The high-level expression of the xylanase GH11 gene from Aspergillus niger IA-001 called xynB was successfully completed in Pichia pastoris. The xynB gene encoding a mature xylanase of 225 amino acid was subcloned into the pPICZαA vector and was transformed into P. pastoris X-33 under the control of the alcohol oxidase I (AOX1) promoter. The xynB gene was ligated with a sequence encoding modified α-factor signal peptide (pPICZαmA) and the recombinant xylanase activity, which was measured 1280 U ml(-1) , was 1.5-fold higher than when it was inserted into pPICZαA and was 19.39-fold greater than the native xylanase in the original strain. In a 10 L fermenter, the recombinant xylanase activity measured 10,035 U ml(-1) after 114 h. The SDS-PAGE analysis revealed that the purified xynB protein migrated as a single band with an apparent molecular weight of 24 kDa. The specific activity, using beechwood xylan as a substrate, was 1916 U mg(-1) . The xylanase activity was optimal at pH 5.0 and at 50 °C. In addition, the xynB was active over a pH range of 2.2 to 10.0. The apparent Km and Vmax values were 4.429 mg ml(-1) and 1429 U mg(-1) , respectively.
Degrading hydrocarbon by Streptococcus sp. BT-003 as a kind of microbe for oil recovery was analyzed in this paper. The Streptococcus sp. BT-003 showed that it could use crude oil as the sole source of carbon and produce organic acid, bio-gas and polysaccharide which were propitious to emulsify and reduce the viscosity of crude oil. After cultivating 8-14 h, the viscosity of crude oil reduced from 8000-15000 mPa . s to 50-250 mPa . s. The content of organic acid increased 8-15 times, and carbon dioxide and polysaccharide reached 55 ml/l and 8 g/l respectively. Paraffin and resin reduced by 60-95%, and light components increased obviously. Fluid rheology was better than before, the interfacial tension between the crude oil and water were reduced effectively.
In this study, diisodecyl phthalate (DIDP) was efficiently degraded by Bacillus sp. SB-007. The optimal conditions for DIDP (100 mg l(-1)) degradation by Bacillus sp. SB-007 in a mineral salts medium were found to be pH 7.0 at 30 degrees C, stirring at 200 rpm. The specific rate of DIDP degradation was found to be concentration dependent with a maximum of 4.87 mg DIDP l(-1) h(-1). DIDP was transformed rapidly by Bacillus sp. SB-007 with the formation of monoisodecyl phthalate and phthalic acid, which subsequently degraded further. These results highlight the potential of this bacterium for removing DIDP contaminated waste in the environment.
Extracellular lipase production by Aspergillus sp. (RBD-01) was monitored by modulating pH of the growth medium, ambient temperature for growth, source of nitrogen and percentage of carbon (virgin cottonseed oil). This strain was observed to be viable and produces lipase even up to 50% oil as a main carbon source. Maximum lipase activity of 21.8 U/ml was obtained with 50% (v/v) oil acting as the main carbon source and peptone (0.5% w/v) as nitrogen source. The optimum pH and temperature for enzymatic activity were observed to be 7.5 and 35 degrees C, respectively. The observations are of significance due to limited reports on use of 50% of oil as the main carbon source while obtaining significant lipase activity of 21.8 U/ml.
Escherichia coli 0164 is the most prevalent serotype among the enteroinvasive E. coli in Bulgaria and is annually isolated in sporadic, as well in epidemic cases. We studied 243 strains of this serotype, isolated in different regions of the country over the period 1954-1988. The plasmid analysis performed revealed that the presence of large plasmid DNA with molecular size of 140 MDa was related to the virulence of the strains, assessed by the test of SERENY. The absence of this plasmid or its deletion led to the loss of virulence. In 92% of the strains two small plasmids were demonstrated with molecular size of 6.0 MDa and 4.8 MDa, which appeared to be serotype-specific. On the basis of the plasmid content of the strains, 7 plasmid profiles were differentiated. The stability of the plasmid profile within a given epidemic focus was confirmed.
The new compound lignoren (1) was isolated from Trichoderma lignorum HKI 0257 by chromatographic methods. This metabolite has a santalane-like structure, which was elucidated by mass spectrometric and NMR spectroscopic investigations. Lignoren (1) shows a moderate narrow-spectrum of antibacterial and antifungal activity.
ECO-0501 is a novel linear polyene antibiotic, which was discovered from Amycolatopsis orientalis. Recent study of ECO-0501 biosynthesis pathway revealed the presence of regulatory gene: ECO-orf4. The A. orientalis ECO-orf4 gene from the ECO-0501 biosynthesis cluster was analyzed, and its deduced protein (ECO-orf4) was found to have amino acid sequence homology with large ATP-binding regulators of the LuxR (LAL) family regulators. Database comparison revealed two hypothetical domains, a LuxR-type helix-turn-helix (HTH) DNA binding motif near the C-terminal and an N-terminal nucleotide triphosphate (NTP) binding motif included. Deletion of the corresponding gene (ECO-orf4) resulted in complete loss of ECO-0501 production. Complementation by one copy of intact ECO-orf4 restored the polyene biosynthesis demonstrating that ECO-orf4 is required for ECO-0501 biosynthesis. The results of overexpression ECO-orf4 on ECO-0501 production indicated that it is a positive regulatory gene. Gene expression analysis by reverse transcription PCR of the ECO-0501 gene cluster showed that the transcription of ECO-orf4 correlates with that of genes involved in polyketide biosynthesis. These results demonstrated that ECO-orf4 is a pathway-specific positive regulatory gene that is essential for ECO-0501 biosynthesis.
Sites co-contaminated with heavy metals and 1,2-DCA may pose a greater challenge for bioremediation, as the heavy metals could inhibit the activities of microbes involved in biodegradation. Therefore, this study was undertaken to quantitatively assess the effects of heavy metals (arsenic, cadmium, mercury, and lead) on 1,2-DCA biodegradation in co-contaminated water. The minimum inhibitory concentrations (MICs) and concentrations of the heavy metals that caused half-life doubling (HLDs) of 1,2-DCA as well as the degradation rate coefficient (k1 ) and half-life (t½ ) of 1,2-DCA were measured and used to predict the toxicity of the heavy metals in the water microcosms. An increase in heavy metal concentration resulted in a progressive increase in the t½ and relative t½ and a decrease in k1 . The MICs and HLDs of the heavy metals were found to vary, depending on the heavy metals type. In addition, the presence of heavy metals was shown to inhibit 1,2-DCA biodegradation in a dose-dependent manner, with the following order of decreasing inhibitory effect: Hg(2+) > As(3+) > Cd(2+) > Pb(2+) . Findings from this study have significant implications for the development of bioremediation strategies for effective degradation of 1,2-DCA and other related compounds in wastewater co-contaminated with heavy metals.
Media with 1,3-1,4-beta -glucans as selective markers were used for isolation of non-starch-polysaccharide (NSP) degrading bacteria from the intestinal tract of broiler chicken. Formerly unknown 1,3-1,4-beta endoglucanase activities in various bacterial species were identified in this study. E. faecium , Streptococcus , Bacteroides and Clostridium strains seem to be responsible for degradation of mixed linked beta -glucans in the small intestine and in the hind gut of chickens. Strict anaerobic bacteria (Bacteroides ovatus , B. uniformis , presumably B. capillosus and Clostridium perfringens ) as well as an unidentified bacterium with 98% 16S rDNA homology to an uncultered chicken cecum bacterium were isolated. Additionally, Streptococcus bovis with 1,3-1,4-beta -endoglucanase activity was also detected. Different 1,3-1,4-beta -endoglucanase activity profiles were observed in SDS/PAGE zymograms.
An Enterococcus faecium strain with a novel endo 1,3-1,4-endo-beta-glucanase (lichenase, E.C. 18.104.22.168) was isolated from the intestinal tract of broiler chicken. The enzyme was secreted into the culture medium and acted exclusively on mixed linked 1,3-1,4-beta-glucans as determined with a reducing sugar assay. The purified enzyme has its isoelectric point at pI 4.8, maximum activity was determined at pH 6.5 and 40 degrees C. Thermal stability of the enzyme was low, but high pH stability and high residual activity was observed after incubation in digesta samples from the chicken intestine. Multiple lichenase activities were obtained from culture supernatants on SDS/PAGE and native zymograms, but it is concluded that the lichenase consists of one active protein at 30.5 kD and additional polypeptides of unknown function.
A Gram-positive bacterial strain was isolated from oil rich soil in Newfoundland and found to utilize various di- and trihydroxylated aromatic compounds as a source of carbon and energy. This bacterium exhibited rod/coccus dimorphism during its growth cycle. Chemical analysis of cell wall composition (amino acids, sugars, and fatty acids) was performed using gas chromatography-mass spectrophotometry and high pressure liquid chromatography. Comparison of both acid production and growth substrates showed complete homology with Rhodococcus erythropolis. Growth of the isolate on phloroglucinol (1,3,5 trihydroxybenzene) occurred in the pH range 5-8; with a substrate and temperature optima of 8.0 mM and 25 degrees C. The oxidation of PG was examined using whole cells as well as crude cell extracts. PG oxidation was shown to be due to an inducible enzyme system. Tentatively the isolate was identified as Rhodococcus species BPG-8 which is able to utilize phloroglucinol as the sole source of carbon and energy.
A comparative study of the wild type A. nidulans and a mutant strain aco-T69 (lacking conidia and cleistothecia) revealed the better production of beta-1,4-glucosidase in the former. The relative distribution of the enzyme levels in various morphological structures viz. somatic hyphae, spores and cleistothecia also showed a variation. Highest specific activity was found in the cleistothecial extracts. The electrophoretic analysis of the wild type strain demonstrated the presence of three isoenzymes of beta-1,4-glucosidase i.e. beta-GLU I, beta-GLU II and beta-GLU III, the number of which varies from one to three during the growth of the organism. The parallel study of the mutant strain showed complete absence of beta-GLU II. All three forms were present in the somatic hyphae and cleistothecial extracts while spore extracts depicted the absence of beta-GLU III in the wild type.
The white-rot fungus Trametes versicolor was used to study the influence of extracellular laccase activity on the degradation of 2-chlorophenol (2-CP) and the formation of metabolites under conditions, characterized by the absence of other phenol-oxidizing enzymes. 2-CP enhanced the production of extracellular laccase by fungal mycelia. The formation of the metabolite 2-chloro-1,4-benzoquinone (2-CIBQ) was found to be correlated with extracellular laccase activity. In cell-free crude culture liquids laccase was responsible for the oxidation of 2-CP. In contrast to this, the disappearance of 2-CP caused by the entire organism did not correlate with extracellular laccase activity. The primary oxidative step during the degradation of this compound can thus only partially be attributed to extracellular laccase; indicating the involvement of cell-bound processes.
In crude extracts of Candida maltosa, about 12 proteins are phosphorylated in the presence of cAMP or of a catalytic subunit of cAMP-dependent protein kinase.
A strongly labelled protein spot occurred in the position of fructose-1,6-bisphosphatase both after electrophoresis of crude extracts incubated with cAMP and of a partially purified fructose-1,6-bisphosphatase incubated with a catalytic subunit of cAMP-dependent protein kinase. No phosphorylation of the cytoplasmic malate dehydrogenase could be detected.
From these results it was concluded that cAMP-dependent phosphorylation plays an important role in the catabolite inactivation of fructose-1,6-bisphosphatase in Candida maltosa, as described for Saccharomyces cerecisiae.
A soluble, NADP+-dependent alcohol dehydrogenase (ADH) with a pH optimum at 8.7 was found in A. calcoaceticus EB 104 after growth on different carbon sources. n-Alkanols with short and medium chain length were employed as test substrates. The Km values decreased with increasing chain length. The Vmax values remained nearly unchanged. The activities determined were independent of the carbon source. Furthermore, a n-alkanol-dependent reduction of DCPIP was measured in membrane fractions of cells grown on different carbon sources. The optimum pH for this reaction was at 7.5. Further proof for the presence of a pyridine nucleotide-independent ADH was derived from the oxidation of 14C-decanol in the absence of NADP+ or NAD+.
The content of cytochrome P-450 as a function of oxygen supply was studied during growth of Acinetobacter on n-hexadecane in batch cultures at constant pH and agitation. The rate of growth and the content of cytochrome P-450 were not affected as long as the dissolved oxygen tension ranged above 3 to 5% of saturation. The amount of cytochrome P-450 increased when the oxygen tension declined to zero. Cytochrome P-450 levels of about 0.3 to 0.4 nmol/mg protein, i.e. a more than a threefold increase, were observed under conditions where oxygen supply was strictly limited and allowed to maintain only a minimum of metabolism or growth. Limited oxygen supply exerted a special effect on the induction of the cytochrome P-450 as concluded from an increasing ratio between cytochrome P-450 and cytochrome o, and from the absence of cytochrome d in cells with elevated content of cytochrome P-450. The increased formation of cytochrome P-450 was a reversible process.
The highly solvent-tolerant bacterium Pseudomonas sp. BCNU 106 was investigated to elucidate the solvent tolerance under specific culture conditions with the presence of solvents and its adaptive mechanisms to those conditions with reference to the antioxidant system. When exposed to 10% toluene, Pseudomonas sp. BCNU 106 increased the generation of reactive oxygen species assessed by monitoring the oxidation of 2',7'-dichlorofluorescein. Typical antioxidant enzymes (viz. catalase, superoxide dismutase, and glutathione reductase) showed increased activity with prolonged incubation in 10% toluene. In addition, the levels of these antioxidant proteins were higher during exposure to 10% toluene than in toluene-free condition. The present study indicates that antioxidant defense activity is one of the adaptive and protective mechanisms developed to avoid the deleterious damage of organic solvents, especially toluene.
The isolation and identification of several anthracyclinones (designated as G44-K4/5, G44-G1, G44-G2) and anthracyclines (G44-A, B, C, D, E, F, G) produced by the mutant strain IMET JA 3933/G44 of the daunomycin-producing Streptomyces griseus strain IMET JA 3933 are described. G44-K4/5 was found to be identical with 7,11-dideoxy-13-dihydrodaunomycinone previously isolated from a mutant strain of Streptomyces coeruleorubidus. Compound G44-G1 was identified as 11-deoxydaunomycinone, the aglycone of the antibiotic 11-deoxydaunomycin. G44-G2 was found to be a stereoisomer of G44-G1. The NMR and CD spectral data suggest strongly that the compound is 7-epi-11-deoxydaunomycinone. Of the 7 isolated G44-glycosides only the major component G44-B could be identified. Comparison with an authentic sample revealed that this compound is 11-deoxydaunomycin which had previously been isolated from cultures of Streptomyces peucetius var. aureus and Micromonospora peucetica. As reported for S. coeruleorubidus, S. peucetius var. aureus, and Micromonospora peucetica the 11-deoxydaunomycinone derivatives described in this paper were isolated from the fermentation broth of a mutant of a daunomycin-producing wild type strain. This suggest that in general the accumulation of 11-deoxydaunomycinone derivatives may be the result of a block of C11-hydroxylation in the normal biosynthetic pathway of daunomycin and its analogues.
There are two conflicting primary nucleotide sequences of the Staphylococcus aureus bacteriophage phi 11 amidase gene in Genbank. Nucleotide sequence differences as well as alternative translational start site assignments result in three non-identical protein sequence predictions for this amidase. Therefore, it is prudent to verify the correct phi11 amidase protein sequence, especially since multiple versions of the amidase gene have been subcloned, deletion analysis performed, and their experimental use described. There is also a resurgence of interest in the expression and use of bacteriophage lytic proteins as bactericidal agents and the phi 11 amidase has a high antimicrobial potential. The correct amidase sequence is identified through a combination of DNA sequence analysis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis of the recombinant purified phi11 amidase protein.
Nine fungal strains showing ability of cortexolone hydroxylation to epicortisol and/or cortisol were screened in this work for anthracene and phenanthrene elimination (250 mg/l). All of the strains (Cylindrocladium simplex IM 2358, C. simplex IM 2358/650, Monosporium olivaceum IM 484, Curvularia lunata IM 2901, C. lunata IM 2901/366, C. tuberculata IM 4417, Cunninghamella elegans IM 1785, C. elegans IM 1785/21Gp, C. elegans IM 1785/10Gi) significantly removed anthracene and phenanthrene. During incubation with anthracene formation of intermediate products was observed. The amount of the main intermediate product, identified as 9, 10-anthraquinone, was not greater than 22.2% of the anthracene introduced to the fungal cultures. C. elegans IM 1785/21Gp was the best degrader of both anthracene and phenanthrene, removing 81.6 and 99.4% of these compounds after 7 days, respectively. Phenanthrene removal by C. elegans IM 1785/21Gp was preceded by PAHs accumulation in mycelium and growth inhibition. Elimination of phenanthrene started after one day of incubation and was related to the fungus growth.
We have constructed double mutants carrying either ssb-1 or ssb-113 alleles, which encode temperature-sensitive single strand DNA binding proteins (SSB), and the uvrD::Tn5 allele causing deficiency in DNA helicase II, and have examined sensitivity to ultraviolet light (UV), recombination and spontaneous as well as UV-induced mutagenesis. We have found in a recA+ background that (i) none of the ssb uvrD double mutants was more sensitive to UV than either single mutant; (ii) the ssb-1 allele partially suppressed the strong UV sensitivity of uvrD::Tn5 mutants; (iii) in the recA730 background with constitutive SOS expression, the ssb-1 and ssb-113 alleles suppressed the strong UV-sensitivity caused by the uvrD::Tn5 mutation; (iv) in ssb-113 mutants, the level of recombination was reduced only 10-fold but 100-fold in ssb-1 mutants, showing that there was no correlation between the DNA repair deficiency and the recombination deficiency; (v) the hyper-recombination phenotype of the uvrD::Tn5 mutant was suppressed by the addition of either the ssb-1 or the ssb-113 allele; (vi) no addition of the spontaneous mutator effects promoted by the uvrD::Tn5 and the ssb-113 alleles was observed. These results suggest a possible functional interaction between SSB and Helicase II in DNA repair and mutagenesis.
The filamentous fungus Aspergillus ochraceus NCIM-1146 was found to degrade kerosene, when previously grown mycelium (96 h) was incubated in the broth containing kerosene. Higher levels of NADPH-DCIP reductase, aminopyrine N-demethylase and kerosene biodegradation activities were found to be present after the growth in potato dextrose broth for 96 h, when compared with the activities at different time intervals during the growth phase. NADPH was the preferred cofactor for enzyme activity, which was inhibited by CO, indicating cytochrome P450 mediated reactions. A significant increase in all the enzyme activities was observed when mycelium incubated for 18 h in mineral salts medium, containing cholesterol, camphor, naphthalene, 1,2-dimethoxybenzene, phenobarbital, n-hexane, kerosene or saffola oil as inducers. Acetaldehyde produced by alcohol dehydrogenase could be used as an indicator for the kerosene biodegradation.
The present investigation deals with the promotry effect of different additives and metallic micro minerals on citric acid production by Aspergillus niger MNNG-115 using different carbohydrate materials. For this, sugar cane bagasse was fortified with sucrose salt medium. Ethanol and coconut oil at 3.0% (v/w) level increased citric acid productivity. Fluoroacetate at a concentration of 1.0 mg/ml bagasse enhanced the yield of citric acid significantly. However, the addition of ethanol and fluoroacetate after 6 h of growth gave the maximum conversion of available sugar to citric acid. In another study, influence of some metallic micro-minerals viz. copper sulphate, molybdenum sulphate, zinc sulphate and cobalt sulphate on microbial synthesis of citric acid using molasses medium was also carried out. It was found that copper sulphate and molybdenum sulphate remarkably enhanced the production of citric acid while zinc sulphate was not so effective. However, cobalt sulphate was the least effective for microbial biosynthesis of citric acid under the same experimental conditions. In case of CuSO(4), the strain of Aspergillus niger MNNG-115 showed enhanced citric productivity with experimental (9.80%) over the control (7.54%). In addition, the specific productivity of the culture at 30 ppm CuSO(4) (Q(p) = 0.012a g/g cells/h) was several folds higher than other all other concentrations. All kinetic parameters including yield coefficients and volumetric rates revealed the hyper productivity of citric acid by CuSO(4) using blackstrap molasses as the basal carbon source.
Unorganized collections and over exploitation of naturally occurring medicinal plant Bacopa monniera is leading to rapid depletion of germplasm and is posing a great threat to its survival in natural habitats. The species has already been listed in the list of highly threatened plants of India. This calls for micropropagation based multiplication of potential accessions and understanding of their mycorrhizal associations for obtaining plants with enhanced secondary metabolite contents. The co-cultivation of B. monniera with axenically cultivated root endophyte Piriformospora indica resulted in growth promotion, increase in bacoside content, antioxidant activity and nuclear hypertrophy of this medicinal plant.
A variety of arylsulfonates were examined for their ability to support growth of Clostridium pasteurianum as sole source of sulfur. Among the eleven different arylsulfonates tested, six of them (benzenesulfonate, 4-toluenesulfonate, 4-xylene-2-sulfonate, 4-aminobenzenesulfonate, 4-sulfobenzoic acid, 1,3-benzenedisulfonate) could serve as sole sulfur source for C. pasteurianum DSM 12136. None of the sulfonates tested could serve as sole sulfur source for C. pasteurianum ATCC 6013. The two C. pasteurianum in this study could not utilize any of these sulfonates as sole carbon and energy source. We demonstrated that desulfonation of arylsulfonates could take place under anoxic conditions and the sulfur atom of these compounds could be utilized as sole source of sulfur by anaerobic bacteria.
A psychrotrophic bacterium isolated from polychlorinated biphenyls (PCBs)-contaminated soil grew on biphenyl as sole carbon and energy source, and actively cometabolized PCBs at low temperature. Analysis of cellular fatty acids indicate that the bacterium is most closely related to Hydrogenophaga taeniospiralis. Resting cells incubated with 10 ppm of Aroclor 1221 at 5 or 30 degrees C for 48 h removed all mono-, most di-, and several trichlorobiphenyls. At 5 degrees C, removal of MCBs (monochlorobiphenyls) was between 63 to 89%, DCBs (dichlorobiphenyls) was between 30 to 78%, and TCBs (trichlorobiphenyls) was between 30 to 75%. At 30 degrees C, removal of MCBs was 100%, DCBs was between 30 to 100%, and TCBs was between 27 to 59%. Congeners with two or more ortho chlorine were generally resistant to degradation. However, removal of di-ortho plus para-substituted congeners at 30 degrees C and not at 5 degrees C, suggest that the presence of a para-chlorine enhanced the cometabolism of these congeners at 30 degrees C. Furthermore, after 72 h, resting cells removed 68 and 83% of 500 microM of 2,4'-dichlorobiphenyl (2,4'-DCB) and, 35 and 44% of 500 microM of 2,3-dichlorobiphenyl (2,3-DCB) at 5 and 30 degrees C, respectively. Analysis of metabolites by GC-MS indicates that the cometabolized 2,3-DCB was completely recovered as 2,3-chlorobenzoic acid (2,3-CBA), while the cometabolized 2,4'-DCB was not completely recovered as chlorobenzoic acid. To our knowledge, it is the first strain of Hydrogenophaga taeniospiralis found to degrade an organic pollutant, and also the first psychrotrophic strain of a member of the genus Hydrogenophaga to grow on biphenyl or cometabolize PCBs at low temperature. Results suggest that the bacterium has potential use in the bioremediation of PCB-contaminated sites in cold regions.
Statistically-based experimental designs were applied to optimize the fermentation for the production of laccase by Pleurotus florida NCIM 1243. Eleven components were screened for their significant effect on laccase production using Plackett-Burman factorial design. Glucose (carbon source), asparagine (nitrogen source), CuSO(4)(inducer) and incubation period were found to have highest positive influence on the laccase production. The combined effect of these factors on laccase production was studied using central composite design of Response surface methodology. The optimal point of variables for maximum laccase production using Response surface methodology are glucose (15.21 g/l), asparagine (6.40 g/l), CuSO(4) (91.78 microM) and incubation period (178.55 h), respectively. The maximum enzyme activity predicted by the model was 5.0 U/ml which was in perfect agreement with the actual experimental value (4.8 U/ml). Further, partially purified laccase from the optimized cultural condition was used for the decolorization of reactive dyes, Reactive Blue 198 and Reactive Red 35.
We collected 97 non-repetitive carbapenemases-sensitive clinical isolates of Pseudomonas aeruginosa in Human Province, China, during the period of October 2006 to January 2007. From these isolates, we identified two novel oxacillin-hydrolysing (OXA) type extended-spectrum-β-lactamases (ESBLs): bla OXA-128 and bla OXA-129, which contain the mutations of I89V from bla OXA-56 and K134N from bla OXA-10, respectively. Clinical isolates containing either bla OXA-128 or bla OXA-129 show resistance to cephamycin-class antibiotics but sensitive to carbapenem-class antibiotics. The occurrence of novel OXA-type lactamases suggests a regional prevalent pattern of ESBLs Pseudomonas aeruginosa in this area.
A novel natural niphimycin analog, N'-methylniphimycin was isolated from the culture broth of the Streptomyces spec. 57-13. The chemical constitution was elucidated from the physico-chemical properties, NMR techniques and mass spectrometry to be a 36-membered macrolide related to azalomycin F5a, shurimycin B and RS-22C. N'-methylniphimycin displayed moderate activity against some yeasts and filamentous fungi.
Calcium alginate-entrapped mycelium of Penicillium raistrickii i 477 was used for the 15 alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione. The system was tested in 10 batches during 40 days of operation, the catalytic activity could be completely regenerated by incubation in a nutrient medium between several batches.
A halotolerant strain FP-133, able to grow at concentrations of 0-12.5% (w/v) NaCl, was isolated from a fish paste and identified as Bacillus subtilis . B. subtilis strain FP-133 produced an intracellular protease which showed catalytic activity under saline conditions. The enzyme was purified to homogeneity 143-fold with a yield of 0.9%. The purified enzyme showed an optimum activity at a concentration of 5% (w/v) NaCl. After storage in 7.5% (w/v) NaCl at 4 degrees C for 24 h, the enzyme kept 100% of its activity. The molecular mass of the protease was determined to be 59 kDa by gel filtration; the protein consisted of four subunits each with a molecular mass of 14 kDa. The enzyme showed aminopeptidase activity. It acted on L-leucyl-p-nitroanilide, L-leucyl-beta-naphthylamide, and oligopeptides containing glycine, L-histidine, or L-leucine. The K(m ) and V (max) values for L-leucyl-p-nitroanilide were 18 microm and 2.2 mm/h mg, respectively. The enzyme was activated by Fe(2+), Fe(3+), and Ni(2+) in synergism with Mg(2+).
Under the current assay conditions, lipase production in mineral medium was only detected in the presence of vegetable oils, reaching the highest specific activity with olive oil. In this way, effect of different environmental conditions on fungal morphology and olive oil-induced extracellular lipases production from Aspergillus niger MYA 135 was studied. It was observed that addition of 1.0 g l(-1) FeCl(3)to the medium encouraged filamentous growth and increased the specific activity 6.6 fold after 4 days of incubation compared to the control. However, major novelty of this study was the satisfactory production of an acidic lipase at initial pH 3 of the culture medium (1.74 +/- 0.06 mU microg(-1)), since its potencial applications in food and pharmaceutical industry are highly promising.
Syngas utilizing bacterium Clostridium ljungdahlii DSM 13528 is a promising platform organism for a whole variety of different biofuels and biochemicals production from syngas. During syngas fermentation, C. ljungdahlii DSM 13528 could convert butanol into butyrate, which significantly reduces productivity of butanol. However, there has been no any enzyme involved in the degradation of butanol characterized in C. ljungdahlii DSM 13528. In this study two genes, CLJU_c24880 and CLJU_c39950, encoding putative butanol dehydrogenase (designated as BDH1 and BDH2) were identified in the genome of C. ljungdahlii DSM 13528 and qRT-PCR analysis showed the expression of bdh1 and bdh2 was significantly upregulated in the presence of 0.25% butanol. And the deduced amino acid sequence for BDH1 and BDH2 showed 69.85 and 68.04% identity with Clostridium acetobutylicum ADH1, respectively. Both BDH1 and BDH2 were oxygen-sensitive and preferred NADP(+) as cofactor and butanol as optimal substrate. The optimal temperature and pH for BDH1 were at 55 °C and pH 7.5 and specific activity was 18.07 ± 0.01 µmol min(-1) mg(-1) . BDH2 was a thermoactive dehydrogenase with maximum activity at 65 °C and at pH 7.0. The specific activity for BDH2 was 11.21 ± 0.02 µmol min(-1) mg(-1) . This study provided important information for understanding the molecular mechanism of butanol degradation and determining the targets for gene knockout to improve the productivity of butanol from syngas in C. ljungdahlii DSM 13528 in future.
Although many strains of Bacillaceae are considered nonpathogenic, Bacillus cereus is recognized worldwide as a bacterial pathogen in a variety of foods. The ability of B. cereus to cause gastroenteritis following ingestion of contaminated food is due to the production of enterotoxins. The ubiquity of this genus makes it a persistent problem for quality assurance in food processing environments. The primary objective of this study was to develop and apply a multiplex real-time PCR-based assay for rapid and sensitive detection of enterotoxigenic B. cereus. Template DNA was separately extracted from tryptic soy broth (TSB)-grown and 2.5% Nonfat Dry Milk (NFDM)-grown B. cereus using a commercial system. Three enterotoxin gene fragments (hblC, nheA, and hblA) were simultaneously amplified in real-time followed by melting curve analysis to confirm amplicon identity. Resolution of melting curves (characteristic T(m)) was achieved for each amplicon (hblC = 74.5 °C; nheA = 78 °C; and hblA = 85.5 °C in TSB and 84 °C in NFDM) with an assay sensitivities of 10(1) CFU/ml for both TSB and NFDM-grown B. cereus compared to 10(4) CFU/ml in either matrix using gel electrophoresis. The results demonstrate the potential sensitivity of real-time bacterial detection methods in a heterogenous food matrix using real-time PCR.
Trichoderma atroviride (T-15603.1) is a promising fungal agent for biological control of wood decay fungi in urban tree wounds. The aim of this work was to determine the combined effects of water activity (a(w), 0.998-0.892), temperature (10-30 °C) and pH (3-7) on the development of conidia on low-nutrient agar (LNA). Lag phase prior to germination (h), germination rates (µ(m)) and germ-tube elongation were obtained at each set of conditions. The experimental data were used to fit a response surface model for predicting the germination rates of T-15603.1 and to analyze the effect of the abiotic parameters tested. The polynomial response surface model was mathematically evaluated using graphical plots and several statistical indices (RMSE, %SEP, A(f), B(f), pRE). Data analyses showed a highly significant effect on conidial development of a(w) and temperature (P < 0.001), whereas no significant effect of pH (P ≥ 0.05) was observed. The germination rate dropped and the lag prior to germination increased as the temperature and a(w) decreased, but T-15603.1 appeared to be more sensitive to a(w) reduction than to temperature. The minimum a(w) level for germination was 0.910 at 15-25 °C, and maximum germination rates were obtained at a(w) = 0.998, 25-30 °C and pH 5. The response surface model was useful and widely accurate (R² = 0.983) for predicting the germination rate of T-15603.1 and complemented the experimental results. These findings contribute to better understanding how combined environmental factors affect the environmental parameter tolerance levels of T-15603.1 and to the development of an adequate delivery system for optimized application of T-15603.
Experiments on the incorporation of D- and L-[alanine-3-13C,2-15N]tryptophan into the antibiotic pyrrolnitrin in Pseudomonas aureofaciens confirmed earlier conclusions about the conversion of L-tryptophan into pyrrolnitrin. They also demonstrated that a fraction of the D isomer is incorporated without breakage of the 15N-carbon bond, consistent with the operation of a second pathway from D-tryptophan to pyrrolnitrin. Cell-free experiments confirmed the conversion of 3-(o-aminophenyl)pyrrole into aminopyrrolnitrin but failed to detect enzymatic oxidation of the latter to pyrrolnitrin.
Because of its structural similarity to nucleoside, toyocamycin exhibits potential of wide application and various biological activities. Streptomyces diastatochromogenes 1628, capable of producing toyocamycin, has exhibited a potential biocontrol effect in inhibiting the development of phytopathogens in the agriculture field. An efficient transformation system is a prerequisite for genetic and molecular study of S. diastatochromogenes 1628. In this study, we optimized experimental factors involved in the electroporation transformation process. Key features of this procedure, including collection of cells at the mid-log phase stage and the treatment of cells with lysozyme and penicillin G prior to the electroporation and recovery medium and time, produced the greatest increase in the efficiency and consistency of results. The transformation efficiency also depends on field strength, cell concentration, and plasmid DNA quantity. Under the optimal conditions, a maximal efficiency of (3 ± 0.4) × 10(4) µg(-1) DNA was obtained. The development of transformation method for S. diastatochromogenes 1628 will foster genetic manipulation of this important strain.
Two-dimensional gel electrophoresis was used to demonstrate the synthesis of approximately 65 [35S]-methionine-labelled soluble proteins between 0 and 10 min after the start of germination, of approximately 210 proteins at 10-20 min, and of approximately 260 proteins during vegetative growth of Bacillus subtilis. When actinomycin D and [35S]-methionine were administered at the onset of germination and the proteins synthesized during the subsequent 15 min were analyzed, two proteins were detected, and were designated protein I and protein II. Immunoblot analysis with an antiserum raised against RNA polymerase from Escherichia coli demonstrated that protein II corresponded to the sigma A factor of Bacillus subtilis. Thus, the sigma A factor is synthesized during early germination of Bacillus subtilis in the presence of actinomycin D.
In this study, two designed primers were evaluated to identify soil Streptomyces and to detect streptomycin production by strb1 targeted PCR. Potential Streptomyces-specific signatures were identified in their 16S rDNA sequences in regions located around nucleotide positions 576 and 995. Primer pair RI7/RI8 derived from these regions was investigated for its specificity in detecting and identifying Streptomyces isolates by PCR assays using DNA from pure cultures. The constructed primer pair showed high specificity in detecting and identifying Streptomyces type strains as well as soil isolates. Streptomycin-producers were detected by PCR assays through the selective amplification of streptomycin biosynthetic gene (strb1). Results suggest that PCR assay facilitates the differential identification of Streptomyces-specific antibiotic producers and a resident population of Streptomyces in Jordan with the capacity of streptomycin-production is present.
A multi-drug resistant Escherichia coli C21 was isolated from a chicken in China. It was shown to be positive for the presence of the bla(TEM-1) , bla(CTX-M-55) and rmtB genes by PCR. This strain was examined by phylogenetic grouping, conjugation experiments, plasmid analysis, PCR-based replicon typing and multi-locus sequence typed (MLST). The genetic environment of bla(CTX-M-55) was investigated by PCR mapping. The strain belonged to phylogroup A, ST156. The bla(CTX-M-55) and rmtB genes were found to be present in separate plasmids that belonged to the IncI1 and IncN families, respectively. These antibiotic-resistant plasmids could be transferred to the recipient strain alone or together. A new arrangement of ISEcp1Δ-IS1294-ΔISEcp1-bla(CTX-M-55) -ORF477, in which the ISEcp1 element was disrupted by another IS1294 element, was identified initially. Conjugative transfer and IS elements found in this study could lead to the rapid dissemination of bla(CTX-M-55) and rmtB among strains of Enterobacteriaceae, which could pose a threat to animal husbandry and public health.
A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries.