Characterization and evaluation of two Bacillus strains,SS-12.6 and SS-13.1, as potential agents for the control of phytopathogenic bacteria and fungi

Two strains of Bacillus sp., SS-12.6 and SS-13.1, showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed that both strains have the genes for biosynthesis of iturin, bacillomycin and surfactin. Kinetics of production of antimicrobial substances in these strains showed that synthesis started at the beginning of exponential phase of growth. Maximum of activity was slowly reached at the beginning of stationary growth phase and was maintained until the end of observed period. Ethyl acetate extracts of cell-free supernatants of both strains were particularly active against several postharvest fungal pathogens, in vitro and in vivo, in the experiment with apple fruits. Mass spectrometry analysis of ethyl acetate extract of the supernatant of strain SS-12.6 confirmed the presence of antimicrobial lipopeptide surfactin.     

Physiology and ultrastructure of Leptothrix discophora SS-1

Leptothrix discophora strain SS-1 (ATCC 43182) is a Gram-negative, Mn²⁺-oxidizing, aerobic heterotroph which lost its sheath-forming ability after 18 months of cultivation on laboratory media. SS-1 possesses high 6-phosphogluconate dehydratase and KDPG aldolase activities, and a very low level of phosphofructokinase, indicating carbohydrate catabolism by the Entner-Doudoroff pathway. The strain is polarly flagellated, accumulates PHB up to 67% of its dry weight when grown in pyruvate-containing medium, and has a G+C content of 69.8 mol%. These properties indicate that L. discophora is essentially a pseudomonad which can form a sheath and oxidize Mn²⁺. Ultrastructural observations made before SS-1 lost its sheath-forming ability indicated two cell types. Short, flagellated, non-sheathed cells seen under the electron microscope probably corresponded to swarmer cells observed under phase-contrast microscopy. These cells contained plate organelles and PHB granules, and produced extracellular blebs approx. 25–50 nm in diameter. Larger sheathed cells also contained plate organelles, PHB granules, and blebs that were often sandwiched between the outer membrane and the sheath. Cells grown in the presence of added Mn²⁺ were surrounded by an extensive fibrillar matrix, rendered electron dense by precipitation of manganic oxide. The matrix was connected to various points of the cell by outer membrane evaginations or electron dense threads. We propose that the outer membrane blebs represent vehicles for excretion of unorganized sheath material and/or Mn²⁺-oxidizing protein produced by L. discophora.Parts of this work were presented previously (W.C. Ghiorse, Abst. Annu. Meet. Am. Soc. Microbiol., 1981, N 64, p 183)     

Temperature profile ofCandida cacaoi SS-2566

Summary Candida cacaoi SS-2566 was found to have a dissociative temperature profile and thermotolerant growth yield behaviour. With temperature-independent growth rates up to 42°C, its utilization at relatively high temperature for the production of cells (SCP, SCL) and/or metabolites may well be of interest, particularly in view of its hydrocarbon utilization.     

Kluyveromyces fragilis SS-437: An associatively-profiled thermotolerant yeast

The lactose-utilizing Kluyveromyces fragilis SS-437 was found to have an associative temperature profile, but a thermotolerant growth yield behaviour. Cardinal growth temperatures were: 3°C minimum for growth; 41.5°C optimum; 44.5°C final maximum (growth and death rates equalize); 46.1°C initial maximum (maximum limit for growth).     

Cytochrome P450 cam: SS- dimer and -SH derivative reactivities.

N-ethyl-maleimide alkylation converts ferric P450_cam to a (succinimido-cys)₄ protein with native optical and EPR spectra but insensitive to substrate induced shift of iron to high spin with Soret absorption to higher energy and inactive in putidaredoxin ferric-ferrous reduction. On photo or chemical reduction the ferrous protein oxygenates and, with reduced putidaredoxin, converts substrate to product. Mild oxidation of P450_cam yields a disulfide dimer whose properties on alkylation of 3 sulfhydryls equal the succinimido₄ monomer; additional alkylation converts either monomer or dimer to a P420.     

Direct and Mn-Controlled Indirect Iron Oxidation by Leptothrix discophora SS-1 and Leptothrix cholodnii

Direct biotic and homogeneous abiotic Fe(II) oxidation rates as well as oxidation rates of Fe(II) with MnO_X were determined in laboratory experiments and compared with biotic Mn(II) oxidation rates. In groundwaters and thermal installation waters, parameters for both Fe(II) oxidation steps were studied and products of biotic and abiotic Fe(II) and Mn(II) oxidation were analyzed. Direct Fe(II) oxidation of active Leptothrix cholodnii cultures can reach first-order rates of up to 1.17 ± 0.90 h^?1. Second-order rates of Fe(II) oxidation with biogenic, Leptothrix-cholodnii- and Leptothrix-discophora-SS-1-originating MnO_X lead to rates comparable with those as obtained with abiotic H⁺-birnessite.     

Protective effects of mitochondrion-targeted peptide SS-31 against hind limb ischemia-reperfusion injury

Hind limb ischemia-reperfusion injury is an important pathology in vascular surgery. Reactive oxygen species are thought to be involved in the pathogenesis of hind limb ischemia-reperfusion injury. SS-31, which belongs to a family of mitochondrion-targeted peptide antioxidants, was shown to reduce mitochondrial reactive oxygen species production. In this study, we investigated whether the treatment of SS-31 could protect hind limb from ischemia-reperfusion injury in a mouse model. The results showed that SS-31 treatment either before or after ischemia exhibited similar protective effects. Histopathologically, SS-31 treatment prevented the IR-induced histological deterioration compared with the corresponding vehicle control. SS-31 treatment diminished oxidative stress revealed by the reduced malondialdehyde level and increased activities and protein levels of Sod and catalase. Cellular ATP contents and mitochondrial membrane potential increased and the level of cytosolic cytC was decreased after SS-31 treatment in this IR model, demonstrating that mitochondria were protected. The IR-induced increase of levels of inflammatory factors, such as Tnf-α and Il-1β, was prevented by SS-31 treatment. In agreement with the reduced cytosolic cytC, cleaved-caspase 3 was kept at a very low level after SS-31 treatment. Overall, the effect of SS-31 treatment before ischemia is mildly more effective than that after ischemia. In conclusion, our results demonstrate that SS-31 confers a protective effect in the mouse model of hind limb ischemia-reperfusion injury preventatively and therapeutically.     

Siderophores of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis

Three Bacillus anthracis Sterne strains (USAMRIID, 7702, and 34F2) and Bacillus cereus ATCC 14579 excrete two catecholate siderophores, petrobactin (which contains 3,4-dihydroxybenzoyl moieties) and bacillibactin (which contains 2,3-dihydroxybenzoyl moieties). However, the insecticidal organism Bacillus thuringiensis ATCC 33679 makes only bacillibactin. Analyses of siderophore production by previously isolated [Cendrowski et al., Mol. Microbiol. 52 (2004) 407-417] B. anthracis mutant strains revealed that the B. anthracis bacACEBF operon codes for bacillibactin production and the asbAB gene region is required for petrobactin assembly. The two catecholate moieties also were synthesized by separate routes. PCR amplification identified both asbA and asbB genes in the petrobactin producing strains whereas B. thuringiensis ATCC 33679 retained only asbA. Petrobactin synthesis is not limited to the cluster of B. anthracis strains within the B. cereus sensu lato group (in which B. cereus, B. anthracis, and B. thuringiensis are classified), although petrobactin might be prevalent in strains with pathogenic potential for vertebrates.     

Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis

Strains of Bacillus cereus can produce a heat-stable toxin (cereulide). In this study, 101 Bacillus strains representing 7 Bacillus species were tested for production of heat-stable toxins. Strains of B. megaterium, B. firmus and B. simplex were found to produce novel heat-stable toxins, which showed varying levels of toxicity. B. cereus strains (18 out of 54) were positive for toxin production. Thirteen were of serovar H1, and it was of interest that some were of clinical origin. Two were of serovars 17B and 20, which are not usually implicated in the emetic syndrome. Partial purification of the novel B. megaterium, B. simplex and B. firmus toxins showed they had similar physical characteristics to the B. cereus emetic toxin, cereulide.     

Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis

Three species of the Bacillus cereus group (Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis) have a marked impact on human activity. Bacillus cereus and B. anthracis are important pathogens of mammals, including humans, and B. thuringiensis is extensively used in the biological control of insects. The microbiological, biochemical, and genetic characteristics of these three species are reviewed, together with a discussion of several genomic studies conducted on strains of B. cereus group. Using bacterial systematic concepts, we speculate that to understand the taxonomic relationship within this group of bacteria, special attention should be devoted also to the ecology and the population genetics of these species.     

Mitochondria-targeted antioxidant peptide SS-31 mediates neuroprotection in a rat experimental glaucoma model

To investigate the neuroprotective effects of the mitochondria-targeted antioxidant Szeto-Schiller peptide 31 (SS-31) in a rat experimental glaucoma model, SS-31 was intraperitoneally (IP) injected into Sprague-Dawley rats, followed by intracameral injection of polystyrene microspheres to induce elevated intraocular pressure (IOP). After 6 weeks, electroretinography (ERG) and flash visual-evoked potentials (F-VEPs) were recorded to assess retinal function. Hematoxylin-eosin staining was performed on retinal cross-sections to measure ganglion cell complex (GCC) thickness. Apoptotic retinal cells were assessed by TUNEL staining. Brn3a-positive retinal ganglion cells (RGCs) were counted in retinal flat mounts via immunofluorescence. The retinal total SOD, SOD2, and MDA expression levels were assessed in retinal tissue homogenates. The cyt c, Baxz and Bcl-2 protein levels in rat retinas were detected by western blot analysis. Bax and Bcl-2 expressions were also evaluated using immunohistochemistry in paraffinized sections. Our results showed that the rats that received microsphere injection developed elevated IOP. SS-31 ameliorated the reductions in the a- and b-wave amplitudes on ERG and the F-VEP amplitude in glaucomatous eyes. GCC thickness was preserved, TUNEL-positive cells were decreased in the retina, and Brn3a-positive RGCs were in creased in the SS-31-treated glaucoma group compared with those in the non-treated glaucoma group. SS-31 significantly reduced MDA levels and increased SOD2 levels after glaucoma induction. Significant suppression of cyt c release, upregulation of Bcl-2, and downregulation of Bax were observed following SS-31 administration. In summary, SS-31 exerts neuroprotective effects in this experimental glaucoma model by inhibiting mitochondrial dysfunction and therefore represents a promising therapeutic agent for glaucoma.     

Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis

Eight strains of highly amylolytic, sporeforming bacilli (hereafter referred to as Bacillus amyloliquefaciens) were compared with respect to their taxonomic relationship to B. subtilis. The physiological-biochemical properties of these two groups of organisms showed that B. amyloliquefaciens differed from B. subtilis by their ability to grow in 10% NaCl, characteristic growth on potato plugs, increased production of alpha-amylase, and their ability to ferment lactose with the production of acid. The base compositions of the deoxyribonucleic acid (DNA) of the B. subtilis strains consistently fell in the range of 41.5 to 43.5% guanine + cytosine (G + C), whereas that of the B. amyloliquefaciens strains was in the 43.5 to 44.9% G + C range. Hybrid formation between B. subtilis W23 and B. amyloliquefaciens F DNA revealed only a 14.7 to 15.4% DNA homology between the two species. Transducing phage, SP-10, was able to propagate on B. subtilis W23 and B. amyloliquefaciens N, and would transduce B. subtilis 168 (indole(-)) and B. amyloliquefaciens N-10 (arginine(-)) to prototrophy with a frequency of 3.9 x 10(-4) and 2.4 x 10(-5) transductants per plaque-forming unit, respectively. Attempts to transduce between the two species were unsuccessful. These data show that Bacillus amyloliquefaciens is a valid species and should not be classified as a strain or variety of B. subtilis.     

Fatty Acids in Bacillus larvae, Bacillus lentimorbus, and Bacillus popilliae

The types of fatty acids produced by two strains each of Bacillus larvae, B. lentimorbus, and B. popilliae, and their distribution patterns, were studied by gas-liquid chromatography. All six organisms produced eight major fatty acids: six branched (iso-C(14), -C(15), -C(16), and -C(17), and anteiso-C(15) and -C(17)), two normal (n-C(14) and -C(16)), and two minor (n-C(15) and monounsaturated n-C(16)). In addition, some other trace acids were produced. Branched-chain fatty acids accounted for 54 to 85% of the total fatty acids. These compositions are similar to those previously found with 26 strains of 12 species of the genus Bacillus. Thus, an abundance of branched-chain fatty acids seems to be a characteristic of the biochemical nature of the genus Bacillus. It is noteworthy that marked differences between the nutritional requirements of the three insect pathogens used in the present study and those of the other 12 species of the genus Bacillus studied previously are not significantly reflected in their fatty acid composition.     

Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis

The three species of the group 1 bacilli, Bacillus anthracis, B. cereus, and B. thuringiensis, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis. Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B. anthracis Ames. Ninety-three DNA sequences that were present in B. anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B. anthracis strains. These sequences map to distinct loci on the B. anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B. anthracis.     

Methionine regeneration and aminotransferases in Bacillus subtilis,Bacillus cereus,and Bacillus anthracis

The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L. C. Berger, J. Wilson, P. Wood, and B. J. Berger, J. Bacteriol. 183:4421-4434, 2001). The genome of Bacillus subtilis has been found to contain no subfamily Ia aminotransferase sequences. Instead, the analogous enzymes in B. subtilis were found to be members of the If subfamily. These putative aspartate aminotransferases, the yugH, ywfG, ykrV, aspB, and patA gene products, have been cloned, expressed, and characterized for methionine regeneration activity. Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor. In contrast, subcellular homogenates of B. subtilis and Bacillus cereus utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors. The two putative branched-chain aminotransferase genes in B. subtilis, ybgE and ywaA, were also cloned, expressed, and characterized. Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine. The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. subtilis ybgE gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa. Examination of B. cereus and Bacillus anthracis genome data found that there were no subfamily IIIa homologues in these organisms. In both B. cereus and B. anthracis, two putative branched-chain aminotransferases and two putative D-amino acid aminotransferases were discovered as members of subfamily IIIb. These four sequences were cloned from B. cereus, expressed, and characterized. Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. anthracis homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity. The aminooxy compound canaline was found to be an uncompetitive inhibitor of B. subtilis YbgE and also inhibited growth of B. subtilis and B. cereus in culture.     

Genome Differences That Distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis

The three species of the group 1 bacilli, Bacillus anthracis, B. cereus, and B. thuringiensis, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis. Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B. anthracis Ames. Ninety-three DNA sequences that were present in B. anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B. anthracis strains. These sequences map to distinct loci on the B. anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B. anthracis.