Cloning and expression of the erythromycin resistance determinant from Campylobacter jejuni in Escherichia coli

The erythromycin resistance gene (Em^r) from Campylobacter jejuni ABA94 plasmid DNA was cloned into the pUC18 vector and then expressed in Escherichia coli. The location of the Em^r determinant on the chimeric plasmid was determined by restriction endonuclease mapping within a 0.8-kb EcoRI fragment. This fragment then hybridized to the 78-kb plasmid DNA but not to the 3.3-or 12.6-kb plasmid DNA of Campylobacter jejuni ABA94. Em^r in Campylobacter jejuni is therefore probably plasmid-mediated.The authors are with the Department of Genetics and Cellular Biology, University of Malaya, 59100 Kuala Lumpur, Malaysia     

空肠弯曲菌fliD基因突变株的构建及其生物学特性

【目的】研究fliD基因对空肠弯曲菌生物学特性的影响,为阐明该基因的功能和作用机制奠定基础。【方法】利用同源重组技术构建fliD基因的插入失活突变株NCTC11168△fliD,并通过与野生株比较,对fliD突变株生长速率、运动力、黏附力和侵袭力等生物学特性进行研究。【结果】与野生型NCTC11168相比,突变株NCTC11168△fliD的生理生化特性不变;突变株的生长速率无明显变化;MH半固体穿刺实验中,突变株只能在接种处生长,运动力明显减弱;在Caco-2细胞黏附、侵袭实验中,fliD突变株的黏附率和侵袭率分别为164.00±19.49、55.00±6.09,fliD基因失活使得突变株的黏附率和侵袭率显著降低(0P0.01)。【结论】fliD基因是空肠弯曲菌运动能力重要的分子基础,与空肠弯曲菌感染细胞的黏附侵袭作用密切相关,即与空肠弯曲菌的致病性密切相关。     

Nucleotide sequence and regulated expression of the Salmonella fljA gene encoding a repressor of the phase 1 flagellin gene

Summary The nucleotide sequence of Salmonella abortus-equi fljA, which together with the phase 2 flagellin gene constitutes the fljBA operon and encodes the repressor for the phase 1 flagellin gene fliC, was determined. The repressor was predicted to be a basic protein consisting of 179 amino acid residues (M_r = 20419 Da) encoded by ORFII. This was confirmed by the fact that host fliC is repressed by plasmid-encoded ORFII, which indeed expresses a 20 kDa product as determined by urea SDS-polyacrylamide gel electrophoresis. An amino acid sequence capable of forming a helix-turn-helix type of structure was predicted in the C-terminal region of FljA. A rho-independent intercistronic terminator was detected between fljB and ftjA. Chloramphenicol acetyltransferase (CAT) assays of fusions indicated that the terminator is capable of reducing expression of fljA to the level of a few percent, relative to fljB in broth cultures and to 1 % in M9 glycerol cultures.     

Molecular cloning and transposition of a kanamycin resistance determinant from Campylobacter jejuni between replicons in Escherichia coli

This paper reports a restriction map of a fragment of DNA encoding kanamycin resistance cloned from plasmid DNA of Campylobacter jejuni ABA94 in the recombinant plasmid pRS9421-1. In transposition experiments, kanamycin-resistant R751::km9421 transconjugants appeared at frequencies of 10^-7 per donor cell. These transconjugants harboured a plasmid 4 kb larger than the parental 49 kb plasmid R751. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that the kanamycin resistant determinant had transposed from recombinant plasmid pRS9421-1 to plasmid R751.The authors are with the Department of Genetics and Cellular Biology, Faculty of Science, University of Malaya, 59100 Kuala Lumpur, Malaysia     

A newly constructed primer pair for the PCR amplification,cloningand sequencing of the flagellin (<Emphasis Type="Italic">flaA</Emphasis>) gene from isolatesof urease-negative <Emphasis Type="Italic">Campylobacter lari</Emphasis>

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564–572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70–75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.     

Different alleles of the flagellin gene hagB in Escherichia coli standard H test strains

Abstract The genes determining flagellar antigen specificities H36, H47 and H53 in the respective E. coli standard H test strains were found to be alleles of the flagellin gene hagB . Until now, only the allele encoding the flagellar antigen H3 has been identified. The chromosomal regions of flagellin genes hagB in E. coli and H2 in Salmonella were non-homologous as these genes integrated at different sites in the E. coli K-12 chromosome and were unable to replace each other. The hagA allele encoding E. coli flagellar antigen H48 was insensitive to the repressor produced by Salmonella gene rhl or by its putative analog in E. coli .     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.     

Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants

We have investigated the use of the tetracycline-dependent gene expression system to regenerate and propagate tobacco plants transformed with a gene whose product — when highly expressed — interferes with regeneration and/or further reproduction. Plants transformed with the Agrobacterium rhizogenes rolB gene under the control of the tetracycline-dependent expression system were phenotypically indistinguishable from wild type owing to efficient repression of the promoter. Induction of the rolB gene with tetracycline led to high-level expression of the rolB mRNA, which resulted in extremely stunted plants with necrotic and wrinkled leaves that did not develop a floral meristem. Upon cessation of tetracycline treatment healthy shoots developed even from severely affected meristems. Data on the dose response of the rolB phenotype as a function of tetracycline concentration demonstrate that the tetracycline-dependent gene expression system can be used to modulate the manifestation of a particular phenotype.     

Changes in cell morphology and polyamine composition during growth of Campylobacter jejuni

Resting cells of Campylobacter jejuni were spherical whereas growing cells were mainly spiral. Content of cadaverine increased with the decrease in spherical forms prior to growth commencing but production of spermidine increased in early log phase. Cadaverine and spermidine are possibly involved in changes in cell morphology and growth, respectively.S. Suzuki is with the Faculty of Agriculture, Kochi University, Nankoku, Kochi 783, Japan; Y. Horikoshi and K. Takama are with the Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, Japan.     

Analysis of variants affecting the catalase developmental program in maize scutellum

Summary The catalase of maize scutella is coded for by two loci, Cat1 and Cat2, which are differentially expressed in this tissue during early seedling growth. Two variant lines have been previously identified in which the developmental program for the expression of the Cat2 structural gene in the scutellum has been altered. Line R6–67 exhibits higher than normal levels of CAT-2 catalase in this tissue after four days of postgerminative growth. This phenotype is controlled by a temporal regulatory gene designated Car1. Line A16 exhibits a CAT-2 null phenotype. Further analysis of Car1 verifies the initial indication that it is trans-acting and exhibits strict tissue (scutellum) specificity. A screen of other available inbred lines uncovered eight additional catalase high-activity lines. All eight lines exhibit significantly higher than normal levels of CAT-2 protein. Two of these lines have been shown to be regulated by Car1 as in R6–67. Another line (A338) uncovered during the screen exhibits a null phenotype for CAT-2 protein and resembles A16. Catalase activity levels are low in the scutellum and no CAT-2 CRM (cross-reacting material) is present in the tissues of this line. Also, unlike most maize lines, CAT-2 cannot be induced in the leaf tissue of A338 upon exposure to light. Finally, a single line (A337), demonstrating a novel catalase developmental program, was identified.     

Regulation of nodulin gene expression

The expression of plant genes specifically induced during rhizobial infection and the early stages of nodule ontogeny (early nodulin genes) and those induced in the mature, nitrogen-fixing nodule (late nodulin genes) is differentially regulated and tissue/cell specific. We have been interested in the signal transduction pathway responsible for symbiotic, temporal and spatial control of expression of an early (Enod2) and a late (Leghemoglobin;lb) nodulin gene from the stem-nodulated legumeSesbania rostrata, and in identifying thecis-acting elements andtrans-acting factors involved in this process (De Bruijn and Schell, 1992). By introducing chimericS. rostrata lb promoter-gus reporter gene fusions into transgenicLotus corniculatus plants, we have been able to show that thelb promoter directs an infected-cell-specific expression pattern inLotus nodules. We have been able to delimit thecis-acting element responsible for nodule-infected-cell-expression to a 78 pb region of thelb promoter (NICE Element) and have analyzed this element in detail by site-specific mutagenesis. We have studied the interaction of the NICE element, and further upstreamcis-acting elements, withtrans-acting factors of both plant- and rhizobial origin. We have obtained evidence for the involvement of rhizobial proteins in infected-cell-specific plant gene expression (Welters et al., 1993). We have purified one of the bacterial binding proteins from theS. rostrata symbiontAzorhizobium caulinodans (AcBBP1), and cloned and mutated the corresponding gene, in order to examine its symbiotic phenotype. We have also found that theS. rostrata Enod2 gene is rapidly induced by physiologically significant concentrations of cytokinins, suggesting the role of cytokinin as a potential secondary signal involved in nodulation (Dehio and De Bruijn, 1992). We are examining whether the observed cytokinin induction, as well as the nodule-specific expression pattern, are modulated by theSrEnod2 promoter.     

不同产地浙贝母生物碱含量及其合成相关基因表达研究

为了探究浙贝母(Fritillaria thunbergii)药效成分积累量与生物碱合成相关基因表达水平之间的关系,该研究采用UPLC-MS、qPCR技术分别测定不同产地11个样品中总生物碱(贝母素甲和贝母素乙之和)含量和3个参与生物碱合成途径相关基因(HMGR、FPS和DXR)的表达量,同时运用生物统计学方法分析成熟期鳞茎生物碱含量与各基因表达量之间的相关性。结果表明:不同产地浙贝母成熟期鳞茎总生物碱含量存在显著差异(P<0.05),为0.2105%～0.4612%; HMGR和FPS基因在盛花期组织表达、盛花期至成熟期鳞茎表达变化趋势同生物碱含量变化趋势基本一致; DXR基因在成熟期鳞茎中表达量最高,盛花期组织表达、盛花期至成熟期鳞茎表达变化趋势同生物碱含量变化趋势大体不一致; HMGR、FPS基因表达量分别与贝母素甲、贝母素乙和总生物碱含量呈显著或极显著正相关性(P<0.05或P<0.01),以FPS基因表达量与生物碱含量相关系数为最高,相关系数分别为0.672,0.631,0.664,DXR基因与生物碱含量呈低度相关性。由此可以推断,生物碱的积累受MVA途径中...     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.