Kinetics of two complementary hydrogen sink reactions in a defined 3-chlorobenzoate degrading methanogenic co-culture

Abstract The interrelationships between an obligate hydrogen-producing and two different hydrogen-scavenging populations grown as synthrophic members of a 3-chlorobenzoate degrading methanogenic consortium were studied. The hydrogen producer was a benzoate degrader (strain BZ-2), and the hydrogen consumers were a 3-chlorobenzoate dechlorinating bacterium ( Desulfomonile tiedjei ) and a hydrogenotropic methanogen ( Methanospirillum strain PM-1). When a mixture of 3-chlorobenzoate plus benzoate was added to this consortium, the rate of benzoate degradation was 50% higher, at slightly lower H₂ concentrations, than when benzoate alone was added. The enhanced benzoate degradation rate was apparantly triggered by the lower H₂ concentration, as the rate of benzoate degradation was shown to be a function of the H₂ concentration. By offering a hydrogen sink, in addition to methanogenesis, the dechlorinating hydrogen-scavenging population stimulated the rate of benzoate degradation. The lowering of the H₂ concentration was very small, which was in agreement with the observation that the rate of methanogenesis was hardly affected by this lower hydrogen concentration. Thus there was no significant competition for H₂ between the two hydrogen-scavenging populations in the consortium, as they practically complemented each other's hydrogen-scavenging potential at in situ hydrogen concentrations during the degradation of 3-chlorobenzoate. The H₂ concentrations at which hydrogen driven methanogenesis by Methanospirillum occurred in the consortium were well below the threshold concentration extrapolated for this methanogen after growth at high H₂ concentrations.     

Production and cross-reactivity patterns of a panel of high affinity monoclonal antibodies to Vibrio cholerae O139 Bengal

Abstract A series of monoclonal antibodies of different isotypes specific for Vibrio cholerae O139, the new pandemic strain of cholera, was produced. These mAbs reacted only with the reference strain (MO45) representing serovar O139 but did not react with any of the other reference strains representing serovars O1 to O140. Significantly, the mAbs did not agglutinate the R-cultures of V. cholerae (CA385, 20–93) which demonstrated the exceptional specificity of these mAbs and indicated that the mAbs recognized antigenic determinants unique for the O139 serovar. There was heterogeneity in the intensity of reactivity of the mAbs with strains of V. cholerae O139 isolated from diverse sources. Apart from 4H6, the other mAbs agglutinated all the O139 strains examined. 2D12 and 2F8 were the best mAbs based on the intensity of agglutination with all the O139 strains. Evaluation of 3A10 in comparison with a polyclonal anti-O139 antibody raised in rabbit using the slide agglutination format revealed that 3A10 fared as well as the polyclonal antibody for the laboratory identification of the O139 serovar. The acquisition of these mAbs provide reagents which would be very useful in the development of simple immunodiagnostic assays for the diagnosis of V. cholerae O139 infections.     

Antibodies (IgG) to lipopolysaccharide of Vibrio cholerae O1 mediate protection through inhibition of intestinal adherence and colonisation in a mouse model

An antiserum raised against purified lipopolysaccharide (LPS) of a Vibrio cholerae O1 strain (Co366) induced passive protection against challenge with the parent as well as other O1 organisms but not against O139 or non-O1/non-O139 organisms. A considerable level of protection against O1 strains was also observed with the IgG fraction of the antiserum which inhibited intestinal adherence and colonisation. The monovalent Fab(IgG) fragment, on the other hand, showed only a low level of protection. Interestingly, purified LPS failed to inhibit intestinal colonisation by the parent strain (Co366), thereby suggesting that the cell surface LPS moieties of vibrios may not be directly involved in the colonisation process. It may be concluded that the anti-LPS antibodies induce passive protection through microagglutination and/or immobilisation of vibrios which do not allow the organisms to adhere to and colonise the intestine.     

Production of α-amylase by Myxococcus coralloides D

M.E. FÁREZ-VIDAL, A. FERNANDEZ-VIVAS AND J.M. ARIAS. 1992. Myxococcus coralloides D secreted amylase into a liquid growth medium containing 1% starch. Amylase activity was highest at the end of the exponential growth phase. Of the nitrogen sources tested, the greatest growth and amylase production were obtained with trypticase peptone, casitone, probion L and probion F. When starch was replaced by other carbon sources, amylase production was reduced; trisaccharide produced better results than disaccharide while monosaccharide reduced amylase production to basal levels. Maltose repressed amylase production. Amylase production was greater in stirred flasks, at pH between 6.5 and 7.5, and at temperatures from 28C to 33C. The activity of partially purified M. coralloides D amylase was used to determine the products released from the hydrolysis of starch with thin-layer chromatography, paper chromatography and nuclear magnetic resonance. These products were maltose and glucose and limit dextrins.     

Expression of the Streptomyces griseusα-amylase gene in Escherichia coli

Abstract The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular α-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no α-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.     

Induction of α-amylase by maltooligosaccharides in Thermomonospora curvata

The action pattern of the α-amylase produced by Thermomonospora curvata is unique. Maltooligosaccharides (maltose to maltopentaose) were tested individually for their ability to induce α-amylase in this thermophilic actinomycete. Maltotetraose was the most inductive followed by maltotriose. Maltose was a good inducer of amylase production when used as sole carbon source, but had relatively little inductive capacity in the presence of either glucose or cellobiose. When cellobiose was added during exponential growth on maltose, maltose utilization and extracellular α-amylase accumulation were transiently inhibited. With maltotriose as the initial carbon source, addition of cellobiose did not inhibit the utilization of the trisaccharide; however, cellobiose, whether added during exponential growth or stationary phase, resulted in the rapid degradation of amylase when maltotriose was depleted from the medium. This inactivation did not appear to be a growth phase-induced phenomenon because stationary phase cells in the absence of cellobiose maintained their peak extracellular amylase level. This cellobiose-mediated α-amylase inactivation would be particularly important during production of the enzyme on a complex lignocellulosic substrate.     

Monoclonal antibodies against O and K antigens of uropathogenic Escherichia coli O4 : K12 : H− as opsonins

Abstract Monoclonal antibodies of subclasses IgG1 and IgG2b and specific for the O4 antigen of Escherichia coli 20025 (O4 : K12 : H⁻) and the capsular K12 polysaccharide of the same strain (IgM) were obtained with the hybridoma technique using spleen cells from Balb/c mice, immunized with a crude bacterial extract, and Sp2/O-Ag8 myeloma cells. The anti-O4 antibodies reacted exclusively with the O4 lipopolysaccharide and not with those from serologically O-cross reactive E. coli . The anti-K12 antibodies recognized as epitope (part of) the KDO moiety of the capsular K12 polysaccharide. Not only anti-K12, but also anti-O4 antibodies effectively phagoopsonized encapsulated E. coli 20025. The opsonized bacteria were killed in subsequent in vitro phagocytosis by human leokocytes in the presence of human serum complement.     

The nucleotide sequence of an α-amylase gene from an alkalopsychrotrophic Micrococcus sp

An alpha-amylase gene from Micrococcus sp. 207 was cloned into Escherichia coli JM101 using the vector pHSG399. The constructed recombinant plasmid pYK63 contained a 4.8 kb chromosomal DNA fragment derived from strain 207 DNA. The cloned amylase isolated from E. coli JM101 (pYK63) produced mainly maltotetraose from starch, and exhibited temperature and pH activity profiles closely similar to those of the enzyme from the original strain. Nucleotide sequence analysis of the cloned DNA fragment revealed one open reading frame containing the gene which consisted of 3312 bp (1104 amino acids). When compared with several other alpha-amylases, three consensus sequences were identified in the region of the active site. About 300 amino acid residues were present both upstream and downstream of the active site region.     

Cloning and expression of a Clostridium acetobutylicumα-amylase gene in Escherichia coli

A gene library of Clostridium acetobutylicum ATCC824 was constructed in the plasmid vector pEcoR251. The library was tested for the presence of starch hydrolyzing clones. One clone in which the recombinant plasmid, pVP101, conferred alpha-amylase activity to the Escherichia coli host cell, was detected. The gene is carried on a 3.45-kbp BglII restriction fragment. A detailed physical map of pVP101 is presented.     

Cloning of a thermostable α-amylase gene from Bacillus licheniformis and its expression in Escherichia coli and Bacillus subtilis

Abstract The gene coding for the thermostable α-amylase Bacillus licheniformis has been isolated from a direct shotgun in Escherichia coli using the bacteriophage lambda as a vector. The fragment containing the α-amylase gene has been sub-cloned in pBR322 and its restriction map determined. The α-amylase produced by the E. coli clones retained the thermostability of the B. licheniformis enzyme. Expression and properties of the gene product in E. coli and Bacillus subtilis have been examined.     

Cloning and expression of the thermostable α-amylase gene from Clostridium thermosulfurogenes (DSM 3896) in Escherichia coli

Abstract The gene coding for a thermostable α-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulforogenes carrying the α-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium.     

霍乱弧菌脂多糖O抗原基因在大肠杆菌中的克隆及表达

经典生物型及埃尔托生物型霍乱弧菌的染色体DNA片段分别与载体质粒pUC18,B.S(M13~+)进行克隆,从克隆株中筛选到能表达霍乱弧菌脂多糖O抗原基因的重组子。它们所表达的脂多糖O抗原具有很好的抗原性及免疫原性,其重组质粒pMG-301、pMG-302经酶切分析表明,外源片段大小分别为8.4kb,7.6kb,较文献报道的16kb要小,而且基因结构之间也存在很大差异。     

Investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O1, serotype Inaba

A free amino group present on the acid-detoxified lipopolysaccharide (pmLPS) of V. cholerae O1 serotype Inaba was investigated for site-specific conjugation. Chemoselective pmLPS biotinylation afforded the corresponding mono-functionalized derivative, which retained antigenicity. Thus, pmLPS was bound to carrier proteins using thioether conjugation chemistry. Induction of an anti-LPS antibody (Ab) response in BALB/c mice was observed for all conjugates. Interestingly, the sera had vibriocidal activity against both Ogawa and Inaba strains opening the way to a possible bivalent vaccine. However, the level of this Ab response was strongly affected by both the nature of the linker and of the carrier. Furthermore, no switch from IgM to IgG, i.e. from a T cell-independent to a T cell-dependent immune response was detected, a result tentatively explained by the possible presence of free polysaccharide in the formulation. Taken together, these results encourage further investigation towards the development of potent pmLPS-based neoglycoconjugate immunogens, fully aware of the challenge faced in the development of a cholera vaccine that will provide efficient serogroup coverage.     

Nucleotide sequence of the extracellular α-amylase gene in the yeast Schwanniomyces occidentalis ATCC 26077

The Schwanniomyces occidentalis (formerly castellii) ATCC 26077 (CBS 2863) alpha-amylase (AMY 26077) gene was cloned in Saccharomyces cerevisiae and sequenced. An open-reading frame encoding the AMY consists of 1536 base pairs and contains 512 amino-acid residues, which is almost the same in size as the AMY of Sch. occidentalis ATCC 26076 and CCRC 21164. The amino-acid sequence of AMY 26077 differed from that of ATCC 26076 alpha-amylase (AMY 26076) at two residues and from that of CCRC 21164 alpha-amylase (AMY 21164) at three residues. Comparison of the AMY 26077 gene with its homologues from two other strains (Sch. alluvius CBS 1153 and Sch. persoonii CBS 2169) using several restriction enzymes revealed that the AMY 26077 was very similar to AMY CBS 1153 but different from that of CBS 2169.     

Properties and significance of an α-amylase produced by Myxococcus coralloides D

M.E.FÁREZ-VIDAL, A. FERNÁNDEZ-VIVAS, F. GONZÁLEZ AND J.M. ARIAS. 1995. The extracellular amylase activity from Myxococcus coralloides D was purified by Sephacryl S-200 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-25. The molecular weight was estimated by SDS-PAGE and by gel filtration as 22.5 kDa. The optimum temperature was 45°C. The pH range of high activity was between 6.5 and 8.5, with an optimum at pH 8.0. Activity was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ag⁺, Pb²⁺, Fe²⁺ and Fe³⁺, EDTA and glutardialdehyde, but was less affected by Ni²⁺ and Cd²⁺. Li⁺, Mg²⁺, Ba²⁺, Ca²⁺, N -ethylmaleimide, carbodiimide and phenyl methyl sulphonyl fluoride had almost no affect. The K _m (45°C, pH 8) for starch hydrolysis was 2.0 times 10^-3 gl^-1. Comparison of the blue value-reducing curves with the time of appearance of maltose identified the enzyme produced by M. coralloides D as an α-amylase.     

Localization of antigenic determinants on P-fimbriae of uropathogenic Escherichia coli

Abstract Construction and analysis of mutations in the hypervariable regions of the F7₁, F9 and F11 fimbrillin genes are described. The results show that mutations in the hypervariable regions of the fimbrillin can be made without abolishing the ability of these fimbrillins to assemble into filaments. The mutant fimbriae show binding patterns with specific monoclonal antibodies that differed from those of the corresponding wild-type fimbriae. These results confirm that antigenic determinants of the P-fimbriae are, at least partly, located in the hypervariable regions, and suggest that more than one antigenic determinant is involved in the F7₁ and F11 specificity.     

Specificity of insertion of IS91, an insertion sequence present in α-haemolysin plasmids of Escherichia coli

We have determined the DNA sequences of eight different insertions of IS91 in a specifically engineered recipient plasmid of known DNA sequence (pSU300). The sequences at the termini of IS91 are 5'-CGAGTAGG...CCTATCGAT. IS91 inserts specifically 5' to either one of the tetranucleotides 5'-GAAC or 5'-CAAG, and always in the same relative orientation with respect to the sequence of the target. Except in one special case, no duplications of the recipient DNA were produced at the site of insertion.     

Production, purification and characterization of an α-amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α-amylase synthesis. The enzyme was purified 118-fold from the culture supernatant fluid by isopropanol precipitation and DEAE-Sephadex A50 chromatography. The purified enzyme was characterized as an α-amylase. The α-amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; K_m (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.