Jochen A. Müller

Molecular Identification of the Catabolic Vinyl Chloride Reductase from Dehalococcoides sp. Strain VS and Its Environmental Distribution

ABSTRACT Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene (vcrA) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.

Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi.

Six obligately anaerobic bacterial isolates (195(T), CBDB1, BAV1, VS, FL2 and GT) with strictly organohalide-respiring metabolisms were obtained from chlorinated solvent-contaminated aquifers, contaminated and uncontaminated river sediments or anoxic digester sludge. Cells were non-motile with a disc-shaped morphology, 0.3-1 µm in diameter and 0.1-0.2 µm thick, and characteristic indentations on opposite flat sides of the cell. Growth occurred in completely synthetic, reduced medium amended with a haloorganic electron acceptor (mostly chlorinated but also some brominated compounds), hydrogen as electron donor, acetate as carbon source, and vitamins. No other growth-supporting redox couples were identified. Aqueous hydrogen consumption threshold concentrations were <1 nM. Growth ceased when vitamin B(12) was omitted from the medium. Addition of sterile cell-free supernatant of Dehalococcoides-containing enrichment cultures enhanced dechlorination and growth of strains 195 and FL2, suggesting the existence of so-far unidentified stimulants. Dechlorination occurred between pH 6.5 and 8.0 and over a temperature range of 15-35 °C, with an optimum growth temperature between 25 and 30 °C. The major phospholipid fatty acids were 14 : 0 (15.7 mol%), br15 : 0 (6.2 mol%), 16 : 0 (22.7 mol%), 10-methyl 16 : 0 (25.8 mol%) and 18 : 0 (16.6 mol%). Unusual furan fatty acids including 9-(5-pentyl-2-furyl)-nonanoate and 8-(5-hexyl-2-furyl)-octanoate were detected in strains FL2, BAV1 and GT, but not in strains 195(T) and CBDB1. The 16S rRNA gene sequences of the six isolates shared more than 98 % identity, and phylogenetic analysis revealed an affiliation with the phylum Chloroflexi and more than 10 % sequence divergence from other described isolates. The genome sizes and G+C contents ranged from 1.34 to 1.47 Mbp and 47 to 48.9 mol% G+C, respectively. Based on 16S rRNA gene sequence comparisons, genome-wide average nucleotide identity and phenotypic characteristics, the organohalide-respiring isolates represent a new genus and species, for which the name Dehalococcoides mccartyi gen. nov., sp. nov. is proposed. Isolates BAV1 ( = ATCC BAA-2100  = JCM 16839  = KCTC 5957), FL2 ( = ATCC BAA-2098  = DSM 23585  = JCM 16840  = KCTC 5959), GT ( = ATCC BAA-2099  = JCM 16841  = KCTC 5958), CBDB1, 195(T) ( = ATCC BAA-2266(T)  = KCTC 15142(T)) and VS are considered strains of Dehalococcoides mccartyi, with strain 195(T) as the type strain. The new class Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov. are described to accommodate the new taxon.

Genomic analysis of anaerobic respiration in the archaeon Halobacterium sp. strain NRC-1: dimethyl sulfoxide and trimethylamine N-oxide as terminal electron acceptors.

We have investigated anaerobic respiration of the archaeal model organism Halobacterium sp. strain NRC-1 by using phenotypic and genetic analysis, bioinformatics, and transcriptome analysis. NRC-1 was found to grow on either dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as the sole terminal electron acceptor, with a doubling time of 1 day. An operon, dmsREABCD, encoding a putative regulatory protein, DmsR, a molybdopterin oxidoreductase of the DMSO reductase family (DmsEABC), and a molecular chaperone (DmsD) was identified by bioinformatics and confirmed as a transcriptional unit by reverse transcriptase PCR analysis. dmsR, dmsA, and dmsD in-frame deletion mutants were individually constructed. Phenotypic analysis demonstrated that dmsR, dmsA, and dmsD are required for anaerobic respiration on DMSO and TMAO. The requirement for dmsR, whose predicted product contains a DNA-binding domain similar to that of the Bat family of activators (COG3413), indicated that it functions as an activator. A cysteine-rich domain was found in the dmsR gene, which may be involved in oxygen sensing. Microarray analysis using a whole-genome 60-mer oligonucleotide array showed that the dms operon is induced during anaerobic respiration. Comparison of dmsR+ and DeltadmsR strains by use of microarrays showed that the induction of the dmsEABCD operon is dependent on a functional dmsR gene, consistent with its action as a transcriptional activator. Our results clearly establish the genes required for anaerobic respiration using DMSO and TMAO in an archaeon for the first time.

Initiation of Anaerobic Degradation of p-Cresol by Formation of 4-Hydroxybenzylsuccinate in Desulfobacterium cetonicum

The anaerobic bacterium Desulfobacterium cetonicum oxidized p-cresol completely to CO(2) with sulfate as the electron acceptor. During growth, 4-hydroxybenzylsuccinate accumulated in the medium. This finding indicated that the methyl group of p-cresol is activated by addition to fumarate, analogous to anaerobic toluene, m-xylene, and m-cresol degradation. In cell extracts, the formation of 4-hydroxybenzylsuccinate from p-cresol and fumarate was detected at an initial rate of 0.57 nmol min(-1) (mg of protein)(-1). This activity was specific for extracts of p-cresol-grown cells. 4-Hydroxybenzylsuccinate was degraded further to 4-hydroxybenzoyl-coenzyme A (CoA), most likely via beta-oxidation. 4-Hydroxybenzoyl-CoA was reductively dehydroxylated to benzoyl-CoA. There was no evidence of degradation of p-cresol via methyl group oxidation by p-cresol-methylhydroxylase in this bacterium.

Extremely Radiation-Resistant Mutants of a Halophilic Archaeon with Increased Single-Stranded DNA-Binding Protein (RPA) Gene Expression

Abstract DeVeaux, L. C., Müller, J. A., Smith, J. R., Petrisko, J., Wells, D. P. and DasSarma, S. Extremely Radiation-Resistant Mutants of a Halophilic Archaeon with Increased Single-Stranded DNA-Binding Protein (RPA) Gene Expression. Radiat. Res. 168, 507–514 (2007). Extremely halophilic archaea are highly resistant to multiple stressors, including radiation, desiccation and salinity. To study the basis of stress resistance and determine the maximum tolerance to ionizing radiation, we exposed cultures of the model halophile Halobacterium sp. NRC-1 to four cycles of irradiation with high doses of 18–20 MeV electrons. Two independently obtained mutants displayed an LD50 > 11 kGy, which is higher than the LD50 of the extremely radiation-resistant bacterium Deinococcus radiodurans. Whole-genome transcriptome analysis comparing the mutants to the parental wild-type strain revealed up-regulation of an operon containing two single-stranded DNA-binding protein (RPA) genes, VNG2160 (rfa3) and VNG2162, and a third gene of unknown function, VNG2163. The putative transcription start site for the rfa3 operon was mapped ∼40 bp upstream of the ATG start codon, and a classical TATA-box motif was found centered about 25 bp further upstream. We propose that RPA facilitates DNA repair machinery and/or protects repair intermediates to maximize the ionizing radiation resistance of this archaeon.

In-situ biodegradation of tetrachloroethene and trichloroethene in contaminated aquifers monitored by stable isotope fractionation.

Stable carbon isotope analysis of tetrachloroethene (PCE) and trichloroethene (TCE) was applied to evaluate natural attenuation processes in the upper Quaternary and lower Tertiary aquifer in the area of a former dry-cleaning plant located in Leipzig, Germany. Groundwater samples were taken during one monitoring campaign in 2001. The 13C enrichment in contaminants along the water flow path suggested that both, PCE and TCE were degraded in the Quaternary aquifer. The enrichment of 13C in the residual PCE fraction and an isotope fractionation factor from laboratory experiments were used to calculate the extent of biodegradation in the Quaternary aquifer. These calculations indicated that a major portion of PCE was biodegraded in the course of the plume. In the Tertiary aquifer the carbon isotope ratios of PCE and TCE indicated that the decreasing concentrations of these contaminants were probably not caused by microbial processes.

ipso-Hydroxylation and subsequent fragmentation — a novel microbial strategy to eliminate sulfonamide antibiotics

ABSTRACT Sulfonamide antibiotics have a wide application range in human and veterinary medicine. Because they tend to persist in the environment, they pose potential problems with regard to the propagation of antibiotic resistance. Here, we identified metabolites formed during the degradation of sulfamethoxazole and other sulfonamides in Microbacterium sp. strain BR1. Our experiments showed that the degradation proceeded along an unusual pathway initiated by ipso-hydroxylation with subsequent fragmentation of the parent compound. The NADH-dependent hydroxylation of the carbon atom attached to the sulfonyl group resulted in the release of sulfite, 3-amino-5-methylisoxazole, and benzoquinone-imine. The latter was concomitantly transformed to 4-aminophenol. Sulfadiazine, sulfamethizole, sulfamethazine, sulfadimethoxine, 4-amino-N-phenylbenzenesulfonamide, and N-(4-aminophenyl)sulfonylcarbamic acid methyl ester (asulam) were transformed accordingly. Therefore, ipso-hydroxylation with subsequent fragmentation must be considered the underlying mechanism; this could also occur in the same or in a similar way in other studies, where biotransformation of sulfonamides bearing an amino group in the para-position to the sulfonyl substituent was observed to yield products corresponding to the stable metabolites observed by us.

Fermentative degradation of 3-hydroxybenzoate in pure culture by a novel strictly anaerobic bacterium, Sporotomaculum hydroxybenzoicum gen. nov., sp. nov.

A strictly anaerobic bacterium, strain BT, from termite hindgut homogenates, was isolated in pure culture and grew on 3-hydroxybenzoate as sole source of carbon and energy. No other substrate tested was degraded, sulfate, sulfite, thiosulfate, nitrate, ferric iron, oxygen or fumarate were not reduced, and no electron transfer to partner organisms was observed. 3-Hydroxybenzoate was fermented to butyrate, acetate and CO2. Benzoate was detected in the culture supernatant as an intermediate. The isolate was a slightly motile, endosporeforming Gram-positive rod; 16S rDNA sequence analysis revealed a high similarity to members of the genus Desulfotomaculum. The G + C content of the DNA was 48 mol%. Strain BT differs from the members of the genus Desulfotomaculum significantly due to its lack of dissimilatory sulfate reduction, and is therefore described as the type strain of a new genus and species. Sporotomaculum hydroxybenzoicum gen. nov., sp. nov.

Sanitation in constructed wetlands: A review on the removal of human pathogens and fecal indicators

Removal of human pathogens from wastewater is a critical factor with linkage to human health. Constructed Wetlands (CWs) are environmental friendly ecosystems that are applicable not only for chemical pollution control, but also for the reduction of pathogens from wastewater. Yet the knowledge on the fate and removal of such indicator bacteria in CWs is still not sufficient due to the complexity of removal mechanisms and influencing factors. This review serves to provide a better understanding of this state-of-the-art technology, which is necessary for further investigations and design development. The fecal indicator bacteria in CWs mainly come from three sources, namely, influent wastewaters, regrowth within the CWs, and animal activities. The properties of microbial contamination vary depending on the different sources. The removal of pathogens is a complex process that is influenced by operational parameters such as hydraulic regime and retention time, vegetation, seasonal fluctuation, and water composition. The most frequent and well-validated removal mechanisms include natural die-off due to starvation or predation, sedimentation and filtration, and adsorption. The concentration of the main fecal indicator bacteria in the effluent was found to be exponentially related to the loading rate. Generally, horizontal subsurface flow CWs have better reduction capacity than free water surface flow CWs, and hybrid wetland systems were found to be the most efficient due to a longer retention time. Further improvement of fecal indicator bacteria removal in CWs is needed, however, levels in CW effluents are still higher than most of the regulation standards for reuse.

In situ protein-SIP highlights Burkholderiaceae as key players degrading toluene by para ring hydroxylation in a constructed wetland model

In constructed wetlands, organic pollutants are mainly degraded via microbial processes. Helophytes, plants that are commonly used in these systems, provide oxygen and root exudates to the rhizosphere, stimulating microbial degradation. While the treatment performance of constructed wetlands can be remarkable, a mechanistic understanding of microbial degradation processes in the rhizosphere is still limited. We investigated microbial toluene removal in a constructed wetland model system combining 16S rRNA gene sequencing, metaproteomics and (13) C-toluene in situ protein-based stable isotope probing (protein-SIP). The rhizospheric bacterial community was dominated by Burkholderiales and Rhizobiales, each contributing about 20% to total taxon abundance. Protein-SIP data revealed that the members of Burkholderiaceae, the proteins of which showed about 73% of (13) C-incorporation, were the main degraders of toluene in the planted system, while the members of Comamonadaceae were involved to a lesser extent in degradation (about 64% (13) C-incorporation). Among the Burkholderiaceae, one of the key players of toluene degradation could be assigned to Ralstonia pickettii. We observed that the main pathway of toluene degradation occurred via two subsequent monooxygenations of the aromatic ring. Our study provides a suitable approach to assess the key processes and microbes that are involved in the degradation of organic pollutants in complex rhizospheric ecosystems.