Isolation and characterization of Zymomonas mobilis mutants resistant to octadecyltrimethylammonium chloride,a detergent acting on hopanoid-producing bacteria

Growth of the hopanoid-producing bacterium Zymomonas mobilis was inhibited at low concentrations of the cationic detergent octadecyltrimethylammoniumchloride (OTAC). A relationship between sensitivity of Zymomonas mobilis to OTAC, presence of hopanoids and ethanol tolerance was postulated. Mutants resistant to OTAC were isolated from strains ZM1 and ZM4. They did not present any alteration of the hopanoid content and their squalene cyclases showed the same sensitity to OTAC as the parent enzymes. Resistance to OTAC paralleled pleiotropic effects including, enhanced accessibility of the membrane-bound alkaline phosphatase, important release of proteins from cells by Tris/HCl treatment, increased resistance to antibiotics and increased sensitivity to ethanol. In addition, OTAC^R mutants were also characterized by the synthesis or the overproduction of an outer membrane protein (F53) not detected on 2D-PAGE maps of parent strains and by a normal heat shock response. The role of hopanoids, heat shock proteins, protein F53 and membrane organization in ethanol tolerance is discussed.Abbreviations OTAC octadecyltrimethylammoniumchloride - SLS sodium lauryl sarcosinate     

Energy coupling sites in the electron transport chain of Zymomonas mobilis

Abstract Energy-coupling sites in the electron transport chain of the obligately fermentative aerotolerant bacterium Zymomonas mobilis were examined. The H⁺ /O stoichiometry of the electron transport chain in intact bacteria oxidizing ethanol was close to 3.3. Cytoplasmic membrane vesicles coupled NADH oxidation to ATP synthesis. With ascorbate/phenazine methosulfate they showed oxygen uptake which was sensitive to antimycin A, but no significant ATP synthesis could be detected. Cells with a defective coupling site I, prepared by cultivation on a sulfate-deficient medium, showed a decreased rotenone sensitivity of respiration, and they lacked almost all the respiration-driven proton translocation and ATP synthesis. We conclude that, despite the reported composition of the electron transport chain, only energy coupling site 1 was functional in Z. mobilis .     

Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis

Summary Insertion of the transposable element Ty at the ADH4 locus results in increased levels of a new alcohol dehydrogenase (ADH) activity in Saccharomyces cerevisiae. The DNA sequence of this locus has been determined. It contains a long open reading frame which is not homologous to the other ADH isozymes that have been characterized in S. cerevisiae nor does it show obvious homology to Drosophila ADH. The hypothetical ADH does, however, show strong homology to the sequence of an iron-activated ADH from the bacterium Zymomonas mobilis. Thus ADH4 appears to encode an ADH structural gene which, along with the Zymomonas enzyme, may define a new family of alcohol dehydrogenases.Now The Plant Cell Research Institute, Inc., 6560 Trinity Court, Dublin, CA 94568, USA     

Formation of acetyl-CoA in Zymomonas mobilis by a pyruvate dehydrogenase complex

In the gram negative, obligately ethanologenic bacterium Zymomonas mobilis a pyruvate dehydrogenase complex was identified and the complex was enriched from cell extracts. This multienzyme complex is responsible for acetyl-CoA biosynthesis from pyruvate. No activities of related multienzyme complexes, 2-ketoglutarate dehydrogenase and branched chain keto acid dehydrogenase, could be detected.     

The crustacean lacinia mobilis: a reconsideration of its origin, function and phylogenetic implications

The lacinia mobilis of the Crustacea Malacostraca is a more or less spine-like movable appendage of the medial mandibular edge, inserted near the base of the incisor process. It occurs in two or possibly three eumalacostracan superorders but is retained in the adult stage only in the Peracarida. The lacinia has been interpreted as the distal member of the spine-row found in many adult Malacostraca and/or their larvae, or alternatively as a derivative of a certain cusp ('cusp b') of the biting edge of the primitive lophogastrids. The distribution, ontogeny and function of the lacinia were studied in a variety of Eumalacostraca. There is great variability in the guiding and locking mechanisms involved in biting, within the subclass and even within single orders. A lacinia-based guiding and locking system is likely to function only in weak mandibles. New evidence is produced in favour of derivation of the lacinia from the spine-row, and the 'cusp b' derivation hypothesis is rejected, 'cusp b' being only a highly specialized lacinia. Doubt is cast upon the unity of the superorder Peracarida mainly because the place of the order Amphipoda within it is regarded as insecure.     

A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonas mobilis and invertase

A new biosensor for specific determination of sucrose was developed using an oxidoreductase of Zymomonas mobilis and invertase. Cells of Z. mobilis were permeabilized with toluene in order to utilize the enzymes of glucose-fructose oxidoreductase and gluconolactonase inside the intact cells. Permeabilized cells and invertase were coimmobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on a pH electrode. The production of hydrogen ion was detected using the biosensor-connected microcomputer, and the concentration of sucrose was determined by using both the initial rate and the steady-state methods. Optimum conditions for biosensor response were pH 6.2 and temperature 35 degrees C. The effect of interfering compounds on the electrode response was investigated, and the interference by various sugars was eliminated by determining sucrose concentration using the steady-state method. The biosensor developed is simple and reproducible, and the calibration curve for sucrose is linear up to 70 g/L.     

EVALUATION OF BIOETHANOL PRODUCTION FROM CAROB PODS BY Zymomonas mobilis AND Saccharomyces cerevisiae IN SOLID SUBMERGED FERMENTATION

Bioethanol production from carob pods has attracted many researchers due to its high sugar content. Both Zymomonas mobilis and Saccharomyces cerevisiae have been used previously for this purpose in submerged and solid-state fermentation. Since extraction of sugars from the carob pod particles is a costly process, solid-state and solid submerged fermentations, which do not require the sugar extraction step, may be economical processes for bioethanol production. The aim of this study is to evaluate the bioethanol production in solid submerged fermentation from carob pods. The maximum ethanol production of 0.42 g g^?1 initial sugar was obtained for Z. mobilis at 30°C, initial pH 5.3, and inoculum size of 5% v/v, 9 g carob powder per 50 mL of culture media, agitation rate 0 rpm, and fermentation time of 40 hr. The maximum ethanol production for S. cerevisiae was 0.40 g g^?1 initial sugar under the same condition. The results obtained in this research are comparable to those of Z. mobilis and S. cerevisiae performance in other culture mediums from various agricultural sources. Accordingly, solid submerged fermentation has a potential to be an economical process for bioethanol production from carob pods.     

A simple method for the purification of over-expressed extracellular sucrase of Zymomonas mobilis from a recombinant Escherichia coli

The over-expressed extracellular sucrase (SacC) of Zymomonas mobilisfrom a recombinant Escherichia coli (pZSP62) carrying the sacC gene was purified partially by repeated cycles of freezing and thawing. This method separated the highly expressed recombinant protein from the bulk of endogenous E. coli proteins. The enzyme was further purified 14 fold with a 55% yield from the cellular extract of E. coli by hydroxyapatite chromatography. The purified enzyme had a Mr of 46 kDa by SDS-PAGE. Its km value for sucrose was 86 mM and was optimal at pH 5.0 and at 36°C.     

产乙醇大肠杆菌的构建及其活性检测方法的改进

adhB和pdc是运动发酵单胞菌产乙醇途径的关键基因,分别编码乙醇脱氢酶和丙酮酸脱羧酶,将添加有聚球藻PCC7942rbcLS基因RBS序列的adhB和pdc基因插入pUC18载体,经双重菌液PCR检验和酶切检验得到分别含有pUC-adhB、pUC-pdc和pUC-adhB-pdc载体的3个重组菌株。活性检测实验表明聚球藻PCC7942的rbcLS基因的RBS序列能有效介导运动发酵单胞菌的adhB和pdc基因在大肠杆菌中表达,摇瓶发酵实验表明重组大肠杆菌的产乙醇能力较出发菌株大幅提升。鉴于乙醛指示平板法存在着对希夫试剂的要求较高、易产生较强的背景色等缺点,对定性检测丙酮酸脱羧酶和乙醇脱氢酶表达菌株的方法做了改进,即:将菌液诱导表达,然后分别添加对应于两种酶的底物,让酶与底物反应0.5至1小时,之后再加希夫试剂进行显色反应,结果表明改进后的方法比乙醛指示平板法更加简便、快速、可靠。