求助主题Diversity of Methylotrophy Pathways in the Genus Paracoccus
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Diversity of MethylotrophyPathways 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.
Paracoccus denitrifcans Pd 1222 is a model methy-lotrophic bacterium. Its methylotrophy is basedon autotrophic growth （enabled by the Calvincycle） supported by energy from the oxidation ofmethanol or methylamine. Te growing availabil-ity of genome sequence data has made it possibleto investigate methylotrophy in other Paracoccusspecies. Te examination of a large number of Para-coccus spp. genomes reveals great variability in C1metabolism, which have been shaped by difer-ent evolutionary mechanisms. Surprisingly, themethylotrophy schemes of many Paracoccus strainsappear to have quite diferent genetic and bio-chemical bases. Besides the expected 'autotrophicmethylotrophs', many strains of this genus possessanother C1assimilatory pathway, the serine cycle,which seems to have at least three independentorigins. Analysis of the co-occurrence of diferentmethylotrophic pathways indicates, on the onehand, evolutionary linkage between the Calvincycle and the serine cycle, and, on the other hand,that genes encoding some C1substrate-oxidizingenzymes occur more frequently in association withone or the other. Tis suggests that some geneticmodule combinations form more harmoniousenzymatic sets, which act with greater efciency inthe methylotrophic process and thus undergo posi-tive selection.
Te genus Paracoccus （class Alphaproteobacteria,order Rhodobacterales, family Rhodobacteraceae）currently includes around 70 defned species andhundreds of strains whose taxonomic position hasyet to be precisely assigned （NCBI Taxonomy,10 January 2018）。 Representatives of this genushave been identifed in perse environments. Teoriginal isolate and the type species, P. denitrifcans,was isolated from soil （Beijerinck, 1910）， like manyother Paracoccus spp. （Urakami et al., 1990; Silleret al., 1996; Tsubokura et al., 1999）。 Numerousstrains have been isolated from fresh water （Sheuet al., 2018）， seawater （Kim and Lee, 2015）， sedi-ments （G. Zhang et al., 2016）， activated sludge （Liuet al., 2006）， bioflters （Lipski et al., 1998）， or fromenvironments linked to higher organisms, such asplant roots （rhizosphere） （Doronina et al., 2002），marine bryzoans （Pukall et al., 2003）， insects （S.Zhang et al., 2016）， and human tissues （opportun-istic pathogens P. yeei and P. sanguinis） （Funke et al.,2004; McGinnis et al., 2015）。
Te ubiquity of Paracoccus spp. is due to theirgreat metabolic persity and fexibility. All para-cocci have an aerobic respiratory metabolism andutilize multi-carbon compounds. However, manyof them can switch between diferent growthmodes, using diferent carbon and energy sources,and employing various fnal electron acceptors. In the absence of oxygen, some Paracoccus spp. con-duct nitrate respiration （Baker et al., 1998; Kellyet al., 2006）。 Tis process leads to denitrifcationand has been applied for the removal of nitratesfrom wastewater （Liu et al., 2012）。 In addition to'standard' carbon sources, like sugars, amino acidsand succinate, Paracoccus strains isolated frompolluted environments can utilize xenobiotics, e.g.polycyclic aromatic hydrocarbons （PAHs）， makingthem useful in bioremediation （Sun et al., 2013）。
Numerous representatives of the genus can growchemolithoautotrophically, coupling CO2assimila-tion with the oxidation of inorganic compounds orelements, such as thiosulfate, thiocyanate, elemen-tal sulfur, molecular hydrogen, or ferrous ions（Kelly et al., 2006）。 Finally, many Paracoccus spp.are methylotrophs. Most utilize methanol （MeOH）and methylamine （MA） as sole carbon and energysources, e.g. P. denitrifcans and closely related P.versutus and P. kondratievae （Kelly et al., 2006）。
However, other strains isolated from environmentspolluted with C1 compounds are able to metabolizedimethylamine （DMA）， trimethylamine （TMA），N,N-dimethylformamide （DMF） （Urakami et al.,1990; Kim et al., 2001; Sanjeevkumar et al., 2013）or dichloromethane （Doronina et al., 1998）。
Te purpose of this study is to describe thepersity of methylotrophy in the genus Paracoccusat the biochemical and genetic levels, includingan examination of the origin and evolution of C1metabolism in this group of bacteria.
Paracoccus denitrifcansPd 1222 as an example of an'autotrophic' methylotroph
Since its isolation at the beginning of the 20thcentury （Beijerinck, 1910）， P. denitrifcans hasbeen extensively studied and diferent aspects of itsenergy metabolism have been revealed. Te compo-sition of its core respiratory chain closely resemblesthat of the classic mitochondrial respiratory chain（unlike respiratory chains of many other bacteria,including E. coli）， which made it a valuable modelfor studies on the energetic processes in eukaryotes（ John and Whatley, 1975）。 However, the electrontransport chain of P. denitrifcans also has manybranches at both the entrance and exit sides of thecore. On the one hand, this allows the bacterium toutilize alternative fnal electron receptors, namelynitrate, nitrite, nitric oxide and nitrous oxide （whichleads to denitrifcation – P. denitrifcans is an impor-tant model in studies on this process （Baker et al.,1998）， thus permiting growth when oxygen is lim-ited. On the other hand, diferent electron donorsmay be used. As a consequence, P. denitrifcans hasthe ability to grow chemolithoautotrophically oninorganic energy sources such as hydrogen andthiosulphate （Friedrich and Mitrenga, 1981）。 Itsautotrophic growth may also be supported by theoxidation of some organic C1 compounds, namelyMeOH, MA and formate （Baker et al., 1998）。 Tesecompounds are oxidized to CO2, the released elec-trons are used for oxidative phosphorylation, andthe ATP and CO2produced are used in the Calvincycle for biomass production. Tus, P. denitrifcansis an example of an 'autotrophic methylotroph',which lacks a 'heterotrophic' pathway dedicatedto the assimilation of reduced C1units （such as theserine cycle or ribulose monophosphate pathway），but it can assimilate carbon from C1compoundsafer their total oxidation （Baker et al., 1998; Chis-toserdova, 2011）（ Fig. 6.1）。
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 pathwaysdiscussed in this study. The enzymes and pathways present in P. denitrifcans Pd 1222 are shown in red, thosepresent 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-formylglutathionehydrolase; 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, TMAmonooxygenase; XoxF, XoxF-type MeOH dehydrogenase. *Methylated amines – TMA, DMA and MA.
Since the 1970s, numerous studies have soughtto understand the details of C1metabolism inP. denitrifcans （Harms et al., 1985; Baker et al.,1998）， especially in strain Pd 1222, which is read-ily transformed by conjugation to enable geneticmanipulation （Devries et al., 1989）。 Te resultsof these studies have uncovered the properties ofmany P. denitrifcans proteins involved in methylo-trophy （mainly MeOH and MA dehydrogenases, aswell as associated proteins, i.e. those involved in thetransfer of electrons from the dehydrogenases tothe respiratory chain）， and have shed light on theregulation of their expression （Baker et al., 1998）。
Te whole genome sequence of P. denitrifcansPd 1222 was obtained in 2006 （NCBI Genomes）。
It has an unusual structure consisting of twochromosomes （chromosome 1, 2.8 Mb, and chro-mosome 2, 1.7 Mb） and one large plasmid （plasmid1 1650 kb）。 Te availability of this sequence haspermited elucidation of the genetic basis of itsmethylotrophy. P. denitrifcans Pd 1222 carries sev-eral gene clusters responsible for C1metabolism,dispersed across the three replicons. Genes for theenzymes involved in the oxidation of primary C1substrates to formaldehyde are located on chromo-some 2 （gene cluster encoding MxaFI-type MeOHdehydrogenase and associated proteins） and plasmid 1 （the mau genes encoding small and largesubunits of MA dehydrogenase and associated pro-teins）。 Te second step in the methylotrophy of P. denitrifcans Pd 1222 is oxidation of formaldehydeto formate in the glutathione-dependent pathway,which is essential for growth of this strain on C1compounds （Harms et al., 1996）。 Tree enzymesof this pathway, S-（hydroxymethyl）glutathionesynthase （Gfa）， S-（hydroxymethyl）glutathionedehydrogenase （FlhA）， and S-formylglutathionehydrolase （FghA）， are encoded within chromo-some 1. Interestingly, these glutathione-dependentformaldehyde oxidation genes occur in the imme-diate vicinity of genes encoding XoxF-type MeOHdehydrogenase and associated proteins. XoxF wasrecently confrmed as a MeOH-oxidizing enzyme（Keltjens et al., 2014; Chistoserdova, 2016）。
However, its involvement in MeOH metabolismin P. denitrifcans was suggested many years before（Harms et al., 1996）， although its redundancywith a MxaFI-type system remains unexplained.
Formate is oxidized to CO2 by two multi subunit（encoded in chromosomes 1 and 2） or one singlesubunit （encoded in chromosome 1） formate dehy-drogenase. Finally, the Calvin cycle gene cluster,which is required for assimilation of CO2, is locatedon chromosome 1 and consists of genes encod-ing two subunits of RuBisCO （rbcL and rbcS），as well as genes for fructose-1,6-bisphosphatase（fp）， phosphoribulokinase （prk）， transketolase（tkt）， fructose-1,6-bisphosphate aldolase （fa），RuBisCO activating protein （cbbX）， and the Calvincycle regulator （cbbR）。
Parallel studies on the methylotrophy of theclosely related P. versutus have shown that this spe-cies utilizes similar routes of C1 metabolism. Teassimilatory pathway required for growth on C1substrates includes highly similar MA dehydro-genase and Calvin cycle enzymes （Karagouni andKelly, 1989; Baker et al., 1998）。
Paracoccus aminophilusJCM 7686 and Paracoccusaminovorans JCM 7685 asserine cycle methylotrophsspecialized in DMF utilization
In 1990, the isolation of two DMF-degradingstrains from a sample of DMF-polluted soil inJapan was reported （Urakami et al., 1990）。 Tesestrains, designated JCM 7686 and JCM 7685 wererecognized as representatives of two new Paracoccusspecies: P. aminophilus and P. aminovorans （Urakamiet al., 1990）。 Te methylotrophic pathways of theseisolates were shown to be more complex than thoseof P. denitrifcans, because they include enzymes required for the utilization of a wider range of C1compounds. Both strains are able to decomposeDMF to formate and DMA, and oxidize TMA viatrimethylamine N-oxide （TMAO） to DMA, andthen degrade the resultant DMA to MA （Urakamiet al., 1990）。
Te entire genome sequences of P. aminophilusJCM 7686 and P. aminovorans JCM 7685 wereobtained by our group （Dziewit et al., 2014; Czar-necki et al., 2017）， facilitating the reconstruction oftheir methylotrophic pathways. As expected, genesrequired for the metabolism of additional C1sub-strates were identifed in both strains. Tese encodesmall and large subunits of DMFase （DmfA1A2），as well as TMA monooxygenase （Tmm）， TMAOdemethylase （Tmd）， and multi-subunit DMAmonooxygenase （DmmABCD）。 Besides TMA andDMA monooxygenases, the P. aminovorans JCM7685 genome also encodes TMA and DMA dehy-drogenases （Tdh and Ddh, respectively）， whichmay catalyse the oxidation of TMA and DMA （ Fig.6.1）。 Te role of these genes in the metabolism ofspecifc C1 substrates has been confrmed in bothstrains （Dziewit et al., 2010, 2015; Czarnecki et al.,2017）。
Surprisingly, the enzymes involved in themetabolism of MeOH and MA by P. aminophilusand P. aminovorans difer from those employed byP. denitrifcans. In the case of MeOH utilization,P. aminophilus and P. aminovorans do not possessa MxaFI-type MeOH dehydrogenase, and theirgrowth on this compound relies fully on a XoxF-type dehydrogenase, as has been confrmed bymutational analysis （Dziewit et al., 2015）。 In thecase of MA, both strains have genes for an alterna-tive MA oxidation pathway: the N-methylglutamate（NMG） pathway. In P. aminovorans the NMG path-way is the only pathway for MA oxidation, while inP. aminophilus it co-exists with the MA dehydroge-nase pathway, which was previously characterized inP. denitrifcans （ Fig. 6.1）。 Furthermore, both strainslack RuBisCO genes, so cannot assimilate CO2.
Tus, their methylotrophy has to be supported byanother pathway of C1 unit assimilation. A serinecycle gene cluster was found in both genomes; itsinvolvement in methylotrophy has been confrmedin P. aminovorans, and the role of transcriptionalregulator ScyR in its regulation was revealed（Czarnecki et al., 2017）。 Te serine cycle requiresglyoxylate regeneration, which is accomplished bythe action of the ethylmalonyl-CoA pathway in P. aminovorans JCM 7685 （Czarnecki et al., 2017）。 Allgenes required for this pathway are also present inP. aminophilus, in the non-serine cycle methylotrophP. denitrifcans and even in non-methylotrophicstrains, as they are used for other purposes, such asgrowth on C2 compounds （Schneider et al., 2012）。
Te genes required for another glyoxylate-regen-erating process, the glyoxylate shunt, are foundin P. aminophilus and P. aminovorans, but, as theywere unable to support methylotrophic growth ina strain with a blocked ethylmalonyl-CoA pathway（Czarnecki et al., 2017）， their role remains unclear.
Owing to the presence of diferent enzymes oxi-dizing primary C1 substrates and the serine cycle,the fate of C1 units released during methylotrophicmetabolism is more complex in P. aminophilus andP. aminovorans than in P. denitrifcans. In the serinecycle, carbon is assimilated in the form of methyl-ene group bound to tetrahydrofolate （THF） andCO2（ Fig. 6.1）。 Methylene-THF may be delivereddirectly by the C1-substrate-oxidizing enzymesTMAO demethylase and DMA monooxygenaseor the NMG pathway, which do not release freeformaldehyde, but transfer the C1 unit directly toTHF （ Fig. 6.1）。 On the other hand, the XoxF-typeMeOH dehydrogenase, TMA dehydrogenase,DMA dehydrogenase and MA dehydrogenaserelease free formaldehyde, which has to be oxidizedto formate by the glutathione-dependent pathwaypresent in P. aminophilus, P. aminovorans and P. deni-trifcans. To feed the serine cycle, the formate has tobe bound to THF, and then it has to be reduced toa methylene group in an energy-requiring process.
Te THF-dependent formate reduction pathway isfound in all three Paracoccus spp., and requires theaction of two enzymes: formate-THF ligase （Ftf L）and 5,10-methylene-tetrahydrofolate dehydroge-nase/methenyl-tetrahydrofolate cyclohydrolase（FolD）。 Tis pathway may also act in the oppositedirection to oxidize the methylene group to formate.
In this case, FolD promotes the reverse reactionitself, while the second reaction is catalysed by for-myltetrahydrofolate deformylase （PurU）， which isalso present in all three species （ Fig. 6.1）。 A specialsituation occurs during growth on DMF, whereDMFase releases a C1 unit directly in the form offormate and the second product is DMA. Te fateof C1 units has been analysed experimentally in P. aminovorans, where growth of a strain lacking the glutathione-dependent pathway for formaldehydeoxidation on diferent C1 substrates was examined.
As expected, this strain was unable to utilize MeOH（it has the XoxF-type MeOH dehydrogenasereleasing free formaldehyde, which cannot be fur-ther oxidized without the glutathione-dependentpathway）， but no efect on growth on MA wasdetected （it has the NMG pathway which producesmethylene-THF, which directly enters the serinecycle or is oxidized in the THF-dependent pathway,without involvement of the glutathione-dependentpathway）。 Intermediate phenotypes were observedduring growth on DMF, TMA or DMA, sincemonooxygenases can oxidize some portion of thesecompounds to produce methylene-THF, whileanother portion is oxidized by dehydrogenases toproduce free formaldehyde （Czarnecki et al., 2017）（ Fig. 6.1）。 Like P. denitrifcans, P. aminophilus andP. aminovorans have formate dehydrogenases todeal with an excess of formate （Dziewit et al., 2015;Czarnecki et al., 2017）。
Te genomic localization and clustering ofmethylotrophy genes of P aminophilus and P. amino-vorans suggests that many of them could have beenacquired horizontally to confer increased ftness forgrowth in DMF-polluted soil. Some DMFase genesof P. aminophilus are located on the small plasmidpAMI2 （18.6 kb）， which also carries a geneticmodule putatively involved in its mobilization forconjugal transfer （Dziewit et al., 2010）。 A closelyrelated DMFase is encoded in the chromosomesof both P. aminovorans and P. aminophilus （84%aa identity of large subunits and 73% aa identityof small subunits）。 However, these chromosomalDMFase genes and their adjacent transcriptionalregulator genes are surrounded by genes encodingtransposases and other proteins typical of mobilegenetic elements, which indicates their recentacquisition. Similarly, the TMA and DMA dehydro-genase genes of P. aminovorans JCM 7685, that arenot found in any other Paracoccus strain （Czarneckiet al., 2017）， are located on the large extrachromo-somal replicon pAMV3 （740 kb）， which, like otherreplicons of this type, seems to be a reservoir ofvarious horizontally transmited genes.
A notable example of methylotrophy genesacquired by HGT are those clustered withina 40-kb methylotrophy island （MEI） locatedon the extrachromosomal replicon pAMV1 ofP. aminovorans （ Fig. 6.2）。 Te genes present on thisisland are involved in all steps of methylotrophy: （i）oxidation of primary C1 substrates with methylene-THF generation （TMA monooxygenase, TMAOdemethylase, DMA monooxygenase, the NMGpathway）， （ii） oxidation of methylene-THF toformate （FolD and PurU）， and assimilation of C1units in the form of methylene-THF and CO2（theserine cycle）。 Te closest homologue of this MEIwas identifed in Paracoccus sp. N5 （Beck et al.,2015; Dziewit et al., 2015; Czarnecki et al., 2017），and similarly clustered genes are located in thechromosomes of many bacteria of the Roseobacterclade, including Ruegeria pomeroyi （Dziewit et al.,2015）。 Te MEI genes are also present in the P. ami-nophilus genome. However, in this case the island ispided in two, with one part encoding the serine cycle enzymes located in the chromosome, andthe second part, comprising the rest of the MEI,in extrachromosomal replicon pAMI6 （207 kb）（Dziewit et al., 2015）， whose genetic load is 40%homologous to that of P. aminovorans pAMV1. Itappears that acquisition of the MEI （most prob-ably from representatives of the Roseobacter clade（Dziewit et al., 2015） could have enhanced themethylotrophic ability of P. aminophilus and P. aminovorans, which made them beter adapted toliving in DMF-polluted soil. To fully reconstructthe evolution of C1 pathways in the genus Paracoc-cus, deeper analyses are required, which considernot only the presence of given pathways （which hasalready been done （Dziewit et al., 2015; Czarneckiet al., 2017）， but also their phylogenetic relation-ships.
Occurrence and phylogeneticrelationships of methylotrophygenes in Paracoccus spp
Wepreviouslyexamined44Paracoccusspp.genomesto determine the persity of methylotrophy genesin this genus （Dziewit et al., 2015; Czarnecki et al.,2017）。 In this study we have broadened this analysisusing additional recently deposited Paracoccus spp. sequences （in total, 62 Paracoccus genomes wereavailable in NCBI GenBank on 11 May 2018） plus10 Paracoccus spp. genomes obtained by our group,which will be fully described in a forthcoming pub-lication （P. bengalensis DSM 17099, P. ferrooxidansNCCB 1300066, P. haundaensis LGM P-21903, P. kondratievae NCIBM 13773, P. pantotrophus DSM11072, P. solventivorans DSM 11592, P. sulfuroxidansJCM 14013, P. thiocyanatus JCM 20756, P. versutusUW1 and P. yeei CCUG 32053）。 Te majority of thestudied Paracoccus strains （almost 65%）， containsets of genetic modules involved in all three steps ofmethylotrophy （Chistoserdova, 2011）， which arepotentially sufcient for methylotrophic growth.
Comparative analysis of this large number ofParacoccus genomes allowed us to distinguish twogroups of methylotrophy-related genes based ontheir degree of conservation: （i） highly conserved,vertically transmited genes, present in almost all ofthe genomes, and （ii） genes present only in somestrains because of selective loss, horizontal genetransfer, or a combination of these two evolution-ary mechanisms. Te genes of the frst group areusually located on chromosomes, while many of thegenes of the second group are found on extrachro-mosomal replicons, or in the company of mobilegenetic elements when present on chromosomes.
Te frst group includes genes comprisingfundamental pathways, that are important fornon-methylotrophic metabolism, to which furtherC1 pathways are appended. Te frst exampleof this group is a cluster consisting of genes forglutathione-dependent formaldehyde oxidation（gfa, fhA, fghA） and for MeOH oxidation by XoxF（ Fig. 6.3）。 Te compact and conserved nature ofthe gene cluster comprising both pathways suggeststheir cooperation. As mentioned above, both theXoxF and the glutathione-dependent pathways maybe essential for some methylotrophic processesin Paracoccus spp. （Ras et al., 1995; Harms et al.,1996）。 However, the ubiquity of this gene cluster innon-methylotrophs indicates that its signifcance ismore general. Its probable function in non-methyl-otrophic organisms is in the utilization of MeOH asan additional source of energy, without its assimila-tion into biomass. Despite its overall conservation,the cluster is truncated in some Paracoccus spp. andlacks the xox genes. For example, P. halophilus JCM14014 does not have any homologues of xoxF. InP. alcaliphilus JCM 7364 the xox genes and thegenes for the glutathione-dependent pathway areseparated, being located on a large （430 kb） extra-chromosomal replicon and on the chromosome,respectively.
Another example of genes of the conservedgroup are those encoding the numerous enzymesof the ethylmalonyl-CoA pathway. Tese donot form a single gene cluster but are scateredthroughout Paracoccus genomes. Enzymes of theethylmalonyl-CoA pathway are involved in variousprocesses, such as the utilization of C2 compoundsas carbon and energy sources or synthesis of poly-hydroxyalkanoates. As mentioned above, in someParacoccus spp., the ethylmalonyl-CoA pathway isresponsible for glyoxylate regeneration, which iscrucial for functioning of the serine cycle duringmethylotrophic growth （Chistoserdova, 2011）。
A few Paracoccus spp. lack some enzymes of thispathway, e.g. P. chinensis CGMCC 1.7655 or strainsof P. sanguinis. Nevertheless, a complete set ofethylmalonyl-CoA pathway genes was found in allstrains that employ the serine cycle.
Te next examples of conserved methylotrophy-linked genes in Paracoccus spp. are those involvedin THF-dependent transformations of C1 units: folD, purU and ffL. Te encoded enzymes areimportant for the generation of C1-THF interme-diates required by biosynthetic pathways, such aspurine synthesis. As in the serine cycle C1 units areincorporated into biomass in the form of methyl-ene-THF, the THF-dependent pathway constitutesa central metabolic process in Paracoccus serinecycle methylotrophs （ Fig. 6.1）。 It should be notedthat besides the conserved set of folD, purU andffL genes, there are also numerous homologuesthat are horizontally transferred, for example in thecompany of serine cycle genes or NMG pathwaygenes. It is possible that the serine cycle-associatedand the NMG pathway-associated homologuesare beter adapted to co-operate with the methylo-trophic pathways, while the conserved homologuesare mainly responsible for anabolic housekeepingfunctions, but members of these groups are likelyto be interchangeable to some extent.
Telastrepresentativesofthegroupofconservedgenes are gene clusters encoding multi-subunitformate dehydrogenases, which are responsiblefor formate detoxifcation in both methylotrophsand non-methylotrophs, and form part of the coregenome of Paracoccus spp. Te second group of genes, which only occursin some Paracoccus strains, includes gene setsinvolved in C1 unit assimilation pathways, theCalvin cycle and the serine cycle, as well as genesinvolved in primary oxidation of C1 substrates. TeCalvin cycle is widespread among Paracoccus spe-cies （ Fig. 6.4） and a complete set of genes requiredfor this process was identifed in 56% of analysedstrains. Phylogenetic analysis of the RuBisCO largesubunit （RbcL） and phosphoribulokinase （Prk）indicates that there are at least two evolutionarilydistinct lineages of Calvin cycle genes in this genus（Figs. 6.5–6.7）。 Tis is refected in variations in theorganization of Calvin cycle gene clusters in dif-ferent Paracoccus spp. （ Fig. 6.5）。 Te Calvin cyclegenes are generally found in chromosomes, withsome exceptions, e.g. in Paracoccus sp. N5 and P. kondratievae they are located on large extrachromo-somal replicons （1 Mb and 423 kb, respectively），most probably as a result of translocation from thechromosome.
Compared with the Calvin cycle, the serine cycleis less prevalent in Paracoccus spp. and its occur-rence was predicted in 17% of the analysed strains.
Serine cycle gene sets are present in a few diferentlineages of the genus （ Fig. 6.4）， and three types ofgene organization were identifed （ Fig. 6.5）。 Teoccurrence of three gene organization schemes isprobably an efect of the independent acquisitionof the serine cycle from diferent sources. Teseseparate events are refected in the phylogenetictree of phosphoenolpyruvate carboxylase （Ppc），the key enzyme of the cycle （ Fig. 6.8）。 Te frst typeof serine cycle gene organization occurs in the MEIof P. aminovorans JCM 7685 and, as mentionedabove, is also found in many representatives of theRoseobacter clade, in the order Rhodobacterales, asare Paracoccus spp. Te second type of serine cyclegene organization, found on the chromosome of P. zhejiangensis J6, for example, is also most similar toarrangements present in members of the Roseobacterclade. Te third type of serine cycle gene organiza-tion, found in P. denitrifcans ISTOD1, is typical forstrains of the Aminobacter and Labrys genera （Becket al., 2015）， within the order Rhizobiales. In someParacoccus spp., two types of serine cycle modulecoexist in the same genome （ Fig. 6.4）。 Te serinecycle clusters may be located within extrachromo-somal replicons, as in pAMV1 in P. aminovoransJCM 7685 and probably also ISTOD1 in P. deni-trifcans （the unfnished genome sequence of thisstrain does not allow confrmation of the genomiclocalization of this gene cluster, although it is pre-sent in a sequence scafold containing a repABC replication-partitioning module, typical for largeplasmids of Alphaproteobacteria）。 Te serine cyclegenes have a chromosomal location in P. aminophi-lus JCM 7685 or P. sulfuroxidans JCM 14013. Tedistribution of the serine cycle genes indicates thatthey were acquired horizontally several times indiferent lineages of the genus. Nevertheless, theirprevalence could also have been shaped by geneloss. Te best example is the incomplete serinecycle cluster of P. alcaliphilus JCM 7364, whichlacks the pcc gene （there are no pcc homologues inthe entire P. alcaliphilus genome）。
One particularly interesting aspect is co-occurrence of the Calvin cycle and the serine cycle,and co-evolution of these two cycles in Paracoccusgenomes. Te phylogeny and distribution pat-terns of the key enzymes of these cycles （Figs 6.4,6.6–6.8） indicate that the Calvin cycle is the moreancient in this group of bacteria, and that the serinecycle has been acquired independently in severallineages. In some strains, these two cycles coexist（ Fig. 6.4）， but in others, the Calvin cycle seems tohave been lost afer serine cycle acquisition. Tisphenomenon is perfectly illustrated by a group ofthree isolates: Paracoccus sp. N5, P. aminovoransHPD-2 and P. aminovorans JCM 7685. Te frststrain has the intact Calvin cycle gene cluster, thesecond one possesses an incomplete cluster lacking the rbcS gene but containing rbcL and cbbX pseu-dogenes, while the third strain does not possessthe rbcS, rbcL nor cbbX genes （ Fig. 6.4）。 However,not all of the Calvin cycle genes are lost in Paracoc-cus serine cycle methylotrophs lacking this cycle.
Tese strains still have a truncated version of theCalvin cycle gene cluster, including its three frstgenes encoding the Calvin cycle regulator （CbbR），fructose-1,6-bisphosphatase （Fbp） and phosphori-bulokinase （Prk）。 Te phylogeny of the Paracoccusphosphoribulokinases （ Fig. 6.7） indicates that thesame reduction of the Calvin cycle gene clusterarose in diferent lineages where the serine cycleappeared. Moreover, retention of a reduced Calvincycle gene cluster is typical only for the serine cyclemethylotrophs, whereas the cbbR, fp and prk genesare not present in Paracoccus non-methylotrophs.
An identically organized gene cluster was detectedin another serine cycle methylotroph, Methylo-bacterium extorquens PA1. Tis includes the geneencoding QscR, a transcriptional regulator withhomology to CbbR, which is a global regulatorof the serine cycle genes （Kalyuzhnaya and Lid-strom, 2003, 2005）。 Recently, QscR was shown tobe regulated by phosphoribulokinase, and it wasdemonstrated that both the qscR and prk genes areessential for the methylotrophy of M. extorquens（Ochsner et al., 2017）。 Parallel evolution of thesame gene set in Paracoccus serine cycle methylo-trophs （and M. extorquens） implies that there is auniversal evolutionary linkage between the Calvincycle and the serine cycle （Ochsner et al., 2017）。 Asthe role of the cbbR–fp–prk cluster is only regula-tory, some strains may have evolved towards theloss of these genes. In the analysed strain set thereis one example where partial loss of this regulatorycluster has occurred: P. saliphilus DSM 18447 hastwo diferent serine cycle gene sets, plus a remnantof the cbbR–fp–prk cluster （it lacks cbbR, has a fppseudogene and an intact prk gene）。 However, the ability of this strain to grow methylotrophically hasyet to be tested.
Among the genes involved in C1 metabolism,those encoding enzymes participating in the pri-mary oxidation of C1 substrates, the frst stage ofmethylotrophy, show the greatest variability intheir occurrence. One prominent exception is theaforementioned conserved XoxF-type MeOHdehydrogenase. Conversely, a second type ofMeOH dehydrogenase, the two subunit enzymeMxaFI, is found only in a few Paracoccus spp. （ Fig.6.4）。 A similarly limited distribution is observedin the case of MA dehydrogenase （ Fig. 6.4）。 Bothenzymes, which were thought to be reliable mark-ers of methylotrophy in this genus based on studieson P. denitrifcans Pd 1222, seem to be rather rareamong Paracoccus spp. whose genomes have beensequenced. MxaFI-type MeOH dehydrogenaseand MA dehydrogenase usually co-exist with theCalvin cycle （ Fig. 6.4）， which may result fromadaptation of their mode of action to interact beterwith the Calvin cycle than with the serine cycle（release of the free formaldehyde, which is thenoxidized to formate in the glutathione-dependent pathway, but not direct binding of the methylenegroup to THF）。 Genes of the NMG pathway forMA oxidation are mainly found in strains that pos-sess the serine cycle （ Fig. 6.4）。 Te NMG pathwayenzymes appear to be evolutionarily bound to theserine cycle enzymes, since their phylogenetictrees show a similar topology （ Fig. 6.9）。 While thethird type of serine cycle gene cluster （ Fig. 6.4） isfound in the company of only the NMG pathwaygenes, the frst and the second types co-occur withother methylene-THF-producing enzymes, TMAmonooxygenase, TMAO demethylase and DMAmonooxygenase （ Fig. 6.4）， e.g. within the MEI ofP. aminovorans JCM 7685 （ Fig. 6.2） （Czarnecki etal., 2017）。 Te exception are strains of P. yeei whichlack the serine cycle, but have the NMG pathwayaccompanied by DMA monooxygenase, TMAmonooxygenase and TMA N-oxide demethylasegenes （ Fig. 6.4）。
Te DMFase genes dmfA1A2 were only foundin three strains, P. aminophilus JCM 7686, P. ami-novorans JCM 7685 and P. aminovorans HPD-2,and always in association with serine cycle, NMGpathway, TMA monooxygenase, TMAO demethyl-ase and DMA monooxygenase genes （ Fig. 6.4）。 Temethylotrophic potential of Paracoccus spp. doesnot seem to have been fully determined. Genes forTMA and DMA dehydrogenases were found onlyin P. aminovorans JCM 7685, but these enzymaticactivities had already been identifed in other Para-coccus isolates, and they may be important for theutilization of methylated amines when the oxygenconcentration is changeable （Kim et al., 2001,2003）。 Moreover, one strain whose genome has yetto be sequenced, P. methylutens DM 12, expressesdichloromethane halogenase, which is required fordichloromethane utilization. Tus, further inves-tigation of the content of Paracoccus spp. genomesmay reveal interesting and unexpected features oftheir C1pathways.
From our detailed analysis of numerous Paracoccusspp. genome sequences, it is now clear that the arche-typal autotrophic methylotroph, P. denitrifcans Pd1222, represents only a small part of the methylo-trophic capacity present in the genus. Besides somegenerally conserved features, like the presence ofthe glutathione-dependent formaldehyde oxida-tion pathway and the ethylmalonyl-CoA pathway,methylotrophic Paracoccus spp. vary greatly in thegenetic and biochemical basis of their C1metabo-lism. As anticipated there are diferences in the setsof enzymes responsible for the primary oxidation ofC1compounds, which are located at the periphery of the C1metabolic net, and thus may be most'exposed' to evolutionary changes. However, thereis also considerable variation in the very nucleus ofthe C1metabolism: the C1assimilatory pathways.
Tis variability is an efect of diferent evolution-ary mechanisms, which are very hard to retrace.
As was proposed previously, genetic modules rep-resenting diferent steps of methylotrophy may bereassembled in diferent genomes, generating newmetabolic capabilities （Chistoserdova, 2011）。 Tisappears to be the case in Paracoccus spp. Te morefrequent coexistence of certain modules indicatesthat they may 'ft' together beter, probably due tomore efcient cooperation. Tis explains why themethylene THF-generating NMG pathway for MAoxidation is typically found with the methyleneTHF-consuming serine cycle, whereas the formal-dehyde-producing MA dehydrogenase is foundwith the Calvin cycle. Te opposite combinationsof these pathways occur in some Paracoccus spp.,but they are much less common.
Te evolutionary tendencies in methylotrophyof the genus Paracoccus are the same as in the classAlphaproteobacteria as a whole, where the polyphy-letic origins of C1metabolism have already beendescribed （Beck et al., 2015）。 Interestingly, thesetendencies may be observed even on a microscalelevel, within strains of the same species. For exam-ple, comparison of two strains of P. denitrifcans,Pd 1222 and ISTOD1, shows how evolutionaryprocesses can easily rebuild a metabolic net. It islikely that the discovery of additional genetic mod-ules associated with methylotrophy in this specieswill follow the sampling of new strains, especiallythose from environments where C1compounds arepresent.
Although analysis of the increasing body of genomicdata from Paracoccus spp. can give many interestingresults, there is a need for experimental studies onthe C1metabolism of these bacteria. Te predictedfunctions of many genes have to be verifed, theinvolvement of others confrmed （e.g. genes encod-ing putative transporters located in vicinity of methylotrophy genes）， and their regulatory mecha-nisms characterized. It would also be informativeto determine whether serine cycle-based methylo-trophy can be transmited to other strains, e.g. viathe transfer of the pAMV1 methylotrophy island.
Paracoccus spp. seem to be ideal models for study-ing the evolution of methylotrophy. Studies onthese bacteria may shed light on some unexploredmolecular aspects of C1compound utilization,such as the relationship between metabolic routesrepresenting diferent steps of methylotrophy （e.g. the serine cycle and the NMG pathway）， and theregulatory dependencies between the Calvin cycleand the serine cycle. Such studies may also helpclarify some ecological aspects, such as diferencesin ftness in particular niches between 'autotrophic' and 'heterotrophic' methylotrophs. A greaterunderstanding of the Paracoccus C1metabolism willnot only broaden general knowledge on methylo-trophy, but may also assist the construction of novelmethylotrophic strains that are adapted to performindustrially important processes.
Tis work was supported by the National ScienceCentre （NCN）， Poland, on the basis of decision no.
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