Paenibacillus etheri sp. nov., able to grow on media supplemented with methyl tert-butyl ether (MTBE) and isolated from hydrocarbon-contaminated soil.

A bacterial strain, designated SH7T, was isolated from the hydrocarbon-contaminated soil of a pilot plant (Granada, Spain). The strain was selected for its capacity to grow in media supplemented with methyl tert-butyl ether (MTBE) as sole energy and carbon source. Strain SH7T was a Gram-stain-positive, facultatively anaerobic, spore-forming, rod-shaped bacterium. Phylogenetic analysis using 16S rRNA gene sequences showed that strain SH7T belongs to a cluster comprising species of the genus Paenibacillus and was closely related to Paenibacillus borealis KK19T (97 % 16S rRNA gene sequence similarity) and Paenibacillus odorifer TOD45T (98 %). DNA-DNA hybridization tests showed low relatedness of strain SH7T with the type strains of Paenibacillus borealis (16.9 ± 1.5 %) and Paenibacillus odorifer (16.6 ± 2.1 %). The cell wall of strain SH7T contained meso-diaminopimelic acid. The predominant respiratory quinone was MK-7, and anteiso-C15 : 0 (32.9 %) and C16 : 0 (29.0 %) were the predominant cellular fatty acids. Phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and three unknown aminophospholipids were the major phospholipids. The DNA G+C content was 44.3 mol%. Data obtained in this study indicate that strain SH7T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus etheri sp. nov. is proposed. The type strain is SH7T ( = CECT 8558T = DSM 29760T).

Constitutive expression of the cytochrome P450 EthABCD monooxygenase system enables degradation of synthetic dialkyl ethers in Aquincola tertiaricarbonis L108.

In Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005, and Gordonia sp. strain IFP 2009, the cytochrome P450 monooxygenase EthABCD catalyzes hydroxylation of methoxy and ethoxy residues in the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). The expression of the IS3-type transposase-flanked eth genes is ETBE dependent and controlled by the regulator EthR (C. Malandain et al., FEMS Microbiol. Ecol. 72:289-296, 2010). In contrast, we demonstrated by reverse transcription-quantitative PCR (RT-qPCR) that the betaproteobacterium Aquincola tertiaricarbonis L108, which possesses the ethABCD genes but lacks ethR, constitutively expresses the P450 system at high levels even when growing on nonether substrates, such as glucose. The mutant strain A. tertiaricarbonis L10, which is unable to degrade dialkyl ethers, resulted from a transposition event mediated by a rolling-circle IS91-type element flanking the eth gene cluster in the wild-type strain L108. The constitutive expression of Eth monooxygenase is likely initiated by the housekeeping sigma factor σ(70), as indicated by the presence in strain L108 of characteristic -10 and -35 binding sites upstream of ethA which are lacking in strain IFP 2001. This enables efficient degradation of diethyl ether, diisopropyl ether, MTBE, ETBE, TAME, and tert-amyl ethyl ether (TAEE) without any lag phase in strain L108. However, ethers with larger residues, n-hexyl methyl ether, tetrahydrofuran, and alkyl aryl ethers, were not attacked by the Eth system at significant rates in resting-cell experiments, indicating that the residue in the ether molecule which is not hydroxylated also contributes to the determination of substrate specificity.

Assembled mixed co-cultures for emerging pollutant removal using native microorganisms from sewage sludge.

The global pharmaceutical pollution caused by drug consumption (>100,000 tonnes) and its disposal into the environment is an issue which is currently being addressed by bioremediation techniques, using single or multiple microorganisms. Nevertheless, the low efficiency and the selection of non-compatible species interfere with the success of this methodology. This paper proposes a novel way of obtaining an effective multi-domain co-culture, with the capacity to degrade multi-pharmaceutical compounds simultaneously. To this end, seven microorganisms (fungi and bacteria) previously isolated from sewage sludge were investigated to enhance their degradation performance. All seven strains were factorially mixed and used to assemble different artificial co-cultures. Consequently, 127 artificial co-cultures were established and ranked, based on their fitness performance, by using the BSocial analysis web tool. The individual strains were categorized according to their social behaviour, whose net effect over the remaining strains was defined as 'Positive', 'Negative' or 'Neutral'. To evaluate the emerging-pollutant degradation rate, the best 10 co-cultures, and those which contained the social strains were then challenged with three different Pharmaceutical Active compounds (PhACs): diclofenac, carbamazepine and ketoprofen. The co-cultures with the fungi Penicillium oxalicum XD-3.1 and Penicillium rastrickii were able to degrade PhACs. However, the highest performance (>80% degradation) was obtained by the minimal active microbial consortia consisting of both Penicillium spp., Cladosporium cladosporoides and co-existing bacteria. These consortia transformed the PhACs to derivate molecules through hydroxylation and were released to the media, resulting in a low ecotoxicity effect. High-throughput screening of co-cultures provides a quick, reliable and efficient method to narrow down suitable degradation co-cultures for emerging PhAC contaminants while avoiding toxic metabolic derivatives.

Comparative analysis of microbial DNA extraction protocols for groundwater samples.

A comparative analysis of four different DNA extraction protocols was performed to determine the best choice for groundwater microbial diversity studies using temperature gradient gel electrophoresis (TGGE) analysis. The methods used were a chelex-based method, a modified salting out procedure (MSOP), and the commercial kits Epicentre and FastDNA. Both commercial kits exhibited the greatest reproducibility in their methods; however, their band patterns were very different. The protocol that showed the highest diversity was the chelex-based method, and the one that showed the lowest diversity was the FastDNA kit.

Actinobacterial Degradation of 2-Hydroxyisobutyric Acid Proceeds via Acetone and Formyl-CoA by Employing a Thiamine-Dependent Lyase Reaction.

The tertiary branched short-chain 2-hydroxyisobutyric acid (2-HIBA) has been associated with several metabolic diseases and lysine 2-hydroxyisobutyrylation seems to be a common eukaryotic as well as prokaryotic post-translational modification in proteins. In contrast, the underlying 2-HIBA metabolism has thus far only been detected in a few microorganisms, such as the betaproteobacterium Aquincola tertiaricarbonis L108 and the Bacillus group bacterium Kyrpidia tusciae DSM 2912. In these strains, 2-HIBA can be specifically activated to the corresponding CoA thioester by the 2-HIBA-CoA ligase (HCL) and is then isomerized to 3-hydroxybutyryl-CoA in a reversible and B12-dependent mutase reaction. Here, we demonstrate that the actinobacterial strain Actinomycetospora chiangmaiensis DSM 45062 degrades 2-HIBA and also its precursor 2-methylpropane-1,2-diol via acetone and formic acid by employing a thiamine pyrophosphate-dependent lyase. The corresponding gene is located directly upstream of hcl, which has previously been found only in operonic association with the 2-hydroxyisobutyryl-CoA mutase genes in other bacteria. Heterologous expression of the lyase gene from DSM 45062 in E. coli established a 2-hydroxyisobutyryl-CoA lyase activity in the latter. In line with this, analysis of the DSM 45062 proteome reveals a strong induction of the lyase-HCL gene cluster on 2-HIBA. Acetone is likely degraded via hydroxylation to acetol catalyzed by a MimABCD-related binuclear iron monooxygenase and formic acid appears to be oxidized to CO2 by selenium-dependent dehydrogenases. The presence of the lyase-HCL gene cluster in isoprene-degrading Rhodococcus strains and Pseudonocardia associated with tropical leafcutter ant species points to a role in degradation of biogenic short-chain ketones and highly branched organic compounds.

Polyphasic study of microbial communities of two Spanish farmhouse goats' milk cheeses from Sierra de Aracena.

The microbial communities present in 2 different types of farmhouse goats' milk cheese from the Aracena mountains (southwest Spain), Quesailla Arochena (hard cheese) and Torta Arochena (soft cheese), have been studied using both culture-dependent and culture-independent techniques. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR and multiplex PCR. Thus a total of 26 different species were identified, the majority belonging to the lactic-acid bacteria (LAB), mainly represented by Lactococcus lactis and Lactobacillus species such as Lactobacillus plantarum and Lactobacillus paracasei, together with a significant proportion of enterococci. Amongst the non-lactic-acid bacteria (NLAB), which represented 37% of the isolates in Torta Arochena, enterobacteria were the most important, Hafnia alvei and Serratia liquefaciens being the predominant species in Quesailla Arochena and Torta Arochena respectively. Moreover, RAPD analysis of the isolates revealed that most of the genotypes were specific to one of the cheeses, although a few genotypes common to both cheeses were found. The culture-independent study carried out by temporal-temperature-gradient gel electrophoresis (TTGE) with 2 target genes, rRNA 16S and rpoB, revealed less species diversity but L. lactis and Lb. plantarum were also predominant. Nevertheless, TTGE carried out using RNAr 16S also detected some organisms that had not been isolated by the culture-dependent method, such as Leuconostoc lactis and Mycoplasma agalactie in Quesailla Arochena. Although TTGE of the rpoB gene revealed less species diversity, it did lead to the detection of previously non-isolated species, such as Ln. lactis in Quesailla Arochena. Apart from this, the fingerprinting of Lactobacillus populations by length-heterogeneity PCR showed the predominance of the Lb. plantarum group, followed by Lactobacillus curvatus and, in smaller quantities, Lb. paracasei in Torta Arochena. From our results we may conclude that both types of methods complement each other and offer a more complete vision of the microbial diversity of these ecosystems.

Social microbial inocula confer functional stability in a methyl tert-butyl ether extractive membrane biofilm bioreactor.

Methyl tert-butyl ether (MTBE) degradation technologies based on two-phase partitioning systems such as extractive membrane biofilm reactors (EMBFR) permit separation of biological and contaminant compartments, thus allowing optimization of the biological section. In this study, we set-up an EMBFR with three MTBE-degrading and cooperating strains (termed social biofilm: Agrobacterium sp. MS2, Paenibacillus etheri SH7T and Rhodococcus ruber EE6). The removal efficiency of the social-biofilm EMBFR was 80%, and functional stability was observed in the reactor, i.e. more efficient than previous studies (single-strain inoculated EMBFR, <50% removal efficiency and unstable function). Metabolite tert-butyl alcohol was not observed, and the EC50 values were higher than those observed in single-strain EMBFRs. Comparative analysis of the MTBE enzymatic pathway and the social-biofilm was performed, where the mechanism of cooperation observed within the social-biofilm is likely due to enzymatic redundancy. Functional outcomes were equal to previous batch tests, hence 100% scalability was obtained. Overall, higher functional and stability outcomes are obtained with the use of the social-biofilm in an MTBE-EMBFR.

Selection and identification of bacterial strains with methyl-tert-butyl ether, ethyl-tert-butyl ether, and tert-amyl methyl ether degrading capacities.

Nine bacterial strains isolated from two hydrocarbon-contaminated soils were selected because of their capacity for growth in culture media amended with 200 mg/L of one of the following gasoline oxygenates: Methyl-tert-butyl ether (MTBE), ethyl-tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). These strains were identified by amplification of their 16S rRNA gene, using fDl and rD1 primers, and were tested for their capacity to grow and biotransform these oxygenates in both mineral and cometabolic media. The isolates were classified as Bacillus simplex, Bacillus drentensis, Arthrobacter sp., Acinetobacter calcoaceticus, Acinetobacter sp., Gordonia amicalis (two strains), Nocardioides sp., and Rhodococcus ruber. Arthrobacter sp. (strain MG) and A. calcoaceticus (strain M10) consumed 100 (cometabolic medium) and 82 mg/L (mineral medium) of oxygenate TAME in 21 d, respectively, under aerobic conditions. Rhodococcus ruber (strain E10) was observed to use MTBE and ETBE as the sole carbon and energy source, whereas G. amicalis (strain T3) used TAME as the sole carbon and energy source for growth. All the bacterial strains transformed oxygenates better in the presence of an alternative carbon source (ethanol) with the exception of A. calcoaceticus (strain M10). The capacity of the selected strains to remove MTBE, ETBE, and TAME looks promising for application in bioremediation technologies.

Active site alanine preceding catalytic cysteine determines unique substrate specificity in bacterial CoA-acylating prenal dehydrogenase.

In detoxification and fermentation processes, acylating dehydrogenases catalyze the reversible oxidation of aldehydes to their corresponding acyl-CoA esters. Here, we characterize an enzyme from Aquincola tertiaricarbonis L108 responsible for prenal (3-methyl-2-butenal) to 3-methylcrotonyl-CoA oxidation. Enzyme kinetics demonstrate a preference for C5 substrates not yet observed in aldehyde dehydrogenases. Compared to acetaldehyde and acetyl-CoA, conversion of valeraldehyde and valeryl-CoA is > 100- and 8-fold more efficient, respectively. Enzyme variants with A254I, A254P, and A254G mutations indicate that active site Ala preceding the catalytic C255 is crucial for this unique specificity. These results shed new light on evolutionary adaptation of aldehyde dehydrogenases toward xenobiotics and structure-guided design of highly specific enzymes for production of biofuels, such as linear or iso-branched butanols and pentanols.

BSocial: Deciphering Social Behaviors within Mixed Microbial Populations.

Ecosystem functionality depends on interactions among populations, of the same or different taxa, and these are not just the sum of pairwise interactions. Thus, know-how of the social interactions occurring in mixed-populations are of high interest, however they are commonly unknown due to the limitations posed in tagging each population. The limitations include costs/time in tediously fluorescent tagging, and the number of different fluorescent tags. Tag-free strategies exist, such as high-throughput sequencing, but ultimately both strategies require the use of expensive machinery. Our work appoints social behaviors on individual strains in mixed-populations, offering a web-tool (BSocial http://m4m.ugr.es/BSocial.html) for analyzing the community framework. Our quick and cheap approach includes the periodic monitoring of optical density (OD) from a full combinatorial testing of individual strains, where number of generations and growth rate are determined. The BSocial analyses then enable us to determine how the addition/absence of a particular species affects the net productivity of a microbial community and use this to select productive combinations, i.e., designate their social effect on a general community. Positive, neutral, or negative assignations are applied to describe the social behavior within the community by comparing fitness effects of the community against the individual strain. The usefulness of this tool for selection of optimal inoculum in biofilm-based methyl tert-butyl ether (MTBE) bioremediation was demonstrated. The studied model uses seven bacterial strains with diverse MTBE degradation/growth capacities. Full combinatorial testing of seven individual strains (triplicate tests of 127 combinations) were implemented, along with MTBE degradation as the desired function. Sole observation of highest species fitness did not render the best functional outcome, and only when strains with positive and neutral social assignations were mixed (Rhodococcus ruber EE6, Agrobacterium sp. MS2 and Paenibacillus etheri SH7), was this obtained. Furthermore, the use of positive and neutral strains in all its combinations had a significant higher degradation mean (x1.75) than exclusive negative strain combinations. Thus, social microbial processes benefit bioremediation more than negative social microbial combinations. The BSocial webtool is a great contributor to the study of social interactions in bioremediation processes, and may be used in other natural or synthetic habitat studies.

Draft Genome Sequence of Paenibacillus etheri sp. nov. SH7T, a Methyl Tert-Butyl Ether Degrader.

We report here the draft genome sequence of Paenibacillus etheri sp. nov. SH7(T) (= CECT 8558(T) = DSM 29760(T)), isolated from a hydrocarbon-contaminated soil pilot plant in Granada, Spain. The bacterium was isolated and sequenced due to its methyl tert-butyl ether (MTBE)-degrading properties.

Distribution and microbial community structure analysis of a single-stage partial nitritation/anammox granular sludge bioreactor operating at low temperature.

In the last decade, autotrophic nitrogen removal technologies based on anammox metabolism have become state of the art in urban and industrial wastewater treatment systems, due to their advantages over traditional nitrogen removal processes. However, their application is currently limited to the treatment of warm wastewater (25-40°C) mainly due to the low growth rate of the anammox bacteria. The extension of the application field to wastewater characterized by lower temperatures (8-20°C), such as those typical for municipal sewage, allows the design of treatment systems with a net energy production. In this study, the distribution and bacterial community structure of a lab-scale single-stage partial nitritation/anammox (PN/A) granular sludge bioreactor operating at low temperatures was analysed using next-generation sequencing techniques. The presence of ammonium-oxidizing bacteria and anammox bacteria was found, but the appearance of other bacterial species shows a complex microbial ecosystem. Evaluation of ecological roles of representative species inside the single-stage PN/A bioreactor was accomplished. Results obtained will be helpful for the future design and operation of PN/A systems performing at low temperatures.

A novel pair of inducible expression vectors for use in Methylobacterium extorquens.

BACKGROUND: Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used γ-proteobacteria, such as Escherichia coli or Pseudomonas species. To this end, we have sought to develop a pair of inducible expression vectors for use in the α-proteobacterium Methylobacterium extorquens, a model methylotroph. FINDINGS: We found that the P(R) promoter from rhizobial phage 16-3 was active in M. extorquens and engineered the promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybrid promoters, P(R/cmtO) and P(R/tetO), were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively. Compared to an existing cumate-inducible (10-fold range), high-level expression system for M. extorquens, P(R/cmtO) and P(R/tetO) have 33% of the maximal activity but were able to repress gene expression 3 and 8-fold greater, respectively. Both promoters were observed to exhibit homogeneous, titratable activation dynamics rather than on-off, switch-like behavior. The utility of these promoters was further demonstrated by complementing loss of function of ftfL--essential for growth on methanol--where we show P(R/tetO) is capable of not only fully complementing function but also producing a conditional null phenotype. These promoters have been incorporated into a broad-host-range backbone allowing for potential use in a variety of bacterial hosts. CONCLUSIONS: We have developed two novel expression systems for use in M. extorquens. The expression range of these vectors should allow for increased ability to explore cellular physiology in M. extorquens. Further, the P(R/tetO) promoter is capable of producing conditional null phenotypes, previously unattainable in M. extorquens. As both expression systems rely on the use of membrane permeable inducers, we suspect these expression vectors will be useful for ectopic gene expression in numerous proteobacteria.

Biofilm formation and microbial activity in a biofilter system in the presence of MTBE, ETBE and TAME.

Emerging water contaminants derived from unleaded gasoline such as methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), are in need of effective bioremediation technologies for restoring water resources. In order to design the conditions of a future groundwater bioremediating biofilter, this work assesses the potential use of Acinetobacter calcoaceticus M10, Rhodococcus ruber E10 and Gordonia amicalis T3 for the removal of MTBE, ETBE and TAME in consortia or as individual strains. Biofilm formation on an inert polyethylene support material was assessed with scanning electron microscopy, and consortia were also analysed with fluorescent in situ hybridisation to examine the relation between the strains. A. calcoaceticus M10 was the best coloniser, followed by G. amicalis T3, however, biofilm formation of pair consortia favoured consortium M10-E10 both in formation and activity. However, degradation batch studies determined that neither consortium exhibited higher degradation than individual strain degradation. The physiological state of the three strains was also determined through flow cytometry using propidium iodide and 3'-dihexylocarbocyanine iodide thus gathering information on their viability and activity with the three oxygenates since previous microbial counts revealed slow growth. Strain E10 was observed to have the highest physiological activity in the presence of MTBE, and strain M10 activity with TAME was only maintained for 24 h, thus we believe that biotransformation of MTBE occurs within the active periods established by the cytometry analyses. Viable cell counts and oxygenate removal were determined in the presence of the metabolites tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA), resulting in TBA biotransformation by M10 and E10, and TAA by M10. Our results show that A. calcoaceticus M10 and the consortium M10-E10 could be adequate inocula in MTBE and TAME bioremediating technologies.

Yeast GTB1 encodes a subunit of glucosidase II required for glycoprotein processing in the endoplasmic reticulum.

Glucosidase II is essential for sequential removal of two glucose residues from N-linked glycans during glycoprotein biogenesis in the endoplasmic reticulum. The enzyme is a heterodimer whose alpha-subunit contains the glycosyl hydrolase active site. The function of the beta-subunit has yet to be defined, but mutations in the human gene have been linked to an autosomal dominant form of polycystic liver disease. Here we report the identification and characterization of a Saccharomyces cerevisiae gene, GTB1, encoding a polypeptide with 21% sequence similarity to the beta-subunit of human glucosidase II. The Gtb1 protein was shown to be a soluble glycoprotein (96-102 kDa) localized to the endoplasmic reticulum lumen where it was present in a complex together with the yeast alpha-subunit homologue Gls2p. Surprisingly, we found that Deltagtb1 mutant cells were specifically defective in the processing of monoglucosylated glycans. Thus, although Gls2p is sufficient for cleavage of the penultimate glucose residue, Gtb1p is essential for cleavage of the final glucose. Our data demonstrate that Gtb1p is required for normal glycoprotein biogenesis and reveal that the final two glucose-trimming steps in N-glycan processing are mechanistically distinct.