Construction of a Temperature-Sensitive Shuttle Vector between Lactic Acid Bacteria and Escherichia coli and Its Application to a Tn10-Based Random Mutagenesis Tool in Lactic Acid Bacteria.

In the present study, we have obtained a temperature-sensitive replication mutant in the Escherichia (E.) coli-lactic acid bacterium (LAB) shuttle vector pLES003-b carrying erythromycin-resistance gene by error-prone PCR technique. Among 858 clones obtained in the construction of the random mutation libraries of pLES003-b in the ori and repA regions, three clones could grow normally at 28 °C but not at 42 °C. One of the clones was designated as pLES003-b TS1. The sequencing analysis of pLES003-b TS1 revealed that the plasmid has four substitution mutations (376G > A, 435A > T, 914C > A, and 1996T > A) and one insertional mutation (1806_1807insA). Among those mutations, substitution mutation 914C > A, which leads to a CGC-to-AGC codon change at position 44 of the RepA protein (arginine-to-serine substitution mutation: R44S in RepA), was predicted to be a cause of temperature sensitivity. Therefore, the C-to-A substitution was introduced into the repA gene in pLES003-b using a site-directed mutagenesis method, and the resultant plasmid was electroporated into a Lactobacillus (L.) plantarum cell. The resultant transformant cannot grow at 42 °C in the presence of erythromycin, which is used as a selective marker, indicating that the R44S point mutation in the RepA protein may be crucial for temperature sensitivity. Furthermore, we have developed a new plasmid as an efficient genetic engineering tool for random insertional mutagenesis in LABs using a combination of transposon Tn10 and the temperature-sensitive replication system in pLES003-b. The resultant plasmid vector, which was designated pLES-Tn10-TS1, would be useful for genetic analysis of the functional molecule in lactic acid bacterial strains.

Expression of Genes Involved in Bacteriocin Production and Self-Resistance in Lactobacillus brevis 174A Is Mediated by Two Regulatory Proteins.

We have previously shown that the lactic acid bacterium Lactobacillus brevis 174A, isolated from Citrus iyo fruit, produces a bacteriocin designated brevicin 174A, which is comprised of two antibacterial polypeptides (designated brevicins 174A-ß and 174A-γ). We have also found a gene cluster, composed of eight open reading frames (ORFs), that contains genes for the biosynthesis of brevicin 174A, self-resistance to its own bacteriocin, and two transcriptional regulatory proteins. Some lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth. Generally, the system consists of a membrane-bound histidine protein kinase (HPK) that senses a specific environmental stimulus and a corresponding response regulator (RR) that mediates the cellular response. We have previously shown that although the HPK- and RR-encoding genes are not found on the brevicin 174A biosynthetic gene cluster in the 174A strain, two putative regulatory genes, designated breD and breG, are in the gene cluster. In the present study, we demonstrate that the expression of brevicin 174A production and self-resistance is positively controlled by two transcriptional regulatory proteins, designated BreD and BreG. BreD is expressed together with BreE as the self-resistance determinant of L. brevis 174A. DNase I footprinting analysis and a promoter assay demonstrated that BreD binds to the breED promoter as a positive autoregulator. The present study also demonstrates that BreG, carrying a transmembrane domain, binds to the common promoter of breB and breC, encoding brevicins 174A-ß and 174A-γ, respectively, for positive regulation.IMPORTANCE The problem of the appearance of bacteria that are resistant to practical antibiotics and the increasing demand for safe foods have increased interest in replacing conventional antibiotics with bacteriocin produced by the lactic acid bacteria. This antibacterial substance can inhibit the growth of pathogenic bacteria without side effects on the human body. The bacteriocin that is produced by a Citrus iyo-derived Lactobacillus brevis strain inhibits the growth of pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, and Streptococcus mutans In general, lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth, which is called a quorum-sensing system. The system consists of a membrane-bound histidine protein kinase that senses a specific environmental stimulus and a corresponding response regulator that mediates the cellular response. The present study demonstrates that the expression of the genes encoding bacteriocin biosynthesis and the self-resistance determinant is positively controlled by two transcriptional regulatory proteins.

Characterization of the SN35N Strain-Specific Exopolysaccharide Encoded in the Whole Circular Genome of a Plant-Derived Lactobacillus plantarum.

Lactobacillus plantarum SN35N, which has been previously isolated from pear, secretes exopolysaccharide (EPS). The aim of the present study is to characterize the EPS chemically and to find the EPS-biosynthesizing gene cluster. The present study demonstrates that the strain produces an acidic EPS carrying phosphate residue, which is composed of glucose, galactose, and mannose at a molecular ratio of 15.0 : 5.7 : 1.0. We also show that acidic EPS strongly inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35), promoting an inflammatory reaction. In the present study, we also determined the complete genome sequence of the SN35N strain, demonstrating that the genome is a circular DNA with 3267626 bp, and the number of predicted coding genes is 3146, with a GC content of 44.51%. In addition, the strain harbors four plasmids, designated pSN35N-1, -2, -3, and -4. Although four EPS-biosynthesizing genes, designated lpe1, lpe2, lpe3, and lpe4, are present in the SN35N chromosomal DNA, another EPS gene cluster, lpe5, is located in the pSN35N-3 plasmid, composed of 35425 bp. EPS low-producing mutants, which were obtained by treating SN35N cells with novobiocin, lost the lpe5 gene cluster in the plasmid-curing experiment, suggesting that the gene cluster for the biosynthesis of acidic EPS is present in the plasmid. The present study shows the chemical characterization of the acidic EPS and its inhibitory effect to the hyaluronidase.

Uric Acid in the Follow-Up Determines 30% Decline in Estimated GFR Over 2 Years: a Propensity Score Analysis.

BACKGROUND/AIMS: Higher level of serum uric acid (SUA) predicts early entry to dialysis in chronic kidney disease (CKD) patients. However, a short-term effect of SUA remains to be elucidated using a novel surrogate endpoint. METHODS: Japanese CKD stage 3 to 4 patients were retrospectively examined (n= 701). The follow-up level of SUA was estimated as time-averaged uric acid (TA-UA). A propensity score for 6.0, 6.5 or 7.0 mg/dL of TA-UA was respectively calculated using baseline 23 covariates. The time-to-event analysis was performed for 30% decline in estimated GFR over 2 years. RESULTS: Incidence rates over 2 years were 90 of 440 in men and 36 of 261 in women (p = 0.03). Despite the negative result of baseline SUA, stratified Cox regression on the quintiles of the estimated propensity score showed that higher TA-UA of the three thresholds were all significant (crude HR 2.10 to 2.44) even after adjusting for the confounders. Kaplan-Meier analysis after propensity score matching likewise showed worse survival in the patients with the higher TA-UA (HR 3.11 to 4.26). CONCLUSION: Higher SUA increases likelihood of reaching a surrogate endpoint over 2 years. Early intervention for SUA less than 6.0 mg/dL is recommended for slowing CKD progression.

Characterization of Exopolysaccharides Produced by Thermophilic Lactic Acid Bacteria Isolated from Tropical Fruits of Thailand.

In the present study, we have obtained two exopolysaccharide (EPS)-producing thermophilic lactic acid bacteria (LAB) that were isolated from tropical fruits of Thailand. The two strains, designated LY45 and PY45, were identified as Pediococcus pentosaceus and Lactobacillus amylovorus, respectively. Both plant-derived LAB strains, which produce neutral EPSs together with the acidic one, can grow vigorously at 45°C and even at 50°C. Hyaluronidase (EC 3.2.1.35), which catalyzes the degradation of hyaluronic acid, activates an inflammatory reaction. Interestingly, EPSs produced by the LY45 and PY45 strains were found to inhibit hyaluronidase activity at the same order of IC50 values as did sodium cromoglicate and dipotassium glycyrrhizinate, which are well-known as anti-inflammatory agents. The LY45-derived neutral EPS consists of glucose and mannose as monosaccharide components, whereas the acidic one contains mainly mannose, together with glucose and galactose. On the other hand, although Lactobacillus amylovorus PY45 also produces neutral and acidic EPSs, the main monosaccharide in both EPSs is mannose, and glucose is a minor component. Furthermore, the PY45 strain may be probiotically and industrially useful because the microorganism can utilize starch and glycogen as carbon sources.

Time to target uric acid to retard CKD progression.

Uric acid (UA) remains a possible risk factor of chronic kidney disease (CKD) but its potential role should be elucidated given a fact that multidisciplinary treatments assure a sole strategy to inhibit the progression to end-stage renal disease (ESRD). In clinical setting, most observational studies showed that elevation of serum uric acid (SUA) independently predicts the incidence and the development of CKD. The meta-analysis showed that SUA-lowering therapy with allopurinol may retard the progression of CKD but did not reach conclusive results due to small-sized studies. Larger scale, randomized placebo-controlled trials to assess SUA-lowering therapy are needed. Our recent analysis by propensity score methods has shown that the threshold of SUA should be less than 6.5 mg/dL to abrogate ESRD. In animal models an increase in SUA by the administration of oxonic acid, uricase inhibitor, or nephrectomy can induce glomerular hypertension, arteriolosclerosis including afferent arteriolopathy and tubulointerstitial fibrosis. The ever-growing discoveries of urate transporters prompt us to learn UA metabolism in the kidney and intestine. One example is that the intestinal ABCG2 may play a compensatory role at face of decreased renal clearance of UA in nephrectomized rats, the trigger of which is not a uremic toxin but SUA itself. This review will summarize the recent knowledge on the relationship between SUA and the kidney and try to draw a conclusion when and how to treat asymptomatic hyperuricemia accompanied by CKD. Finally we will address a future perspective on UA study including a Mendelian randomization approach.

Stimulation of V1a receptor increases renal uric acid clearance via urate transporters: insight into pathogenesis of hypouricemia in SIADH.

BACKGROUND: Hypouricemia is pathognomonic in syndrome of inappropriate secretion of antidiuretic hormone (SIADH) but the underlying mechanism remains unclear. Based on the previous studies, we hypothesized that V1a receptor may play a principal role in inducing hypouricemia in SIADH and examined uric acid metabolism using a rat model. METHODS: Terlipressin (25 ng/h), a selective V1a agonist, was subcutaneously infused to 7-week-old male Wistar rats (n = 9). Control rats were infused with normal saline (n = 9). The rats were sacrificed to obtain kidney tissues 3 days after treatment. In addition to electrolyte metabolism, changes in expressions of the urate transporters including URAT1 (SLC22A12), GLUT9 (SLC2A9), ABCG2 and NPT1 (SLC17A1) were examined by western blotting and immunohistochemistry. RESULTS: In the terlipressin-treated rats, serum uric acid (UA) significantly decreased and the excretion of urinary UA significantly increased, resulting in marked increase in fractional excretion of UA. Although no change in the expression of URAT1, GLUT9 expression significantly decreased whereas the expressions of ABCG2 and NPT1 significantly increased in the terlipressin group. The results of immunohistochemistry corroborated with those of the western blotting. Aquaporin 2 expression did not change in the medulla, suggesting the independence of V2 receptor stimulation. CONCLUSION: Stimulation of V1a receptor induces the downregulation of GLUT9, reabsorption urate transporter, together with the upregulation of ABCG2 and NPT1, secretion urate transporters, all changes of which clearly lead to increase in renal UA clearance. Hypouricemia seen in SIADH is attributable to V1a receptor stimulation.

Time-dependent risk factors associated with the decline of estimated GFR in CKD patients.

BACKGROUND: Targeting the modifiable risk factors may help halt the progression of CKD, thus risk factor analysis is better performed using the parameters in the follow-up. This study aimed to examine the time-dependent risk factors for CKD progression using time-averaged values and to investigate the characteristics of rapid progression group. METHODS: This is a retrospective cohort study enrolling 770 patients of CKD stage 3-4. Time-dependent parameters were calculated as time-averaged values by a trapezoidal rule. % decline of estimated GFR (eGFR) per year from entry was divided to three groups: <10% (stable), 10-25% (moderate progression), and ≥25% (rapid progression). Multivariate regression analyses were employed for the baseline and the time-averaged datasets. RESULTS: eGFR decline was 2.83 ± 4.04 mL/min/1.73 m(2)/year (8.8 ± 12.9 %) in male and 1.66 ± 3.23 mL/min/1.73 m(2)/year (5.4 ± 11.0%) in female (p < 0.001). % decline of eGFR was associated with male, proteinuria, phosphorus, and systolic blood pressure as risk factors and with age, albumin, and hemoglobin as protective factors using either dataset. Baseline eGFR and diabetic nephropathy appeared in the baseline dataset, while uric acid appeared in the time-averaged dataset. The rapid progression group was associated with proteinuria, phosphorus, albumin, and hemoglobin in the follow-up. CONCLUSION: These results suggest that time-averaged values provide insightful clinical guide in targeting the risk factors. Rapid decline of eGFR is strongly associated with hyperphosphatemia, proteinuria, and anemia indicating that these risk factors should be intervened in the follow-up of CKD.

Protein tyrosine phosphatase 1B inhibition protects against podocyte injury and proteinuria.

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed nonreceptor protein-tyrosine phosphatase that regulates various cellular functions, including migration. Recent studies suggest that an increased migratory phenotype of podocytes may be responsible for proteinuria and foot process effacement. The current study addresses the role of PTP1B in podocyte injury and proteinuria. PTP1B was markedly up-regulated in the glomerulus, notably in podocytes, in three rodent models of podocyte injury. Podocyte-specific ablation of the PTP1B gene ameliorated proteinuria induced by lipopolysaccharide and Adriamycin (doxorubicin). The use of a specific PTP1B inhibitor also protected against lipopolysaccharide-induced proteinuria. In contrast, podocyte-specific PTP1B transgenic male mice developed spontaneous proteinuria and foot process effacement. In cultured mouse podocytes, PTP1B knockdown and/or pretreatment with the PTP1B inhibitor blunted lipopolysaccharide-induced cell migration, activation of Src-family kinases (SFKs), and phosphorylation of focal adhesion kinase at Y397 (pFAK(Y397)), the latter being crucial for cell migration. Lipopolysaccharide-injected mice showed increased glomerular expression of active SFKs and pFAK(Y397), both of which were inhibited by podocyte-specific PTP1B knockout and the PTP1B inhibitor. Moreover, podocyte-specific PTP1B transgenic mice showed increased glomerular expression of active SFKs and pFAK(Y397). In summary, PTP1B up-regulation in podocytes induces a migratory response by activating SFKs and FAK, leading to foot process effacement and proteinuria. Pharmacological inhibition of PTP1B may have therapeutic potential in the treatment of proteinuric diseases.

High-Level Heterologous Production of D-Cycloserine by Escherichia coli.

Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 µM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 µM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 µM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 µM).

Characterization and Mutational Analysis of a Two-Polypeptide Bacteriocin Produced by Citrus Iyo-Derived Lactobacillus brevis 174A.

In the present study, we isolated a lactic acid bacterium (LAB) from a citrus iyo fruit and identified it as Lactobacillus brevis. This plant-derived LAB strain, designated 174A, produces bacteriocin consisting of two polypeptides designated brevicin 174A-ß and 174A-γ. Although each polypeptide itself displays antibacterial activity, the ability is enhanced 100 fold by mixing both polypeptides at a 1 : 1 ratio. Significantly, brevicin 174A inhibits even the growth of several pathogenic bacteria that are more resistant to a lantibiotic bacteriocin, nisin A, which is commonly utilized as a preservative added to foodstuffs. Structural analysis of the 174A bacteriocin using a program that predicts secondary structure suggests that both component polypeptides have a positively charged N-terminal region, as well as two cysteine residues in both the N- and C-terminals. Judging from a mutational analysis of the antibacterial polypeptides, these unique amino acid sequences of 174A-ß might be important for the expression of the synergistic activity that occurs in the presence of the two polypeptides combined.

Crystallographic study to determine the substrate specificity of an L-serine-acetylating enzyme found in the D-cycloserine biosynthetic pathway.

DcsE, one of the enzymes found in the d-cycloserine biosynthetic pathway, displays a high sequence homology to l-homoserine O-acetyltransferase (HAT), but it prefers l-serine over l-homoserine as the substrate. To clarify the substrate specificity, in the present study we determined the crystal structure of DcsE at a 1.81-Å resolution, showing that the overall structure of DcsE is similar to that of HAT, whereas a turn region to form an oxyanion hole is obviously different between DcsE and HAT: in detail, the first and last residues in the turn of DcsE are Gly(52) and Pro(55), respectively, but those of HAT are Ala and Gly, respectively. In addition, more water molecules were laid on one side of the turn region of DcsE than on that of HAT, and a robust hydrogen-bonding network was formed only in DcsE. We created a HAT-like mutant of DcsE in which Gly(52) and Pro(55) were replaced by Ala and Gly, respectively, showing that the mutant acetylates l-homoserine but scarcely acetylates l-serine. The crystal structure of the mutant DcsE shows that the active site, including the turn and its surrounding waters, is similar to that of HAT. These findings suggest that a methyl group of the first residue in the turn of HAT plays a role in excluding the binding of l-serine to the substrate-binding pocket. In contrast, the side chain of the last residue in the turn of DcsE may need to form an extensive hydrogen-bonding network on the turn, which interferes with the binding of l-homoserine.

Crystallographic and mutational analyses of tannase from Lactobacillus plantarum.

Tannin acylhydrolase (EC 3.1.1.20) referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannins to release gallic acid. Although the enzyme is useful for various industries, the tertiary structure is not yet determined. In this study, we determined the crystal structure of tannase produced by Lactobacillus plantarum. The tannase structure belongs to a member of α/ß-hydrolase superfamily with an additional "lid" domain. A glycerol molecule derived from cryoprotectant solution was accommodated into the tannase active site. The binding manner of glycerol to tannase seems to be similar to that of the galloyl moiety in the substrate.

Establishment of an in vitro D-cycloserine-synthesizing system by using O-ureido-L-serine synthase and D-cycloserine synthetase found in the biosynthetic pathway.

We have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic, D-cycloserine. The gene cluster is composed of 10 open reading frames, designated dcsA to dcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization of O-ureidoserine. DcsD is similar to O-acetylserine sulfhydrylase, which generates L-cysteine using O-acetyl-L-serine with sulfide, and therefore, DcsD may be a synthase to generate O-ureido-L-serine using O-acetyl-L-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase converting O-ureido-D-serine into D-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed in Escherichia coli and purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substrates O-acetyl-L-serine and hydroxyurea, synthesis of D-cycloserine was successfully attained. These in vitro studies yield the conclusion that DcsD and DcsG are necessary for the syntheses of O-ureido-L-serine and D-cycloserine, respectively. DcsD was also able to catalyze the synthesis of L-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclic d-amino acid analogs, such as D-homocysteine thiolactone.

Whole-Genome and Plasmid Comparative Analysis of Campylobacter jejuni from Human Patients in Toyama, Japan, from 2015 to 2019.

Campylobacter jejuni is a major causative agent of food poisoning, and increasing antimicrobial resistance is a concern. This study investigated 116 clinical isolates of C. jejuni from Toyama, Japan, which were isolated from 2015 to 2019. Antimicrobial susceptibility testing and whole-genome sequencing were used for phenotypic and genotypic characterization to compare antimicrobial resistance (AMR) profiles and phylogenic linkage. The multilocus sequence typing approach identified 37 sequence types (STs) and 15 clonal complexes (CCs), including 7 novel STs, and the high frequency CCs were CC21 (27.7%), CC48 (10.9%), and CC354 (9.9%). The AMR profiles and related resistant factors were as follows: fluoroquinolones (51.7%), mutation in quinolone resistance-determining region (QRDRs) (GyrA T86I); tetracyclines (27.6%), acquisition of tet(O); ampicillin (7.8%), harboring blaOXA184 or a promoter mutation in blaOXA193; aminoglycosides (1.7%), acquisition of ant(6)-Ia and aph(3')-III; chloramphenicol (0.9%), acquisition of cat. The acquired resistance genes tet(O), ant(6)-Ia, aph(3')-III, and cat were located on pTet family plasmids. Furthermore, three pTet family plasmids formed larger plasmids that incorporated additional genes such as the type IV secretion system. Sequence type 4526 (ST4526; 10.9%), which is reported only in Japan, was the most predominant, suggesting continued prevalence. This study reveals the sequences of the pTet family plasmids harbored by C. jejuni in Japan, which had been unclear, and the acquisition of the insertion sequences in a part of the pTet family plasmids. Because pTet family plasmids can be horizontally transmitted and are a major factor in acquired resistance in Campylobacter, the risk of spreading pTet that has acquired further resistance should be considered. IMPORTANCE Campylobacter jejuni is among the major causes of enteritis and diarrhea in humans in many countries. Drug-resistant Campylobacter is increasing in both developing and developed countries, and in particular, fluoroquinolone-resistant Campylobacter was one of the species included on the priority list of antibiotic-resistant bacteria. Campylobacter drug resistance surveillance is important and has been conducted worldwide. In this study, we performed whole-genome analysis of Campylobacter jejuni isolated from diarrhea patients at a hospital in Toyama, Japan. This revealed the continued prevalence of Campylobacter jejuni ST4526, which has been reported to be prevalent in Japan, and the acquisition of resistance and virulence factors in the pTet family plasmids. The diversity of pTet family plasmids, the major resistance transmission factor, is expected to potentially increase the risk of Campylobacter. The usefulness of whole-genome sequencing in Campylobacter surveillance was also demonstrated.

Effects of the order of exposure to antimicrobials on the incidence of multidrug-resistant Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa (MDRP) is one of the most important pathogens in clinical practice. To clarify the mechanisms contributing to its emergence, we isolated MDRPs using the P. aeruginosa PAO1, the whole genome sequence of which has already been elucidated. Mutant strains resistant to carbapenems, aminoglycosides, and new quinolones, which are used to treat P. aeruginosa infections, were isolated; however, none met the criteria for MDRPs. Then, PAO1 strains were exposed to these antimicrobial agents in various orders and the appearance rate of MDRP varied depending on the order of exposure; MDRPs more frequently appeared when gentamicin was applied before ciprofloxacin, but were rarely isolated when ciprofloxacin was applied first. Exposure to ciprofloxacin followed by gentamicin increased the expression of MexCD-OprJ, an RND-type multidrug efflux pump, due to the NfxB mutation. In contrast, exposure to gentamicin followed by ciprofloxacin resulted in more mutations in DNA gyrase. These results suggest that the type of quinolone resistance mechanism is related to the frequency of MDRP and that the risk of MDRP incidence is highly dependent on the order of exposure to gentamicin and ciprofloxacin.

A molecular mechanism for copper transportation to tyrosinase that is assisted by a metallochaperone, caddie protein.

The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated "caddie," which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16-1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.

Heme protein and hydroxyarginase necessary for biosynthesis of D-cycloserine.

We have recently cloned a D-cycloserine (DCS) biosynthetic gene cluster that consists of 10 genes, designated dcsA~dcsJ, from Streptomyces lavendulae ATCC 11924 (16). In the predicted pathway of hydroxyurea (HU) formation in DCS biosynthesis, L-arginine (L-Arg) must first be hydroxylated, prior to the hydrolysis of N(ω)-hydroxy-L-arginine (NHA) by DcsB, an arginase homolog. The hydroxylation of L-Arg is known to be catalyzed by nitric oxide synthase (NOS). In this study, to verify the supply route of HU, we created a dcsB-disrupted mutant, ΔdcsB. While the mutant lost DCS productivity, its productivity was restored by complementation of dcsB, and also by the addition of HU but not NHA, suggesting that HU is supplied by DcsB. A NOS-encoding gene, nos, from S. lavendulae chromosome was cloned, to create a nos-disrupted mutant. However, the mutant maintained the DCS productivity, suggesting that NOS is not necessary for DCS biosynthesis. To clarify the identity of an enzyme necessary for NHA formation, a dcsA-disrupted mutant, designated ΔdcsA, was also created. The mutant lost DCS productivity, whereas the DCS productivity was restored by complementation of dcsA. The addition of NHA to the culture medium of ΔdcsA mutant was also effective to restore DCS production. These results indicate that the dcsA gene product, DcsA, is an enzyme essential to generate NHA as a precursor in the DCS biosynthetic pathway. Spectroscopic analyses of the recombinant DcsA revealed that it is a heme protein, supporting an idea that DcsA is an enzyme catalyzing hydroxylation.

Catalytic mechanism of bleomycin N-acetyltransferase proposed on the basis of its crystal structure.

Bleomycin (Bm) N-acetyltransferase, BAT, is a self-resistance determinant in Bm-producing Streptomyces verticillus ATCC15003. In our present study, we crystallized BAT under both a terrestrial and a microgravity environment in the International Space Station. In addition to substrate-free BAT, the crystal structures of BAT in a binary complex with CoA and in a ternary complex with Bm and CoA were determined. BAT forms a dimer structure via interaction of its C-terminal domains in the monomers. However, each N-terminal domain in the dimer is positioned without mutual interaction. The tunnel observed in the N-terminal domain of BAT has two entrances: one that adopts a wide funnel-like structure necessary to accommodate the metal-binding domain of Bm, and another narrow entrance that accommodates acetyl-CoA (AcCoA). A groove formed on the dimer interface of two BAT C-terminal domains accommodates the DNA-binding domain of Bm. In a ternary complex of BAT, BmA(2), and CoA, a thiol group of CoA is positioned near the primary amine of Bm at the midpoint of the tunnel. This proximity ensures efficient transfer of an acetyl group from AcCoA to the primary amine of Bm. Based on the BAT crystal structure and the enzymatic kinetic study, we propose that the catalytic mode of BAT takes an ordered-like mechanism.

Genome Sequence-Guided Finding of Lucensomycin Production by Streptomyces achromogenes Subsp. streptozoticus NBRC14001.

Information on microbial genome sequences is a powerful resource for accessing natural products with significant activities. We herein report the unveiling of lucensomycin production by Streptomyces achromogenes subsp. streptozoticus NBRC14001 based on the genome sequence of the strain. The genome sequence of strain NBRC14001 revealed the presence of a type I polyketide synthase gene cluster with similarities to a biosynthetic gene cluster for natamycin, which is a polyene macrolide antibiotic that exhibits antifungal activity. Therefore, we investigated whether strain NBRC14001 produces antifungal compound(s) and revealed that an extract from the strain inhibited the growth of Candida albicans. A HPLC analysis of a purified compound exhibiting antifungal activity against C. albicans showed that the compound differed from natamycin. Based on HR-ESI-MS spectrometry and a PubChem database search, the compound was predicted to be lucensomycin, which is a tetraene macrolide antibiotic, and this prediction was supported by the results of a MS/MS analysis. Furthermore, the type I polyketide synthase gene cluster in strain NBRC14001 corresponded well to lucesomycin biosynthetic gene cluster (lcm) in S. cyanogenus, which was very recently reported. Therefore, we concluded that the antifungal compound produced by strain NBRC14001 is lucensomycin.