Definición de las variables socioeconómicas

A total of 49 genes identified in the H. sulfonivorans transcriptome show substantial upregulation greater than 10-fold when cultivated on DMSO2 instead of MeOH, most of which do not map onto the metabolic pathways of MSC metabolism outlined above. A combination of NCBI annotation (Haft et al., 2018) and KEGG orthology (Kanehisa et al., 2016) has been used to infer the roles of these proteins and attempt to match this function back to MSC metabolism. This list of highly upregulated proteins includes several of the enzymes already mapped to the pathways of MSC metabolism discussed above, as well as a potential DMSO2 transporter and several enzymes of cofactor biosynthesis related to the activity of FMNH2- dependent monooxygenases.

To begin with the upregulated enzymes that have already been described in Section 4.2, both pairs of subunits for the DmoAB-type DMS monooxygenase (dmoA and dmoB) and the

SfnFG-type DMSO2 monooxygenase (sfnF2 and sfnG2) appear on the list as highly upregulated on DMSO2 versus MeOH. The Sox system enzyme SoxCD subunits soxC and soxD are also present, as is one of the hydrogen peroxide detoxifying catalases.

Located in the same cluster as dmoA, dmoB, sfnF2 and sfnG2 is a porin encoding gene C6Y62_13180, upregulated by ~350 times. A BLAST search for the product of this gene against the Uniprot and NCBI databases reveal no homologues that have been functionally characterised, only that the sequence encodes a porin; porins form transmembrane protein channels for the passive diffusion of molecules across bacterial membranes (Jap & Walian, 1996). As C6Y62_13180 so highly upregulated on DMSO2 and the porin is localised so close to dmoA, it raises the possibility that this gene encodes a porin based DMSO2 transporter.

Three of the substantially upregulated genes in the DMSO2 data appear to encode rib genes involved in riboflavin biosynthesis. The first is C6Y62_13255, upregulated 50 times and encoding a putative RibH-like 6,7-dimethyl-8-ribityllumazine synthase (K00794) (García- Ramírez et al., 1995). Two more genes, C6Y62_13195 and C6Y62_13230, are upregulated 27 and 35 times respectively and both encode a putative RibBA-like 3,4-dihydroxy 2-butanone 4-phosphate synthase/ GTP cyclohydrolase II (K14652) based on KEGG annotation (Herz et al., 2000). All three of these genes are in close proximity to the FMN-dependent monooxygenases sfnG (C6Y62_13190) and dmoA (C6Y62_13210) in the H. sulfonivorans genome, suggesting that the purpose of this upregulation in riboflavin synthesis is to provide cofactors for these MSC oxidising enzymes.

Looking beyond MSC metabolism, the data suggests an upregulation on DMSO2 of various other proteins relating to cofactor biosynthesis, oxidative stress, DNA repair, transcription, translation and amino acid metabolism, as well as various other hypothetical proteins and enzymes of unknown function. These will be discussed in further detail in Section 4.3 if they are also found to be associated with the metabolism of DMSO2 by comparative proteomics. A full list of these upregulated genes is displayed below in Table 4.2-1.

Table 4.2-1a: Transcriptomics of H. sulfonivorans, most highly induced genes on DMSO2 versus MeOH. Table displaying the 25 most upregulated genes identified in the transcriptomics data on a sole carbon source of DMSO2 versus the MeOH control condition. Genes that have previously been discussed are highlighted in bold.

Accession No. gene name NCBI annotation strand position KEGG ID versus Control fold change condition C6Y62_11255 - 30S ribosomal protein S3 Reverse contig4 654015:654812 K02982 3,805.29 C6Y62_11335 - 50S ribosomal protein L1 Reverse contig4 673440:674192 K02863 2,601.43 C6Y62_11345 nusG transcription termination/antitermination protein NusG Reverse contig4 674903:675439 K02601 1,471.37

C6Y62_13210 dmoA 5%2C10-methylene tetrahydromethanopterin reductase Forward contig5 390172:391614 K20938 815.81

C6Y62_01205 - DUF1134 domain-containing protein Reverse contig1 273831:274784 687.27

C6Y62_13175 - hypothetical protein Forward contig5 382414:383469 583.27

C6Y62_13810 pal peptidoglycan-associated lipoprotein Pal Forward contig6 78769:79278 K03640 402.87

C6Y62_13220 soxC sulfite dehydrogenase Forward contig5 392325:393596 K17225 379.97

C6Y62_02865 - hypothetical protein Forward contig1 632242:632514 373.59

C6Y62_13180 - porin Forward contig5 383678:384979 352.49

C6Y62_04965 - two-component sensor histidine kinase Reverse contig1 1074640:1075902 K15011 272.40 C6Y62_01165 - formate dehydrogenase accessory sulfurtransferase FdhD Forward contig1 261573:262346 K02379 215.13

C6Y62_06250 - hypothetical protein Forward contig2 244023:244811 K03589 151.31

C6Y62_09975 - GlsB/YeaQ/YmgE family stress response membrane protein Reverse contig4 366228:366500 145.86 C6Y62_04595 - hydroxymethylbilane synthase Reverse contig1 991731:992660 K01749 122.69

C6Y62_07635 - DUF159 family protein Forward contig2 553254:553943 92.85

C6Y62_13225 soxD cytochrome c Forward contig5 393589:394167 K22622 90.47

C6Y62_02050 - hypothetical protein Forward contig1 456531:458135 85.80

C6Y62_03365 - disulfide bond formation protein B Reverse contig1 734913:735434 85.33

C6Y62_10455 - DNA helicase Forward contig4 472991:473695 73.36

C6Y62_08715 - NADH-quinone oxidoreductase subunit L Forward contig4 77351:79294 K00341 68.77

C6Y62_01595 - hypothetical protein Reverse contig1 363545:363979 64.60

C6Y62_09035 - 2Fe-2S ferredoxin Forward contig4 159959:160279 K04755 55.70

C6Y62_13200 dmoB flavin reductase Forward contig5 388235:388765 53.98

C6Y62_03540 hisH imidazole glycerol phosphate synthase subunit HisH Forward contig1 768671:769324 K02501 53.71 scale (fold change versus control condition) 1 >1 >10 >100 >1000

Table 4.2-1b: Transcriptomics of H. sulfonivorans, most highly induced genes on DMSO2 versus MeOH (continued). Table displaying the 50- 26th most upregulated genes identified in the transcriptomics data on a sole carbon source of DMSO2 versus the MeOH control condition. Genes that have previously been discussed are highlighted in bold.

Accession No. gene name NCBI annotation strand position KEGG ID versus Control fold change condition C6Y62_13255 ribH 6%2C7-dimethyl-8-ribityllumazine synthase Forward contig5 398527:399039 K00794 48.05 C6Y62_12935 - precorrin-6A synthase (deacetylating) Forward contig5 324245:325006 K02228 46.96

C6Y62_04345 - DUF1751 domain-containing protein Forward contig1 932412:933131 37.68

C6Y62_12010 xth exodeoxyribonuclease III Reverse contig5 124364:125146 K01142 36.66

C6Y62_11970 - protein-L-isoaspartate O-methyltransferase Forward contig5 111101:111814 K00573 36.62 C6Y62_13195 ribB 3%2C4-dihydroxy-2-butanone-4-phosphate synthase Forward contig5 387078:388208 K14652 35.43

C6Y62_01955 - methyltransferase Reverse contig1 437406:438245 34.38

C6Y62_13190 sfnG2 dimethyl sulfone monooxygenase SfnG Forward contig5 385844:386962 K17228 34.11

C6Y62_12715 - DUF192 domain-containing protein Forward contig5 273943:274443 K09005 33.89

C6Y62_04975 - SCO family protein Forward contig1 1076709:1077308 K07152 31.86

C6Y62_13240 - acyl dehydratase Forward contig5 396128:396577 30.62

C6Y62_13185 sfnF2 FMN reductase Forward contig5 385211:385756 K00299 30.25

C6Y62_13230 - peptide ABC transporter substrate-binding protein Forward contig5 394185:394805 K14652 27.16 C6Y62_15670 trmB tRNA (guanosine(46)-N7)-methyltransferase TrmB Forward contig8 18941:19630 K03439 25.71

C6Y62_13245 - MFS transporter Forward contig5 396627:397856 23.21

C6Y62_00510 - hypothetical protein Forward contig1 114428:114784 21.24

C6Y62_05850 - hypothetical protein Forward contig2 158304:159068 K14998 20.11

C6Y62_09015 - MFS transporter Forward contig4 153060:154478 K05820 18.90

C6Y62_09325 - phosphoribosyl-AMP cyclohydrolase Reverse contig4 226035:226481 K01496 18.54

C6Y62_12260 - P-II family nitrogen regulator Forward contig5 179270:179605 12.62

C6Y62_08345 - transfer Agent Forward contig4 7220:7594 11.68

C6Y62_14210 - alkylhydroperoxidase Reverse contig6 178821:179174 10.64

C6Y62_07450 - hypothetical protein Forward contig2 518848:520296 10.59

C6Y62_09260 - catalase Forward contig4 214166:215644 K03781 10.27

C6Y62_12775 - TonB-dependent receptor Forward contig5 288326:290635 K02014 9.48

4.3.8 Conclusions

The comparative transcriptomics of DMSO2 oxidation in H. sulfonivorans S1 has

yielded a large data set covering ~90% of the predicted coding sequences of the H. sulfonivorans genome. A small but significant group of these genes show substantial differential expression between DMSO2 and MeOH, several of which can be traced to either

MSC metabolism, the dmoA gene cluster and/or the biosynthesis of cofactors for DmoAB. Although many of the enzymes of MSC metabolism in H. sulfonivorans remain unknown, the trends exhibited in differential expression data of the few genes that have already been identified is quite promising. For example, the dmoA gene encoding the DMS monooxygenase that previous characterised by Boden et al. (2011) is massively upregulated on DMSO2, while the putative MeOH dehydrogenase mxaF is substantially upregulated on the