Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.     

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.     

Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase

The genes for FokI, a type-IIS restriction-modification system from Flavobacterium okeanokoites (asymmetric recognition sequence: 5'-GGATG/3'-CCTAC), were cloned into Escherichia coli. Recombinants carrying the fokIR and fokIM genes were found to modify their DNA completely, and to restrict lambdoid phages weakly. The nt sequences of the genes were determined, and the probable start codons were confirmed by aa sequencing. The FokI endonuclease (R · FokI) and methyltransferase (M · FokI are encoded by single, adjacent genes, aligned in the same orientation, in the order M then R. The genes are large by the standards of type-II systems, 1.9 kb for the M gene, and 1.7 kb for the R gene. Preceding each gene is a pair of FokI recognition sites; it is conceivable that interactions between the sites and the FokI proteins could regulate expression of the genes. The aa sequences of the N- and C-terminal halves of M · FokI are similar to one another, and to certain other DNA-adenine methyltransferases, suggesting that the enzyme has a ‘tandem’ structure, such as could have arisen by the fusion of a pair of adjacent, ancestral M genes. Truncated derivatives of M · FokI were constructed by deleting the 5'- or 3' -ends of the fokIM gene. Deleting most of the C-terminus of M · FokI produced derivatives that methylated only the top (GGATG) strand of the recognition sequence. Conversely, deleting most of the N-terminus produced derivatives that methylated only the bottom (CATCC) strand of the recognition sequence. These results indicate that the domains in M · FokI for methylating the two strands of the recognition sequence are largely separate.     

The tyrosyl-tRNA synthetase from Escherichia coli: Complete nucleotide sequence of the structural gene

The structural component of the tyrS gene of Escherichia coli, comprising 1269 base pairs, has been fully sequenced by the combined M13/dideoxychain termination approach. The gene has a codon usage pattern which is typical of highly expressed proteins and similar to other Escherichia coli aminoacyl-tRNA synthetase genes. Peptide purification and sequencing has been used to locate the N-terminus and to provide confirmation of 95% of the translated protein sequence. This latter yields on M_r of 47 403 for the Escherichia coli tyrosyl-tRNA synthetase, and reveals considerable homology with the primary structure of the analogous enzyme isolated from Bacillus staerothermophilus.     

Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes

The nucleotide sequences of the Escherichia coli genome between the glycogen biosynthetic genes glgB and glgC, and 1170 bp of DNA which follows glgA have been determined. The region between glgB and glgC contains an open reading frame (ORF) of 1521 bp which we call glgX. This ORF is capable of coding for an M_r 56 684 protein. The deduced amino acid (aa) sequence for the putative product shows significant similarity to the E. coli glycogen branching enzyme, and to several different glucan hydrolases and transferases. The regions of sequence similarity include residues which have been reported to be involved in substrate binding and catalysis by taka-amylase. This suggests that the proposed product may catalyze hydrolysis or glycosyltransferase reactions. The cloned region which follows glgA contains an incomplete ORF (1149 bp), glgY, which appears to encode 383 aa of the N terminus of glycogen phosphorylase, based upon sequence similarity with the enzyme from rabbit muscle (47% identical aa residues) and with maltodextrin phosphorylase from E. coli (37% identical aa residues). Results suggest that neither ORF is required for glycogen biosynthesis. The localization of glycogen biosynthetic and degradative genes together in a cluster may facilitate the regulation of these systems in vivo.     

A novel DNA microarray design for accurate and straightforward identification of Escherichia coli safety and laboratory strains

Escherichia coli K-12, B, C and W strains and their derivates are declared in biological safety guidelines as risk group 1 organisms as they are unable to colonise the human gut.

Differentiation and identification of these safety strains is mainly based on pulsed-field gel electrophoresis (PFGE), phage sensitivity tests or PCR-based methods. However, these methods are either tedious and time consuming (phage sensitivity, PFGE) or based on single specific fragments (PCR) or patterns (PFGE) lacking additional information for further differentiation of the strains.

In the current study, subtractive hybridisation techniques were applied to detect specific DNA fragments which were used to design a microarray (chip) for accurate and simple identification of these organisms, and to differentiate them from other E. coli strains. The chip can be used to identify E. coli safety strains and monitor them during ongoing experiments for changes in their genome and culture purity. The hybridisation layout of the microarray was arranged in such a way that the respective lineages of safety strains could be easily identified as distinct letters (K, B, C or W). Differentiation of single strains or subtyping was possible with further probes. In addition, a set of probes targeting genes coding for common virulence factors has been included, both to differentiate safety strains from pathogenic variants and to make sure that no transfer of these genes happens during handling or storage. The reliability of the approach has been tested on a comprehensive selection of E. coli laboratory strains and pathogenic representatives.     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

The Mycobacterium tuberculosis mysB gene product is a functional equivalent of the Escherichia coli sigma factor, KatF

Mycobacterium tuberculosis, the causative agent of tuberculosis, may remain dormant within its host for many years. The nature of this dormant or latent state is not known, but it may be a specialized form of the stationary growth phase. In Escherichia coli, KatF (or RpoS) is the major stationary phase sigma factor regulating an array of genes expressed in this phase of growth. A potential M. tuberculosis katF homologue was cloned using a fragment of the E. coli katF gene as a probe. DNA sequence analysis of a resultant clone showed 100% identity to a fragment of DNA encoding the M. tuberculosis mysA and mysB genes. Overexpression of mysB in M. bovis BCG resulted in an increase in katG mRNA and catalase and peroxidase activity, and an increase in sensitivity of the cells to isoniazid. An increase in katG promoter activity from a reporter vector was demonstrated when mysB was overexpressed from the same plasmid, indicating a direct relationship between MysB and katG expression.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5′-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3′ end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

Isolation and characterization of the Neisseria meningitidis lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis

The lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis in Neisseria meningitidis has been cloned and sequenced. By complementation of a temperature-sensitive E. coli lpxD mutant, we first cloned a meningococcal chromosomal fragment that carries the lpxD homologue. Cloning and sequence analysis of chromosomal DNA downstream of lpxD revealed the presence of the fabZ and lpxA genes. This gene cluster shows high homology to the corresponding genes from several other bacterial species. The LpxA and LpxD proteins catalyze early steps in the lipid A biosynthetic pathway, adding the O- and N-linked 3-OH fatty acyl chains, respectively. In E. coli and N. meningitidis, LpxD has the same specificity, in both cases adding 3-OH myristoyl chains; in contrast to E. coli, the meningococcal LpxA protein is presumed to add 3-OH lauroyl chains instead. The established sequence points the way to further experiments to define the basis for this difference in specificity, and should allow modification of meningococcal lipid A biosynthesis through gene exchange.     

基于转录物组学的大肠杆菌血红素过氧化物酶EfeB的生理功能解析

大肠杆菌血红素过氧化物酶EfeB属于染料脱色过氧化物酶超家族。该家族的酶类一般具有良好的合成染料降解能力,但是其在生物体内的功能尚不清楚。为了深入研究EfeB的生理功能,本文通过同源重组的方法构建了efeB敲除菌株Eco△efeB,比较了亲本菌株E.coli BL21和Eco△efeB在基因组转录水平上、不同条件下的细胞生长以及对铁离子应答上的差异。结果表明:efeB基因的缺失引起了菌体1 765个基因的差异表达,这些基因主要与菌体的细胞代谢途径、细胞膜合成和鞭毛运动有关;在pH为7.0时,BL21和EcoΔefeB的生长无显著差异,但在pH4.5时BL21的生长明显优于EcoΔefeB,efeB基因的功能性表达可以支持大肠杆菌在低pH下的生存;当培养环境中有Fe²⁺存在时,efeB显著上调。以上结果为EfeB生理功能的认知和利用提供了一定的理论依据。     

Biocatalytic synthesis of monocyclic arene-dihydrodiols and -diols by Escherichia coli cells expressing hybrid toluene/biphenyl dioxygenase and dihydrodiol dehydrogenase genes

The hybrid toluene/biphenyl dioxygenase, which is encoded by the todC1 gene of Pseudomonas putida F1 and the bphA2A3A4 genes of Pseudomonas pseudoalcaligenes KF707, has substrate ranges wider than toluene dioxygenase endoced by the todC1C2BA genes of P. putida F1. We carried out growing cell reactions by Escherichia coli expressing the todC1-bphA2A3A4 genes for the comprehensive production of monocyclic arene-dihydrodiols. As a result, we successfully biotranformed acetophenone-related compounds (acetophenone, propiophenone, and butyrophenone) to the corresponding cis-dihydrodiols. Furthermore, we performed the bioconversion experiments by E. coli cells expressing the bphB (dihydrodiol dehydrogenase) gene in addition to todC1-bphA2A3A4 to produce a series of monocyclic arene-diols. Consequently, toluene, benzene, stylene, p-xylene, acetophenone, propiophenone, butyrophenone, and trifluoroacetophenone were converted to the corresponding vicinal diols. The antioxidative activity of these generated diol compounds was markedly higher than that of the substrate used.     

真核表达猪白细胞介素17及其生物活性研究

目的:为真核表达猪白细胞介素17(IL-17),研究产物在细胞培养下的免疫生物活性。方法:通过PCR扩增出猪IL-17基因并插入到真核表达载体p VAX1,然后转染到IPEC-J2细胞、Ha Ca T细胞和L02细胞中。在转染后第24、48和72h收集细胞,第48h收集上清液。收集细胞通过实时荧光定量PCR检测相关免疫基因的表达水平,收集上清液通过抑菌试验检测相关抗菌肽的生物活性。结果:采用p VAX1载体构建了表达猪IL-17的重组质粒,转染到细胞中。证实IL-17基因能诱导抗菌肽基因(RegⅢ、S100A8和BD2)的表达,显著上调JAK-STAT信号通路基因(JAK1、STAT1和STAT3)和细胞因子基因(IL-6、IL-12和TNF-α)的表达。此外,细胞上清液能够在不同程度上抑制大肠杆菌及金黄色葡萄球菌的增殖。结论:成功将猪IL-17基因真核表达,其表达产物能诱导效应细胞表达多种细胞因子,产生多种抗菌肽,具有抑菌能力;这为进一步研发猪IL-17作为抗菌免疫分子制剂奠定了初步基础。     

Chlorambucil-induced high mutation rate and suicidal gene downregulation in a base excision repair-deficient Escherichia coli strain

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating agent widely used as an anticancer drug and also as an immunosuppressant. Its chemical structure and clinical experience indicate that CLB is mutagenic and carcinogenic. We have investigated the ability of CLB to induce mutations and gene expression changes in the wild-type (WT) Escherichia coli strain AB1157 and in the base excision repair-deficient (alkA1, tag-1) E. coli strain MV1932 using a rifampicin (rif) forward mutation system and a cDNA array method. The results showed that CLB is a potent mutagen in MV1932 cells compared with the E. coli WT strain AB1157, emphasizing the role of 3-methyladenine DNA glycosylases I and II in protecting the cells from CLB-induced DNA damage and subsequent mutations. Global gene expression profiling revealed that nine genes in WT E. coli and 100 genes in MV1932, of a total of 4290 genes, responded at least 2.5-fold to CLB. Interestingly, all of these MV1932 genes were downregulated, while 22% were upregulated in WT cells. The downregulated genes in MV1932 represented most (19/23) functional categories, and unexpectedly, many of them code for proteins responsible for genomic integrity. These include: (i) RecF (SOS-response, adaptive mutation), (ii) RecC (resistance to cross-linking agents), (iii) HepA (DNA repair, a possible substitute of RecBCD), (iv) Ssb (DNA recombination repair, controls RecBCD), and (v) SbcC (genetic recombination). Our results strongly suggest that in addition to the DNA damage itself, the downregulation of central protecting genes is responsible for the decreased cell survival (demonstrated in a previous work) and the increased mutation rate (this work) of DNA repair-deficient cells, when exposed to CLB.     

Monitoring of transcriptome and proteome profiles to investigate the cellular response of E. coli towards recombinant protein expression under defined chemostat conditions

The use of strong promoter systems for recombinant protein production generates high product yields, but also overburdens the host cell metabolism and compromises production. Escherichia coli has highly developed regulatory pathways that are immediately responsive to adverse conditions. To gain insight into stress response mechanisms and to detect marker genes and proteins for stress specific monitoring time course analysis of controlled chemostat cultivations was performed using E. coli total microarray and difference gel electrophoresis (Ettan™ DIGE). In order to detect differences and consistencies of stress response as well as the impact of the inducer isopropyl-β-d-thiogalactopyranosid on cells, expression of two recombinant proteins (hSOD and GFPmut3.1) was investigated. Genes involved in aerobic metabolism under control of the ArcB/ArcA two component system were found to be down-regulated, and the interplay of the psp operon, ArcA system and guanosine tetraphosphate is suggested to be involved in stress regulatory mechanisms. A distinct impact of the two recombinant proteins was observed, particularly on levels of known stress regulatory genes and proteins, as well as on the response associated with ArcA and psp. Altogether, 62 genes as well as seven proteins showed consistent expression levels due to recombinant gene expression, and are therefore suggested to be appropriate monitoring targets.     

Hydroxylations of substituted naphthalenes by Escherichia coli expressing aromatic dihydroxylating dioxygenase genes from polycyclic aromatic hydrocarbon-utilizing marine bacteria

Bioconversion experiments of various mono- or di-substituted naphthalenes such as dimethylnaphthalenes were carried out using the cells of Escherichia coli that expressed aromatic dihydroxylating dioxygenase genes (phnA1A2A3A4 and phdABCD) from polycyclic aromatic hydrocarbon-utilizing marine bacteria, Nocardioides sp. KP7 and Cycloclasticus sp. A5, respectively. We found that the former dioxygenase PhnA1A2A3A4 had broad substrate preference for these compounds and often was able to hydroxylate their methyl groups. Specifically, 1,4-dimethylnaphthalene was predominantly bioconverted into 1,4-dihydroxymethylnaphthalene.     

钝齿棒杆菌中异源表达N-乙酰鸟氨酸脱乙酰基酶合成L-鸟氨酸的研究

目的:对一株产鸟氨酸的钝齿棒杆菌Corynebacterium crenatum SYPA5-5/△proB/△argF(SYPO-1)进行代谢工程改造,筛选不同细菌来源的N-乙酰鸟氨酸脱乙酰基酶在大肠杆菌中克隆与表达,纯化后对其进行酶学性质的比较;将黏质沙雷氏菌Serratia marcescens Y213来源的Smarg E基因编码的N-乙酰鸟氨酸脱乙酰基酶在L-鸟氨酸生产菌株C.crenatum SYPO-1中过量表达,进一步提高L-鸟氨酸的产量。方法:通过利用pDXW10穿梭质粒对不同来源的N-乙酰鸟氨酸脱乙酰化酶进行克隆表达和酶学性质比较,选择性质最优来源的N-乙酰鸟氨酸脱乙酰基酶编码基因Smarg E在产L-鸟氨酸重组钝齿棒杆菌中表达,考察重组菌株发酵过程中参数的变化。结果:来源于S.marcescens Y213的N-乙酰鸟氨酸脱乙酰基酶比酶活最高为798.98U/mg,最适pH为7,最适温度为37℃,0.1mmol/L的Mg~(2+)、Li~+、Mn~(2+)促进酶的比酶活提高了50%;在钝齿棒杆菌中表达N-乙酰鸟氨酸脱乙酰基酶酶活达到128.4U/ml,显著提高了钝齿棒杆菌中胞内乙酰基循环水平;5L发酵罐发酵重组菌株96h,L-鸟氨酸的产量达到38.5g/L,比出发菌株,L-鸟氨酸的产量提高了33.2%,产率达0.401g/(L·h)。结论:筛选出最佳来源的N-乙酰鸟氨酸脱乙酰基酶,在鸟氨酸生产菌株C.crenatum(SYPO-1)中过量表达,可以促进鸟氨酸的前体物质N-乙酰鸟氨酸的快速消耗,实现鸟氨酸的积累。