Genes for albicidin biosynthesis and resistance span at least 69 kb in the genome of Xanthomonas albilineans

All Tn 5 insertion mutants of Xanthomonas albilineans , the cause of leaf scald disease of sugar cane, which failed to produce albicidin antibiotics failed to cause chlorosis in inoculated sugar cane but remained resistant to albicidin. Southern analysis revealed that mutants deficient in albicidin production carried the transposon on different chromosomal restriction fragments spanning at least 50 kb in the X. albilineans genome, which is larger than any reported cluster of genes involved in the production of a bacterial phytotoxin. Albicidin-resistant cosmid clones from a Tox⁻ Tn 5 insertion mutant did not carry the transposon, and the subcloned albicidin resistance gene did not hybridize to any of the restriction fragments carrying Tn 5 in the Tox⁻ mutants, indicating that the albicidin biosynthesis and resistance genes are not closely linked in X. albilineans .     

Identification of new candidate pathogenicity factors in the xylem-invading pathogen Xanthomonas albilineans by transposon mutagenesis

Xanthomonas albilineans is a xylem-invading pathogen that produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms of sugarcane leaf scald. In contrast to other xanthomonads, X. albilineans does not possess a hypersensitive response and pathogenicity type III secretion system and does not produce xanthan gum. Albicidin is the only previously known pathogenicity factor in X. albilineans, yet albicidin-deficient mutant strains are still able to efficiently colonize sugarcane. To identify additional host adaptation or pathogenicity factors, sugarcane 'CP80-1743' was inoculated with 1,216 independently derived Tn5 insertions in X. albilineans XaFL07-1 from Florida. Sixty-one Tn5 mutants were affected in development of leaf symptoms or in stalk colonization. The Tn5 insertion sites of these mutants were determined and the interrupted genes were identified using the recently available genomic DNA sequence of X. albilineans GPE PC73 from Guadeloupe. Several pathogenicity-related loci that were not previously reported in Xanthomonas spp. were identified, including loci encoding hypothetical proteins, a membrane fusion protein conferring resistance to novobiocin, transport proteins, TonB-dependent outer-membrane transporters, and an OmpA family outer-membrane protein.     

Xanthomonas albilineans HtpG is required for biosynthesis of the antibiotic and phytotoxin albicidin

Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane, produces a highly potent polyketide-peptide antibiotic and phytotoxin called albicidin. Previous studies established the involvement of a large cluster of genes in the biosynthesis of this toxin. We report here the sub-cloning and sequencing of an additional gene outside of the main cluster and essential for albicidin biosynthesis. This gene encodes a 634-amino-acid protein that shows high identity with the Escherichia coli heat shock protein HtpG. Complementation studies of X. albilineans Tox- mutants confirmed the requirement of htpG for albicidin biosynthesis and revealed functional interchangeability between E. coli and X. albilineans htpG genes. HtpG was co-localised with albicidin in the cellular membrane, i.e., the cellular fraction where the toxin is most probably biosynthesised. Here we show the requirement of an HtpG protein for the biosynthesis of a polyketide-peptide antibiotic.     

Albicidin antibiotic and phytotoxin biosynthesis in Xanthomonas albilineans requires a phosphopantetheinyl transferase gene

Xanthomonas albilineans produces a family of highly potent antibiotics and phytotoxins called albicidins, which are key pathogenesis factors in the systemic development of leaf scald disease of sugarcane. A gene (xabA) required for albicidin biosynthesis encodes a peptide of 278 amino acids, including the signature sequence motifs for phosphopantetheinyl transferases (PPTases) that activate polyketide and non-ribosomal peptide synthetases. The Escherichia coli entD gene, which encodes a PPTase involved in biosynthesis of enterobactin (a siderophore), restored biosynthesis of albicidin (a DNA replication inhibitor) in X. albilineans Tox- LS156 (xabA::Tn5). We conclude that XabA is a PPTase required for post-translational activation of synthetases in the albicidin biosynthetic pathway. This is the first antibiotic or toxin biosynthesis gene characterized from the genus Xanthomonas, and the first demonstration that antibiotic synthetases in the Pseudomonadaceae, like those in the Enterobacteriaceae and in Gram-positive bacteria, can require activation by a PPTase. Coupled with the recent demonstration of a separate albicidin biosynthetic gene cluster, the results indicate the possibility for overproduction of albicidins,which allows better understanding and application of these potent inhibitors of prokaryote DNA replication.     

Identification and cloning of genes involved in phaseolotoxin production by Pseudomonas syringae pv. "phaseolicola"

Genes involved in the production of phaseolotoxin by Pseudomonas syringae pv. "phaseolicola" NPS3121 were identified by Tn5 mutagenesis and cosmid cloning. A total of 5,180 kanamycin-resistant colonies were screened for the loss of phaseolotoxin production by a microbiological assay. Six independent, prototrophic, Tox- mutants were isolated that had Tn5 insertions in five different EcoRI fragments. All six mutants had Tn5 inserted in the same KpnI fragment, which had a length of ca. 28 kilobases including Tn5. The mutants produced residual toxin in vitro. An EcoRI fragment containing Tn5 and flanking sequences from mutant NPS4336 was cloned and used to probe a wild-type genomic library by colony hybridization. Seven recombinant plasmids showing homology to this probe were identified. Each Tox- mutant was restored in OCTase-specific toxin production by two or more of the recombinant plasmids. The data suggest that at least some of the genes involved in phaseolotoxin production were clustered in a large KpnI fragment. No homology was detected between the Tn5 target fragment cloned from mutant NPS4336 and the total genomic DNA from closely or distantly related bacteria that do not produce phaseolotoxin.     

Substrate specificity-conferring regions of the nonribosomal peptide synthetase adenylation domains involved in albicidin pathotoxin biosynthesis are highly conserved within the species Xanthomonas albilineans

Albicidin is a pathotoxin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald disease of sugarcane. Albicidin is synthesized by a nonribosomal pathway via modular polyketide synthase and nonribosomal peptide synthetase (NRPS) megasynthases, and NRPS adenylation (A) domains are responsible for the recognition and activation of specific amino acid substrates. DNA fragments (0.5 kb) encoding the regions responsible for the substrate specificities of six albicidin NRPS A domains from 16 strains of X. albilineans representing the known diversity of this pathogen were amplified and sequenced. Polymorphism analysis of these DNA fragments at different levels (DNA, protein, and NRPS signature) showed that these pathogenicity loci were highly conserved. The conservation of these loci most likely reflects purifying selective pressure, as revealed by a comparison with the variability of nucleotide and amino acid sequences of two housekeeping genes (atpD and efp) of X. albilineans. Nevertheless, the 16 strains of X. albilineans were differentiated into several groups by a phylogenetic analysis of the nucleotide sequences corresponding to the NRPS A domains. One of these groups was representative of the genetic diversity previously found within the pathogen by random fragment length polymorphism and amplified fragment length polymorphism analyses. This group, which differed by three single synonymous nucleotide mutations, contained only four strains of X. albilineans that were all involved in outbreaks of sugarcane leaf scald. The amount of albicidin produced in vitro in agar and liquid media varied among the 16 strains of X. albilineans. However, no relationship among the amount of albicidin produced in vitro and the pathotypes and genetic diversity of the pathogen was found. The NRPS loci contributing to the synthesis of the primary structure of albicidin apparently are not involved in the observed pathogenicity differences among strains of X. albilineans.     

Transposon mutagenesis and complementation of the fructokinase gene in Rhizobium leguminosarum biovar trifolii

Transposon Tn5 was used to generate a fructokinase mutation in Rhizobium leguminosarum biovar trifolii BAL. The section of the genome containing Tn5 was cloned into the EcoRI site of the vector pHC79 and isolated by direct selection on medium containing kanamycin and tetracycline. Total EcoRI digestion was used to obtain a single fragment containing Tn5 and flanking DNA sequences. The flanking DNA was used as a probe to isolate an intact fructokinase gene from a pLAFR1 cosmid clone bank of the parental strain. A cosmid showing homology to the probe was tri-parentally conjugated into the fructokinase-negative strain, complementing the mutation. The complemented mutant exhibited the wild-type phenotype, with an increase in fructokinase production presumably due to multiple copies of the gene.     

浑球红细菌谷氨酸合酶基因(glt)的克隆和图谱分析

利用转座子Tn5随机插入诱变筛选得到12株浑球红细菌(Rhodobacter sphaeroides)氨同化缺陷突变株(Asm~-)。这些突变株胞内均无GOGAT活性,同时它们均无固氮酶活性(Nif~-),并且具有氮代谢多效性缺失表型(Ntr~-)。将含有Azorhizobium sesbaniae ORS571的完整glt基因的质粒pHB10转入突变株中能互补上述表型。通过筛选携带Tn5的R-prime质粒克隆了glt::Tn5片段。Southern杂交证明所克隆glt::Tn5片段与E. coli的gltBD基因有同源性。用此片段与以pLAFR3为载体所构建的R. sphaeroides 601基因文库进行菌落原位杂交筛选到了携带glt基因的cosmid pLT27。pLT27能互补所有12株R.sphaeroides氨同化缺陷突变株。酶切分析表明在该cosmid中插人的染色体DNA片段大小约为26.5kb。以pRK415为载体亚克隆了4.0kb与10.5kh的pLT27的Hindlll酶切片段,分别命名为pLTRK271与pLTRK272。pLTRK272能互补变种GT6、GT10、GT11,pLTRK…     

Transposon mutagenesis of Pseudomonas aeruginosa exoprotease genes.

Transposon Tn5 was used to generate protease-deficient insertion mutants of Pseudomonas aeruginosa. The presence of Tn5 in the chromosome of P. aeruginosa was demonstrated by transduction and DNA-DNA hybridization. The altered protease production and kanamycin resistance were cotransduced into a wild-type P. aeruginosa strain. A radiolabeled probe of Tn5 DNA hybridized to specific BamHI fragments isolated from the insertion mutants. Two independently isolated Tn5 insertion mutants had reduced protease production, partially impaired elastase activity, and no immunologically reactive alkaline protease.     

Transposon Tn5-259 mutagenesis of Pseudomonas cepacia to isolate mutants deficient in antifungal activity.

Transposon Tn5-259 was inserted into the chromosome of Pseudomonas cepacia by mating with an Escherichia coli strain harboring a self-mobilizable, temperature-sensitive plasmid, pME12. Data from Southern blots and auxotroph analyses indicated that a single copy of the transposon was inserted in several places into the chromosome of P. cepacia. Among 1500 Tn5-259 transconjugants, only one mutant was found to be defective in the production of an antifungal compound, pyrrolnitrin. In addition, this mutant lost its ability to antagonize fungal phytopathogens. Using flanking DNA of the mutated gene as a probe, we have isolated four overlapping cosmid clones from a genomic library of P. cepacia. However, we were unable to complement the mutant because of difficulty in mobilizing the cosmids from E. coli to P. cepacia.     

Analysis of the genes flanking xabB: a methyltransferase gene is involved in albicidin biosynthesis in Xanthomonas albilineans

Transposon mutagenesis and complementation studies previously identified a gene (xabB) for a large (526kDa) polyketide-peptide synthase required for biosynthesis of albicidin antibiotics and phytotoxins in the sugarcane leaf scald pathogen Xanthomonas albilineans. A cistron immediately downstream from xabB encodes a polypeptide of 343aa containing three conserved motifs characteristic of a family of S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases. Insertional mutagenesis and complementation indicate that the product of this cistron (designated xabC) is essential for albicidin production, and that there is no other required downstream cistron. The xabB promoter region is bidirectional, and insertional mutagenesis of the first open reading frame (ORF) in the divergent gene also blocks albicidin biosynthesis. This divergent ORF (designated thp) encodes a protein of 239aa displaying high similarity to several IS21-like transposition helper proteins. The thp cistron is not located in a recognizable transposon, and is probably a remnant from a past transposition event that may have contributed to the development of the albicidin biosynthetic gene cluster. Failure of 'in trans' complementation of thp indicates that a downstream cistron transcribed with thp is required for albicidin biosynthesis.     

Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity

Summary Three nodulation-deficient (nod) mutants of Rhizobium leguminosarum were isolated following insertion of the transposon Tn5 into pRL1JI, the R. leguminosarum plasmid known to carry the nodulation genes. DNA adjacent to the nod: Tn5 alleles was subcloned and used to probe a cosmid clone bank containing DNA from a Rhizobium strain carrying pRL1JI. Two cosmid clones which showed homology with the probe contained about 10 kb of DNA in common. The R. leguminosarum host-range determinants were found to be present within this 10 kb common region since either of the cosmid clones could enable a cured R. phaseoli strain to nodulate peas instead of Phaseolus beans, its normal host. Electron microscopy of nodules induced by Rhizobium strains cured of their normal symbiotic plasmid but containing either of the two cosmid clones showed bacteroid-forms surrounded by a peri-bacteroid membrane, indicating that normal infection had occurred. Thus it is clear that this 10 kb region of nodDNA carries the genes that determine host range and that relatively few bacterial genes may be involved in nodule and bacteroid development.     

Mechanisms of biocontrol by Pantoea dispersa of sugar cane leaf scald disease caused by Xanthomonas albilineans

Albicidins, a family of phytotoxins and antibiotics produced by Xanthomonas albilineans , are important in sugar cane leaf scald disease development. The albicidin detoxifying bacterium Pantoea dispersa (syn. Erwinia herbicola ) SB1403 provides very effective biocontrol against leaf scald disease in highly susceptible sugar cane cultivars. The P. dispersa gene ( albD ) for enzymatic detoxification of albicidin has recently been cloned and sequenced. To test the role of albicidin detoxification in biocontrol of leaf scald disease, albD was inactivated in P. dispersa by site-directed mutagenesis. The mutants, which were unable to detoxify albicidin, were less resistant to the toxin and less effective in biocontrol of leaf scald disease than their parent strain. This indicates that albicidin detoxification contributes to the biocontrol capacity of P. dispersa against X. albilineans . Rapid growth and ability to acidify media are other characteristics likely to contribute to the competitiveness of P. dispersa against X. albilineans at wound sites used to invade sugar cane.     

Genes for phaseolotoxin synthesis are located on the chromosome ofPseudomonas syringae pv.phaseolicola

Transposon mutagenesis was used to locate the genes for phaseolotoxin biosynthesis inPseudomonas syringae pathovar.phaseolicola. Mutants unable to produce toxin were obtained that carried Tn5 on different chromosomal restriction fragments. None of the Tn5-induced nontoxigenic mutants carried the transposon in plasmid DNA. The insertion of Tn5 intotox DNA was confirmed by site-directed mutagenesis. The results reported here suggest the involvement of at least five chromosomal genes in phaseolotoxin biosynthesis. All of the toxinminus mutants retained both full pathogenicity on beans and resistance to the toxin.     

Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization.

Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.     

Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419.

Four Tn5-induced mutants of Rhizobium meliloti WSM419 were unable to grow or maintain intracellular pH at an external pH of 5.6. Restriction analysis of DNA fragments carrying Tn5 and flanking sequences cloned from these mutants indicated that all four cloned mutations are unique and that the two strains (TG1-6 and TG1-11) carry Tn5 insertions which are within 4.4 kilobases of each other on a single EcoRI fragment. Southern analysis of total mutant DNA indicated a single copy of Tn5 in each mutant. A limited cosmid gene bank of wild-type WSM419 DNA was probed for homology to mutant DNA cloned from the acid-sensitive mutants. Dot hybridization experiments identified one cosmid element within this bank carrying wild-type DNA sequences corresponding to DNA implicated in acid tolerance. This cosmid was able to complement defects in growth and intracellular pH maintenance in TG1-11 but not TG1-6.     

Isolation and characterization of pathogenicity genes of Pseudomonas syringae pv. tabaci.

Pseudomonas syringae pv. tabaci BR2 produces tabtoxin and causes wildfire disease on tobacco and bean plants. Approximately 2,700 Tn5 insertion mutants of a plasmid-free strain, PTBR 2.024, were generated by using suicide plasmid pGS9. Of these Tn5 mutants, 8 were no longer pathogenic on tobacco plants and 10 showed reduced symptoms. All of the eight nonpathogenic mutants caused typical wildfire disease symptoms on bean plants. Two of the nonpathogenic mutants failed to produce tabtoxin. The eight nonpathogenic mutants have Tn5 insertions into different EcoRI and SalI restriction fragments. The EcoRI fragments containing Tn5 from the eight nonpathogenic mutants were cloned into vector pTZ18R or pLAFR3. A genomic library of the parent strain was constructed in the broad-host-range cosmid pLAFR3. Three different cosmid clones that hybridized to the cloned Tn5-containing fragment from one of the nonpathogenic mutants, PTBR 4.000, were isolated from the genomic library. These clones contained six contiguous EcoRI fragments (a total of 57 kilobases [kb]). A 7.2-kb EcoRI fragment common to all three restored pathogenicity to mutant PTBR 4.000. None of the six EcoRI fragments hybridized to Tn5-containing fragments from the other seven mutants. The 7.2-kb fragment was conserved in P. syringae pv. tabaci and P. syringae pv. angulata, but not in other pathovars or strains. Because the mutants retained pathogenicity on bean plants and because of the conservation of the 7.2-kb EcoRI fragment only in pathovars of tobacco, we suggest that genes on the fragment might be related to host specificity.     

Identification and sequence analysis of the Rhizobium meliloti dctA gene encoding the C4-dicarboxylate carrier

Transposon Tn5-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could be complemented by cosmid pRmSC121 were subdivided into two classes. Class I mutants (RMS37 and RMS938) were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation. They were mutated in the structural gene dctA, which codes for the C4-dicarboxylate carrier. Class II mutants (RMS11, RMS16, RMS17, RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogen fixation. These mutants were mutated in regulatory dct genes which do not play an essential role in the symbiotic state. Thin sections of alfalfa nodules induced by the wild type and class I and class II mutants were analyzed by light microscopy. Class mutants induced typical Fix- nodules, showing a large senescent zone, whereas nodules induced by class II mutants only differed in an enhanced content of starch granules compared with wild-type nodules. Class I mutants could be complemented by a 2.1-kilobase SalI-HindIII subfragment of cosmid pRmSC121. DNA sequencing of this fragment resulted in the identification of an open reading frame, which was designated dctA because Tn5 insertion sites of the class I mutants mapped within this coding region. The dctA gene was preceded by a nif consensus promoter and an upstream NifA-binding element. Upstream of the dctA promoter, the 5' end of the R. meliloti dctB gene could be localized. The amino acid sequence of the N-terminal part of the R. meliloti DctB protein shared 49% homology with the corresponding part of the R. leguminosarum DctB protein. The DctA protein consisted of 441 or 453 amino acids due to two possible ATG start codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively. The hydrophobicity plot suggests that DctA is a membrane protein with several membrane passages. The amino acid sequences of the R. meliloti and the R. leguminosarum DctA proteins were highly conserved (82%).