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Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C.

We found that, under iron-limiting conditions, Aeromonas hydrophila ATCC 7966T could utilize the xenosiderophore desferrioxamine B (DFOB) for growth by inducing the expression of its own outer membrane receptor. Two consecutive genes, desR and desA, were selected as candidates involved in DFOB utilization. The presence of the ferric-uptake regulator boxes in their promoters suggested that these genes are under iron-dependent regulation. Mutation of desA, a gene that encodes the outer membrane receptor of ferrioxamine B, disrupted the growth of the amonabactin-deficient mutant in the presence of DFOB. β-Galactosidase reporter assays and reverse transcriptase-quantitative PCR demonstrated that desR, a gene that encodes an AraC-like regulator homolog is required for the induction of desA transcription in the presence of DFOB and under iron-limiting conditions. The functions of desA and desR were analyzed using complementation experiments. Our data provided evidence that DesA is powered primarily by the TonB2 system.

Acinetobacter haemolyticus ATCC 17906(T) is known to produce the siderophore acinetoferrin under iron-limiting conditions. Here, we show that an operon consisting of eight consecutive genes, named acbABCD and actBCAD, participates in the biosynthesis and transport of acinetoferrin, respectively. Transcription of the operon was found to be iron-regulated by a putative Fur box located in the promoter region of the first gene, acbA. Homology searches suggest that acbABCD and actA encode enzyme proteins involved in acinetoferrin biosynthesis and an outer-membrane receptor for ferric acinetoferrin, respectively. Mutants defective in acbA and actA were unable to produce acinetoferrin or to express the ferric acinetoferrin receptor under iron-limiting conditions. These abilities were rescued by complementation of the mutants with native acbA and actA genes. Secondary structure analysis predicted that the products of actC and actD may be inner-membrane proteins with 12 membrane-spanning helices that belong to the major facilitator superfamily proteins. ActC showed homology to Sinorhizobium meliloti RhtX, which has been characterized as an inner-membrane importer for ferric rhizobactin 1021 structurally similar to acinetoferrin. Compared to the parental ATCC 17906(T) strain, the actD mutant displayed about a 35 % reduction in secretion of acinetoferrin, which was restored by complementation with actD, suggesting that ActD acts as an exporter of the siderophore. Finally, the actB product was significantly similar to hypothetical proteins in certain bacteria, in which genes encoding ActBCA homologues are arranged in the same order as in A. haemolyticus ATCC 17906(T). However, the function of ActB remains to be clarified.

Aeromonas hydrophila ATCC 7966T produces a catecholate siderophore amonabactin in response to iron starvation. In this study, we determined that this strain utilizes exogenously supplied enterobactin (Ent) for growth under iron-limiting conditions. A homology search of the A. hydrophila ATCC 7966T genomic sequence revealed the existence of a candidate gene encoding a protein homologous to Vibrio parahaemolyticus IrgA that functions as the outer membrane receptor for ferric Ent. SDS–PAGE showed induction of IrgA under iron-limiting conditions. The growth of the double mutant of irgA and entA (one of the amonabactin biosynthetic genes) was restored when it was complemented with irgA in the presence of Ent. Moreover, a growth assay of three isogenic tonB mutants indicated that the tonB2 system exclusively provides energy for IrgA to transport ferric Ent. Finally, reverse transcriptase-quantitative PCR revealed that the transcription of irgA and the TonB2 system cluster genes is iron-regulated, consistently w...

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator.

In this study, we found that Acinetobacter baumannii utilized exogenously supplied desferricoprogen, rhodotorulic acid, and desferrioxamine B for growth under iron-limiting conditions. The ferric uptake regulator (Fur) titration assay method was then successfully applied to select iron-regulated genes in A. baumannii genomic libraries. Part of the nucleotide sequence homologous to Escherichia coli, fhuE, obtained from one of the positive clones allowed us to clone the entire gene, which was named fhuE. The fhuE gene had an amino acid sequence consistent with the N-terminal amino acid sequence of the 76-kDa iron-repressible outer membrane proteins in A. baumannii. Reverse transcription-polymerase chain reaction analysis demonstrated that fhuE mRNA is transcribed under iron-limiting conditions, consistent with the presence of a sequence homologous to the consensus Fur box in the promoter region. Disruption of fhuE resulted in the loss of expression of the 76-kDa protein. In addition, the double disruptant of fhuE and basD, which encodes one of the biosynthetic genes for the cognate siderophore acinetobactin, was unable to grow in the presence of desferricoprogen, rhodotorulic acid or desferrioxamine B. However, growth of the double disruptant was restored by complementation with fhuE, demonstrating that A. baumannii FhuE functions as the receptor common to coprogen, ferric rhodotorulic acid and ferrioxamine B.

Gabexate (GB), a serine protease inhibitor that is widely used for the treatment of acute pancreatitis and disseminated intravascular coagulation, has been reported to be partly hydrolyzed by human serum albumin. However, other enzymes responsible for GB hydrolysis in human blood remain unclear. In this study, we examined in vitro metabolism of GB with human blood samples. The hydrolytic activities of the plasma and erythrocytes at 100µM of GB were 167 ± 26 and 151 ± 9n mol/min/ml blood fraction (mean ± S.D., n = 8), respectively. Kinetic analysis indicated that Vmax and Km values were 1.75µmol/min/ml blood fraction and 529µ Mf or the plasma and 10.6µmol/min/ml blood fraction and 7360µ Mf or the erythrocytes, respectively. The activity of human plasma wa si nhibited by ethopropazine, a butyrylcholinesterase inhibitor (27% inhibition at 100µM). Furthermore, the plasma activity was inhibited by 5,5 � -dithiobis(2-nitrobenzoic acid) (DTNB) (40% inhibition at 5000µM), suggesting the involvement of albumin in the plasma GB hydrolysis. The erythrocyte activity was also decreased by DTNB (56% inhibition at 5000µM), implying that this activity was dependent on the presence of sulfhydryl group(s), while little or no inhibition of the activity was seen with phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, and BW284C51. Butyrylcholinesterase from human serum showed GB hydrolytic activity with Vmax of 363 nmol/min/m gp rotein and Km of 263µM. These results suggest that, in addition to albumin, butyrylcholinesterase and erythrocyte sulfhydryl-dependent enzyme are responsible for GB hydrolysis in human blood.

Metabolism of nafamostat, a clinically used serine protease inhibitor, was investigated with human blood and liver enzyme sources. All the enzyme sources examined (whole blood, erythrocytes, plasma and liver microsomes) showed nafamostat hydrolytic activity. V(max) and K(m) values for the nafamostat hydrolysis in erythrocytes were 278 nmol/min/mL blood fraction and 628 microM; those in plasma were 160 nmol/min/mL blood fraction and 8890 microM, respectively. Human liver microsomes exhibited a V(max) value of 26.9 nmol/min/mg protein and a K(m) value of 1790 microM. Hydrolytic activity of the erythrocytes and plasma was inhibited by 5, 5'-dithiobis(2-nitrobenzoic acid), an arylesterase inhibitor, in a concentration-dependent manner. In contrast, little or no suppression of these activities was seen with phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), bis(p-nitrophenyl)phosphate (BNPP), BW284C51 and ethopropazine. The liver microsomal activity was markedly inhibited by PMSF, DFP and BNPP, indicating that carboxylesterase was involved in the nafamostat hydrolysis. Human carboxylesterase 2 expressed in COS-1 cells was capable of hydrolyzing nafamostat at 10 and 100 microM, whereas recombinant carboxylesterase 1 showed significant activity only at a higher substrate concentration (100 microM). The nafamostat hydrolysis in 18 human liver microsomes correlated with aspirin hydrolytic activity specific for carboxylesterase 2 (r=0.815, p

The properties of ES46.5K, an esterase from mouse hepatic microsomes, were compared with those of carboxylesterases from rabbit and porcine liver. The inhibitory profile with a serine hydrolase inhibitor (bis-p-nitrophenylphosphate) and detergents (sodium dodecylsulfate, Emulgen 911) was different between ES46.5K and the carboxylesterases. Bis-p-nitrophenylphosphate (0.1 mM) markedly inhibited the catalytic activity of the carboxylesterases but not that of ES46.5K. Emulgen 911 (0.05-0.25%) inhibited the catalytic activity of the carboxylesterases, whereas the detergent conversely stimulated that of ES46.5K by 150%. The two carboxylesterases catalyzed the hydrolysis of acetate esters of tetrahydrocannabinol (THC) analogues with different side chain lengths (C1-C5), although ES46.5K showed marginal activity only against the acetate of Delta8-tetrahydrocannabiorcol, a methyl side chain derivative of Delta8-THC. ES46.5K hydrolyzed cannabinoid esters stereospecifically and regioselectively. The esterase hydrolyzed 8alpha-acetoxy-Delta9-tetrahydrocannabinol (8alpha-acetoxy-Delta9-THC, 5.62 nmol/min/mg protein), while the enzyme did not hydrolyze 8beta-acetoxy-Delta9-THC, 7alpha-acetoxy-, and 7beta-acetoxy-Delta8-THC at all. In contrast, the carboxylesterases from rabbit and porcine liver hydrolyzed 8beta-acetoxy-Delta9-THC efficiently but not 8alpha-acetoxy-Delta9-THC. ES46.5K hydrolyzed side chain acetoxy derivatives of Delta8-THC at the 3'- and 4'-positions, and a methyl ester of 5'-nor-Delta8-THC-4'-oic acid. The enzyme, however, could not hydrolyze methyl esters of Delta8- and Delta9-THC-11-oic acid, while both carboxylesterases hydrolyzed side chain acetoxy derivatives of Delta8-THC and three methyl esters of THC-oic acids. These differences in stereospecificity and regioselectivity between ES46.5K and carboxylesterases suggest that the configurations of important amino acids for the catalytic activities of these enzymes are different from each other.

To assess the safety of the food flavoring agents, methyl anthranilate (MA) and methyl N-methylanthranilate (MNMA), their relationships with metabolism and allergenicity were examined in guinea pigs. Both MA and MNMA were hydrolyzed to anthranilic acid (AA) and N-methylanthranilic acid (N-methylAA), respectively, by guinea pig liver microsomes. These hydrolytic activities at 1000 µM were 320 and 35 nmol/min/mg protein, respectively. Moreover, MNMA was N-demethylated to MA by the liver microsomes; the oxidative activity at 1000 µM of MNMA was 2.8 nmol/min/mg protein. The N-demethylase activity for N-methylAA at 1000 µM in the liver microsomes was 3.9 nmol/min/mg protein. Kinetic analysis indicated that the Vmax/Km values of MA and MNMA hydrolyses were 15and 7.4-fold greater in guinea pig liver microsomes than in the cytosol, respectively, suggesting that these hydrolytic activities were predominantly localized in the microsomes. Liver microsomal activities for the hydrolysis of these flavoring esters were markedly inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and bis(pnitrophenyl)phosphate but not by physostigmine. These hydrolytic activities were suppressed by aspirin, a substrate of carboxylesterase, in a concentration-dependent manner. The N-demethylations of MNMA and N-methylAA were inhibited by SKF 525-A, a nonselective inhibitor of cytochrome P450. At the same time as the metabolic study described above, skin reactions in guinea pigs were investigated using MA, MNMA, N-methylAA, and AA. All compounds examined elicited positive skin reactions, although MNMA and AA exhibited relatively greater sensitizing properties. These results may provide useful information about metabolism in the toxicologic evaluations of MA and MNMA.

Microsomal alcohol oxygenase catalyzes the stereoselective oxidation of 7alpha- and 7beta-hydroxy-delta8-tetrahydrocannabinol (7alpha- and 7beta-hydroxy-delta8-THC) to 7-oxo-delta8-THC in monkey liver, and the activity for 7beta-hydroxy-delta8-THC is relatively higher than that for 7alpha-hydroxy-delta8-THC. We previously reported that purified P450JM-E, assumed to be CYP3A8, is a major enzyme responsible for the oxidation of 7-hydroxy-delta8-THC to 7-oxo-delta8-THC in monkey liver and is capable of catalyzing the oxidative reaction by NADH as well as NADPH. In the present study, we demonstrated that some steroids such as testosterone and progesterone stimulated both the NADH- and NADPH-dependent conversions of 7beta-hydroxy-delta8-THC to 7-oxo-delta8-THC in monkey liver microsomes. Kinetic analyses revealed that both the NADH- and NADPH-dependent 7-oxo-delta8-THC formation showed sigmoid kinetics. Testosterone caused a decrease in S50 and an increase in V(max) for the NADH-dependent activity, and resulted in a decrease in S50 without changing the V(max) for the NADPH-dependent activity. On the other hand, NADH-dependent testosterone 6beta-hydroxylation activity showed Michaelis-Menten kinetics and was also inhibited by 7beta-hydroxy-delta8-THC, resulting in a decrease in V(max) with no effect on the K(m). NADPH-dependent testosterone 6beta-hydrozylation activity was also inhibited by 7beta-hydroxy-delta8-THC, resulting in a decrease in both S50 and V(max). In order to explain the metabolic interaction between 7beta-hydroxy-delta8-THC and testosterone, we propose a kinetic model involving at least three binding sites, for the mechanism of activation by testosterone.

In order to assimilate iron,Acinetobacter baumanniiATCC 19606Tproduces a siderophore named acinetobactin (Ab) that is composed of equimolar quantities of 2,3-dihydroxybenzoic acid (DHBA),l-threonine andN-hydroxyhistamine. Application of the Fur titration assay system toA. baumanniigenomic libraries, followed by further cloning of the regions surrounding the candidate genes, led to the identification of the Ab cluster, which harbours the genetic determinants necessary for the biosynthesis and transport of the siderophore. However, anentAhomologue essential for DHBA biosynthesis was not found in this cluster. Functions of potential biosynthetic genes inferred by homology studies suggested that the precursors, DHBA,l-threonine andN-hydroxyhistamine, are linked in steps resembling those of bacterial non-ribosomal peptide synthesis to form Ab. Genes responsible for the two-step biosynthesis ofN-hydroxyhistamine from histidine were also identified in this cluster. Their genetic organization suggests that five genes involved in the transport system of ferric Ab into the cell cytosol form an operon. Construction of disruptants of some selected genes followed by phenotypic analysis supported their predicted biological functions. Interestingly, three additional genes probably involved in the intracellular release of iron from ferric Ab and the secretion of nascent Ab are contained in this cluster. Primer extension and RT-PCR analyses suggested that the Ab cluster, which includes 18 genes, is organized in seven transcriptional units originating from respective Fur-regulated promoter-operator regions.

Hepatic microsomes from human liver catalyzed oxidation of the allyl aldehydes such as 11-oxo-Delta(8)-tetrahydrocannabinol and 9-anthraldehyde to the corresponding carboxylic acid metabolites. The oxygenation mechanism was confirmed by GC-MS that molecular oxygen was exclusively incorporated into Delta(8)-tetrahydrocannabinol-11-oic acid and 9-anthracene carboxylic acid formed under oxygen-18 gas. The microsomal aldehyde oxygenase (named MALDO) activities of 11-oxo-Delta(8)-tetrahydrocannabinol and 9-anthraldehyde were significantly inhibited by the antibody against CYP2C and CYP3A, respectively. MALDO activity for 11-oxo-Delta(8)-tetrahydrocannabinol was significantly inhibited by sulfaphenazole whereas that for 9-anthraldehyde was markedly inhibited by troleandomycin, but not by sulfaphenazole. CYP2C9 expressed in human B-lymphoblastoid cells catalyzed efficiently the MALDO activity for 11-oxo-Delta(8)-tetrahydrocannabinol (10.1 nmol/min/nmol P450), while the catalytic activities of other human CYPs expressed in the cells were lesser extents. In MALDO activity for 9-anthraldehyde, CYP3A4 expressed in the cells had the highest catalytic activity (7.72 nmol/min/nmol P450). These results indicate that CYP2C9 and CYP3A4 are major enzymes responsible for the MALDO activity in human liver for 11-oxo-Delta(8)-tetrahydrocannabinol and 9-anthraldehyde, respectively.

Metabolism of cannabinol (CBN) was studied in vitro using hepatic microsomes from human livers. The metabolites formed were analyzed by thinlayer chromatography (TLC) and identified by gas chromatography-mass spectrometry as their trimethylsilyl derivatives. 11-Hydroxy-CBN, the major metabolite, was detected together with a smaller amount of another mono-hydroxylated metabolite. The minor metabolite was identified as 8-hydroxy-CBN, after comparing its Rf value by TLC, retention time by GC, and the mass spectrum with those of the authentic compound. 8-Hydroxy-CBN was confirmed to be a new metabolite of CBN formed by human hepatic microsomes.

The present authors have previously reported that Vibrio mimicus expresses 77-kDa and 80-kDa outer membrane proteins in response to iron-limited conditions, and that the 77-kDa protein serves as the receptor for ferriaerobactin. In this study, it was found that V. mimicus can use heme and hemoglobin as iron sources. FURTA was then applied to V. mimicus 7PT to obtain candidate gene fragments involved in utilization of heme and hemoglobin. One FURTA-positive clone was shown to contain a partial gene, whose predicted amino acid sequence correlated with the N-terminal amino acid sequence determined for the 80-kDa outer membrane protein and also shared homology with heme/hemoglobin receptors of Gram-negative bacteria. Based on this information, the entire gene (named mhuA), and a gene upstream of mhuA (named mhuB) encoding a LysR family of transcriptional activator, were cloned and analyzed. RNA analysis indicated that mhuA and mhuB are each transcribed from individual Fur-regulated promoters. Moreover, RNA analysis of an mhuB deletion mutant and a promoter reporter assay coupled with β-galactosidase suggested that MhuB could function as an activator for mhuA transcription. Finally, the role of MhuA as the heme/hemoglobin receptor was confirmed by construction of an mhuA deletion mutant and its complemented strain followed by growth assay.

Although the authors recently reported that nafamostat, a clinically used serine protease inhibitor, was mainly hydrolysed by carboxylesterase in human liver microsomes, the involvement of human liver cytosol has not been elucidated. The current study examined the in vitro metabolism of nafamostat with human liver cytosols. Kinetic analysis indicated that the Vmax and Km values in the liver cytosols were 9.82 nmolmin(-1) mg(-1) protein and 197 microM for a liver sample HL-1, and 15.1 nmolmin(-1) mg(-1) protein and 157 microM for HL-2, respectively. The Vmax/Km values in both cytosols were at least threefold higher than those in the corresponding microsomes. The liver cytosolic activity for nafamostat hydrolysis was inhibited by phenylmethylsulfonyl fluoride (PMSF) (43% inhibition at 100 microM), whereas diisopropyl fluorophosphate (DFP) and bis(p-nitrophenyl)phosphate (BNPP) failed to inhibit the activity. Furthermore, the hydrolytic activity was also reduced by palmitoyl-CoA (67% inhibition at 100 microM) but not by acetyl-CoA. Effects of PMSF, DFP and BNPP on cytosolic palmitoyl-CoA hydrolytic activity were comparable with those of the cytosolic nafamostat hydrolytic activity. In addition, the palmitoyl-CoA hydrolytic activity was competitively inhibited by nafamostat with the apparent Ki value of 164 microM for the liver cytosol from HL-2. These results suggest that an isoform of long-chain acyl-CoA hydrolase may be responsible for the nafamostat hydrolysis in human liver cytosol.

In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, l -alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named pvsABCDE , constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore. Another gene in the same operon, pvsC , encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named pvuBCDE , encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA . Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.