Sucrose metabolism in halotolerant methanotroph Methylomicrobium alcaliphilum 20Z

Supplementary material

Sergey Y. But1, Valentina N. Khmelenina1,Alexander S. Reshetnikov1,

Ildar I. Mustakhimov2, Marina G. Kalyuzhnaya3,4, andYuri A. Trotsenko1*

Present Address: 1Laboratory of methylotrophy, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, 142290 Russia

2Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino, 142290 Russia

3Biology Department, San Diego State University, San Diego, CA 92182-4614

4Department of Microbiology, University of Washington, Box 331750, Seattle, WA 98195-1750, USA

Suppl. Fig. 1Dependence of the total specific activity of the recombinant amylosucrase on sucrose concentrations

Suppl. Fig. 2 Polyglycan formation by E. coli Top10 cells transformed by the pHSG575/ams plasmid or the intact pHSG575 vector. The cells were grown on LB plates containing 150 mM sucrose and 1 mM IPTG and stained with by iodine vapors

Suppl. Fig. 3 The IGV-based visualization of the RNA-seq mapping coverage for the genome restore coding for sucrose-phosphate synthase (sps), sucrose-phosphate phosphatase (spp), fructokinase (fruK), amylosucrase (ams), putative ABC-transporter (orf1), and a conserved membrane protein (orf2) was obtained from. The putative transcriptional start site and the -10/-35 regions are in black bold. Gene expression data used for this study were downloaded from NCBI (GEO gene accession numbers are GSM 1239656-1239658). IGV plots were generated as described (Matson et al., 2013)

Suppl. Fig. 4 Phylogenetic tree of the amino acid sequences of sucrose phosphate synthases, fructokinases and amylosucrases combined in one polypeptide. α-Proteobacteria are blue, β-Proteobacteria are light green, γ-Proteobacteria are orange, Planctomycetes are brown, Cyanobacteria are dark green

Suppl. Fig. 5 Phylogenetic tree of the translated amino acid sequences of sucrose phosphate phosphotases. α-Proteobacteria are blue, β-Proteobacteria are light green, γ-Proteobacteria are orange, Planctomycetes are brown, Cyanobacteria are dark green, plants are red

Suppl. Fig. 6Organizationof the genes for sucrose metabolism in some Proteobacteria

Suppl. Table 1Primers used in the work

Primer / Sequence (5’-3’)
Spp20z-F*
Spp20z-R*
Ams20z-F*
Ams-20z-R*
sps-F
spp2R
sppF
fruk2R
AS-F
AS-R
ABC-R2
ABC-R1
Spp1R
Fruk1R
AS-RT
ABCrc / ataccatggatgaccgatcggcttttgctttgt
tatcaagctttcgccaattatccagtgtttga
taacatatgatgtacgaacagatttcgca
ttaaagcttatactcagttaaccaata
ttgcccggtttgactttacagccca
tccattgcgaccactcttgccattcat
tgttttaccggcttgcgcgact
ttcatcaatataatcgtaggcttgtt
ttggcggcgctttatccggtttc
cgcttgaaacccgctgcaaatctt
tcaacccgataaggctaatgacagtc
cgtatggaccgatgcaggagtcaac
tgccgatacataataactgagcttgaaacgat
ccaccaaggttcgcgcaaattgacatccat
ttcatgctcgtccgaggtatgattcaacaca
gctacatttccgctttaaaatatcgaccgtcagatgc

*Primer pairsSpp20zF-Spp20zR and Ams20zF-Ams20zRwere used for amplification of spp and ams genes in PCRs carried out under the following conditions: initial denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 20 s, extension at 72 °C for 2 min for 25 cycles, and final extension at 72 °C for 4 min.

Suppl. Table 2 The presence of the putative genes for sucrose metabolism in bacteria

Group / Organism / sps / spp / fruK / ams
Alphaproteobacteria / Magnetococcus marinus / + / + / + / +
Betaproteobacteria / Methylobacillus flagellatus / + / - / + / +
Methylophilus methylotrophus / + / - / + / +
Methylophilus sp. 1 / + / - / + / +
Methylophilus sp. 42 WGS / + / - / + / +
Methylotenera versatilis / + / - / + / +
Methylotenera sp. 73s WGS / + / - / + / +
Methylovorusglucosetrophus / + / - / + / +
Methylovorus sp.MP688 / + / - / + / +
Nitrosomonas europaea / + / - / + / -
Nitrosomonas eutropha / + / - / + / -
Nitrosomonas sp. AL212 / + / - / + / +
Nitrosomonas sp. Is79A3 / + / - / + / +
Nitrosospira multiformis / + / - / + / -
Thiobacillus denitrificans / + / - / + / -
Thiobacillus thioparus / + / - / + / -
Gammaproteobacteria / Methylobacter luteus / + / + / + / +
Methylocaldum szegediense / + / + / + / -
Acidithiobacillus caldus / + / - / + / -
Acidithiobacillus ferrivorans / + / - / + / -
Acidithiobacillus ferrooxidans / + / - / + / -
Acidithiobacillus thiooxidans / + / + / + / -
Allochromatium vinosum / + / + / + / +
Ectothiorhodospira sp. PHS-1 / + / - / + / -
Halothiobacillus neapolitanus / + / + / - / -
Marichromatium purpuratum / + / + / + / +
Methylobacter marinus / + / + / + / +
Methylobacter tundripaludum / + / + / + / +
Methyloglobulus morosus KoM1 / + / + / + / +
Methylohalobius crimeensis / + / - / + / -
Methylomicrobium album / + / + / + / +
Methylomicrobium alcaliphlum / + / + / + / +
Methylomonas methanica / + / + / + / +
Methylomonas sp. LW13 / + / + / + / +
Methylophaga aminisulfidivorans / + / + / + / +
Methylophaga lonarensis / + / + / + / +
Methylophaga sp. JAM1 / + / + / + / +
Methylophaga sp. JAM7 / + / + / + / +
Methylophaga thiooxidans / + / + / + / +
Methylosarcina fibrata / + / + / + / +
Nitrosococcus halophilus / + / - / + / -
Nitrosococcus multiformis / + / - / + / -
Nitrosococcus watsonii / + / - / + / -
Nitrosococcus oceani / + / - / + / -
Thioalkalivibrio thiocyanoxidans / + / + / + / +
Thioalkalivibrio nitratireduce / + / + / + / +
Thioalkalivibrio sulfidophilus / + / - / + / -
Thiocapsa marina / + / + / + / +
Thiocystis violascens / + / + / + / +
Thioflavicoccus mobilis / + / + / + / +
Thiomicrospira arctica / + / + / + / +
Thiomicrospira halophila / + / + / + / +
Thiorhodococcus drewsii / + / + / + / +
Thiorhodovibrio sp 970 / + / + / + / +
Thiothrix nivea / + / + / + / +
Deltaproteobacteria / Geopsychrobacter electrodiphilus / + / - / + / +
Desulfobacterium autotrophicum / + / + / + / +
Desulfuromonas acetoxidans / + / - / + / -
Desulfonatronospira thiodismutans / + / - / + / -
Desulfocapsa sulfexigens / + / + / - / +
Desulfomicrobium baculatum / + / + / + / +
Desulfurivibrio alkaliphilus / + / + / + / -
Desulfobacter postgatei / + / + / + / +
Desulfococcus multivorans / + / - / + / +
Cyanobacteria / Anabaena cylindrica / + / + / - / -
Anabaena sp. 90 / + / - / - / -
Calothrix sp. PCC 6303 / + / + / + / -
Coleofasciculus chthonoplastes / + / + / + / -
Cyanobium gracile / + / + / + / +
Cyanobium sp. PCC 7001 / + / + / - / -
Cylindrospermum stagnale / + / + / - / -
Geminocystis herdmanii / + / + / + / +
Microcoleus sp. PCC7113 / + / + / - / -
Moorea producens / + / + / + / -
Nodularia spumigena / + / + / + / -
Nostoc punctiforme / + / + / - / -
Oscillatoria acuminata / + / + / - / -
Prochlorococcus marinus / + / - / - / -
Richelia intracellularis / + / + / + / -
Synechococcus elongatus / + / + / + / -
Synechococcus sp. WH5701 / + / - / + / +
Synechococcus sp. WH7803 / + / - / + / -
Synechococcus sp. PCC 7002 / + / + / + / +
Synechococcus sp. RS9916 / + / - / + / -
Synechocystis sp. 6803 / + / + / + / -
Thermosynechococcus elongatus / + / - / + / -
Planctomycetes / Blastopirellula marina / + / + / + / +
Planctomyces brasiliensis / + / + / - / +
Planctomyces maris / + / - / + / +
Rhodopirellula europaea / + / + / + / +
Rhodopirellula maiorica / + / + / + / +
Rhodopirellula sallentina / + / + / + / +
Firmicutes / Caloramator australicus / + / - / + / -
Halanaerobium hydrogeniformans / + / - / + / -
Halanaerobium saccharolyticum / + / - / + / -
Halobacteroides halobius / + / - / + / -
Halothermothrix orenii / + / + / + / -
Petrotoga mobilis / + / - / + / -
Deferribacteres / Denitrovibrio acetiphilus / + / - / + / -
Nitrospinae / Nitrospina gracilis / + / - / + / -
Chrysiogenetes / Desulfurispirillum indicum / + / - / + / -

Suppl. Table 3Relative expression levels of genes for sucrose metabolism and glycogen synthesis in Methylomicrobium alcaliphilum 20Z cells grown on methane.

Locus tag / Gene name / Description / Expression level
(RPKM)
MALCv4_0614 / sps / sucrose-phosphate synthase / 274±7
MALCv4_0615 / spp / sucrose-phosphate phosphatase / 260±70
MALCv4_0616 / fruK / fructokinase / 220±47
MALCv4_0617 / ams / amylosucrase / 140±14
MALCv4_3499 / glgB2 / 1,4-alpha-glucan branching enzyme / 300±60
MALCv4_3500 / amyA / alpha amylase family protein / 270±22
MALCv4_3501 / glgA2 / glycogen synthase 2 / 260±17
MALCv4_3502 / amyC / alpha-amylase/Glycoside hydrolase, family 57 / 135±10
MALCv4_3506 / glgC / glucose-1-phosphate adenylyltransferase / 640±66
MALCv4_3507 / glgB1 / 1,4-alpha-glucan branching enzyme / 227±9
MALCv4_3508 / glgA1 / glycogen synthase / 183±26

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