1  

Supplementary Material Metabolic model for the filamentous “Candidatus Microthrix parvicella” based on genomic and metagenomic analyses

Simon Jon McIlroy, Rikke Kristiansen, Mads Albertsen, Søren Michael Karst,

Simona Rossetti, Jeppe Lund Nielsen, Valter Tandoi, Robert James Seviour and Per

Halkjær Nielsen

Contents: Supplementary Figure 1 page 1 Supplementary Figure 2 page 2 Supplementary Figure 3 page 3 Supplementary Text page 4 Supplementary Table 1 page 8 Supplementary Table 2 page 11 Supplementary Table 3 page 12 Supplementary Table 4 page 18 References page 20

Supplementary Figure 1: Maximum likelihood phylogenetic tree of the 16S rRNA genes from M. parvicella isolates and related sequences. The RN1 strain is represented in bold typeface. Scale bar corresponds to 0.1 substitutions per nucleotide position. T. flocculiformis was selected as the root sequence.

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Supplementary Figure 2: Explorative mapping visualisation for the RN1 genome. Each contig is represented by a circle. Lines connecting circles indicate associations between the represented contigs. Circle size represents relative contig size (max= 442 525 bp). Circle shade indicates relative coverage (shade range: black = min to white = max).

a.

b.

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NAD(P)H( NAD(P)+(

PolyP( PPi(

CoASH(

ATP( ADP( AMP(

Polyphosphate( Pyrophosphate(

Coenzyme(A(

TAG(

Triacylglycerol(

Quinone( Ferredoxin(

Q( Fd(

GTP( GDP(

CO2/HCO

3H(

CO2(Pi(

Orthophosphate(

Oxaloacetate(Citrate(

Malate(

SuccinylHCoA(

PropionylHCoA(

Glyoxylate(

Glycerol(GlycerateH3P(

βHmethylmalylHCoA(

AcetylHCoA(

TCA$cycle$

FaJy(acylHCoA(

β*oxida0on$

EMP$pathway$

Diacylglycerol(

Long(chain(faJy(acid((LCFA)(

Monoacylglycerol(

Glycerol(

Triacylglycerol(

(TAG)(

GlyceroneHP(GlyceraldehydeH3P(

Phosphoenolpyruvate(

Succinate(

2HOxoglutarate( Glutamate(

NH3(

GABA( Succinate(semialdehyde((Q(

(Q(

GABA$shunt$

Succinate(

Ethylmalonyl*CoA$pathway$

Polyphosphate(

(n+1)(

LCFA(

LCFA(

AcetylHCoA(

AcetylHCoA(Pyruvate(

UDPHGlucoseH1P(

βHDHFructoseH6P(

Trehalose(

43*$αHDHGlucoseH6P(

44$

45$47$25$

27$

28$

29$30$

31$

32$

33$

34$

35$

72$

71$70*$69*$68$

1-3$

4$5$

6$

7$

11-12$ Fd(

15-16$17-19$

20$

21$

14$

118$

(119)$GABA(

111/112$

AcetylHCoA(

AcetylHCoA(

54*$(55)$ 56*$

57$

58$ 59*$ (60)$ 61$

Glyoxylate(

62*$

64-65*$58$

66-67$

62*$63*$

86*$ 83$

85*$(84)$93*$

93*$

94*$

120/123$

123$120$

121$

Fd(

82*$

133-140$

FHATPase(

H+(

Pst(transporter(

128-131$

Na+(

Na+(

132*$

NH3(

NH3(

110$

Oxaloacetate(Citrate(

Malate(

SuccinylHCoA(

PropionylHCoA(

Glyoxylate(

GlycerolH3P(

GlycerateH3P(

βHmethylmalylHCoA(

AcetylHCoA(

TCA$cycle$

EMP$pathway$

Diacylglycerol(

LCFA(

Monoacylglycerol(

GlycerolH3P(

Triacylglycerol(

(TAG)(

GlyceroneHP(GlyceraldehydeH3P(

Phosphoenolpyruvate(

2HOxoglutarate(

Ethylmalonyl*CoA$pathway$

AcetylHCoA(Pyruvate(

βHDHFructoseH6P(

26$

28$

29$30$

31$

32$

33$

34$

35$

72$

1-3$

4$5$

6$

7$

11-12$ Fd(

15-16$

21$

AcetylHCoA(

AcetylHCoA(

54*$(55)$ 56*$

57$

58$ 59*$ (60)$ 61$

Glyoxylate(

62*$

64-65*$58$

66-67$

62*$63*$

91$

(88)$

(87)$

133-140$

FHATPase(

141$

H+(

H+(

Trehalose(

βHDHGlucose(

βHDHGlucoseH1P(

βHDHGlucoseH6P(

(48)$

49$25$

24$23*$

Fd(

Succinate(

Propionate(

Acetate(

FaJy(acylHCoA(

FaJy(acylHCoA(

FaJy(acylHCoA(

90*$

FaJy(acylHCoA(

β*oxida0on$

AcetylHCoA(

86*$ 83$

85*$(84)$

82*$

Glycerol(

71$

TAG( Diacylglycerol( Monoacylglycerol(

LCFA(

Glycerol(

Exocellular(lipases(

LCFA(

LCFA(Glycerol(

Glycerol(

GlycerolH3P(

GlycerolH3P(

H+(

H+(

Proton(pump(

74-77$

78$

???(

Polyphosphate(

(nH1)(

121$

120/123$

123$

122*$

80-81*$

80-81*$

92*$92*$

a.$Anaerobic$

b.$Aerobic$

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Supplementary Figure 3: Diagrammatic representation of the proposed metabolic model for M. parvicella in EBPR activated sludge treatment plants a. in the absence of electron acceptors (anaerobic) and b. in the presence of the electron acceptors oxygen (aerobic) or nitrate. Underlined products indicate putative storage or end products of metabolism. Numbers correspond to gene assignments in Table S3.

Supplementary Text LCFA transport in M. parvicella The mechanism whereby M. parvicella assimilates long chain fatty acids (LCFA) is unclear, as it is for Gram positive bacteria in general. However, putative fadD genes coding for the inner membrane associated fatty acyl-CoA synthases (FACS) (EC 6.2.1.3) were identified in its genome. These are thought to play a role in the required activation of LCFAs to their CoA esters for subsequent metabolism, a process concomitant in E. coli with their transport into the cell by the fadL transporter (Overath et al., 1969; Black et al., 1992). Substrate uptake profiles for M. parvicella The narrow substrate uptake profiles observed using in situ FISH-MAR for M. parvicella often disagree fundamentally with the corresponding pure culture data which suggest that VFAs are in fact utilized as sole carbon sources supporting its growth (Tandoi et al., 1998; Tomei et al., 1999). With the ‘Dutch isolate’ VFA utilization appeared to require the presence of esterified LCFAs (Slijkhuis, 1983). More specifically, acetate has been reported repeatedly to support growth of the RN1 strain in pure culture (Tandoi et al., 1998; Tomei et al., 1999), yet in situ analyses have never shown any capability for M. parvicella to assimilate this substrate (Andreasen & Nielsen, 1997; 1998). Analysis of the RN1 strain genome failed to reveal any candidate gene for an acetate transporter, although it is possible that it is present but very different in sequence to the gene/s for transporters in other organisms characterized in this manner. This contradiction may reflect the inability of M. parvicella to adapt to, and compete effectively for, VFAs with other populations in situ in a highly competitive environment where substrate concentrations are likely to be much lower than those used in pure culture studies. It is now known that some bacteria under the dynamic conditions of activated sludge demonstrate much more specialized carbon utilization profiles than they do in pure culture (Nielsen et al., 2010; Kindaichi et al., In press). TAG metabolism in M. parvicella Putative genes encoding enzymes for the synthesis of the glycerol backbone and the two acyltransferases, plsB and plsC catalysing the first two acylation steps in the pathway were located. Although evidence for the latter two genes was not totally convincing, these acylation steps are shared with phospholipid synthesis. The

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diacylglycerol (DAG) is released from the resultant phosphatidate by the action of a phosphatidate phosphatase (PAP) (EC 3.1.3.4). A candidate gene coding for this enzyme in members of the bacteria is yet to be identified (Kalscheuer, 2010), although one gene was located in the RN1 genome encoding a putative PAP domain that may possibly catalyse such a reaction. Strongly indicative of the ability of M. parvicella to store TAG is the location of at least ten putative copies of a bi-functional enzyme having waxy ester synthesis and acyl-CoA: DAG acyltransferase activities (WS/DGAT) (Kalscheuer & Steinbüchel, 2003) which catalyses the final step in TAG synthesis. The potential for PHA storage by M. parvicella Whether M. parvicella can accumulate intracellularly polyhydroxyalkanoates (PHA) could not be determined. An unambiguous candidate gene for a PHA synthetase (phaC), the key enzyme in the synthesis of polyhydroxyalkanoates (PHA), was not located in its genome. However, similarity appears to exist between a region of one gene with putative PHA synthetases identified in other organisms including Tetrasphaera japonica (Kristiansen et al., In press). The remainder of this gene is more closely related to that encoding acyl-CoA synthetases which are involved in the acylation of LCFA for β-oxidation or TAG synthesis. Whether this gene is a fused multifunctional gene is not clear. Putative hydrolases (3.1.1.-) that may act as PHA depolymerases were also located. Therefore it is possible that M. parvicella has some ability to store PHA, although several findings support the suggestion that TAG is the principal storage compound in this organism. Firstly, at least 10 putative copies of the WS/DGAT genes, encoding the key enzyme catalysing the ultimate step in TAG synthesis, were recognised to one putative PHA synthetase. In situ and pure culture studies both agree that LCFAs are probably stored in their esterified form (Slijkhuis et al., 1984; Nielsen et al., 2002; Rossetti et al., 2005). Additionally, the only direct evidence for PHA storage in M. parvicella is staining with Nile blue A, and this may stain other lipidic compounds including TAG (Serafim et al., 2002). Phosphorus metabolism The key enzyme in polyP biosynthesis is polyphosphate kinase (ppk), catalyzing the reversible transfer of phosphate from ATP to the growing polyphosphate chain (Ahn & Kornberg, 1990). The RN1 genome contains one putative ppk1 gene and three copies of the ppk2 gene, where the latter encodes an enzyme with a preference for GDP as its phosphate acceptor. It also favours the reverse reaction where polyP is degraded (Ishige et al., 2002; Zhang et al., 2002). The possible importance of the higher copy number of the ppk2 gene is unclear, although the phosphophenolpyruvate carboxykinase (EC 4.1.1.32) encoding gene annotated in the RN1 genome utilises GTP and serves as an important link between the TCA cycle and gluconeogenesis to synthesise trehalose and the glycerol backbone for TAG synthesis. As with the Tetrasphaera and Accumulibacter PAO, the M. parvicella genome also contains a gene encoding an exopolyphosphatase (ppx), able to hydrolyse polyP to orthophosphate (Kornberg et al., 1999). One clear difference between these organisms

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is the absence in the M. parvicella RN1 genome of any candidate gene encoding a dedicated polyphosphate:AMP transferase (PAP), although genes for polyphosphate and adenylate kinases were detected, and these enzymes are reported to form a complex exhibiting some PAP activity (Ishige & Noguchi, 2000). Putative genes encoding for the high affinity Pst transport system genes were located, but the low affinity Pit system was not (Kornberg et al., 1999). An additional putative phosphate-sodium symporter gene was also located. In Accumulibacter a putative F0F1-type ATP synthase (F-ATPase) is probably involved, at least theoretically, in the phosphorylation of ADP to generate ATP with the import of a proton, and where equilibrium is maintained by the action of a proton exporting transport pyrophosphatase and the Pit system (García-Martín et al., 2006). Candidate genes for the ATPase and the pyrophosphatase were located in the RN1 genome. Nitrogen metabolism The M. parvicella genome contains putative genes for a nitrate transporter, as well as narG, narH and narI genes encoding enzymes involved in a nitrate reductase complex (EC 1.7.99.4). The fdnG, fdnH and fdnI genes coding for the formate dehydrogenase complex (EC 1.2.1.2) were also detected. Interestingly this formate dehydrogenase gene cluster is preceded by a putative nrfD/PrsC type gene, which may encode an enzyme facilitating the formate dependent reduction of nitrite (Dietrich & Klimmek, 2002), or polysulphides respectively (Hussain et al., 1994). However, homology values were too low to allow confidence in reaching such conclusions. The location of these putative genes enabling nitrite reduction is at odds with the ‘inverse metabolic selection’ hypothesis that attempted to explain why filamentous bacteria appear to be favored selectively over the floc forming organisms under conditions of high nitrate. The suggestion of Casey et al. (1994; 1992) was that the latter eventually inhibited their aerobic growth by reducing nitrate to nitric oxide. A putative norA/ytfE type gene, considered to be important potentially in nitric oxide detoxification, was located in the M. parvicella genome. Such gene products may be involved in repairing oxidative or nitrosative damage to iron-sulphur clusters in enzymes involved in central metabolic pathways (Justino et al., 2007), which may themselves also bind free nitric oxide, reducing levels in the cytoplasm (Strube et al., 2007). Also located were putative mrsA and mrsB genes encoding peptide methionine sulfoxide reductases (EC. 1.8.4.11 and 1.8.4.12, respectively). These enzymes are hypothesized to repair damage to the cell caused by peroxynitrite, which can be formed from nitric oxide reacting with the superoxide generated during aerobic metabolism (St John et al., 2001). Furthermore, several genes involved in TAG synthases appeared to be up-regulated when M. tuberculosis was exposed to nitric oxide, possibly when the cells entered a dormant state allowing their survival under conditions unfavourable for growth (Daniel et al., 2004). Thus the ‘inverse metabolic selection’ explanation of why M. parvicella may prevail at relatively high nitrate levels (Casey et al., 1992; 1994) may not reflect an inability to reduce nitrite to nitric oxide, but instead an ability to reduce nitric oxide toxicity. Speculatively, M.

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parvicella may also produce the nitric oxide for its toxic effects on competing bacteria as it has now been suggested that truncated forms of the denitrification pathway are used by pathogens against host cells during infection (Mocca & Wang, 2012). It is also suggested that M. parvicella is also advantaged under conditions of high ammonia. Slijkhuis and colleagues (1983) reported that ammonia was essential for the growth of their morphologically identified isolate, although strain RN1 is capable of growing on media with only organic nitrogen sources (Rossetti et al., 1997). The genome sequence data support the ability of this filament to utilize ammonia as a nitrogen source, containing all the genes necessary for its uptake and subsequent conversion to glutamate. Beside a possible role for ammonia in GABA synthesis, the reported proliferation of M. parvicella in systems with relatively high levels of influent ammonia (Kruit et al., 2002; Tsai et al., 2003) may reflect its indirect influence on this organism, where low oxygen levels, which would favour its microaerophilic growth, also lead to a deterioration in aerobic nitrification capacity and the associated accumulation of ammonia (Slijkhuis & Deinema, 1988). Sulphur metabolism The ‘Dutch’ isolate of M. parvicella could not utilize sulphate as its source of sulphur, requiring instead reduced sulphur compounds for growth (Slijkhuis, 1983), a requirement that has never been tested in other M. parvicella pure cultures (Rossetti et al., 2005). However, the genome of RN1 contains candidate genes for sulphate transporters as well as most of those required for the assimilatory reduction of sulphate to hydrogen sulphide via 3'-phosphoadenylyl sulphate (PAPS). It is also not clear how closely related phylogenetically the Dutch isolate is to RN1, given that its 16S rRNA gene sequence has not been published.

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Supplementary Table 1: Summary of ‘single copy essential genes’ (Dupont et al., 2012) in the M. parvicella genomes No. Gene Protein RN1 1 alaS Alanyl-tRNA synthetase BN381_50129 2 argS Arginyl-tRNA synthetase BN381_680005 3 aspS Aspartyl-tRNA synthetase BN381_50123 4 cgtA Obg family GTPase Cgta BN381_10207 5 coaE Dephospho-CoA kinase BN381_430004 6 cysS Cysteinyl-tRNA ligase BN381_210089

BN381_50169 7 dnaA Chromosomal replication initiator protein BN381_100166 8 dnaG DNA primase BN381_330110 9 dnaK Chaperone protein BN381_150056 10 dnaN DNA polymerase III, beta sub-unit BN381_100168 11 dnaX DNA polymerase III, gamma/tau sub-unit BN381_150040 12 engA Ribosome-associated GTPase BN381_40072 13 era GTP-binding protein Era BN381_330098 14 ffh Signal recognition particle protein BN381_330034 15 fmt Methionyl-tRNA formyltransferase BN381_50155 16 frr Ribosome recycling factor BN381_330054 17 ftsY Signal recognition particle-docking protein BN381_330032 18 glySQ Glycyl-tRNA synthetase BN381_330109 19 gmk Guanylate kinase BN381_50147 20 grpE Co-chaperone BN381_150055 21 gyrA DNA gyrase, alpha subunit BN381_100172

BN381_60010 22 gyrB DNA gyrase, beta subunit BN381_100171

BN381_60013

23 hisS Histidyl-tRNA synthetase BN381_50120 24 ileS Isoleucyl-tRNA synthetase BN381_170043 25 infB Translation initiation factor IF-2 BN381_330070 26 infC Translation initiation factor IF-3 BN381_290132 27 ksgA Dimethyladenosine transferase BN381_20097 28 lepA GTP-binding protein LepA BN381_80270 29 leuS Leucyl-tRNA synthetase BN381_100077 30 ligA DNA ligase, NAD-dependent BN381_10084 31 mnmA tRNA (5-methylaminomethyl-2-thiouridylate)-

methyltransferase BN381_10079

32 mraW mraW methylase family BN381_140015 33 nusA Transcription termination factor BN381_330069 34 nusG Transcription termination/antitermination factor BN381_350058 35 pgk Phosphoglycerate kinase BN381_80402 36 pheS Phenylalanyl-tRNA synthetase, alpha sub-unit BN381_290128 37 pheT Phenylalanyl-tRNA synthetase, beta sub-unit BN381_290127 38 prfA Peptide chain release factor BN381_10020 39 proS Prolyl-tRNA synthetase BN381_330063 40 pyrG CTP synthase BN381_290083 41 recA recA protein BN381_80311 42 rfbA Ribosome binding factor A BN381_330072 43 rnc Ribonuclease III BN381_330021 44 rplA Ribosomal protein L1 BN381_350061 45 rplB Ribosomal protein L2 BN381_450094 46 rplC Ribosomal protein L3 BN381_450097

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47 rplD Ribosomal protein L4 BN381_450096 48 rplE Ribosomal protein L5 BN381_450085 49 rplF Ribosomal protein L6 BN381_450082 50 rplI Ribosomal protein L9 BN381_100140 51 rplJ Ribosomal protein L10 BN381_350062 52 rplK Ribosomal protein L11 BN381_350060 53 rplL Ribosomal protein L7/L12 BN381_350063 54 rplM Ribosomal protein L13 BN381_130023 55 rplN Ribosomal protein L14 BN381_450087 56 rplO Ribosomal protein L15 BN381_450078 57 rplP Ribosomal protein L16 BN381_450090 58 rplQ Ribosomal protein L17 BN381_450070 59 rplR Ribosomal protein L18 BN381_450081 60 rplS Ribosomal protein L19 BN381_330041 61 rplT Ribosomal protein L20 BN381_290130 62 rplU Ribosomal protein L21 BN381_80103 63 rplV Ribosomal protein L22 BN381_450092 64 rplW Ribosomal protein L23 BN381_450095 65 rplX Ribosomal protein L24 BN381_450086 66 rpmA Ribosomal protein L27 BN381_80104 67 rpmB Ribosomal protein L28 BN381_10342 68 rpmC Ribosomal protein L29 BN381_450089 69 rpmF Ribosomal protein L32 BN381_330016 70 rpmH Ribosomal protein L34 - 71 rpmI Ribosomal protein L35 BN381_290131 72 rpoA DNA-directed RNA polymerase, alpha subunit BN381_450071 73 rpoB DNA-directed RNA polymerase, beta subunit BN381_350066 74 rpoC DNA-directed RNA polymerase, beta’ subunit BN381_350067 75 rpsB Ribosomal protein S2 BN381_330051 76 rpsC Ribosomal protein S3 BN381_450091 77 rpsD Ribosomal protein S4 BN381_450072 78 rpsE Ribosomal protein S5 BN381_450080 79 rpsF Ribosomal protein S6 BN381_100143 80 rpsG Ribosomal protein S7 BN381_180002 81 rpsH Ribosomal protein S8 BN381_450083 82 rpsI Ribosomal protein S9 BN381_130024 83 rpsJ Ribosomal protein S10 BN381_450099 84 rpsK Ribosomal protein S11 BN381_450073 85 rpsL Ribosomal protein S12 BN381_180003 86 rpsM Ribosomal protein S13 BN381_450074 87 rpsO Ribosomal protein S15 BN381_330077 88 rpsP Ribosomal protein S16 BN381_330035 89 rpsQ Ribosomal protein S17 BN381_450088 90 rpsR Ribosomal protein S18 BN381_100141 91 rpsS Ribosomal protein S19 BN381_450093 92 rpsT Ribosomal protein S20 BN381_80262 93 secA Preprotein translocase, secA subunit BN381_80106 94 secE Preprotein translocase, secE subunit BN381_350057 95 secG Preprotein translocase, secG subunit BN381_80404 96 secY Preprotein translocase, secY subunit BN381_450077 97 secS Seryl-tRNA synthetase BN381_450047 98 smpB smpB protein BN381_80115 99 thrS Threonyl-tRNA synthetase BN381_50171 100 tig Trigger factor BN381_80089 101 tilS tRNA(Ile)-lysidine synthetase BN381_110021

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102 tsf Translation elongation factor Ts BN381_330052 103 tyrS Tyrosyl-tRNA synthetase BN381_290105 104 uvrB Exinuclease ABC, Beta subunit BN381_420007 105 valS Valyl-tRNA synthetase BN381_250104 106 ybeY Conserved hypothetical protein YbeY BN381_330096 107 ychF GTP-binding protein YchF BN381_20071

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Supplementary Table 2: Clusters of orthologous groups (COG)1 ID Description % CDS D Cell cycle control, cell division, chromosome partitioning 1.6 M Cell wall/membrane/envelope biogenesis 4.5 N Cell motility 1.0 O Posttranslational modification, protein turnover, chaperones 3.2 T Signal transduction mechanisms 3.2 U Intracellular trafficking, secretion, and vesicular transport 1.1 V Defence mechanisms 2.4 W Extracellular structures 0.1 Z Cytoskeleton 0.2 A RNA processing and modification <0.1 B Chromatin structure and dynamics <0.1 J Translation, ribosomal structure and biogenesis 4.3 K Transcription 5.0 L Replication, recombination and repair 5.8 C Energy production and conversion 5.8 E Amino acid transport and metabolism 10.6 F Nucleotide transport and metabolism 2.2 G Carbohydrate transport and metabolism 5.9 H Coenzyme transport and metabolism 3.0 I Lipid transport and metabolism 5.8 P Inorganic ion transport and metabolism 7.0 Q Secondary metabolites biosynthesis, transport and catabolism 4.5 R General function prediction only 15.8 S Function unknown 4.8 1. Assigned by the MaGe COG automatic classifier function

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Supplementary Table 3: Summary of putative gene assignments for genes relevant to selected pathways of interest in the M. parvicella genomes No. Gene Protein EC No. RN1 TCA cycle related 1 ace Pyruvate dehydrogenase (acetyl-

transferring) 1.2.4.1 BN381_210097

2 ipd Dihydrolipoyl dehydrogenase 1.8.1.4 BN381_10137 BN381_210107

3 pdhC Dihydrolipoyllysine-residue acetyltransferase

2.3.1.12 BN381_210108*

4 pckG Phosphoenolpyruvate carboxykinase (GTP) 4.1.1.32 BN381_10340 5 gltA Citrate (Si)-synthase 2.3.3.1 BN381_430036 6 acnA Aconitate hydratase 4.2.1.3 BN381_430007 7 icd Isocitrate dehydrogenase (NADP(+)) 1.1.1.42 BN381_100187 8 sucA Oxoglutarate dehydrogenase (succinyl-

transferring) 1.2.4.2 BN381_10317*

9 sucB Dihydrolipoyllysine-residue succinyltransferase

2.3.1.61 BN381_210108*

10 kgd 2-oxoglutarate decarboxylase 4.1.1.71 BN381_10317* 11 korA 2-oxoglutarate:ferredoxin oxidoreductase,

alpha sub-unit 1.2.7.3 BN381_80371

12 korB 2-oxoglutarate:ferredoxin oxidoreductase, beta sub-unit

1.2.7.3 BN381_80372

13 fpr Ferredoxin--NADP(+) reductase 1.18.1.2 BN381_130168 14 gabD Succinate-semialdehyde dehydrogenase

(NAD(P)(+)) 1.2.1.16 BN381_290124

15 sucD Succinate--CoA ligase (ADP-forming), alpha sub-unit

6.2.1.5 BN381_130058

16 sucC Succinate--CoA ligase (ADP-forming), beta sub-unit

6.2.1.5 BN381_130057

17 sdhA Succinate dehydrogenase/fumarate reductase, flavoprotein subunit

1.3.99.1 BN381_20048* BN381_130078*

18 sdhB Succinate dehydrogenase/fumarate reductase, iron sulphur sub-unit

1.3.99.1 BN381_20047* BN381_130077*

19 sdhC Succinate dehydrogenase/fumarate reductase, cytochrome B sub-unit

1.3.99.1 BN381_130079* BN381_20049*

20 fumC Fumarate hydratase 4.2.1.2 BN381_40065 21 mdh Malate dehydrogenase 1.1.1.37 BN381_130070 Glycolysis/gluconeogenesis 22 galM Aldose 1-epimerase 5.1.3.3 BN381_250111 23 glk Glucokinase 2.7.1.2 (BN381_10326)

(BN381_80234) 24 ppgK Polyphosphate glucokinase 2.7.1.63 BN381_10130 25 pgi Glucose-6-phosphate isomerase 5.3.1.9 BN381_300009 26 pfkA 6-phosphofructokinase 2.7.1.11 BN381_140032 27 glpX Fructose-bisphosphatase 3.1.3.11 BN381_10054 28 fbaB Fructose-bisphosphate aldolase 4.1.2.13 BN381_290092 29 tpiA Triose-phosphate isomerase 5.3.1.1 BN381_80403 30 gapA Glyceraldehyde-3-phosphate

dehydrogenase 1.2.1.12 BN381_80401

31 pgk Phosphoglycerate kinase 2.7.2.3 BN381_80402 32 gpm Phosphoglycerate mutase 5.4.2.1 BN381_150058

BN381_10014

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BN381_130014 33 eno Phosphopyruvate hydratase 4.2.1.11 BN381_500005

BN381_20129 34 pyk Pyruvate kinase 2.7.1.40 BN381_50094 35 pps Pyruvate, water dikinase 2.7.9.2 BN381_10302 Pentose phosphate pathway (PPP) 36 zwf Glucose-6-phosphate dehydrogenase 1.1.1.49 BN381_30024 37 pgl 6-phosphogluconolactonase 3.1.1.31 BN381_30023 38 gnd Phosphogluconate dehydrogenase

(decarboxylating) 1.1.1.44 BN381_30025

39 rpe Ribulose-phosphate 3-epimerase 5.1.3.1 BN381_80063 40 rpiB Ribose-5-phosphate isomerase 5.3.1.6 BN381_80386*

BN381_10026* 41 tkt Transketolase 2.2.1.1 BN381_410021 42 tal Transaldolase 2.2.1.2 BN381_410022

BN381_330086 Trehalose metabolism 43 pgm Phosphoglucomutase 5.4.2.2 BN381_20094* 44 galU UTP--glucose-1-phosphate

uridylyltransferase 2.7.7.9 BN381_90066

45 otsA α,α-trehalose-phosphate synthase 2.4.1.15 BN381_30004 46 treC α,α-phosphotrehalase 3.2.1.93 (BN381_100217) 47 otsB Trehalose-phosphatase 3.1.3.12 BN381_30003 48 - α,α-trehalose phosphorylase 2.4.1.64 (BN381_80260) 49 pgmB β-phosphoglucomutase 5.4.2.6 BN381_80261 Fructose metabolism 50 fruK 1-phosphofructokinase 2.7.1.56 BN381_80021 51 frk Fructokinase 2.7.1.4 BN381_80023 52 glmS Glutamine--fructose-6-phosphate

transaminase (isomerizing) 2.6.1.16 BN381_130026

53 glmM Phosphoglucosamine mutase 5.4.2.10 BN381_130025 Ethylmalonyl-CoA pathway related 54 phaA Acetyl-CoA C-acetyltransferase 2.3.1.9 BN381_150043*

BN381_330040* 55 phaB Acetoacetyl-CoA reductase 1.1.1.36 (BN381_130298)

(BN381_10287) (BN381_100125)

56 crt 3-hydroxybutyryl-CoA dehydratase 4.2.1.55 BN381_400021* 57 ccr Crotonyl-CoA carboxylase/reductase 1.3.1.85 BN381_150046 58 epi Ethylmalonyl-CoA/methylmalonyl-CoA

epimerase - BN381_150047

59 ecm (2R)-ethylmalonyl-CoA mutase - BN381_150069* 60 mcd (2S)-methylsuccinyl-CoA dehydrogenase - (BN381_210057) 61 mch Mesaconyl-CoA hydratase - BN381_130227 62 mcl-1 β-methylmalyl-CoA/(3S)-malyl-CoA lyase -/4.1.3.24 BN381_10090* 63 mcl-2 (3S)-Malyl-CoA thioesterase - BN381_10091* 64 pccA Propionyl-CoA carboxylase, alpha subunit 6.4.1.3 (BN381_10171) 65 pccB Propionyl-CoA carboxylase, beta subunit 6.4.1.3 BN381_150062 66 mutA Methylmalonyl-CoA mutase, small sub unit 5.4.99.2 BN381_100017 67 mutB Methylmalonyl-CoA mutase, large sub unit 5.4.99.2 BN381_70007 Glycerol metabolism 68 garK Glycerate kinase 2.7.1.31 BN381_150091 69 - Aldehyde dehydrogenase (NAD(+)) 1.2.1.3 BN381_20120*

BN381_50055*

  14  

BN381_130067* BN381_130126* BN381_170023* BN381_100111* BN381_250074*

70 - Alcohol dehydrogenase 1.1.1.1 BN381_250084* BN381_30019* BN381_310008*

71 glpK Glycerol kinase 2.7.1.30 BN381_140033 BN381_170037

72 gpsA Glycerol-3-phosphate dehydrogenase (NAD(P)(+))

1.1.1.94 BN381_80156

73 glpD Glycerol-3-phosphate oxidase 1.1.3.21 BN381_170034* BN381_290062*

74 ugpA Glycerol-3-phosphate transporter subunit; membrane component

- BN381_20064

75 ugpB Glycerol-3-phosphate transporter subunit; periplasmic-binding component

- BN381_20066

76 ugpC Glycerol-3-phosphate transporter subunit; ATP-binding protein

3.6.3.20 BN381_20063

77 ugpE Glycerol-3-phosphate transporter subunit; membrane component

- BN381_20065

78 aqpZ Aquaporin - BN381_330118 β-oxidation - related 79 acs Acetate--CoA ligase 6.2.1.1 BN381_160039 80 acdA Acetate--CoA ligase (ADP-forming), alpha

sub-unit 6.2.1.13 BN381_410014*

81 acdB Acetate--CoA ligase (ADP-forming), beta sub-unit

6.2.1.13 BN381_410015*

82 fadD Long-chain-fatty-acid--CoA ligase 6.2.1.3 BN381_130266* BN381_70027* BN381_60045* BN381_70075*

83 - Acyl-CoA dehydrogenase 1.3.8.- BN381_80083 BN381_250102 BN381_290144 BN381_210057 BN381_80237 BN381_40022 BN381_10292 BN381_510008 BN381_130250 BN381_80379 BN381_510006 BN381_510007 BN381_350084 BN381_130282 BN381_210093 BN381_250066 BN381_400017 BN381_430058 BN381_40056 BN381_420004 BN381_150138

  15  

BN381_40061 BN381_290148 BN381_300026 BN381_210110 BN381_90091 BN381_350101

84 3-hydroxyacyl-CoA dehydrogenase 1.1.1.35 (BN381_100132) (BN381_210100) (BN381_250119) (BN381_310013) (BN381_350105) (BN381_80232)

85 Enoyl-CoA hydratase 4.2.1.17 BN381_10064* BN381_80395* BN381_630012* BN381_130132* BN381_60005* BN381_250035* BN381_330019* BN381_350099* BN381_310045* BN381_110089* BN381_130358* BN381_310046* BN381_290012* BN381_130337* BN381_130222* BN381_90027*

86 Acetyl-CoA C-acyltransferase 2.3.1.16 BN381_70026* BN381_90096* BN381_350093*

Triacylglycerol (TAG) metabolism 87/ 88

plsB/ plsC

Glycerol-3-phosphate 1-O-acyltransferase/ 1-acylglycerol-3-phosphate O-acyltransferase

2.3.1.15/ 2.3.1.51

(BN381_130301) (BN381_40033) (BN381_50170) (BN381_150060) (BN381_430032) (BN381_630010)

89 dagK Diacylglycerol kinase 2.7.1.107 BN381_310070 90 - Phosphatidate phosphatase 3.1.3.4 BN381_290038* 91 dgat Diacylglycerol O-acyltransferase 2.3.1.20 BN381_70077

BN381_80224 BN381_250088 BN381_250038 BN381_360020 BN381_360018 BN381_130180 BN381_310062 BN381_10328 BN381_10279

92 Extracellular lipase 3.1.1.3 BN381_10063* BN381_870003* BN381_360050* BN381_80050*

  16  

93 lipA Triacylglycerol lipase 3.1.1.3 BN381_10029* BN381_110007*

94 lipB Acylglycerol lipase 3.1.1.23 BN381_180024* BN381_150081*

Polyhydroxyalkanoate (PHA) metabolism 54 phaA Acetyl-CoA C-acetyltransferase 2.3.1.9 BN381_150043*

BN381_330040* 55 phaB Acetoacetyl-CoA reductase 1.1.1.36 (BN381_130298)

(BN381_10287) (BN381_100125)

95 phaC Polyhydroxyalkanoate synthase 2.3.1.- (BN381_10327) 96 phaZ poly(3‐hydroxyalkanoate)

depolymerase 3.1.1.- (BN381_230027)

(BN381_310018) (BN381_80360)

Nitrogen metabolism related 97 nasA Nitrate transporter - BN381_50092* 98 narG Nitrate reductase 1.7.99.4 BN381_100092* 99 narH Nitrate reductase 1.7.99.4 BN381_100091* 100 narI Nitrate reductase 1.7.99.4 BN381_100093* 101 fdnG Formate dehydrogenase, alpha sub-unit 1.2.1.2 BN381_110061* 102 fdnH Formate dehydrogenase, beta sub-unit 1.2.1.2 BN381_110060* 103/104

nrfD Polysulfide reductase/formate dependent nitrite reductase

1.97.1.3/- BN381_110059*

105 nirK Nitrite reductase 1.7.2.1 BN381_80376 106 norA Putative NO response regulator - BN381_70126* 107 msrA Peptide methionine sulfoxide reductase A 1.8.4.11 BN381_40088 108 msrB Peptide methionine sulfoxide reductase B 1.8.4.11 BN381_40089 109 glnB Nitrogen regulatory protein P-II - BN381_150106 110 amt Ammonium transporter - BN381_150105

BN381_350049 111 gdh Glutamate dehydrogenase (NAD(P)(+)) 1.4.1.3 BN381_250090* 112 gdhA Glutamate dehydrogenase (NADP(+)) 1.4.1.4 BN381_290066 113 gltB Glutamate synthase (NADPH), large sub-

unit 1.4.1.13 BN381_50068*

114 gltD Glutamate synthase (NADPH), small sub-unit

1.4.1.13 BN381_50069*

115 glsF Glutamate synthase (ferredoxin) 1.4.7.1 BN381_90004* 116 glnA Glutamate--ammonia ligase 6.3.1.2 BN381_100101

BN381_100100 BN381_50037 BN381_80316

117 gls Glutaminase 3.5.1.2 BN381_290112 118 gad Glutamate decarboxylase 4.1.1.15 BN381_230020 119 gabT 4-aminobutyrate transaminase 2.6.1.19 (BN381_130303) Phosphorus metabolism related 120 ppk1 Polyphosphate kinase 2.7.4.1 BN381_130089 121 ppk2 Polyphosphate kinase 2 2.7.4.- BN381_310091

BN381_310092 BN381_10053

122 ppx Exopolyphosphatase 3.6.1.11 BN381_80138* BN381_630014*

123 adk Adenylate kinase 2.7.4.3 BN381_450076 24 ppgK Polyphosphate glucokinase 2.7.1.63 BN381_10130 124 ppnK Polyphosphate/ATP-NAD kinase 2.7.1.23 BN381_290089

  17  

125 phoB DNA-binding response regulator - BN381_30042 126 phoR Sensory histidine kinase 2.7.13.3 BN381_30043 127 phoU Phosphate transport system regulator - BN381_30044 128 pstA Phosphate transport system, permease

component - BN381_30046

129 pstB Phosphate transport system, ATP binding component

3.6.3.27 BN381_30045

130 pstC Phosphate transport system, permease component

- BN381_30047

131 pstS Phosphate transport system, phosphate binding component

- BN381_30048

132 - Phosphate:sodium symporter - BN381_150112* 133 atpB ATP synthase F0, subunit A 3.6.3.14 BN381_10042 134 atpF ATP synthase F0, subunit B 3.6.3.14 BN381_10044

BN381_10045 135 atpE ATP synthase F0, subunit C 3.6.3.14 BN381_10043 136 atpA ATP synthase F1, alpha subunit 3.6.3.14 BN381_10047 137 atpD ATP synthase F1, beta sub-unit 3.6.3.14 BN381_10049 138 atpG ATP synthase F1, gamma sub-unit 3.6.3.14 BN381_10048 139 atpH ATP synthase F1, delta sub-unit 3.6.3.14 BN381_10046 140 atpC ATP synthase F1, epsilon sub-unit 3.6.3.14 BN381_10050 141 hppA Pyrophosphate-energized proton pump 3.6.1.1 BN381_130116 142 ppa Inorganic diphosphatase 3.6.1.1 BN381_310039 Sulfur metabolism 143 nodQ Bi-functional enzyme: sulfate

adenylyltransferase, sub unit 1/ Adenylyl-sulfate kinase

2.7.7.4/ 2.7.1.25

BN381_350087

144 cysD Sulfate adenylyltransferase, sub unit 2 2.7.7.4 BN381_350088 145 cysH Phosphoadenylyl-sulfate reductase

(thioredoxin) 1.8.4.8 BN381_180013

146 cysJ Sulfite reductase (NADPH), beta sub-unit 1.8.1.2 - 147 cysI Sulfite reductase (NADPH), beta sub-unit 1.8.1.2 BN381_180012* 148 sir Sulfite reductase (ferredoxin) 1.8.7.1 (BN381_180012) * Indicates substantial evidence for an alternative gene assignment. Gene IDs in parenthesis indicates that there was some evidence for this assignment but it was either relatively very poor or higher evidence was evident for a different gene assignment.

  18  

Supplementary Table 4: Details of selected regions of the RN1 genome underrepresented in closely related community strains (see Fig. 3). CDS Protein Section 1 BN381_10258 Membrane protein of unknown function BN381_10259 Protein of unknown function BN381_10260 Putative site-specific recombinase BN381_10261 Protein of unknown function BN381_10262 DNA methylase domain protein BN381_10263 Protein of unknown function BN381_10264 Protein of unknown function BN381_10265 Protein of unknown function BN381_10266 Putative antirestriction protein ArdA BN381_10267 Protein of unknown function BN381_10268 Putative relaxase domain protein BN381_10269 Protein of unknown function BN381_10270 Protein of unknown function BN381_10271 Protein of unknown function BN381_10272 Putative DNA primase/helicase domain protein BN381_10273 Putative site-specific recombinase BN381_10274 Putative competence related protein Section 2 BN381_50001 Protein of unknown function BN381_50002 Protein of unknown function BN381_50003 MBOAT domain protein BN381_50004 Putative glycolsyl transferase BN381_50005 Protein of unknown function BN381_50006 Putative acyl-CoA N-acyltransferase domain protein BN381_50007 Protein of unknown function BN381_50008 Protein of unknown function BN381_50009 Protein of unknown function BN381_50010 Putative bifunctional protein: Glutamate-1-semialdehyde 2,1-

aminomutase, Acylneuraminate cytidylyltransferase BN381_50011 Putative aldo/keto reductase BN381_50012 Putative pseudaminic acid biosynthesis-associated protein BN381_50013 Protein of unknown function BN381_50014 Protein of unknown function BN381_50015 Putative N-acetylneuraminic acid synthase related BN381_50016 Putative aminotransferase BN381_50017 Putative UDP-galactose-4-epimerase BN381_50018 Protein of unknown function BN381_50019 Putative ABC transporter, ATP-binding/permease protein BN381_50020 Putative SAM dependent methyltransferase BN381_50021 Putative glycosyl transferase BN381_50022 Protein of unknown function BN381_50023 Membrane protein of unknown function BN381_50024 Putative non-specific protein-tyrosine kinase BN381_50025 Putative polysaccharide deacetylase BN381_50026 Putative glycosyl transferase BN381_50027 Putative phosphotransferase involved in extracellular matrix synthesis BN381_50028 putative protein-tyrosine phosphatase BN381_50029 Membrane protein of unknown function BN381_50030 Putative NAD-dependent epimerase/dehydratase BN381_50031 Exported protein of unknown function

  19  

BN381_50032 UDP-glucose/GDP-mannose dehydrogenase family protein BN381_50033 Putative acyltransferase BN381_50034 Protein of unknown function BN381_50035 Putative polysaccharide biosynthesis protein CapD BN381_50036 Putative Mg2+ transporter protein, CorA-like Section 3 BN381_80001 Protein of unknown function BN381_80002 Protein of unknown function BN381_80003 Protein of unknown function BN381_80004 Protein of unknown function BN381_80005 Radical SAM domain protein BN381_80006 Putative oxireductase BN381_80007 Protein of unknown function BN381_80008 Protein of unknown function BN381_80009 Putative nitroreductase BN381_80010 Protein of unknown function BN381_80011 Protein of unknown function BN381_80012 Protein of unknown function BN381_80013 Protein of unknown function BN381_80014 Protein of unknown function BN381_80015 Protein of unknown function BN381_80016 Protein of unknown function BN381_80017 Exported protein of unknown function BN381_80018 Membrane protein of unknown function BN381_80019 Protein of unknown function BN381_80020 Exported protein of unknown function BN381_80021 Putative 1-phosphofructokinase BN381_80022 Putative transcriptional regulator involved in sugar metabolism BN381_80023 Putative fructokinase Section 4 BN381_500009 Putative transcriptional regulator BN381_500010 Protein of unknown function BN381_500011 Protein of unknown function BN381_500012 Protein of unknown function BN381_500013 Putative transcriptional regulator MerR BN381_500014 Putative alkylmercury lyase BN381_500015 Exported protein of unknown function BN381_500016 Putative glutothionine reductase BN381_500017 Putative methyltransferase BN381_500018 Putative adenylate cyclase BN381_500019 Putative lipoprotein signal peptidase BN381_500020 Putative heavy metal translocating P-type ATPase BN381_500021 Putative transcriptional regulator involved in heavy metal resistance BN381_500022 Putative transport protein (related to putative mercury transporter) BN381_500023 Putative protein of unknown function BN381_500024 Putative protein of unknown function BN381_500025 IstB domain protein ATP-binding protein BN381_500026 Putative protein of unknown function BN381_500027 Putative transposase/integrase (fragment)

  20  

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