Diversity of Methylotrophy Pathways in the Genus Paracoccus (Alphaproteobacteria)

Jakub Czarnecki1,2* and Dariusz Bartosik1

1Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland. 2Bacterial Genome Plasticity, Department of Genomes and Genetics, Institut Pasteur, Paris, France. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.033.117

Abstract Introduction Paracoccus denitrifcans Pd 1222 is a model methy- Te genus Paracoccus (class Alphaproteobacteria, lotrophic bacterium. Its methylotrophy is based order Rhodobacterales, family Rhodobacteraceae) on autotrophic growth (enabled by the Calvin currently includes around 70 defned species and cycle) supported by energy from the oxidation of hundreds of strains whose taxonomic position has methanol or methylamine. Te growing availabil- yet to be precisely assigned (NCBI Taxonomy, ity of genome sequence data has made it possible 10 January 2018). Representatives of this genus to investigate methylotrophy in other Paracoccus have been identifed in diverse environments. Te species. Te examination of a large number of Para- original isolate and the type species, P. denitrifcans, coccus spp. genomes reveals great variability in C1 was isolated from soil (Beijerinck, 1910), like many metabolism, which have been shaped by difer- other Paracoccus spp. (Urakami et al., 1990; Siller ent evolutionary mechanisms. Surprisingly, the et al., 1996; Tsubokura et al., 1999). Numerous methylotrophy schemes of many Paracoccus strains strains have been isolated from fresh water (Sheu appear to have quite diferent genetic and bio- et al., 2018), seawater (Kim and Lee, 2015), sedi- chemical bases. Besides the expected ‘autotrophic ments (G. Zhang et al., 2016), activated sludge (Liu methylotrophs’, many strains of this genus possess et al., 2006), bioflters (Lipski et al., 1998), or from another C1 assimilatory pathway, the serine cycle, environments linked to higher organisms, such as which seems to have at least three independent plant roots (rhizosphere) (Doronina et al., 2002), origins. Analysis of the co-occurrence of diferent marine bryzoans (Pukall et al., 2003), insects (S. methylotrophic pathways indicates, on the one Zhang et al., 2016), and human tissues (opportun- hand, evolutionary linkage between the Calvin istic pathogens P. yeei and P. sanguinis) (Funke et al., cycle and the serine cycle, and, on the other hand, 2004; McGinnis et al., 2015). that genes encoding some C1 substrate-oxidizing Te ubiquity of Paracoccus spp. is due to their enzymes occur more frequently in association with great metabolic diversity and fexibility. All para- one or the other. Tis suggests that some genetic cocci have an aerobic respiratory metabolism and module combinations form more harmonious utilize multi-carbon compounds. However, many enzymatic sets, which act with greater efciency in of them can switch between diferent growth the methylotrophic process and thus undergo posi- modes, using diferent carbon and energy sources, tive selection. and employing various fnal electron acceptors. In

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the absence of oxygen, some Paracoccus spp. con- nitrate, nitrite, nitric oxide and nitrous oxide (which duct nitrate respiration (Baker et al., 1998; Kelly leads to denitrifcation – P. denitrifcans is an impor- et al., 2006). Tis process leads to denitrifcation tant model in studies on this process (Baker et al., and has been applied for the removal of nitrates 1998), thus permiting growth when oxygen is lim- from wastewater (Liu et al., 2012). In addition to ited. On the other hand, diferent electron donors ‘standard’ carbon sources, like sugars, amino acids may be used. As a consequence, P. denitrifcans has and succinate, Paracoccus strains isolated from the ability to grow chemolithoautotrophically on polluted environments can utilize xenobiotics, e.g. inorganic energy sources such as hydrogen and polycyclic aromatic hydrocarbons (PAHs), making thiosulphate (Friedrich and Mitrenga, 1981). Its them useful in bioremediation (Sun et al., 2013). autotrophic growth may also be supported by the Numerous representatives of the genus can grow oxidation of some organic C1 compounds, namely

chemolithoautotrophically, coupling CO2 assimila- MeOH, MA and formate (Baker et al., 1998). Tese tion with the oxidation of inorganic compounds or compounds are oxidized to CO2, the released elec- elements, such as thiosulfate, thiocyanate, elemen- trons are used for oxidative phosphorylation, and

tal sulfur, molecular hydrogen, or ferrous ions the ATP and CO2 produced are used in the Calvin (Kelly et al., 2006). Finally, many Paracoccus spp. cycle for biomass production. Tus, P. denitrifcans are methylotrophs. Most utilize methanol (MeOH) is an example of an ‘autotrophic methylotroph’, and methylamine (MA) as sole carbon and energy which lacks a ‘heterotrophic’ pathway dedicated

sources, e.g. P. denitrifcans and closely related P. to the assimilation of reduced C1 units (such as the versutus and P. kondratievae (Kelly et al., 2006). serine cycle or ribulose monophosphate pathway),

However, other strains isolated from environments but it can assimilate carbon from C1 compounds polluted with C1 compounds are able to metabolize afer their total oxidation (Baker et al., 1998; Chis- dimethylamine (DMA), trimethylamine (TMA), toserdova, 2011) (Fig. 6.1). N,N-dimethylformamide (DMF) (Urakami et al., Since the 1970s, numerous studies have sought

1990; Kim et al., 2001; Sanjeevkumar et al., 2013) to understand the details of C1 metabolism in or dichloromethane (Doronina et al., 1998). P. denitrifcans (Harms et al., 1985; Baker et al., Te purpose of this study is to describe the 1998), especially in strain Pd 1222, which is read- diversity of methylotrophy in the genus Paracoccus ily transformed by conjugation to enable genetic at the biochemical and genetic levels, including manipulation (Devries et al., 1989). Te results an examination of the origin and evolution of C1 of these studies have uncovered the properties of metabolism in this group of bacteria. many P. denitrifcans proteins involved in methylo- trophy (mainly MeOH and MA dehydrogenases, as well as associated proteins, i.e. those involved in the Paracoccus denitrifcans transfer of electrons from the dehydrogenases to Pd 1222 as an example of an the respiratory chain), and have shed light on the ‘autotrophic’ methylotroph regulation of their expression (Baker et al., 1998). Since its isolation at the beginning of the 20th Te whole genome sequence of P. denitrifcans century (Beijerinck, 1910), P. denitrifcans has Pd 1222 was obtained in 2006 (NCBI Genomes). been extensively studied and diferent aspects of its It has an unusual structure consisting of two energy metabolism have been revealed. Te compo- chromosomes (chromosome 1, 2.8 Mb, and chro- sition of its core respiratory chain closely resembles mosome 2, 1.7 Mb) and one large plasmid (plasmid that of the classic mitochondrial respiratory chain 1 1650 kb). Te availability of this sequence has (unlike respiratory chains of many other bacteria, permited elucidation of the genetic basis of its including E. coli), which made it a valuable model methylotrophy. P. denitrifcans Pd 1222 carries sev-

for studies on the energetic processes in eukaryotes eral gene clusters responsible for C1 metabolism, (John and Whatley, 1975). However, the electron dispersed across the three replicons. Genes for the

transport chain of P. denitrifcans also has many enzymes involved in the oxidation of primary C1 branches at both the entrance and exit sides of the substrates to formaldehyde are located on chromo- core. On the one hand, this allows the bacterium to some 2 (gene cluster encoding MxaFI-type MeOH utilize alternative fnal electron receptors, namely dehydrogenase and associated proteins) and

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Figure 6.1 Summary of the methylotrophic pathways of Paracoccus spp. P. denitrifcans Pd 1222 and P. aminovorans JCM 7685 are used as examples because these strains possess all of the methylotrophic pathways discussed in this study. The enzymes and pathways present in P. denitrifcans Pd 1222 are shown in red, those present in P. aminovorans JCM 7685 are shown in blue, and those present in both strains are shown in violet. Ddh, DMA dehydrogenase; DmfA1A2, DMFase; DmmABCD, DMA monooxygenase; FghA, S-formylglutathione hydrolase; FlhA, S-(hydroxymethyl)glutathione dehydrogenase; FolD, methylenetetrahydrofolate dehydrogenase (NADP+)/methenyltetrahydrofolate cyclohydrolase; FtfL, formate-tetrahydrofolate ligase; Gfa, S-(hydroxymethyl) glutathione synthase; MauAB, MA dehydrogenase; MxaFI, MxaFI-type MeOH dehydrogenase; PurU, formyltetrahydrofolate deformylase; Tdh, TMA dehydrogenase; Tmd, TMA N-oxide demethylase; Tmm, TMA monooxygenase; XoxF, XoxF-type MeOH dehydrogenase. *Methylated amines – TMA, DMA and MA. plasmid 1 (the mau genes encoding small and large as well as genes for fructose-1,6-bisphosphatase subunits of MA dehydrogenase and associated pro- (fp), phosphoribulokinase (prk), transketolase teins). Te second step in the methylotrophy of P. (tkt), fructose-1,6-bisphosphate aldolase (fa), denitrifcans Pd 1222 is oxidation of formaldehyde RuBisCO activating protein (cbbX), and the Calvin to formate in the glutathione-dependent pathway, cycle regulator (cbbR). which is essential for growth of this strain on C1 Parallel studies on the methylotrophy of the compounds (Harms et al., 1996). Tree enzymes closely related P. versutus have shown that this spe- of this pathway, S-(hydroxymethyl)glutathione cies utilizes similar routes of C1 metabolism. Te synthase (Gfa), S-(hydroxymethyl)glutathione assimilatory pathway required for growth on C1 dehydrogenase (FlhA), and S-formylglutathione substrates includes highly similar MA dehydro- hydrolase (FghA), are encoded within chromo- genase and Calvin cycle enzymes (Karagouni and some 1. Interestingly, these glutathione-dependent Kelly, 1989; Baker et al., 1998). formaldehyde oxidation genes occur in the imme- diate vicinity of genes encoding XoxF-type MeOH dehydrogenase and associated proteins. XoxF was Paracoccus aminophilus recently confrmed as a MeOH-oxidizing enzyme JCM 7686 and Paracoccus (Keltjens et al., 2014; Chistoserdova, 2016). aminovorans JCM 7685 as However, its involvement in MeOH metabolism serine cycle methylotrophs in P. denitrifcans was suggested many years before specialized in DMF utilization (Harms et al., 1996), although its redundancy In 1990, the isolation of two DMF-degrading with a MxaFI-type system remains unexplained. strains from a sample of DMF-polluted soil in

Formate is oxidized to CO2 by two multi subunit Japan was reported (Urakami et al., 1990). Tese (encoded in chromosomes 1 and 2) or one single strains, designated JCM 7686 and JCM 7685 were subunit (encoded in chromosome 1) formate dehy- recognized as representatives of two new Paracoccus drogenase. Finally, the Calvin cycle gene cluster, species: P. aminophilus and P. aminovorans (Urakami which is required for assimilation of CO2, is located et al., 1990). Te methylotrophic pathways of these on chromosome 1 and consists of genes encod- isolates were shown to be more complex than those ing two subunits of RuBisCO (rbcL and rbcS), of P. denitrifcans, because they include enzymes

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required for the utilization of a wider range of C1 the action of the ethylmalonyl-CoA pathway in P. compounds. Both strains are able to decompose aminovorans JCM 7685 (Czarnecki et al., 2017). All DMF to formate and DMA, and oxidize TMA via genes required for this pathway are also present in trimethylamine N-oxide (TMAO) to DMA, and P. aminophilus, in the non-serine cycle methylotroph then degrade the resultant DMA to MA (Urakami P. denitrifcans and even in non-methylotrophic et al., 1990). strains, as they are used for other purposes, such as Te entire genome sequences of P. aminophilus growth on C2 compounds (Schneider et al., 2012). JCM 7686 and P. aminovorans JCM 7685 were Te genes required for another glyoxylate-regen- obtained by our group (Dziewit et al., 2014; Czar- erating process, the glyoxylate shunt, are found necki et al., 2017), facilitating the reconstruction of in P. aminophilus and P. aminovorans, but, as they their methylotrophic pathways. As expected, genes were unable to support methylotrophic growth in

required for the metabolism of additional C1 sub- a strain with a blocked ethylmalonyl-CoA pathway strates were identifed in both strains. Tese encode (Czarnecki et al., 2017), their role remains unclear. small and large subunits of DMFase (DmfA1A2), Owing to the presence of diferent enzymes oxi- as well as TMA monooxygenase (Tmm), TMAO dizing primary C1 substrates and the serine cycle, demethylase (Tmd), and multi-subunit DMA the fate of C1 units released during methylotrophic monooxygenase (DmmABCD). Besides TMA and metabolism is more complex in P. aminophilus and DMA monooxygenases, the P. aminovorans JCM P. aminovorans than in P. denitrifcans. In the serine 7685 genome also encodes TMA and DMA dehy- cycle, carbon is assimilated in the form of methyl- drogenases (Tdh and Ddh, respectively), which ene group bound to tetrahydrofolate (THF) and

may catalyse the oxidation of TMA and DMA (Fig. CO2 (Fig. 6.1). Methylene-THF may be delivered 6.1). Te role of these genes in the metabolism of directly by the C1-substrate-oxidizing enzymes specifc C1 substrates has been confrmed in both TMAO demethylase and DMA monooxygenase strains (Dziewit et al., 2010, 2015; Czarnecki et al., or the NMG pathway, which do not release free 2017). formaldehyde, but transfer the C1 unit directly to Surprisingly, the enzymes involved in the THF (Fig. 6.1). On the other hand, the XoxF-type metabolism of MeOH and MA by P. aminophilus MeOH dehydrogenase, TMA dehydrogenase, and P. aminovorans difer from those employed by DMA dehydrogenase and MA dehydrogenase P. denitrifcans. In the case of MeOH utilization, release free formaldehyde, which has to be oxidized P. aminophilus and P. aminovorans do not possess to formate by the glutathione-dependent pathway a MxaFI-type MeOH dehydrogenase, and their present in P. aminophilus, P. aminovorans and P. deni- growth on this compound relies fully on a XoxF- trifcans. To feed the serine cycle, the formate has to type dehydrogenase, as has been confrmed by be bound to THF, and then it has to be reduced to mutational analysis (Dziewit et al., 2015). In the a methylene group in an energy-requiring process. case of MA, both strains have genes for an alterna- Te THF-dependent formate reduction pathway is tive MA oxidation pathway: the N-methylglutamate found in all three Paracoccus spp., and requires the (NMG) pathway. In P. aminovorans the NMG path- action of two enzymes: formate-THF ligase (FtfL) way is the only pathway for MA oxidation, while in and 5,10-methylene-tetrahydrofolate dehydroge- P. aminophilus it co-exists with the MA dehydroge- nase/methenyl-tetrahydrofolate cyclohydrolase nase pathway, which was previously characterized in (FolD). Tis pathway may also act in the opposite P. denitrifcans (Fig. 6.1). Furthermore, both strains direction to oxidize the methylene group to formate.

lack RuBisCO genes, so cannot assimilate CO2. In this case, FolD promotes the reverse reaction Tus, their methylotrophy has to be supported by itself, while the second reaction is catalysed by for- another pathway of C1 unit assimilation. A serine myltetrahydrofolate deformylase (PurU), which is cycle gene cluster was found in both genomes; its also present in all three species (Fig. 6.1). A special involvement in methylotrophy has been confrmed situation occurs during growth on DMF, where in P. aminovorans, and the role of transcriptional DMFase releases a C1 unit directly in the form of regulator ScyR in its regulation was revealed formate and the second product is DMA. Te fate (Czarnecki et al., 2017). Te serine cycle requires of C1 units has been analysed experimentally in P. glyoxylate regeneration, which is accomplished by aminovorans, where growth of a strain lacking the

Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Methylotrophy in the Genus Paracoccus | 121 glutathione-dependent pathway for formaldehyde of small subunits). However, these chromosomal oxidation on diferent C1 substrates was examined. DMFase genes and their adjacent transcriptional As expected, this strain was unable to utilize MeOH regulator genes are surrounded by genes encoding (it has the XoxF-type MeOH dehydrogenase transposases and other proteins typical of mobile releasing free formaldehyde, which cannot be fur- genetic elements, which indicates their recent ther oxidized without the glutathione-dependent acquisition. Similarly, the TMA and DMA dehydro- pathway), but no efect on growth on MA was genase genes of P. aminovorans JCM 7685, that are detected (it has the NMG pathway which produces not found in any other Paracoccus strain (Czarnecki methylene-THF, which directly enters the serine et al., 2017), are located on the large extrachromo- cycle or is oxidized in the THF-dependent pathway, somal replicon pAMV3 (740 kb), which, like other without involvement of the glutathione-dependent replicons of this type, seems to be a reservoir of pathway). Intermediate phenotypes were observed various horizontally transmited genes. during growth on DMF, TMA or DMA, since A notable example of methylotrophy genes monooxygenases can oxidize some portion of these acquired by HGT are those clustered within compounds to produce methylene-THF, while a 40-kb methylotrophy island (MEI) located another portion is oxidized by dehydrogenases to on the extrachromosomal replicon pAMV1 of produce free formaldehyde (Czarnecki et al., 2017) P. aminovorans (Fig. 6.2). Te genes present on this (Fig. 6.1). Like P. denitrifcans, P. aminophilus and island are involved in all steps of methylotrophy: (i) P. aminovorans have formate dehydrogenases to oxidation of primary C1 substrates with methylene- deal with an excess of formate (Dziewit et al., 2015; THF generation (TMA monooxygenase, TMAO Czarnecki et al., 2017). demethylase, DMA monooxygenase, the NMG Te genomic localization and clustering of pathway), (ii) oxidation of methylene-THF to methylotrophy genes of P aminophilus and P. amino- formate (FolD and PurU), and assimilation of C1 vorans suggests that many of them could have been units in the form of methylene-THF and CO2 (the acquired horizontally to confer increased ftness for serine cycle). Te closest homologue of this MEI growth in DMF-polluted soil. Some DMFase genes was identifed in Paracoccus sp. N5 (Beck et al., of P. aminophilus are located on the small plasmid 2015; Dziewit et al., 2015; Czarnecki et al., 2017), pAMI2 (18.6 kb), which also carries a genetic and similarly clustered genes are located in the module putatively involved in its mobilization for chromosomes of many bacteria of the Roseobacter conjugal transfer (Dziewit et al., 2010). A closely clade, including Ruegeria pomeroyi (Dziewit et al., related DMFase is encoded in the chromosomes 2015). Te MEI genes are also present in the P. ami- of both P. aminovorans and P. aminophilus (84% nophilus genome. However, in this case the island is aa identity of large subunits and 73% aa identity divided in two, with one part encoding the serine

Figure 6.2 Genetic organization of the methylotrophy island (MEI) of P. aminovorans JCM 7685. The general functions of genes are indicated above their names. Potential operons are indicated by thin arrows. dmmABCD, DMA monooxygenase; folD, methylenetetrahydrofolate dehydrogenase (NADP+)/methenyltetrahydrofolate cyclohydrolase; gck, glycerate 2-kinase; glyA, serine hydroxymethyltransferase; gmaS, glutamate-methylamine ligase; hpr, hydroxypyruvate reductase; mcl, malyl-CoA lyase; mgdABCD, N-methylglut amate dehydrogenase; mgsABCD, N-methylglutamate synthase; mtkAB, malate-CoA ligase; scyR, serine cycle transcriptional regulator; sga, serine-glyoxylate aminotransferase; tmd, TMA N-oxide demethylase; tmm, TMA monooxygenase; ppc, phosphoenolpyruvate carboxylase; purU, formyltetrahydrofolate deformylase.

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cycle enzymes located in the chromosome, and usually located on chromosomes, while many of the the second part, comprising the rest of the MEI, genes of the second group are found on extrachro- in extrachromosomal replicon pAMI6 (207 kb) mosomal replicons, or in the company of mobile (Dziewit et al., 2015), whose genetic load is 40% genetic elements when present on chromosomes. homologous to that of P. aminovorans pAMV1. It Te frst group includes genes comprising appears that acquisition of the MEI (most prob- fundamental pathways, that are important for ably from representatives of the Roseobacter clade non-methylotrophic metabolism, to which further (Dziewit et al., 2015) could have enhanced the C1 pathways are appended. Te frst example methylotrophic ability of P. aminophilus and P. of this group is a cluster consisting of genes for aminovorans, which made them beter adapted to glutathione-dependent formaldehyde oxidation living in DMF-polluted soil. To fully reconstruct (gfa, fhA, fghA) and for MeOH oxidation by XoxF the evolution of C1 pathways in the genus Paracoc- (Fig. 6.3). Te compact and conserved nature of cus, deeper analyses are required, which consider the gene cluster comprising both pathways suggests not only the presence of given pathways (which has their cooperation. As mentioned above, both the already been done (Dziewit et al., 2015; Czarnecki XoxF and the glutathione-dependent pathways may et al., 2017), but also their phylogenetic relation- be essential for some methylotrophic processes ships. in Paracoccus spp. (Ras et al., 1995; Harms et al., 1996). However, the ubiquity of this gene cluster in non-methylotrophs indicates that its signifcance is Occurrence and phylogenetic more general. Its probable function in non-methyl- relationships of methylotrophy otrophic organisms is in the utilization of MeOH as genes in Paracoccus spp. an additional source of energy, without its assimila- We previously examined 44 Paracoccus spp. genomes tion into biomass. Despite its overall conservation, to determine the diversity of methylotrophy genes the cluster is truncated in some Paracoccus spp. and in this genus (Dziewit et al., 2015; Czarnecki et al., lacks the xox genes. For example, P. halophilus JCM 2017). In this study we have broadened this analysis 14014 does not have any homologues of xoxF. In using additional recently deposited Paracoccus spp. P. alcaliphilus JCM 7364 the xox genes and the sequences (in total, 62 Paracoccus genomes were genes for the glutathione-dependent pathway are available in NCBI GenBank on 11 May 2018) plus separated, being located on a large (430 kb) extra- 10 Paracoccus spp. genomes obtained by our group, chromosomal replicon and on the chromosome, which will be fully described in a forthcoming pub- respectively. lication (P. bengalensis DSM 17099, P. ferrooxidans NCCB 1300066, P. haundaensis LGM P-21903, P. kondratievae NCIBM 13773, P. pantotrophus DSM 11072, P. solventivorans DSM 11592, P. sulfuroxidans JCM 14013, P. thiocyanatus JCM 20756, P. versutus UW1 and P. yeei CCUG 32053). Te majority of the studied Paracoccus strains (almost 65%), contain sets of genetic modules involved in all three steps of methylotrophy (Chistoserdova, 2011), which are potentially sufcient for methylotrophic growth. Figure 6.3 Genetic organization of the gene cluster Comparative analysis of this large number of for the glutathione-dependent formaldehyde Paracoccus genomes allowed us to distinguish two oxidation pathway and the XoxF-type methanol dehydrogenase of P. denitrifcans Pd 1222. The groups of methylotrophy-related genes based on general functions of genes are indicated above their degree of conservation: (i) highly conserved, their names. fghA, S-formylglutathione hydrolase; vertically transmited genes, present in almost all of fhA, S-(hydroxymethyl)glutathione dehydrogenase; the genomes, and (ii) genes present only in some gfa, S-(hydroxymethyl)glutathione synthase; xoxF, XoxF-type MeOH dehydrogenase; xoxG, c-type strains because of selective loss, horizontal gene cytochrome; xoxI, SRPBCC family protein; xoxJ, transfer, or a combination of these two evolution- quinoprotein dehydrogenase-associated putative ary mechanisms. Te genes of the frst group are ABC transporter substrate-binding protein.

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Another example of genes of the conserved involved in primary oxidation of C1 substrates. Te group are those encoding the numerous enzymes Calvin cycle is widespread among Paracoccus spe- of the ethylmalonyl-CoA pathway. Tese do cies (Fig. 6.4) and a complete set of genes required not form a single gene cluster but are scatered for this process was identifed in 56% of analysed throughout Paracoccus genomes. Enzymes of the strains. Phylogenetic analysis of the RuBisCO large ethylmalonyl-CoA pathway are involved in various subunit (RbcL) and phosphoribulokinase (Prk) processes, such as the utilization of C2 compounds indicates that there are at least two evolutionarily as carbon and energy sources or synthesis of poly- distinct lineages of Calvin cycle genes in this genus hydroxyalkanoates. As mentioned above, in some (Figs. 6.5–6.7). Tis is refected in variations in the Paracoccus spp., the ethylmalonyl-CoA pathway is organization of Calvin cycle gene clusters in dif- responsible for glyoxylate regeneration, which is ferent Paracoccus spp. (Fig. 6.5). Te Calvin cycle crucial for functioning of the serine cycle during genes are generally found in chromosomes, with methylotrophic growth (Chistoserdova, 2011). some exceptions, e.g. in Paracoccus sp. N5 and P. A few Paracoccus spp. lack some enzymes of this kondratievae they are located on large extrachromo- pathway, e.g. P. chinensis CGMCC 1.7655 or strains somal replicons (1 Mb and 423 kb, respectively), of P. sanguinis. Nevertheless, a complete set of most probably as a result of translocation from the ethylmalonyl-CoA pathway genes was found in all chromosome. strains that employ the serine cycle. Compared with the Calvin cycle, the serine cycle Te next examples of conserved methylotrophy- is less prevalent in Paracoccus spp. and its occur- linked genes in Paracoccus spp. are those involved rence was predicted in 17% of the analysed strains. in THF-dependent transformations of C1 units: Serine cycle gene sets are present in a few diferent folD, purU and ffL. Te encoded enzymes are lineages of the genus (Fig. 6.4), and three types of important for the generation of C1-THF interme- gene organization were identifed (Fig. 6.5). Te diates required by biosynthetic pathways, such as occurrence of three gene organization schemes is purine synthesis. As in the serine cycle C1 units are probably an efect of the independent acquisition incorporated into biomass in the form of methyl- of the serine cycle from diferent sources. Tese ene-THF, the THF-dependent pathway constitutes separate events are refected in the phylogenetic a central metabolic process in Paracoccus serine tree of phosphoenolpyruvate carboxylase (Ppc), cycle methylotrophs (Fig. 6.1). It should be noted the key enzyme of the cycle (Fig. 6.8). Te frst type that besides the conserved set of folD, purU and of serine cycle gene organization occurs in the MEI ffL genes, there are also numerous homologues of P. aminovorans JCM 7685 and, as mentioned that are horizontally transferred, for example in the above, is also found in many representatives of the company of serine cycle genes or NMG pathway Roseobacter clade, in the order Rhodobacterales, as genes. It is possible that the serine cycle-associated are Paracoccus spp. Te second type of serine cycle and the NMG pathway-associated homologues gene organization, found on the chromosome of P. are beter adapted to co-operate with the methylo- zhejiangensis J6, for example, is also most similar to trophic pathways, while the conserved homologues arrangements present in members of the Roseobacter are mainly responsible for anabolic housekeeping clade. Te third type of serine cycle gene organiza- functions, but members of these groups are likely tion, found in P. denitrifcans ISTOD1, is typical for to be interchangeable to some extent. strains of the Aminobacter and Labrys genera (Beck Te last representatives of the group of conserved et al., 2015), within the order Rhizobiales. In some genes are gene clusters encoding multi-subunit Paracoccus spp., two types of serine cycle module formate dehydrogenases, which are responsible coexist in the same genome (Fig. 6.4). Te serine for formate detoxifcation in both methylotrophs cycle clusters may be located within extrachromo- and non-methylotrophs, and form part of the core somal replicons, as in pAMV1 in P. aminovorans genome of Paracoccus spp. JCM 7685 and probably also ISTOD1 in P. deni- Te second group of genes, which only occurs trifcans (the unfnished genome sequence of this in some Paracoccus strains, includes gene sets strain does not allow confrmation of the genomic involved in C1 unit assimilation pathways, the localization of this gene cluster, although it is pre- Calvin cycle and the serine cycle, as well as genes sent in a sequence scafold containing a repABC

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Figure 6.4 Occurrence of methylotrophy genes in Paracoccus spp. The maximum likelihood phylogenetic tree of Paracoccus spp. is based on the conserved gene for ethylmalonyl-CoA mutase (ecm). Where not indicated, the bootstrap support value is 100. Three sub-clades of Paracoccus spp., classifed on the basis of 16S rDNA, dnaA (data not shown) and ecm comparisons, are indicated in light blue, dark blue and red. Ddh, DMA dehydrogenase; DmfA1A2, DMFase; DmmABCD, DMA monooxygenase; MauAB, MA dehydrogenase; MxaFI, MxaFI-type MeOH dehydrogenase; NMGP, N-methylglutamate pathway; Tdh, TMA dehydrogenase; Tmd, TMA N-oxide demethylase; Tmm, TMA monooxygenase. *Truncated gene clusters.

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Figure 6.5 Organization of genetic modules for the Calvin cycle and the serine cycle in Paracoccus spp. The composition of the Calvin cycle gene cluster type 1 varies in some strains, e.g. it lacks the rpe gene in P. denitrifcans, P. pantotrophus, P. versutus, P. kondratievae and P. thiocyanatus. It is also truncated in Paracoccus sp. N5 (lacks the tkt, fba and rpe genes) and in P. alkenifer DSM 11593 (lacks the tkt gene). In all these cases, homologues of the absent genes are present at other locations in the genome. An incomplete Calvin cycle gene cluster type 1 is found in P. aminovorans HPD-2 (lacks rbcS, pseudogenes of rbcL and cbbX). The serine cycle gene cluster type 1 occurs without the gck gene in P. aminophilus JCM 7686 and P. sulfuroxidans JCM 14013. In both cases, other homologues of gck are present in the genome. cbbR, Calvin cycle transcriptional regulator; cbbX, RuBisCO activating protein; fba, fructose-bisphosphate aldolase; fbp, fructose-bisphosphatase; ffL, formate-THF ligase; folD, 5,10-methylene-tetrahydrofolate dehydrogenase/methenyl-tetrahydrofolate cyclohydrolase (FolD); gck, glycerate 2-kinase; glyA, serine hydroxymethyltransferase; hpr, hydroxypyruvate reductase; mcl, malyl-CoA lyase; mtkA, malate-CoA ligase subunit alpha; mtkB, malate-CoA ligase subunit beta; ppc, phosphoenolpyruvate carboxylase; prk, phosphoribulokinase; rbcL, RuBisCO large subunit; rbcS, RuBisCO small subunit; rpe, ribulose-phosphate 3-epimerase; scyR, serine cycle transcriptional regulator; sga, serine-glyoxylate aminotransferase; tkt, transketolase.

replication-partitioning module, typical for large and co-evolution of these two cycles in Paracoccus plasmids of Alphaproteobacteria). Te serine cycle genomes. Te phylogeny and distribution pat- genes have a chromosomal location in P. aminophi- terns of the key enzymes of these cycles (Figs 6.4, lus JCM 7685 or P. sulfuroxidans JCM 14013. Te 6.6–6.8) indicate that the Calvin cycle is the more distribution of the serine cycle genes indicates that ancient in this group of bacteria, and that the serine they were acquired horizontally several times in cycle has been acquired independently in several diferent lineages of the genus. Nevertheless, their lineages. In some strains, these two cycles coexist prevalence could also have been shaped by gene (Fig. 6.4), but in others, the Calvin cycle seems to loss. Te best example is the incomplete serine have been lost afer serine cycle acquisition. Tis cycle cluster of P. alcaliphilus JCM 7364, which phenomenon is perfectly illustrated by a group of lacks the pcc gene (there are no pcc homologues in three isolates: Paracoccus sp. N5, P. aminovorans the entire P. alcaliphilus genome). HPD-2 and P. aminovorans JCM 7685. Te frst One particularly interesting aspect is co- strain has the intact Calvin cycle gene cluster, the occurrence of the Calvin cycle and the serine cycle, second one possesses an incomplete cluster lacking

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Figure 6.6 Maximum likelihood phylogenetic tree of RuBisCO large subunits (RbcL) of Paracoccus spp. Two phylogenetic groups matching diferent types of genetic organization of the Calvin cycle genes are indicated. Three sub-clades of Paracoccus spp. are coloured as in Fig. 6.4.

the rbcS gene but containing rbcL and cbbX pseu- encoding QscR, a transcriptional regulator with dogenes, while the third strain does not possess homology to CbbR, which is a global regulator the rbcS, rbcL nor cbbX genes (Fig. 6.4). However, of the serine cycle genes (Kalyuzhnaya and Lid- not all of the Calvin cycle genes are lost in Paracoc- strom, 2003, 2005). Recently, QscR was shown to cus serine cycle methylotrophs lacking this cycle. be regulated by phosphoribulokinase, and it was Tese strains still have a truncated version of the demonstrated that both the qscR and prk genes are Calvin cycle gene cluster, including its three frst essential for the methylotrophy of M. extorquens genes encoding the Calvin cycle regulator (CbbR), (Ochsner et al., 2017). Parallel evolution of the fructose-1,6-bisphosphatase (Fbp) and phosphori- same gene set in Paracoccus serine cycle methylo- bulokinase (Prk). Te phylogeny of the Paracoccus trophs (and M. extorquens) implies that there is a phosphoribulokinases (Fig. 6.7) indicates that the universal evolutionary linkage between the Calvin same reduction of the Calvin cycle gene cluster cycle and the serine cycle (Ochsner et al., 2017). As arose in diferent lineages where the serine cycle the role of the cbbR–fp–prk cluster is only regula- appeared. Moreover, retention of a reduced Calvin tory, some strains may have evolved towards the cycle gene cluster is typical only for the serine cycle loss of these genes. In the analysed strain set there methylotrophs, whereas the cbbR, fp and prk genes is one example where partial loss of this regulatory are not present in Paracoccus non-methylotrophs. cluster has occurred: P. saliphilus DSM 18447 has An identically organized gene cluster was detected two diferent serine cycle gene sets, plus a remnant in another serine cycle methylotroph, Methylo- of the cbbR–fp–prk cluster (it lacks cbbR, has a fp bacterium extorquens PA1. Tis includes the gene pseudogene and an intact prk gene). However, the

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Figure 6.7 Maximum likelihood phylogenetic tree of phosphoribulokinases (Prk) of Paracoccus spp. Two phylogenetic groups matching diferent types of genetic organization of the Calvin cycle genes are indicated. Three sub-clades of Paracoccus spp. are coloured as in Fig. 6.4. *Strains with the serine cycle, which have truncated Calvin cycle gene clusters (and lack RuBisCO). ability of this strain to grow methylotrophically has in the case of MA dehydrogenase (Fig. 6.4). Both yet to be tested. enzymes, which were thought to be reliable mark- Among the genes involved in C1 metabolism, ers of methylotrophy in this genus based on studies those encoding enzymes participating in the pri- on P. denitrifcans Pd 1222, seem to be rather rare mary oxidation of C1 substrates, the frst stage of among Paracoccus spp. whose genomes have been methylotrophy, show the greatest variability in sequenced. MxaFI-type MeOH dehydrogenase their occurrence. One prominent exception is the and MA dehydrogenase usually co-exist with the aforementioned conserved XoxF-type MeOH Calvin cycle (Fig. 6.4), which may result from dehydrogenase. Conversely, a second type of adaptation of their mode of action to interact beter MeOH dehydrogenase, the two subunit enzyme with the Calvin cycle than with the serine cycle MxaFI, is found only in a few Paracoccus spp. (Fig. (release of the free formaldehyde, which is then 6.4). A similarly limited distribution is observed oxidized to formate in the glutathione-dependent

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Figure 6.8 Maximum likelihood phylogenetic tree of phosphoenolpyruvate carboxylases (Ppc) of Paracoccus spp. Three phylogenetic groups matching diferent types of genetic organization of the serine cycle genes are indicated. Three sub-clades of Paracoccus spp. are coloured as in Fig. 6.4.

pathway, but not direct binding of the methylene in P. aminovorans JCM 7685, but these enzymatic group to THF). Genes of the NMG pathway for activities had already been identifed in other Para- MA oxidation are mainly found in strains that pos- coccus isolates, and they may be important for the sess the serine cycle (Fig. 6.4). Te NMG pathway utilization of methylated amines when the oxygen enzymes appear to be evolutionarily bound to the concentration is changeable (Kim et al., 2001, serine cycle enzymes, since their phylogenetic 2003). Moreover, one strain whose genome has yet trees show a similar topology (Fig. 6.9). While the to be sequenced, P. methylutens DM 12, expresses third type of serine cycle gene cluster (Fig. 6.4) is dichloromethane halogenase, which is required for found in the company of only the NMG pathway dichloromethane utilization. Tus, further inves- genes, the frst and the second types co-occur with tigation of the content of Paracoccus spp. genomes other methylene-THF-producing enzymes, TMA may reveal interesting and unexpected features of

monooxygenase, TMAO demethylase and DMA their C1 pathways. monooxygenase (Fig. 6.4), e.g. within the MEI of P. aminovorans JCM 7685 (Fig. 6.2) (Czarnecki et al., 2017). Te exception are strains of P. yeei which Conclusions lack the serine cycle, but have the NMG pathway From our detailed analysis of numerous Paracoccus accompanied by DMA monooxygenase, TMA spp. genome sequences, it is now clear that the arche- monooxygenase and TMA N-oxide demethylase typal autotrophic methylotroph, P. denitrifcans Pd genes (Fig. 6.4). 1222, represents only a small part of the methylo- Te DMFase genes dmfA1A2 were only found trophic capacity present in the genus. Besides some in three strains, P. aminophilus JCM 7686, P. ami- generally conserved features, like the presence of novorans JCM 7685 and P. aminovorans HPD-2, the glutathione-dependent formaldehyde oxida- and always in association with serine cycle, NMG tion pathway and the ethylmalonyl-CoA pathway, pathway, TMA monooxygenase, TMAO demethyl- methylotrophic Paracoccus spp. vary greatly in the

ase and DMA monooxygenase genes (Fig. 6.4). Te genetic and biochemical basis of their C1 metabo- methylotrophic potential of Paracoccus spp. does lism. As anticipated there are diferences in the sets not seem to have been fully determined. Genes for of enzymes responsible for the primary oxidation of

TMA and DMA dehydrogenases were found only C1 compounds, which are located at the periphery

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Figure 6.9 Maximum likelihood phylogenetic tree of N-methylglutamate dehydrogenase subunits C (MgdC) of Paracoccus spp. Three phylogenetic groups corresponding to the diferent types of serine cycle gene clusters in Paracoccus spp. (compare with Fig. 6.8) are indicated. Three sub-clades of Paracoccus spp. are coloured as in Fig. 6.4. #Strains with the NMG pathway, which do not have the serine cycle; *strains with orphan mgd operons, which occur without other genes of the NMG pathway.

of the C1 metabolic net, and thus may be most Alphaproteobacteria as a whole, where the polyphy- ‘exposed’ to evolutionary changes. However, there letic origins of C1 metabolism have already been is also considerable variation in the very nucleus of described (Beck et al., 2015). Interestingly, these the C1 metabolism: the C1 assimilatory pathways. tendencies may be observed even on a microscale Tis variability is an efect of diferent evolution- level, within strains of the same species. For exam- ary mechanisms, which are very hard to retrace. ple, comparison of two strains of P. denitrifcans, As was proposed previously, genetic modules rep- Pd 1222 and ISTOD1, shows how evolutionary resenting diferent steps of methylotrophy may be processes can easily rebuild a metabolic net. It is reassembled in diferent genomes, generating new likely that the discovery of additional genetic mod- metabolic capabilities (Chistoserdova, 2011). Tis ules associated with methylotrophy in this species appears to be the case in Paracoccus spp. Te more will follow the sampling of new strains, especially frequent coexistence of certain modules indicates those from environments where C1 compounds are that they may ‘ft’ together beter, probably due to present. more efcient cooperation. Tis explains why the methylene THF-generating NMG pathway for MA oxidation is typically found with the methylene Future trends THF-consuming serine cycle, whereas the formal- Although analysis of the increasing body of genomic dehyde-producing MA dehydrogenase is found data from Paracoccus spp. can give many interesting with the Calvin cycle. Te opposite combinations results, there is a need for experimental studies on of these pathways occur in some Paracoccus spp., the C1 metabolism of these bacteria. Te predicted but they are much less common. functions of many genes have to be verifed, the Te evolutionary tendencies in methylotrophy involvement of others confrmed (e.g. genes encod- of the genus Paracoccus are the same as in the class ing putative transporters located in vicinity of

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methylotrophy genes), and their regulatory mecha- metabolism of the methylotrophic bacterium Paracoccus aminovorans JCM 7685. Environ. Microbiol. 19, 4536– nisms characterized. It would also be informative 4550. htps://doi.org/10.1111/1462-2920.13901 to determine whether serine cycle-based methylo- Devries, G.E., Harms, N., Hoogendijk, J., and Stouthamer, trophy can be transmited to other strains, e.g. via A.H. (1989). Isolation and characterization of Paracoccus the transfer of the pAMV1 methylotrophy island. denitrifcans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA- Paracoccus spp. seem to be ideal models for study- modifying property. Arch. Microbiol. 152, 52–57. ing the evolution of methylotrophy. Studies on htps://doi.org/10.1007/Bf00447011. these bacteria may shed light on some unexplored Doronina, N.V., Trotsenko, Y.A., Krausova, V.I., and Suzina, molecular aspects of C compound utilization, N.E. (1998). Paracoccus methylutens sp. nov. – a new 1 aerobic facultatively methylotrophic bacterium utilizing such as the relationship between metabolic routes dichloromethane. Syst. Appl. Microbiol. 21, 230–236. representing diferent steps of methylotrophy (e.g. htps://doi.org/10.1016/S0723-2020(98)80027-1. the serine cycle and the NMG pathway), and the Doronina, N.V., Trotsenko, Y.A., Kuznetzov, B.B., and regulatory dependencies between the Calvin cycle Tourova, T.P. (2002). Emended description of Paracoccus kondratievae. Int. J. Syst. Evol. Microbiol. 52, and the serine cycle. Such studies may also help 679–682. htps://doi.org/10.1099/00207713-52-2- clarify some ecological aspects, such as diferences 679. in ftness in particular niches between ‘autotrophic’ Dziewit, L., Dmowski, M., Baj, J., and Bartosik, D. (2010). and ‘heterotrophic’ methylotrophs. A greater Plasmid pAMI2 of Paracoccus aminophilus JCM 7686 carries N,N-dimethylformamide degradation-related understanding of the Paracoccus C1 metabolism will genes whose expression is activated by a LuxR family not only broaden general knowledge on methylo- regulator. Appl. Environ. Microbiol. 76, 1861–1869. trophy, but may also assist the construction of novel htps://doi.org/10.1128/AEM.01926-09 methylotrophic strains that are adapted to perform Dziewit, L., Czarnecki, J., Wibberg, D., Radlinska, M., Mrozek, P., Szymczak, M., Schlüter, A., Pühler, A., and industrially important processes. Bartosik, D. (2014). Architecture and functions of a multipartite genome of the methylotrophic bacterium Acknowledgements Paracoccus aminophilus JCM 7686, containing primary Tis work was supported by the National Science and secondary chromids. BMC Genomics 15, 124. htps://doi.org/10.1186/1471-2164-15-124 Centre (NCN), Poland, on the basis of decision no. 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