Genomic and Transcriptomic Analysis of Growth-Supporting Dehalogenation of Chlorinated Methanes in Methylobacterium

Abstract

Bacterial adaptation to growth with toxic halogenated chemicals was explored in the context of methylotrophic metabolism of Methylobacterium extorquens, by comparing strains CM4 and DM4, which show robust growth with chloromethane and dichloromethane, respectively. Dehalogenation of chlorinated methanes initiates growth-supporting degradation, with intracellular release of protons and chloride ions in both cases. The core, variable and strain-specific genomes of strains CM4 and DM4 were defined by comparison with genomes of non-dechlorinating strains. In terms of gene content, adaptation toward dehalogenation appears limited, strains CM4 and DM4 sharing between 75 and 85% of their genome with other strains of M. extorquens. Transcript abundance in cultures of strain CM4 grown with chloromethane and of strain DM4 grown with dichloromethane was compared to growth with methanol as a reference C1 growth substrate. Previously identified strain-specific dehalogenase-encoding genes were the most transcribed with chlorinated methanes, alongside other genes encoded by genomic islands (GEIs) and plasmids involved in growth with chlorinated compounds as carbon and energy source. None of the 163 genes shared by strains CM4 and DM4 but not by other strains of M. extorquens showed higher transcript abundance in cells grown with chlorinated methanes. Among the several thousand genes of the M. extorquens core genome, 12 genes were only differentially abundant in either strain CM4 or strain DM4. Of these, 2 genes of known function were detected, for the membrane-bound proton translocating pyrophosphatase HppA and the housekeeping molecular chaperone protein DegP. This indicates that the adaptive response common to chloromethane and dichloromethane is limited at the transcriptional level, and involves aspects of the general stress response as well as of a dehalogenation-specific response to intracellular hydrochloric acid production. Core genes only differentially abundant in either strain CM4 or strain DM4 total 13 and 58 CDS, respectively. Taken together, the obtained results suggest different transcriptional responses of chloromethane- and dichloromethane-degrading M. extorquens strains to dehalogenative metabolism, and substrate- and pathway-specific modes of growth optimization with chlorinated methanes.

Dichloromethane Degradation Pathway from Unsequenced Hyphomicrobium sp. MC8b Rapidly Explored by Pan-Proteomics

“…On the other hand, the small number of differentially abundant proteins shared by strains MC8b and DM4 growing with DCM contrasts with the fact that the two strains share 1136 homologous proteins with over 50% identity at the protein level, with a similar number of detected proteins in the two studies (2453 proteins for strain MC8b versus 2878 for strain DM4 [39]). To us, this suggests that adaptation to dehalogenation of DCM involves specific changes in expression of the taxonomically defined core genome following acquisition of genes for DCM utilisation [49,50], in keeping with the broad functional categories associated with transformation of DCM (Supplementary Table S5), and as suggested by transcriptional studies [51]. In other words, genes involved in adaptation to DCM are not limited to a specific set of genes of DCM-degrading strains, as evidenced by differentially abundant proteins shared by many and sometimes all 13 Hyphomicrobium strains of the pan-proteomics database (Supplementary Figure S1).…”

Chloromethane formation and degradation in the fern phyllosphere

“…Bacteria able to degrade CH 3 Cl have been isolated from plant leaves and may utilize the cmu chloromethane utilization pathway (Nadalig et al, 2014). The only CH 3 Cl degradation pathway characterized in detail, was mainly studied in Methylobacterium extorquens CM4, an aerobic methylotrophic strain able to use CH 3 Cl as the sole source of carbon and energy (Chaignaud et al, 2017;Roselli et al, 2013;Vanelli et al, 1999). In this pathway, the CH 3 Cl dehalogenase includes a corrinoid methyltransferase (CmuA) and a tetrahydrofolate-dependent methyltransferase (CmuB) (Studer et al, 2001(Studer et al, , 1999.…”

Methanol consumption drives the bacterial chloromethane sink in a forest soil

“…Such low concentrations likely do not yield sufficient energy for substantial bacterial growth with CH 3 Cl. However, many known alphaproteobacterial CH 3 Cl degraders also grow with methanol (CH 3 OH) [ 13 , 21 ], and this is also true in situ for soil methylotrophs of a deciduous forest [ 22 ]. Abundance of methylotrophs in O and A soil horizons is high, and ranges from 10 6 to 3 × 10 8 cells g soil −1 , consistent with their frequent isolation from soils [ 23 , 24 ].…”

“…Which organisms define the bacterial CH 3 Cl sink in soils is largely unknown at present. The only biochemically characterized pathway for CH 3 Cl utilization is the cmu pathway, characterized in detail for Methylobacterium extorquens CM4 [ 21 ]. It has been found in various CH 3 Cl-degrading bacterial strains, including several strains from forest soil [ 7 , 18 , 25 – 27 ].…”