Suresh S. Patil

Preliminary characterization of an antibiotic produced by Xanthomonas albilineans which inhibits DNA synthesis in Escherichia coli.

Chlorosis-inducing isolates of Xanthomonas albilineans, the sugarcane leaf scald pathogen, produced a mixture of antibacterial compounds in culture. The antibiotic mixture, which eluted as a single strongly retarded peak from Sephadex LH-20 in methanol, was bactericidal to Escherichia coli. Inhibition of E. coli was not reversed by added nutrients, and affected cells were not lysed but many accumulated polyphosphate granules. The major antibacterial component, isolated in crystalline form after HPLC, is given the trivial name albicidin. Near the minimum inhibitory concentration, albicidin caused a complete block to DNA synthesis, followed by partial inhibition of RNA and protein synthesis, as assessed by incorporation of radioactive precursors. Spontaneous antibiotic-resistant mutants of E. coli showed no cross-resistance between albicidin and inhibitors of either subunit of DNA gyrase. Mixing albicidin with purified DNA from E. coli did not alter the thermal denaturation behaviour of the DNA, or the absorption spectrum of the antibiotic. PolA+ and PolA - strains of E. coli were equally sensitive to albicidin, indicating that the antibiotic does not bind to or modify DNA. Selective inhibition of DNA synthesis without evidence of DNA binding suggests a specific interaction of albicidin with an essential replication protein.

Inhibitors of Ornithine Carbamoyltransferase from Pseudomonas syringae pv. phaseolicola. st]Revised Structure of Phaseolotoxin

Abstract The structure of phaseolotoxin, a toxin produced by cultured Pseudomonas syringae pv. phaseolicola , the causal agent of halo blight disease in bean plants is revised to 3. The structure of octicidin, isolated from leaves of bean plants infected with pv. phaseolicola . has been determined to be 4, a protease degradation product of 3 which is formed in the plant.

Cutinase deficient mutants of Colletotrichum gloeosporioides are nonpathogenic to papaya fruit

Mutants of Colletotrichum gloeosporioides , the causal agent of papaya anthracnose, which are deficient in cutinase production and are nonpathogenic to papaya fruit have been obtained. Survivors (5285) of treatment of pathogen spores with four different mutagens (UV radiation, N -methyl- N -nitro- N -nitrosoguanidine, ethylmethane sulphonate, HNO 2 ) were screened for cutinase production in a rapid and selective plate assay. Two isolates were apparently identical to the wild-type except in cutinase production. To further confirm their Cut − phenotype, the spores of the two mutants were inoculated into a liquid medium containing papaya cutin as the sole carbon source. No visual growth by either mutant was observed and filtrates from neither culture reacted with rabbit anticutinase nor showed esterase activity as determined by hydrolysis of PNB. In a laboratory fruit bioassay, the cutinase deficient mutants failed to infect intact papaya surfaces; however, they produced normal lesions when papaya fruit surfaces were either artificially wounded or treated with purified C. gloeosporioides cutinase prior to inoculation. These studies offer further confirming evidence that C. gloeosporioides breaches papaya cuticle with cutinase.

Evidence that an albicidin-like phytotoxin induces chlorosis in sugarcane leaf scald disease by blocking plastid DNA replication

Abstract Albicidin is the best characterized member of a family of antibiotics produced by Xanthomonas albilineans which inhibit prokaryote DNA replication. Results with mutants of X. albilineans have suggested that a toxin biosynthetically closely related to albicidin causes the blocked chloroplast differentiation which leads to chlorosis in sugarcane leaf scald. This paper describes effects of albicidin on sugarcane plants and plastids. Albicidin was detected in white leaf stripes surrounding invaded vascular bundles in diseased plants. Time-course and dose-response data showed that in isolated sugarcane proplastids inhibition of DNA replication is a primary action of albicidin, followed by partial inhibition of protein synthesis. Albicidin caused permanent white chlorosis of shoots re-differentiating from treated callus, but did not cause chlorosis when injected into the spindles of intact sugarcane plants. These results coupled with earlier ultrastructural and mutant studies strongly support the hypothesis that a member of the albicidin family of compounds produced by X. albilineans in invaded xylem causes chlorosis in sugarcane leaf scald disease by preferentially inhibiting plastid DNA replication, resulting in blocked chloroplast differentiation.

Molecular strategies of pathogens and host plants

The field of the molecular basis of plant disease is rapidly developing. The nineteen chapters of current information in Molecular Strategies of Pathogens and Host Plants are written by well known experts in the United States and Japan, and cover recent progress in the genetics and molecular biology of bacteria and fungi which are pathogens of plants. This research emphasizes the genes which are responsible for production of toxins, enzymes, and hormones that lead to pathogenicity and specificity in plant-pathogen interactions. Several chapters also examine the biochemistry of the plants response to microbial attack.

Correlation between albicidin production and chlorosis induction by Xanthomonas albilineans, the sugarcane leaf scald pathogen

Abstract Chlorosis of emerging leaves of sugarcane plants invaded by Xanthomonas albilineans results from blocked chloroplast differentiation, apparently caused by a phytotoxin. Chlorosis inducing isolates of X. albilineans produce a family of antimicrobial compounds, including albicidin which inhibits prokaryote DNA replication. The objective of this study was to determine whether albicidin, or a related compound produced in diseased plants, causes chlorosis of uninvaded tissues characteristic of sugarcane leaf scald disease. Spontaneous loss of albicidin production occurs at a high rate but does not involve extrachromosomal genetic elements. Results with mutants and revertants in albicidin production show a close correlation between albicidin production and ability to cause chlorosis, strongly suggesting that the chlorosis inducing toxin is biosynthetically closely related to albicidin. Plasmid pJB4JI is transmitted at high frequency from Escherichia coli to X. albilineans, where it is stably maintained as a conjugative plasmid, providing a basis for further genetic analysis of X. albilineans using well-characterized P1 group plasmids.

Multiple copies of a DNA sequence from Pseudomonas syringae pathovar phaseolicola abolish thermoregulation of phaseolotoxin production

Phaseolotoxin, a phytotoxin of Pseudomonas syringae pv. phaseolicola, is produced at 18°C but not at 28°C. Here we report that a fragment (24.4 kb) cloned from the wild‐type strain, which does not harbour a gene(s) involved in phaseolotoxin biosynthesis, abolishes this thermoregulation in the wild type and suppresses a Tox− mutant at both temperatures. A subclone harbouring a 465bp fragment contains motifs that are characteristic of DNA‐binding sites. In mobility shift assays we have detected a protein(s) from the wild‐type and the mutant strains, grown at appropriate temperatures, that specifically binds to the fragment containing the DNA‐binding motifs. We propose that the binding protein is a repressor which is ‘titrated’ by this fragment when it is present in the cell on a multiple copy plasmid, thus allowing expression of phaseolotoxin genes.

Accumulation of antibacterial isoflavonoids in hypersensitively responding bean leaf tissues inoculated with Pseudomonas phaseolicola

Abstract Red Kidney bean ( Phaseolus vulgaris L.) plants inoculated with Pseudomonas phaseolicola (isolate HB-36) showed water-soaked infection centers on the inoculated leaf surfaces about 48 h after inoculation. In contrast, resistant bean plants (GN Nebraska 27) inoculated with the same isolate responded by developing a hypersensitive reaction (HR) after roughly the same period after inoculation. High concentrations of phenolic compounds were detected in the ethanolic extracts of leaf tissues showing HR. Among these were identified the isoflavonoid phytoalexins phaseollin (pterocarpan), phaseollinisoflavan (isoflavan), coumestrol (coumestan) and kievitone (isoflavanone). These isoflavonoids were detected in substantial quantities 24 h after inoculation and their concentrations increased further up to 72 h, after which time they began to decrease. In susceptible Red Kidney leaves, isolate HB-36 induced much lower concentrations of these compounds at all stages of disease development. In repeated in vitro bioassays the above four compounds consistently restricted colony formation by P. phaseolicola isolates. However, the race 2 isolates, HB-36 and G-50, were found to be comparatively more tolerant to these compounds than the race 1 isolates, HB-33 and HB-20. Colony formation in race 1 isolates was completely inhibited by phaseollin and phaseollinisoflavan at 42 and 21 μg/ml; colony formation of race 2 isolates was retarded (90 to 98%) but not completely suppressed at 52·5 μg/ml of phaseollin and 52 μg/ml of phaseollinisoflavan. Coumestrol and kievitone at 21 and 33 μg/ml respectively were totally toxic to race 1 isolates but they were relatively less toxic to race 2 isolates even at higher test concentrations. All four phytoalexins also restricted the growth of P. glycinea isolates, R-2 and 724 (Leben). Isolate 724, however, exhibited a greater degree of tolerance than isolate R-2. P. fluorescens , a saprophyte, was relatively insensitive to the phytoalexins and showed only 26 to 30% retardation in colony formation at the maximum test concentrations used. These studies show that the virulent isolates of P. phaseolicola are not as sensitive to bean phytoalexins as the relatively less virulent isolates of P. phaseolicola .

Phaseotoxin A: An antimetabolite from pseudomonas phaseolicola

Abstract Phaseotoxin, the exotoxin of the bean pathogen Pseudomonas phaseolicola , has been resolved into four biologically active fractions by DEAE cellulose chromatography. Phaseotoxin A, the toxin in the first fraction decomposed to give glutamic acid and inorganic orthophosphate only, and was chromatographically identical with synthetic N-phosphoglutamic acid. Like phaseotoxin A, the synthetic compound inhibits ornithine carbamoyltransferase and induces chlorosis in bean leaves. To our knowledge this is the first report of occurrence of an N-phosphorylated primary amine in nature.

Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases

Many peptide antibiotics in prokaryotes and lower eukaryotes are produced non-ribosomally by multi-enzyme complexes. Analysis of gene-derived amino acid sequences of some peptide synthetases of bacterial and fungal origins revealed a high degree of conservation (35-50% identity). The genes encoding those peptide synthetases are clustered into large operons with repetitive domains (about 600 amino acids), in the case of synthetases activating more than one amino acid. We used two 35-mer oligonucleotides derived from two highly conserved regions of known peptide synthetases to identify the surfactin synthetase operon in Bacillus subtilis ATCC 21332, a strain not accessible to genetic manipulation. We show that the derived oligonucleotides can be used not only for the identification of unknown peptide synthetase genes by hybridization experiments but also in sequencing reactions as primers to identify internal domain sequences. Using this method, a 25.8-kb chromosomal DNA fragment bearing a part of the surfactin biosynthesis operon was cloned and partial sequences of two internal domains were obtained.