Cloning of a locus involved in pathogenicity and production of extracellular polysaccharide and protease in Xanthomonas campestris pv. campestris

Abstract A Tn5-induced mutant of Xanthomonas campestris pv campestris deficient in protease and extracellular polysaccharide production, and non-pathogenic to turnip seedlings, was isolated. Recombinant clones which restored all three characteristics concomitantly were obtained from a wild-type genomic library. One of the plasmid clones, pIJ3090, showed no hybridization to previously isolated clones involved In protease production. The mutant was not complemented by other known clones involved in pathogenicity, in xanthan gum production or in protease production.     

Differential expression of conserved protease genes in crucifer-attacking pathovars of Xanthomonas campestris.

Strains of Xanthomonas campestris pathovars armoraciae and raphani, which cause leaf spotting diseases in brassicas, produce a major extracellular protease in liquid culture which was partially purified. The protease (PRT 3) was a zinc-requiring metalloenzyme and was readily distinguishable from the two previously characterized proteases (PRT 1 and PRT 2) of X. campestris pv. campestris by the pattern of degradation of beta-casein and sensitivity to inhibitors. PRT 3 was produced at a low level in the vascular brassica pathogen X. campestris pv. campestris (five strains tested), in which PRT 1 and PRT 2 predominate. In contrast, expression of PRT 1, a serine protease, could not be detected in the six tested strains of the leaf spotting mesophyll pathogens. However, all these strains had DNA fragments which hybridized to a prtA probe and which probably carry a functional prtA (the structural gene for PRT 1). The structural gene for PRT 3 (prtC) was cloned by screening a genomic library of X. campestris pv. raphani in a protease-deficient X. campestris pv. campestris strain. Subcloning and Tn5 mutagenesis located the structural gene to 1.2 kb of DNA. DNA fragments which hybridized to the structural gene were found in all strains of the crucifer-attacking X. campestris pathovars tested as well as in a number of other pathovars. Experiments in which the pattern of protease production of the pathovars was manipulated by introduction of cloned genes into heterologous pathovars suggested that no determinative relationship exists between the pattern of protease gene expression and the (vascular or mesophyllic) mode of pathogenesis.     

一种来源于链霉菌的纤溶酶的纯化及其基因的克隆

链霉菌C3662的发酵液上清经 80 %硫酸铵沉淀 ,DEAE Sepharose和CM Sepharose层析分离后纯化出一种纤溶酶。SDS PAGE显示为单一的条带 ,分子量约为 30kD。以 pIJ699为载体 ,S .lividansTK2 4为宿主菌 ,鸟枪法克隆纤溶酶基因 ,从 30 0 0个转化子中挑选到 1个具活性转化子 ,经亚克隆 ,序列测定得到一个 90 3bp的完整ORF ,其GC %为 68.33% ,密码子第三位GC %为 95.6% ,符合链霉菌基因的典型特征。与多种蛋白酶具有较高的同源性     

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.     

Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris.

Nonpathogenic mutants of Xanthomonas campestris pv. campestris, generated from transposon mutagenesis, accumulated extracellular polygalacturonate lyase, alpha-amylase, and endoglucanase in the periplasm. The transposon Tn5 was introduced by a mobilizable, suicidal plasmid, pSUP2021 or pEYDG1. Genomic banks of wild-type X. campestris pv. campestris, constructed on the broad-host-range, mobilizable cosmid pLAFR1 or pLAFR3, were conjugated with one of the mutants, designated XC1708. Recombinant plasmids isolated by their ability to complement XC1708 can be classified into two categories. One, represented by pLASC3, can complement some mutants, whereas the other, represented by a single plasmid, pLAHH2, can complement all of the other mutants. Restriction mapping showed that the two recombinant plasmids shared an EcoRI fragment of 8.9 kb. Results from subcloning, deletion mapping, and mini-Mu insertional mutation of the 8.9-kb EcoRI fragment suggested that a 4.2-kb fragment was sufficient to complement the mutant XC1708. Sequence analysis of this 4.2-kb fragment revealed three consecutive open reading frames (ORFs), ORF1, ORF2, and ORF3. Hybridization experiments showed that Tn5 in the genome of XC1708 and other mutants complemented by pLASC3 was located in ORF3, which could code for a protein of 83.5 kDa. A signal peptidase II processing site was identified at the N terminus of the predicted amino acid sequence. Sequence homology of 51% was observed between the amino acid sequences predicted from ORF3 and the pulD gene of Klebsiella species.     

The zinc uptake regulator Zur is essential for the full virulence of Xanthomonas campestris pv. campestris

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 microM and in nonrich medium supplemented with Zn2+ at a concentration of 110 microM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 microM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.     

Nucleotide sequence of the engXCA gene encoding the major endoglucanase of Xanthomonas campestris pv. campestris

The nucleotide sequence of the gene (engXCA) encoding the major extracellular endoglucanase (ENGXCA) of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (X. c. campestris) was determined and compared with the N-terminal amino acid (aa) sequence of the purified enzyme. An open reading frame of 1479 bp encoding 493 aa was identified, of which the N-terminal 25 aa represent a potential signal peptide. Determination of the exact position of a Tn5 insertion within engXCA, which did not reduce the encoded enzyme activity, indicated that the C-terminal region of the protein is not crucial for ENGXCA activity. Comparison of the complete deduced aa sequence with those deduced from other endoglucanase- and exoglucanase-encoding genes revealed a region with a high degree of homology, located towards the C terminus of the protein. These data indicate that the X. c. campestris ENGXCA may have a domain structure similar to that of many other bacterial and fungal cellulolytic enzymes. Hydrophobic cluster analysis was performed on the deduced aa sequence. Comparison of this analysis with those of 30 other cellulase sequences belonging to six different families indicated that the X. c. campestris enzyme can be classified in family A. The two aa residues which had previously been identified as 'potentially catalytic' within this family of cellulases, are conserved in the X. c. campestris ENGXCA.     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.     

野油菜黄单胞菌野油菜致病变种中一个与EPS合成有关的新基因的鉴定

用转座子Tn5gusA5对野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris,简称Xcc)野生型菌株8004进行诱变,分离到一批胞外多糖(EPS)合成减少的突变体。采用TAIL-PCR(thermal asymmetric interlaced PCR)分析突变体的Tn5gusA5插入位点,发现其中一株编号为151D09的突变体的插入位点位于Xcc 8004菌株的基因组编号为XC3695的ORF内,该ORF功能尚未见报道。序列分析表明,该ORF演绎的编码产物与Serratia marcescens的kdtX基因和Klebsiella pneumoniae的waaE基因演绎的编码产物分别具有52%和50%的相似性,并具有第2家族糖基转移酶的功能域, 因此暂将该ORF命名为waxE基因。用同源双交换方法构建了waxE基因的缺失突变体,并采用PCR和Southern杂交的方法对突变体进行了验证。waxE基因缺失突变体在营养丰富培养基的生长繁殖不受影响,但其EPS产量与野生型菌株8004相比,降低35%左右,并且一段PCR合成的包含waxE基因的DNA片段能反式互补waxE基因缺失突变体,恢复缺失突变体的EPS产量,表明Xcc waxE基因与EPS的生物合成有关。     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen.

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.     

Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene.

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.     

Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.     

n-Terminal Amino Acid Sequences of Neutral Proteases from Bacillus amyloliquefaciens and Bacillus subtilis: Identification of a Neutral Protease Gene Cloned in Bacillus subtilis

Bacillus subtilis 1A20 transformed with a hybrid plasmid, pNP150, to which a DNA fragment from Bacillus amyloliquefaciens F was attached, produced a large amount of a neutral protease. To identify the origin of the gene specifying this neutral protease, neutral proteases from B. amyloliquefaciens F, B. subtilis NP58 (a derivative of Marburg 6160), and B. subtilis 1A20 transformed with pNP150 were purified. We investigated their immunological properties and primary structures.

The proteases from these two species were indistinguishable by chromatography, but they were distinguishable from each other by SDS-polyacrylamide gel electrophoresis and double immunodiffusion. Amino acid sequencing of these two proteases by Edman degradation showed that there were four substitutions in the 20-residue amino acid sequence from the N-termini.

Neutral protease from the transformant had the same immunological characteristics and N-terminal amino acid sequence as that from B. amyloliquefaciens. These results meant that the gene in question was derived from a gene specifying the neutral protease in this bacterium.     

The extracellular proteases of the phytopathogenic bacterium Xanthomonas campestris

The culture liquids of three Xanthomonas campestris pv. campestris strains were found to possess proteolytic activity. The culture liquid of strain B-611 with the highest proteolytic activity was fractionated by salting-out with ammonium sulfate, gel filtration, and ion-exchange chromatography. The electrophoretic analysis of active fractions showed the presence of two proteases in the culture liquid of strain B-611, the major of which being serine protease. The treatment of cabbage seedlings with the proteases augmented the activity of peroxidase in the cabbage roots by 28%.     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

MarR家族转录因子HpaR和XC0449协同调控野油菜黄单胞菌致病性

MarR家族转录因子广泛存在于细菌及古生菌中,并灵活、精细地调控多种毒力、抗胁迫及抗生素相关的生理生化途径。在野油菜黄单胞菌中,MarR家族转录因子HpaR (XC2827)的失活会显著降低细菌对于寄主甘蓝的致病力,同时会导致胞外蛋白酶的过量表达。本研究进一步发现,Xcc 8004基因组一共编码9个MarR家族转录因子。表达并纯化其中的HpaR (XC2827)和XC0449,体外微量热泳动(MST)实验及Pull-down实验证明二者可以在体外特异性结合。同时,表型检测发现XC0449突变会导致细菌致病力显著下降。通过体外凝胶迁移阻滞试验(EMSA)、体内qRT-PCR和GUS检测证明,XC0449和HpaR均作为转录激活子协同调控下游致病相关基因XC0705的表达,最终调控细菌毒力及胞外酶合成。     

The gene encoding dipeptidyl aminopeptidase BI from Pseudomonas sp. WO24: cloning,sequencing and expression in Escherichia coli

We have isolated the dipeptidyl aminopeptidase BI (DAP BI) gene from the plasmid library of Pseudomonas sp. WO24 chromosomal DNA by the enzymatic plate asaay using a chromogenic substrate. The DAP BI gene, designated dap b1, was further subcloned and sequenced. Sequence analysis of an approx. 3-kb fragment revealed an open reading frame of 2169 nucleotides, which was assigned to the dap b1 gene by N-terminal and internal amino acid sequences. The predicted amino acid sequence of DAP BI containing a serine protease Gly–X–Ser–X–Gly consensus motif displays extensive homologies to the several proteases belonging to the prolyl oligopeptidase family, a novel serine protease family possessing the catalytic triad with a specific array of Ser, Asp and His in this order, which is the hallmark of the member of this family including DAP IV. The dap b1 gene was expressed in Escherichia coli and the expressed enzyme was purified about 230-fold with 2.6% recovery from the cell-free extracts. The enzymatic properties such as molecular mass, substrate specificity and effect of inhibitor were similar to the native enzyme from Pseudomonas sp. WO24.     

Characterisation of an extracellular serine protease gene (nasp gene) from Dermatophilus congolensis

A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases.     

Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.

Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.