Purification and physicochemical properties of malate dehydrogenase from bacteria of the genus Beggiatoa

Homogeneous malate dehydrogenase (MDH) with a specific activity of 20-24 units per mg protein was purified from the sulfur bacterium Beggiatoa leptomitiformis strain D-402 grown organotrophically and lithotrophically and from the organotrophic bacterium Beggiatoa alba. MDHs from the B. leptomitiformis strain D-402 grown under organotrophic conditions and from B. alba are homodimers with the subunit molecular weight of 40 kD. Tetrameric MDH is formed in B. leptomitiformis strain D-402 grown under lithotrophic conditions. The dimeric and tetrameric forms of MDH from B. leptomitiformis D-402 display some differences in kinetic properties.     

Xylitol Production by a Corynebacterium Species

The washed cells of a gluconate-utilizing Corynebacterium strain grown in a gluconate- xylose medium produced xylitol from D-xylose in the presence of gluconate. The amount of xylitol was progressively increased with increasing gluconate concentration.

An extract of cells grown in the gluconate-xylose medium showed NADPH-dependent D-xylose reductase activity and NADP-dependent 6-phosphogluconate dehydrogenase activity.

These enzymes in the cell-free extract were purified by Sephadex G–100 gel filtration.

The reduction of D-xylose to xylitol was demonstrated by the coupling the D-xylose reductase activity to the 6-phosphogluconate dehydrogenase activity with NADP as a cofactor using the cell-free extract and the fractionated enzymes.     

A New Intact Cell System for Determination of the Rate of Vitamin B6 Biosynthesis

To establish an intact cell system to determine the rate of vitamin B6 (B6) biosynthesis, conditions suitable for the reaction were examined. Cells of Escherichia coli B WG2, a pdxH mutant, were used for the reaction. Pyridoxal-starved cells of a stationary phase culture showed a negligible change of cell mass and a constant increase of B6 in the reaction. The rate of B6 biosynthesis was reliably determined with a 2-hr reaction initiated with cells of 0.10 to 0.15mg per ml. The influence of endogenous metabolites was completely abolished by the use of such a low concentration of the starved cells.

When glucose was used as a sole carbon substrate in the reaction system, the maximum rate of B6 biosynthesis was determined to be 0.5 to 0.6 nmol per mg cells per hr. The maximum rate increased to 0.8 to 0.9 nmol per mg cells per hr on supplementation of glycolaldehyde to the reaction mixture. The result led to the conclusion that the rate-limiting step in the reaction sequence of B6 biosynthesis lies the process of incorporation of the 5–5′ carbon fragment of the B6 molecule. This was also confirmed by a growth experiment with the wild type strain.     

Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of investigation of alicyclic hydrocarbon-utilizing microorganisms, five strains of ethylcyclohexane-utilizing bacteria were isolated from soil samples.

Among those bacteria, the strain S6B1 that was identified as Alcaligenes faecalis, showed the best growth in shaking culture.

The strain S6B1 was found to produce 4-ethylcyclohexanol from ethylcyclohexane.

This substance separated from culture broth was purified and identified to be trans-4-ethylcyclohexanol by the use of NMR.     

Tetramethylammonium:coenzyme M methyltransferase system from Methanococcoides sp.

A methanogen (strain NaT1) that belongs to the family of Methanosarcinaceae and that can grow on tetramethylammonium as the sole energy source has recently been isolated. We report here that cell extracts of the archaeon catalyze the formation of methyl-coenzyme M from coenzyme M and tetramethylammonium. The activity was dependent on the presence of Ti(III) citrate and ATP, and was rapidly lost under oxic conditions. Anoxic chromatography on DEAE-Sepharose revealed that two fractions, fractions 3 and 4, were required for activity. A 50-kDa protein that together with fraction 3 catalyzed methyl-coenzyme M formation from tetramethylammonium and coenzyme M was purified from fraction 4. From fraction 3, a 22-kDa corrinoid protein and a 40-kDa protein exhibiting methylcobalamin:coenzyme M methyltransferase (MT2) activity were purified. The N-terminal amino acid sequences of these purified proteins were determined. The 40-kDa protein showed sequence similarity to MT2 isoenzymes from Methanosarcina barkeri. Cell extract of strain NaT1 grown on trimethylamine rather than on tetramethylammonium did not exhibit tetramethylammonium:coenzyme M methyltransferase activity. The strain was identified as belonging to the genus of Methanococcoides, its closest relative being Methanococcoides methylutens. Received: 7 April 1998 / Accepted: 26 June 1998     

Effect of nitrogen starvation on the level of adenosine 3′,5′-monophosphate in Anabaena variabilis

Low levels of adenosine 3′,5′-monophosphate (cyclic AMP) were detected in the cyanobacterium Anabaena variabilis using a protein binding assay and two radioisotopic labelling methods. The basal concentration of intracellular cyclic AMP ranged from 0.27 pmol/mg protein in A. variabilis Kutz grown under heterotrophic conditions to 1.0–2.7 pmol/mg protein in A. variabilis strain 377 grown autotrophically. Extracellular cyclic AMP was found to comprise as much as 90% of the total cyclic AMP in rapidly growing cultures. When A. variabilis strain 377 was starved of nitrogen, a 3–4-fold increase in intracellular cyclic AMP was observed during the 24 h period coincident with early heterocyst development.     

S-adenosylmethionine and morphogenesis inMucor racemosus

The intracellular concentration of S-adenosylmethionine (SAM) and the specific activity of S-adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, EC 2.5.1.6) were examined in wild-typeMucor racemosus, as well as a morphological mutant termedcoy, under conditions designed to prevent the morphogenesis of yeasts to hyphae. When the mutant was grown in a defined medium supplemented with methionine and induced to shift by exposure to air, there was an increase in intracellular SAM analogous to that previously reported with the wild type. However, when thecoy mutant was grown in the absence of methionine, the intracellular concentration decreased dramatically and the mutant failed to undergo the yeast to hypha transition. An inhibitor of SAM synthetase activity, cycloserine, was used to lower the intracellular concentration of SAM in the wild-type organisms. Under these conditions, wild-typeM. racemosus failed to undergo the transition from yeasts to hyphae when exposed to air.     

Identification of Fruity Aroma-Producing Compounds from <Emphasis Type="Italic">Chryseobacterium</Emphasis> sp. Isolated from the Western Ghats,India

A fruity aroma-producing strain WG4 was isolated from a water sample collected from the Western Ghats, India. The 16S rRNA gene sequence analysis of strain WG4 indicated that Chryseobacterium indologenes, a member of the family ‘Flavobacteriaceae’ is the closest related species with a pair-wise sequence similarity of 98.6%. Strain WG4 produces a fruity aroma when grown on nutrient or trypticase soy agar plates. The fruity aroma is more when the strain WG4 is grown on agar plates compared to their growth in broth. The aromatic compounds produced by the strain WG4 were identified as ester compounds and were confirmed as ethyl-2-methylbutyrate and ethyl-3-methylbutyrate based on Gas Chromatography–Mass Spectrometry (GC–MS) analysis and using standard reference compounds. Even after repeated subcultures strain WG4 produced the same aroma in high intensity. Thus, strain WG4 could serve as a source for the production of these flavour compounds.     

Functional Activity of Exoglycans from Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Resistant Mutant M-71 during the Formation of Legume–Rhizobia Symbiosis against a High-Nitrogen Background

The functional activity of the exoglycan complex (EGC) polysaccharides from Rhizobium leguminosarum bv. viciae 250a and its nitrogen-resistant mutant M-71, capable of inducing the formation of nitrogen-fixing nodules on pea roots against a high-nitrogen background (4.8 mM NO₃ ^–), was studied in vegetation tests. For this purpose, the bacterial inoculum washed free of its own exoglycans was supplemented with EGC of the same or another strain grown in the presence of 6 or 20 mM nitrate. The best symbiotic characteristics (nodule number and nitrogenase activity, mass of the roots and aerial parts of plants) were recorded when the inoculum cells and exoglycans were obtained from strain M-71 grown in the presence of 20 mM nitrate. When the plants were inoculated with the cells (grown at 6 mM nitrate) + EGC (obtained at 6 mM nitrate) of this strain, the nodulation characteristics and the effectiveness of symbiosis decreased 1.5- to 2-fold. Partial recovery of the symbiotic potential of strain M-71 was observed when EGC (obtained at 20 mM nitrate) was substituted for its exoglycans (obtained at 6 mM nitrate). In the presence of exoglycans of the parent strain 250a (obtained at 6 or 20 mM nitrate), the mutant formed a substantially lesser number of nodules with a very low nitrogen-fixing activity. In turn, the mutant exoglycans synthesized in medium with either high or low nitrate nitrogen concentration did not recover the fix⁺ phenotype of strain 250a, capable of forming symbiosis with pea plants only against a low-nitrogen background. In study of the relative content of high-molecular-weight exopolysaccharide components and low-molecular-weight glycans in the exoglycan complex, it was established that, in strain 250a (grown at 6 and 20 mM nitrate), as well as in its mutant M-71 (grown at 6 mM nitrate), exopolysaccharides prevailed, accounting for 72–75% of the sum of both types of glycopolymers, while low-molecular-weight glycans accounted for 25–28%. In contrast, in the EGC of strain M-71 obtained at 20 mM nitrate, which was the most active inducer of the formation of the symbiotrophic system by strain M-71 in the presence of a high mineral nitrogen concentration, low-molecular-weight glycans were the main component, accounting for 61% of total glycopolymers, while the polysaccharide content was 39%. Low-molecular-weight exoglycans are supposed to be involved in maintaining the physiological activity and the symbiotic status of rhizobia under unfavorable environmental conditions.     

Evaluation of the Biotechnological Potential of Pure and Mixture of Indigenous Iron-Oxidizing Bacteria to Dissolve Trace Metals from Cu Bearing Ore

Abstract

This study aimed to investigate the ability of pure and consortia of indigenous iron-oxidizing bacteria to enhance the dissolution of trace metals from Cu and Zn-bearing ore. Three bacterial strains Acidithiobacillus ferrooxidans strain WG101, Leptospirillum ferriphilum strain WG102, Leptospirillum ferrooxidans strain WG103 isolated from Baiyin copper mine, China were used in this study. The biotechnological potential of these indigenous isolates was evaluated both in pure and in consortia to extract cobalt, chromium, and lead from the copper and zinc bearing ore. The sulfur and iron-oxidizing bacterial isolate Acidithiobacillus ferrooxidans strain WG101 exhibited efficient dissolution compared to sole iron-oxidizing Leptospirillum ferriphilum strain WG102, and Leptospirillum ferrooxidans strain WG103. Initial medium pH, pulp density, and temperature were studied as influential parameters in bioleaching carried out by bacterial consortia. The achieved optimum conditions were; initial pH of 1.5, 10% of pulp density, and temperature 30?°C with 68.7?±?3.9% cobalt, 56.6?±?3.9% chromium, and 36?±?3.7% lead recovery. Analytical study of oxidation-reduction potential and pH fluctuation were observed during this whole process that shows the metal dissolution efficiency of bacterial consortia. Alterations in spectral bands of processed residues were reported through FTIR analysis compared with control ore sample. Mössbauer spectroscopy analysis showed the influence of bacterial consortia on iron speciation in bioleached samples. The findings confirm that the indigenous acidophilic iron-oxidizing bacterial strains are highly effective in the dissolution of trace elements present in ore samples. This study not only supports the notion that indigenous bacterial strains are highly effectual in metal dissolution but provides the basic vital conditions to upscale the bioleaching technique for metals dissolution.     

Genetic and physiological analysis of the CO2-concentrating system of Chlamydomonas reinhardii

When grown photoautotrophically at air levels of CO₂, Chlamydomonas reinhardii possesses a system involving active transport of inorganic carbon which increases the intracellular CO₂ concentration considerably above ambient, thereby stimulating photosynthetic CO₂ assimilation. In previous investigations, two mutant strains of this unicellular green alga deficient in some component of this CO₂-concentrating system were recovered as strains requiring high levels of CO₂ to support photoautotrophic growth. One of the mutants, ca-1-12-1C, is a leaky (nonstringent) CO₂-requiring strain deficient in carbonic anhydrase (EC 4.2.1.1) activity, while the other, pmp-1-16-5K, is a stringent CO₂-requiring strain deficient in inorganic carbon transport. In the present study a double mutant (ca pmp) was constructed to investigate the physiological and biochemical interaction of the two mutations. The two mutations are unlinked and inherited in a Mendelian fashion. The double mutant was found to have a leaky CO₂-requiring phenotype, indicating that the mutation ca-1 overcomes the stringent CO₂-requirement conferred by the mutation pmp-1. Several physiological characteristics of the double mutant were very similar to the carbonic-anhydrase-deficient mutant, including high CO₂ compensation concentration, photosynthetic CO₂ response curve, and deficiency of carbonic-anhydrase activity. However, the labeling pattern of metabolites during photosynthesis in ¹⁴CO₂ was more like that of the bicarbonatetransport-deficient mutant, and accumulation of internal inorganic carbon was intermediate between that of the two original mutants. These data indicate a previously unsuspected complexity in the Chlamydomonas CO₂-concentrating system.     

Impact of PGPR inoculation on growth and antioxidant status of wheat under saline conditions

Two plant growth‐promoting rhizobacterial (PGPR) strains, Bacillus subtilis SU47 and Arthrobacter sp. SU18, were found to tolerate 8% NaCl. Wheat co‐inoculated with these two PGPR strains, and grown under different salinity regimes (2–6 dS m^?1), showed an increase in dry biomass, total soluble sugars and proline content. Wheat sodium content was reduced under co‐inoculated conditions but not after single inoculation with either strain or in the control. The activity of antioxidant enzymes in wheat leaves decreased under salinity stress after PGPR co‐inoculation, suggesting these PGPR species could be used for amelioration of stress in wheat plants. Activity of three antioxidant enzymes in wheat grown with both PGPR strains was reduced, most notably that of catalase activity at a salinity of 6 dS m^?1, when compared with the control. The results indicate that co‐inoculation with B. subtilis and Arthrobacter sp. could alleviate the adverse effects of soil salinity on wheat growth.     

Effects of temperature and relative humidity on Aflatoxin B1 reduction in corn grains and antagonistic activities against Aflatoxin-producing Aspergillus flavus by a volatile organic compound-producing yeast,Kwoniella heveanensis DMKU-CE82

As shown in our previous study, Kwoniella heveanensis DMKU-CE82, a volatile organic compound (VOC)-producing yeast, demonstrated promising antagonistic activity against aflatoxin-producing strain of Aspergillus flavus. This yeast’s volatile organic compounds (VOCs) could reduce Aflatoxin B1 (AFB1) in corn grains. In the current study, we evaluated the effect of temperatures and relative humidity on AFB1 reduction during grain storage when co-incubated with this VOC-producing yeast. The VOCs produced by K. heveanensis DMKU-CE82 could promote reduction of AFB1 to less than 20 part per billion (ppb) in the fungal contaminated corn grains under most storage conditions at 35 °C. The major VOCs produced by 2-, 4-, and 6-day-old yeast cultures were closely matched to 3-methyl-1-butanol, 2-methyl-1-butanol, hydrazine-1-1-dimethyl, and butanoic acid-3-methyl. In addition, this yeast strain had the ability to produce β-1,3-glucanase, amylase, cellulase, chitinase, siderophores, and biofilms. Scanning electron microscopy also confirmed the antagonistic activity of K. heveanensis DMKU-CE82 as it caused structural damage to conidia and inhibited the development of mycelia and conidiophores in both direct fungal–yeast interaction and the VOC method in corn grains. These results demonstrated that this yeast strain could be a promising biocontrol agent against aflatoxin-producing fungi in agricultural products.

     

Influence of cultural conditions on growth and lipolytic activity inNocardia asteroides

The growth and the production of extracellular and intracellular lipases were measured fromNocardia asteroides grown under different cultural conditions. Maximal growth and intracellular and extracellular activities were observed at 3 d after inoculation. Among the tested media, synthetic medium induced maximal growth and extracellular activity, whereas tryptic soy broth induced the maximal intracellular lipase activity. The best carbon and nitrogen sources for growth and lipolytic activity were glucose, fructose, glutamate and nitrate, respectively. The optimal C∶N ratio for growth was in the range of 1∶4 to 2∶3 and for lipase activity the range was 2∶3 to 3∶2. Anything above or below this range was detrimental to the organism and its enzyme activity. Under the conditions of this study,N. asteroides grew best and had the highest lipase activity when compared toN. brasiliensis andN. caviae.     

Studies on the Utilization of Hydrocarbons by Microorganisms

During the course of an investigation of the microbial assimilation of aromatic hydrocarbons, several strains were found to produce a large amount of cumic acid from p-cymene.

Five strains, S449B1, B2, B3, B4 and B6, were isolated from soil with the aromatic hydrocarbon substrates. They all assimilated both p-cymene and cumene. The strain S449B3 grew also on p-xylene, and S449B6 on p-xylene, toluene, and ethylbenzene.

They were all shown to be capable of producing an ultraviolet-absorbing substance from p-cymene. This substance was isolated in crystalline form and identified as cumic acid by infrared absorption spectrum and other observations.

The superior strain, S449B6, produced the acid as much as 1000 mg/1 in shaking culture at 30°C after 24 hours. The yields were increased up to approximately 1700 mg/1 after further investigations. Addition of calcium carbonate and considerable agitation were favorable conditions for the acid production.

The taxonomical studies of these strains were carried out, and they were all identified as closely resembling Pseudomonas desmolytica.     

EFFECT OF SPECTRAL QUALITY ON GROWTH AND PIGMENTATION OF PICOCYANOBACTERIA1

The influence of spectral quality on growth and pigmentation was compared among five strains of marine and freshwater picocyanobacteria grown under the same photon flux density (28 μE · m^?2·s^?1). Growth and phycoerythrin (PE) concentration per unit carbon increased when marine Synechococcus WH7803 was grown under green light as compared to red light, but no change in phycocyanin concentration occurred. Marine Synechococcus strain 48B66 also showed greater levels of PE when grown under green light than under red light, but no concomitant growth increase occurred. Both strains thus exhibited Group II chromatic adaptation. Additionally, strain 48B66 increased the relative level of phycourobilin compared to phycoerythrobilin when grown under red light. In contrast, both marine and freshwater Synechococcus strains containing no PE showed decreased growth under green light. Chlorophyll a concentrations were greatest or among the greatest in all strains grown under green light. These results suggest that light quality, through its effects on growth rate, may be an important factor controlling the distribution and abundance of the various pigment types of Synechococcus.     

Oligo(cis-1,4-isoprene) aldehyde-oxidizing dehydrogenases of the rubber-degrading bacterium Gordonia polyisoprenivorans VH2

The actinomycete Gordonia polyisoprenivorans strain VH2 is well-known for its ability to efficiently degrade and catabolize natural rubber [poly(cis-1,4-isoprene)]. Recently, a pathway for the catabolism of rubber by strain VH2 was postulated based on genomic data and the analysis of mutants (Hiessl et al. in Appl Environ Microbiol 78:2874–2887, 2012). To further elucidate the degradation pathway of poly(cis-1,4-isoprene), 2-dimensional-polyacrylamide gel electrophoresis was performed. The analysis of the identified protein spots by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry confirmed the postulated intracellular pathway suggesting a degradation of rubber via β-oxidation. In addition, other valuable information on rubber catabolism of G. polyisoprenivorans strain VH2 (e.g. oxidative stress response) was provided. Identified proteins, which were more abundant in cells grown with rubber than in cells grown with propionate, implied a putative long-chain acyl-CoA-dehydrogenase, a 3-ketoacyl-CoA-thiolase, and an aldehyde dehydrogenase. The amino acid sequence of the latter showed a high similarity towards geranial dehydrogenases. The expression of the corresponding gene was upregulated > 10-fold under poly(cis-1,4-isoprene)-degrading conditions. The putative geranial dehydrogenase and a homolog were purified and used for enzyme assays. Deletion mutants for five aldehyde dehydrogenases were generated, and growth with poly(cis-1,4-isoprene) was investigated. While none of the mutants had an altered phenotype regarding growth with poly(cis-1,4-isoprene) as sole carbon and energy source, purified aldehyde dehydrogenases were able to catalyze the oxidation of oligoisoprene aldehydes indicating an involvement in rubber degradation.

     

Bioleaching of copper- and zinc-bearing ore using consortia of indigenous iron-oxidizing bacteria

Indigenous iron-oxidizing bacteria were isolated on modified selective 9KFe²⁺ medium from Baiyin copper mine stope, China. Three distinct acidophilic bacteria were isolated and identified by analyzing the sequences of 16S rRNA gene. Based on published sequences of 16S rRNA gene in the GenBank, a phylogenetic tree was constructed. The sequence of isolate WG101 showed 99% homology with Acidithiobacillus ferrooxidans strain AS2. Isolate WG102 exhibited 98% similarity with Leptospirillum ferriphilum strain YSK. Similarly, isolate WG103 showed 98% similarity with Leptospirillum ferrooxidans strain L15. Furthermore, the biotechnological potential of these isolates in consortia form was evaluated to recover copper and zinc from their ore. Under optimized conditions, 77.68 ± 3.55% of copper and 70.58 ± 3.77% of zinc were dissolved. During the bioleaching process, analytical study of pH and oxidation–reduction potential fluctuations were monitored that reflected efficient activity of the bacterial consortia. The FTIR analysis confirmed the variation in bands after treatment with consortia. The impact of consortia on iron speciation within bioleached ore was analyzed using Mössbauer spectroscopy and clear changes in iron speciation was reported. The use of indigenous bacterial consortia is more efficient compared to pure inoculum. This study provided the basic essential conditions for further upscaling bioleaching application for metal extraction.

     

Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04?mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14?mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778?U/L at 34?hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164?U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.     

Biochemical and molecular characterization of a tetrachloroethene dechlorinating Desulfitobacterium sp. strain Y51: a review

A strict anaerobic bacterium, Desulfitobacterium sp. strain Y51, is capable of very efficiently dechlorinating tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) at concentrations as high as 960 μM and as low as 0.06 μM. Dechlorination was highly susceptible to air oxidation and to potential alternative electron acceptors, such as nitrite, nitrate or sulfite. The PCE reductive dehalogenase (encoded by the pceA gene and abbreviated as PceA dehalogenase) of strain Y51 was purified and characterized. The purified enzyme catalyzed the reductive dechlorination of PCE to cis-DCE at a specific activity of 113.6 nmol min⁻¹  mg protein⁻¹ . The apparent K _m values for PCE and TCE were 105.7 and 535.3 μM, respectively. In addition to PCE and TCE, the enzyme exhibited dechlorination activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane and 1,1,2,2-tetrachloroethane. An 8.4-kb DNA fragment cloned from the Y51 genome revealed eight open reading frames, including the pceAB genes. Immunoblot analysis revealed that PceA dehalogenase is localized in the periplasm of Y51 cells. Production of PceA dehalogenase was induced upon addition of TCE. Significant growth inhibition of strain Y51 was observed in the presence of cis-DCE, More interestingly, the pce gene cluster was deleted with high frequency when the cells were grown with cis-DCE.