Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene.

The protein NfxB, involved in conferring resistance to quinolones in Pseudomonas aeruginosa, has a helix-turn-helix motif which is similar to that of other DNA-binding proteins. It appears to affect the membrane-associated energy-driven efflux of some antibiotics (H. Nikaido, Science 264:382-388, 1994). We constructed a plasmid that overproduced NfxB in Escherichia coli and purified the protein. Two species of NfxB (23 and 21 kDa), which are probably translated from different initiation codons, were isolated. Both proteins are also expressed in vivo in P. aeruginosa, with the 23-kDa NfxB being the major species. NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting. Expression of the phi (nfxB'-lacZ+) (Hyb) gene was repressed in the presence of the nfxB gene product provided by a second compatible plasmid in E. coli. In the P. aeruginosa wild-type strain (PAO2142), NfxB was undetectable by immunoblotting; however, it was detected in the nfxB missense mutant (PK1013E). These results suggested that NfxB negatively autoregulates the expression of nfxB itself. Since the 54-kDa outer membrane protein (OprJ) (N. Masuda, E. Sakagawa, and S. Ohya, Antimicrob. Agents Chemother. 39:645-649, 1995) was overproduced in nfxB mutants, NfxB may also regulate the expression of membrane proteins that are involved in the drug efflux machinery of P. aeruginosa.     

Crystal structures of OprN and OprJ,outer membrane factors of multidrug tripartite efflux pumps of Pseudomonas aeruginosa

The genome of Pseudomonas aeruginosa encodes tripartite efflux pumps that extrude functionally and structurally dissimilar antibiotics from the bacterial cell. MexAB‐OprM, MexCD‐OprJ, MexEF‐OprN, and MexXY‐OprM are the main tripartite efflux pumps responsible for multidrug resistance in P. aeruginosa. The outer membrane factors OprN, OprJ, and OprM are essential components of functional tripartite efflux pumps. To elucidate the structural basis of multidrug resistance, we determined the crystal structures of OprN and OprJ. These structures revealed several features, including tri‐acylation of the N‐terminal cysteine, a small pore in the β‐barrel domain, and a tightly sealed gate in the α‐barrel domain. Despite the overall similarity of OprN, OprJ, and OprM, a comparison of their structures and electrostatic distributions revealed subtle differences at the periplasmic end of the α‐barrel domain. These results suggested that the overall structures of these outer membrane factors are specifically optimized for particular tripartite efflux pumps. Proteins 2016; 84:759–769. © 2016 Wiley Periodicals, Inc.     

Autoinduction of RpoS Biosynthesis in the Biocontrol Strain <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18

The rpoS gene from Pseudomonas sp. M18, which encodes predicted protein (an alternative sigma factor s, σ^S, or σ³⁸) with 99.5% sequence identity with RpoS from Pseudomonas aeruginosa PAO1, was first cloned. In order to investigate the mechanism of rpoS expression, an rpoS null mutant, named M18S, was constructed with insertion of aacC1 cassette bearing a gentamycin resistance gene. With introduction of a plasmid containing an rpoS′–′lacZ translational fusion (pMERS) to wild-type strain M18 or M18S, it was first found that β-galactosidase activity expressed in strain M18S (pMERS) decreased to fourfold of that expressed in the strain M18 (pMERS). When strain M18S (pMERS) was introduced with another plasmid pBBS containing the wild-type rpoS gene, its β-galactosidase expression level was enhanced and almost restored to that in strain M18 (pMERS). Similarly, expression of β-galactosidase from a chromosomal fusion of the promoter of the wild-type rpoS gene with lacZ (rpoS–lacZ) was enhanced fivefold in the presence of a plasmid with the wild-type rpoS gene. With these findings, it is suggested that RpoS sigma factor may be involved in autoinducing its own gene expression in Pseudomonas sp. M18.     

Unconventional Integration of the <Emphasis Type="Italic">bla</Emphasis> Gene from Plasmid pIT2 During IS<Emphasis Type="Italic">lacZ</Emphasis>/hah Transposon Mutagenesis in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

The ISlacZ/hah transposon carried by pIT2 and derived originally from Tn5 has been a popular system in the generation of random insertion mutants of Pseudomonas aeruginosa. Using this system in the current study, two transconjugants were identified as conferring high levels of carbenicillin resistance. Analyses by gene complementation tests and site-specific gene knockout experiments support the conclusion that carbenicillin resistance in these two mutants is not due to the insertion of ISlacZ/hah transposon into the affected genes. Instead, the production of a TEM β-lactamase was detected, and integration of the bla gene from pIT2 to the chromosome of the recipient strain was confirmed by polymerase chain reaction. This surprising event was reproducible, with an estimated frequency among the transconjugants of 4% to 10%, and it may cause a potential complication in the interpretation of mutant phenotypes without notice.     

HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from <Emphasis Type="Italic">P. aeruginosa</Emphasis> cells

To establish a novel strategy of P. aeruginosa control, we acquired recombination HNP-3 by gene recombination. Then we examined HNP-3 bio-activities and the influences of antimicrobial peptide on the efflux of ATP. Consequently, we obtained target protein with a molecular mass of 3,000 D consistent with the Anticipation. FIC index of Ciprofloxacin added HNP-3 was less than 0.5 and HNP3 synergistically cooperated with CIP to suppress P. aeruginosa colony formation revealed that there was significant synergy. ATP efflux was however up-regulated by low concentrations of HNP-3, although CIP did not exert any influence on ATP efflux. Conclusively, recombination protein HNP-3 displayed antimicrobial and synergic effects. HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from P. aeruginosa cells and decreasing efflux of the drugs, which could have useful clinical applications.     

A convenient method for multiple insertions of desired genes into target loci on the <Emphasis Type="Italic">Escherichia coli</Emphasis> chromosome

We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and β-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF ^fbr and tyrA ^fbr or aroF ^fbr and pheA ^fbr ) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 Resistance to Zinc Pyrithione: Phenotypic Changes Suggest the Involvement of Efflux Pumps

The aim of this study is to investigate the involvement of an efflux pump in the development of Pseudomonas aeruginosa resistance to zinc pyrithione (ZnPT). In the presence of efflux inhibitor carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), the minimum inhibitory concentration of ZnPT for P. aeruginosa resistant cells is reduced significantly (p < 0.05). In addition, the concentration of ZnPT excluded by the resistant bacteria was reduced significantly (p < 0.01). However, the above reductions did not reach the levels measured for P. aeruginosa PAO1 sensitive strain. Furthermore, such changes in P. aeruginosa resistant cells were correlated with the overexpression of outer membrane proteins, reduced sensitivity toward imipenem (p < 0.01) and increased sensitivity toward sulphatriad and chloramphenicol (p < 0.05). In a continuation to a previous study, we conclude that P. aeruginosa resistance to ZnPT is multifactorial and involves induced efflux systems. Suzanne Abdel Malek is currently on leave from Petra University, and a member at the Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.     

Identification of Two Multidrug-Resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Clonal Lineages with a Countrywide Distribution in Hungary

The aim of this study was to identify class 1 integrons from extended-spectrum and metallo-β-lactamase-negative, multidrug-resistant Pseudomonas aeruginosa clinical isolates from Hungary and to characterize the isolates by phenotypic and molecular methods. Fourteen selected P. aeruginosa isolates resistant to ceftazidime, gentamicin, and ciprofloxacin were subjected to serotyping, random amplification of polymorphic DNA (RAPD), integron content analysis, and a phenotypic test to detect high-level production of AmpC. Four representative isolates were further analyzed by multilocus sequence typing. Two P. aeruginosa multidrug-resistant clonal lineages were identified with a countrywide distribution. The first lineage is characterized by serotype O4, RAPD genotype A, sequence type ST175, and the presence of a class 1 integron harbouring aadB and aadA13 gene cassettes in its variable region. The second lineage is characterized by serotype O6, RAPD genotype B, sequence type ST395, and a class 1 integron carrying a single aadB cassette. The corresponding isolates were recovered from altogether 11 towns in Hungary. ST175 and ST395 are the presently calculated founders of two distinct P. aeruginosa clonal complexes that appear to have a wide geographical distribution also outside Hungary. The multidrug-resistant phenotype associated with these two clonal lineages might have contributed to an increase in their frequency and to their subsequent diversification. Both P. aeruginosa lineages displayed ≥8-fold synergy with boronic acid/ceftazidime combinations, suggesting an AmpC-mediated resistance to ceftazidime. Our observations underscore the role of class 1 integrons in the spread of aminoglycoside resistance by clonal dissemination among P. aeruginosa clinical isolates in Hungary.     

Effects of Brazilian green propolis extract on planktonic cells and biofilms of multidrug-resistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa

Abstract

Propolis could represent an alternative therapeutic agent for targeting multidrug-resistant bacteria due to its antimicrobial potential. The effect of Brazilian green propolis (BGP) aqueous extract (AqExt) was evaluated on eight multidrug-resistant clinical strains of Klebsiella pneumoniae and Pseudomonas aeruginosa, as well as on one reference strain for each bacterial species. The minimum bactericidal concentration (MBC) was determined and optimal concentrations were further evaluated in comparison with 0.12% chlorhexidine. The natural extract was chemically characterized by HPLC-DAD analysis. The MBC values ranged between 3.12 and 27.5?mg ml^?1. Analysis of bacterial metabolic activity after treatment for 5?min with BGP-AqExt revealed a strong antimicrobial potential, similar to chlorhexidine. The extract comprised several active compounds including quercetin, gallic acid, caffeic and p-coumaric acid, drupani, galangin, and artepillin C. Altogether, the findings suggest that BGP-AqExt is fast and effective against multidrug-resistant strains of K. pneumoniae and P. aeruginosa in planktonic cultures and biofilms.     

Gene-specific silencing induced by parallel complementary RNA in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

To investigate whether parallel complementary RNA (pRNA) could induce gene-specific silencing in Pseudomonas aeruginosa, pRNA of the mexA gene was expressed in it. Compared to the control strains, the strain expressing pRNA of mexA showed a 50% decrease in minimum inhibitory concentrations (MICs) of several antimicrobial agents and a twofold increase in the initial accumulation rate of ethidium bromide, all of which are substrates of the MexAB-OprM efflux pump. These results suggest that gene-specific silencing was induced by pRNA. This is the first time that such a route for gene silencing has been reported in a bacterium other than Escherichia coli. Gene-specific silencing induced by pRNA may be useful as a novel biotechnology tool for gene regulation in prokaryotes.     

Effect of the proton motive force inhibitor carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP) on Pseudomonas aeruginosa biofilm development

Aims: Proton motive force (PMF) inhibition enhances the intracellular accumulation of autoinducers possibly interfering with biofilm formation. We evaluated the effect of the PMF inhibitor carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP) on Pseudomonas aeruginosa biofilm development. Methods and Results: Four epidemiologically unrelated P. aeruginosa isolates were studied. A MexAB‐oprM overproducing strain was used as control. Expression of gene mexB was examined and biofilm formation after incubation with 0, 12·5 and 25 μmol l^?1 of CCCP was investigated. Mean values of optical density were analysed with one‐way analysis of variance and t‐test. Two isolates subexpressed mexB gene and only 25 μmol l^?1 of CCCP affected biofilm formation. Biofilms of the other two isolates and control strain PA140 exhibited significantly lower absorbance (P ranging from <0·01 to <0·05) with either 12·5 or 25 μmol l^?1 of CCCP. Conclusions: The PMF inhibitor CCCP effect was correlated with the expression of MexAB‐OprM efflux system and found to compromise biofilm formation in P. aeruginosa. Significance and Impact of the Study: These data suggest that inhibition of PMF‐dependent trasporters might decrease biofilm formation in P. aeruginosa.     

Influence of <Emphasis Type="Italic">Pseudomonas aeruginosa pvdQ</Emphasis> Gene on Altering Antibiotic Susceptibility Under Swarming Conditions

In Pseudomonas aeruginosa PAO1, the pvdQ gene has been shown to have at least two functions. It encodes the acylase enzyme and hydrolyzes 3-oxo-C12-HSL, the key signaling molecule of quorum sensing system. In addition, pvdQ is involved in swarming motility. It is required for up-regulated during swarming motility, which is triggered by high cell densities. As high-density bacterial populations also display elevated antibiotic resistance, studies have demonstrated that swarm-cell differentiation in P. aeruginosa promotes increased resistance to various antibiotics. PvdQ acts as a signal during swarm-cell differentiation, and thus may play a role in P. aeruginosa antibiotic resistance. The aim of this study is to examine whether pvdQ was involved in modifying antibiotic susceptibility during swarming conditions, and to investigate the mechanism by which this occurred. We constructed the PAO1pMEpvdQ strain, which overproduced PvdQ. PAO1pMEpvdQ promotes swarming motility, while PAO1ΔpvdQ abolishes swarming motility. In addition, both PAO1 and PAO1pMEpvdQ acquired resistance to ceftazidime, ciprofloxacin, meropenem, polymyxin B, and gentamicin, though PAO1pMEpvdQ exhibited a two to eightfold increase in antibiotic resistance compared to PAO1. These results indicate that pvdQ plays an important role in elevating antibiotic resistance via swarm-cell differentiation and possibly other mechanisms as well. We analyzed outer membrane permeability. Our data also suggest that pvdQ decreases P. aeruginosa outer membrane permeability, thereby elevating antibiotic resistance under swarming conditions. Our results suggest new approaches for reducing P. aeruginosa resistance.     

Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR

In Pseudomonas aeruginosa many of the clinically relevant resistance mechanisms result from changes in gene expression as exemplified by the Mex drug efflux pumps, the AmpC beta-lactamase and the carbapenem-specific porin OprD. We used quantitative real-time-PCR to analyze the expression of these genes in susceptible and antibiotic-resistant laboratory and clinical strains. In nalB mutants, which overexpress OprM, we observed a four- to eightfold increase in the expression of mexA, mexB, and oprM genes. MexX and mexY genes were induced eight to 12 times in the presence of 2 mg L(-1) tetracycline. The mexC/oprJ and mexE/oprN gene expression levels were increased 30- to 250-fold and 100- to 760-fold in nfxB and nfxC mutants, respectively. We further found that in defined laboratory strains expression levels of ampC and oprD genes paralleled beta-lactamase activity and OprD protein levels, respectively. Our data support the use of quantitative real-time-PCR chain reaction for the analysis of the antimicrobial resistance gene expression in P. aeruginosa.     

Molecular investigation of carbapenem resistance among multidrug‐resistant Pseudomonas aeruginosa isolated clinically in Thailand

Carbapenem resistant Pseudomonas aeruginosa were isolated among multidrug‐resistant (CR‐MDR) organisms from tertiary hospitals in Thailand. Decreased expression of oprD mRNA (93.65%) was predominant followed by increased expression of mexAB‐oprM mRNA (92.06%) and mexXY mRNA (63.49%). Interestingly, 23 of 126 (18.25%) isolates were susceptible to imipenem with down‐regulated oprD expression and non‐up‐regulated mexCD‐oprJ mRNA expression. Metallo‐β‐lactamases production was clearly positive in 24 isolates (18.46%) and weakly positive in 12 isolates (9.23%). Among both of these sets of isolates, imp‐1, imp‐14 and vim‐2 were identified. Hyperproduction of AmpC β‐lactamase had the lowest prevalence rate (3.97%). It was concluded that CR‐MDR P. aeruginosa clinical isolates in Thailand possess multifactorial resistance mechanisms.     

Isolation and transposon mutagenesis of a Pseudomonas putida KT2442 toluene-resistant variant: involvement of an efflux system in solvent resistance

A toluene-resistant variant of Pseudomonas putida KT2442, strain TOL, was isolated after liquid cultivation under xylene followed by toluene for 1 month in each condition. Almost all the populations of the variant strain formed small but readily visible colonies under toluene within 24 h at 30°C. The toluene-resistant strain also showed an increase in resistance to some unrelated antibiotics. Several toluene-sensitive Tn5 mutants have been isolated from the toluene-resistant strain and showed various levels of sensitivity. Most of these mutations did not cause significant changes in antibiotic resistance; however, one of the mutants (TOL-4) was highly susceptible to both organic solvents and various antibiotics, especially β-lactams. Sequencing analysis revealed that the mutation in TOL-4 had been introduced into a gene that may encode a transporter protein of an efflux system. This efflux system is very similar to one of the multidrug efflux systems of Pseudomonas aeruginosa. These observations indicate that a multidrug efflux system plays a major role in the organic solvent resistance of P. putida TOL. However, several other genes may also be involved. Received: December 18, 1997 / Accepted: March 16, 1998     

Investigation of saturated and aromatic hydrocarbon resistance mechanisms in Pseudomonas aeruginosa IBBML1

Pseudomonas aeruginosa IBB_ML1, isolated from Poeni petroleum sludge, was able to tolerate and degrade both saturated (n-hexane, n-heptane, n-hexadecane, cyclohexane) and aromatic (benzene, ethylbenzene, propylbenzene, xylene isomers, styrene) hydrocarbons. Molecular studies have revealed that the high hydrocarbon resistance of Pseudomonas aeruginosa IBB_ML1 could be due to the action of members of the HAE1 (hydrophobe/amphiphile efflux 1) family of transporters. It is further possible that additional mechanisms may account for the tolerance of Pseudomonas aeruginosa IBB_ML1 to hydrocarbons, and a combination of short-term and long-term mechanisms may act together in the adaptation of Pseudomonas aeruginosa IBB_ML1 cells to saturated and aromatic hydrocarbons. β-galactosidase activity measurements revealed that there was significant induction of the lacZ gene in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of either 5% and 10% (v/v) saturated or aromatic hydrocarbons, compared with control (cells incubated without hydrocarbons). Rhodamine 6G accumulation in Pseudomonas aeruginosa IBB_ML1 cells grown in the presence of 5% and 10% (v/v) saturated hydrocarbons was higher than rhodamine 6G accumulation in cells grown in the presence of 5% and 10% (v/v) aromatic hydrocarbons. The study of cellular and molecular modifications to Pseudomonas aeruginosa IBB_ML1 induced by 5% and 10% (v/v) saturated and aromatic hydrocarbons revealed a complex response of bacterial cells to the presence of different hydrophobic substrates in the culture medium.     

Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr

Succinate dehydrogenase (SDH) of Escherichia coli, the sole membrane-bound enzyme of the tricarboxylic acid cycle, participates in the aerobic electron-transport pathway to generate energy via oxidative phosphorylation reactions. Previous studies have established that succinate dehydrogenase (SDiH) synthesis is elevated by aerobiosis and supressed during growth with glucose. To examine how the sdhCDAB genes that encode SDH are regulated by changes in the environment, sdh–lacZ fusions were constructed and analysed in vivo following cell growth under a variety of alternative culture conditions. Expression of sdh–lacZ was highest under aerobic conditions and was decreased 10-foid in the absence of oxygen. The fnr and arcA gene products are required for this oxygen control and each acts to repress sdhC–lacZ expression. Expression of sdh–lacZ also varied 10- to 14-foid depending on the type of carbon substrate used or the medium richness. This control was shown to be independent of the crp and fruR gene products, and indicates that some other regulatory element exists in the ceil to adjust SDH enzyme levels accordingly. Iron and haem availability affected sdhC–lacZ expression by two- to threefold. Lastly, fold. Lastly, sdhC–lacZ expression was shown to vary with the cell growth rate during aerobic and anaerobic conditions.