Metaproteomics reveals methyltransferases implicated in dichloromethane and glycine betaine fermentation by 'Candidatus Formimonas warabiya' strain DCMF.

Dichloromethane (DCM; CH2Cl2) is a widespread pollutant with anthropogenic and natural sources. Anaerobic DCM-dechlorinating bacteria use the Wood-Ljungdahl pathway, yet dechlorination reaction mechanisms remain unclear and the enzyme(s) responsible for carbon-chlorine bond cleavage have not been definitively identified. Of the three bacterial taxa known to carry out anaerobic dechlorination of DCM, 'Candidatus Formimonas warabiya' strain DCMF is the only organism that can also ferment non-chlorinated substrates, including quaternary amines (i.e., choline and glycine betaine) and methanol. Strain DCMF is present within enrichment culture DFE, which was derived from an organochlorine-contaminated aquifer. We utilized the metabolic versatility of strain DCMF to carry out comparative metaproteomics of cultures grown with DCM or glycine betaine. This revealed differential abundance of numerous proteins, including a methyltransferase gene cluster (the mec cassette) that was significantly more abundant during DCM degradation, as well as highly conserved amongst anaerobic DCM-degrading bacteria. This lends strong support to its involvement in DCM dechlorination. A putative glycine betaine methyltransferase was also discovered, adding to the limited knowledge about the fate of this widespread osmolyte in anoxic subsurface environments. Furthermore, the metagenome of enrichment culture DFE was assembled, resulting in five high quality and two low quality draft metagenome-assembled genomes. Metaproteogenomic analysis did not reveal any genes or proteins for utilization of DCM or glycine betaine in the cohabiting bacteria, supporting the previously held idea that they persist via necromass utilization.

Evaluating Enzymatic Productivity-The Missing Link to Enzyme Utility.

Kinetic productivity analysis is critical to the characterization of enzyme catalytic performance and capacity. However, productivity analysis has been largely overlooked in the published literature. Less than 0.01% of studies which report on enzyme characterization present productivity analysis, despite the fact that this is the only measurement method that provides a reliable indicator of potential commercial utility. Here, we argue that reporting productivity data involving native, modified, and immobilized enzymes under different reaction conditions will be of immense value in optimizing enzymatic processes, with a view to accelerating biotechnological applications. With the use of examples from wide-ranging studies, we demonstrate that productivity is a measure of critical importance to the translational and commercial use of enzymes and processes that employ them. We conclude the review by suggesting steps to maximize the productivity of enzyme catalyzed reactions.

The use of isothermal titration calorimetry for the assay of enzyme activity: Application in higher education practical classes.

Determination of enzyme activity is crucial for discovery, research, and development in life sciences. The activity of enzymes is routinely determined using spectrophotometric assays that measure rates of substrate consumption or product formation. Though colorimetric-based detection systems are simple, rapid, and economical to perform, the majority of enzymes are unsuitable for this technique as their substrates/products do not absorb in the UV or visible range. This limitation can be addressed by the use of coupled-enzyme assays or artificial chromogenic substrates; however these approaches have their own drawbacks. Here, we describe a method based on the use of an isothermal titration calorimeter (ITC) to measure the heat produced or absorbed during any enzyme-catalyzed reaction. The concept of calorimetric enzyme assays was demonstrated for the determination of enzyme hexokinase activity, which cannot be monitored colorimetrically without first coupling it to another enzymatic reaction. The assay is suitable for incorporation into undergraduate laboratory classes, providing students with an appreciation for; the versatility and ease of use of ITC assays; ITC as a flexible generic method for exploring the functional characteristics of uncharacterized enzymes; an activity detection parameter suitable for enzymes that either have no straightforward colorimetric methods available or require the use of nonartificial chromogenic substrates.

Novel dichloromethane-fermenting bacteria in the Peptococcaceae family.

Dichloromethane (DCM; CH2Cl2) is a toxic groundwater pollutant that also has a detrimental effect on atmospheric ozone levels. As a dense non-aqueous phase liquid, DCM migrates vertically through groundwater to low redox zones, yet information on anaerobic microbial DCM transformation remains scarce due to a lack of cultured organisms. We report here the characterisation of DCMF, the dominant organism in an anaerobic enrichment culture (DFE) capable of fermenting DCM to the environmentally benign product acetate. Stable carbon isotope experiments demonstrated that the organism assimilated carbon from DCM and bicarbonate via the Wood-Ljungdahl pathway. DCMF is the first anaerobic DCM-degrading population also shown to metabolise non-chlorinated substrates. It appears to be a methylotroph utilising the Wood-Ljungdahl pathway for metabolism of methyl groups from methanol, choline, and glycine betaine. The flux of these substrates from subsurface environments may either directly (DCM, methanol) or indirectly (choline, glycine betaine) affect the climate. Community profiling and cultivation of cohabiting taxa in culture DFE without DCMF suggest that DCMF is the sole organism in this culture responsible for substrate metabolism, while the cohabitants persist via necromass recycling. Genomic and physiological evidence support placement of DCMF in a novel genus within the Peptococcaceae family, 'Candidatus Formimonas warabiya'.

Whole genome sequencing of a novel, dichloromethane-fermenting Peptococcaceae from an enrichment culture.

Bacteria capable of dechlorinating the toxic environmental contaminant dichloromethane (DCM, CH2Cl2) are of great interest for potential bioremediation applications. A novel, strictly anaerobic, DCM-fermenting bacterium, "DCMF", was enriched from organochlorine-contaminated groundwater near Botany Bay, Australia. The enrichment culture was maintained in minimal, mineral salt medium amended with dichloromethane as the sole energy source. PacBio whole genome SMRTTM sequencing of DCMF allowed de novo, gap-free assembly despite the presence of cohabiting organisms in the culture. Illumina sequencing reads were utilised to correct minor indels. The single, circularised 6.44 Mb chromosome was annotated with the IMG pipeline and contains 5,773 predicted protein-coding genes. Based on 16S rRNA gene and predicted proteome phylogeny, the organism appears to be a novel member of the Peptococcaceae family. The DCMF genome is large in comparison to known DCM-fermenting bacteria. It includes an abundance of methyltransferases, which may provide clues to the basis of its DCM metabolism, as well as potential to metabolise additional methylated substrates such as quaternary amines. Full annotation has been provided in a custom genome browser and search tool, in addition to multiple sequence alignments and phylogenetic trees for every predicted protein, http://www.slimsuite.unsw.edu.au/research/dcmf/.

Isolation and Characterization of Dehalobacter sp. Strain TeCB1 Including Identification of TcbA: A Novel Tetra- and Trichlorobenzene Reductive Dehalogenase.

Dehalobacter sp. strain TeCB1 was isolated from groundwater near Sydney, Australia, that is polluted with a range of organochlorines. The isolated strain is able to grow by reductive dechlorination of 1,2,4,5-tetrachlorobenzene to 1,3- and 1,4-dichlorobenzene with 1,2,4-trichlorobenzene being the intermediate daughter product. Transient production of 1,2-dichlorobenzene was detected with subsequent conversion to monochlorobenzene. The dehalogenation capability of strain TeCB1 to respire 23 alternative organochlorines was examined and shown to be limited to the use of 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene. Growth on 1,2,4-trichlorobenzene resulted in the production of predominantly 1,3- and 1,4-dichlorobenzene. The inability of strain TeCB1 to grow on 1,2-dichlorobenzene indicated that the production of monochlorobenzene during growth on 1,2,4,5-tetarchlorobezene was cometabolic. The annotated genome of strain TeCB1 contained only one detectable 16S rRNA gene copy and genes for 23 full-length and one truncated Reductive Dehalogenase (RDase) homologs, five unique to strain TeCB1. Identification and functional characterization of the 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene RDase (TcbA) was achieved using native-PAGE coupled with liquid chromatography tandem mass spectrometry. Interestingly, TcbA showed higher amino acid identity with tetrachloroethene reductases PceA (95% identity) from Dehalobacter restrictus PER-K23 and Desulfitobacterium hafniense Y51 than with the only other chlorinated benzene reductase [i.e., CbrA (30% identity)] functionally characterized to date.

Genome Sequence of Dehalobacter sp. Strain TeCB1, Able To Respire Chlorinated Benzenes.

Dehalobacter sp. strain TeCB1 was isolated from groundwater contaminated with a mixture of organohalides and is able to respire 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene. Here, we report its 3.13-Mb draft genome sequence.

Genomic, transcriptomic and proteomic analyses of Dehalobacter UNSWDHB in response to chloroform.

Organohalide respiring bacteria (ORB) are capable of utilising organohalides as electron acceptors for the generation of cellular energy and consequently play an important role in the turnover of natural and anthropogenically-derived organohalides. In this study, the response of a Dehalobacter sp. strain UNSWDHB to the addition of trichloromethane (TCM) after a 50 h period of its absence (suffocation) was evaluated from a transcriptomic and proteomic perspective. The up-regulation of TCM reductive dehalogenase genes (tmrABC) and their gene products (TmrABC) was confirmed at both transcriptional and proteomic levels. Other findings include the upregulation of various hydrogenases (membrane-associated Ni-Fe hydrogenase complexes and soluble Fe-Fe hydrogenases), formate dehydrogenases, complex I and a pyrophosphate-energized proton pump. The elevated expression of enzymes associated with carbon metabolism, including complete Wood Ljungdahl pathway, during TCM respiration raises interesting questions on possible fates of intracellular formate and its potential role in the physiology of this bacterium. Overall, the findings presented here provide a broader view on the bioenergetics and general physiology of Dehalobacter UNSWDHB cells actively respiring with TCM.

Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation.

Organohalides are recalcitrant pollutants that have been responsible for substantial contamination of soils and groundwater. Organohalide-respiring bacteria (ORB) provide a potential solution to remediate contaminated sites, through their ability to use organohalides as terminal electron acceptors to yield energy for growth (i.e., organohalide respiration). Ideally, this process results in non- or lesser-halogenated compounds that are mostly less toxic to the environment or more easily degraded. At the heart of these processes are reductive dehalogenases (RDases), which are membrane bound enzymes coupled with other components that facilitate dehalogenation of organohalides to generate cellular energy. This review focuses on RDases, concentrating on those which have been purified (partially or wholly) and functionally characterized. Further, the paper reviews the major bacteria involved in organohalide breakdown and the evidence for microbial evolution of RDases. Finally, the capacity for using ORB in a bioremediation and bioaugmentation capacity are discussed.

Isolation and characterization of Dehalobacter sp. strain UNSWDHB capable of chloroform and chlorinated ethane respiration.

Dehalobacter sp. strain UNSWDHB can dechlorinate up to 4 mM trichloromethane at a rate of 0.1 mM per day to dichloromethane and 1,1,2-trichloroethane (1 mM, 0.1 mM per day) with the unprecedented product profile of 1,2-dichloroethane and vinyl chloride. 1,1,1-trichloroethane and 1,1-dichloroethane were slowly utilized by strain UNSWDHB and were not completely removed, with minimum threshold concentrations of 0.12 mM and 0.07 mM respectively under growth conditions. Enzyme kinetic experiments confirmed strong substrate affinity for trichloromethane and 1,1,2-trichloroethane (Km = 30 and 62 µM respectively) and poor substrate affinity for 1,1,1-trichloroethane and 1,1-dichloroethane (Km = 238 and 837 µM respectively). Comparison of enzyme kinetic and growth data with other trichloromethane respiring organisms (Dehalobacter sp. strain CF and Desulfitobacterium sp. strain PR) suggests an adaptation of strain UNSWDHB to trichloromethane. The trichloromethane RDase (TmrA) expressed by strain UNSWDHB was identified by BN-PAGE and functionally characterized. Amino acid comparison of homologous RDases from all three organisms revealed only six significant amino acid substitutions/deletions, which are likely to be crucial for substrate specificity. Furthermore, strain UNSWDHB was shown to grow without exogenous supply of cobalamin confirming genomic-based predictions of a fully functional cobalamin synthetic pathway.

Reductive Dehalogenases Come of Age in Biological Destruction of Organohalides.

Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant toxic, and carcinogenic environmental pollutants. Select microorganisms encode enzymes known as reductive dehalogenases (EC 1.97.1.8) that catalyze reductive dehalogenation reactions resulting in the generation of lesser-halogenated compounds that may be less toxic and more biodegradable. Recent breakthroughs in enzyme structure determination, elucidation of the mechanisms of reductive dehalogenation, and in heterologous expression of functional reductive dehalogenase enzymes have substantially increased our understanding of this fascinating class of enzymes. This knowledge has created opportunities for more versatile (in situ and ex situ) biologically-mediated organohalide destruction strategies.

Destructuring plant biomass: focus on fungal and extremophilic cell wall hydrolases.

The use of plant biomass as feedstock for biomaterial and biofuel production is relevant in the current bio-based economy scenario of valorizing renewable resources. Fungi, which degrade complex and recalcitrant plant polymers, secrete different enzymes that hydrolyze plant cell wall polysaccharides. The present review discusses the current research trends on fungal, as well as extremophilic cell wall hydrolases that can withstand extreme physico-chemical conditions required in efficient industrial processes. Secretomes of fungi from the phyla Ascomycota, Basidiomycota, Zygomycota and Neocallimastigomycota are presented along with metabolic cues (nutrient sensing, coordination of carbon and nitrogen metabolism) affecting their composition. We conclude the review by suggesting further research avenues focused on the one hand on a comprehensive analysis of the physiology and epigenetics underlying cell wall degrading enzyme production in fungi and on the other hand on the analysis of proteins with unknown function and metagenomics of extremophilic consortia. The current advances in consolidated bioprocessing, altered secretory pathways and creation of designer plants are also examined. Furthermore, recent developments in enhancing the activity, stability and reusability of enzymes based on synergistic, proximity and entropic effects, fusion enzymes, structure-guided recombination between homologous enzymes and magnetic enzymes are considered with a view to improving saccharification.

Versatile peroxidase degradation of humic substances: use of isothermal titration calorimetry to assess kinetics, and applications to industrial wastes.

The kinetic constants of a hybrid versatile-peroxidase (VP) which oxidizes complex polymeric humic substances (HS) derived from lignin (humic and fulvic acids) and industrial wastes were determined for the first time using isothermal titration calorimetry (iTC). The reaction conditions were manipulated to enable manganese-peroxidase (MnP) and/or lignin-peroxidase (LiP) activities to be evaluated. The peroxidase reactions exhibited varying degrees of product inhibition or activation; properties which have not previously been reported for VP enzymes. In contrast to previous work (Ertan et al., 2012) on small non-polymeric substrates (MnSO4, veratryl alcohol and dyes), all kinetic plots for polymeric HS were sigmoidal, lacked Michaelis-Menten characteristics, and were indicative of positive cooperativity. Under conditions when both LiP and MnP were active, the kinetic data fitted to a novel biphasic Hill Equation, and the rate of enzymatic reaction was significantly greater than the sum of individual LiP plus MnP activities implying synergistic activation. By employing size-exclusion chromatography and electrospray ionization mass spectrometry, the characteristics of the oxidative degradation products of the HS were also monitored. Our study showed that the allosteric behaviour of the VP enzyme promotes a high level of regulation of activity during the breakdown of model and industrial ligninolytic substrates. The work was extended to examine the kinetics of breakdown of industrial wastes (effluent from a pulp and paper plant, and fouled membrane solids extracted from a ground water treatment membrane) revealing unique, VP-mediated, kinetic responses. This work demonstrates that iTC can be successfully employed to study the kinetic properties of VP enzymes in order to devise reaction conditions optimized for oxidative degradation of HS present in materials used in a wide range of industries.

Kinetic and thermodynamic characterization of the functional properties of a hybrid versatile peroxidase using isothermal titration calorimetry: Insight into manganese peroxidase activation and lignin peroxidase inhibition.

Isothermal titration calorimetry (ITC) was developed for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. Developing an ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Although VP from Bjerkandera adjusta is a hybrid enzyme, specific conditions of [Mn+2] and pH were defined that limited activity to either LiP or MnP activities, or enabled both to be active simultaneously. MnP activity was found to be more efficient than LiP activity, with activity increasing with increasing concentrations of Mn+2. These properties of MnP were explained by a second metal binding site involved in homotropic substrate (Mn+2) activation. The activation of MnP was also accompanied by a decrease in both activation energy and substrate (Mn) affinity, reflecting a flexible enzyme structure. In contrast to MnP activity, LiP activity was inhibited by high dye (substrate) concentrations arising from uncompetitive substrate inhibition caused by substrate binding to a site distinct from the catalytic site. Our study provides a new level of understanding about the mechanism of substrate regulation of catalysis in VP from B. adjusta, providing insight into a class of enzyme, hybrid class II peroxidases, for which little experimental data is available.

Defining the response of a microorganism to temperatures that span its complete growth temperature range (-2°C to 28°C) using multiplex quantitative proteomics.

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses.

The RNA polymerase subunits E/F from the Antarctic archaeon Methanococcoides burtonii bind to specific species of mRNA.

RNA polymerase in Archaea is composed of 11 or 12 subunits - 9 or 10 that form the core, and a heterodimer formed from subunits E and F that associates with the core and can interact with general transcription factors and facilitate transcription. While the ability of the heterodimer to bind RNA has been demonstrated, it has not been determined whether it can recognize specific RNA targets. In this study we used a recombinant archaeal MbRpoE/F to capture cellular mRNA in vitro and a microarray to determine which transcripts it specifically binds. Only transcripts for 117 genes (4% of the total) representing 48 regions of the genome were bound by MbRpoE/F. The transcripts represented important genes in a number of functional classes: methanogenesis, cofactor biosynthesis, nucleotide metabolism, transcription, translation, import/export. The arrangement and characteristics (e.g. codon and amino acid usage) of genes relative to the putative origin of replication indicate that MbRpoE/F preferentially binds to mRNA of genes whose expression may be important for cellular fitness. We also compared the biophysical properties of RpoE/F from M. burtonii and Methanocaldococcus jannaschii, demonstrating a 50°C difference in their apparent melting temperatures. By using MbRpoE/F to capture and characterize cellular RNA we have identified a previously unknown functional property of the MbRpoE/F heterodimer.

Global proteomic analysis of the insoluble, soluble, and supernatant fractions of the psychrophilic archaeon Methanococcoides burtonii. Part II: the effect of different methylated growth substrates.

Methanococcoides burtonii is a cold-adapted methanogenic archaeon from Ace Lake in Antarctica. Methanol and methylamines are the only substrates it can use for carbon and energy. We carried out quantitative proteomics using iTRAQ of M. burtonii cells grown on different substrates (methanol in defined media or trimethylamine in complex media), using techniques that enriched for secreted and membrane proteins in addition to cytoplasmic proteins. By integrating proteomic data with the complete, manually annotated genome sequence of M. burtonii, we were able to gain new insight into methylotrophic metabolism and the effects of methanol on the cell. Metabolic processing of methanol and methylamines is initiated by methyltransferases specific for each substrate, with multiple paralogs for each of the methyltransferases (similar to other members of the Methanosarcinaceae). In M. burtonii, most methyltransferases appear to have distinct roles in the metabolism of methylated substrates, although two methylamine methyltransferases appear to be nonfunctional. One set of methyltransferases for trimethylamine catabolism appears to be membrane associated, potentially providing a mechanism to directly couple trimethylamine uptake to demethylation. Important roles were highlighted for citrate synthase, glutamine synthetase, acetyl-CoA decarbonylase/synthase, and pyruvate synthase in carbon and nitrogen metabolism during growth on methanol. M. burtonii had only a marginal response to the provision of exogenous amino acids (from yeast extract), indicating that it is predisposed to the endogenous synthesis of amino acids. Growth on methanol appeared to cause oxidative stress in the cell, possibly through the formation of reactive nonoxygen species and formaldehyde, and the oxidative inactivation of corrinoid proteins, with the cell responding by elevating the synthesis of universal stress (Usp) proteins, several nucleic acid binding proteins, and a serpin. In addition, changes in levels of cell envelope proteins were linked to counteracting the disruptive solvent effects of methanol on cell membranes. This is the first global proteomic study to examine the effects of different carbon sources on the growth of an obligately methylotrophic methanogen.

The genomic basis of trophic strategy in marine bacteria.

Many marine bacteria have evolved to grow optimally at either high (copiotrophic) or low (oligotrophic) nutrient concentrations, enabling different species to colonize distinct trophic habitats in the oceans. Here, we compare the genome sequences of two bacteria, Photobacterium angustum S14 and Sphingopyxis alaskensis RB2256, that serve as useful model organisms for copiotrophic and oligotrophic modes of life and specifically relate the genomic features to trophic strategy for these organisms and define their molecular mechanisms of adaptation. We developed a model for predicting trophic lifestyle from genome sequence data and tested >400,000 proteins representing >500 million nucleotides of sequence data from 126 genome sequences with metagenome data of whole environmental samples. When applied to available oceanic metagenome data (e.g., the Global Ocean Survey data) the model demonstrated that oligotrophs, and not the more readily isolatable copiotrophs, dominate the ocean's free-living microbial populations. Using our model, it is now possible to define the types of bacteria that specific ocean niches are capable of sustaining.

Carbon and nitrogen substrate utilization in the marine bacterium Sphingopyxis alaskensis strain RB2256.

Sphingopyxis alaskensis is a marine member of the Alphaproteobacteria that is adapted to heterotrophic growth under nutrient-depleted (oligotrophic) conditions. S. alaskensis strain RB2256 is an ultramicrobacterium (cell volume <0.1 microm(3)), and has a genome size larger than that of the ultramicrobacterium 'Candidatus Pelagibacter ubique' HTCC1062 (SAR11 clade of Alphaproteobacteria): 3.35 versus 1.31 Mbp. In this study, we investigate the carbon and nitrogen metabolism of strain RB2256 using an integrated approach that combines growth and enzyme assays, proteomics and genome analysis. S. alaskensis is able to use specific amino acids and putrescine as a sole carbon and nitrogen source, and higher energy-yielding substrates such as glucose and trehalose as carbon sources. Alanine, in particular, emerges as a very important substrate in S. alaskensis metabolism. In an oligotrophic environment where competition for nutrients is intense, our data support a simplified metabolism for S. alaskensis in which the fate of certain substrates is constrained, especially at the intersections of central carbon and nitrogen metabolism, in order to ensure optimal disposition of scarce resources. This is the first investigation of central metabolism for an oligotrophic ultramicrobacterium that possesses a relatively large genome size. In contrast to the behavior so far observed for SAR11 oligotrophic bacteria, S. alaskensis shows a physiological capacity to exploit increases in ambient nutrient availability and thereby achieve high-population densities.

A proteomic determination of cold adaptation in the Antarctic archaeon, Methanococcoides burtonii.

A global view of the biology of the cold-adapted archaeon Methanococcoides burtonii was achieved using proteomics. Proteins specific to growth at 4 degrees C versus T(opt) (23 degrees C) were identified by mass spectrometry using the draft genome sequence of M. burtonii. mRNA levels were determined for all genes identified by proteomics, and specific enzyme assays confirmed the protein expression results. Key aspects of cold adaptation related to transcription, protein folding and metabolism, including specific roles for RNA polymerase subunit E, a response regulator and peptidyl prolyl cis/trans isomerase. Heat shock protein DnaK was expressed during growth at T(opt), indicating that growth at 'optimal' temperatures was stressful for this cold-adapted organism. Expression of trimethylamine methyltransferase involves contiguous translation of two open reading frames, which is likely to result from incorporation of pyrrolysine at an amber stop codon. Thermal regulation in M. burtonii is achieved through complex gene expression events involving gene clusters and operons, through to protein modifications.