<Emphasis Type="Italic">Escherichia coli</Emphasis> malate dehydrogenase,a novel solubility enhancer for heterologous proteins synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Using 2-dimensional gel electrophoresis, the Escherichia coli proteome response to a heat-shock stress was analyzed and a 1.6-fold increase of malate dehydrogenase was observed even under the heat-shock condition where the total number of soluble proteins decreased by about 5%. We subsequently demonstrated that, as an N-terminus fusion expression partner, malate dehydrogenase facilitated the folding of, and dramatically increased the solubility of, many aggregation-prone heterologous proteins in E. coli cytoplasm. Therefore, malate dehydrogenase is well suited for production of a biologically active fusion mutant of cutinase (Pseudomonas putida origin) that is currently of considerable to biotechnology and commercial industries.     

Role of oxygen in the utilization of phenol by Pseudomonas CF600 in continuous culture

The dissolved oxygen (DO) level is the key factor which decides the rate of degradation of the organic load in aerobic growth conditions. In this study the role of DO levels on the utilization of phenols has been reported using the continuous culture system. A phenol-utilizing strain, Pseudomonas CF600 has been used as a model. Its phenol-degrading capacity was studied using continuous cultivation for a period of 60 days. The bioreactor was kept at a dilution rate of 0.006 h^–1 with DO levels maintained at 2, 3, and 4 ppm keeping all the other cultivation conditions constant. Physiological variations under the cultivation conditions were studied by monitoring off-line phenol utilization and respirometric analysis of harvested culture against different substrates. It was observed that the accumulation of 2-hydroxymuconate semialdehyde (HMS), an intermediate in the phenol degradation pathway, depends on the DO level. The maximum level of HMS in the medium observed was 3.92 M when DO was maintained at 2 ppm whereas with 3 ppm of DO, HMS level was below 0.4 M. Oxygen uptake data of the cells harvested from cultures grown at different DO levels showed that the uptake was highest at 3 ppm DO for all the substrates tried. When phenol was used as substrate, the oxygen uptake rate was 42.66, 66.36 and 35.55 nM/min/mg dry weight of cells at 4, 3 and 2 ppm DO respectively. Results show that DO levels influence the rate of phenol utilization in Pseudomonas CF600.     

Molecular analysis of the <Emphasis Type="Italic">fur</Emphasis> (ferric uptake regulator) gene of a pathogenic <Emphasis Type="Italic">Edwardsiella tarda</Emphasis> strain

The gene encoding the Edwardsiella tarda ferric uptake regulator (Fur(Et)) was cloned from a pathogenic E. tarda strain isolated from diseased fish. Fur(Et) shares 90% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and was able to complement the mutant phenotype of a fur (Ec)-defective E. coli strain. Mutational analysis indicated that C92S and C95S mutations inactivated Fur(Et) whereas E112K mutation resulted in a superactive Fur(Et) variant. Fur(Et) negatively regulated its own expression; interruption of this regulation impaired bacterial growth, altered the production of certain outer membrane proteins, and attenuated bacterial virulence.     

The molybdate-binding protein (ModA) of the plant pathogen Xanthomonas axonopodis pv. citri

The modABC operon of phytopathogen Xanthomonas axonopodis pv. citri (X. citri) encodes a putative ABC transporter involved in the uptake of the molybdate and tungstate anions. Sequence analyses showed high similarity values of ModA orthologs found in X. campestris pv. campestris (X. campestris) and Escherichia coli. The X. citri modA gene was cloned in pET28a and the recombinant protein, expressed in the E. coli BL21 (DE3) strain, purified by immobilized metal affinity chromatography. The purified protein remained soluble and specifically bound molybdate and tungstate with K(d) 0.29+/-0.12 microM and 0.58+/-0.14 microM, respectively. Additionally binding of molybdate drastically enhanced the thermal stability of the recombinant ModA as compared to the apoprotein. This is the first characterization of a ModA ortholog expressed by a phytopathogen and represents an important tool for functional, biochemical and structural analyses of molybdate transport in Xanthomonas species.     

Effect of lactic bacteria metabolic products on the transmission of R-plasmids in enterobacteria in vitro]

The authors present data on the study of lactobacteria used in the production of dry lactobacterin (lactobacterinum siccum). Metabolic products (lactocidin) were extracted with lactic and acetic acid after Vincent et al. Two donor strains (E. coli K12 J5-3 R1-19 and E. coli K12 W1845 R26) were crossed in the conjugation process in various conbinations with six recipient strains (E. coli K12S, E. coli Su 3912/41, Sh. sonnei 263B, Sh. sonnel 3470, S. heidelberg, A161, and S. typhimurium SH3 his-). The frequency of R-plasmide transmission in enterobacteria was decreased in vitro under the effect of L. plantarum 8R-A3 and L. fermentum 90T-S4 metabolites.     

Cloning and characterization of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene of the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus aidingensis</Emphasis> AD-6<Superscript>T</Superscript>

A gene encoding a Na(+)/H(+) antiporter was obtained from the genome of Halobacillus aidingensis AD-6(T), which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na(+)/H(+) antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K(+)/H(+) antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na(+)/H(+) antiporter activity.     

Preliminary characterisation of an Escherichia coli K5 lyase-deficient strain producing the K5 polysaccharide

Escherichia coli K5 polysaccharide has structural analogies with N-acetylheparosan, a non-sulphated precursor of heparin and, for this reason, can be considered an attractive precursor for the production of semi-synthesis heparin analogues. This polysaccharide has two components: a high molecular weight (HMW) one and a low molecular weight (LMW) one, whose ratio varies depending on the action of a lyase enzyme synthesized by the same K5 producer strain. The present paper reports the production of the K5 polysaccharide by a spontaneous E. coli mutant strain lacking the lyase activity. Similar K5 polysaccharide yields, 180 mg l(-1) after 16 h fermentation, were obtained by both the wild and mutant strains, though K5 lyase activity was only observed in the culture filtrates from the wild strain. The time course of the specific filtrate volume (1 m(-2)) and of the specific filtrate flux rate (1 m(-2) h(-1)) during ultrafiltration (UF) of culture filtrates where the lyase enzyme acted on the K5 chain, showed a decrease of UF performance, probably because of membrane fouling by the LMW K5 fraction. In particular, the specific filtrate volume and specific filtrate flux rate of wild strain samples reached respectively 13 l m(-2) and 4 l m(-2) h(-1), compared to 25 l m(-2) and 15 l m(-2) h(-1) obtained from the mutant strain samples. PCR molecular analysis of the DNA region encoding for the lyase enzyme showed that, in the mutant strain, molecular rearrangements occurred in both regulatory and structural regions.     

Purification and in vitro characterization of the maltose-binding protein of the plant pathogen Xanthomonas citri

The uptake of maltose and maltodextrins in gram-negative bacteria is mediated by an ATP-dependent transport complex composed of a periplasmic maltose-binding protein (MBP) and membrane-associated proteins responsible for the formation of a membrane pore and generation of energy to drive the translocation process. In this work, we report the purification and in vitro functional analysis of MBP, encoded by the malE gene, of the plant pathogen Xanthomonas citri, responsible for the canker disease affecting citrus plants throughout the world. The X. citri MBP is composed of 456 amino acids, displaying a low amino acid identity (16% throughout the sequence) compared to the Escherichia coli K12 ortholog. The X. citri malE gene was cloned into a pET28a vector, and the encoded protein was expressed and purified by affinity chromatography as a His-tag N-terminal fusion peptide produced by the E. coli BL21 strain. Enhanced levels of soluble protein were achieved with static cultures kept overnight at 23 degrees C. Ability to bind immobilized amylose, the emission of intrinsic fluorescence and circular dichroism spectra indicated that the purified recombinant protein preserved both conformation and biological activity of the native protein. The availability of the recombinant MBP will contribute to the functional and structural analysis of the maltose and maltodextrin uptake system of the plant pathogen X. citri.     

Subunit fusion of two yeast d-amino acid oxidases enhances their thermostability and resistance to H2O2

D-amino acid oxidases from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) are both yeast homodimeric flavoenzymes. Two of their cDNA genes were connected by a hexanucleotide linker and heterologously expressed in E. coli to produce the corresponding double DAOs (dRtDAO and dTvDAO) with two subunits fused into a single polypeptide. The specific activities of double DAOs remained similar to those of native dimeric DAOs, although the catalytic efficiencies (k(cat)/K(M)) were decreased due to higher K(M) values. The T(m) value for dRtDAO was shifted 5 degrees C higher while that for dTvDAO was increased only by 2 degrees C, in comparison with the corresponding native counterparts. In the presence of 10 mM H(2)O(2), dRtDAO and dTvDAO exhibited half-lives of about 60 and 40 min, respectively, which were 2- and 1.5-fold, respectively, longer than their native DAOs. These yeast DAOs can therefore be thermally and oxidatively stabilized by linking their subunits together.     

Cloning and characterization of the gene cluster for biosynthesis of ectoine from <Emphasis Type="Italic">Nesterenkonia halobia</Emphasis> DSM 20541

The ectABC genes encoding the biosynthesis of ectoine were identified from Nesterenkonia halobia DSM 20541. The intergenic regions of the ectABC genes from N. halobia DSM 20541 were more loosely spaced than those that had been reported before. The amino acid sequence deduced from ectABC of the strain was highly homologous to the EctABC of Brevibacterium linens BL2 (EctA 50%, EctB 70%, and EctC 68% identities). The osmoprotection of ectABC was studied in the Escherichia coli KNabc and E. coli XL1-Blue. The results revealed that ectABC could shorten the lag phase and enhance the final OD600 of E. coli XL1-Blue in MM63 medium containing 0.68 M NaCl, and could initiate KNabc growth in 0.2 M NaCl. Ectoine was proven to be accumulated in E. coli KNabc/pGEM-Nect using HPLC-UV, and validated by LC-MSD-Trap-VL.     

Axial dispersion in respiratory bronchioles and alveolar ducts

The mixing of gases in the pulmonary acinus was characterized by analyzing axial gas dispersion during steady flow in models of respiratory bronchioles and alveolar ducts. An analysis (method of moments) developed for addressing dispersion in porous media was used to derive an integral expression for the axial dispersion coefficient (D*). Evaluation of D* required solving the Navier-Stokes equations for the flow field and a convection-diffusion type equation arising from the analysis. D* was strongly dependent on alveolar volume per central duct volume, the aperture size through which the alveoli communicate with the central duct, and the Péclet number (Pe). At smaller Pe (flow rate) D* was substantially smaller than the molecular diffusion coefficient, whereas at larger Pe (flow rate) D* was much greater than the Taylor-Aris result for flow-enhanced dispersion in straight tubes. Also, flow-enhanced dispersion became appreciable at smaller Pe than indicated by the Taylor-Aris result. These behaviors transcend both the lower and upper limits established previously for gas mixing in the pulmonary acinus.     

Inactivation of barotolerant strains of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 by ultra high pressure and <Emphasis Type="Italic">tert</Emphasis>-butylhydroquinone combination

Antimicrobial efficacy of ultra-high-pressure (UHP) can be enhanced by application of additional hurdles. The objective of this study was to systematically assess the enhancement in pressure lethality by TBHQ treatment, against barotolerant strains of Escherichia coli O157:H7 and Listeria monocytogenes. Two L. monocytogenes Scott A and the barotolerant OSY-328 strain, and two E. coli O157:H7 strains, EDL-933 and its barotolerant mutant, OSY-ASM, were tested. Cell suspensions containing TBHQ (50 ppm, dissolved in dimethyl sulfoxide) were pressurized at 200 to 500 MPa (23+/-2 degrees C) for 1 min, plated on tryptose agar and enumerated the survivors. The TBHQ-UHP combination resulted in synergistic inactivation of both pathogens, with different degrees of lethality among strains. The pressure lethality threshold, for the combination treatment, was lower for E. coli O157:H7 (> or = 200 MPa) than for L. monocytogenes (> 300 MPa). E. coli O157:H7 strains were extremely sensitive to the TBHQ-UHP treatment, compared to Listeria strains. Interestingly, a control treatment involving DMSO-UHP combination consistently resulted in higher inactivation than that achieved by UHP alone, against all strains tested. However, sensitization of the pathogens to UHP by the additives (TBHQ in DMSO) was prominently greater for UHP than DMSO. Differences in sensitivities to the treatment between these two pathogens may be attributed to discrepancies in cellular structure or physiological functions.     

Involvement of <Emphasis Type="Italic">soxRS</Emphasis> Regulon in Response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Oxidative Stress Induced by Hydrogen Peroxide

The effect of hydrogen peroxide on the activity of soxRS and oxyR regulon enzymes in different strains of Escherichia coli has been studied. Treatment of bacteria with 20 μM H₂O₂ caused an increase in catalase and peroxidase activities (oxyR regulon) in all strains investigated. It is shown for the first time that oxidative stress induced by hydrogen peroxide causes in some E. coli strains a small increase in activity of superoxide dismutase and glucose-6-phosphate dehydrogenase (soxRS regulon). This effect is cancelled by chloramphenicol, an inhibitor of protein synthesis in prokaryotes. The increase in soxRS regulon enzyme activities was not found in the strain lacking the soxR gene. These results provide evidence for the involvement of the soxRS regulon in the adaptive response of E. coli to oxidative stress induced by hydrogen peroxide. __________ Translated from Biokhimiya, Vol. 70, No. 11, 2005, pp. 1506–1513. Original Russian Text Copyright ? 2005 by Semchyshyn, Bagnyukova, Lushchak.     

山东省某地区奶牛源大肠埃希菌的血清型、耐药特性及分子特性

【目的】旨在对从山东省某地区4个健康奶牛养殖场分离到的大肠埃希菌进行优势血清型、耐药特性、Ⅰ类整合子基因盒携带情况以及系统进化群分析。【方法】采集194份来自山东省某地区4个规模化奶牛场奶牛新鲜粪便样品,进行大肠埃希菌分离和鉴定,利用常用大肠埃希菌诊断血清进行血清型鉴定;利用10%的绵羊血平板检测溶血性;利用K-B法检测对14种常规抗菌药物的敏感性;利用聚合酶链式反应(PCR)检测革兰阴性菌常见的6大类24种耐药基因、Ⅰ类整合子基因盒结构并对目的条带测序分析;利用细菌多位点序列分型(Multilocus sequence typing,MLST)技术分析大肠埃希菌的ST型并使用eBURST v3软件分析菌株之间的克隆关系。【结果】从194份新鲜粪便样品中分离到171株大肠埃希菌,其中主要为致病性(19.9%)和侵袭性大肠埃希菌(17.0%),优势血清型分别为O128:K67(12/171)和O143:K7(12/171)。另外,具有溶血性的大肠埃希菌阳性率为9.4%(16/171);药敏试验结果显示多重耐药菌株的比率为22.2%,其中对氨苄西林耐药率最高为33.9%,四环素次之,为24.0%;PCR检测耐药基因和整合子结果显示,59.1%的菌株携带β-内酰胺类耐药基因blaTEM,59.1%的菌株携带氨基糖苷类耐药基因ant(2′),未检测到四环素耐药基因tetA和tetB;Ⅰ类整合子的阳性率为4.1%(7/171),dfrA12-aadA2-sul1为优势基因盒结构(4/171);MLST将大肠埃希菌分为8种ST型,其中,ST155(10/171)和ST58(45/171)形成一个克隆复合物且没有发现新的ST型。【结论】本研究证实,从该地区规模化健康奶牛场新鲜粪便中分离到的大肠埃希菌优势血清型为O128:K67和O143:K7;少部分大肠埃希菌具有溶血性;仅对氨苄西林、四环素等具有较高的耐药率;优势基因盒结构为dfrA12-aadA2-sul1;MLST分型显示不同奶牛场分离出亲缘关系较近的菌株,其分布具有多态性,血清型与ST型之间无相关性。本研究表明源自表观健康的奶牛的大肠埃希菌存在多重耐药现象,具有食品公共卫生安全隐患,该研究对于提升规模化奶牛场奶制品的安全生产与质量评估具有一定的理论指导意义。