Evidence for clustered pathogenicity genes in Xanthomonas campestris pv. campestris

Summary Plasmid pIJ3000 contains DNA from the plant pathogen Xanthomonas campestris pv. campestris which restores symptom production to a non-pathogenic mutant. pIJ3000 was mutagenised with the kanamycin resistance transposon Tn5 in Escherichia coli, and the sites of insertion were mapped physically. Derivatives of pIJ3000 containing Tn5 inserted in X. c. campestris DNA were introduced into a wild-type X. c. campestris strain, and the Tn5 insertions recombined into the genome by a marker exchange process. The resulting strains were tested for pathogenicity to turnip seedlings.The parts of the cloned DNA required for symptom production by the wild-type were defined by correlating the phenotype of each Tn5 insertion in the genome with its position in pIJ3000. This led to the identification of a region of approximately 10 kb consisting of a cluster of genes involved in pathogenicity.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Cloning of genes involved in negative regulation of production of extracellular enzymes and polysaccharide of Xanthomonas campestris pathovar campestris

Summary A recombinant plasmid pIJ3079 contains DNA sequences from Xanthomonas campestris pv campestris involved in coordinate negative regulation of production of the extracellular enzymes protease, endoglucanase, amylase and polygalacturonate lyase, and extracellular polysaccharide (EPS). Wild-type bacteria harbouring pIJ3079 and therefore carrying extra copies of the gene(s) therein showed reduced enzyme and EPS production and reduced aggresiveness to plants. Localised Tn5 mutagenesis of the corresponding region of the genome gave mutants producing higher levels of enzymes and EPS than the wild type, suggesting that the gene(s) may negatively regulate production in the normal cell. Enzyme and EPS production in the mutants was still dependent on previously characterised positive regulatory genes.     

茄科作物青枯菌NADH脱氢酶F亚基在细胞运动和致病性中的功能研究

[目的]劳尔氏菌（Ralstonia solanacearum）在茄科作物上引起严重的细菌性青枯病,本研究旨在发掘青枯劳尔氏菌与致病相关的基因。[方法]利用Tn5转座子构建随机插入突变体,分析生物膜形成、细胞运动和致病性;对有表型变化的突变体,运用TAIL-PCR方法鉴定Tn5插入位点,确定所突变的基因。[结果]以模式菌株GMI000为出发菌,总共获得了400个突变体,其中2个突变体不能形成生物膜,在软琼脂平板上的运动能力下降;接种感病番茄植物,这2个突变体都不能引起萎焉症状。TAIL-PCR结果显示,2个突变体的Tn5插入位点都在NADH脱氢酶F亚基（nuoF）中,距离翻译起始位点分别为103-bp和225-bp。ripAY基因启动子推动的nuoF基因互补载体,完全恢复了2个突变体的表型。[结论]NADH脱氢酶复合物是微生物呼吸电子传递链中的第一步催化酶。我们的结果表明,NADH脱氢酶复合物对R.solanacearum生物膜形成、细胞运动和致病性也有重要作用。     

Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris

Summary The cosmid clone. pIJ3020 containing DNA from the plant pathogenic bacterium Xanthomonas campestris pathovar campestris has previously been shown to complement a non-pathogenic mutant defective in synthesis of extracellular enzymes. The DNA cloned in pIJ3020 was analysed by mutagenesis with Tn5 and Tn5lac and by nucleotide sequencing. The results indicate that this region of the genome contains a cluster of genes, mutation in any of which results in failure of the enzymes and extracellular polysaccharide to be synthesized. The designation rpf (regulation of pathogenicity factors) is proposed for these genes. The nucleotide sequence of one gene (rpfC) predicts a protein product with homology to conserved domains of both sensor and regulator proteins of prokaryotic two-component regulatory systems, which are usually involved in regulating gene expression in response to environmental stimuli.     

Behaviour of the transposons Tn5 and Tn7 in Xanthomonas campestris pv. campestris

Summary Xanthomonas campestris pv. campestris was tested for its ability to maintain various plasmids after they had been transferred by conjugation from Escherichia coli donors. Broad host-range plasmids belonging to incompatibility groups P and Q could be maintained but X. campestris was unable to support replication of narrow host-range ColE1, pACYC184 and pBR325 replicons. Delivery systems based on E. coli donors of suicide plasmids and on X. campestris Hfrs were used to introduce Tn7 and Tn5 into X. campestris. Tn7 insertions were recovered at high frequency while Tn5 transposed at low frequency. Three auxotrophic Tn5 insertions were isolated but transposition of Tn7 into the X. campestris genome did not generate any auxotrophs. DNA hybridization analysis showed that Tn7 had inserted into the same hot spot(s) in all cases tested.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Transposon mutagenesis in Azospirillum brasilense: isolation of auxotrophic and Nif- mutants and molecular cloning of the mutagenized nifDNA

Summary We report the successful mutagenesis of Azospirillum brasilense 29710 Rif Sm with transposon Tn5. The narrow host-range plasmid pGS9 (p15A replicon), which possesses broad host-range N-type transfer genes, was used as the suicide vehicle to deliver Tn5 in Azospirillum. Out of 900 colonies tested, 0.8% proved to be auxotrophic. One mutant altered in indoleacetic acid (auxin) biosynthesis was isolated and, in addition, three mutants completely defective in nitrogen fixation (nif) were obtained. All the mutants tested contained a single copy of Tn5 integrated randomly in the genome. The Tn5-mutagenized EcoRI fragments were cloned from the three Nif^- mutants. Physical analysis of cloned DNA showed that Tn5 was present on a different EcoRI fragment in each case, ranging in size from 15–17 kb. The nitrogenase structural genes (nifHDK) in A. brasilense 29710 Rif Sm were localized on a 6.7 kb EcoRI fragment. We found that Tn5 is not inserted in the nifHDK genes in the Nif^- mutants reported here. Site-directed mutagenesis using the cloned, Tn5-containing DNA from mutant Nif27(pMS188), produced a large number of Nif^- transconjugants of the A. brasilense 29710 Rif wild-type strain, showing the linkage between Tn5 insertion and the Nif^- phenotype. This is the first time that transposon-mutagenized auxotrophic, Nif^- and other mutants have been available for genetic analysis in Azospirillum. This should greatly facilitate the cloning and mapping of genes involved in nitrogen fixation as well as in many other phenotypic characteristics of Azospirillum.     

A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not of beans by R. phaseoli and is corrected by cloned DNA from Rhizobium or the phytopathogen Xanthomonas

Summary A Tn5-induced mutant strain of R. phaseoli which failed to synthesize exopolysaccharide (EPS) was isolated and was shown to induce normal nitrogen-fixing nodules on Phaseolus beans, the host of this Rhizobium species. The corresponding wild-type Rhizobium DNA was cloned in a wide host-range vector and by isolating Tn5 insertions in this cloned DNA, mutations in a gene termed pss (polysaccharide synthesis) were isolated. These were introduced by marker exchange into near-isogenic strains of R. leguminosarum and R. phaseoli which differed only in the identity of their symbiotic plasmids. Whereas the EPS-deficient mutant strain of R. phaseoli induced normal nitrogen-fixing nodules on Phaseolus beans, the same mutation prevented nodulation of peas by a strain of R. leguminosarum which normally nodulates this host. Further, it was found that DNA cloned from the plant pathogen Xanthomonas campestris pathover campestris could correct the defect in EPS synthesis in R. leguminosarum and R. phaseoli and also restored the ability to nodulate peas to the pss::Tn5 mutant strain of R. leguminosarum.     

Conservation of xcp genes, involved in the two-step protein secretion process, in different Pseudomonas species and other gram-negative bacteria

Summary The two-step protein secretion pathway in Pseudomonas aeruginosa is dependent on the xcp genes. We investigated whether a similar secretion mechanism is present in non-pathogenic Pseudomonas spp. and in other gram-negative bacteria. The plant growth stimulating Pseudomonas strains P. putida WCS358, P. fuorescens WCS374 and Pseudomonas 1310 appeared to secrete proteins into the extracellular medium. Southern hybridization experiments showed the presence of xcp genes in these strains and also in other gram-negative bacteria, including Xanthomonas campestris. Complementation experiments showed that the xcp gene cluster of P. aeruginosa restored protein secretion in an X. campestris secretion mutant. The secretion gene cluster of X. campestris however, restored secretion capacity in P. aeruginosa mutants only to a low degree. Two heterologous proteins were not secreted by P. fuorescens and P. aeruginosa. The results suggest the presence of a similar two-step protein secretion mechanism in different gram-negative bacteria, which however, is not always functional for heterologous proteins.     

Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1

A genomic library was prepared in Escherichia coli from DNA of wild-type Xanthomonas campestris pv. campestris (aetiological agent of crucifer black rot), partially digested with endonuclease EcoRI, using the mobilisable broad host range cosmid vector pLAFR1. Recombinant plasmids contained inserts ranging in size from 19.1 to 32.3 kb (mean 26.6). Certain of the clones complemented E. coli auxotrophic markers. Using the narrow host range plasmid pRK2013 as a helper the pooled recombinant plasmids were transferred conjugally to X. c. campestris mutants, and clones were identified which restored yellow pigmentation to white mutants, prototrophy to amino acid auxotrophs and pathogenicity towards turnip plants to two non-pathogenic mutants. The lesion in one mutant (8288, complemented by the plasmid pIJ3000) is unknown. However mutant 8237 is defective in production of extracellular protease and polygalacturonate lyase and restoration of pathogenicity by complementation with the plasmid pIJ3020 concomitantly restored both enzyme levels to wild-type values.     

蛋白-O-岩藻糖基转移酶1基因CfPOFUT1在果生炭疽菌中的功能分析

【目的】蛋白-O-岩藻糖基转移酶1 (protein O-fucosyltransferase 1,POFUT1)是催化蛋白质O-岩藻糖基化的关键酶,在动物和人体内被证明调控一系列的生理病理过程,然而POFUT1基因在果生炭疽菌乃至真菌中还未见报道。本研究旨在克隆果生炭疽菌中CfPOFUT1基因,并分析其生物学功能。【方法】利用RT-PCR技术扩增CfPOFUT1的基因并进行生物信息学分析,构建了CfPOFUT1基因的沉默和过表达载体,通过PEG介导法将载体导入原生质体中获得CfPOFUT1基因的沉默和过表达突变体。测定了野生型菌株、CfPOFUT1沉默菌株和过表达菌株在PDA上的菌丝生长、分生孢子产生、萌发与附着胞形成、胁迫应答和致病力、杀菌剂敏感性等生物学表型。【结果】与野生型菌株相比,基因过表达突变体产孢量显著增加,致病力增强,对嘧菌酯敏感性降低,但对多菌灵和咪鲜胺敏感性增强。基因沉默突变体产孢量减少,细胞壁完整性、内质网应激敏感性提高,致病力减弱,对嘧菌酯敏感性提高,但对多菌灵和咪鲜胺敏感性降低。【结论】CfPOFUT1基因参与调控果生炭疽菌分生孢子产量,细胞壁完整性、内质网对应激和药剂敏感性,并对其致病性也具有一定的影响。     

Insertions of Tn5 linked to mutations affecting carotenoid synthesis inMyxococcus xanthus

Summary We have characterized severalMyxococcus xanthus mutants in which carotenoid synthesis is affected. Six of them produce carotenoids in the absence of visible light, an absolute requirement for carotenogenesis in wild-type strains, and thus will be referred to as constitutive mutants. The six corresponding mutations have been mapped by transductional analysis mediated by linked Tn5 insertions. Five of the mutations have been localized to a single locus, closely linked to Tn5 insertion ΩMR136 and loosely linked to ΩDK4611. The sixth mutation, present in strain MR7, is linked to Tn5 insertion ΩMR134. Another Tn5 insertion site (ΩDK2836) has been characterized and found to be linked to the MR7 colour mutation and to ΩMR134. Darkor light-grown cultures of strains carrying the Tn5 insertion ΩDK2836 do not produce carotenoids even if they simultaneously carry any of the constitutive mutations.     

Effect of ogt expression on mutation induction by methyl-, ethyl- and propylmethanesulphonate in Escherichia coli K12 strains

We have previously reported the isolation of an Escherichia coli K12 mutant that is extremely sensitive to mutagenesis by low doses of ethylating agents. We now show by Southern analysis that the mutation involves a gross deletion covering at least the ogt and fnr genes and that no O⁶-alkylguanine-DNA-alkyltransferase activity is present in cell-free extracts of an ada::Tn10 derivative of these bacteria. Confirmation that sensitisation to ethylation-induced mutagenesis was attributable to ogt and not to any other loci covered by the deletion was obtained by constructing derivatives. Thus an ogt::kan^r disruption mutation was introduced into the parental ogt ⁺ bacteria, and the ogt::kan^r mutation was then eliminated by cotransduction of ogt ⁺ with the closely linked Tet ^r marker (zcj::Tn10). The (ogt-fnr) deletion or ogt::kan^r disruption mutants were highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by the induction of forward mutations to l-arabinose resistance (Ara¹). Furthermore, the number of Ara^r mutants increased linearly with dose, unlike the case in ogt ⁺ bacteria, which had a threshold dose below which no mutants accumulated. Differences in mutability were even greater with propyl methanesulphonate. Overproduction of the ogt alkyltransferase from a multicopy plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt mutant strains and also methylmethanesulphonate mutagenesis in ada ^– bacteria. A sample of AB 1157 obtained from the E. coli K12 genetic stock centre also had a deletion covering the ogt and fnr genes. Since such deletions greatly influence the mutagenic responses to alkylating agents, a survey of the presence of the ogt gene in the E. coli K12 strain being used is advisable.     

Molecular genetics of met 17 and met 25 mutants of Saccharomyces cerevisiae: intragenic complementation between mutations of a single structural gene

Summary A method for transposon mutagenesis in Azospirillum lipoferum 29708 is reported with transposon Tn5. The suicide plasmid pSUP2021 was used to deliver Tn5 in A. lipoferum using Escherichia coli SM10 as the donor. Neomycin-resistant transconjugants were detected at a frequency of 6x10^-6 per recipient. Different types of mutants were isolated, e.g. auxotrophic, coloured, IAA-negative, and IAA-overproducers. Among the auxotrophic mutants, cysteine and methionine requirers prevailed. Random Tn5-insertion with only one copy per mutant was demonstrated by Southern blotting and hybridization. Tn5-induced mutants are relatively stable, with reversion rates of 2–20×10^-8. A gene which is a part of the carotenoid pathway is closely linked to the histidine genes. The existence of two pathways for IAA production in A. lipoferum is discussed.     

苹果树腐烂病菌细胞色素P450基因Vmcyp5的功能

【目的】研究表明,细胞色素P450(CYP)在死体营养型真菌的毒素合成代谢中发挥重要作用,预测可能与病原菌致病相关。论文对苹果树腐烂病菌(Valsa mali)毒素合成基因簇中的1个上调表达的CYP基因Vmcyp5进行生物学功能研究,明确CYP基因对病原菌致病力影响,为细胞色素P450基因家族对苹果树腐烂病菌致病机理的进一步研究提供依据。【方法】通过Double-joint PCR和PEG介导的原生质体转化技术获得具有G418抗性的突变体,并对突变体进行PCR检测及Southern blotting验证得到单拷贝敲除突变体。将目的基因片段重新导入敲除突变体,筛选获得互补突变体。最终对野生型菌株及敲除突变体、互补突变体进行菌落、产孢及致病力观察,利用SPSS软件对数据进行差异显著性分析,并利用q RT-PCR技术分析突变体黑色素基因簇的表达水平。【结果】通过基因敲除技术获得1个Vmcyp5基因的敲除突变体。与野生型菌株相比,Vmcyp5基因的敲除突变体菌落呈白色,产孢量减少51.3%。q RT-PCR分析发现敲除突变体黑色素基因簇基因表达量降低。重要的是,敲除突变体致病力较野生型菌株降低24.5%。互补突变体菌落颜色、产孢及致病力近似恢复至野生型菌株水平。【结论】Vmcyp5基因与病原菌黑色素合成、子实体的产生和致病力相关。     

Marking of a Sym plasmid of Rhizobium with Tn7

Summary Transposon Tn7 was inserted into wide host range plasmid pSUP202 and used as a suicide plasmid vehicle for transposon mutagenesis in Rhizobium leguminosarum. Tn7 is transposed with high frequency into the self-transmissible plasmid pJB5JI without affecting the transfer, nodulation and nitrogen fixation functions. Tn7 transposition provides a useful tool for marking symbiotic plasmids.     

Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence

Summary The blackleg pathogen Erwinia carotovora subsp. atroseptica (Eca) causes an economically important disease of potatoes. We selected a genetically amenable Eca strain for the genetic analysis of virulence. Tn5 mutagenesis was used to generate nine mutants which exhibited reduced virulence (Rvi^-) of strain SCRI1043. Following physiological characterisation, mutants were divided into three classes: (1) auxotrophs; (2) extracellular enzyme mutants; and (3) a growth rate mutant. The isolation of these Rvi^- mutants has allowed us to consider some factors that affect Eca virulence.     

Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

Summary Random Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS571 mutants disturbed in ammonium assimilation (Asm^-). Both Asm^- mutant strains were shown to lack NADPH-glutamate synthase (NADPH-GOGAT) activity and to carry Tn5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn5-containing region of one of the GOGAT^- mutant strains was cloned in pACYC184 and used to identify the wild-type glt (GOGAT) locus in a phage clone bank of ORS571. The cloned region was shown to have DNA homology with the Escherichia coli glt locus and to complement the Asm^- phenotype of E. coli and ORS571 GOGAT^- strains. The ORS571 GOGAT^- mutations were found to interfere with free-living as well as symbiotic nitrogen fixation. Expression of ORS571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation (ntr) system.     

Resistance in tomato to Xanthomonas campestris pv vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3

Bacterial spot disease of tomato and pepper caused by Xanthomonas campestris pv vesicatoria is prevented by resistance genes in the plant that match genes for avirulence in the bacterium. Based on DNA homology to the avirulence gene avrBs3, which induces the resistance response on pepper, we have isolated another avirulence gene from X. c. vesicatoria, designated avrBs3-2. This gene differs in specificity from avrBs3 in inducing the hypersensitive response on tomato but not on pepper. Sequence analysis of the avrBs3-2 gene revealed a high degree of conservation: the 3480 by open reading frame contains an internal region of 17.5 nearly identical 102 bp repeat units that differ in their order from those present in the avrBs3 gene. The coding region is 97% identical to avrBs3 and expresses constitutively a 122 kDa protein, thus representing a natural allele of this gene. The previously isolated 1.7 kb avrBsP gene from X. c. vesicatoria is 100% identical to the corresponding avrBs3-2 sequence, indicating that these genes might be identical. Interestingly, derivatives of avrBs3-2 lacking the C-terminal region and part of the repetitive region are still able to confer incompatibility in tomato. The avrBs3-2 gene is compared with the sequence of avrBs3 derivatives generated by deletion of repeat units that also have avirulence activity on tomato. Both genes, avrBs3 and avrBs3-2, are flanked by a 62 by long inverted repeat, which prompts speculations about the origin of the members of the avrBs3 gene family.     

Localization of his genes on the Rhizobium trifolii RS800 linkage map

Summary We have studied N-methyl-N-nitro-N-nitrosoguanidine (NTG)- and Tn5-induced histidine auxotrophic mutants in Rhizobium trifolii. HisGE, hisD and hisH mutants have been characterized. Using the Kemper's equation we have located them on the R. trifolii linkage map. The hisGE and hisD genes are clustered in the same region and are closely linked to the spectinomycin marker. The hisH gene is located in another region equidistant from the streptomycin and rifampicin markers. The two regions carrying his genes are separated by a distance approximately one-third of the length of the chromosome.