. 20:4253-4262, 2001).     

Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number.

The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kilodaltons (kDa) are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were o overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 +/- 0.6 ng of TrfA-32 and 1.8 +/- 0.2 ng of TrfA-43 per unit of optical density at 600 nm (mean +/- standard deviation). On the basis of determinations of the number of cells per unit of optical density at 600 nm, this corresponds to about 220 molecules of TrfA-32 and 80 molecules of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy number of RK2 by about 30%. Additional increases in TrfA protein concentration had no further effect on copy number, even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concentration. These results demonstrate that TrfA protein concentration is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concentration functions to limit RK2 copy number in the presence of excess TrfA.     

TrfA-dependent inner membrane-associated plasmid RK2 DNA synthesis and association of TrfA with membranes of different gram-negative hosts

TrfA, the replication initiator protein of broad-host-range plasmid RK2, was tested for its ability to bind to the membrane of four different gram-negative hosts in addition to Escherichia coli: Pseudomonas aeruginosa, Pseudomonas putida, Salmonella enterica serovar Typhimurium, and Rhodobacter sphaeroides. Cells harboring TrfA-encoding plasmids were fractionated into soluble, inner membrane, and outer membrane fractions. The fractions were subjected to Western blotting, and the blots were probed with antibody to the TrfA proteins. TrfA was found to fractionate with the cell membranes of all species tested. When the two membrane fractions of these species were tested for their ability to synthesize plasmid DNA endogenously (i.e., without added template or enzymes), only the inner membrane fraction was capable of extensive synthesis that was inhibited by anti-TrfA antibody in a manner similar to that of the original host species, E. coli. In addition, although DNA synthesis did occur in the outer membrane fraction, it was much less extensive than that exhibited by the inner membrane fraction and only slightly affected by anti-TrfA antibody. Plasmid DNA synthesized by the inner membrane fraction of one representative species, P. aeruginosa, was characteristic of supercoil and intermediate forms of the plasmid. Extensive DNA synthesis was observed in the soluble fraction of another representative species, R. sphaeroides, but it was completely unaffected by anti-TrfA antibody, suggesting that such synthesis was due to repair and/or nonspecific chain extension of plasmid DNA fragments.     

Mutational analysis of the TonB1 energy coupler of Pseudomonas aeruginosa

Siderophore-mediated iron transport in Pseudomonas aeruginosa is dependent upon the cytoplasmic membrane-associated TonB1 energy coupling protein for activity. To assess the functional significance of the various regions of this molecule and to identify functionally important residues, the tonB1 gene was subjected to site-directed mutagenesis, and the influence on iron acquisition was determined. The novel N-terminal extension of TonB1, which is absent in all other examples of TonB, was required for TonB1 activity in both P. aeruginosa and Escherichia coli. Appending it to the N terminus of the nonfunctional (in P. aeruginosa) Escherichia coli TonB protein (TonB(Ec)) rendered TonB(Ec) weakly active in P. aeruginosa and did not compromise the activity of this protein in E. coli. Elimination of the membrane-spanning, presumed membrane anchor sequence of TonB1 abrogated TonB1 activity in P. aeruginosa and E. coli. Interestingly, however, a conserved His residue within the membrane anchor sequence, shown to be required for TonB(Ec) function in E. coli, was shown here to be essential for TonB1 activity in E. coli but not in P. aeruginosa. Several mutations within the C-terminal end of TonB1, within a region exhibiting the greatest similarity to other TonB proteins, compromised a TonB1 contribution to iron acquisition in both P. aeruginosa and E. coli, including substitutions at Tyr264, Glu274, Lys278, and Asp304. Mutations at Pro265, Gln293, and Val294 also impacted negatively on TonB1 function in E. coli but not in P. aeruginosa. The Asp304 mutation was suppressed by a second mutation at Glu274 of TonB1 but only in P. aeruginosa. Several TonB1-TonB(Ec) chimeras were constructed, and assessment of their activities revealed that substitutions at the N or C terminus of TonB1 compromised its activity in P. aeruginosa, although chimeras possessing an E. coli C terminus were active in E. coli.