Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance

Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.     

A tetracycline efflux gene on Bacteroides transposon Tn4400 does not contribute to tetracycline resistance.

Previously, we demonstrated that the Bacteroides transposon Tn4351, which confers tetracycline resistance only on aerobically grown Escherichia coli, carries a gene that codes for a tetracycline-inactivating enzyme (B. S. Speer and A. A. Salyers, J. Bacteriol. 170:1423-1429, 1988). However, Park et al. (B. H. Park, M. Hendricks, M. H. Malamy, F. P. Tally, and S. B. Levy, Antimicrob. Agents Chemother. 31:1739-1743, 1987) showed that E. coli carrying a closely related transposon, Tn4400, exhibits energy-dependent efflux of tetracycline as well as tetracycline-inactivating activity (B. H. Park and S. B. Levy, Antimicrob. Agents Chemother. 32:1797-1800, 1988). This result raised the question of whether efflux or inactivation or a combination of the two was necessary for resistance conferred by both transposons. We showed that cells carrying Tn4351 did not exhibit the clear-cut efflux activity seen with cells carrying Tn4400 but rather exhibited a tetracycline accumulation profile which could be explained solely on the basis of inactivation of tetracycline in the cytoplasm and rapid diffusion of altered tetracycline out of the cell. Additionally, we were able to clone the efflux and tetracycline-modifying genes of Tn4400 separately. The region carrying the efflux gene spanned one of the two regions in which Tn4400 differs from Tn4351. A clone containing the corresponding region of Tn4351 did not exhibit efflux. Thus, it appears that Tn4351 does not have the efflux gene and that efflux makes no contribution to the resistance conferred by Tn4351. The MIC for cells carrying the subclone from Tn4400 that contained only the gene for tetracycline inactivation was the same that for cells carrying both the inactivation and efflux genes. Cells carrying only the gene for tetracycline efflux were tetracycline sensitive. This was true even when the efflux gene was on a high-copy-number plasmid which increased the level of efflux to that associated with the Tcr gene on pBR328. These results indicate that efflux activity does not contribute significantly to the tetracycline resistance conferred by Tn4400.     

SR-BI mediates cholesterol efflux via its interactions with lipid-bound ApoE. Structural mutations in SR-BI diminish cholesterol efflux

Apolipoprotein E (apoE) and the lipoprotein receptor SR-BI play critical roles in lipid and lipoprotein metabolism. We have examined the cholesterol efflux from wild-type (WT) and mutant forms of SR-BI expressed in ldlA-7 cells using reconstituted discoidal particles consisting of apoE, 1-palmitoyl-2-oleoyl-l-phospatidylcholine (POPC), and cholesterol (C) as acceptors. POPC/C-apoE particles generated using apoE2, apoE3, apoE4, or carboxy-terminally truncated forms apoE4-165, apoE4-202, apoE4-229, and apoE4-259 caused similar (20-25%) cholesterol efflux from WT SR-BI. Cholesterol efflux mediated by POPC/C-apoE was not enhanced in the presence of lipid-free apoE. The rate of cholesterol efflux mediated by particles containing the WT or carboxy-terminally truncated forms of apoE was decreased to approximately 30% of the WT control with the Q402R/Q418R mutant SR-BI form that is unable to bind native HDL normally but binds LDL. The rate of cholesterol efflux was further decreased to approximately 7% of the WT control with another SR-BI mutant (M158R) that binds neither HDL nor LDL. The level of binding of POPC/C-apoE particles (150 microg/mL) to SR-BI mutant forms Q402R/Q418R and M158R was 70 and 8% of the WT control, respectively. SR-BI-dependent binding of lipid-free apoE to cells was undetectable, and cholesterol efflux was less than 0.5%. The findings establish that only lipid-bound apoE promotes SR-BI-mediated cholesterol efflux and that the amino-terminal region of residues 1-165 of apoE is sufficient for both receptor binding and cholesterol efflux. The SR-BI-apoE interactions may contribute to overall cholesterol homeostasis in cells and tissues that express SR-BI and apoE.     

An H(+)-coupled multidrug efflux pump, PmpM, a member of the MATE family of transporters, from Pseudomonas aeruginosa

We cloned the gene PA1361 (we designated the gene pmpM), which seemed to encode a multidrug efflux pump belonging to the MATE family, of Pseudomonas aeruginosa by the PCR method using the drug-hypersensitive Escherichia coli KAM32 strain as a host. Cells of E. coli possessing the pmpM gene showed elevated resistance to several antimicrobial agents. We observed energy-dependent efflux of ethidium from cells possessing the pmpM gene. We found that PmpM is an H(+)-drug antiporter, and this finding is the first reported case of an H(+)-coupled efflux pump in the MATE family. Disruption and reintroduction of the pmpM gene in P. aeruginosa revealed that PmpM is functional and that benzalkonium chloride, fluoroquinolones, ethidium bromide, acriflavine, and tetraphenylphosphonium chloride are substrates for PmpM in this microorganism.     

Evidence that phosphate specific transporter is amplified in a fluoroquinolone resistant Mycobacterium smegmatis.

We reported in an earlier study that active efflux of drug has a predominant role in conferring resistance in a laboratory-generated ciprofloxacin-resistant mutant of Mycobacterium smegmatis. This mutant exhibited mRNA level overexpression, as well as chromosomal amplification, of the gene pstB, encoding the putative ATPase subunit of phosphate specific transport (Pst) system. We demonstrate here that this mutant shows enhanced phosphate uptake and that inactivation of pstB in the parental strain results in loss of high affinity phosphate uptake and hypersensitivity to fluoroquinolones. These findings suggest a novel role of the Pst system in active efflux, in addition to its involvement in phosphate transport.     

Roles of MexXY- and MexAB-multidrug efflux pumps in intrinsic multidrug resistance of Pseudomonas aeruginosa PAO1

To envisage the roles of MexXY- and MexAB-multidrug efflux pumps in the intrinsic multidrug resistance of wild-type strain Pseudomonas aeruginosa PAO1, we constructed mutants lacking either individual or both efflux pumps. A mutant lacking MexXY showed increased susceptibility to aminoglycosides, erythromycin, and tetracycline, but not to beta-lactams, chloramphenicol, or quinolones. A mutant lacking MexAB showed increased susceptibility to beta-lactams, chloramphenicol, and nalidixic acid, but not to aminoglycosides, erythromycin, tetracycline, or fluoroquinolones. A mutant lacking both MexXY and MexAB showed an increased susceptibility to all antimicrobial agents tested compared with the wild type. Very similar results were obtained with a mutant lacking MexAB-OprM and a mutant lacking both MexXY and MexAB-OprM. Thus it is clear that OprM is essential not only for the function of MexAB, but also for the function of MexXY. Furthermore, we found that each pump compensated to some extent for the lack of another pump with respect to the common substrates (tetracycline, quinolones, and cefpirome). The introduction of a plasmid carrying the mexXY genes into P. aeruginosa PAO1 cells increased the resistance to fluoroquinolones. This suggests that the mexXY genes could be involved in acquired resistance to fluoroquinolones in P. aeruginosa PAO1.     

Salt-inducible multidrug efflux pump protein in the moderately halophilic bacterium Chromohalobacter sp

It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.     

Involvement of a novel efflux system in biofilm-specific resistance to antibiotics

Bacteria growing in biofilms are more resistant to antibiotics than their planktonic counterparts. How this transition occurs is unclear, but it is likely there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. We have identified a novel efflux pump in Pseudomonas aeruginosa that is important for biofilm-specific resistance to a subset of antibiotics. Complete deletion of the genes encoding this pump, PA1874 to PA1877 (PA1874-1877) genes, in an P. aeruginosa PA14 background results in an increase in sensitivity to tobramycin, gentamicin, and ciprofloxacin, specifically when this mutant strain is growing in a biofilm. This efflux pump is more highly expressed in biofilm cells than in planktonic cells, providing an explanation for why these genes are important for biofilm but not planktonic resistance to antibiotics. Furthermore, expression of these genes in planktonic cells increases their resistance to antibiotics. We have previously shown that ndvB is important for biofilm-specific resistance (T. F. Mah, B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A. O'Toole, Nature 426:306-310, 2003). Our discovery that combining the ndvB mutation with the PA1874-1877 gene deletion results in a mutant strain that is more sensitive to antibiotics than either single mutant strain suggests that ndvB and PA1874-1877 contribute to two different mechanisms of biofilm-specific resistance to antibiotics.     

Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide

Pyrazinamide (PZA) is an important antituberculosis drug. Unlike most antibacterial agents, PZA, despite its remarkable in vivo activity, has no activity against Mycobacterium tuberculosis in vitro except at an acidic pH. M. tuberculosis is uniquely susceptible to PZA, but other mycobacteria as well as nonmycobacteria are intrinsically resistant. The role of acidic pH in PZA action and the basis for the unique PZA susceptibility of M. tuberculosis are unknown. We found that in M. tuberculosis, acidic pH enhanced the intracellular accumulation of pyrazinoic acid (POA), the active derivative of PZA, after conversion of PZA by pyrazinamidase. In contrast, at neutral or alkaline pH, POA was mainly found outside M. tuberculosis cells. PZA-resistant M. tuberculosis complex organisms did not convert PZA into POA. Unlike M. tuberculosis, intrinsically PZA-resistant M. smegmatis converted PZA into POA, but it did not accumulate POA even at an acidic pH, due to a very active POA efflux mechanism. We propose that a deficient POA efflux mechanism underlies the unique susceptibility of M. tuberculosis to PZA and that the natural PZA resistance of M. smegmatis is due to a highly active efflux pump. These findings may have implications with regard to the design of new antimycobacterial drugs.     

Energy-dependent efflux of cadmium coded by a plasmid resistance determinant in Staphylococcus aureus.

Resistance of Staphylococcus aureus strain 17810R to Cd2+ appears to be due to a plasmid-coded Cd2+ efflux system. Complete efflux of Cd2+ after transfer of preloaded cells into Cd2+-free medium occurred in the resistant strain 17810R, but not in the plasmidless derivative strain 17810S. Net efflux was blocked by 2,4-dinitrophenol, N,N,-dicyclohexylcarbodiimide (DCCD), and incubation at 4 degrees C. The inhibition of Cd2+ efflux by DCCD paralleled a stimulation of net uptake in the resistant cells by this agent. Cd2+ efflux by the resistant strain was accompanied by a reversal of inhibition of respiration, whereas in the sensitive strain, inhibition of respiration was not reversed after transfer to Cd2+-free medium. Net Cd2+ uptake by strain 17810R was inhibited by p-chloromercuribenzoate. In Cd2+ contrast, Cd2+ uptake by the plasmidless strain 17810S was affected neither by p-chloromercuribenzoate nor by DCCD when added alone, but was blocked by a combination of these two agents. Valinomycin had no effect on the reduced Cd2+ uptake by the resistant strain, whereas nigericin stimulated uptake to values comparable to those of the untreated sensitive cells. With sensitive cells, valinomycin reduced Cd2+ uptake by about 50%, whereas nigericin was without effect. A possible mechanism of Cd2+ movements in both strains is discussed.     

Characterization of the twin-arginine translocase secretion system of Mycobacterium smegmatis

The twin-arginine translocation (TAT) system secretes fully folded proteins that contain a twin-arginine motif within their signal sequence across the cytoplasmic membrane in bacteria. Using a green fluorescent protein fused with a TAT signal sequence, we demonstrated that Mycobacterium smegmatis contains a TAT system. By inactivating individual genes, we showed that three genes (tatA, tatB, and tatC) are required for a functional TAT system in M. smegmatis. The tat mutants exhibited a decreased growth rate and altered colony morphology compared to the parent strain. Comparison of the secreted proteins of the deltatatC and parent strain by two-dimensional polyacrylamide gel electrophoresis revealed an alteration in the secretion of at least five proteins, and one of the major TAT-dependent secreted proteins was identified as beta-lactamase (BlaS). The genome of M. smegmatis was analyzed with the TATFIND program, and 49 putative TAT substrates were identified, including the succinate transporter DctP. Because disruption of the TAT secretion system has a direct effect on the physiology of M. smegmatis and homologs of the TAT proteins are also present in the genome of Mycobacterium tuberculosis, the TAT secretion system or its substrates may be good candidates for drug or vaccine development.     

AcrD of Escherichia coli is an aminoglycoside efflux pump

AcrD, a transporter belonging to the resistance-nodulation-division family, was shown to participate in the efflux of aminoglycosides. Deletion of the acrD gene decreased the MICs of amikacin, gentamicin, neomycin, kanamycin, and tobramycin by a factor of two to eight, and DeltaacrD cells accumulated higher levels of [(3)H]dihydrostreptomycin and [(3)H]gentamicin than did the parent strain.     

Compounds of Alpinia katsumadai as potential efflux inhibitors in Mycobacterium smegmatis

Efflux pumps are one of the well established mechanisms that contribute to antibiotic resistance in bacteria, such as mycobacteria. As a result, the identification of efflux pump inhibitors is an attractive target in antimicrobial therapy. The isolated compounds, three diarylheptanoids, trans,trans-1,7-diphenylhepta-4,6-dien-3-one (1), (5R)-trans-1,7-diphenyl-5-hydroxyhept-6-en-3-one (2), (3S,5S)-trans-1,7-diphenylhept-1-ene-3,5-diol (3) and the flavonoid pinocembrin (4), from Alpinia katsumadai, Zingiberaceae, were examined for their antimycobacterial activity and their synergistic effects with different antibiotics against M. smegmatis mc(2) 155. Furthermore, these compounds were evaluated as potential EtBr efflux inhibitors. Although they showed weak antimycobacterial activities (MIC ≥ 64 mg/L), especially compound 1 revealed a significant activity on the EtBr accumulation and efflux as well as a synergistic effect in combination with rifampicin.     

Serum amyloid A promotes cholesterol efflux mediated by scavenger receptor B-I

Serum amyloid A (SAA) is an acute phase protein whose expression is markedly up-regulated during inflammation and infection. The physiological function of SAA is unclear. In this study, we reported that SAA promotes cellular cholesterol efflux mediated by scavenger receptor B-I (SR-BI). In Chinese hamster ovary cells, SAA promoted cellular cholesterol efflux in an SR-BI-dependent manner, whereas apoA-I did not. Similarly, SAA, but not apoA-I, promoted cholesterol efflux from HepG2 cells in an SR-BI-dependent manner as shown by using the SR-BI inhibitor BLT-1. When SAA was overexpressed in HepG2 cells using adenovirus-mediated gene transfer, the endogenously expressed SAA promoted SR-BI-dependent efflux. To assess the effect of SAA on SR-BI-mediated efflux to high density lipoprotein (HDL), we compared normal HDL, acute phase HDL (AP-HDL, prepared from mice injected with lipopolysaccharide), and AdSAA-HDL (HDL prepared from mice overexpressing SAA). Both AP-HDL and AdSAA-HDL promoted 2-fold greater cholesterol efflux than normal HDL. Lipid-free SAA was shown to also stimulate ABCA1-dependent cholesterol efflux in fibroblasts, in line with an earlier report (Stonik, J. A., Remaley, A. T., Demosky, S. J., Neufeld, E. B., Bocharov, A., and Brewer, H. B. (2004) Biochem. Biophys. Res. Commun. 321, 936-941). When added to cells together, SAA and HDL exerted a synergistic effect in promoting ABCA1-dependent efflux, suggesting that SAA may remodel HDL in a manner that releases apoA-I or other efficient ABCA1 ligands from HDL. SAA also facilitated efflux by a process that was independent of SR-BI and ABCA1. We conclude that the acute phase protein SAA plays an important role in HDL cholesterol metabolism by promoting cellular cholesterol efflux through a number of different efflux pathways.     

Role of Mycobacterium tuberculosis Ser/Thr kinase PknF: implications in glucose transport and cell division

Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3H-labeled and 14C-labeled substrates. A significant increase in the uptake of D-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.     

Participation of fad and mbt genes in synthesis of mycobactin in Mycobacterium smegmatis

Colonies of Mycobacterium smegmatis LR222 on iron-limiting (0.1 micro M Fe) minimal medium agar fluoresce under UV light due to the accumulation in the cells of the deferri form of the siderophore mycobactin. Two mutants with little or no fluorescence, designated LUN8 and LUN9, were isolated by screening colonies of transposon (Tn611)-mutagenized M. smegmatis. Ferrimycobactin prepared from iron-restricted cells of the wild type had an R(f) of 0.62 on high-performance thin-layer chromatography (HPTLC) and a characteristic visible absorption spectrum with a peak near 450 nm. Similar extracts from LUN8 cells contained a small amount of ferrimycobactin with an R(f) of 0.58 on HPTLC and an absorption spectrum with the peak shifted to a wavelength lower than that of the wild-type ferrimycobactin. Nuclear magnetic resonance spectroscopy studies suggested that the LUN8 mycobactin may have an altered fatty acid side chain. Mutant strain LUN9 produced no detectable mycobactin. Neither mutant strain produced measurable amounts of excreted mycobactin, although both excreted exochelin (the mycobacterial peptido-hydroxamate siderophore), and both mutants were more sensitive than the wild-type strain to growth inhibition by the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The transposon insertion sites were identified, and sequence analyses of the cloned flanking chromosome regions showed that the mutated gene in LUN9 was an orthologue of the Mycobacterium tuberculosis mycobactin biosynthetic gene mbtE. The mutated gene in LUN8 had homology with M. tuberculosis fadD33 (Rv1345), a gene that may encode an acyl-coenzyme A synthase and which previously was not known to participate in synthesis of mycobactin.     

Acceleration of glutathione efflux and inhibition of gamma-glutamyltranspeptidase sensitize metastatic B16 melanoma cells to endothelium-induced cytotoxicity

Highly metastatic B16 melanoma (B16M)-F10 cells, as compared with the low metastatic B16M-F1 line, have higher GSH content and preferentially overexpress BCL-2. In addition to its anti-apoptotic properties, BCL-2 inhibits efflux of GSH from B16M-F10 cells and thereby may facilitate metastatic cell resistance against endothelium-induced oxidative/nitrosative stress. Thus, we investigated in B16M-F10 cells which molecular mechanisms channel GSH release and whether their modulation may influence metastatic activity. GSH efflux was abolished in multidrug resistance protein 1 knock-out (MRP-/-1) B16M-F10 transfected with the Bcl-2 gene or in MRP-/-1 B16M-F10 cells incubated with l-methionine, which indicates that GSH release from B16M-F10 cells is channeled through MRP1 and a BCL-2-dependent system (likely related to an l-methionine-sensitive GSH carrier previously detected in hepatocytes). The BCL-2-dependent system was identified as the cystic fibrosis transmembrane conductance regulator, since monoclonal antibodies against this ion channel or H-89 (a protein kinase A-selective inhibitor)-induced inhibition of cystic fibrosis transmembrane conductance regulator gene expression completely blocked the BCL-2-sensitive GSH release. By using a perifusion system that mimics in vivo conditions, we found that GSH depletion in metastatic cells can be achieved by using Bcl-2 antisense oligodeoxynucleotide- and verapamil (an MRP1 activator)-induced acceleration of GSH efflux, in combination with acivicin-induced inhibition of gamma-glutamyltranspeptidase (which limits GSH synthesis by preventing cysteine generation from extracellular GSH). When applied under in vivo conditions, this strategy increased tumor cytotoxicity (up to approximately 90%) during B16M-F10 cell adhesion to the hepatic sinusoidal endothelium.     

The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a

Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of ¹⁴C-phenanthrene to radiolabeled polar metabolites and ¹⁴CO₂. The emhB ^? WEN strain produced approximately twice as much ¹⁴CO₂ and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of ¹⁴C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.     

Identification of the monooxygenase gene clusters responsible for the regioselective oxidation of phenol to hydroquinone in mycobacteria

Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.