Kunthala Jayaraman

Spontaneous loss of a high molecular weight plasmid and the biocide of Bacillusthuringiensis var. israeliensis

Abstract A rapid loss of biocidal potency of B. thuringiensis var israeliensis against mosquito larvae has been correlated to the appearance of acrystalliferous variants at a high frequency. This irreversible loss is attributed to the loss of a large molecular weight plasmid. Segregation of asporogenic variants has also been noticed although at a much lower frequency.

Biocide gene(s) and biocidal activity in different strains of Bacillus sphaericus. Expression of the gene(s) in E. coli maxicells

SummaryThe recently cloned biocidal determinant of the highly toxic strain of B. sphaericus 1593M (Ganesan et al. 1983) was used as probe to investigate homologous sequences in different toxic and non-toxic strains of B. sphaericus. It was found that the potent strains we have analysed are characterised by the presence of DNA sequences (6.6, 6.4, 5.8, 1.6, 1.3 and 0.6 Kb) not found in the non-toxic strains. These results further show that one of the two weakly toxic strains analysed presents a hybridisation pattern completely different from that observed with the highly potent strains of B. sphaericus. When the DNA of the two non-toxic strains was analysed, SSII-I failed to hybridise to the probe and Rem4 exhibited mainly one hybridisable sequence of 2.3 Kb not detectable in the toxic strains.No region of homology to the probe was found in the DNA of two strains of B. thuringiensis (var. berliner and var. israeliensis) analysed.By dot blot hybridisation experiments it was estimated that the larvicidal determinant might be present in about one to three copies per genome.With the use of E. coli maxicells we have shown further that the toxin gene(s) encoded four polypeptides with molecular weights of 21, 19, 15, and 12 Kd. The significance of these findings is discussed.

Identification of the peptides of the crystals of Bacillusthuringiensis var israelensis involved in the mosquito larvicidal activity

Tryptic digestion of the proteins from the purified crystals of B.thuringiensis var israelensis resulted in the decline of high molecular weight peptides without the loss of mosquito larvicidal activity, measured after immobilization of the digests with DEAE- Sephadex A 50 beads. Amongst the peptides generated (less than 44 kDa), a 21 kDa peptide was immunoreactive to the crystal antiserum. Analysis of the peptides released from spores of the toxic (Cry+) and non-toxic (Cry-) strains has revealed a pattern in which only the 26kDa peptide was missing in the Cry-strain. Sporulation and crystal formation were dissociated by the addition of the antibiotic netropsin, which could also inhibit the crystal assembly, without considerable decrease of the larvicidal activity and retention of the 26kDa peptide. These results implicate the 26kDa peptide in the larvicidal action.

Biosynthesis of polymyxin by Bacillus polymyxa: I. The status of the biosynthetic multienzyme complex during active antibiotic synthesis and sporulation

Abstract The involvement of membrane fractions of Bacillus polymyxa in the early stages of the biosynthesis of the peptide antibiotic polymyxin, was established by (a) incorporation of the precursor amino acid in an acid precipitable form in the absence of protein synthesis, (b) presence of all the component amino acid-activating enzymes, and (c) association of bioassayable polymyxin, in the purified membrane fraction. Polymyxin negative mutants that were also blocked at stage 0 of sporulation were shown to be defective in one or more of their membrane-bound amino acid-activating enzymes. A strong correlation between sporulation and antibiotic production had been indicated by the isolation of these mutants which are revertible simultaneously to Ab + and Spo + traits. During the onset of the rapid phase of the elaboration of polymyxin, a delocalization of one of the membrane-bound enzymes, 2,4-diaminobutyric acid-activating enzyme, to the soluble fraction was observed. Concomitant with this change, the levels of the intracellular protein-bound and free polymyxin was increased in the soluble fraction.

Biosynthesis of polymyxin by Bacillus polymyxa: II. On the nature and interaction of the multienzyme complex with the end product polymyxin☆

Abstract The interaction of polymyxin with the producer organism Bacillus polymyxa has been shown to be at the level of membranes, resulting in an enhancement of the activities of its own biosynthetic enzymes. This enhancement has been shown to be due to the solubilization of the membrane-associated multienzyme complex by polymyxin in a specific manner. The relevance of this physiological feature was also indicated by the appearance of the soluble multienzyme complex activity only in cells, which synthesize maximal amounts of polymyxin. Purification of the polymyxin released multienzyme complex from the membranes and the soluble form of the complex from the stationary phase cells has revealed several similarities between them. Both contain two major fractions of the molecular weights of 300,000 and 170,000, harboring all the polymyxin component amino acid-activating enzymes. Their multisubunit nature was established by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Using mutants blocked in sporulation and/or antibiotic synthesis, it was shown that the interaction of polymyxin with the producer organism was inoperative when antibiotic production was curtailed. This interaction has been suggested as one of the early sporulation-specific phenemenon.

Appearance of polyadenylated RNA species during sporulation in Bacillus polymyxa

Abstract Two main features of altered transcriptional programming during sporulation are reported; 1. The occurrence of the polyA RNA species exclusively during sporulation. 2. The localisation in the membranes of regions of DNA that may be enriched in spore genes.

Localization of (dA-dT)-rich sequences in the membrane-bound DNA and their possible role in sporulation of Bacillus polymyxa

SummaryThe appearance of poly(A)RNA species at the onset of sporulation has been demonstrated in B. polymyxa. These RNA species, characterized by short oligo(A) stretches, are synthesized at the end of exponential growth (t0) and their level reaches a maximum two hours later (t2 of sporulation). This new feature of sporulation appears relevant to this process as indicated by the lack of formation of poly(A)RNA in mutants blocked at the early stages of sporulation. In addition, the antibiotic netropsin, which is known to inhibit sporulation without affecting the vegetative growth of B. subtilis, also inhibits poly(A) RNA synthesis and sporulation in B. polymyxa. The selective inhibitory effect of netropsin on the poly(A)RNA formation, as reflected in the reduced incorporation of [3H]-adenosine into RNA by sporulating cells, is traced to the inhibition of sporulation-specific RNA polymerase under in vitro conditions. The inhibition of transcription concerns mainly the DNA regions that are firmly associated with membranes. Moreover, [32P]-labeled poly(A)RNA isolated from sporulating cells at t2 hybridizes preferentially with membrane bound DNA (mDNA). Further evidence that the mDNA is enriched in d(AT) sequences was obtained by demonstration of the resistance of the mDNA-netropsin complex to DNase action. Based on the property of netropsin to bind to AT sequences in DNA, it is proposed that early spore genes might be enriched in d(AT) sequences and are localized in mDNA.

Evidence for the presence of two D-amino acid oxidases inPseudomonas aeruginosa PAO

By the isolation of mutants that were unable to grow on L-hydroxyproline or DL-valine, it has been possible to demonstrate the presence of two different types of D-amino acid oxidase activities inPseudomonas aeruginosa PAO. One was the D-amino acid dehydrogenase, probably involved in the oxidation of a number of D-amino acids such as D-alanine, D-phenylalanine and D-valine. The other was the inducible oxidase, specific to the oxidation of allohydroxy-D-proline formed from L-hydroxyproline during its oxidation. Thus, it has been possible to delink the involvement of the general D-amino acid dehydrogenase in the oxidative breakdown of allohydroxy-Dsproline.

Mapping of the loci involved in the catabolic oxidation of L-hydroxyproline in Pseudomonas aeruginosa PAO.

SummaryGenes specifying the oxidative utilization of hydroxyproline (Hyp) in P. aeruginosa PAO were located on the chromosome, around 19th minute by conjugation experiments. A map order of his-68-his-07-Hyp was assigned. Confirmation of this gene order was also demonstrated by transductional mapping studies. All the genes determining the enzymes of Hyp dissimiliatory pathway were closely linked.

Demonstration of the formation of 1,3-diphosphoglyceric acid as an intermediate in the high-energy phosphate metabolism during reinitiation of development in Artemia

Abstract Extensive labelling of the glycolytic intermediate 2,3-diphosphoglycerate by 32PO3−4 during the early periods of development in Artemia is reported. At 30 min of activation this is the major labelled compound. The mobilization of inorganic phosphate through glycolysis leading to the formation of 1,3-diphosphoglycerate results in the formation of a high-energy phosphate donor. The label from this compound could be chased to high-energy phosphates (adenine derivatives). The location and subsequent high degree of labelling of 2,3-diphosphoglycerate in the yolk platelets further demonstrate the important role played by this organelle in the metabolic events accompanying the breakdown of dormancy in Artemia.