Identification and characterization of novel ColE1-type, high-copy number plasmid mutants in Legionella pneumophila

ColE1-type plasmids are commonly used in bacterial genetics research, and replication of these plasmids is regulated by interaction of RNA I and RNA II. Although these plasmids are narrow-host-range, they can be maintained in Legionella pneumophila under antibiotic selection, with low-copy number and instability. Here, we have described the isolation of two novel spontaneous mutants of pBC(gfp)Pmip, pBG307 and pBG309, which are able to mark the L. pneumophila with strong green fluorescence when exposed to visible light. One of the mutants, pBG307, has a single CG-->TA mutation in RNA II promoter located 2-bases upstream the - 10 region. Another one, pBG309, has the same mutation, as well as an additional CG-->AT mutation in the 76th nucleotide of RNA I, or in the 6th nucleotide of RNA II. A plasmid with the single mutation in RNA I, pBG308, was also constructed. Characterization of these plasmids carrying the enhanced green fluorescent protein (gfpmut2) gene revealed that the green fluorescence intensities of these plasmids were 2- to 30-fold higher than that of the wild type and both of the mutations contribute to increase the plasmid copy number and/or plasmid stability. The mutation located in RNA II promoter played a more dominant role in elevating the copy number, compared to the mutation in RNA I. We also tested the mutant plasmids for replication in Escherichia coli, and found that their copy number and stability were dramatically decreased, except pBG307. Our data suggest that these plasmids might be useful and convenient in genetic studies in L. pneumophila.     

A two-plasmid Escherichia coli system for expression of Dr adhesins

This paper presents a very efficient expression system for production of Dr adhesins. The system consists of two plasmids. One is the pACYCpBAD-DraC-C-His, which contains the draC gene under the control of the arabinose promoter (pBAD), encoding the DraC usher. The second is the pET30b-syg-DraBE, which contains the draB and draE genes under the control of the T7lac promoter, encoding the DraB chaperone and the DraE adhesin, respectively. Those plasmids have different origin of replication and can therefore coexist in one cell. Since different promoters are present, the protein expression can be controlled. The Dr adhesion expression system constructed opens up a lot of possibilities, and could be very useful in experiments focusing on understanding the biogenesis of Gram-negative bacteria adhesins. For this purpose we showed that the AfaE-III adhesin (98.1% identity between the DraE and the AfaE-III adhesins, with three divergent amino acids within the sequences) was able to pass through the DraC channel in the Escherichia coli BL21(DE3) strain. Immunoblotting analysis and immunofluorescence microscopy showed the presence of AfaE-III on the bacterial cell surface. In addition, the system described can be useful for displaying the immune-relevant sectors of foreign proteins on the bacterial cell. The heterologous epitope sequence of the HSV1 glycoprotein D was inserted into the draE gene in place of the N-terminal region of surface exposed domain 2. Chimeric proteins were exposed on the bacterial surface as evidenced by immunoblotting and immunofluorescence microscopy. The effective display of peptide segments on Dr fimbriae expressed at the bacterial cell surface, can be used for the development of a fimbrial vaccine.     

pPSY: a vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli

pPSY is a 12kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10kb. In the present study three different genes were amplified by PCR, cloned into pGEM-T Easy and sub-cloned into the EcoRI site of pPSY. The first gene, vioA, is a FAD-dependent l-tryptophan amino acid oxygenase from the high G+C Gram-negative bacterium Chromobacterium violaceum. VioA is involved in the synthesis of the indolocarbazole antitumour antibiotic violacein. It was found that vioA was strongly expressed in Escherichia coli from its native promoter. Two other genes encoding recombinase A (recA) and an amylase (amyA), derived from the high G+C Gram-positive streptomycete, Streptomyces lividans, were also tested. Despite recA lacking its native promoter sequence, it was strongly expressed in E. coli using the lac promoter of pGEM-T Easy. Similar to vioA, S. lividansamyA was strongly expressed in E. coli from its native promoter. Unlike pGEM-T Easy, pPSY stably maintained all three genes without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable amplicon cloning vector for the expression of heterologous genes in E. coli.     

High-level expression of bovine rotavirus VP4 protein in Escherichia coli using a hepatitis B core antigen vector

Six regions of the VP4 protein of bovine rotavirus strain UKtc were expressed using hepatitis B core antigen (HBcAg) as a carrier. Following induction by IPTG, the six fusion proteins, AHBcAg through FHBcAg, were expressed in Escherichia coli to a level of 20–37% of total cellular protein. Soluble fusion proteins in a particulate form were partially purified with yields ranging from 0.5–6.4 mg l^–1 of culture.     

大肠埃希菌生物被膜基因调控研究进展

大肠埃希菌(Escherichia coli)是一种兼性厌氧、有鞭毛的革兰氏阴性短杆菌,常寄生于人和动物肠道内,是常见的人畜共患病病原之一。大肠埃希菌易形成生物被膜,这是一种由细菌群落分泌能够包裹自身的胞外基质与细菌结合形成的特殊聚集体,也是临床细菌感染疾病难以治愈的主要原因。生物被膜的形成不仅帮助细菌逃避宿主的防御系统,还可以降低或阻止药物发挥作用,从而诱发生物被膜相关感染(biofilm-associated infections, BAI)。本文从生物被膜形成的基因调控系统和相关调控蛋白等角度,归纳总结调控大肠埃希菌生物被膜形成的分子机制,并对防治BAI的策略进行了概述,为寻找合适的药物靶点以及防治BAI提供参考。     

A novel expression vector, designated as pHisJM, for producing recombinant His-fusion proteins

Compared to glutathione S -transferase (GST), tagging with hexahistidine residues (His) has several merits: low levels of toxicity and immunogenicity, a smaller size and no electric charge. We have constructed a novel expression vector, designated as pHisJM (EMBL/GenBank/DDJB accession no. AB116367), for producing recombinant His-fusion proteins. This vector was constructed by replacing GST and multiple cloning site (MCS) cassettes in pGEX-5X-3 with those of hexahistidine and MCS derived from pRSET C vector. Human annexin IV (Anx IV) was used as target protein. His-Anx IV fusion protein was expressed using pHisJM and gave a 40 kDa band when immuno-stained with anti-His mAb or anti-Anx IV mAb as predicted. To compare expression efficiency, a Anx IV cDNA inserted-pHisJM or pGEX-5X-3 was transformed into Escherichia coli DH5alpha, JM109, BL21 and BL21(DE3). Using pHisJM, Anx IV protein was highly expressed in all cell strains. In addition to the merits of using His-tag, pHisJM has several advantages: 1) it has high expression efficiency; 2) it can be used in any Escherichia coli strain; and 3) it can be used in a single strain of Escherichia coli in all steps from plasmid construction to the expression of the target gene.     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacteriumAnabaena sp. strain PCC 7120

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacteriumAnabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of theAnabaena ADPGlc PPase gene and its expression inEscherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48347 Da which is in agreement with the molecular mass determined by SDS-PAGE for theAnabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and theE. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in theAnabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in anE. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the nativeAnabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be theAnabaena enzyme.     

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.     

Characterization of a cryptic plasmid from a Greenland ice core Arthrobacter isolate and construction of a shuttle vector that replicates in psychrophilic high G+C Gram-positive recipients

Over 60 Greenland glacial isolates were screened for plasmids and antibiotic resistance/sensitivity as the first step in establishing a genetic system. Sequence analysis of a small, cryptic, 1,950 bp plasmid, p54, from isolate GIC54, related to Arthrobacter agilis, showed a region similar to that found in theta replicating Rhodococcus plasmids. A 6,002 bp shuttle vector, pSVJ21, was constructed by ligating p54 and pUC18 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. Candidate Gram-positive recipients were chosen among glacial isolates based on phylogenetic relatedness, relatively short doubling times at low temperatures, sensitivity to antibiotics, and absence of indigenous plasmids. We developed an electroporation protocol and transformed seven isolates related to members of the Arthrobacter, Microbacterium, Curtobacterium, and Rhodoglobus genera with pSVJ21. Plasmid stability was demonstrated by successive transformation into Escherichia coli and four Gram-positive isolates, growth without antibiotic, and plasmid re-isolation. This shuttle vector and our transformation protocol provide the basis for genetic experiments with different high G+C Gram-positive hosts to study cold adaptation and expression of cold-active enzymes at low temperatures.     

滇龙胆GrWRKY5基因的克隆和植物表达载体构建

WRKY蛋白是目前被广泛研究的一类DNA特异结合转录因子,在植物次生代谢物生物合成、植物生长和发育及衰老等生理过程以及生物、非生物防御反应中起重要的调控作用。该研究从滇龙胆幼叶中克隆萜类合成的关键转录因子基因Gr WRKY5,并利用生物信息学方法对基因功能进行预测,构建植物过表达载体。结果表明:根据三年生滇龙胆转录组Gr WRKY5基因序列,设计特异性引物,通过RT-PCR扩增Gr WRKY5 ORF序列,并进行TA克隆、测序和序列分析;构建入门载体p ENTRTM2B-Gr WRKY5,经LR反应后构建植物过表达载体。Gr WRKY5 ORF全长591 bp,编码196个氨基酸,Gen Bank登录号为KF922375,其中第167与170之间的色氨酸(W)、精氨酸(R)、赖氨酸(K)、酪氨酸(Y)组成WRKY蛋白所特有的"WRKY"结构。序列分析表明Gr WRKY5是WRKY超家族的成员。经生物信息学在线软件分析发现,Gr WRKY5的等电点为6.29,脂肪族指数为61.37,不稳定指数为57.80。总平均疏水性为-0.708,为亲水蛋白;含有20种氨基酸,其中天冬氨酸(Asp)和脯氨酸(Pro)含量最高,为8.1%;半胱氨酸(Cys)含量最低,仅为1.0%。氨基酸序列系统发育分析表明,Gr WRKY5与拟南芥中WRKY家族中遗传距离最接近的是WRKY27,属于Ⅱe类成员;与Cr WRKY22和Vv WRKY22蛋白的亲缘关系较近;与Jc WRKY47和Tc WRKY27亲缘关系较远;BLASTp结果显示,Gr WRKY5与欧洲油菜Bn WRKY27-1的同源性最高(为69%);与拟南芥Aa WRKY22的一致性最低(仅为31%)。以Gateway入门载体p ENTR2B和目的载体p K2GW7为基础,成功构建了植物过表达载体p K2-35S-Gr WRKY5,该载体的成功构建为该基因在拟南芥、滇龙胆等植物中的遗传转化奠定了基础,同时为WRKY基因功能的深入研究提供依据。     

Co-expression of a prophage system and a plasmid system in Bacillus subtilis

A dual expression system for overexpressing two proteins by a single cell strain has been developed in Bacillus subtilis. This dual expression system combines the phi105MU331 prophage system and a plasmid system within a single cell. Protein expression by the prophage system is heat inducible, while that of the plasmid system is constitutive. Three candidate genes, BPN, BT, and amyE, all of Bacillus origin, were used as test models. Seven strains (BPN, BT, AMY, BS168K, MU331K, BPNK, and BTK) were constructed to investigate the influences of the prophage system and the plasmid system on each other, and to compare the efficiency of the individual expression systems with that of the dual expression system. Individually, the yield of the plasmid system is higher than that of the prophage system, which could be attributed to the constitutive nature of the expression of the plasmid system. Nonetheless, for the dual expression strains, the expression of two enzymes in a single fermentation run can reduce costs in facilities, manpower, and utilities. Fed-batch fermentation of BPNK strains confirmed the feasibility of applying this dual expression system in industrial-scale production.     

Effects of fermentation feeding strategies prior to induction of expression of a recombinant malaria antigen inEscherichia coli

Summary A variety of feeding strategies have been described for attaining high cell densities in fed-batch fermentors. Although cell density is an important component in the produtivity of recombinant fermentations, it must be achievable with high product expression levels. Experiments were conducted to study the influence of fermentation feeding strategies on the production of a recombinant malaria antigen inEscherichia coli. C-source feeding profiles were calculated to maintain specific growth rates at 0.1, 0.2, 0.35, and 0.5 l/h prior to induction in defined and complex media using an exponential growth model. Fed-batch fermentations employing these feeding profiles effectively controlled the specific growth rates prior to induction. Antigen yields per dry cell weight did not vary with specific growth rate. Antigen yields from fed-batch fermentations achieving high cell densities were similar to batch fermentations achieving low cell densities. These results show that C-feeding policies can limit growth without reducing expression levels in some systems, and suggest applications in managing oxygen demand and catabolic by-product formation during process scale-up.     

Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene

Summary An integration vector for use in Methanococcus voltae was constructed, based on the Escherichia coli vector pUC18. It carries the structural gene for puromycin transacetylase from Streptomyces alboniger, which is flanked by expression signals of M. voltae structural genes and hisA gene sequences of this bacterium. Transformed M. voltae cells are puromycin resistant. Several types of integration of the vector into the chromosome were found. Only one case was due to nonhomologous recombination. The integrated sequences were stable under selective pressure but were slowly lost in some cases in the absence of the selective drug. The vector could be excised from M. voltae chromosomal DNA, recircularized and transformed back into E. coli.The order of the other authors is not indicative of the relative importance of their experimental contributions which are considered to be equivalentWe mourn the loss of our colleague and friend Lionel Sibold, who died while this work was still in progress