EXOPOLYSACCHARIDE PRODUCTION BY

Sphingomonas elodea AND Pseudomonas

aeruginosa

Fialho et al. (2008). Occurrence, production and applications of gellan: current state
and perspectives. Appl Microbiol Biotechnol.79:889-900.
 Bacterial exopolysaccharides

Bacterial exopolysaccharides (EPS) are long-chain,

high-molecular-mass polymers

These polymers play a protective role in the native state

Established applications of bacterial EPS:

Emulsion stabilization
Film formation
Flocculant agent
Gelling agent
Suspending agent
Viscosity control agent
Sphingomonas elodea ATCC 31461

• Gram-negative rod-shaped
bacteria

• First isolated: surface of a

plant (Elodea genus)

• Previously designated
Pseudomonas elodea, also
Sphingomonas paucimobilis
and recently, Sphingomonas
elodea
 THE GELLAN GUM:
an exopolysaccharide produced by Sphingomonas elodea ATCC 31461

Ac (1/2)

6
3)-βD-Gluc-(1 4)-βD-GlcA-(1 4)-βD-Glc-(1 4)-αL-Rha-(1
2

≈ 12–15 g/l
L-glyceric

Linear polymer of
500 kDa
Production of Gellan gum
Sphingomonas elodea ATCC 31461
 Gellan gum: industrial applications

KELCOGEL  and GELRITE® is the commercial form of gellan

 Biosynthesis of gellan

Carbon source
OM

IM

1st-Synthesis of the sugar-nucleotides;

2nd- Assembly of the tetrasaccharidic unit;

3rd- Polymerization and export of the polymer

Gellan
 Peptigoglycan
OM
IM
PgmG
PgmG
Glucose-6-P Glucose-1-P
ugpG Glucose -6 -P Glucose - 1 -P
pgmG UgpG
RmlA UgpG
T gpG
pgmG ugpG

dTDP-D-Glucose dTDP -D - UDP Trealose

-D -
UDP-D-Glucose
ugdG Glucose Glucose
RmlB,C,D
ugdG TRS UgdG Ug d G

dTDP-L-Rhamnose UDP-D-Glucuronic
dTDP -L - acid GelB
UDP - D - ác .
Ramnose Glucurónico

Cluster gel
atr rhs GelB
Q I K L J F gel
D cluster
C E 32 26 D B A CBD
atr rml GelL ? GelK GelK
Q I K L J F D C E 32 26 D B A CBD

GelL ?
GelD GelQ ?
GelQ ?
GelC ,E
? ?
GelC,GelE ? ?
?
?

Gellan
n
Model for the polymerization and secretion of
gellan

GelD Outer membrane

Peptidoglycan
P
P P
P

Inner membrane
P
GelS GelC
P
GelG GelE P
ATP
Pi PTPase?
 Genes involved in the formation of nucleotide sugar
precursors

pgmG - encodes a bifunctional protein with phosphoglucomutase and

phosphomannomutase activities

Gene amplified from the S. elodea genome using degenerated

primers, followed by sequence determination

Box I Box II Box III

Active site Metal-ion-binding Sugar binding
PgmG S. paucimobilis (AF167367) QITGSHNPGN-109 GADGDGDRID-249 LAGEMSGH-326
ExoC Azospirillum brasilense (P45632) MITGSHNPPD-111 GFDGDGDRIG-247 LAGEMSGH-327
PmmA Prochlorothrix hollandica (AAC44309) QITGSHNPPE-110 AFDGDGDRIG-251 LAGEMSGH-331
ExoC Rickettsia prowazekii (CAA14961) MVTGSHNPRD-116 AFDGDGDRIG-296 LAGEMSGH-376
AlgC Pseudomonas aeruginosa (P26276) MLTGSHNPPD-113 AFDGDGDRVG-249 LAGEMSGH-329
Pgm Neisseria gonorrhoeae (P40390) MITGSHNPPD-108 AFDGDADRLG-246 VAGEMSGH-326
XanA Xanthomonas campestris (AAA27610) MVTASHNPMD-102 AWDGDFDRCF-244 YGGEMSAH-324
ManB Escherichia coli (P24175) EVTASHNPMD-103 AFDGDFDRCF-252 YGGEMSAH-332
RFK7 E. coli (P37742) EVTASHNPMD-101 AFDGDFDRCF-250 YGGEMSAH-329
CelB A.xylinum (P38569) VITPSHNPPE-153 ANDTDADRHG-313 FGGEESAG-397
Pgm E. coli (P36938) VITPSHNPPE-151 ANDPDYDRHG-311 FGGEESAG-394
Pgm1 Homo sapiens (P36871) ILTASHNPGG-122 AFDGDGDRNM-295 LCGEESFG-380
Pgm1 Saccharomyces cerevisiae (S41199) ILTASHNPGG-125 ASDGDGDRNM-298 ICGEESFG-383
PgmU Oryctolagus cuniculus (P00949) ILTASHNPGG-122 AFDGDGDRNM-295 LCGEESFG-380
* **** * * ** ** *
(Videira et al.2000, Appl. Environ. Microbiol. 66:2252-2258)
GelE is a putative tyrosine autokinase involved in
polysaccharide chain length regulation

(Moreira et al.2004, J Mol Microbiol Biotechnol 8:43-57).

 gelE non polar mutant is defective in gellan
biosynthesis
ATCC

gelF D C E M N

∆gelE

gelF D C aacC1 M N

gelE deletion mutant (SpLM21-4) can be complemented by transformation

with plasmid pHA010-3 containing gelE gene in the replicative vector
pBBR1MCS (homologous complementation)
(Moreira et al.2004, J Mol Microbiol Biotechnol 8:43-57).
 Biosynthesis of Alginate: genes and enzymes

Alginate (mannuronate, guluronate)

is produced by Pseudomonas
aeruginosa
algC from Pseudomonas is homologous to pgmG from Sphingomonas
 Triparental mating
Helper strain Donor strain
E. coli /pRK2013 E. coli /pLC100 or pNZ49
mob+ tra+ mob+; tra-; pgmG+ or algC+

pRK2013-
narrow host specificity
Recipient strain
Pseudomonas 8822
algC-; Alg-

Selection: PIA supplemented

with carbenicilin
Homologous vs heterologous complementation

P. aeruginosa 8822 P. aeruginosa 8822

pMMB66HE (cloning pLC100 (pgmG)
vector)
P. aeruginosa 8822
pNZ49 (algC)