PUL ID

PUL0459

PubMed

31915221, mSphere. 2020 Jan 8;5(1). pii: 5/1/e00792-19. doi: 10.1128/mSphere.00792-19.

Characterization method

RNA-Seq, analysis of reaction products, enzyme activity assay

Genomic accession number

NZ_PJAI02000001.1

Nucelotide position range

312863-400633

Substrate

agar

Loci

CWS31_RS01305-CWS31_RS01540

Species

Colwellia echini/1982103

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

- CWS31_RS01305 WP_148747674.1 312863 - 315437 (+) NZ_PJAI02000001.1:625726-628300 -
- CWS31_RS01310 WP_148747638.1 315525 - 317088 (+) NZ_PJAI02000001.1:628388-629951 -
- CWS31_RS01315 WP_148747639.1 317316 - 319605 (+) NZ_PJAI02000001.1:630179-632468 -
fdhD CWS31_RS01320 WP_148747640.1 319785 - 320526 (+) NZ_PJAI02000001.1:632648-633389 -
- CWS31_RS01325 WP_148747641.1 320825 - 323114 (+) NZ_PJAI02000001.1:633688-635977 -
- CWS31_RS01330 WP_148747642.1 323189 - 323867 (+) NZ_PJAI02000001.1:636052-636730 -
- CWS31_RS01335 WP_148747643.1 324567 - 328530 (+) NZ_PJAI02000001.1:637430-641393 -
- CWS31_RS01340 WP_148747644.1 329137 - 332644 (+) NZ_PJAI02000001.1:642000-645507 -
- CWS31_RS01345 WP_148747645.1 332791 - 333865 (-) NZ_PJAI02000001.1:645654-646728 -
- CWS31_RS01350 WP_148747646.1 334000 - 334762 (-) NZ_PJAI02000001.1:646863-647625 -
- CWS31_RS01355 WP_148747647.1 335028 - 336648 (-) NZ_PJAI02000001.1:647891-649511 -
- CWS31_RS01360 WP_148747675.1 337191 - 339561 (+) NZ_PJAI02000001.1:650054-652424 -
- CWS31_RS01365 WP_148747648.1 339550 - 341875 (+) NZ_PJAI02000001.1:652413-654738 -
- CWS31_RS01370 WP_148747649.1 341993 - 343706 (+) NZ_PJAI02000001.1:654856-656569 -
- CWS31_RS01380 WP_148747676.1 346644 - 348210 (+) NZ_PJAI02000001.1:659507-661073 -
- CWS31_RS01385 WP_148747677.1 348551 - 348662 (+) NZ_PJAI02000001.1:661414-661525 -
- CWS31_RS01390 WP_148747650.1 348662 - 349949 (-) NZ_PJAI02000001.1:661525-662812 -
- CWS31_RS01395 WP_148747651.1 350260 - 351031 (-) NZ_PJAI02000001.1:663123-663894 -
- CWS31_RS01400 WP_148747652.1 351545 - 352658 (+) NZ_PJAI02000001.1:664408-665521 -
- CWS31_RS01405 WP_148747653.1 352670 - 353693 (+) NZ_PJAI02000001.1:665533-666556 -
- CWS31_RS01410 WP_148747678.1 353741 - 354506 (+) NZ_PJAI02000001.1:666604-667369 1.1.1.127
aldA CWS31_RS01415 WP_148747654.1 354512 - 355976 (+) NZ_PJAI02000001.1:667375-668839 -
- CWS31_RS01420 WP_148747655.1 356258 - 360656 (+) NZ_PJAI02000001.1:669121-673519 -
- CWS31_RS01430 WP_148747679.1 361278 - 362823 (+) NZ_PJAI02000001.1:674141-675686 -
- CWS31_RS01435 WP_148747680.1 363069 - 364467 (-) NZ_PJAI02000001.1:675932-677330 -
- CWS31_RS01440 WP_148747681.1 364562 - 365990 (-) NZ_PJAI02000001.1:677425-678853 -
- CWS31_RS01445 WP_148747656.1 366077 - 366773 (-) NZ_PJAI02000001.1:678940-679636 -
- CWS31_RS01450 WP_148747657.1 367011 - 367752 (-) NZ_PJAI02000001.1:679874-680615 -
- CWS31_RS01455 WP_148747658.1 367768 - 369322 (-) NZ_PJAI02000001.1:680631-682185 -
- CWS31_RS01460 WP_148747682.1 369449 - 372224 (-) NZ_PJAI02000001.1:682312-685087 -
- CWS31_RS01465 WP_148747659.1 372803 - 373925 (-) NZ_PJAI02000001.1:685666-686788 -
- CWS31_RS01475 WP_148747661.1 375386 - 377753 (-) NZ_PJAI02000001.1:688249-690616 -
- CWS31_RS01480 WP_148747662.1 378225 - 381903 (+) NZ_PJAI02000001.1:691088-694766 -
- CWS31_RS01485 WP_148747663.1 381967 - 384112 (+) NZ_PJAI02000001.1:694830-696975 -
- CWS31_RS01490 WP_148747664.1 384573 - 386124 (+) NZ_PJAI02000001.1:697436-698987 -
- CWS31_RS01495 WP_148747665.1 386156 - 386876 (+) NZ_PJAI02000001.1:699019-699739 -
- CWS31_RS01500 WP_148747666.1 387090 - 389538 (-) NZ_PJAI02000001.1:699953-702401 -
- CWS31_RS01505 WP_148747667.1 389782 - 390202 (-) NZ_PJAI02000001.1:702645-703065 -
- CWS31_RS01510 WP_148747683.1 390204 - 391182 (-) NZ_PJAI02000001.1:703067-704045 -
- CWS31_RS01515 WP_148747684.1 391209 - 392226 (-) NZ_PJAI02000001.1:704072-705089 -
- CWS31_RS01520 WP_148747668.1 392260 - 393772 (-) NZ_PJAI02000001.1:705123-706635 -
- CWS31_RS01525 WP_148747669.1 393842 - 394802 (-) NZ_PJAI02000001.1:706705-707665 -
- CWS31_RS01530 WP_148747685.1 394992 - 398085 (-) NZ_PJAI02000001.1:707855-710948 -
- CWS31_RS01535 WP_148747670.1 398425 - 399367 (+) NZ_PJAI02000001.1:711288-712230 -
- CWS31_RS01540 WP_148747671.1 399547 - 400633 (+) NZ_PJAI02000001.1:712410-713496 -

Cluster number

2

Gene name

Gene position

Gene type

Found by CGCFinder?

- 312864 - 315437 (+) CAZyme: GH2 Yes (cluster 1)
- 315526 - 317088 (+) TC: gnl|TC-DB|P96169|2.A.21.3.2 Yes (cluster 1)
- 317317 - 319605 (+) other Yes (cluster 1)
- 319786 - 320526 (+) other Yes (cluster 1)
- 320826 - 323114 (+) TC: gnl|TC-DB|P07658|3.D.1.9.2 Yes (cluster 1)
- 323190 - 323867 (+) other Yes (cluster 1)
- 324568 - 328530 (+) CAZyme: GH96|CBM6 Yes (cluster 1)
- 329138 - 332644 (+) CAZyme: GH96|CBM6 Yes (cluster 1)
- 332792 - 333865 (-) other Yes (cluster 1)
- 334001 - 334762 (-) other Yes (cluster 1)
- 335029 - 336648 (-) TC: gnl|TC-DB|P96710|2.A.1.1.55 Yes (cluster 1)
- 337192 - 339561 (+) CAZyme: GH50 Yes (cluster 1)
- 339551 - 341875 (+) CAZyme: GH50 Yes (cluster 1)
- 341994 - 343706 (+) other Yes (cluster 1)
- 344252 - 346543 (+) other Yes (cluster 1)
- 346645 - 348210 (+) CAZyme: GH29 Yes (cluster 1)
- 348552 - 348662 (+) CDS No
- 348663 - 349949 (-) STP: STP|SBP_bac_1 No
- 350261 - 351031 (-) TF: DBD-Pfam|TrmB No
- 351546 - 352658 (+) CDS No
- 352671 - 353693 (+) CDS No
- 353742 - 354506 (+) CDS No
- 354513 - 355976 (+) CDS No
- 356259 - 360656 (+) CDS No
- 360804 - 361037 (+) CDS No
- 361279 - 362823 (+) CDS No
- 363070 - 364467 (-) CDS No
- 364563 - 365990 (-) CDS No
- 366078 - 366773 (-) CDS No
- 367012 - 367752 (-) CDS No
- 367769 - 369322 (-) CDS No
- 369450 - 372224 (-) TC: gnl|TC-DB|Q9A608|1.B.14.12.1 Yes (cluster 2)
- 372804 - 373925 (-) other Yes (cluster 2)
- 374451 - 375065 (-) TF: DBD-Pfam|GntR,DBD-SUPERFAMILY|0039384 Yes (cluster 2)
- 375387 - 377753 (-) CAZyme: GH50 Yes (cluster 2)
- 378226 - 381903 (+) TC: gnl|TC-DB|Q9AAZ6|1.B.14.12.2 Yes (cluster 2)
- 381968 - 384112 (+) other Yes (cluster 2)
- 384574 - 386124 (+) other Yes (cluster 2)
- 386157 - 386876 (+) other Yes (cluster 2)
- 387091 - 389538 (-) CAZyme: GH86 Yes (cluster 2)
- 389783 - 390202 (-) other Yes (cluster 2)
- 390205 - 391182 (-) TC: gnl|TC-DB|G4FGN4|3.A.1.2.20 Yes (cluster 2)
- 391210 - 392226 (-) TC: gnl|TC-DB|Q0HIQ6|3.A.1.2.14 Yes (cluster 2)
- 392261 - 393772 (-) TC: gnl|TC-DB|Q8G847|3.A.1.2.23 Yes (cluster 2)
- 393843 - 394802 (-) TC: gnl|TC-DB|P39325|3.A.1.2.25 Yes (cluster 2)
- 394993 - 398085 (-) other Yes (cluster 2)
- 398426 - 399367 (+) STP: STP|PfkB Yes (cluster 2)
- 399548 - 400633 (+) CAZyme: GH117 Yes (cluster 2)

PUL ID

PUL0459

PubMed

31915221, mSphere. 2020 Jan 8;5(1). pii: 5/1/e00792-19. doi: 10.1128/mSphere.00792-19.

Title

A Multifunctional Polysaccharide Utilization Gene Cluster in Colwellia echini Encodes Enzymes for the Complete Degradation of kappa-Carrageenan, iota-Carrageenan, and Hybrid beta/kappa-Carrageenan.

Author

Christiansen L, Pathiraja D, Bech PK, Schultz-Johansen M, Hennessy R, Teze D, Choi IG, Stougaard P

Abstract

Algal cell wall polysaccharides constitute a large fraction in the biomass of marine primary producers and are thus important in nutrient transfer between trophic levels in the marine ecosystem. In order for this transfer to take place, polysaccharides must be degraded into smaller mono- and disaccharide units, which are subsequently metabolized, and key components in this degradation are bacterial enzymes. The marine bacterium Colwellia echini A3(T) is a potent enzyme producer since it completely hydrolyzes agar and kappa-carrageenan. Here, we report that the genome of C. echini A3(T) harbors two large gene clusters for the degradation of carrageenan and agar, respectively. Phylogenetical and functional studies combined with transcriptomics and in silico structural modeling revealed that the carrageenolytic cluster encodes furcellaranases, a new class of glycoside hydrolase family 16 (GH16) enzymes that are key enzymes for hydrolysis of furcellaran, a hybrid carrageenan containing both beta- and kappa-carrageenan motifs. We show that furcellaranases degrade furcellaran into neocarratetraose-43-O-monosulfate [DA-(alpha1,3)-G4S-(beta1,4)-DA-(alpha1,3)-G], and we propose a molecular model of furcellaranases and compare the active site architectures of furcellaranases, kappa-carrageenases, beta-agarases, and beta-porphyranases. Furthermore, C. echini A3(T) was shown to encode kappa-carrageenases, iota-carrageenases, and members of a new class of enzymes, active only on hybrid beta/kappa-carrageenan tetrasaccharides. On the basis of our genomic, transcriptomic, and functional analyses of the carrageenolytic enzyme repertoire, we propose a new model for how C. echini A3(T) degrades complex sulfated marine polysaccharides such as furcellaran, kappa-carrageenan, and iota-carrageenan.IMPORTANCE Here, we report that a recently described bacterium, Colwellia echini, harbors a large number of enzymes enabling the bacterium to grow on kappa-carrageenan and agar. The genes are organized in two clusters that encode enzymes for the total degradation of kappa-carrageenan and agar, respectively. As the first, we report on the structure/function relationship of a new class of enzymes that hydrolyze furcellaran, a partially sulfated beta/kappa-carrageenan. Using an in silico model, we hypothesize a molecular structure of furcellaranases and compare structural features and active site architectures of furcellaranases with those of other GH16 polysaccharide hydrolases, such as kappa-carrageenases, beta-agarases, and beta-porphyranases. Furthermore, we describe a new class of enzymes distantly related to GH42 and GH160 beta-galactosidases and show that this new class of enzymes is active only on hybrid beta/kappa-carrageenan oligosaccharides. Finally, we propose a new model for how the carrageenolytic enzyme repertoire enables C. echini to metabolize beta/kappa-, kappa-, and iota-carrageenan.