Due to the apparent relevance of the genus Variovorax to methanol oxidation in the rhizosphere environment (Chapter 5 and 6), the strain of Variovorax paradoxus isolated
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from the CF soil (Section 3.2.1) was sent for genome sequencing. This genome adds to the growing list of Variovorax paradoxus genomes which are publicly available (16 at time of writing). The genome of Variovorax paradoxus MM1 has a genome size of 7.1 Mb with GC 67.2 Mol%. The other Variovorax paradoxus genomes vary in size between 6.5 – 9.6 Mb with GC 66.5 -69.2 Mol%. The genus Variovorax, and specifically the species Varivorax paradoxus, has been shown to be metabolically versatile (Kim et al., 2006; Miwa et al., 2008; Im et al., 2010; Satola et al., 2013; Brandt et al., 2014). Strains have been isolated from varied environments, including marine and terrestrial, as well as pristine and contaminated (Anesti et al., 2005; Kim et al., 2006; Yoon et al., 2006b; Miwa et al., 2008; Im et al., 2010; Jin et al., 2012; Schreiter et al., 2014). The organism and its diverse metabolism makes it an ideal study system for the degradation of several compounds (Satola et al., 2013; Brandt et al., 2014). Plant growth promoting traits have also been shown to be present in the species (Han et al., 2011; Satola et al., 2013).
3.3.1 General metabolic pathways
Variovorax paradoxus MM1 represents a facultative methylotroph, as it is able to utilise C1 and multicarbon compounds for growth and energy (Anthony 1983). Variovorax paradoxus MM1, and additional methylotrophic strains of Variovorax paradoxus, can be further classified as a less restricted facultative methylotroph due to the broad range of substrates on which they are able to grow.
Similar to the genomes of other sequenced Variovorax paradoxus species (Satola et al., 2013; Brandt et al., 2014), the genome of Variovorax paradoxus MM1 contains genes which encode for a complete TCA cycle. The genome also contains all of the genes required for assimilation of formaldehyde through the serine cycle. All of the genes comprising the complete Calvin-Benson-Bassham cycle for the assimilation of carbon from carbon dioxide are also present within the genome. The genome also contains genes that encode for the glyoxylate shunt, possessing both an isocitrate lyase and a malate synthase. In addition to its role in two-carbon assimilation this, or the alternative EMC pathway, is essential for the regeneration of glyoxylate in methylotrophs that utilise the serine cycle (Korotkova et al., 2002; Chistoserdova et al., 2009; Peyraud et al., 2011; Keltjens et al., 2014).
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The genome of MM1 contains one copy of a xoxF methanol dehydrogenase gene. The sequence of this gene was aligned at the amino acid level with a database of xoxF sequences to identify the clade of this methanol dehydrogenase. The sequence clustered with xoxF5, the most genetically diverse and phylogenetically distributed of the five clades of xoxF methanol dehydrogenase (Keltjens et al., 2014). The strain with the highest identity to MM1 at the 16S rRNA gene level, Variovorax paradoxus S110, possesses a xoxF3 and a xoxF5 gene. Due to the draft nature of the genome of MM1, which does not contain a xoxF3, DNA extracted from MM1 was used as template in a PCR assay to confirm the absence of a xoxF3 gene in this organism and no product was obtained. Therefore, it is presumed that this strain of Variovorax paradoxus only has a xoxF5. The xoxF5 sequence has high identity (96-99%) to xoxF5 genes encoded in the genomes of five other strains of Varivorax (Figure 3.2). The genetic region upstream and downstream of the xoxF5 methanol dehydrogenase encoding gene is conserved between the genomes of Variovorax paradoxus MM1, S110 and B4 (Figure 3.3). This region includes accessory genes known or predicted to play a role in methanol dehydrogenase formation (Keltjens et al., 2014). In addition, the genes that encode ribulose-1,5-bisphosphate, the key enzyme of the CBB pathway, are upstream of the xoxF5 gene. However, the question of whether expression of this enzyme could be linked to the expression of xoxF5, and the relative contribution of the serine cycle and CBB cycle to the growth of MM1 when grown on methanol as a carbon source would be need to be validated through further physiological characterisation of this strain.
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Figure 3.2 Phylogenetic analysis of xoxF5 gene sequences from Variovorax paradoxus MM1 aligned with additional xoxF5 genes, aligned at the deduced amino acid level, with the phylogenetic tree constructed from nucleotide sequences. The blue bracket marks the Variovorax paradoxus xoxF5 sequences. The evolutionary history was inferred using the Neighbour-Joining method with a bootstrap value of 500. The scale bar represents nucleotide substitution per position.
Variovorax paradoxus MM3 Variovorax sp. Root318D1 Variovorax paradoxus S11 Variovorax paradoxus B4 Variovorax sp. OV7 Variovorax sp. CF79 Methylibium petroleiphilum PM1 Leptothrix cholodnii SP-6
Rubrivivax gelatinosus IL144 Variovorax sp. URHB2 Variovorax sp. OV329 Methyloversatilis universalis FAM5
Methyloversatilis sp. RZ18-153
Methyloversatilis sp. FAM1 unannotated mdh only Verminephrobacter eiseniae EF1-2
Burkholderia sp. YI23
Burkholderia xenovorans LB4 Methylomicrobium album BG8 Methylovulum miyakonense HT12
Hyphomicrobium denitrificans ATCC 51888 Hyphomicrobium denitrificans 1NES1
Azospirillum lipoferum 4B Rhodopseudomonas palustris BisA53
Bradyrhizobium diazoefficiens USDA 11 Methylobacterium extorquens AM1
Methylocella silvestris BL2 Sinorhizobium meliloti Rm41
Paracoccus denitrificans PD1222 Sagittula stellata E-37
Roseobacter litoralis Och 149
Variovorax paradoxus MM1 Variovorax paradoxus S110 Variovorax paradoxus B4 Variovorax sp. CF79 Variovorax sp. Root318D1 Variovorax sp. OV7 Methylibium petroleiphilum PM1 Leptothrix cholodnii SP-6
Rubrivivax gelatinosus IL144 Variovorax sp. URHB2 Variovorax sp. OV329 Methyloversatilis universalis FAM5
Methyloversatilis sp. RZ18-153 Methyloversatilis sp. FAM1
Verminephrobacter eiseniae EF1-2 Burkholderia sp. Y123
Burkholderia xenovorans LB4 Methylomicrobium album BG8 Methylovulum miyakonense HT12
Hyphomicrobium denitrificans ATCC 51888 Hyphomicrobium denitrificans 1NES1
Azosporillium lipoferum 4B Rhodopseudomonas palustris BisA53
Bradyrhizobium diazoefficiens USDA11 Methylobacterium extorquens AM1
Methylocella silvestris BL2 Sinorhizobium meliloti Rm41
Paracoccus denitrificans PD1222 Sagittula stellate E-37
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Figure 3.3 Gene cluster surrounding the region surrounding the xoxF5 methanol dehydrogenase gene of Variovorax paradoxus MM1. A-B, ribulose bisphosphate carboxylase small subunit and large subunit; 31, LysR family transcriptional regulator; 44, Outer membrane receptor protein; 17, tricarboxylate transport protein; 15, multridrug transport system; 6, hypothetical protein; 3, hypothetical protein; 4, ATP binding protein; C, hypothetical protein; 1, methanol dehydrogenase xoxF5; 16, Cytochrome c55; 2, hypothetical protein; 12, hypothetical protein; 23, hypothetical protein; 30, moxR.
The genome of MM1 contains genes encoding enzymes of two formaldehyde oxidation pathways. It possesses the glutathione-dependent formaldehyde oxidation pathway (Wilson et al., 2008). Initially, a glutathione formaldehyde activating enzyme (Gfa) converts formaldehyde to hydroxymethyl-glutathione. A glutathione dependent formaldehyde dehydrogenase (GSH-FALDH) then oxidises this to S-formyl GSH, which is then converted to formate by a formyl-glutathione hydrolase. Further analysis of the genome showed that it contained the genes required for the tetrahydrofolate (H4F) linked pathway of formaldehyde assimilation (Vorholt, 2002). The reaction between H4F and formaldehyde produces methylene-H4F, which can be inserted into the serine cycle for assimilation or oxidised further to formate. Genes required for the dissimilation of formaldehyde from methylene-H4F are present. FolD, a bifunctional enzyme capable of methylene-H4F dehydrogenase and methenyl-H4F cyclohydrolase activity would convert the methyl-H4F to 10-formyltetrahydrofolate. This would then be converted to formate and tetrahydrofolate by the enzyme 10-formly-H4F hydrolase (Chistoserdova et al., 2009; Keltjens et al., 2014). The genome also contains genes encoding for three formate dehydrogenases, FDH1, FDH2 and FDH3.
3.3.2 Further metabolic traits
The genome of Variovorax paradoxus MM1 contains genes encoding for an assimilatory nitrate reductase (NasAB) and two dissimilatory nitrite reductases (NirBD). In addition to this, the genome contains genes that encode for a 2-nitropropane dioxygenase, an enzyme that converts 2-nitropropane to acetone and nitrite. The genome also contains
B
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genes encoding two nitrilases, converting nitriles to a carboxylate and ammonia (Howden et al., 2009). Both of these enzymes would require experimental validation to determine their functionality, but would expand the metabolic capability of the strain with regards to nitrogen. The genome of Variovorax paradoxus MM1 was also predicted to contain two inactive prophages.
As previously mentioned, Variovorax is considered an important genus for the degradation of natural and polluting aromatic compounds. Analysis of the genome of MM1 showed it to contain genes encoding for the degradation of aromatic compounds to acetyl-CoA and succinyl CoA, allowing for subsequent utilisation by central metabolic pathways (Satola et al., 2013; Liang et al., 2014). The degradation pathways present are for nitrobenzene and naphthalene as are the pathways for the subsequent utilisation of catechol and 3-oxoadipate. These pathways would require experimental testing to confirm their functionality, however other closely related strains of Varivorax have been implicated in the degradation these compounds (Brandt et al., 2014; Liang et al., 2014; Posman et al., 2016).
3.4 Enrichment and isolation of methylotrophs using soil from CF using dNMS and