Degradation in oxic conditions

The first described strain able to utilize chloromethane as sole source of carbon and energy was Hyphomicrobium sp. MC1 (Hartmans et al., 1986). Like this strain, the majority of the chloromethane-degrading strains isolated so far utilize chloromethane under oxic conditions. They were isolated from various environments such as petrochemical factory soil, wastewater treatment plans, forest soils, freshwater and marine environments (Tableau 1.5). They are affiliated to the class of Alphaproteobacteria of the genera Methylobacterium, Hyphomicrobium and Aminobacter. As of this date, the cmu pathway is the best-characterized bacterial chloromethane-degradation pathway with a majority of the studies performed in M. extorquens CM4, but uncharacterized cmuA-independent pathway exist (see below).

− Chloromethane degradation pathway in Methylomicrobium album BG8

The methanotroph Methylomicrobium album BG8 degrades chloromethane in oxic conditions, independently of the cmu pathway, only in presence of another carbon source such as methanol (Figure 1.13) (Han et Semrau, 2000).

Figure 1.13. Proposed mechanism of chloromethane oxidation in Methylomicrobium album BG8

Chloromethane can be oxidized by the particulate methane monooxygenase (pMMO) since M. album BG8 stops degrading chloromethane in presence of acetylene, a pMMO inhibitor.

Methanol is oxidizes into formaldehyde (HCHO) by the methanol dehydrogenase (MDH).

Part of the produced formaldehyde enters the C1 metabolism to produce biomass and the rest is oxidized into CO2 and reducing equivalents (2[H]) for energy production via the

41 formaldehyde deshydrogenase (FADH) and the formate dehydrogenase (FDH). Compared to methanol, chloromethane degradation generates 2 reducing equivalents less (From Han and Semrau, 2000).

Chloromethane stimulates M. album BG8 growth with methanol, and when using [¹⁴ C]-labeled chloromethane, it was demonstrated that the C]-labeled carbon was assimilated into the biomass (38%) and released as CO2 (50%). This strongly suggested that chloromethane can be used as carbon and energy source when methanol was present (Han and Semrau, 2000). When provided alone, chloromethane is unable to sustain growth and serve as the sole source of carbon. The authors suggested that M. album BG8 would not be able to generate enough reducing equivalents from chloromethane degradation to sustain growth with chloromethane. In presence of another carbon source, in larger quantities, the chloromethane utilization was observed (5 mM of methanol and 2.6 mM of chloromethane) (Han and Semrau, 2000).

− The cmu pathway

Combined biochemical and genetic studies in M. extorquens CM4 were used to identify the cmu (chloromethane utilization) pathway under oxic conditions (Vannelli et al., 1998, 1999).

M. extorquens CM4 metabolizes one mole of CO2 and one mole of chloride (Vannelli et al., 1998). Using random mutagenesis with the minitransposon Tn5, 9 mutants were detected with wild-type growth with methanol or methylamine, but impaired growth with chloromethane (Vannelli et al., 1998). Analysis of the Tn5 insertion sites in mutants that lost the ability to grow with chloromethane, demonstrated the essential role of cmuABC, purU and metF2 (Studer et al., 2001, 2002). In addition, cmuAB genes were demonstrated to be essential for chloromethane dechlorination (Studer et al., 2001b, 2002). CmuA and CmuB catalyze the methyl group transfer from chloromethane to H4F to form methyl-H4F (CH3-H4F) (Figure 1.14) (Studer et al., 2001). CH3-H4F is oxidized either into formate and then CO2 for energy production, or into methylene-H4F for biomass production via the serine cycle (Vannelli et al., 1999).

Figure 1.14 The cmu pathway of Methylobacterium extorquens CM4

The methyl group of chloromethane is transferred to tetrahydrofolate (H4F) by cmuA and cmuB. The methylene-H4F produced by the oxidation of methyl-H4F enters the serine cycle to produce biomass and is also oxidized to CO2 to produce energy. CmuA, protein with a methyltransferase domain and a corrinoid-binding domain (Studer et al., 2001), CmuB, methyltransferase methylcobalamin: H4F (Studer et al., 1999); MetF2, methylene reductase;

FolD, bifunctional enzyme methylene-H4F dehydrogenase/ methenyl-H4F cyclohydrolase;

PurU, 10-formyl-H4F hydrolase.

All the genes of cmu pathway in M. extorquens CM4 are plasmid pCMU01-borne in a region of 180 kb (Figure 1.15). The cmu genes are clustered in two groups 30 kb apart that displays an atypical GC content and is devoid of detectable mobile elements. Upstream of purU and folD, gene folC2 encoded a bi-functional folylpolyglutamate synthase / dihydrofolate synthase protein. This region contains also paaE-like, fmdB and hutI, encoding respectively a putative oxidoreductase, a putative transcriptional regulator and a putative imidazole hydrolase.

43 Figure 1.15 The chloromethane utilization pCMU01 plasmid in M. extorquens CM4

The 21 kb distant clusters 1 and 2 contain the essential genes for chloromethane utilization (cmu) known before the genome has been sequenced (Vannelli et al., 1999; Studer et al., 2002). The cmu region covers 40 kb (cluster 1 and 2). The S1, S2, S3 regions correspond to a region of 69 kb conserved between pCMU01 and the p1METDI plasmid of M. extorquens DM4. The S4 region shows a synteny with a duplicated region on M. extorquens DM4 chromosome. Circles represent (from the outside): 1, percentage of deviation of the GC content in a 1 kb window; 2, predicted CDS transcribed in the clockwise direction; 3, predicted CDS transcribed in the counterclockwise direction; 4, GC skew (G+C/G-C) in a 1 kb window; 5, transposable elements (in pink) and pseudogenes (in grey) (adapted from the PhD manuscript of Sandro Roselli, 2009).

This region contains 23 genes involved in biosynthesis and transport of cobalamin, those involved on H4F biosynthesis, both cofactors are essential for chloromethane utilization via the cmu pathway (Figure 1.15). Some of these plasmid-borne genes have chloromsomal homologs (Roselli et al., 2013). Genome comparative analysis of strains harboring cmu genes, demonstrated two types of cmu gene organization; one found in M. extorquens CM4

where genes are localized in two clusters spaced by 30 kb and a second one, found in others bacteria, where cmu are contiguous (Figure 1.15). A high degree of similarity in cmu genes organization where found in chloromethane-degrading strains (Figure 1.15, Schäfer et al., 2007), all affiliated to the class of Alphaproteobacteria. The genes order into the cmu cluster is highly conserved (Nadalig et al., 2011). In M. extorquens CM4, cmuB and cmuC genes are part of the same transcriptional unit, unlike gene cmuA, which has its own promoter (Studer et al., 2002). The genome of some chloromethane degrading-strains lack detectable cmu genes as in Roseovarius sp. 217 and Leisingera methylohalidovorans MB2 (Schäfer et al., 2007). Chloromethane stable isotope analysis revealed different isotopic fractionation signatures for the carbon and hydrogen in cmu-containing strains and L. methylohalidovorans MB2 which not possess those genes (Nadalig et al., 2014). The cmu-independent chloromethane degradation pathway in L. methylohalidovorans MB2, needs to be solved.

45 Figure 1.16. Comparison of cmu gene organization in bacteria harboring gene cmuA

Encoding genes are represented with arrows. Genes with an incomplete determined sequenced are represented by incomplete arrows. The gene reference colors are based on those from M. extorquens CM4 representation, where they are separated by 30 kb.

Homologous genes are represented by the same color, the percentage of homology with proteins encoded by M. extorquens CM4 genes are written into arrows. The genetic representation is represented at the scale. To note that in the strain MB2, only one part of cmuA, corresponding to corrinoid cofactor binding-domain is present.