Cloning and sequencing of genes encoding the beta subunits of the ATP-synthases from Enterobacter aerogenes and Flavobacterium ferrugineum

Abstract The genes encoding the β-subunit of the ATPase from Enterobacter aerogenes and Flavobacterium ferrugineum were cloned and their sequences determined. The predicted amino acid sequences were compared with the corresponding proteins from other eubacteria. Homology values of 58–98% confirmed the highly conserved character of the ATPase β-subunit. The enterobacterial ( Escherichia coli, E. aerogenes ) β-subunits represent the shortest sequences, whereas the corresponding F. ferrugineum protein exhibits an additional 33 amino acid residues as insertions at three different locations.     

Bacterial Reduction of Hexavalent Chromium: Kinetic Aspects of Chromate Reduction by Enterobacter cloacae HO1

Kinetic aspects of the bacterial reduction of hexavalent chromium (chromate: CrO^2-₄) were investigated using Enterobacter cloacae strain HO1. E. cloacae strain HO1 could reduce hexavalent chromium to the trivalent form (Cr³⁺) anaerobically. High concentrations of CrO^2-₄ inhibited the reduction, and a substrate inhibition model gave a good fit to the observed data. The rate of chromate reduction was proportional to cell density. The effect of temperature on the reduction rate followed the Arrhenius equation. The rate of chromate reduction was also dependent on pH and the concentrations of carbon and energy sources in the culutre medium. Amino acids including asparagine, methionine, serine and threonine were utilized effectively as carbon and energy sources for chromate reduction.     

Development of Enterobacter aerogenes fuel cells: From in situ biohydrogen oxidization to direct electroactive biofilm

This study described an Enterobacter aerogenes-catalyzed microbial fuel cell (MFC) with a carbon-based anode that exhibited a maximum power density of 2.51 W/m³ in the absence of artificial electron mediators. The MFC was started up rapidly, within hours, and the current generation in the early stage was demonstrated to result from in situ oxidation of biohydrogen produced by E. aerogenes during glucose fermentation. Over periodic replacement of substrate, both planktonic biomass in the culture liquid and hydrogen productivity decreased, while increased power density and coulombic efficiency and decreased internal resistance were unexpectedly observed. Using scanning electron microscopy and cyclic voltammetry, it was found that the enhanced MFC performance was associated with the development of electroactive biofilm on the anodic surface, proposed to involve an acclimation and selection process of E. aerogenes cells under electrochemical tension. The significant advantage of rapid start-up and the ability to develop an electroactive biofilm identifies E. aerogenes as a suitable biocatalyst for MFC applications.     

Imipenem resistance in Enterobacter aerogenes is associated with derepression of chromosomal cephalosporinases and impaired permeability

Enterobacter aerogenes mutants with high-level resistance to imipenem were studied. They were derived from strains characterized by stable over-production of a class-I beta-lactamase. This enzyme (pI = 8.2) exhibited high affinity toward imipenem and hydrolysed the drug slowly. Imipenem-resistant mutants lacked a major 43-kDa outer membrane protein and displayed decreased permeability to cephaloridine. Introduction of a plasmid coding for the regulatory ampD gene abolished beta-lactamase production and rendered the mutants susceptible to imipenem.     

Isolation and characterisation of a new mosquitocidal bacterium strain of Enterobacter cloacae VCRC-B519 from marine soil

A novel mosquitocidal bacterium was isolated from marine soil. 16S rRNA gene sequence alignment depicted that this isolate belonged to the strain, Enterobacter cloacae VCRC-B519 (NCBI: KC119193). Biochemical studies such as bacterial growth, biomass production and protein (toxin) synthesis showed that the strain is plausibly useful for mosquito control. It showed an increasing pattern of toxicity for Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti, without negative effects for non-targeted organisms Chironomus riparius, Daphnia cephalata and Notonecta glauca. The qualitative analysis of the E. cloacae showed that three polypeptides (M.wt: 25, 30 and 50 kDa) were associated to the toxicity observed. The characterisation of these polypeptides (M/S MALDI-TOF)showed that they are enzymatic in nature. Consequently, the peptide sequences are identified to be polysugar degrading enzymes (25 kDa), cell wall associated hydrolases (30 kDa) and amino peptidase (50 kDa). Phylogenetic analyses of 16S rDNA gene sequence of E. cloacae revealed the occurrence of homology with closely related Enterobacter species. Therefore, it is concluded that the marine bacterium (Enterobacter cloacae)is possibly of use for the biological control of mosquito immatures.     

Regulation of glycerol metabolism in <Emphasis Type="Italic">Enterobacter aerogenes</Emphasis> NBRC12010 under electrochemical conditions

Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD⁺/NADH. At a NAD⁺/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD⁺/NADH. Exogenous NADH was oxidized to NAD⁺ by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.     

New approach to use ethidium bromide monoazide as an analytical tool

Aims: Ethidium bromide monoazide (EMA) has been determined to cause delay in DNA amplification from dead bacteria at real‐time PCR. However, there is concern that the increasing EMA concentration to suppress amplification from high number of dead bacteria also affects live bacteria. The aim is to disclose a novel application of EMA for food hygienic test. Methods and Results: We performed a low‐dose double EMA treatment. Live or heat‐dead Enterobacter sakazakii (reclassified as Cronobacter spp.) in 10% powdered infant formula (PIF) solution was subjected to a treatment with 20 μg ml^?1 of EMA followed by a treatment with 10 μg ml^?1 of EMA without washing, and direct real‐time PCR. We observed that DNA amplification from 10⁷ cells ml^?1 of dead Ent. sakazakii was completely suppressed within 50 cycles of PCR, whereas 10²–10³ cells ml^?1 of viable cells could be detected. When a 3‐h enrichment step in liquid medium was included after the first EMA treatment, live Ent. sakazakii could be detected at initial levels of 10⁰–10² cells ml^?1. We compared the low‐dose double‐treated EMA‐PCR with the culture method using 80 samples of PIF, and completely correlative results were obtained for both methods. Conclusions: We concluded that the newly developed low‐dose double‐treated EMA‐PCR is a very effective tool for live Ent. sakazakii detection in PIF. Significance and Impact of the Study: We focused on the specific nature of photoreactive compound that residual EMA is cancelled by irradiation. We were successful in treating bacteria with EMA in gradient concentration to increase live and dead distinction ability.     

Improvement of Biohydrogen Production Under Decreased Partial Pressure of H2 by Enterobacter cloacae

When the partial pressure of H₂ was decreased by lowering the total pressure in the headspace of the reactor in a batch fermentation process from 760 mm Hg to 380 mm Hg containing Enterobacter cloacae, the molar yield of H₂ increased from 1.9 mol to 3.9 mol H₂/mol glucose. The maximum production rate was 0.017 mmol H₂/h l at 380 mm Hg. The lag period as well as total batch time of H₂ production decreased using a decreased partial pressure.     

Molecular characterization of the alpha-glucosidase activity in Enterobacter sakazakii reveals the presence of a putative gene cluster for palatinose metabolism

Enterobacter sakazakii is considered an opportunistic pathogen for premature infants and neonates. Although E. sakazakii has been isolated from various types of food, recontaminated dried infant formula has been epidemiologically identified as the major source of infection. Amongst others, alpha-glucosidase activity is one of the most important biochemical features, which differentiates E. sakazakii from other species in the family Enterobacteriaceae and has therefore been used as a selective marker in the development of differential media. However, it has been shown, that methods based on this biochemical feature are prone to producing false-positive results for presumptive E. sakazakii colonies due to the presence of this enzymatic activity in other species of the Enterobacteriaceae. Therefore, elucidation of the molecular basis responsible for the biochemical feature in E. sakazakii would provide novel targets suitable for the development of more specific and direct identification systems for this organism. By applying the bacterial artificial chromosome (BAC) approach, along with heterologous gene expression in Escherichia coli, the molecular basis of the alpha-glucosidase activity in E. sakazakii was characterized. Here we report the identification of two different alpha-glucosidase encoding genes. Homology searches of the deduced amino acid sequences revealed that the proteins belong to a cluster of gene products putatively responsible for the metabolism of isomaltulose (palatinose; 6-O-alpha-d-glucopyranosyl-d-fructose). The glycosyl-hydrolyzing activity of each protein was demonstrated by subcloning the respective open reading frames and screening of E. coli transformants for their ability to hydrolyze 4-methyl-umbelliferyl-alpha-d-glucoside. Analysis at the protein level revealed that both enzymes belong to the intracellular fraction of cell proteins. The presence of the postulated palatinose metabolism was proven by growth experiments using this sugar as a sole carbon source.     

酶法合成抗病毒药物阿糖腺苷

目的:为了开发一种生产阿糖腺苷的有效方法。方法:研究了以产气肠杆菌完整细胞为催化剂酶法合成阿糖腺苷,优化了菌体培养条件以及酶反应条件。结果:在培养基中添加0.5%葡萄糖,33℃下培养16h,既能得到较多菌体,又能使菌体的催化活性保持较高。酶反应在pH7.0、25mmol/L的磷酸钾缓冲液中进行,底物浓度为阿糖尿苷30mmol/L,腺嘌呤10mmol/L,加入10%湿菌体,在60℃下振荡反应48h,腺嘌呤转化率可达90%。结论:酶法合成阿糖腺苷可应用于大规模工业化生产。