Zwittermicin A biosynthetic cluster.

The goal of this study was to identify the biosynthetic cluster for zwittermicin A, a novel, broad spectrum, aminopolyol antibiotic produced by Bacillus cereus. The nucleotide sequence of 2.7kb of DNA flanking the zwittermicin A self-resistance gene, zmaR, from B. cereus UW85 revealed three open reading frames (ORFs). Of these ORFs, two had sequence similarity to acyl-CoA dehydrogenases and polyketide synthases, respectively. Insertional inactivation demonstrated that orf2 is necessary for zwittermicin A production and that zmaR is necessary for high-level resistance to zwittermicin A but is not required for zwittermicin A production. Expression of ZmaR was temporally associated with zwittermicin A production. The results suggest that zmaR is part of a cluster of genes that is involved in zwittermicin A biosynthesis, representing the first biosynthetic pathway for an aminopolyol antibiotic.

rosR, a determinant of nodulation competitiveness in Rhizobium etli.

We previously described a Tn5 mutant of Rhizobium etli strain CE3, designated CE3003, that is decreased in nodulation competitiveness, reduced in competitive growth in the rhizosphere, and has a hydrophobic cell surface (R. S. Araujo, E. A. Robleto, and J. Handelsman, Appl. Environ. Microbiol., 60:1430-1436, 1994). To determine the molecular basis for the mutant phenotypes, we identified a 1.2-kb fragment of DNA derived from the parent that restored the wild-type phenotypes to the mutant. DNA sequence analysis indicated that this 1.2-kb fragment contained a single open reading frame that we designated rosR. The Tn5 insertion in CE3003 was within rosR. We constructed a derivative of CE3 that contained a deletion in rosR, and this mutant was phenotypically indistinguishable from CE3003 in cell surface and competitive characteristics. Based on the nucleotide sequence, the deduced RosR amino acid sequence is 80% identical to that of the Ros protein from Agrobacterium tumefaciens and the MucR protein from Rhizobium meliloti. Both Ros and MucR are transcriptional repressors that contain a putative zinc-finger DNA-binding domain. This study defines a gene, rosR, that is homologous to a family of transcriptional regulators and contributes to nodulation competitiveness of R. etli. Moreover, we established that a single gene affects nodulation competitiveness, competitive growth in the rhizosphere, and cell surface hydrophobicity.

Genotypic and phenotypic analysis of zwittermicin A-producing strains of Bacillus cereus.

Many strains of Bacillus cereus produce zwittermicin A, a novel antibiotic that contributes to the ability of B. cereus to suppress certain plant diseases. The purpose of this study was to identify molecular indicators of zwittermicin A production in B, cereus strains, contribute to an understanding of the ecology and evolution of this group of bacteria, and identify potential agents for control of plant disease. The fatty acid composition of 20 strains known to be zwittermicin A producers and 20 strains known to be non-producers was determined. Cluster analysis of the fatty acid methyl ester (FAME) profiles revealed that zwittermicin A producers grouped together in two clusters, apart from most non-producers. Discriminant analysis of the FAME profiles generated models that correctly predicted the zwittermicin A-production phenotype in 17 of 20 zwittermicin A producers and 17 of 20 non-producers. Sixteen random oligonucleotide primers were tested in PCR, and one primer was identified that generated a fragment of 0.48 kb or 0.49 kb from total DNA from 26 of 28 strains known to produce zwittermicin A, whereas PCR with this primer did not generate bands of that size from 16 of 20 non-producing strains. PCR with primers designed to amplify zmaR, a gene from B. cereus that confers resistance to zwittermicin A, generated DNA fragments of 1.1 kb and 1.0 kb in all 29 zwittermicin A-producing strains tested, amplified a fragment of 0.3 kb in some of the zwittermicin A-producing strains, and amplified no fragments in 20 of 23 non-producing strains in a stock collection of B. cereus strains. The zmaR primers were tested for their ability to identify new zwittermicin A-producing isolates of B. cereus from two soils. All 12 of the isolates that produced the banding pattern characteristic of this primer pair produced zwittermicin A, and none of the 12 isolates that did not have the banding pattern produced detectable zwittermicin A. Seven of the 12 isolates initially identified as zwittermicin A producers with the zmaR primers significantly suppressed damping-off of alfalfa, whereas only one of the non-producers suppressed this disease. The results show that FAME and PCR analyses distinguish B. cereus strains that produce zwittermicin A from other B. cereus strains, that PCR with the primers designed to amplify zmaR is the most reliable method of those tested for identification of zwittermicin A producers, and that this method can be used to identify new strains with disease-suppressive activity.

Production of kanosamine by Bacillus cereus UW85.

Bacillus cereus UW85 produces two antibiotics that contribute to its ability to suppress certain plant diseases (L. Silo-Suh, B. Lethbridge, S. J. Raffel, H. He, J. Clardy, and J. Handelsman, Appl. Environ. Microbiol. 60:2023-2030, 1994). To enhance the understanding of disease suppression by UW85, we determined the chemical structure, regulation, and the target range of one of the antibiotics. The antibiotic was identified as 3-amino-3-deoxy-D-glucose, also known as kanosamine. Kanosamine was highly inhibitory to growth of plant-pathogenic oomycetes and moderately inhibitory to certain fungi and inhibited few bacterial species tested. Maximum accumulation of kanosamine in B. cereus UW85 culture supernatants coincided with sporulation. Kanosamine accumulation was enhanced by the addition of ferric iron and suppressed by addition of phosphate to rich medium. Kanosamine accumulation was also enhanced more than 300% by the addition of alfalfa seedling exudate to minimal medium.

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.

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 accumulation 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP.

Transporters encoded in genetic loci putP, proP and proU mediate proline and/or betaine accumulation by Escherichia coli K-12. The ProP and ProU systems are osmoregulatory. Activation of ProP in response to hyperosmotic stress has been demonstrated both in vivo and in vitro. It therefore serves as a model experimental system for the analysis of osmosensory and osmoregulatory mechanisms. We developed methodologies which will facilitate the identification of proline transporter genes by functional complementation of putP proP proU bacteria. E. coli gene proP was isolated and located within a chromosomal DNA fragment. Deletion, complementation and sequence analysis revealed putative promoter and transcription termination signals flanking a 1500 base-pair open reading frame. The predicted 55 kDa ProP protein was hydrophobic. In vitro expression of proP yielded a protein whose apparent molecular mass was determined to be 42 kDa by polyacrylamide gel electrophoresis under denaturing conditions. Database searches and cluster analysis defined relationships among the ProP sequence and those of integral membrane proteins that comprise a transporter superfamily. Members of the superfamily catalyze facilitated diffusion or ion linked transport of organic solutes in prokaryotes and eukaryotes. Multiple alignment revealed particularly close correspondence among the ProP protein, citrate transporters from E. coli and Klebsiella pneumoniae and an alpha-ketoglutarate transporter from E. coli. The predicted ProP sequence differed from those closely similar sequences in possessing an extended central hydrophilic loop and a carboxyl terminal extension. Unlike other protein sequences within the transporter superfamily, the carboxyl terminal extension of ProP was strongly predicted to participate in formation of an alpha-helical coiled coil. These data suggest that the ProP protein catalyzes solute-ion cotransport. Its unusual structural features may be related to osmoregulation of its activity.

Molecular and symbiotic characterization of exopolysaccharide-deficient mutants of Rhizobium tropici strain CIAT899.

We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non-mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS-deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS-deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R. tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.

Insertion proQ220::Tn5 alters regulation of proline porter II, a transporter of proline and glycine betaine in Escherichia coli.

Mutation pro-220::Tn5, which increases the resistance of Escherichia coli to 3,4-dehydroproline (M. E. Stalmach, S. Grothe, and J. M. Wood, J. Bacteriol. 156:481-486, 1983), is not linked to putP, proP, or proU. It was located at 40.4 min on the E. coli chromosomal linkage map, by conjugational and transductional mapping, and is now denoted proQ220::Tn5. Proline porter II was not detectable when proQ220::Tn5 proP+ bacteria were cultivated under optimal conditions or with nutritional stress (amino acid limitation). Toxic proline analog sensitivity and proline porter II activity were partially restored to proQ220::Tn5 proP+ bacteria, but not to a proQ220::Tn5 proP219 strain, by a hyperosmotic shift and by growth under osmotic stress. Elevated expression of a proP::lacZ gene fusion, for bacteria grown under osmotic stress, was not influenced by the proQ220::Tn5 insertion. We propose that the proQ locus encodes a positive regulatory element which elevates proline porter II activity.

Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12.

Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R. L., McClellan, D. J., Milner, J. L., and Wood, J. M. (1986) J. Bacteriol. 166, 253-259). Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress. That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP. Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift. In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase. Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake. These results suggested that proline porter II was respiration-dependent and probably ion-linked. Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient. Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles. Thus, activation of proline porter II was reversible. It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift.

Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12.

Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/delta 1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.

Proline transport and osmotic stress response in Escherichia coli K-12.

Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.