Zwittermicin A resistance gene from Bacillus cereus.

Zwittermicin A is a novel aminopolyol antibiotic produced by Bacillus cereus that is active against diverse bacteria and lower eukaryotes (L.A. Silo-Suh, B.J. Lethbridge, S.J. Raffel, H. He, J. Clardy, and J. Handelsman, Appl. Environ. Microbiol. 60:2023-2030, 1994). To identify a determinant for resistance to zwittermicin A, we constructed a genomic library from B. cereus UW85, which produces zwittermicin A, and screened transformants of Escherichia coli DH5alpha, which is sensitive to zwittermicin A, for resistance to zwittermicin A. Subcloning and mutagenesis defined a genetic locus, designated zmaR, on a 1.2-kb fragment of DNA that conferred zwittermicin A resistance on E. coli. A DNA fragment containing zmaR hybridized to a corresponding fragment of genomic DNA from B. cereus UW85. Corresponding fragments were not detected in mutants of B. cereus UW85 that were sensitive to zwittermicin A, and the plasmids carrying zmaR restored resistance to the zwittermicin A-sensitive mutants, indicating that zmaR was deleted in the zwittermicin A-sensitive mutants and that zmaR is functional in B. cereus. Sequencing of the 1.2-kb fragment of DNA defined an open reading frame, designated ZmaR. Neither the nucleotide sequence nor the predicted protein sequence had significant similarity to sequences in existing databases. Cell extracts from an E. coli strain carrying zmaR contained a 43.5-kDa protein whose molecular mass and N-terminal sequence matched those of the protein predicted by the zmaR sequence. The results demonstrate that we have isolated a gene, zmaR, that encodes a zwIttermicin A resistance determinant that is functional in both B. cereus and E. coli.     

Cloning and partial characterization of zwittermicin A resistance gene cluster from Bacillus thuringiensis subsp. kurstaki strain HD1

AIM: The study seeks to shed light on the aminopolyol, broad-spectrum antibiotic zwittermicin A gene cluster of Bacillus thuringiensis subsp. kurstaki HD1 and to identify any new uncharacterized genes with an eventual goal to establish a better understanding of the resistance gene cluster. METHODS AND RESULTS: We screened 51 serovars of B. thuringiensis by PCR and identified 12 zmaR-positive strains. The zmaR-positive B. thuringiensis subsp. kurstaki HD1 strain displayed inhibition zones against indicator fungal strain Phytophthora meadii and bacterial strain Erwinia herbicola as well as against Rhizopus sp., Xanthomonas campestris and B. thuringiensis subsp. finitimus. The zmaR gene cluster of strain HD1 was partially cloned using a lambda library and was extensively characterized based on the information available from a study performed on a similar group of genes in Bacillus cereus. CONCLUSIONS: Three of the five genes in the zwittermicin gene cluster, including the zmaR gene, had counterparts in B. cereus, and the other two were new members of the B. thuringiensis zmaR gene cluster. SIGNIFICANCE AND IMPACT OF THE STUDY: The two new genes were extensively analysed and the data is presented. Understanding antifungal activity of B. thuringiensis may help us to design suitable Cry toxin delivery agents with antifungal activity as well as enhanced insecticidal activity.     

Zwittermicin A-producing strains of Bacillus cereus from diverse soils.

Bacillus cereus UW85 produces a novel aminopolyol antibiotic, zwittermicin A, that contributes to the ability of UW85 to suppress damping-off of alfalfa caused by Phytophthora medicaginis. UW85 produces a second antibiotic, provisionally designated antibiotic B, which also contributes to suppression of damping-off but has not been structurally defined yet and is less potent than zwittermicin A. The purpose of this study was to isolate genetically diverse strains of B. cereus that produce zwittermicin A and suppress disease. We found that most isolates of B. cereus that were sensitive to phage P7 or inhibited the growth of Erwinia herbicola produced zwittermicin A; therefore, phage typing and E. herbicola inhibition provided indirect, but rapid screening tests for identification of zwittermicin A-producing isolates. We used these tests to screen a collection of 4,307 B. cereus and Bacillus thuringiensis isolates obtained from bacterial stock collections and from diverse soils collected in Honduras, Panama, Australia, The Netherlands, and the United States. A subset of the isolates screened by the P7 sensitivity and E. herbicola inhibition tests were assayed directly for production of zwittermicin A, leading to the identification of 57 isolates that produced zwittermicin A; 41 of these isolates also produced antibiotic B. Eight isolates produced antibiotic B but not zwittermicin A. The assay for phage P7 sensitivity was particularly useful because of its simplicity and rapidity and because 22 of the 23 P7-sensitive isolates tested produced zwittermicin A. However, not all zwittermicin A-producing isolates were sensitive to P7, and the more labor-intensive E. herbicola inhibition assay identified a larger proportion of the zwittermicin A producers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of antibiotic-related genes from bacterial biocontrol agents with polymerase chain reaction

Pseudomonas chlororaphis PA23, Pseudomonas spp. strain DF41, and Bacillus amyloliquefaciens BS6 consistently inhibit infection of canola petals by Sclerotinia sclerotiorum in both greenhouse and field experiments. Bacillus thuringiensis BS8, Bacillus cereus L, and Bacillus mycoides S have shown significant inhibition against S. sclerotiorum on plate assays. The presence of antibiotic biosynthetic or self-resistance genes in these strains was investigated with polymerase chain reaction and, in one case, Southern blotting. Thirty primers were used to amplify (i) antibiotic biosythetic genes encoding phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin, and (ii) the zwittermicin A self-resistance gene. Our findings revealed that the fungal antagonist P. chlororaphis PA23 contains biosynthetic genes for phenazine-1-carboxylic acid and pyrrolnitrin. Moreover, production of these compounds was confirmed by high performance liquid chromatography. Pseudomonas spp. DF41 and B. amyloliquefaciens BS6 do not appear to harbour genes for any of the antibiotics tested. Bacillus thuringiensis BS8, B. cereus L, and B. mycoides S contain the zwittermicin A self-resistance gene. This is the first report of zmaR in B. mycoides.     

ZmaR, a novel and widespread antibiotic resistance determinant that acetylates zwittermicin A.

ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of Bacillus. The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro. Cell extracts of E. coli containing zmaR inactivated zwittermicin A by covalent modification. Chemical analysis of inactivated zwittermicin A by 1H NMR, 13C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated. Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with [14C]acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases. Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function.     

Biological activities of two fungistatic antibiotics produced by Bacillus cereus UW85.

Cultures and culture filtrates of Bacillus cereus UW85 suppress damping-off of alfalfa caused by Phytophthora medicaginis. We studied the role in disease suppression of two antibiotics from culture filtrates of UW85 that reversibly inhibited growth of P. medicaginis. We purified the two antibiotics by cation-exchange chromatography and high-voltage paper electrophoresis and showed that one of them, designated zwittermicin A, was an aminopolyol of 396 Da that was cationic at pH 7.0; the second, designated antibiotic B, appeared to be an aminoglycoside containing a disaccharide. Both antibiotics prevented disease of alfalfa seedlings caused by P. medicaginis. Purified zwittermicin A reversibly reduced elongation of germ tubes derived from cysts of P. medicaginis, and antibiotic B caused swelling of the germ tubes. Mutants generated with Tn917 or mitomycin C treatment were screened either for antibiotic accumulation in an agar plate diffusion assay or for the ability to suppress damping-off disease of alfalfa. Of 2,682 mutants screened for antibiotic accumulation, 5 mutants were substantially reduced in antibiotic accumulation and disease-suppressive activity. Of the 1,700 mutants screened for disease-suppressive activity, 3 mutants had reduced activity and they accumulated less of both antibiotics than did the parent strain. The amount of antibiotic accumulated by the mutants was significantly correlated with the level of disease suppression. Addition of either zwittermicin A or antibiotic B to alfalfa plants inoculated with a culture of a nonsuppressive mutant resulted in disease suppression. These results demonstrate that B. cereus UW85 produces two fungistatic antibiotics that contribute to suppression of damping-off disease of alfalfa.     

Cloning and analysis of the simocyclinone biosynthetic gene cluster of <Emphasis Type="Italic">Streptomyces antibioticus</Emphasis> Tü 6040

The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusTü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.     

Construction of a bacterial artificial chromosome library for a myxobacterium of the genus Cystobacter and characterization of an antibiotic biosynthetic gene cluster

A bacterial artificial chromosome (BAC) library was constructed to isolate the biosynthetic gene cluster for the polyketide/peptide hybrid-type antibiotic cystothiazole A from the myxobacterium Cystobacter fuscus strain AJ-13278. Sequence analysis of a 63.9 kb contiguous region that encompasses the biosynthetic gene cluster (cta) led to the identification of a polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) hybrid gene cluster 32.1 kb in size, which consists of six open reading frames (ORFs), ctaB to ctaG, as well as downstream genes ctaJ and ctaK (1.0 and 0.9 kb, respectively) responsible for the final biosynthetic steps. The genes ctaB, ctaE, and ctaF encode PKSs, the genes ctaC and ctaG encode NRPSs, and ctaD encodes an NRPS-PKS hybrid enzyme. Disruption of ctaD impaired cystothiazole A production. Additionally, two downstream genes, ctaJ and ctaK, which encode a nitrilase and an O-methyltransferase, respectively, must be responsible for the final methyl ester formation in the cystothiazole A biosynthesis.     

Preliminary phenotypic and molecular screening for potential bacterial biocontrol agents of Leptosphaeria maculans, the blackleg pathogen of canola

Leptosphaeria maculans causes blackleg disease of canola (Brassica napus L.). Bacteria isolated from soil, canola stubble and plant parts were assayed for suppression of blackleg. In plate assays, the bacteria isolated from canola stubble had the highest agar-diffusible antifungal activity (75%), which was fungitoxic. In plant cotyledon assays, endophytes had the highest disease suppression. Bacteria with the highest disease suppression in cotyledon assays also had significant disease suppression at the three- to four-leaf stage. PCR screening for bacterial biosynthetic genes, commonly thought to be involved in plant disease suppression, revealed 22 bacteria to be positive for pyrrolnitrin. Pseudomonas chlororaphis and P. aurantiaca isolates contained the phenazine biosynthetic gene. Three Bacillus cereus isolates had the zmaR resistance gene. This study generated a novel set of primers specific to the zwittermicin A biosynthetic cluster. The PCR screening has confirmed the presence of genes encoding pyrrolnitrin (55%), phenazine (10%), zwittermicin A biosynthesis (7.5%) and zwittermicin A resistance (7.5%) from the canola phyllosphere and rhizosphere, which seems more widely distributed than genes for 2,4-diacetylphloroglucinol and pyoluteorin.     

Novel roles of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> to control plant diseases

Bacillus thuringiensis is well known as an effective bio-insecticidal bacterium. However, the roles of B. thuringiensis to control plant diseases are not paid great attention to. In recent years, many new functions in protecting plants from pathogen infection have been discovered. For example, acyl homoserine lactone lactonase produced by B. thuringiensis can open the lactone ring of N-acyl homoserine lactone, a signal molecule in the bacterial quorum-sensing system. This in turn, significantly silences bacterial virulence. This finding resulted in the development of a new strategy against plant bacterial diseases by quenching bacterial quorum sensing. Another new discovery about B. thuringiensis function is zwittermicin A, a linear aminopolyol antibiotic with high activity against the Oomycetes and their relatives, as well as some gram-negative bacteria. This paper summarized the relative progresses of B. thuringiensis in plant disease control and its favorable application prospects.     

Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius

The biosynthetic gene cluster for tobramycin, a 2-deoxystreptamine-containing aminoglycoside antibiotic, was isolated from Streptomyces tenebrarius ATCC 17920. A genomic library of S. tenebrarius was constructed, and a cosmid, pST51, was isolated by the probes based on the core regions of 2-deoxy-scyllo-inosose (DOI) synthase, and L-glutamine:DOI aminotransferase and L-glutamine:scyllo-inosose aminotransferase. Sequencing of 33.9 kb revealed 24 open reading frames (ORFs) including putative tobramycin biosynthetic genes. We demonstrated that one of these ORFs, tbmA, encodes DOI synthase by in vitro enzyme assay of the purified protein. The catalytic residues of TbmA and dehydroquinate synthase were studied by homology modeling. The gene cluster found is likely to be involved in the biosynthesis of tobramycin.     

Cloning and heterologous expression of the vibrioferrin biosynthetic gene cluster from a marine metagenomic library

A biosynthetic gene cluster of siderophore consisting of five open reading frames (ORFs) was cloned by functional screening of a metagenomic library constructed from tidal-flat sediment. Expression of the cloned biosynthetic genes in Escherichia coli led to the production of vibrioferrin, a siderophore originally reported for the marine bacterium Vibrio parahaemolyticus. To the best of our knowledge, this is the first example of heterologous production of a siderophore by biosynthetic genes cloned from a metagenomic library. The cloned cluster was one of the largest of the clusters obtained by functional screening. In this study, we demonstrated and extended the possibility of function-based metagenomic research.     

DNA sequence and mutational analysis of rhizobitoxine biosynthesis genes in Bradyrhizobium elkanii.

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94 Delta rtx::Omega 1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94 Delta rtx::Omega 1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.     

Cloning and Heterologous Expression of the Vibrioferrin Biosynthetic Gene Cluster from a Marine Metagenomic Library

A biosynthetic gene cluster of siderophore consisting of five open reading frames (ORFs) was cloned by functional screening of a metagenomic library constructed from tidal-flat sediment. Expression of the cloned biosynthetic genes in Escherichia coli led to the production of vibrioferrin, a siderophore originally reported for the marine bacterium Vibrio parahaemolyticus. To the best of our knowledge, this is the first example of heterologous production of a siderophore by biosynthetic genes cloned from a metagenomic library. The cloned cluster was one of the largest of the clusters obtained by functional screening. In this study, we demonstrated and extended the possibility of function-based metagenomic research.     

The entire nogalamycin biosynthetic gene cluster of Streptomyces nogalater: characterization of a 20-kb DNA region and generation of hybrid structures

Fragments spanning 20 kb of Streptomyces nogalater genomic DNA were characterized to elucidate the molecular genetic basis of the biosynthetic pathway of the anthracycline antibiotic nogalamycin. Structural analysis of the products obtained by expression of the fragments in S. galilaeus and S. peucetius mutants producing aclacinomycin and daunomycin metabolites, respectively, revealed hybrid compounds in which either the aglycone or the sugar moiety was modified. Subsequent sequence analysis revealed twenty ORFs involved in nogalamycin biosynthesis, of which eleven could be assigned to the deoxysugar pathway, four to aglycone biosynthesis, while the remaining five express products with unknown function. On the basis of sequence similarity and experimental data, the functions of the products of the newly discovered genes were determined. The results suggest that the entire biosynthetic gene cluster for nogalamycin is now known. Furthermore, the compounds obtained by heterologous expression of the genes show that it is possible to use the genes in combinatorial biosynthesis to create novel chemical structures for drug screening purposes.     

Culture conditions that influence accumulation of zwittermicin A by Bacillus cereus UW85

Bacillus cereus strain UW85 produces an antibiotic, designated zwittermicin A, that is associated with the ability of UW85 to suppress damping-off disease of alfalfa (Medicago sativa) caused by the oomycete pathogen, Phytophthora medicaginis, in a laboratory bioassay. We have identified certain culture conditions that promote or suppress zwittermicin A accumulation by UW85. Maximum accumulation was detected in supernatants of trypticase soy broth cultures after sporulation, which is when cultures of UW85 provide the greatest suppression of damping-off on alfalfa. Inorganic amendments to trypticase soy broth cultures had the following effects on zwittermicin A accumulation and disease suppression: phosphate (50 mM or more) reduced zwittermicin A accumulation and disease suppression; ferric iron (0.25–1.0 mM) enhanced zwittermicin A accumulaiton and disease suppression; micronutrients (manganese, boron, copper, molybdenum, zinc) had no effect on zwittermicin A accumulation or disease suppression. Cultures of UW85 grown in chemically defined minimal medium supplemented with casein hydrolysate or grown in defined medium containing the minimal requirements for growth supplemented with five amino acids (Gln, Arg, Met, Phe, Ile) accumulated zwittermicin A. In minimal medium, alfalfa seed exudate inhibited growth of UW85, whereas alfalfa sprout exudate enhanced zwittermicin A accumulation by 40%. These data indicate that the accumulation of zwittermicin A can be modulated by specific nutrients, inorganic compounds, and plant-derived factors. These results will facilitate the improvement of large-scale purification of zwittermicin A, suggest appropriate conditions under which to conduct further genetic and biochemical analyses, and further substantiate the association between antibiotic accumulation and disease suppression by UW85.     

Isolation and characterization of bluensomycin biosynthetic genes from Streptomyces bluensis

The biosynthetic gene cluster for bluensomycin, a member of the aminoglycoside family of antibiotics, was isolated and characterized from the bluensomycin producing strain, Streptomyces bluensis ATCC27420. PCR primers were designed specifically to amplify a segment of the dTDP-glucose synthase gene based on its conserved sequences among several actinomycete strains. By screening a cosmid library using amplified PCR fragments, a 30-kb DNA fragment was isolated. Sequence analysis identified 15 open reading frames (ORFs), eight of which had previously been identified by Piepersberg et al. But seven are novel to this study. We demonstrated that one of these ORFs, blmA, confers resistance against the antibiotic dihydrostreptomycin, and another, blmD, encodes a dTDP-glucose synthase. These findings suggest that the isolated gene cluster is very likely to be responsible for the biosynthesis of bluensomycin.