Cloning, sequencing, and expression in Escherichia coli of the new gene encoding beta-1,3-xylanase from a marine bacterium, Vibrio sp. strain XY-214

The Vibrio sp. strain XY-214 beta-1,3-xylanase gene cloned in Escherichia coli DH5alpha consisted of an open reading frame of 1, 383 nucleotides encoding a protein of 460 amino acids with a molecular mass of 51,323 Da and had a signal peptide of 22 amino acids. The transformant enzyme hydrolyzed beta-1,3-xylan to produce several xylooligosaccharides.     

Cloning and expression in Escherichia coli of a recA-like gene from Vibrio cholerae

Summary A library containing more than 80% of the Vibrio cholerae genome was constructed by cloning BamH1 restriction fragments into pBR322. Using interspecific complementation of an Escherichia coli recA mutant with plasmids containing the gene bank of V. cholerae, a recA-like gene was identified. The recombinant plasmid, designated as pDP145, contained a 1.45 kb segment of V. cholerae DNA which codes for a protein of molecular weight 39,000. The product of this gene confers methyl methane sulphonate resistance on the E. coli recA mutant, suppresses its ultraviolet (UV) light sensitive phenotype and has proteolytic activity on the phage repressor. Induction of a 39,000 dalton protein in UV-irradiated V. cholerae cells was demonstrated.     

Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli

A cosmid gene bank of Vibrio cholerae 395, classical Ogawa, was screened in Escherichia coli HB101 for expression of the vibrio neuraminidase (NANase) gene nanH (N-acylneuraminate glycohydrolase). Positive clones were identified by their ability to cleave the fluorogenic NANase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Seven NANase-positive clones were detected after screening 683 cosmid isolates with a rapid, qualitative plate assay method. The nanH gene was subcloned from one of the cosmids and was located within a 4.8-kilobase-pair BglII restriction endonuclease fragment. Evidence that nanH was the NANase structural gene was obtained by transposon mutagenesis and by purification and comparison of the cloned gene product with the secreted NANase purified from the parent V. cholerae strain. The sequence of the first 20 amino-terminal amino acids of the secreted NANase purified from V. cholerae was determined by automated Edman degradation and matched perfectly with the amino acid sequence predicted from nucleotide sequencing of nanH. The sequence data also revealed the existence of a potential signal peptide that was apparently processed from NANase in both V. cholerae and E. coli. In contrast to V. cholerae, E. coli nanH+ clones did not secrete NANase into the growth medium, retaining most of the enzyme in the periplasmic compartment. Kinetic studies in V. cholerae showed that nanH expression and NANase secretion were temporally correlated as cells in batch culture entered late-exponential-phase growth. Similar kinetics were observed in at least one of the E. coli nanH+ clones, suggesting that nanH expression in E. coli might be controlled by some of the same signals as in the parent V. cholerae strain.     

Cloning,expression and characterization of xylose isomerase from the marine bacterium Fulvimarina pelagi in Escherichia coli

Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed‐batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a V_max of 0.142 U/mg purified rXI, and a K_M for xylose in the range of 1.75–4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol K_I values for rXI in cell‐free extracts were 2,500 mg/L and >50 mM, respectively. The low K_M of the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca²⁺ and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher V_max at low pH (<6) and temperature (30°C) would be preferable for use in biofuels production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1230–1237, 2016     

Removal of a high load of ammonia by a marine bacterium,Vibrio alginolyticus in biofilter

A newly isolated heterotrophic marine bacterium,Vibrio alginolyticus, was used to remove a high load of ammonia gas under non-sterile condition. The cells were inoculated onto an inorganic packing material in a fixed-bed reactor (biofilter), and a high bad of ammonia, in the range of ammonia gas concentration of 170 ppm to 880 ppm, was introduced continuously. Sucrose solution and 3% NaCl was supplied intermittently to supplement the carbon source and water to the biofilter. The average percentage of gas removed exceeded 85% for 107-day operation. The maximum removal capacity and the complete removal capacity were 19 g-N kg⁻¹ dry packing material day⁻¹ and 16 g-N kg⁻¹ dry packing material day⁻¹, respectively, which were about three times greater than those obtained in nitrifying sludge inoculated onto the same packing material. On day 82, the enhanced pressure drop was restored to the normal one by NaOH treatment, and efficient removal characteristics were later observed. During this operation, the non-sterile condition had no significantly adverse effect on the removability of ammonia byV. alginolyticus.     

Cloning, sequence analysis and expression of gene alyVI encoding alginate lyase from marine bacterium Vibrio sp. QY101.

The gene (alyVI) encoding an alginate lyase of marine bacterium Vibrio sp. QY101, which was isolated from a decaying thallus of Laminaria, was cloned using a strategy of combined degenerate PCR and long range-inverse PCR (LR-IPCR), then sequenced and expressed in Escherichia coli. Gene alyVI was composed of a 1014 bp open reading frame (ORF) encoding 338 amino acid residues. The calculated molecular mass of alyVI product is 38.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 34 kDa. AlyVI was purified from culture supernatants to electrophoretic homogeneity using affinity chromatography. AlyVI was most active at pH 7.5 and 40 degrees C in the presence of 1 mM ZnCl2. A nine-amino-acid consensus region (YXRESLREM), which was only found in polyguluronate lyases, was also observed in the amino-terminal region of AlyVI. However, AlyVI could degrade both M block and G block. These results indicate that a novel alginate lyase-encoding gene has been cloned.     

Cloning of mnuA, a membrane nuclease gene of Mycoplasma pulmonis, and analysis of its expression in Escherichia coli

Membrane nucleases of mycoplasmas are believed to play important roles in growth and pathogenesis, although no clear evidence for their importance has yet been obtained. As a first step in defining the function of this unusual membrane activity, studies were undertaken to clone and analyze one of the membrane nuclease genes from Mycoplasma pulmonis. A novel screening strategy was used to identify a recombinant lambda phage expressing nuclease activity, and its cloned fragment was analyzed. Transposon mutagenesis was used to identify an open reading frame of 1,410 bp, which coded for nuclease activity in Escherichia coli. This gene coded for a 470-amino-acid polypeptide of 53,739 Da and was designated mnuA (for "membrane nuclease"). The MnuA protein contained a prolipoprotein signal peptidase II recognition sequence along with an extensive hydrophobic region near the amino terminus, suggesting that the protein may be lipid modified or that it is anchored in the membrane by this membrane-spanning region. Antisera raised against two MnuA peptide sequences identified an M. pulmonis membrane protein of approximately 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nucleolytic band of the same size. Maxicell experiments with E. coli confirmed that mnuA coded for a nuclease of unknown specificity. Hybridization studies showed that mnuA sequences are found in few Mycoplasma species, suggesting that mycoplasma membrane nucleases display significant sequence variation within the genus Mycoplasma.     

Copper-induced production of copper-binding supernatant proteins by the marine bacterium Vibrio alginolyticus

Growth of the marine bacterium Vibrio alginolyticus is temporarily inhibited by micromolar levels of copper. During the copper-induced lag phase, supernatant compounds which complex and detoxify copper are produced. In this study two copper-inducible supernatant proteins having molecular masses of ca. 21 and 19 kilodaltons (CuBP1 and CuBP2) were identified; these proteins were, respectively, 25 and 46 times amplified in supernatants of copper-challenged cultures compared with controls. Experiments in which chloramphenicol was added to cultures indicated that there was de novo synthesis of these proteins in response to copper. When supernatants were separated by gel permeation chromatography, CuBP1 and CuBP2 coeluted with a copper-induced peak in copper-binding activity. CuBP1 and CuBP2 from whole supernatants were concentrated and partially purified by using a copper-charged immobilized metal ion affinity chromatography column, confirming the affinity of these proteins for copper. A comparison of cell pellets and supernatants demonstrated that CuBP1 was more concentrated in supernatants than in cells. Our data are consistent with a model for a novel mechanism of copper detoxification in which excretion of copper-binding protein is induced by copper.     

Cloning and sequence analysis of the gene encoding 3-hexulose-6-phosphate synthase from the methylotrophic bacterium, Methylomonas aminofaciens 77a, and its expression in Escherichia coli

Abstract A DNA fragment of 550 bp was specifically amplified by PCR with primers based on the N-terminal sequence of the purified 3-hexulose-6-phosphate synthase from Methylomonas aminofaciens 77a and on that of a lysyl endopep(idase-derived peptide. Using this PCR product as a probe, a gene coding for 3-hexulose-6-phosphate synthase in M. aminofaciens 77a chromosomal DNA was cloned in Escherichia coli JM109. Sequencing analysis revealed that the gene encoding 3-hexulose-6-phosphate synthase contained a 624-bp open reading frame, encoding a protein composed of 208 amino acid residues with a calculated relative molecular mass of 21 224.     

N-ethylmaleimide desensitizes pH-dependence of K+/H+ antiporter in a marine bacterium, Vibrio alginolyticus

The K+/H+ antiporter of a marine bacterium, Vibrio alginolyticus, is strongly dependent upon the cytoplasmic pH and functions only at an internal pH above 7.7. In alkaline buffer with an outwardly directed chemical gradient of K+ (delta pK), the internal pH was maintained at about 7.7. Addition of N-ethylmaleimide (NEM) released cellular K+ and acidified the cytosol below pH 7.7. The NEM effect was reversed by the addition of 2-mercaptoethanol: K+ efflux ceased, and the internal pH returned to about 7.7. In acidic buffer, the internal pH was also regulated at about 7.6 even in the absence of delta pK. Following addition of NEM, the internal pH decreased below 7.6, dissipating delta pH. These results suggest that NEM desensitizes the pH-dependence of the K+/H+ antiporter, allowing the antiporter to function at an internal pH below 7.7.     

Cloning, sequencing, expression and characterization of the manganese superoxide dismutase gene from Vibrio alginolyticus.

The sodA gene coding for manganese superoxide dismutase from the marine microorganism Vibrio alginolyticus was cloned, sequenced and over-expressed in Escherichia coli using the pET20b (+) expression vector. The full-length gene was consisted of 603bp open reading frame, which encoded a polypeptide of 201 amino acid residues, with a calculated molecular weight of 22672Da. The deduced amino acid sequence of the sodA showed considerable homology to other Mn-SODs. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by the metal ion affinity chromatography. The recombinant VAMn-SOD resisted thermo-denaturation up to 60 degrees C and was insensitive to inhibitors such as H2O2, NaN3 and diethyldithiocarbamic acid.     

FMN-reductase from Escherichia coli and its effect on the activity of luciferase from marine bacterium Vibrio fischeri]

Interactions of luciferases isolated from Vibrio fischeri 6 and Escherichia coli JM109(pF3) (bearing cloned V. fischeri luxAB genes) with FMN reductase isolated from E. coli JM109 were studied. FMN reductase formed a stable complex with luciferase, suggesting similar properties of the FMN reductases in the taxonomically close families Vibrionaceae and Enterobacteriaceae.     

Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for a beta-mannanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum"

A lambda recombinant phage expressing beta-mannanase activity in Escherichia coli has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." The gene was cloned into pBR322 on a 5-kb BamHI fragment, and its location was obtained by deletion analysis. The sequence of a 2.1-kb fragment containing the mannanase gene has been determined. One open reading frame was found which could code for a protein of Mr 38,904. The mannanase gene (manA) was overexpressed in E. coli by cloning the gene downstream from the lacZ promoter of pUC18. The enzyme was most active at pH 6 and 80 degrees C and degraded locust bean gum, guar gum, Pinus radiata glucomannan, and konjak glucomannan. The noncoding region downstream from the mannanase gene showed strong homology to celB, a gene coding for a cellulase from the same organism, suggesting that the manA gene might have been inserted into its present position on the "C. saccharolyticum" genome by homologous recombination.     

Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli

The complete gene xyn// that encodes endo-1,4-beta-xylanase secreted by Aspergillus usamii E001 was cloned and sequenced. The coding region of the gene is separated by only one intron. It encodes 184 amino acid residues of a protein with a calculated molecular weight of 19.8kDa plus a signal peptide of 27 amino acids. The amino acid sequence of the xyn// gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expressed fusion protein was analyzed by SDS-PAGE and a new specific band with molecular weight of about 20kDa was found when induced by IPTG. Enzyme activity assay verified the recombinant protein as a xylanase. A maximum activity of 49.6Umg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn//. The xylanase had optimal activity at pH 4.6 and 50 degrees C. This is the first report on the cloning of a xylanase gene from A. usamii.     

Regulation of the Escherichia coli secA gene by protein secretion defects: analysis of secA, secB, secD, and secY mutants.

SecA protein synthesis levels were elevated 10- to 20-fold when protein secretion was blocked in secA, secD, and secY mutants or in a malE-lacZ fusion-containing strain but not in a secB null mutant. An active secB gene product was not required to derepress secA, since SecA levels were elevated during protein export blocks in secB secY and secB malE-lacZ double mutants.     

Copper-induced production of copper-binding supernatant proteins by the marine bacterium Vibrio alginolyticus.

Growth of the marine bacterium Vibrio alginolyticus is temporarily inhibited by micromolar levels of copper. During the copper-induced lag phase, supernatant compounds which complex and detoxify copper are produced. In this study two copper-inducible supernatant proteins having molecular masses of ca. 21 and 19 kilodaltons (CuBP1 and CuBP2) were identified; these proteins were, respectively, 25 and 46 times amplified in supernatants of copper-challenged cultures compared with controls. Experiments in which chloramphenicol was added to cultures indicated that there was de novo synthesis of these proteins in response to copper. When supernatants were separated by gel permeation chromatography, CuBP1 and CuBP2 coeluted with a copper-induced peak in copper-binding activity. CuBP1 and CuBP2 from whole supernatants were concentrated and partially purified by using a copper-charged immobilized metal ion affinity chromatography column, confirming the affinity of these proteins for copper. A comparison of cell pellets and supernatants demonstrated that CuBP1 was more concentrated in supernatants than in cells. Our data are consistent with a model for a novel mechanism of copper detoxification in which excretion of copper-binding protein is induced by copper.     

Subunit component and their roles in the sodium-transport NADH: quinone reductase of a marine bacterium,Vibrio alginolyticus

The sodium-transport respiratory chain NADH: quinone reductase of a marine bacterium, Vibrio alginolyticus, was purified by high-performance liquid chromatography. The purified quinone reductase, which catalyses the reduction of ubiquinone to ubiquinol, was composed of three subunits, α, β and γ, with apparent molecular weights of 52 000, 46 000 and 32 000, respectively. The subunit β contained one molecule of FAD per molecule and catalysed the reduction of ubiquinone to ubisemiquinone. The subunit α contained FMN as a prosthetic group. The quinone reductase was reconstituted from α and βγ, but not from α and β, and the maximum activity was obtained at the equimolar amounts of FAD(β) and FMN(α). The molecular weight of quinone reductase complex was estimated to be 254 000, which corresponded to a dimer of αβγ complex or α₂β₂γ₂. The subunit γ increased the affinity of β for ubiquinone-1. The reaction catalysed by FMN-containing α-subunit was essential for the generation of membrane potential in proteoliposomes and the coupling site of sodium pump in the quinone reductase was localised to this reaction step.     

Cloning and expression of the metE gene in Escherichia coli

A lambda-transducing phage was isolated that contains the metE gene. This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis. A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325. This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype. Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.     

Cloning and characterization of motX, a Vibrio alginolyticus sodium-driven flagellar motor gene.

Four motor proteins, MotX, MotY, PomA, and PomB, have been identified as constituents of the Na(+)-driven flagellum of Vibrio species. In this study, the complete motX gene was cloned from Vibrio alginolyticus and shown to complement three mot mutations, motX94, motX115, and motX119, as well as a V. parahaemolyticus motX mutant. The motX94 mutant contains a frameshift at Val86 of MotX, while the motX115 and motX119 mutations comprise substitutions of Ala146 to Val and Gln 194 to amber, respectively. When MotX was overexpressed in Vibrio cells, the amount of MotY detected in the membrane fraction increased, and vice versa, suggesting that MotX and MotY mutually stabilize each other by interacting at the membrane level. When a plasmid containing the motX gene was introduced into motY mutants NMB117 (motY117) and VIO542 (motY542), the mutations were suppressed. In contrast, motY could not cause the recovery of any swarm-defective motX mutants studied. Considering the above evidence, we propose that MotX is more directly involved than MotY in the mechanical functioning of the Na(+)-type flagellar motor, and that MotY may stabilize MotX to support its interaction with other Mot proteins.