Mucoid strains of Pseudomonas aeruginosa that overproduce alginate are associated with chronic pulmonary disease (e.g. cystic fibrosis). Mutants defective in one of several periplasmic proteins (AlgKGX) for alginate secretion release alginate fragments due to the activity of an alginate lyase (AlgL) in the periplasm, which cleaves the newly formed polymers. However, mutants defective in Alg8 or Alg44 did not secrete polymer or alginate fragments, suggesting that both these membrane proteins have a role in the polymerization reaction. A model for the membrane topology of Alg8, a glycosyltransferase (GT), was constructed using PhoA fusions. This provided evidence for a large cytoplasmic loop containing the active domains predicted for β-GTs such as Alg8 and five transmembrane (TM) domains, one of which resembles a cleavable signal peptide. The C-terminal TM domain of Alg8 was critical for the polymerization reaction in vivo. Alanine substitution mutagenesis showed that all of the predicted active site residues in the widely spaced D, DxD, D, LxxRW motif were required for polymerization activity in vivo, and two of these substitutions also affected Alg8 protein stability. A membrane topology model for Alg44 was also constructed using PhoA fusions, and this showed a central TM domain and predicted an N-terminal TM domain that may be a membrane anchor. An N-terminal PilZ domain in Alg44 for c-di-GMP [bis-(3′,5′)-cyclic dimeric GMP] binding, which is required for alginate synthesis, was localized to the cytoplasmic loop. The long periplasmic C terminus of Alg44 contains a region similar to membrane fusion proteins (MFPs) of multi-drug efflux systems, which predicts the possibility of its interaction with another protein in this compartment. A Western blot analysis of the outer-membrane porin AlgE showed reduced AlgE levels in the alg44 mutant, whereas expression of Alg44 in trans restored AlgE within the cell. C-terminal truncations of Alg44 as small as 24 amino acids blocked alginate polymerization in vivo, indicating a critical role for the MFP domain. These studies suggest that Alg44 may act as a co-polymerase in concert with Alg8, the major GT, and that both inner-membrane proteins are required in vivo for the polymerization reaction leading to alginate production.
BarnyM. A.,
SchoonejansE.,
EconomouA.,
JohnstonA. W.,
DownieJ. A.1996; The C-terminal domain of the Rhizobium leguminosarum chitin synthase NodC is important for function and determines the orientation of the N-terminal region in the inner membrane. Mol Microbiol 19:443–453
BoucherJ. C.,
Martinez-SalazarJ.,
SchurrM. J.,
MuddM. H.,
YuH.,
DereticV.1996; Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA. J Bacteriol 178:511–523
BrickmanE.,
BeckwithJ.1975; Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol 96:307–316
CampbellJ. A.,
DaviesG. J.,
BuloneV.,
HenrissatB.1997; A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem J 326:929–939
DeVriesC. A.,
OhmanD. E.1994; Mucoid to nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT , encoding a putative alternative sigma factor, and shows evidence for autoregulation. J Bacteriol 176:6677–6687
ElkinsC. A.,
NikaidoH.2003; Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efflux pump. J Bacteriol 185:5349–5356
FranklinM. J.,
OhmanD. E.2002; Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa . J Bacteriol 184:3000–3007
HaardtM.,
BremerE.1996; Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmoregulated ProU transport system of Escherichia coli . J Bacteriol 178:5370–5381
JainS.,
FranklinM. J.,
ErtesvagH.,
VallaS.,
OhmanD. E.2003; The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa . Mol Microbiol 47:1123–1133
JohnsonJ. M.,
ChurchG. M.1999; Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps. J Mol Biol 287:695–715
LobsanovY. D.,
RomeroP. A.,
SlenoB.,
YuB.,
YipP.,
HerscovicsA.,
HowellP. L.2004; Structure of Kre2p/Mnt1p: a yeast α 1,2-mannosyltransferase involved in mannoprotein biosynthesis. J Biol Chem 279:17921–17931
MaharajR.,
MayT. B.,
WangS. K.,
ChakrabartyA. M.1993; Sequence of the alg8 and alg44 genes involved in the synthesis of alginate by Pseudomonas aeruginosa . Gene 136:267–269
Mejia-RuizH.,
GuzmanJ.,
MorenoS.,
Soberon-ChavezG.,
EspinG.1997; The Azotobacter vinelandii alg8 and alg44 genes are essential for alginate synthesis and can be transcribed from an algD -independent promoter. Gene 199:271–277
MerighiM.,
LeeV. T.,
HyodoM.,
HayakawaY.,
LoryS.2007; The second messenger bis-(3′-5′)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa . Mol Microbiol 65:876–895
NehmeD.,
LiX. Z.,
ElliotR.,
PooleK.2004; Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa : identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB. J Bacteriol 186:2973–2983
OhmanD. E.,
ChakrabartyA. M.1981; Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun 33:142–148
PierG. B.,
ColemanF.,
GroutM.,
FranklinM.,
OhmanD. E.2001; Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis. Infect Immun 69:1895–1901
RemminghorstU.,
RehmB. H.2006a; In vitro alginate polymerization and the functional role of Alg8 in alginate production by Pseudomonas aeruginosa . Appl Environ Microbiol 72:298–305
Robles-PriceA.,
WongT. Y.,
SlettaH.,
VallaS.,
SchillerN. L.2004; AlgX is a periplasmic protein required for alginate biosynthesis in Pseudomonas aeruginosa . J Bacteriol 186:7369–7377
RoychoudhuryS.,
MayT.,
GillJ.,
SinghS.,
FeingoldD.,
ChakrabartyA.1989; Purification and characterization of guanosine diphospho-d-mannose dehydrogenase. A key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa . J Biol Chem 264:9380–9385
SaxenaI. M.,
BrownR. M.Jr1997; Identification of cellulose synthase(s) in higher plants: sequence analysis of processive β -glycosyltransferases with the common motif ‘D,D,D35Q(R,Q)XRW’. Cellulose 4:33–49
SaxenaI. M.,
LinF. C.,
BrownR. M.Jr1990; Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum . Plant Mol Biol 15:673–683
SaxenaI. M.,
BrownR. M.,
FevreM.,
GeremiaR. A.,
HenrissatB.1995; Multidomain architecture of β -glycosyl transferases: implications for mechanism of action. J Bacteriol 177:1419–1424
SaxenaI. M.,
BrownR. M.Jr,
DandekarT.2001; Structure-function characterization of cellulose synthase: relationship to other glycosyltransferases. Phytochemistry 57:1135–1148
SchweizerH. P.1992; Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6:1195–1204