Global approaches in M. extorquens

Our knowledge of methylotrophy in bacteria belonging to the genus Methylobacterium and in particular in the species M. extorquens has increased considerably over last years with high-throughput Next Generation Sequencing (NGS) technologies, allowing the study of genes (genomics), their expression at the RNA level (transcriptomics), at the protein level (proteomics) as well as of cell metabolites (metabolomics).

4.3.1. Genomic studies in M. extorquens

In 1998, the first Methylobacterium genome sequence was obtained from strain AM1 (Chistoserdova et al., 2003). It has been completed and assembled only in 2009 when the genome of strain DM4 was available (Vuilleumier et al., 2009). Later, in 2012, the genome of strain M. extorquens CM4 was sequenced simultaneously with the genomes of other M. extorquens strains unable to degrade chlorinated methanes (BJ001; PA1) (Marx et al., 2012). When I started my PhD thesis, the MaGe platform hosted by the Génoscope (Evry, France), offered easy annotation and comparative analysis of 6 M. extorquens genomes (AM1, BJ001, CM4, DM4, DSM 13060 and PA1), with characterized growth abilities for methanol and chlorinated methanes. Intra-species M. extorquens comparative genomic study has detected the presence of a high number of insertion sequences (IS) in the vicinity of gene clusters involved in methanol utilization, suggesting a role of these mobile elements in the evolution of phylogenetically closely-related genomes of strains AM1 and DM4 (Vuilleumier et al., 2009). Nevertheless, the history of the evolution of M. extorquens genome remains unclear. As a matter of facts, despite the fact that M. extorquens AM1 and PA1 have similar GC content (68.2% and 68.5%, respectively) and share a large number of common genes involved in methylotrophy (90 genes with at least 95% identity between their corresponding encoded proteins), a recent study highlighted differences in growth

rates for a variety of C1 compounds such as methanol, methylamine or formaldehyde, which would suggest the implication of different mechanisms (Nayak et Marx, 2014).

Combined random mutagenesis and comparative proteomic studies have evidenced the growth with chlorinated methanes involved genes localized on genomic islands, in addition to housekeeping genes involved in stress response, and central metabolism (Michener et al., 2014a; Muller et al., 2011a; Roselli et al., 2013). Chloromethane or dichloromethane utilization generates a cellular stress that requires specific adaptive responses (Kayser et Vuilleumier, 2001; Kayser et al., 2002; Roselli et al., 2013). To decipher those adaptive mechanisms, Michener et al. (2014) have tested the ability to utilize dichloromethane of naïve non-degrading strains. The plasmid-borne dcmA gene has been transferred to strain AM1 (unable to utilize dichloromethane for its growth) (Michener et al., 2014b).

Transconjugants displayed poor growth with dichloromethane and but had a detectable dehalogenation activity. The subsequent genome sequencing of adapted clones enabled the identification of mutation within gene clcA encoding an antiporter Cl^-/H⁺. When a plasmid harbouring genes dcmA and the mutated clcA were introduced in M. extorquens PA1 (unable to utilize dichloromethane for its growth), it readily conferred the ability to growth with dichloromethane.

Complementary studies of chloromethane utilization and adaptation in naïve strains (unable to utilize chloromethane for their growth) have been performed (Michener et al., submitted article). A plasmid harbouring genes of the cmu pathway (folD, metF2, paaE-like, purU) and of associated genes (fmdB, hutI), has been inserted by conjugation in M. extorquens AM1, PA1, BJ001, M. radiotolerans and M. nodulans strains. The resulting transconjugants acquired the ability to growth with chloromethane, although cultures were of low optical densities. When the mutated gene clcA, which has been demonstrated to increase cell fitness for growth with dichloromethane, was also present in the transconjugants, no improvement of the fitness was observed for cells grown with chloromethane. This suggest that the effectiveness of heterologous catabolism of chloromethane and dichloromethane are uncorrelated in Methylobacterium strains (Michener et al., submitted article).

There remains a need for a better understanding of the mechanisms involved in methylotrophic growth in M. extorquens using complementary approaches such as transcriptomics, proteomics or metabolomics in addition to the studies already available (Tableau 1.7 completed from Ochsner et al., 2015).

51 Table 1.7. “Omics” studies in M. extorquens strains

« omic » study Reference Description Genome Vuilleumier et al., 2009 AM1, DM4

Roselli et al., 2013 CM4 Muller et al., 2011 DM4

Bai et al., 2015 Isolates from phyllosphere and rhizosphere (24 genomes)

Modelling Peyraud et al., 2011 AM1 Proteomic Bosch et al., 2008;

Laukel et al., 2004 AM1, methanol and succinate comparison Muller et al., 2011 DM4, dichloromethane et methanol

comparison

Roselli et al., 2013 CM4, chloromethane et methanol comparison Guo et Lidstrom, 2008 AM1, « Profiling » of metabolites in methanol

and succinate Transcriptomic

Okubo et al., 2007 AM1, methanol and succinate comparison (microarray)

Francez-Charlot et al., 2009

AM1, phyR mutant, general stress regulation (microarray)

Metabolomic Kiefer et al., 2008, 2011

AM1, central metabolites concentration in methanol and succinate

Peyraud et al., 2012 AM1, [¹³C]-fluxomic, [¹³C]-CH3OH in co-utilization with succinate

Yang et al., 2013 AM1, ¹³C methanol, metabolite labeling during growth

Cui et al., 2016 AM1 with overexpression of ethylmalonyl-CoA mutase in methanol

Reaser et al., 2016 AM1, monitoring over time of metabolite after incorporating [¹³C]-CH3OH

4.3.2. Transcriptomic studies in M. extorquens

Transcriptomics is a qualitative and quantitative study of the transcriptome, which encompasses in principle all RNAs produced by transcription (Wang et al., 2009). In different growth conditions, the spectrum of transcripts and their abundance is modified. Thus, the quantitative inventory of transcripts enables a better understanding of the adaptive processes involved in response to growth in various conditions.

Today, few transcriptomic studies have been performed in M. extorquens, and when performed, it was using DNA chips (microarrays) in strain AM1. These studies demonstrated the role of genes involved in C1 metabolism in comparative studies of cultures grown with methanol versus succinate (Okubo et al., 2007; Francez-Charlot et al., 2009), as well as the involvement in the stress response of the transcriptional gene regulator phyR (Francez-Charlot et al., 2009). Sequencing and analysis of RNA (RNA sequencing, i.e. a global transcriptome analysis) has not been described so far for M. extorquens, and I provide in this doctoral work the first transcriptome study for strains CM4 and DM4.

4.3.3. Proteomic studies in M. extorquens

There are more published proteomic than transcriptomic studies in M. extorquens (Tableau 1.7, Gourion et al., 2006; Muller et al., 2011; Roselli et al., 2013). In M. extorquens AM1, proteins involved in methylotrophy have been detected in cultures grown with methanol (the reference growth substrate for methylotrophy studies) compared to cultures grown with succinate (C4H6O4) (Laukel et al., 2004; Bosch et al., 2008). Other studies were performed in M. extorquens strains able to degrade chlorinated methanes. In M. extorquens DM4, the protein content of cultures grow with dichloromethane compared to methanol was used to identify proteins involved in dichloromethane utilization (see chapter 3) (Muller et al., 2011a). Similarly, M. extorquens AM1 colonization mechanisms of

A. thaliana phyllosphere has been better understood using proteomic approaches (Tableau 1.7, Knief et al., 2010, 2012; Vorholt, 2012). Overexpression of the regulatory protein PhyR was employed to evidence its role in plant colonization (Gourion et al., 2006).

Proteomic analysis in M. extorquens CM4 chloromethane utilization was used to demonstrated that 49 proteins more abundant in cultures grown with chloromethane compared tomethanol (Figure 1.17).

53 Figure 1.17. 2D gel picture of protein extracts of M. extorquens CM4 grown with chloromethane or with methanol

CmuA, CmuB and PurU framed in red, dark green and light green respectively, were identified by mass spectrometry and were more abundant with chloromethane (Sandro Roselli PhD thesis, 2009).

By combining genomic and proteomic approaches, genes involved in chloromethane utilization highlighted the central implication of plasmid pCMU01 in growth with chloromethane in M. extorquens CM4 (Roselli et al., 2013).

4.3.4. Metabolomic studies in M. extorquens

Most of the metabolomic studies were performed with cultures of M. extorquens AM1 to better characterize the methylotrophic and central metabolism of carbon assimilation. In cultures grown with methanol compared tosuccinate, metabolites specifically associated to growth in methylotrophic or involved in glyoxylate regeneration such as β-hydroxybutyrate, methylsuccinyl-CoA or ethylmalonyl-CoA have been detected (Figure 1.11) (Guo et Lidstrom, 2008). Metabolites profiling were also performed in cultures of

M. extorquens AM1 grown with compounds in C2 (ethylamine) and in C4 (succinate) (Yang et al., 2009). Similarly, M. extorquens AM1 grown with acetate (CH3COO⁻)as the sole source of energy and carbon was used to demonstrated that the ethylmalonyl-CoA pathway replaced the isocitrate lyase pathway for the regeneration of glyoxylate, needed for carbon assimilation in biomass via the serine cycle (Schneider et al., 2012, Figure 1.11). These results illustrate metabolic adjustments to growth with different carbon sources.