Isolation and biochemical characterization of a new NADH oxidase from Lactobacillus brevis

A new NADH oxidase, useful for the regeneration of NAD⁺, was isolated and characterized from Lactobacillus brevis. In crude extracts the activity was from 10–15 U mg^–1. After purification by four chromatographic steps, an activity of 116 U mg^–1 was obtained with 14% yield. Highest activity was from pH 5.5–7 and at 40°C. The enzyme requires dithiothreitol to prevent oxidative deactivation. The K _m value for NADH was 24 M.     

Binding characteristics of the Lactobacillus brevis ATCC 8287 surface layer to extracellular matrix proteins

Self-assembling proteins that form crystalline surface layers on many microorganisms can be involved in bacterial-host adhesion via specific interactions with components of the extracellular matrix. Here, we describe the interaction of the Lactobacillus brevis ATCC 8287 surface-layer protein SlpA with fibronectin, laminin, fibrinogen and collagen using surface plasmon resonance. SlpA was found to interact with high affinity to fibronectin and laminin, with a respective binding constant of 89.8 and 26.7 nM. The interaction of SlpA with collagen and fibrinogen was found to be of much lower affinity, with respective binding constants of 31.8 and 26.1 microM. The serine protease inhibitor benzamidine greatly reduced the affinity of SlpA for fibronectin, whereas the affinity for laminin remained unaffected. No protease activity of the purified SlpA protein could be detected. These data suggest that L. brevis may interact with host cells directly through high affinity interactions with laminin and fibronectin predominantly, involving distinct regions of the SlpA protein.     

Three-dimensional structure of the surface protein layer (MW layer) of Bacillus brevis 47

The three-dimensional (3D) structure of one surface protein layer from Bacillus brevis 47, the middle wall (MW) layer, has been reconstructed from tilted-view electron micrographs after correlation averaging to a resolution of 2 nm. The MW layer has p6 symmetry with a center-to-center spacing of 18.3 nm and a minimum thickness of 5.5 nm. The reconstruction reveals a distinct domain structure: the heavier domain of six monomers jointly forms a massive core centered at the sixfold symmetry axis, and lighter domains interconnect adjacent unit cells. In addition, the larger domains collectively form a pore by making contact with each other towards the inner surface, while the smaller domains establish a second connectivity towards the outer surface of the S layer. The MW layer of B. brevis resembles the S layer of Acetogenium kivui in various aspects: they have very similar lattice parameters and highly reminiscent 3D structures; the pores penetrate through the whole core and appear to determine the porosity of the S layers.     

Isolation and characterization of a new bacterium carboxylating phenol to benzoic acid under anaerobic conditions.

A consortium of spore-forming bacteria transforming phenol to benzoic acid under anaerobic conditions was treated with antibiotics to eliminate the four Clostridium strains which were shown to be unable to accomplish this reaction in pure culture and coculture. Clostridium ghonii was inhibited by chloramphenicol (10 micrograms/ml), whereas Clostridium hastiforme (strain 3) and Clostridium glycolicum were inhibited by clindamycin (20 micrograms/ml), without the transformation of phenol being affected. Electron microscopic observations of resulting liquid subcultures revealed the presence of two different bacilli: a dominant C hastiforme strain (strain 2) (width, 1 micron) and an unidentified strain 6 (width, 0.6 micron) which was not detected on solid medium. Bacitracin (0.5 U/ml) changed the ratio of the strains in favor of strain 6. C hastiforme 2 was eliminated from this culture by dilution. The isolated strain 6 transformed phenol to benzoic acid and 4-hydroxybenzoic acid to phenol and benzoic acid in the presence of proteose peptone. Both of these activities are inducible. This strain is a gram- variable, flagellated rod with a doubling time of 10 to 11 h in the presence of phenol. It has a cellular fatty acid composition like that of C. hastiforme. However, strain 6 does not hydrolyze gelatin or produce indole. The 16S rRNA sequence of strain 6 was found to be most similar to that of some Clostridium species, with homology ranging from 80 to 86%. Tbe evolutionary relationships of strain 6 to different groups of Clostridium and Clostridium-related species revealed that it does not emerge from any of these groups. Strain 6 most likely belongs to a new species closely related to Clostridium species.     

嗜酸乳酸杆菌ATCC 4356菌株异源二聚体b-半乳糖苷酶的克隆表达

嗜酸乳酸杆菌(Lactobacillus acidophilus)的异源二聚体β-半乳糖苷酶属于糖苷水解酶2家族,由两个部分重叠、协同翻译的基因编码(lacL和lacM).[目的]克隆表达该酶并测定其酶学特性.[方法]参照已全基因组测序的嗜酸乳酸杆菌NCFM菌株,以嗜酸乳酸杆菌ATCC4356菌株基因组为模板,将lacL的RBS到lacM的终止子之间的序列(2834 bp)克隆到pQE31质粒上,并电转化JM109菌株.以下列步骤纯化表达产物:硫酸铵分级沉淀、阴离子交换、亲和层析和凝胶排阻层析.以凝胶排阻层析测定纯化酶的天然分子量,以邻硝基苯基半乳糖为底物测定其酶学特性.[结果]实现了该酶在JM109菌株中的可溶性表达.其氨基酸序列有一处不同于嗜酸乳酸杆菌NCFM菌株,即其大亚基(LacL)的第512位氨基酸不是组氨酸而是精氨酸.纯化酶比活力为226 U/mg蛋白,天然分子量为96.3±4.6 kDa,最适pH为7,最适温度为49℃,Km和Vmax值分别是:2.18±0.12 mmol/L,273±5 U/mg蛋白.     

Isolation and characterization of a sulfur-oxidizing chemolithotroph growing on crude oil under anaerobic conditions

Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the epsilon subclass of the class Proteobacteria constituted major populations in underground crude-oil storage cavities. In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan. 16S rRNA gene sequence analysis indicated that its closest relative was Thiomicrospira denitrificans (90% identity). Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor. Oxygen also supported its growth only under microaerobic conditions. Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction. Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources. A typical stoichiometry of its energy metabolism followed an equation: S(2-) + 4NO(3)(-) --> SO(4)(2-) + 4NO(2)(-) (Delta G(0) = -534 kJ mol(-1)). In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible. When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth. On the contrary, YK-1 could not utilize crude oil as a carbon source. These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds. Based on its physiology, ecological interactions with other members of the groundwater community are discussed.     

Isolation and characterization of three polyamine oxidase genes from Zea mays

Isolation and sequencing of three genes, MPAO1, MPAO2 and MPAO3, coding for polyamine oxidase (PAO) from maize (Zea mays) are reported here. Gene organization is extremely conserved among these copies, being composed of eight exons and seven introns. Furthermore, these genes encode for a protein of an almost identical amino acid sequence. These data suggest that the three MPAO copies have been derived from gene duplication of a common ancestor gene. Long inverted repeat sequences, also present in other maize genes, have been found within the second intron. Promoter sequences of MPAO1 and MPAO2 genes have been analysed for putative cis-acting elements. According to genomic Southern blot analysis, the MPAO gene family in maize and other monocots is represented by a small number of copies. Northern and western blot analysis have revealed a tissue-specific accumulation of both MPAO mRNA and protein.     

Isolation and characterization of lipid transfer protein (LTP) genes from a wheat-rye translocation line

. Two genes (TaLTP1 and TaLTP2) encoding lipid transfer proteins (LTPs) were isolated from a cDNA library constructed from leaf tissue harvested from 4-week-old seedlings of a wheat-rye near-isogenic line (NIL) involving a translocation of rye chromosome 2RL with wheat 2BS. The spatial and temporal patterns of expression of TaLTP1 and TaLTP2 were examined by Northern blot analysis and in situ hybridization. Both TaLTP1 and TaLTP2 contained a 270-bp open reading frame and encoded a putative LTP precursor molecule of 90 amino acids. Expression of the two LTPs was detected in leaves, stems, and crowns of the NILs but not in the roots. The expression levels of TaLTP1 and TaLTP2 remained constant in response to cold and ABA treatments over a period of 24 h but increased 3 days after the initiation of drought stress. An in situ hybridization study indicated that TaLTP1 was expressed in the cells within the vascular bundles of leaves and in the tissue layers between the vascular bundles in the crowns of the control and drought-treated plants. Expression of TaLTP1 in the tissue layers between the vascular bundles was higher in the drought-treated plants than in the control plants. The results suggested that high levels of expression of TaLTPs in the tissue layers between the vascular bundles might play a role in the drought tolerance response of the wheat crown.     

Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7

A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces. The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus. An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7. It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography. Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE. The bacteriocin was produced constitutively during exponential growth. It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis. The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified.     

Pseudovitamin B(12) is the corrinoid produced by Lactobacillus reuteri CRL1098 under anaerobic conditions

We have reported previously on the ability of Lactobacillus reuteri to produce a compound with vitamin B(12) activity. Here we report on the chemical characterisation of this corrinoid-like molecule. High performance liquid chromatography coupled to an ultraviolet diode array detector, mass spectrometry and nuclear magnetic resonance spectroscopy has enabled us to identify the compound as Coalpha-[alpha-(7-adenyl)]-Cobeta-cyanocobamide or pseudovitamin B(12). This molecule differs from cobalamin in the alpha-ligand, where it has adenine instead of 5,6-dimethylbenzimidazole bound in a alpha-glycosidic linkage to C-1 of ribose. L. reuteri is the first lactic acid bacterium in which the production of a cobalamin-like molecule has been identified and the first microorganism reported to produce exclusively pseudo-B(12).     

Cloning and characterization of three fatty alcohol oxidase genes from Candida tropicalis strain ATCC 20336

Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of omega-hydroxy fatty acids to omega-aldehydes by a fatty alcohol oxidase (FAO). Three FAO genes (one gene designated FAO1 and two putative allelic genes designated FAO2a and FAO2b), have been cloned and sequenced from this strain. A comparison of the DNA sequence homology and derived amino acid sequence homology between these three genes and previously published Candida FAO genes indicates that FAO1 and FAO2 are distinct genes. Both genes were individually cloned and expressed in Escherichia coli. The substrate specificity and K(m) values for the recombinant FAO1 and FAO2 were significantly different. Particularly striking is the fact that FAO1 oxidizes omega-hydroxy fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but not omega-hydroxy fatty acids. Analysis of extracts of strain H5343 during growth on fatty acids indicated that only FAO1 was highly induced under these conditions. FAO2 contains one CTG codon, which codes for serine (amino acid 177) in C. tropicalis but codes for leucine in E. coli. An FAO2a construct, with a TCG codon (codes for serine in E. coli) substituted for the CTG codon, was prepared and expressed in E. coli. Neither the substrate specificity nor the K(m) values for the FAO2a variant with a serine at position 177 were radically different from those of the variant with a leucine at that position.     

Characterization and expression analysis of three cold shock protein (CSP) genes under different stress conditions in the Antarctic bacterium Psychrobacter sp. G

Low temperature is one of the major environmental challenges that Antarctic bacteria must face. Detailed studies of cold shock responses of cold-adapted microorganisms are still insufficient. Here, we cloned three cold shock protein (CSP) genes (Csp1137, Csp2039, and Csp2531) in the Antarctic bacterium Psychrobacter sp. G and their regulatory sequences were identified. The three CSPs were highly conserved with other known CspAs. qRT-PCR was performed to evaluate their expression characteristics under stress conditions, and the potential influence of regulatory sequences also was analyzed. The expression of Csp1137 was enhanced both by low (0, 10?°C) and high temperature (30?°C). The expression of Csp2039 was enhanced by low temperature (0?°C), but was lower than that of Csp1137. This can be explained by the absence in Csp2039 of the AT-rich UP element. Different from Csp1137, the expression of Csp2531 was inhibited by low temperature (0?°C), even with the presence of AT-rich UP element, and it was not sensitive to high temperature (30?°C). The expression of Csp1137 was enhanced by high salinity (90, 120), whereas that of Csp2531was enhanced by low salinity (0, 15). At 0?°C and a salinity of 15, the expression of Csp1137 was repressed initially, but then it increased greatly during the next 10?h. The expressions of Csp2039 and Csp2531 were repressed significantly under four different combinations of stress conditions. Our results showed that the role of the upstream regulation sequences were much more complex than previously thought. Also, gene expressions were also affected by the environmental salinity. These are helpful in further clarification of the adaptation mechanism of Psychrobacter sp. G.     

Isolation of Lactobacillus sakei strain KJ-2008 and its removal of characteristic malodorous gases under anaerobic culture conditions

A number of different sources, such as composts, leachates, and pig feces samples were collected from different pig farms in Korea. Several microorganisms were screened for their ability to deodorize the malodorous gases. As a result, a novel malodorous gas-deodorizing bacterial strain KJ-2008 was isolated due to the most abundant of nitrate-supplemented minimal media under anaerobic conditions. Crimp-sealed serum bottles containing nitrate-supplemented minimal medium (MM-NO(3)(-)) in airtight conditions were inoculated with KJ-2008. Nitrate concentration decreased rapidly after 20 h incubation and nitrite production reached almost zero during the time the experimental was carried out. Taxonomic identification including 16S rDNA base sequencing and phylogenetic analysis indicated that the isolate KJ-2008 had a 99.8% homology in its 16S rDNA base sequence with Lactobacillus sakei. Among the volatile fatty acids, acetic acid contained in large amounts in fresh piggery slurry decreased about 40% after 50 h incubation of the strain KJ-2008. n-Butyric acid, n-valeric acid, and iso-valeric acid gradually decreased, and iso-butyric acid and capronic acid dramatically eliminated at initial time with the treatment. Moreover, NH(3) removal efficiency reached a maximum of 98.5% after 50 h of incubation. The concentration of H(2)S did not change.     

Isolation and characterization of three novel serine protease genes from Xenopus laevis

Three novel cDNAs encoding serine proteases, that may play a role in early vertebrate development, have been identified from Xenopus laevis. These Xenopus cDNAs encode trypsin-like serine proteases and are designated Xenopus embryonic serine protease (Xesp)-1, Xesp-2, and XMT-SP1, a homolog of human MT-SP1. Xesp-1 is likely to be a secreted protein that functions in the extracellular space. Xesp-2 and XMP-SP1 are likely to be type II membrane proteases with multidomain structures. Xesp-2 has eight low density lipoprotein receptor (LDLR) domains and one scavenger receptor cysteine-rich (SRCR) domain, and XMT-SP1 has four LDLR domains and two CUB domains. The temporal expressions of these serine protease genes show distinct and characteristic patterns during embryogenesis, and they are differently distributed in adult tissues. Overexpression of Xesp-1 caused no significant defect in embryonic development, but overexpression of Xesp-2 or XMT-SP1 caused defective gastrulation or apoptosis, respectively. These results suggest that these proteases may play important roles during early Xenopus development, such as regulation of cell movement in gastrulae.     

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting expression of nonregulated genes. The three genes code for poly(A)⁺RNAs that begin to accumulate at different times during conidiation. The brlA-and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.