Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1

Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT).     

The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product.

The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.     

The nucleotide sequence of the amiE gene of Pseudomonas aeruginosa

The nucleotide sequence of the amiE gene, encoding the aliphatic amidase of Pseudomonas aeruginosa, has been determined. The sequence of 1038 nucleotides shows a strong bias in favour of codons with G or C in the third position, and only 44 different codons are utilised.     

Pseudomonas aeruginosa outer membrane lipoprotein I gene: molecular cloning, sequence, and expression in Escherichia coli.

Lipoprotein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. Like porin protein F (OprF), it is a vaccine candidate because it antigenically cross-reacts with all serotype strains of the International Antigenic Typing Scheme. Since lipoprotein I was expressed in Escherichia coli under the control of its own promoter, we were able to isolate the gene by screening a lambda EMBL3 phage library with a mouse monoclonal antibody directed against lipoprotein I. The monocistronic OprI mRNA encodes a precursor protein of 83 amino acid residues including a signal peptide of 19 residues. The mature protein has a molecular weight of 6,950, not including bound glycerol and lipid. Although the amino acid sequences of protein I of P. aeruginosa and Braun's lipoprotein of E. coli differ considerably (only 30.1% identical amino acid residues), peptidoglycan in E. coli, are identical. Using lipoprotein I expressed in E. coli, it can now be tested whether this protein alone, without P. aeruginosa lipopolysaccharide contaminations, has a protective effect against P. aeruginosa infections.     

Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi

The gene coding for the lipase of Pseudomonas fragi was cloned into Escherichia coli JM83 by inserting Sau3A-generated DNA fragments into the BamH I site of pUC9. The plasmid isolated, pKKO, was restriction mapped and the position of the lipase gene on the 2.0 kb insert was pinpointed by subcloning. DNA sequencing revealed that the open reading frame comprises 405 nucleotides and gives a preprotein of 135 amino acids with a predicted Mr of 14643. By comparing the putative lipase amino acid sequence with porcine pancreatic, rat lingual and Staphylococcus hyicus lipases the amino acid sequence around the reactive serine was found to be common among the types of lipase which have been reported.     

Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins.

A number of polyacrylamide gel systems and solubilization procedures were studied to define the number and nature of "major" polypeptide bands in the outer membrane of Pseudomonas aeruginosa. It was shown that five of the eight major outer membrane proteins were "heat modifiable" in that their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the solubilization temperature. Four of these heat-modifiable proteins had characteristics similar to protein II of the Escherichia coli outer membrane. Addition of lipopolysaccharide subsequent to solubilization caused reversal of the heat modification. The other heat-modifiable protein, the porin protein F, was unusually stable to sodium dodecyl sulfate. Long periods of boiling in sodium dodecyl sulfate were required to cause conversion to the heat-modified form. This was demonstrated both with outer membrane-associated and purified lipopolysaccharide-depleted protein F. Furthermore, lipopolysaccharide treatment had no effect on the mobility of heat-modified protein F. Thus it is concluded that protein F represents a new class of heat-modifiable protein. It was further demonstrated that the electrophoretic mobility of protein F was modified by 2-mercaptoethanol and that the 2-mercaptoethanol and heat modification of mobility were independent of one another. The optimal conditions for the examination of the outer membrane proteins of P. aeruginosa by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis are discussed.     

Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin.

It is well established that Pseudomonas aeruginosa cells grown in Mg2+-deficient medium acquire nonmutational resistance to the chelator ethylenediaminetetraacetate and to the cationic antibiotic polymyxin B; this type of resistance can be reversed by transferring the cells to Mg2+-sufficient medium for a few generations. Stable mutants resistant to polymyxin B were isolated and shown to have also gained ethylenediaminetetraacetate resistance. Both the mutants and strains grown on Mg2+-deficient medium had greatly enhanced levels of outer membrane protein H1 when compared with the wild-type strain or with revertants grown in Mg2+-sufficient medium. It was determined that in all strains and at all medium Mg2+ concentrations, the cell envelope Mg2+ concentration varied inversely with the amount of protein H1. In addition, the increase in protein H1 in the mutants was associated with an increase in resistance to another group of cationic antibiotics, the aminoglycosides, e.g., gentamicin. We propose that protein H1 acts by replacing Mg2+ at a site on the lipopolysaccharide which can otherwise be attacked by the cationic antibiotics or ethylenediaminetetraacetate.     

Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa.

Pseudomonas aeruginosa K437 lacks the ferripyoverdine receptor and, as a result, grows poorly on an iron-deficient minimal medium supplemented with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and pyroverdine. By using a phagemid-based in vivo cloning system, attempts were made to clone the receptor gene by complementing this growth defect. Several recombinant phagemids carrying P. aeruginosa chromosomal DNA which provided for good growth on EDDHA-pyoverdine-containing medium and which concomitantly restored production of the ferripyroverdine receptor in strain K437 were isolated. These phagemids contained a common 4.6-kb SphI fragment which similarly restored production of the receptor in K437. Nucleotide sequencing of the SphI fragment revealed a single large open reading frame, designated fpvA (ferripyoverdine uptake), of 2439 bp. The predicted translation product of fpvA has a molecular mass of 89,395 Da. N-terminal amino acid sequence analysis of the purified ferripyoverdine receptor confirmed fpvA as the receptor gene. Moreover, it indicated that the receptor is initially synthesized as a precursor with a signal sequence of 27 amino acids which is cleaved to yield the mature protein. The deduced FpvA polypeptide exhibited homology to regions shown to be conserved in TonB-dependent receptor proteins. FpvA also shared strong homology (41.3% identity) with the PupA protein of Pseudomonas putida WCS358. This protein is the receptor for the iron-bound form of pseudobactin, a compound structurally very similar to pyoverdine.     

Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics.     

Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein

The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino-terminus of the mature protein is located at Lys-26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60,204 Da. The c-heme-binding domain, which contains the only two Cys of the molecule, is located at the amino-terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c-heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.     

Molecular cloning and nucleotide sequence of the streptavidin gene.

Using synthetic oligonucleotides as probes we have cloned the streptavidin gene from a genomic library of Streptomyces avidinii. Nucleotide sequence analysis indicated that a 2 Kb DNA-fragment contained the entire coding region, a signal peptide region and the 3' and 5' flanking regions of the gene. The deduced amino acid sequence shows several interrupted blocks of homology with the amino acid sequence of chicken egg-white avidin. Analysis of the secondary structure suggests a high content of beta-structure in both proteins and considerable overall structural similarity between them.     

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.     

Molecular cloning and nucleotide sequence analysis of the Saccharomyces cerevisiae RAD1 gene.

We have screened a yeast genomic library for complementation of the UV sensitivity of mutants defective in the RAD1 gene and isolated a plasmid designated pNF1000 with an 8.9-kilobase insert. This multicopy plasmid quantitatively complemented the UV sensitivity of two rad1 mutants tested but did not affect the UV resistance of other rad mutants. The location of the UV resistance function in pNF1000 was determined by deletion analysis, and an internal fragment of the putative RAD1 gene was integrated into the genome of a RAD1 strain. Genetic analysis of several integrants showed that integration occurred at the chromosomal RAD1 site, demonstrating that the internal fragment was derived from the RAD1 gene. A 3.88-kilobase region of pNF1000 was sequenced and showed the presence of a small open reading frame 243 nucleotides long that is apparently unrelated to RAD1, as well as a 2,916-nucleotide larger open reading frame presumed to encode RAD1 protein. Depending on which of two possible ATG codons initiates translation, the size of the RAD1 protein is calculated at 110 or 97 kilodaltons.     

Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonas marginalis N6301.

A pectin lyase (PNL;EC4.2.2.10) gene of Pseudomonas marginalis N6301 was cloned and expressed in Escherichia coli. We purified PNL from P. marginalis N6301 and determined N-terminal 33 amino acids sequence. From this sequence, we synthesized two oligonucleotide probes. From the analysis of Southern hybridization, 2. 1kb EcoRI-SmaI fragment from the chromosomal DNA of P. marginalis was found to hybridize with oligonucleotide probes. Then, we cloned the fragment into pUC119 vector and transformed into E. coli DH5 alpha. A plasmid thus obtained was designated as pPNL6301. E. coli DH5 alpha harboring pPNL6301 expressed PNL activity. The nucleotide sequence of pn1 gene in the plasmid pPNL6301 encoding PNL from P. marginalis N6301 was determined. The structural gene of pn1 consisted of 936 base pairs. An open reading frame that encodes a 34,103 dalton polypeptide composed of 312 amino acids was assigned. The molecular weight of the polypeptide predicted from the amino acid composition was close to that of PNL of P. marginalis N6301 determined. The nucleotide sequence of the 5'-flanking region of pn1 gene showed the presence of the consensus sequence of LexA binding site, Pribnow box and ribosome binding site as found in Escherichia coli. The amino acid sequence homology of PNLs and nucleotide sequence homology of pn1 gene between P. marginalis N6301 and E. carotovora Er were 60.8% and 57.2%, respectively.