Bacterial variations on the methionine salvage pathway

Background  

The thiomethyl group of S-adenosylmethionine is often recycled as methionine from methylthioadenosine. The corresponding pathway has been unravelled in Bacillus subtilis. However methylthioadenosine is subjected to alternative degradative pathways depending on the organism.     

Dissimilation of ferulic acid byBacillus subtilis

Bacillus subtilis utilized ferulic acid and its intermediates vanillin, vanillic acid, and protocatechuic acid as sole carbon source. The enzymes of the ferulic acid degradative pathway such as deacetylase, vanillin oxidase, vanillate-o-demethylase, and protocatechuate 3,4-dioxygenase were inducible in nature. Concentration of the inducer profoundly influenced the induction of the enzymes involved in ferulic acid dissimilation.     

Production of mannan-degrading enzymes

Summary Production of mannanase by four hemicellulolytic microorganisms was studied. The highest mannanase activity was produced byBacillus subtilis. -Mannosidase and -galactosidase were not detected inB. subtilis culture filtrate. The hydrolysis of galactomannans was limited by the increasing degree of substitution of the substrate. No monomeric sugars were produced in the hydrolysis withB. subtilis culture filtrate.     

The Oscillopolarographic Behaviour of Deoxyribonucleic Acids Isolated from Bacillus Subtilis and Bacillus brevis

Summary The behaviour of DNA from several strains ofB. subtilis andB. brevis on the dropping mercury electrode in a medium of ammonium formate was studied. Native DNA yields in this medium on the oscillogram dE/dt againstE an anodic indentation for which the residues of deoxyguanylic acid are responsible.B. subtilis DNA produces a substantially smaller indentation thanB. brevis DNA does. It was found that the difference is not conditioned by impurities in the DNA samples, nor by the presence of denatured DNA. The difference in the depth of the indentation produced byB. subtilis andB. brevis DNAs almost disappears after denaturation of these DNAs or in an ammonium formate medium of higher concentration. The assumption was advanced that the different oscillopolarographic behaviour of DNAs obtained fromB. subtilis andB. brevis is connected with the different primary structure of these DNAs.     

Microbial interaction ofAspergillus parasiticus andBacillus subtilis withalternaria alternata. production of alternariol, alternariol monomethylether and tenuazonic acid on sunflower seeds

Production of alternariol, alternariol mono-methylether and tenuazonic acid byAlternaría alternata was studied in competition withAspergillus parasiticus andBacillus subtilis on irradiated sunflower seeds at 0.90 a_w. In cultures co-inoculated withAlternaría alternata andAspergillus parasiticus alternariol production decreased by 64%. Similar results were observed in cultures co-inoculated withAlternaría alternata andBacillus subtilis.     

Effect of oxygen on growth,sporulation, and mosquito larval toxin formation byBacillus sphaericus 1593

The effect of oxygen on growth, sporulation, and mosquito larval toxin synthesis byBacillus sphaericus 1593 grown in a small fermentor was investigated. With air as the source of oxygen, about one-half of the cells sporulated and 1022 units of toxicity/mg of cell dry weight were formed. A shift to 100% oxygen in the gas stream maintained a higher level of dissolved oxygen in the medium, but this produced a late block in sporulation; however, toxin synthesis was normal. The mechanism of oxygen inhibition of sporulation byB. sphaericus is unknown, but the same effect was observed inB. subtilis 168. Stopping of the air flow at 8 h, after forespores were completed in about one-half the cells, inhibited the completion of sporulation, but did not decrease toxin production.     

Effects of bacterial contamination on development ofMicroplitis croceipes [Hym.: Braconidae]

Bacterial contaminants ofHeliothis virescens (F.) influenced the development ofMicroplitis croceipes (Cresson). Among the four bacterial species studied, the most virulent wasPseudomonas maltophilia Hugh and Ryschenkow followed byBacillus subtilis (Ehrenberg) Cohn. Both bacteria caused severe mortality in all stages ofMicroplitis tested.Microplitis larvae were less susceptible toEscherichia coli (Migula) Castellani and Chalmers andLeuconostoc mesenteroides (Tsenkovskii) van Thieghem than toB. subtilis andP. maltophilia. AlthoughE. coli did not affect the number of cocoons produced, adult emergence was lower than in controls. Longevity of adultMicroplitis exposed to bacterially contaminated honey-water was greatly reduced in all bacterial treatments.      

Formation of extracellular neutral proteinase and the stringent response inBacillus subtilis

The kinetics of extracellular neutral proteinase synthesis by an isogenic stringent (IS58) and a relaxed (IS56) strain ofB. subtilis were compared. The specific enzyme formation rate by the stringent strain was higher than that of the relaxed one. Norvaline addition (1 mg/mL) induced the formation of pppGpp and ppGpp, respectively, as well as the appearance of extracellular neutral proteinase activities in cultures of the stringent strain IS58 and a strain with high proteinase production (ZF-178) only. These correlations support the suggestion that (p)ppGpp are involved in the regulation processes responsible for production of extracellular neutral proteinases byB. subtilis. Dedicated to Prof. Dr. F. Mach on the occasion of his 60th birthday.     

Studies on Degradation of d-Biotin by Microorganisms

During the course of investigations on the metabolism of d-biotin by microorganisms, the authors have found that a strain belonging to Endomycopsis effectively converted d-biotin into unknown biotin vitamers. The unknown biotin vitamers formed were isolated in crystalline form from the culture filtrate of a strain of Endomycopsis species and characterized as bisnorbiotin and bisnorbiotin sulfoxide by their physico-chemical and biological properties. The isolated vitamers were shown to support the growth of Bacillus subtilis, but not of Saccharomyces cerevisiae and of Lactobacillus arabinosus. The degradative pathway of d-biotin in microorganisms was also discussed.     

Effect of sodium arsenite on the biosynthesis of mitomycins byStreptomyces caespitosus and mode of action of mitomycin C onBacillus subtilis NRRL B-543

Addition of different concentrations of sodium arsenite to the fermentation medium vised for the production of mitomycin antibiotics byStreptomyces caespitosus hindered the biosynthesis of mitomycins and led to the accumulation of 2-oxoglutarate, pyruvate and acetone. Mitomycin C isolated and purified using thin-layer chromatography in low concentration of about 0.1 μg/ml did not affect the RNA, DNA and protein biosynthesis of the growingBacillus subtilis, while at 10 μg/ml mitomycin C markedly affected RUA, DNA and protein biosynthesis.     

Production of antifungal antibiotic,iturin in a solid state fermentation byBacillus subtilis NB22 using wheat bran as a substrate

Summary Production of a family of lipopeptide antibiotic, iturin byB. subtilis NB22, in solid state fermentation (SSF) of wheat bran (WB) was investigated. The amount of iturin produced per unit weight of wet substrate was 5–6 times more than that in the submerged fermentation (SMF). SSF enabled to produce a homologue of iturin with strong antibiotic activity in a larger fraction compared with the SMF.     

Functional analysis of the response regulator DegU in Bacillus megaterium DSM319 and comparative secretome analysis of degSU mutants

We functionally analysed the two-component regulatory system DegSU (historically SacU) in Bacillus megaterium DSM319 by generating a genetic knock out as well as a sacU32 mutation. The latter—known to cause a hypersecretion phenotype in Bacillus subtilis—had no influence on extracellular protease and amylase activity in B. megaterium. Since the B. megaterium DegU complemented a Bacillus licheniformis ∆degSU mutant, functionality of the protein was proven. Expression of the sacB encoded levansucrase was found to be dependent on DegSU in B. megaterium. Consistently, the fusion of the sacB promoter to gfp revealed a strong increase in GFP-expression in the sacU32 strain. On 2 D-gels of the secretome, a large number of intracellular proteins was seen. The culture medium contained only 42 secreted proteins which can be assigned to polypeptides involved in the metabolism of the cell wall, polypeptides with proteolytic activities and those with unknown functions. Though overall protease activity matches with the wild type, two proteolytic enzymes (Vpr and YwaD) are missing in the secretome of the ∆degSU strain, while other degradative enzymes are not affected. In line with such findings, no increase of proteolytic or other degradative enzymes was seen in the sacU32 mutant. Thus, compared to B. subtilis and B. licheniformis, the number of extracellular proteins influenced by DegSU is surprisingly low in B. megaterium, a feature, probably advantageous as to the use of the sacU32 mutant for production of secreted proteins.     

Cell-free synthesis of the dipeptide antibiotic bacilysin

Summary Bacilysin, a dipeptide antibiotic produced byBacillus subtilis A 14, was synthesized by a cell-free extract of the producing organism from its constitutent amino acids,l-alanine andl-anticapsin. The synthesis required ATP and Mg²⁺ and was optimal at pH 8.1. The same extract also synthesizedl-alanyl-l-alanine. The synthesis of bacilysin was not inhibited by chloramphenicol, DNase or RNase.     

Effects of Commercial and Indigenous Microorganisms on Fusarium Wilt Development in Chickpea

The purpose of this research was to determine whetherBacillus subtilis,nonpathogenicFusarium oxysporum,and/orTrichoderma harzianum,applied alone or in combination to chickpea (Cicer arietinumL.) cultivars ‘ICCV 4’ and ‘PV 61’ differing in their levels of resistance to Fusarium wilt, could effectively suppress disease caused by the highly virulent race 5 ofFusarium oxysporumf. sp.ciceris.Seeds of both cultivars were sown in soil amended with the three microbial antagonists, alone or in combination, and 7 days later seedlings were transplanted into soil infested with the pathogen. All three antagonistic microorganisms effectively colonized the roots of both chickpea cultivars, whether alone or in combination, and significantly suppressed Fusarium wilt development. In comparison with the control, the incubation period for the disease was delayed on average about 3 days and the final disease severity index and standardized area under the disease progress curve were reduced significantly between 14 and 33% and 16 and 42%, respectively, by all three microbial antagonists. Final disease incidence only was reduced byB. subtilis(18–25%) or nonpathogenicF. oxysporum(18%). The extent of disease suppression was higher and more consistent in ‘PV 61’ than in ‘ICCV 4’ whether colonized byB. subtilis,nonpathogenicF. oxysporum,orT. harzianum.The combination ofB. subtilis+T. harzianumwas effective in suppressing Fusarium wilt development but it did not differ significantly from treatments with either of these antagonists alone. In contrast, the combination ofB. subtilis+ nonpathogenicF. oxysporumtreatment was not effective but either antagonist alone significantly reduced disease development.     

Possibility of using a bacterial antagonist against fungal diseases of Piper betle L. and corchorus spp.

Summary A strain ofBacillus subtilis isolated from soil was found effective againstSclerotium rolfsii when grown on 20% potato extract for 48 hours and used along with the medium. Whole boiled potato was also a good medium but the bacteria needed 20 days to grow until insoluble starch was digested.In field trials the antagonist brought down the spread ofSclerotium and anthracnose diseases ofPiper betle. Against seed and air-borne diseases of jute caused byMacrophomina phaseoli,Diplodia corchori andColletotrichum corchori, the antagonist also showed promising results.Concluding part of a scheme supported by Food & Agriculture Council, Pakistan.     

Changes in the Fine Structure of Microbial Cells Induced by Chaotropic Salts

The electron microscopic examination of thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37°C for 3–5 h or at 100°C for 5–6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, the ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

The effects of synthetic protease inhibitors on human proinsulin production by recombinantBacillus subtilis strain

Summary Two inexpensive inhibitors non-toxic to B. subtilis cells, O,O-diethyl-1-(N--hydrohexafluoro-isobutyryl)amino-1-methyl-propylphosphonate and O,O-diísobutyl-1-[2-(ethoxycarbonyl)amino-perfluoroprop-2-yl]-1-methylpropylphosphonate, when added to the culture broth, are able to prevent the proteolytic degradation of recombinant human proinsulin, secreted byB. subtilis AJ73apr73 npr ^– cells.     

Purification of antibiotics produced byLentinus squarrosulus and preliminary characterization of a compound active againstRigidoporus lignosus

Purification and study of the antibiotic substances produced byLentinus squarrosulus have been carried out. The substances, excreted in the culture medium, were extracted withn-butanol. The butanolic extract inhibited growth ofRigidopurus lignosus, the agent of white rot ofHevea brasiliensis, and also ofMucor ramannianus, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, andBacillus subtilis. The antibiotic fractions were purified by silicic acid column chromatography, then by reversed-phase (C18) high performance liquid chromatography (HPLC) followed by preparative adsorption thin-layer chromatography on silica gel. Two purified compounds were obtained: Ls1, which was active againstB. subtilis, and Ls2, which in addition was also active againstR. lignosus, M. ramannianus, and yeasts. Only the Ls2 compound is analyzed in this work. It was characterized by chemical reactions, ultraviolet spectroscopy, infrared spectroscopy, and proton nuclear magnetic resonance spectroscopy, which indicated the hydrophilic character of the molecule and the presence of alcoholic functions as well as a glycosidic moiety. The properties differed from those of already known antibiotics produced by different species ofLentinus.     

Production of saponin in fermentation process of Sanchi (Panax notoginseng) and biotransformation of saponin byBacillus subtilis

An antitumor compound ginsenoside Rh₄ was produced during the fermentation process of Sanchi (Panax notoginseng) byBacillus subtilis. Saponins ginsenosides Rh₁ and Re were transformed byB. subtilis and were produced two main transformed products. the transformed product of ginsenoside Rh₁ was determined to be 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol, and the transformed product of ginsenoside Re had 162 atomic mass units (amu) greater than the substrate. Compard with the substrates, the transformed products had one more glucosyl moiety linked respectively, which indicated that glycosylion reaction occurred in the transformation process.     

The mmgA gene from Bacillus subtilis encodes a degradative acetoacetyl-CoA thiolase

Early in sporulation, the mother cell compartment of Bacillus subtilis transcribes the mother cell metabolic gene (mmg) operon. The gene mmgA was assigned by other workers using sequence homology as an acetyl-CoA acetyltransferase [E.C. 2.3.1.9]. The gene was overexpressed in Escherichia coli, and the protein was purified by Ni²⁺-affinity chromatography. However, the expected MmgA-catalyzed biosynthesis of acetoacetyl-CoA from acetyl-CoA was undetectable by a standard UV assay, HPLC, and mass spectrometry. These methods indicated a preference for the reverse degradative thiolytic reaction, with a k _cat of 80 s⁻¹, and a K _m of 70 and 50 μM for CoA and acetoacetyl-CoA, respectively.