δ-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA

Abstract Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of δ-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis . The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.     

N-Glycosidase activity in extracts of Bacillus subtilis and its inhibition after infection with bacteriophage PBS2.

We have detected in crude extracts of Bacillus subtilis an N-glycosidase activity which catalyzes the release of free uracil from DNA of the subtilis phage PBS2 labeled with [3H]uridine. This DNA contains deoxyuridine instead of thymidine. The enzyme is active in the presence of 1.0 mM EDTA and under these conditions Escherichia coli or T7 DNA labeled with [3H]thymidine is not degraded to labeled acid-soluble products. The activity resembles an N-glycosidase from E. coli which releases free uracil from DNA containing deaminated cytosine residues. Both enzymes in crude extracts are active in the presence of EDTA, do not require dialyzable co-factors, and have the same pH optimum. They differ in that the enzyme from E. coli is more sensitive to heat, sulfhydryl reagents, and salt. The enzyme from B. subtilis is inactive on DNA containing 5-bromouracil or hydroxymethyluracil. Extracts of PBS2-infected B. subtilis lose the N-glycosidase activity within 4 min after infection and contain a factor that inhibits the N-glycosidase activity within 4 min after infection and contain a factor that inhibits the N-glycosidase activity in extracts of uninfected cells in vitro.     

Aspartokinase III, a new isozyme in Bacillus subtilis 168.

A previously undetected Bacillus subtilis aspartokinase isozyme, which we have called aspartokinase III, has been characterized. The new isozyme was most readily detected in extracts of cells grown with lysine, which repressed aspartokinase II and induced aspartokinase III, or in extracts of strain VS11, a mutant lacking aspartokinase II. Antibodies against aspartokinase II did not cross-react with aspartokinase III. Aspartokinases II and III coeluted on gel filtration chromatography at Mr 120,000, which accounts for the previous inability to detect it. Aspartokinase III was induced by lysine and repressed by threonine. It was synergistically inhibited by lysine and threonine. Aspartokinase III activity, like aspartokinase II activity, declined rapidly in B. subtilis cells that were starved for glucose. In contrast, the specific activity of aspartokinase I, the diaminopimelic acid-inhibitable isozyme, was constant under all growth conditions examined.     

Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon

AIMS: The present study evaluated the in vitro and in vivo antagonistic effect of Bacillus against the pathogenic vibrios. METHODS AND RESULTS: Cell-free extracts of Bacillus subtilis BT23 showed greater inhibitory effects against the growth of Vibrio harveyi isolated by agar antagonism assay from Penaeus monodon with black gill disease. The probiotic effect of Bacillus was tested by exposing shrimp to B. subtilis BT23 at a density of 106-108 cfu ml-1 for 6 d before a challenge with V. harveyi at 103-104 cfu ml-1 for 1 h infection. The combined results of long- and short-term probiotic treatment of B. subtilis BT23 showed a 90% reduction in accumulated mortality. CONCLUSIONS: This study reports that pathogenic vibrios were controlled by Bacillus under in vitro and in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicated that probiotic treatment offers a promising alternative to the use of antibiotics in shrimp aquaculture.     

Effect of lipopeptides of antagonistic strains of Bacillus subtilis on the morphology and ultrastructure of the cucurbit fungal pathogen Podosphaera fusca

AIMS: To analyse the morphological and ultrastructural effects of lipopeptides of cell-free liquid cultures from the antagonistic Bacillus subtilis strains, UMAF6614 and UMAF6639, on the cucurbit powdery mildew fungus, Podosphaera fusca, conidial germination. METHODS AND RESULTS: Butanolic extracts from cell-free supernatants of B. subtilis cultures were tested for their ability to arrest P. fusca conidial germination using the zucchini cotyledon disc method. Previously, the occurrence of lipopeptide antibiotics fengycin, iturin/bacillomycin and surfactin in the extracts was verified by diverse chromatographic approaches. Conidial germination was strongly reduced by antifungal extracts obtained from liquid cultures of both B. subtilis strains. Scanning electron microscopy analysis showed morphological damage in conidia characterized by the presence of large depressions and loss of turgidness. Transmission electron microscopy analysis revealed severe modifications in the plasma membrane and disorganization of the P. fusca cell cytoplasm. CONCLUSIONS: The lipopeptides produced by the two strains of B. subtilis are able to reduce cucurbit powdery mildew disease by arresting conidial germination, which seems to result from the induction of important cytological alterations. SIGNIFICANCE AND IMPACT OF THE STUDY: We elucidated the mechanisms employed by these antagonistic strains of B. subtilis to suppress cucurbit powdery mildew disease and delineate the ultrastructural damages responsible for their suppressive effect.     

Expression of the MuIFN alpha 7 gene in Bacillus subtilis using the levansucrase system

The mouse interferon alpha 7 gene, the signal sequence of which has been removed by oligonucleotide-directed mutagenesis, was introduced into a Bacillus subtilis secretion vector containing the promoter and the signal sequence of the B. subtilis levansucrase gene. Different B. subtilis strains were transformed with the fused levansucrase-interferon gene; their cell extracts and culture supernatants tested for antiviral activity and the IFN alpha 7 protein showed the presence of IFN alpha 7 only in the cell extracts. To promote IFN alpha 7 secretion, constructs were realized in order to restore the alpha helix conformation of the signal sequence of levansucrase and interferon protein junction. Our results suggest that factors other than the structure of the peptide around the cleavage site are involved in the secretion of IFN alpha 7 by B. subtilis.     

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extract displayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 micrograms/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 micrograms/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < or = 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 micrograms/ml) and B. subtilis (MIC at 3.9 and 7.8 micrograms/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6 identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 micrograms/ml and 3.12 micrograms/ml, respectively. Both compounds presented MIC of 3.12 micrograms/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 micrograms/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.     

Teichoic acid is an essential polymer in Bacillus subtilis that is functionally distinct from teichuronic acid

Wall teichoic acids are anionic, phosphate-rich polymers linked to the peptidoglycan of gram-positive bacteria. In Bacillus subtilis, the predominant wall teichoic acid types are poly(glycerol phosphate) in strain 168 and poly(ribitol phosphate) in strain W23, and they are synthesized by the tag and tar gene products, respectively. Growing evidence suggests that wall teichoic acids are essential in B. subtilis; however, it is widely believed that teichoic acids are dispensable under phosphate-limiting conditions. In the work reported here, we carefully studied the dispensability of teichoic acid under phosphate-limiting conditions by constructing three new mutants. These strains, having precise deletions in tagB, tagF, and tarD, were dependent on xylose-inducible complementation from a distal locus (amyE) for growth. The tarD deletion interrupted poly(ribitol phosphate) synthesis in B. subtilis and represents a unique deletion of a tar gene. When teichoic acid biosynthetic proteins were depleted, the mutants showed a coccoid morphology and cell wall thickening. The new wall teichoic acid biogenesis mutants generated in this work and a previously reported tagD mutant were not viable under phosphate-limiting conditions in the absence of complementation. Cell wall analysis of B. subtilis grown under phosphate-limited conditions showed that teichoic acid contributed approximately one-third of the wall anionic content. These data suggest that wall teichoic acid has an essential function in B. subtilis that cannot be replaced by teichuronic acid.     

Biotin synthase of Bacillus subtilis shows less reactivity than that of Escherichia coli in in vitro reaction systems

The biotin synthases of Bacillus subtilis and Escherichia coli were compared in a physiological reduction system using cell-free extracts and in a artificial reduction system using photo-reduced deazariboflavin. The biotin synthase of B. subtilis was less active than that of E. coli in both reaction systems and showed at least ten-fold less biotin-forming activity than that of E. coli in the artificial reduction system. The physiological reduction system using the biotin synthases and cell-free extracts of B. subtilis and E. coli showed species specificity. The results suggest that the activity of the physiological reduction system of B. subtilisis weaker than that of E. coli. Addition of excess dethiobiotin inhibited biotin formation by growing cells of B. subtilis, but not by E. coli.     

Peptide Synthesis by Extracts from Bacillus subtilis Spores

Cell-free peptide synthesis by extracts from vegetative cells and spores of Bacillus subtilis was analyzed and compared. The initial rate of phenylalanine incorporation in a polyuridylate-directed system was found to be in a similar range for the two extracts. However, spore extracts frequently incorporated less total phenylalanine as did the vegetative cell system. Optimal conditions for amino acid incorporation by spore extracts were found to be similar to those of vegetative cell extracts. Polyphenylalanine synthesis was stimulated by preincubation of both extracts prior to the addition of polyuridylic acid (poly U) and labeled phenylalanine. Both systems showed a dependence on an energy-generating system and were inhibited by chloramphenicol and puromycin. Ribonuclease, but not deoxyribonuclease, inhibited the reaction significantly. The presence of methionine transfer ribonucleic acid (tRNA(F)) and methionyl-tRNA(F) transformylase was demonstrated in spore extracts. An analysis of several aminoacyl-tRNAs in spores revealed that the relative amounts of these tRNAs were similar to those found in vegetative cells. Only lysine tRNA was found to be present in relatively greater amounts in spores. These results indicate that dormant spores of B. subtilis contain the machinery for the translation of genetic information.     

Antioxidant, radical-scavenging, anti-inflammatory, cytotoxic and antibacterial activities of methanolic extracts of some Hedyotis species

The antioxidant, radical-scavenging, anti-inflammatory, cytotoxic and antibacterial activities of methanolic extracts of seven Hedyotisspecies were investigated. The antioxidant activity was evaluated by the ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods while the radical scavenging activity was measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. The anti-inflammatory activity related to NO inhibition of the plant extracts was measured by the Griess assay while cytotoxicity were measured by the MTT assay against CEM-SS cell line. The antibacterial bioassay (against 4 bacteria, i.e. Bacillus subtilis B28 (mutant), Bacillus subtilis B29 (wild-type), Pseudomonas aeruginosa UI 60690 and methicillin resistant Staphylococcus aureus, (MRSA) was also carried out using the disc-diffusion method. All tested extracts exhibited very strong antioxidant properties when compared to Vitamin E (alpha-tocopherol) with percent inhibition of 89-98% in the FTC and 60-95% in the TBA assays. In the DPPH method, H. herbacea exhibited the strongest radical scavenging activity with an IC50 value of 32 microg/ml. The results from the Griess assay showed that the tested extracts are weak inhibitors of NO synthase. However, all tested extracts exhibited moderate cytotoxic properties against CEM-SS cell line giving CD50 values in the range of 21-41 microg/ml. In the antibacterial bioassay, the stems and the roots of H. capitellata showed moderate activity against the 4 tested bacteria while the leaves showed moderate activity towards B. subtilis B28, MRSA and P. aeruginosa only. The roots of H. dichotoma showed strong antibacterial activity against all 4 bacteria. All other extracts did not exhibit any antibacterial activity.     

Classification and genetic characterization of pattern-forming Bacilli

One of the more natural but less commonly studied forms of colonial bacterial growth is pattern formation. This type of growth is characterized by bacterial populations behaving in an organized manner to generate readily identifiable geometric and predictable morphologies on solid and semi-solid surfaces. In our first attempt to study the molecular basis of pattern formation in Bacillus subtilis , we stumbled upon an enigma: some strains used to describe pattern formation in B. subtilis did not have the phenotypic or genotypic characteristics of B. subtilis . In this report, we show that these strains are actually not B. subtilis , but belong to a different class of Bacilli , group I. We show further that commonly used laboratory strains of B. subtilis can co-exist as mixed cultures with group I Bacilli , and that the latter go unnoticed when grown on frequently used laboratory substrates. However, when B. subtilis is grown under more stringent semiarid conditions, members of group I emerge in the form of complex patterns. When B. subtilis is grown under less stringent and more motile conditions, B. subtilis forms its own pattern, and members of group I remain unnoticed. These findings have led us to revise some of the mechanistic and evolutionary hypotheses that have been proposed to explain pattern growth in Bacilli .     

Physiological studies on cAMP synthesis in Bacillus subtilis

Abstract cAMP was detected in Bacillus subtilis SB 19 and in a temperature sensitive mutant ts 33-6 under oxygen limitation. Growth rate and cAMP content were negatively correlated. Dinitrophenol and α-methylglucoside elicited an increase of the synthesis of cAMP and an increase of the intracellular cAMP content. In response to a decrease was increased. This increase of cAMP concentration did not occur if nitrate was present in the growth medium as an alternative terminal electron acceptor.     

cAMP levels and in situ measurement of cAMP related enzymes during yeast-to-hyphae transition in Candida albicans

Intracellular levels of cAMP and specific activities of adenylate cyclase, cAMP phosphodiesterase and cAMP-dependent protein kinase were measured during filamentation in the dimorphic fungus Candida albicans. Enzymatic assays were performed in permeabilized cells under conditions prevented endogenous proteolysis. The variations observed in cAMP levels were mainly accounted for by variations in the specific activities of adenylate cyclase and cAMP phosphodiesterase at different stages during germ tube formation. cAMP-dependent protein kinase, measured with kemptide as exogenous substrate, was developmental regulated. Some properties of the enzymatic activities from cell-free extracts are described.     

Resistance of bacteriophage H1 to restriction and modification by Bacillus subtilis R

H1, a 5-hydroxymethyluracil (HMU)-containing Bacillus subtilis bacteriophage, was neither restricted nor modified upon infection of B. subtilis R cells. In vitro, H1 DNA was not restricted by BsuR under standard conditions (200 mM salt), although the expected frequency of -GGCC- cleavage sites was approximately 250. However, four specific sites were cleaved under nonstandard conditions (low salt or high pH) or in the presence of organic solvents, like dimethyl sulfoxide and glycerol. After the substitution of thymine for HMU by DNA cloning in B. subtilis, a BsuR cleavage site was restricted and modified under standard conditions. No additional sites were detected after shotgun-cloning of about 11% of the chromosome. The nucleotide sequence of a cleavage site was found to be 5'. .C-A-Hmu-A-A-C-Hmu-Hmu-Hmu-G-G-C-C-Hmu-A-G-. . .3', which shows the presence of a bona fide BsuR (GGCC) recognition sequence, flanked by (Hmu-A)-rich sequences. The results suggested that the resistance of H1 to restriction and modification by B. subtilis R was due to (i) a strong bias against the GGCC-recognition sequence and (ii) protection of the four remaining GGCC sites as a consequence of HMU-A base pairs flanking the sites.     

Sigma 29-like protein is a common sporulation-specific element in bacteria of the genus Bacillus.

A monoclonal antibody specific for an antigenic determinant on the Bacillus subtilis sporulation-induced sigma factor sigma 29 reacted with proteins similar in size to sigma 29 in extracts of sporulating Bacillus licheniformis, Bacillus amyloliquifaciens, Bacillus cereus, Bacillus natto, and Bacillus pumilus but not in extracts prepared from vegetatively growing cultures of these bacteria. These results indicate that RNA polymerase modifications, initially described for B. subtilis, are likely to be common among sporulating Bacillus spp. and that at least some of the specific modifications that are observed in sporulating B. subtilis are conserved among members of this genus.     

Identification of autodigestion target sites in Bacillus subtilis neutral proteinase.

Autocatalytic degradation of purified Bacillus subtilis neutral proteinase was examined under various conditions. At elevated temperatures, under non-inhibitory conditions, mature protein was rapidly degraded, but no accumulation of specific breakdown products occurred. However, by incubating purified neutral proteinase on ice during extended periods of time, specific peptides accumulated. These peptides were analysed by SDS/PAGE and Western blotting, and the N-terminal sequences were determined for the four major peptides, which had sizes of 30, 22, 20 and 15 kDa. Sequence data identified five fission sites in the neutral proteinase, three of which were identical with autodigestion target sites in thermolysin, a thermostable neutral proteinase. Comparison of the identified fission sites of the B. subtilis neutral proteinase with the known substrate-specificity of the enzyme indicated that they were in agreement, showing a preference for the generation of fissions at the N-terminal side of large hydrophobic residues, such as leucine, isoleucine and methionine. These results suggest a high degree of similarity in the three-dimensional structures of B. subtilis neutral proteinase and thermolysin.     

Microbiological Aspects of Ethylene Oxide Sterilization: IV. Influence of Thickness of Polyethylene Film on the Sporicidal Activity of Ethylene Oxide

Spores of Bacillus subtilis var. niger, dried on nonhygroscopic and hygroscopic surfaces, were enclosed in one of four thicknesses of low-density polyethylene film (2, 4, 6, and 20 mils). The surfaces were then placed in a specially designed thermochemical death rate apparatus and exposed to an ethylene oxide concentration of 600 mg/liter (at 54.4 C) and 50% relative humidity. Survival data, including both spore survivor curves and decimal reduction values (expressed as D values at 54.4 C-600 mg of ethylene oxide per liter), demonstrated similar survivor patterns when the B. subtilis var. niger spores were enclosed in low-density polyethylene films 2, 4, and 6 mils thick. A comparison of these patterns with those of spores enclosed in glassine and subjected to identical exposure conditions revealed only slight variations. The use of 20-mil polyethylene film greatly increased the time required to kill B. subtilis var. niger spores under the exposure conditions.