The pilA gene of Xanthomonas campestris pv. citri is required for infection by the filamentous phage cf

Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf.     

Requirement for Phosphoglucose Isomerase of Xanthomonas campestris in Pathogenesis of Citrus Canker

A mutant (XT906) of Xanthomonas campestris pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn5tac1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the pgi gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of X. campestris pv. citri.     

Transposable elements ofXanthomonas campestris pv.citri originating from indigenous plasmids

Summary After introduction of the broad host range plasmid RP4 inXanthomonas campestris pv.citri strain XAS4501 twoXanthomonas transposable elements, ISXC4 and ISXC5, were isolated. These elements were found to be capable of transposition inEscherichia coli. Restriction analysis, DNA hybridization and heteroduplex experiments revealed that ISXC4 and ISXC5 are about 5.55 and 6.95 kb in size, respectively, possess inverted repeats about 50±18 bp in length and share DNA homology in their left (5.0 kb) and right (0.6 kb) ends. ISXC4 and ISXC5 were found to originate from plasmids pXW45N and pXW45J, which are indigenous replicons inX. campestris pv.citri strain XW45.     

Simple Deletion: a vector- and marker-free method to generate and isolate site-directed deletion mutants

We have designed a new vector- and marker-free site-directed deletion system for gram-negative bacteria. In this system, a specific DNA fragment is amplified from a parental strain by using polymerase chain reaction (PCR), then circularized and introduced back into the parental strain for homologous recombination. The recombinant mutant is then detected and isolated by PCR-based sib selection. Unlike conventional methods, our Simple Deletion method requires no cloning procedures, and no foreign genes such as antibiotic-resistance genes are introduced as selection markers. The resulting mutant is, therefore, the same as the parental strain except for the lack of the target region. This method is categorized as a type of “self-cloning,” and the resulting mutant can be used for laboratory research without restrictions. Using this method, we generated a mutant of a plant pathogenic bacterium, Xanthomonas campestris pv. campestris, in which the 20.4-kb hrp gene cluster involved in the type III secretion system and in pathogenicity was deleted. In addition, we proved that this method can also be used to delete smaller DNA regions of X. campestris pv. campestris and to generate deletion mutants of the bacterium Ralstonia solanacearum.     

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     

Xanthomonas campestris pv. campestris lpsI and lpsJ genes encoding putative proteins with sequence similarity to the α- and β-subunits of 3-oxoacid CoA-transferases are involved in LPS biosynthesis

A 2.1-kb SmaI-EcoRI DNA fragment upstream of the xanA and xanB genes of Xanthomonas campestris pv. campestris carries two ORFs encoding putative proteins with sequence similarities to the α- and β-subunits of 3-oxoacid-CoA transferases. The two ORFs were termed lpsI and lpsJ because strains carrying appropriate mutations showed an autoagglutination phenotype and because lipopolysaccharides of these mutant strains were altered according to silver-stained polyacrylamide gels. The monosaccharide composition of the exopolysaccharide xanthan produced by lpsI and lpsJ mutants remained unchanged. Received: 29 March 1997 / Accepted: 21 July 1997     

The Oligopeptide Permease (Opp) of the Plant Pathogen <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

The oligopeptide permease (Opp), a protein-dependent ABC transporter, has been found in the genome of Xanthomonas axonopodis pv. citri (Xac), but not in Xanthomonas campestris pv. campestris (Xcc). Sequence analysis indicated that 4 opp genes (oppA, oppB, oppC, oppD/F), located in a 33.8-kbp DNA fragment present only in the Xac genome, are arranged in an operon-like structure and share highest sequence similarities with Streptomyces roseofulvus orthologs. Nonetheless, analyses of the GC content, codon usage, and transposon positioning suggested that the Xac opp operon does not have an exogenous origin. The presence of a stop codon at one of the ATP-binding domains of OppD/F would render the uptake system nonfunctional, but detection of a single polycistronic mRNA and periplasmic OppA in actively growing bacteria suggests that the Opp permease is active and could contribute to the distinct nutritional requirements and host specificities of the two Xanthomonas species.     

Cross-reactive Antigens between Xanthomonas campestris pv. citri Pathotypes and Citrus Species

Cross-reactive antigens were detected in crude and semi-purified preparations from acetone powder of Citrus aurantifolia and Citrus sinensis leaves with antisera to Xanthomonas campestris pv. citri pathotypes A and C by DAS-ELISA. Antiserum to X. campestris pv. citri pathotype C revealed an antigenic disparity between C. aurantifolia (susceptible host to pathotype C) and C. sinensis (resistant host to pathotype C) whereas antiserum to X, campestris pv. citri pathotype A did not reveal any antigenic disparity between these hosts, both susceptible to pathotype A. The occurrence of “key” cross-reactive antigens in Citrus species and X. campestris pv. citri and their possible involvement in such interaction are discussed.     

Saturation mutagenesis in Escherichia coli of a cloned Xanthomonas campestris DNA fragment with the lux transposon Tn4431 using the delivery plasmid pDS1, thermosensitive in replication

A system allowing transposon mutagenesis of cloned DNA fragments in Escherichia coli with Tn4431, which carries the promotorless luciferase (lux) operon of Vibrio fischeri, has been developed. The transposon delivery plasmid, pDS1, based on an IncF replicon, is thermosensitive in replication and mobilizable to many Gram-negative bacteria. We used pDS1 for Tn4431-saturation mutagenesis of a 10-kb DNA fragment of Xanthomonas campestris pv. campestris (X.c.c.) in E. coli and showed that the expression of the lux operon was dependent on orientation and location of the transposon. Transfer of a specific Tn4431 insertion to X.c.c. allowed the determination of the bioluminescence phenotype in planta. Correspondence to: U. B. Priefer     

The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn 5501 and Tn 5502 , are cryptic transposons of the Tn 3 family

Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1. Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons). The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily. The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons. Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies. Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501. While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors. This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment. It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat). Transposition of phenol degradation genes could not be detected. Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon. Received: 21 July 1997 / Accepted: 7 July 1998     

Cloning and characterization of the glutamate 1-semialdehyde aminomutase gene from Xanthomonas campestris pv. phaseoli

The gene from Xanthomonas campestris pv. phaseoli for glutamate 1-semialdehyde (GSA) aminomutase, which is involved in the C5 pathway for synthesis of -aminolevulinic acid (ALA), was cloned onto a multicopy plasmid, pUC18, by the complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of deletion fragments from the initial 3.5-kb chromosomal fragment allowed the isolation of a 1.7-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.7-kb DNA fragment revealed an open reading frame (ORF) that is located downstream from a potential promoter sequence and a ribosome-binding site. The ORF encodes a polypeptide of 429 amino acid residues, and the deduced molecular mass of this polypeptide is 45,043 Da. The amino acid sequence shows a high degree of homology to the HemL proteins from other organisms, and a putative binding site for pyridoxal 5-phosphate is conserved. Correspondence to: Y. Murooka     

A phagemid shuttle vector based on filamentous phage φLf and theE. coli vector pOK12 for use inXanthomonas campestris

AnE. coli-Xanthomonas shuttle vector, p2GP (3.3 kb), was constructed by fusing pOK12 with a DNA fragment carrying gene II andori from filamentous phage φLf. p2GP retains the advantages afforded by pOK12 and can replicate in differentX. campestris pathovars. Superinfection ofX. c. pv.campestris cells harboring p2GP or its derivatives with φLf causes the release of ssDNA-containing recombinant phage particles which are sources of single-stranded template for sequencing the inserts.     

Increase of xanthan production by self-cloning of a 3.0-kb EcoRI-KpnI chromosomal fragment in Xanthomonas campestris

Summary G76E is a non-mucoid mutant derived from Xanthomonas campestris pv. campestris 17 by Tn mutagenesis. A 3.0-kb EcoRI-KpnI fragment, cloned from a genomic bank of Xc17, was able to complement G76E and caused an increase of xanthan production by ca. 46% over Xc17. This 3.0-kb EcoRI-KpnI fragment insert was different from the two previously cloned EcoRI fragments from Xc17 that also increased xanthan production.     

Identification of a lexA Gene in, and Construction of a lexA Mutant of, Xanthomonas campestris pv. citri

The lexA gene of Xanthomonas campestris pathovar citri (X.c. pv. citri) was cloned and sequenced. The 639-bp open reading frame encodes a protein of 213 amino acids that shares substantial sequence homology with the products of previously characterized lexA genes, sharing 46% identity with the LexA protein of Escherichia coli. Amino acids required for autocatalytic cleavage of LexA are conserved in the X.c. pv. citri protein, whereas domains thought to mediate DNA binding differ markedly from those of LexA proteins from E. coli and other bacteria. The X.c. pv. citri LexA protein was overexpressed in E. coli, and SDS-polyacrylamide gel electrophoresis revealed a molecular size of 23 kDa for the purified protein. A lexA mutant of X.c. pv. citri was constructed by gene replacement, and the basal level of recA expression in this mutant was shown to be similar to that for wild-type cells exposed to a DNA-damaging agent. These results indicate that LexA functions as a repressor of recA expression in X.c. pv. citri. Received: 1 September 1999 / Accepted: 25 October 1999     

Requirement for phosphoglucose isomerase of Xanthomonas campestris in pathogenesis of citrus canker

A mutant (XT906) of Xanthomonas campestris pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn5tac1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the pgi gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of X. campestris pv. citri.     

The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli. Received: 12 March 1998 / Accepted: 30 March 1998     

Molecular cloning of protease gene(s) from Xanthomanas campestris pv. campestris: Expression in Escherichia coli and role in pathogenicity

Summary The recombinant plasmid pIJ3070 isolated from a genomic library of Xanthomonas campestris pv. campestris constructed in the conjugal cosmid pLAFR3 contains protease gene(s) which can be expressed in Escherichia coli. Tn5 mutagenesis and subcloning revealed that the protease structural gene(s) is(are) located in a ca. 10 kb EcoRI fragment. Several protease-minus mutants of X. c. campestris were obtained by Tn5 mutagenesis of pIJ3070 and marker exchange techniques. Studies of pathogenicity of these Tn5 mutants showed that the protease is not critically important for the pathogenicity of X. c. campestris on turnip plants but may play a minor role in disease development.Abbreviations Gm gentamicin - Km kanamycin - Rif rifampicin - Spc spectinomycin - Sm streptomycin - Tc tetracycline     

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Summary Fructose was shown to be phosphorylated by a specific phosphoenolpyruvatc-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose Ell. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.     

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.     

Coronatine analogues produced by Xanthomonas campestris pv Phormiicola

Liquid cultures of Xanthomonas campestris pv phormiicola were found to contain two analogues of coronatine lacking the cyclopropane ring structure, and no trace of either coronatine or norcoronatine. The two compounds were isolated and fully characterised by NMR, MS, hydrolysis and GC of hydrolysis products, as N-coronafacoyl- -valine and N-coronafacoyl- -isoleucine. A survey of 12 strains from 10 other X. campestris pathovars did not locate another source of production of these compounds, whereas all three strains of X. campestris pv phormiicola examined produced comparable levels of both compounds. This is the first report of phytotoxins biosynthetically derived from coronafacic acid outside of the genus Pseudomonas. The implications of these findings to the biosynthesis of the cyclopropane ring structure of coronatine are discussed.