Interesting anaerobes in the environment

Prokaryotes (Bacteria and Archaea) have a wide range of capacities to survive by generating energy in environments and situations lacking oxygen, which abound on Earth. Anaerobic metabolic strategies include anaerobic respiration (numerous types – e.g. nitrate reduction – Paracoccus denitrificans; sulfur respiration – Desulfuromonadales; methanogenesis – Methanosarsina spp.; iron reduction – Geobacter spp.; dehalorespiration – Dehalococcoides ethenogenes) and fermentation (sugars converted to simpler organic compounds like acids, gases and alcohols – e.g. Lactobacillus spp.). Relatively novel environmental anaerobic strategies include anaerobic ammonium oxidation (Anammox – e.g. Brocadia spp.) and anaerobic methane oxidation (AMO)1, which is a syntrophic association between anaerobic methanotrophic archaea (ANME) and sulfate-, iron-, manganese- or nitrate-reducing bacteria2. The classic anaerobic synthrophic example is interspecies hydrogen/formate transfer between a hydrogen/formate producing fatty acid oxidising bacterium (the syntroph) and a hydrogen/formate consumer (methanogen or sulfate-reducer)3. Microbes vary in their oxygen tolerance and are described as obligate anaerobes if they are killed by atmospheric levels of oxygen due to the lack of catalase and superoxide dismutase that provide oxygen radical protection.

Global Transcriptomic and Proteomic Responses of Dehalococcoides ethenogenes Strain 195 to Fixed Nitrogen Limitation

ABSTRACTBacteria of the genusDehalococcoidesplay an important role in the reductive dechlorination of chlorinated ethenes. A systems-level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing cells ofDehalococcoides ethenogenesstrain 195 to fixed nitrogen limitation (FNL), as dechlorination activity and cell yield both decrease during FNL. As expected, the nitrogen-fixing (nif) genes were differentially upregulated in the transcriptome and proteome of strain 195 during FNL. Aside from thenifoperon, a putative methylglyoxal synthase-encoding gene (DET1576), the product of which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and is implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly upregulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally downregulated in response to FNL, and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition, while others were associated with general stress responses. A number of genes related to the functions of nucleotide synthesis, replication, transcription, translation, and posttranslational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number of genes coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate environmental cellular nitrogen status.

Selective Utilization of Exogenous Amino Acids by Dehalococcoides ethenogenes Strain 195 and Its Effects on Growth and Dechlorination Activity

ABSTRACTBacteria of the genusDehalococcoidesare important members of bioremediation communities because of their ability to detoxify chloroethenes to the benign end product ethene. Genome-enabled studies conducted withDehalococcoides ethenogenes195 have revealed that two ATP-binding cassette (ABC)-type amino acid transporters are expressed during its exponential growth stages. In light of previous findings that Casamino Acids enhanced its dechlorination activity, we hypothesized that strain 195 is capable of importing amino acids from its environment to facilitate dechlorination and growth. To test this hypothesis, we applied isotopomer-based dilution analysis with13C-labeled acetate to differentiate the amino acids that were taken up by strain 195 from those synthesizedde novoand to determine the physiological changes caused by the significantly incorporated amino acids. Our results showed that glutamate/glutamine and aspartate/asparagine were almost exclusively synthesized by strain 195, even when provided in excess in the medium. In contrast, phenylalanine, isoleucine, leucine, and methionine were identified as the four most highly incorporated amino acids, at levels >30% of respective proteinogenic amino acids. When either phenylalanine or all four highly incorporated amino acids were added to the defined mineral medium, the growth rates, dechlorination activities, and yields of strain 195 were enhanced to levels similar to those observed with supplementation with 20 amino acids. However, genes for the putative ABC-type amino acids transporters and phenylalanine biosynthesis exhibited insignificant regulation in response to the imported amino acids. This study also demonstrates that using isotopomer-based metabolite analysis can be an efficient strategy for optimizing nutritional conditions for slow-growing microorganisms.

Evidence for Nitrogen Fixation by “Dehalococcoides ethenogenes” Strain 195

ABSTRACT Genome annotation of the chlorinated ethene-respiring “Dehalococcoides ethenogenes” strain 195 indicated the presence of a complete nitrogenase operon. Here, results from long-term growth experiments, gene expression, and 15N2-isotope measurements confirm that strain 195 is capable of fixing atmospheric dinitrogen when a defined fixed-nitrogen source such as ammonium is unavailable.

Isolation and Characterization of “Dehalococcoides” sp. Strain MB, Which Dechlorinates Tetrachloroethene to trans-1,2-Dichloroethene

ABSTRACT In an attempt to understand the microorganisms involved in the generation of trans-1,2-dichloroethene (trans-DCE), pure-culture “Dehalococcoides” sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate cis-DCE as the predominant product, Dehalococcoides sp. strain MB reductively dechlorinates PCE to trans-DCE and cis-DCE at a ratio of 7.3 (±0.4):1. It utilizes H2 as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 μm in diameter and ∼150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with Dehalococcoides ethenogenes strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of trans-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group, Dehalococcoides species.

Investigation of Carbon Metabolism in “Dehalococcoides ethenogenes” Strain 195 by Use of Isotopomer and Transcriptomic Analyses

ABSTRACT Members of the genus “Dehalococcoides” are the only known microorganisms that can completely dechlorinate tetrachloroethene and trichloroethene to the innocuous end product, ethene. This study examines the central metabolism in “Dehalococcoides ethenogenes” strain 195 via 13C-labeled tracer experiments. Supported by the genome annotation and the transcript profile, isotopomer analysis of key metabolites clarifies ambiguities in the genome annotation and identifies an unusual biosynthetic pathway in strain 195. First, the 13C-labeling studies revealed that strain 195 contains complete amino acid biosynthesis pathways, even though current genome annotation suggests that several of these pathways are incomplete. Second, the tricarboxylic acid cycle of strain 195 is confirmed to be branched, and the Wood-Ljungdahl carbon fixation pathway is shown to not be functionally active under our experimental conditions; rather, CO2 is assimilated via two reactions, conversion of acetyl-coenzyme A (acetyl coenzyme A [acetyl-CoA]) to pyruvate catalyzed by pyruvate synthase (DET0724-0727) and pyruvate conversion to oxaloacetate via pyruvate carboxylase (DET0119-0120). Third, the 13C-labeling studies also suggested that isoleucine is synthesized from acetyl-CoA and pyruvate via citramalate synthase (CimA, EC 2.3.1.182), rather than from the common pathway via threonine ammonia-lyase (EC 4.3.1.19). Finally, evidence is presented that strain 195 may contain an undocumented citrate synthase (>95% Re-type stereospecific), i.e., a novel Re-citrate synthase that is apparently different from the one recently reported in Clostridium kluyveri.

Comparative Genomics of “Dehalococcoides ethenogenes” 195 and an Enrichment Culture Containing Unsequenced “Dehalococcoides” Strains

ABSTRACT Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by some “Dehalococcoides” organisms. A Dehalococcoides-organism-containing microbial consortium (referred to as ANAS) with the ability to degrade TCE to ethene, an innocuous end product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of “Dehalococcoides ethenogenes” 195. This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray results revealed that the genes associated with central metabolism, including an apparently incomplete carbon fixation pathway, cobalamin-salvaging system, nitrogen fixation pathway, and five hydrogenase complexes, are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase, tceA, was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of Dehalococcoides genomes will improve our understanding of the physiology and evolution of these bacteria, which is essential in developing effective strategies for the bioremediation of PCE and TCE in the environment.