Use of omic tools to assess methyl tert-butyl ether (MTBE) degradation in groundwater.

This study employed innovative technologies to evaluate multiple lines of evidence for natural attenuation (NA) of methyl tertiary-butyl ether (MTBE) in groundwater at the 22 Area of Marine Corps Base (MCB) Camp Pendleton after decommissioning of a biobarrier system. For comparison, data from the 13 Area Gas Station where active treatment of MTBE is occurring was used to evaluate the effectiveness of omic techniques in assessing biodegradation. Overall, the 22 Area Gas Station appeared to be anoxic. MTBE was detected in large portion of the plume. In comparison, concentrations of MTBE at the 13 Area Gas Station were much higher (42,000 µg/L to 2800 µg/L); however, none of the oxygenates were detected. Metagenomic analysis of the indigenous groundwater microbial community revealed the presence of bacterial strains known to aerobically and anaerobically degrade MTBE at both sites. While proteomic analysis at the 22 Area Gas Station showed the presence of proteins of MTBE degrading microorganisms, the MTBE degradative proteins were only found at the 13 Area Gas Station. Taken together, these results provide evidence for previous NA of MTBE in the groundwater at 22 Area Gas Station and demonstrate the effectiveness of innovative-omic technologies to assist monitored NA assessments.

Correlation of the lung microbiota with metabolic profiles in bronchoalveolar lavage fluid in HIV infection.

BACKGROUND: While 16S ribosomal RNA (rRNA) sequencing has been used to characterize the lung's bacterial microbiota in human immunodeficiency virus (HIV)-infected individuals, taxonomic studies provide limited information on bacterial function and impact on the host. Metabolic profiles can provide functional information on host-microbe interactions in the lungs. We investigated the relationship between the respiratory microbiota and metabolic profiles in the bronchoalveolar lavage fluid of HIV-infected and HIV-uninfected outpatients. RESULTS: Targeted sequencing of the 16S rRNA gene was used to analyze the bacterial community structure and liquid chromatography-high-resolution mass spectrometry was used to detect features in bronchoalveolar lavage fluid. Global integration of all metabolic features with microbial species was done using sparse partial least squares regression. Thirty-nine HIV-infected subjects and 20 HIV-uninfected controls without acute respiratory symptoms were enrolled. Twelve mass-to-charge ratio (m/z) features from C18 analysis were significantly different between HIV-infected individuals and controls (false discovery rate (FDR) = 0.2); another 79 features were identified by network analysis. Further metabolite analysis demonstrated that four features were significantly overrepresented in the bronchoalveolar lavage (BAL) fluid of HIV-infected individuals compared to HIV-uninfected, including cystine, two complex carbohydrates, and 3,5-dibromo-L-tyrosine. There were 231 m/z features significantly associated with peripheral blood CD4 cell counts identified using sparse partial least squares regression (sPLS) at a variable importance on projection (VIP) threshold of 2. Twenty-five percent of these 91 m/z features were associated with various microbial species. Bacteria from families Caulobacteraceae, Staphylococcaceae, Nocardioidaceae, and genus Streptococcus were associated with the greatest number of features. Glycerophospholipid and lineolate pathways correlated with these bacteria. CONCLUSIONS: In bronchoalveolar lavage fluid, specific metabolic profiles correlated with bacterial organisms known to play a role in the pathogenesis of pneumonia in HIV-infected individuals. These findings suggest that microbial communities and their interactions with the host may have functional metabolic impact in the lung.

Mechanism controlling the extended lag period associated with vinyl chloride starvation in Nocardioides sp. strain JS614.

The extended lag period associated with vinyl chloride (VC) starvation in VC- and ethene-assimilating Nocardioides sp. strain JS614 was examined. The extended lag periods were variable (3-7 days), only associated with growth on VC or ethene, and were observed in VC- or ethene-grown cultures following 24 h carbon starvation and mid-exponential phase cultures grown on non-alkene carbon sources (e.g. acetate). Alkene monooxygenase (AkMO) and epoxyalkane:coenzyme M transferase (EaCoMT) are the initial enzymes of VC and ethene biodegradation in strain JS614. Reverse-transcription PCR confirmed that the AkMO gene etnC was expressed in response to epoxyethane, a metabolic intermediate of ethene biodegradation. Epoxyethane (0.5 mM) eliminated the extended lag period in both starved and mid-exponential phase cultures, suggesting that epoxyethane accumulation activates AkMO expression in strain JS614. AkMO activity in ethene-grown cultures was not detected after 6.7 h of carbon starvation, while 40% of the initial EaCoMT activity remained after 24 h. Acetate eliminated the extended lag period in starved cultures but not in mid-exponential phase cultures suggesting that acetate reactivates extant AkMO in starved VC- or ethene-grown cultures. The imbalance between AkMO and EaCoMT activities during starvation likely contributes to the extended lag period by delaying epoxide accumulation and subsequent AkMO induction.

The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamin-dependent mutase pathway.

Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated beta-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.

Cloning and characterization of histamine dehydrogenase from Nocardioides simplex.

Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here, the hdh gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6+/-0.3 mol Fe and 3.7+/-0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histamine reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow substrate specificity, oxidizing only histamine (K(m)=31+/-11 microM, k(cat)/K(m)=2.1 (+/-0.4)x10(5)M(-1)s(-1)), agmatine (K(m)=37+/-6 microM, k(cat)/K(m)=6.0 (+/-0.6)x10(4)M(-1)s(-1)), and putrescine (K(m)=1280+/-240 microM, k(cat)/K(m)=1500+/-200 M(-1)s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K(m) (histamine) and the measurement of a substrate deuterium isotope effect, (D)(k(cat)/K(m) (histamine))=7.0+/-1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat))=1.3+/-0.2.

Physiological and molecular genetic analyses of vinyl chloride and ethene biodegradation in Nocardioides sp. strain JS614.

Nocardioides sp. strain JS614 utilizes vinyl chloride and ethene as carbon and energy sources. JS614 could be influential in natural attenuation and biogeochemical ethene cycling, and useful for bioremediation, biocatalysis and metabolic engineering, but a fundamental understanding of the physiological and genetic basis of vinyl chloride and ethene assimilation in strain JS614 is required. Alkene monooxygenase (AkMO) activity was demonstrated in whole-cell assays and epoxyalkane:coenzyme M transferase (EaCoMT) activity was detected in JS614 cell-free extracts. Pulsed-field gel electrophoresis revealed a 290-kb plasmid (pNoc614) in JS614. Curing experiments and PCR indicated that pNoc614 encodes vinyl chloride/ethene-degradation genes. JS614 vinyl chloride/ethene catabolic genes and flanking DNA (34.8 kb) were retrieved from a fosmid clone. AkMO and EaCoMT genes were found in a putative operon that included CoA transferase, acyl-CoA synthetase, dehydrogenase, and reductase genes. Adjacent to this gene cluster was a divergently transcribed gene cluster that encoded possible coenzyme M biosynthesis enzymes. Reverse transcription-PCR demonstrated the vinyl chloride- and ethene-inducible nature of several genes. Genes encoding possible plasmid conjugation, integration, and partitioning functions were also discovered on the fosmid clone.

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D.

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues-value precursors in the synthesis of modern glucocorticoids starting from 9alpha-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and (1)H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.

Piericidins C5 and C6: new 4-pyridinol compounds produced by Streptomyces sp. and Nocardioides sp.

Piericidins C5 (1) and C6 (2), two new members of the piericidin family, were isolated from a Streptomyces sp. and a Nocardioides sp., together with known piericidins C1 (3), C2 (4), C3 (5), C4 (6), D1 (7), and A3 (8). The structures were determined on the basis of their spectroscopic data. Both new compounds inhibited cell division of fertilized starfish (Asterina pectinifera) eggs at the minimum inhibitory concentration of 0.09 microg/mL.

Patterns of antimicrobial activities from soil actinomycetes isolated under different conditions of pH and salinity.

AIMS: To evaluate the patterns of the production of antimicrobial compounds by diverse collection of actinomycetes isolated from different geographies under alternative conditions of pH and salinity in the media. METHODS AND RESULTS: Actinomycetes were grouped based on their method of isolation and their phenotype diversity was determined by total fatty acid analysis. A total of 335 representative isolates, including 235 Streptomyces species and 100 actinomycetes from other taxa, were screened for the production of antimicrobial activities against a panel of bacteria, filamentous fungi and yeasts, including some of clinical relevance. Production of antimicrobial activities was detected in 230 strains. In the case of the genus Streptomyces, 181 antimicrobial activities (77% of the tested isolates) were recorded. The activities observed among the other actinomycetes taxa were lower (49% of the tested isolates). CONCLUSIONS: The results of this study support the idea that species of actinomycetes isolated in alternative selective conditions of pH and salinity present a significant capacity to produce compounds with antibacterial or antifungal activity. The best group of isolates in terms of production of active secondary metabolites was the one isolated in saline conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrate that these actinomycetes strains isolated in alternative selective conditions of pH and salinity and collected from diverse geographical locations present a significant capacity to produce compounds with antibacterial or antifungal activity.

Microbial conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione acetates by Nocardioides simplex VKM Ac-2033D.

The conversion of pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione 21-acetate (I) and 17,21-diacetate (VI) by Nocardioides simplex VKM Ac-2033D was studied. The major metabolites formed from I were identified as pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV). Pregna-4,9(11)-diene-17alpha,21-diol-3,20-dione (III) and pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V) were formed in minorities. Biotransformation products formed from VI were pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17,21-diacetate (VII), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione (IV), pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII), pregna-1,4,9(11)-triene-17alpha,20beta,21-triol-3-one (V). The conversion pathways were proposed including 1(2)-dehydrogenation, deacetylation, 20beta-reduction and non-enzymatic migration of acyl group from position 17 to 21. The conditions providing predominant accumulation of pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 21-acetate (II) from I and pregna-1,4,9(11)-triene-17alpha,21-diol-3,20-dione 17-acetate (VIII) from VI in a short-term biotransformation were determined.

[The 1(2)-dehydrogenation of steroid substrates by Nocardioides simplex VKM Ac-2033D]. / 1(2)-degidrirovanie steroidnykh substratov shtammom Nocardioides simplex BKM Ac-2033D.

The bacterium formerly known as Arthrobacter globiformis 193 has high 1(2)-dehydrogenase activity toward pharmaceutically important steroids, 9(11)-dehydrocortexolone in particular. The complex analysis of the morphostructural, physiological, biochemical, and phylogenetic properties of this bacterium allowed us to reclassify it into Nocardioides simplex (N. simplex VKM Ac-2033D).

Characterization of JP-7 jet fuel degradation by the bacterium Nocardioides luteus strain BAFB.

In the fall of 1996, numerous bacteria capable of degrading JP-7 jet fuel were isolated from soil collected at Beale Air Force Base in northern California. The most prevalent organism, identified as Nocardioides luteus by16s rRNA sequencing (MIDI Labs, Inc.), was selected for further analysis. Analysis of JP-7 following inoculation with N. luteus demonstrated degradation of the C(11) alkane component of the fuel. Growth rates of N. luteus were determined with alkanes of various lengths as the sole carbon and energy source. The organism grew best on shorter length alkanes (C(8) and C(10)). Growth was measurably slower on C(11), and minimal on C(12), C(13), and C(14).

[Study on production of acrylamide by microbial method (I)--Culture of bacterium cells and expression of high activity of nitrile hydratase].

The cultural conditions for the growth of Norcardia cell were studied in this paper. Controlling pH value, adding nutrient and optimizing the quantity of inducer during cultivation, the activity of nitrile hydratase reached 6567 u/mL (culture medium), which was the highest value appeared in native journals. In the farther hydratase experiments, no by-product, crylic acid, was detected. It showed that the activity of amidase was not promoted obviously while the activity of nitrile hydratase was increased greatly. The results set a strong foundation for the industrial application and the research on new technology.

Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41.

The effect of the presence of an alternate toxic compound (phenol) on the p-nitrophenol (PNP)-degrading activity of freely suspended and calcium alginate immobilized Nocardioides sp. NSP41 was investigated. In the single substrate experiments, when the concentration of phenol and PNP was increased to 1400 mg l(-1) and 400 mg l(-1), respectively, the initial cell concentrations in the freely suspended cell culture should be higher than 1.5 g dry cell weight l(-1) for complete degradation. In the simultaneous degradation experiment, when the initial concentration of phenol was increased from 100 to 400 mg l(-1), the specific PNP degradation rate at the concentration of 200 mg l(-1) was decreased from 0.028 to 0.021 h(-1). A freely suspended cell culture with a high initial cell concentration resulted in a high volumetric degradation rate, suggesting the potential use of immobilized cells for simultaneous degradation. In the immobilized cell cultures, although simultaneous degradation of PNP and phenol was maintained, the specific PNP and phenol degradation rate decreased. However, a high volumetric PNP and phenol degradation rate could be achieved by immobilization because of the high cell concentration. Furthermore, when the immobilized cells were reused in the simultaneous degradation of PNP and phenol, they did not lose their PNP- and phenol-degrading activity for 12 times in semi-continuous cultures. Taken together, the use of immobilized Nocardioides sp. NSP41 for the simultaneous degradation of PNP and phenol at high concentrations is quite feasible because of the high volumetric PNP and phenol degradation rate and the reusability of immobilized cells.

Production of a bioflocculant by mixed culture.

Colony groups that form large amounts of slime externally were obtained from activated sludge among phthalate-assimilating microbes. That slime, which R-3 mixed microbes produced externally, had a high level of flocculation activity. R-3 mixed strains, one of such colony groups, efficiently produced a bioflocculant (APR-3) in liquid cultures of production medium, especially that containing starch and glucose (1:1) as carbon sources. Identification of this group of bioflocculant-producing microbes (R-3 mixed strains) showed that it was comprised of four strains belonging to the genera Oerskovia, Acinetobacter, Agrobacterium, and Enterobacter. The bioflocculant (APR-3) was purified electrophoretically to homogeneity by ethanol and CPC precipitation. Its molecular mass is at least 2 x 10(6) Da. APR-3 is an acidic polysaccharide made of glucose, galactose, succinic acid, and pyruvic acid (molar ratio: 5.6:1:0.6:2.5).

Helvecardins A and B, novel glycopeptide antibiotics. I. Taxonomy, fermentation, isolation and physico-chemical properties.

A strain of actinomycetes identified as Pseudonocardia compacta subsp. helvetica produced new glycopeptide antibiotics, helvecardins A and B. They were isolated from culture broth mainly by affinity chromatography of D-alanyl-D-alanine and preparative HPLC. The physico-chemical properties of helvecardins A and B showed that they resemble each other. Though helvecardin A was structurally related to beta-avoparcin, it clearly differed in the presence of an O-methyl moiety in its NMR spectrum.

Isolation of new minor benanomicins.

Four minor benanomicins, dexylosylbenanomicins A and B, 2'-demethylbenanomicin A and 7-methoxybenanomicinone have been isolated from the culture filtrate of Actinomadura sp. MH193-16F4. Their structures were confirmed by spectral analyses. Dexylosylbenanomicins A and B were derived chemically from benanomicins A and B, respectively.

[Actinomadura species as antibiotic producers]. / Vidy roda Actinomadura kak produtsenty antibiotikov.

Screening of antibiotic-producing cultures among Actinomadura showed that definite species mainly produced antibiotics of the same groups. Thus, carminomycins were produced by all the 4 studied strains of A. carminata, maduramycins were produced by 3 strains of A. rubra, prodigiozines were produced by 3 strains of A. madurae and luzopeptines were produced by 6 strains of A. recticatena. Supposedly, new antibiotics with original spectral characteristics were isolated from 2 strains of A. fulvescens. There was a clear-cut relation of the number of the active strains and their antibiotic productivity in definite media to their species. The liquid nutrient media, such as yeast-sucrose, soya-glucose and soya-glucose with cobalt chloride proved to be the most efficient in the primary screening of antibiotic-producing cultures.

MM 46115, a new antiviral antibiotic from Actinomadura pelletieri. Characteristics of the producing cultures, fermentation, isolation, physico-chemical and biological properties.

Six strains of Actinomadura pelletieri were shown to produce a novel macrolide antibiotic, designated MM 46115. MM 46115 was active against parainfluenza virus 1 and 2 and Gram-positive bacteria. Methods are described for the production of MM 46115 by strain IP 729.63, and for the isolation of the compound from a butanol extract of the culture filtrate.

Pradimicins M, N, O and P, new dihydrobenzo[a]naphthacenequinones produced by blocked mutants of Actinomadura hibisca P157-2.

Four blocked mutants which accumulated new dihydrobenzo[a]naphthacenequinone metabolites, designated pradimicins M, N, O and P, have been isolated from cultures of mutants of Actinomadura hibisca P157-2 resulting from treatment with N-methyl-N'-nitro-N-nitrosoguanidine. The structures of the four compounds were determined by spectral analysis. Pradimicins N, O and P contain D-alanine, while pradimicin M does not. The conformations at C-5 and C-6 of these compounds are different from those of the original pradimicins.