Degradative pathway of p-aminoazobenzene by Bacillus subtilis

Summary p-Aminoazobenzene was degraded by Bacillus subtilis to aniline and p-phenylenediamine by reductive fission of an azo bond. The aniline was then acetylated to acetanilide while the p-phenylenediamine underwent 2 successive acetylations to yield p-aminoacetanilide and p-phenylenediacetanilide. In addition, another pathway was found in Bacillus subtilis in which p-aminoazobenzene was metabolised to p-acetamidoazobenzene.     

Degradation ofp-aminoazobenzene by means of cell-free extracts fromAeromonas hydrophila var. 24B andPseudomonas pseudomallei 13NA

Summary By means of cell-free extracts (supernatant III: crude enzyme, precipitate II: cell debris and precipitate III: microsomal fraction) fromAeromonas hydrophila var. 24B andPseudomonas pseudomallei 13NA,p-aminoazobenzene was effectively degraded. Aniline andp-phenylenediamine were detected in the degradation products. No degradation ofp-aminoazobenzene was found in the supernatant I (extra-cellular enzyme fraction). These results clearly show that thep-aminoazobenzene degradation activity was found in the supernatant III and the precipitate II, III.     

Co-degradation with glucose of four surfactants,CTAB, Triton X-100, SDS and Rhamnolipid,in liquid culture media and compost matrix

Strengthened biodegradation is one of the key means to treat surfactant pollution in environment, and microorganism and surfactant have significant effects on degradation. In this paper, co-degradation of CTAB, Triton X-100, SDS and rhamnolipid with glucose by Pseudomonas aeruginosa, Bacillus subtilis and compost microorganisms in liquid culture media, as well as the degradation of rhamnolipid in compost were investigated. The results showed that CTAB was recalcitrant to degrade by the three microorganisms and it also inhibited microorganisms from utilizing readily degradable carbon source. Non-ionic surfactant Triton X-100 could also hardly be degraded, but it was not toxic to microorganisms and would not inhibit the growth of the microorganisms. Anion surfactant SDS had no toxicity to microorganisms and could be co-degraded as carbon source with glucose. Biosurfactant rhamnolipid was a kind of particular surfactant, which had no toxicity and could be degraded by Bacillus subtilis and compost microorganisms, while it could not be utilized by its producing bacterium Pseudomonas aeruginosa. Among these three bacteria, the compost consortium had the strongest degradation capacity on the tested surfactants due to their microorganisms’ diversity. In compost matrix rhamnolipid could be degraded during composting, but not preferentially utilized.     

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6′‐phosphate phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose‐specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6‐phospho‐α‐glucosidase, which in B. subtilis hydrolyses maltose 6′‐P into glucose and glucose 6‐P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6‐P into glucose 1‐P and glucose 6‐P. However, purified MalP phosphorolyses maltose but not maltose 6′‐P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6′‐P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1‐P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6′‐P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6′‐P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1‐P. MapP therefore connects PTS‐mediated maltose uptake to maltose phosphorylase‐catalysed metabolism. Dephosphorylation assays with a wide variety of phospho‐substrates revealed that MapP preferably dephosphorylates disaccharides containing an O‐α‐glycosyl linkage.     

Degradation of chlorotoluenes and chlorobenzenes by the dual-species biofilm of Comamonas testosteroni strain KT5 and Bacillus subtilis strain DKT

The cooperation of Bacillus subtilis strain DKT and Comamonas testosteroni KT5 was investigated for biofilm development and toluenes and chlorobenzenes degradation. Bacillus subtilis strain DKT and C. testosteroni KT5 were co-cultured in liquid media with toluenes and chlorobenzenes to determine the degradation of these substrates and formation of dual-species biofilm used for the degradation process. Bacillus subtilis strain DKT utilized benzene, mono- and dichlorinated benzenes as carbon and energy sources. The catabolism of chlorobenzenes was via hydroxylation, in which chlorine atoms were replaced by hydroxyl groups to form catechol, followed by ring fission via the ortho-cleavage pathway. The investigation of the dual-species biofilm composed of B. subtilis DKT and C. testosteroni KT5 (a toluene and chlorotoluene-degrading isolate with low biofilm formation) showed that B. subtilis DKT synergistically promoted C. testosteroni KT5 to develop biofilm. The bacterial growth in dual-species biofilm overcame the inhibitory effects caused by monochlorobenzene and 2-chlorotoluene. Moreover, the dual-species biofilm showed effective degradability toward the mixture of these substrates. This study provides knowledge about the commensal relationships in a dual-culture biofilm for designing multispecies biofilms applied for the biodegradation of toxic organic substrates that cannot be metabolized by single-organism biofilms.

     

Formulation and Delivery of the Bacterial Antagonist Bacillus subtilis for Management of Lentil Vascular Wilt Caused by Fusarium oxysporum f. sp. lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log₁₀ CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log₁₀ CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc‐based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.     

The formation of phenol in the degradation ofp-hydroxybenzoic acid byKlebsiella aerogenes (Aerobacter aerogenes)

After growth ofK. aerogenes in chemically defined media consisting of mineral salts andp-hydroxybenzoate with or without glucose, phenol was found in the culture fluid at concentrations inhibiting further growth. Bacteria adapted to mineral salts medium containingp-hydroxybenzoate as sole source of carbon and energy produced small but isolable quantities of 3,4-dihydroxybenzoic acid and catechol and oxidized these substances as rapidly asp-hydroxybenzoate. Bacteria adapted to mineral salts medium containing glucose as sole carbon and energy source did not oxidizep-hydroxybenzoate, 3,4-dihydroxybenzoate or catechol. Bacteria adapted to glucose medium or top-hydroxybenzoate medium did not oxidize or utilize phenol as sole carbon and energy source. A metabolic pathway forp-hydroxybenzoate degradation is proposed and the formation of phenol is attributed to a side reaction.     

Prediction of proton exchange and bacterial growth on various substrates using constraint-based modeling approach

Proton exchange between cells and medium is one of the most important factors affecting culture pH, and hence its prediction is advantageous. In this research, proton exchange flux across the cell membrane was predicted using a genome-scale model. Calculated proton exchange flux was then exploited as a criterion to predict the trends and intensities of pH changes in cultures of Bacillus subtilis containing various C-sources, i.e. glucose, sucrose, glycerol, lactate, and citrate, as well as N-sources, i.e. ammonium chloride, sodium nitrate, urea, and histidine. The results showed that glucose, sucrose, and glycerol lowered culture pH as compared to citrate and lactate, which raised it. With regard to N-sources, the model predicted that ammonium chloride lowered culture pH while other sources raised pH. Furthermore, maximum theoretical biomass yield using the various C&N-sources was calculated, and sensitivity of microbial growth to proton exchange flux was investigated using robustness analysis to identify the effect of pH on growth of B. subtilis using different substrates. Finally, the effect of ammonium nitrate, a widely used nitrogen source, on growth of B. subtilis was studied. Experimental data obtained by cultivation of B. subtilis DSM 3256 on mineral salt media containing various C&N-sources were used to confirm model predictions. Model predictions were in good agreement with the experimental results for all of the examined C-sources as well as ammonium chloride and sodium nitrate as N-sources. However, the predictions for the N-sources urea and histidine showed deviations, possibly because these two compounds serve as both C&N-sources.     

Biodegradation of p-nitrophenol by methyl parathion-degrading Ochrobactrum sp. B2

Ochrobactrum sp. B2, a methyl parathion-degrading bacterium, was proved to be capable of using p-nitrophenol (PNP) as carbon and energy source. The effect of factors, such as temperature, pH value, and nutrition, on the growth of Ochrobactrum sp. B2 and its ability to degrade p-nitrophenol (PNP) at a higher concentration (100 mg l⁻¹) was investigated in this study.The greatest growth of B2 was observed at a temperature of 30 °C and alkaline pH (pH 9–10). pH condition was proved to be a crucial factor affecting PNP degradation. Enhanced growth of B2 or PNP degradation was consistent with the increase of pH in the minimal medium, and acidic pH (6.0) did not support PNP degradation. Addition of glucose (0.05%, 0.1%) decreased the rate of PNP degradation even if increased cell growth occurred. Addition of supplemental inorganic nitrogen (ammonium chloride or ammonium sulphate) inhibited PNP degradation, whereas organic nitrogen (peptone, yeast extract, urea) accelerated degradation.     

Genome-wide mRNA profiling in glucose starved <Emphasis Type="Italic">Bacillus subtilis</Emphasis> cells

In this study global changes in gene expression were monitored in Bacillus subtilis cells entering stationary growth phase owing to starvation for glucose. Gene expression was analysed in growing and starving cells at different time points by full-genome mRNA profiling using DNA macroarrays. During the transition to stationary phase we observed extensive reprogramming of gene expression, with ~1000 genes being strongly repressed and ~900 strongly up-regulated in a time-dependent manner. The genes involved in the response to glucose starvation can be assigned to two main classes: (i) general stress/starvation genes which respond to various stress or starvation stimuli, and (ii) genes that respond specifically to starvation for glucose. The first class includes members of the ^B-dependent general stress regulon, as well as 90 vegetative genes, which are strongly down regulated in the course of the stringent response. Among the genes in the second class, we observed a decrease in the expression of genes encoding proteins required for glucose uptake, glycolysis and the tricarboxylic acid cycle. Conversely, many carbohydrate utilisation systems that depend on phosphotransferase systems (PTS) or ABC transporters were activated. The expression of genes required for utilisation or generation of acetate indicates that acetate constitutes an important energy source for B. subtilis during periods of glucose starvation. Finally, genome wide mRNA profiling data can be used to predict new metabolic pathways in B. subtilis. Thus, our data suggest that glucose-starved cells are able to degrade branched-chain fatty acids to pyruvate and succinate via propionyl-CoA using the methylcitrate pathway. This pathway appears to link lipid degradation to gluconeogenesis in glucose-starved cells.This revised version was published online in May 2005 with corrections to the list of authors     

Properties of an N,N-dimethyl-p-aminoazobenzene Oxide Reductase Purified from Rat Liver Cytosol

Homogenates of all rat tissues examined, except brain, catalyze reduction of N,N-dimethyl-p-aminoazobenzene N-oxide (DMAB N-oxide) to N,N-dimethyl-p-aminoazobenzene by NADPH. Liver is the most active, and about one third of the homogenate activity of this tissue is recovered in the cytosol fraction. The purified cytosol enzyme has the properties of a tetrameric protein (Mr 370,000) consisting of identical subunits free from chromophores that absorb in the visible spectrum and from metals or other detectable prosthetic groups. The purified reductase is also free from NADPH oxidase and from cytochrome c or azo reductase activities. The enzyme is quite specific for NADPH as reductant and DMAB N-oxide as the electron acceptor. Reduction of other N,N-dimethyl-arylamine or alkylamine oxides as well as N-methylheterocyclicamine oxides could not be detected. Analysis of kinetic data indicate that, at saturating concentrations of the other substrate, 21 μM NADPH and 700 μM DMAB N-oxide are required for half maximal velocity. At infinite concentrations of both substrates the turnover is 150 min^?1 at 37 °C.     

Improvement of culture conditions to overproduce β-galactosidase from Escherichia coli in Bacillus subtilis

The effect of some culture variables in the production of β-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), β-galactosidase production was partially growth-associated, as 40%–60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, β-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific β-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-l fermenter scale, a threefold increase in volumetric β-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration. Received: 18 April 1996 / Received revision: 27 August 1996 / Accepted: 6 September 1996     

Resistance to serine in Bacillus subtilis: identification of the serine transporter YbeC and of a metabolic network that links serine and threonine metabolism

The Gram-positive bacterium Bacillus subtilis uses serine not only as a building block for proteins but also as an important precursor in many anabolic reactions. Moreover, a lack of serine results in the initiation of biofilm formation. However, excess serine inhibits the growth of B. subtilis. To unravel the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine concentrations by three targeted and non-targeted genome-wide screens. All screens as well as genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. The screen for serine-resistant mutants also identified mutations that result in increased serine degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine toxicity results from interference with threonine biosynthesis.     

Effects of organic compounds on the degradation ofp-nitrophenol in lake and industrial wastewater by inoculated bacteria

Many microorganisms fail to degrade pollutants when introduced in different natural environments. This is a problem in selecting inocula for bioremediation of polluted sites. Thus, a study was conducted to determine the success of four inoculants to degradep-nitrophenol (PNP) in lake and industrial wastewater and the effects of organic compounds on the degradation of high and low concentrations of PNP in these environments.Corynebacterium strain Z4 when inoculated into the lake and wastewater samples containing 20 µg/ml of PNP degraded 90% of PNP in one day. Addition of 100 µg/ml of glucose as a second substrate did not enhance the degradation of PNP and the bacterium utilized the two substrates simultaneously. Glucose used at the same concentration (100 µg/ml), inhibited degradation of 20 µg of PNP in wastewater byPseudomonas strain MS. However, glucose increased the extent of degradation of PNP byPseudomonas strain GR. Phenol also enhanced the degradation of PNP in wastewater byPseudomonas strain GR, but had no effect on the degradation of PNP byCorynebacterium strain Z4.Addition of 100 µg/ml of glucose as a second substrate into the lake water samples containing low concentration of PNP (26 ng/ml) enhanced the degradation of PNP and the growth ofCorynebacterium strain Z4. In the presence of glucose, it grew from 2×10⁴ to 4×10⁴ cells/ml in 3 days and degraded 70% of PNP as compared to samples without glucose in which the bacterium declined in cell number from 2×10⁴ to 8×10³ cells/ml and degraded only 30% PNP. The results suggest that in inoculation to enhance biodegradation, depending on the inoculant, second organic substrate many play an important role in controlling the rate and extent of biodegradation of organic compounds.Abbreviations PNP p-nitrophenol     

Cloning and expression of the PHA synthase genes phaC1 and phaC1AB into Bacillus subtilis

Summary Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1 and pBE2C1AB. The two plasmids were inserted into Bacillus subtilis DB104 and generated Bacillus subtilis/pBE2C1 and Bacillus subtilis/pBE2C1AB. The two recombinant strains were subjected to fermentation and showed PHA accumulation, the first reported example of medium-chain-length-PHA production in Bacillus subtilis. GC analysis identified the compound produced by Bacillus subtilis/pBE2C1 was a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer while that produced by Bacillus subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer. The results also showed that the recombinant B. subtilis could utilize the malt waste in the medium as a carbon source better than that of glucose and thus could substantially lower the cost of production of PHA.     

Biosynthesis of poly(γ-glutamic acid) from l-glutamic acid,citric acid,and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. When l-glutamic acid, citric acid, and ammonium sulfate were used as carbon and nitrogen sources, a large amount of PGA without a by-product such as a polysaccharide was produced. The time courses of cell growth, PGA, glutamic acid, and citric acid concentrations during cultivation were investigated. It was found that glutamic acid added to the medium was apparently not assimilated. It can be presumed that the glutamic acid unit in PGA is mainly produced from citric acid and ammonium sulfate. The PGA productivity was investigated at various concentrations of ammonium sulfate in the media, which caused the depression of cell growth, high productivity of PGA, and the production of PGA with a high relative molecular mass. The yield of PGA determined by gel permeation chromatography (GPC) reached approximately 20 g/l. This yield was the highest value for PGA production by B. subtilis IFO3335, suggesting that B. subtilis IFO3335 was a bacterium that could produce PGA effectively. Time courses relative to the molecular mass of PGA at various concentrations of ammonium sulfate were investigated. It was suggested that B. subtilis IFO3335 excreted a PGA degradation enzyme with the progress of cultivation and that PGA was degraded by this enzyme. Correspondence to: M. Kunioka     

Growth kinetics under adenine-limiting conditions of Bacillus subtilis KYA 741, an adenine-requiring strain

The growth kinetics of Bacillus subtilis KYA 741, an adenine-requiring strain, was investigated under adenine-limiting conditions. The concentration of adenine (the limiting substrate for cell growth) in the culture filtrate remained constant during the stationary phase. In this phase, DNA turnover was active and the DNA content per cell was constant throughout the cultivation period. When cells were transferred to medium without adenine, the cell concentration began to decrease immediately and then reached a constant level due to the supply of adenine from lysing to growing cells. The rates of degradation of cells and DNA were both found to be 0.2 hr^?1. An equation for cell growth in this pseudostationary phase was obtained by combining Contois' equation, in which the apparent saturation constant was a function of the cell concentration, with a term for cell degradation. This equation satisfactorily expressed the feature of cell growth and adenine consumption by B. subtilis KYA 741 under adenine-limiting conditions.     

Features of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> IMB B-7023 and its streptomycin-resistant strain

Features of phosphate-mobilizing bacteria Bacillus subtilis IMB B-7023 and its streptomycin-resistant strain were investigated. While cultivated in medium with glucose and glycerophosphate, the growth rate of the antibiotic-marked strain was approximately similar to this parameter for Bacillus subtilis IMB B-7023 but cell sizes were 1.3-fold less. Both strains significantly stimulated the germinating of plant seeds, attached to their roots, and insignificantly differed in antagonistic activity toward phytopathogens and quantitative content of cell fatty acids and phosphatase activity. Streptomycin-resistant strain may be used for monitoring of Bacillus subtilis introduced to agroecosystem.     

Kinetics of reactive azo-dye decolorization by Pseudomonas luteola in a biological activated carbon process

A laboratory-scale biological activated carbon (BAC) process was conducted to treat a reactive azo-dye (reactive red 22) by Pseudomonas luteola and the kinetics of azo-dye decolorization was investigated. The BAC-reactor removed 89% of reactive red 22 while P. luteola biofilm and suspended P. luteola reached a maximum growth rate at a steady-state condition. The azo-dye effluent from BAC-reactor met a discharge standard required by Taiwan government. The kinetic BAC-model, based on fundamental mechanisms, including surface diffusion, liquid-film mass transfer, Monod kinetics, growth of biofilm and suspended cells as well as shear loss of biofilm, was developed to describe the performance of biofilm attached on activated carbon in the azo-dye treatment process. The kinetic BAC-model predictions and experimental results for simultaneous adsorption and biodegradation of azo-dye contaminants were compared. It is shown that the fundamental mechanisms of BAC-process for azo-dye decolorization are not the simple addition but the synergetic combination of carbon adsorption and biodegradation of P. luteola strain. The major aspects of such synergism are the bioregeneration of the adsorbent and the reduction of the toxic effect of azo-dye contaminants in textile wastewater on P. luteola strain. The kinetic BAC-model not only provides insights into underlying mechanisms of adsorption and biodegradation but also can be used as a powerful tool to assist the design of a pilot-scale or full-scale BAC-process to treat azo-dye contaminants by P. luteola cells in textile wastewater.