Gene organization for nitric oxide reduction in Alcaligenes faecalis S-6

norB and norC encoding the cytochrome b-containing subunit and the cytochrome c-containing subunit, respectively, of the nitric oxide reductase (NOR) in Alcaligenes faecalis S-6 were cloned and sequenced. Both NorB and NorC showed more than 40% sequence identity to the corresponding subunits of cytochrome bc-type NORs in other denitrifying bacteria. norCB was in a gene cluster containing seven other genes; these were named dnr, orf2, orf3, norE, norF, norQ, and norD on the basis of their similarity with NOR systems in other bacteria. Potential FNR-binding sites were present in front of norCB, norEF, and/or orf2/orf3, suggesting that most of these genes are regulated simultaneously by an FNR-related protein. NorB and NorC proteins produced in the membrane fraction in Escherichia coli showed no enzyme activity, probably due to lack of NorQ and NorD, which appear to perform some essential function for activation of the NorB-NorC complex in the recombinant E. coli.     

Contributions of Aspergillus fumigatus ATP-Binding Cassette Transporter Proteins to Drug Resistance and Virulence

In yeast cells such as those of Saccharomyces cerevisiae, expression of ATP-binding cassette (ABC) transporter proteins has been found to be increased and correlates with a concomitant elevation in azole drug resistance. In this study, we investigated the roles of two Aspergillus fumigatus proteins that share high sequence similarity with S. cerevisiae Pdr5, an ABC transporter protein that is commonly overproduced in azole-resistant isolates in this yeast. The two A. fumigatus genes encoding the ABC transporters sharing the highest sequence similarity to S. cerevisiae Pdr5 are called abcA and abcB here. We constructed deletion alleles of these two different ABC transporter-encoding genes in three different strains of A. fumigatus. Loss of abcB invariably elicited increased azole susceptibility, while abcA disruption alleles had variable phenotypes. Specific antibodies were raised to both AbcA and AbcB proteins. These antisera allowed detection of AbcB in wild-type cells, while AbcA could be visualized only when overproduced from the hspA promoter in A. fumigatus. Overproduction of AbcA also yielded increased azole resistance. Green fluorescent protein fusions were used to provide evidence that both AbcA and AbcB are localized to the plasma membrane in A. fumigatus. Promoter fusions to firefly luciferase suggested that expression of both ABC transporter-encoding genes is inducible by azole challenge. Virulence assays implicated AbcB as a possible factor required for normal pathogenesis. This work provides important new insights into the physiological roles of ABC transporters in this major fungal pathogen.     

Gene structure and expression profile of cytochrome bc nitric oxide reductase from Hydrogenobacter thermophilus TK-6

The genes for a nitric oxide reductase-like cytochrome bc complex were cloned from a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6. The structural genes norC and norB, which encode cytochrome c and cytochrome b subunits of the complex respectively, are probably transcribed as a tricistronic operon with a following gene encoding a putative membrane protein. NorC has, unusually, two hydrophobic transmembrane spans in its N-terminus. Immunoblot analysis showed that expression of NorC was induced by nitrate, nitrite, or sodium nitropurusside, suggesting that the norCB gene product is a denitrification enzyme, nitric oxide reductase. The consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide-sensing regulators of proteobacteria were not found in the norC promoter region, but consensus -35 and -10 sequences were found in this region. These results indicate that strain TK-6 has a nitrogen oxide-sensing regulatory system that differs from proteobacterial systems.     

Two isofunctional nitric oxide reductases in Alcaligenes eutrophus H16.

Two genes, norB and norZ, encoding two independent nitric oxide reductases have been identified in Alcaligenes eutrophus H16. norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%. While norB resides on the megaplasmid pHG1, the norZ gene is located on a chromosomal DNA fragment. Amino acid sequence analysis suggests that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices. The region encompassing helices 3 to 14 shows similarity to the NorB subunit of common bacterial nitric oxide reductases, including the positions of six strictly conserved histidine residues. Unlike the Nor enzymes characterized so far from denitrifying bacteria, NorB and NorZ of A. eutrophus contain an amino-terminal extension which may form two additional helices connected by a hydrophilic loop of 203 amino acids. The presence of a NorB/NorZ-like protein was predicted from the genome sequence of the cyanobacterium Synechocystis sp. strain PCC6803. While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neighboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs. The physiological roles of norB and norZ in A. eutrophus were investigated with mutants disrupted in the two genes. Mutants bearing single-site deletions in norB or norZ were affected neither in aerobic nor in anaerobic growth with nitrate or nitrite as the terminal electron acceptor. Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions. Anaerobic growth was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid. These results show that the norB and norZ gene products are isofunctional and instrumental in denitrification.     

N-caffeoylphenalkylamide derivatives as bacterial efflux pump inhibitors

As part of an ongoing project to identify plant natural products as efflux pump inhibitors (EPIs), bioassay-guided fractionation of the methanolic extract of Mirabilis jalapa Linn. (Nyctaginaceae) led to the isolation of an active polyphenolic amide: N-trans-feruloyl 4'-O-methyldopamine. This compound showed moderate activity as an EPI against multidrug-resistant (MDR) Staphylococcus aureus overexpressing the multidrug efflux transporter NorA, causing an 8-fold reduction of norfloxacin MIC at 292 microM (100 microg/mL). This prompted us to synthesize derivatives in order to provide structure-activity relationships and to access more potent inhibitors. Among the synthetic compounds, some were more active than the natural compound and N-trans-3,4-O-dimethylcaffeoyl tryptamine showed potentiation of norfloxacin in MDR S. aureus comparable to that of the standard reserpine.     

The baeSR two-component regulatory system activates transcription of the yegMNOB (mdtABCD) transporter gene cluster in Escherichia coli and increases its resistance to novobiocin and deoxycholate

Screening of random fragments of Escherichia coli genomic DNA for their ability to increase the novobiocin resistance of a hypersusceptible (Delta)acrAB mutant resulted in the isolation of a plasmid containing baeR, which codes for the response regulator of the two-component regulatory system BaeSR. When induced for expression, baeR cloned in multicopy plasmid pTrc99A significantly increased the resistance of the (Delta)acrAB host strain to novobiocin (16-fold) and to deoxycholate (8-fold). Incubation of cells with novobiocin followed by a chromatographic assay for intracellular drug showed that overproduced BaeR decreased drastically the drug accumulation, presumably via increased active efflux. The genes baeSR are part of a putative operon, yegMNOB baeSR. Direct binding of BaeR to the yegM promoter was demonstrated in vitro by gel retardation assay. The gene yegB, which codes for a major facilitator superfamily transporter, was not necessary for increased resistance, but deletion of yegO or an in-frame deletion of yegN, both of which code for resistance-nodulation-cell division-type multidrug transporters, abolished the BaeR-induced increase in resistance. It is likely that both YegN and YegO produce a complex(es) with the membrane fusion protein family member YegM and pump out novobiocin and deoxycholate. We accordingly propose to rename yegMNOB as mdtABCD (mdt for multidrug transporter). Finally, the expression of two other genes, yicO and ygcL, was shown to be regulated by BaeR, but it is not known if they play any roles in resistance.     

New insights into Staphylococcus aureus stress tolerance and virulence regulation from an analysis of the role of the ClpP protease in the strains Newman, COL, and SA564

In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, and arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.     

An ABC Transporter Plays a Developmental Aggregation Role in Myxococcus xanthus

Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle. Autochemotaxis, a process whereby cells release a self-generated signaling molecule, may be the principal mechanism facilitating directed motility in both the vegetative swarming and developmental aggregation stages of this life cycle. The process requires the Frz signal transduction system, including FrzZ, a protein which is composed of two domains, both showing homology to the enteric chemotaxis response regulator CheY. The first domain of FrzZ (FrzZ1), when expressed as bait in the yeast two-hybrid system and screened against a library, was shown to potentially interact with the C-terminal portion of a protein encoding an ATP-binding cassette (AbcA). The activation domain-AbcA fusion protein did not interact with the second domain of FrzZ (FrzZ2) or with two other M. xanthus response regulator-containing proteins presented as bait, suggesting that the FrzZ1-AbcA interaction may be specific. Cloning and sequencing of the upstream region of the abcA gene showed the ATP-binding cassette to be linked to a large hydrophobic, potentially membrane-spanning domain. This domain organization is characteristic of a subgroup of ABC transporters which perform export functions. Cloning and sequencing downstream of abcA indicated that the ABC transporter is at the start of an operon containing three open reading frames. An insertion mutation in the abcA gene resulted in cells displaying the frizzy aggregation phenotype, providing additional evidence that FrzZ and AbcA may be part of the same signal transduction pathway. Cells with mutations in genes downstream of abcA showed no developmental defects. Analysis of the proposed exporter role of AbcA in cell mixing experiments showed that the ABC transporter mutant could be rescued by extracellular complementation. We speculate that the AbcA protein may be involved in the export of a molecule required for the autochemotactic process.     

Properties of a soluble domain of subunit C of a bacterial nitric oxide reductase

Bacterial nitric oxide reductases are integral membrane proteins that catalyze the reduction of two molecules of nitric oxide to nitrous oxide and water. They are diverged members of the superfamily of heme/copper oxidases. The enzyme from Paracoccus denitrificans (NorBC) contains two subunits; NorB comprises the membrane-integrated active site, which harbors a heme iron/non-heme iron dinuclear center. NorC is a membrane-anchored c-type cytochrome and presumably the site of electron uptake. A DNA construct encoding the water-soluble domain of NorC (NorC(sol)) was coexpressed with the cytochrome c maturation genes in Escherichia coli. Using redox potentiometry, electronic absorption, circular dichroism (CD), magnetic CD (MCD), nuclear magnetic resonance, and electron paramagnetic resonance (EPR) spectroscopy the following observations were made: (i) NorC(sol) was folded into a alpha-helical structure. (ii) The low-spin heme iron was coordinated by histidine and methionine in both redox states. (iii) The midpoint redox potential of the NorC(sol) heme was 183 mV, much lower than the corresponding value of 275 mV in the NorBC complex. This points to an increased solvent exposure of the NorC(sol) heme compared to in the native NorBC complex and shows that the electronic properties of NorC are modulated by NorB in the complex. (iv) The EPR and MCD spectra of NorC(sol) were considered alongside the spectra of NorBC, which has helped to resolve the contribution that different redox centers make in the holo-enzyme complex.     

Candida glabrata ATP-binding cassette transporters Cdr1p and Pdh1p expressed in a Saccharomyces cerevisiae strain deficient in membrane transporters show phosphorylation-dependent pumping properties

The expression and drug efflux activity of the ATP binding cassette transporters Cdr1p and Pdh1p are thought to have contributed to the recent increase in the number of fungal infections caused by Candida glabrata. The function of these transporters and their pumping characteristics, however, remain ill defined. We have evaluated the function of Cdr1p and Pdh1p through their heterologous hyperexpression in a Saccharomyces cerevisiae strain deleted in seven major drug efflux transporters to minimize the background drug efflux activity. Although both Cdr1p- and Pdh1p-expressing strains CDR1-AD and PDH1-AD acquired multiple resistances to structurally unrelated compounds, CDR1-AD showed, in most cases, higher levels of resistance than PDH1-AD. CDR1-AD also showed greater rhodamine 6G efflux and resistance to pump inhibitors, although plasma membrane fractions had comparable NTPase activities. These results indicate that Cdr1p makes a larger contribution than Phd1p to the reduced susceptibility of C. glabrata to xenobiotics. Both pump proteins were phosphorylated in a glucose-dependent manner. Whereas the phosphorylation of Cdr1p affected its NTPase activity, the protein kinase A-mediated phosphorylation of Pdh1p, which was necessary for drug efflux, did not. This suggests that phosphorylation of Pdh1p may be required for efficient coupling of NTPase activity with drug efflux.     

Novel Macrolide-Specific ABC-Type Efflux Transporter in Escherichia coli

In the Escherichia coli genome, five putative open reading frame (ORF) clusters, mdlAB, ybjYZ, yddA, yojHI, and yhiH, have been assumed to be possible genes for ABC drug efflux transporters (I. T. Paulsen, M. K. Sliwinski, and M. H. Saier, Jr., J. Mol. Biol. 277:573-592, 1998). We cloned all of these ORFs in multicopy plasmids and investigated the drug resistance of drug-supersensitive host cells lacking constitutive multidrug efflux transporter genes acrAB. Among them, only ybjYZ gave significant erythromycin resistance and significantly decreased the accumulation of [(14)C]erythromycin. Therefore, ybjYZ was renamed macAB (macrolide-specific ABC-type efflux carrier). Plasmids carrying both the macA and -B genes conferred resistance against macrolides composed of 14- and 15-membered lactones but no or weak resistance against 16-membered ones. Neither of the two genes produced resistance alone. The DNA sequence suggests that MacB is an integral membrane protein with four transmembrane segments and one nucleotide-binding domain, while MacA belongs to a membrane fusion protein (MFP) family with a signal-like sequence at its N terminus. The expression of the histidine-tagged proteins confirmed that MacB is an integral membrane protein and MacA is a peripheral membrane protein. In addition, MacAB required TolC for its function in a way similar to that of most of the MFP-dependent transporters in E. coli. MacB is thus a novel ABC-type macrolide efflux transporter which functions by cooperating with the MFP MacA and the multifunctional outer membrane channel TolC. This is the first case of an experimentally identified ABC antibiotic efflux transporter in gram-negative organisms.     

The Salmonella enterica sv. Typhimurium smvA,yddG and ompD (porin) genes are required for the efficient efflux of methyl viologen

In Gram-negative bacteria, a subset of inner membrane proteins in the major facilitator superfamily (MFS) acts as efflux pumps to decrease the intracellular concentrations of multiple toxic substrates and confers multidrug resistance. The Salmonella enterica sv. Typhimurium smvA gene encodes a product predicted to be an MFS protein most similar to QacA of Staphylococcus aureus. Like mutations in qacA, mutations in smvA confer increased sensitivity to methyl viologen (MV). Mutations in the adjacent ompD (porin) and yddG (drug/metabolite transporter) genes also confer increased sensitivity to MV, and mutations in smvA are epistatic to mutations in ompD or yddG for this phenotype. YddG and OmpD probably comprise a second efflux pump in which the OmpD porin acts as an outer membrane channel (OMC) protein for the efflux of MV and functions independently of the SmvA pump. In support of this idea, the pump dependent on YddG and OmpD has a different substrate specificity from the pump dependent on SmvA. Mutations in tolC, which encodes an OMC protein, confer increased resistance to MV. TolC apparently facilitates the import of MV, and a subset of OMC proteins including the OmpD porin and TolC may facilitate both import and export of distinct subsets of toxic substrates.