The phytopathogen Pseudomonas syringae pv. glycinea produces the exopolysaccharide (EPS) alginate, which is thought to function in epiphytic fitness and virulence. A key regulator for alginate biosynthesis in Pseudomonas aeruginosa and P. syringae is the alternative sigma factor AlgT (σ22). In this study, the contribution of alginate synthesis and AlgT to in planta epiphytic fitness and virulence of P. syringae was examined. Alginate biosynthesis mutants were generated for the P. syringae pv. glycinea strains PG4180 and PG4180.muc, representing a comprehensive set of alginate- and AlgT-positive or -negative derivatives. Analysis of in vitro and in planta phenotypes revealed that AlgT strongly promoted in planta growth, survival and symptom development, but decreased the ability to grow in vitro. In contrast, alginate biosynthesis had only marginal impact. Quantitative in vitro and in planta gene expression analyses for alginate biosynthesis and algT were carried out at two temperatures in AlgT-negative and -positive backgrounds. algT as well as algD gene expression was AlgT-dependent, plant-inducible and temperature-dependent, with higher expression at 18 compared to 28 °C; however, no temperature dependence was observed in vitro. Our data suggest that AlgT may act as a global regulator for virulence and in planta fitness traits of P. syringae independent of its role in EPS biosynthesis.
AusubelF.,
BrentR.,
KingstonR. E.,
MooreD. D.,
SeidmanJ. G.,
SmithJ. A.,
StruhlK.1994Current Protocols in Molecular Biology New York: Wiley;
BeattieG. A.,
LindowS. E.1994; Epiphytic fitness of phytopathogenic bacteria: physiological adaptations for growth and survival. Curr Top Microbiol Immunol 192:1–27
ChitnisC. E.,
OhmanD. E.1993; Genetic analysis of the alginate biosynthetic gene cluster of Pseudomonas aeruginosa shows evidence of an operonic structure. Mol Microbiol 8:583–593
FakhrM. K.,
Peñaloza-VázquezA.,
ChakrabartyA. M.,
BenderC. L.1999; Regulation of alginate biosynthesis in Pseudomonas syringae pv. syringae. J Bacteriol 181:3478–3485
FettW. F.,
DunnM. F.1989; Exopolysaccharides produced by phytopathogenic Pseudomonas syringae pathovars in infected leaves of susceptible hosts. Plant Physiol 89:5–9
FettW. F.,
WijeyC.,
LifsonE. R.1992; Occurrence of alginate gene sequences among members of the pseudomonad rRNA homology groups I–IV. FEMS Microbiol Lett 78:151–157
FialhoA. M.,
ZielinskiN. A.,
FettW. F.,
ChakrabartyA. M.,
BerryA.1990; Distribution of alginate gene sequences in the Pseudomonas rRNA homology group I- Azomonas - Azotobacter lineage of superfamily B procaryotes. Appl Environ Microbiol 56:436–443
FigurskiD. H.,
HelinskiD. R.1979; Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans . Proc Natl Acad Sci U S A 76:1648–1652
FirovedA. M.,
BoucherJ. C.,
DereticV.2002; Global genomic analysis of AlgU ( σ E)-dependent promoters (sigmulon) in Pseudomonas aeruginosa and implications for inflammatory processes in cystic fibrosis. J Bacteriol 184:1057–1064
GrossM.,
RudolphK.1987a; Studies on the extracellular polysaccharides (EPS) produced in vitro by Pseudomonas phaseolicola . I. Indications for a polysaccharide resembling alginic acid in seven P. syringae pathovars. J Phytopathol 118:276–287
GrossM.,
RudolphK.1987b; Studies on the extracellular polysaccharides (EPS) produced in vitro by Pseudomonas phaseolicola . II. Characterization of levan, alginate, and LPS. J Phytopathol 119:206–215
HershbergerC. D.,
YeR. W.,
ParsekM. R.,
XieZ. D.,
ChakrabartyA. M.1995; The algT ( algU ) gene of Pseudomonas aeruginosa , a key regulator involved in alginate biosynthesis, encodes an alternative σ factor ( σ E
. Proc Natl Acad Sci U S A 92:7941–7945
HettwerU.,
JaeckelF. R.,
BochJ.,
MeyerM.,
RudolphK.,
UllrichM. S.1998; Cloning, nucleotide sequence, and expression in Escherichia coli of levansucrase genes from the plant pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola. Appl Environ Microbiol 64:3180–3187
HiranoS. S.,
UpperC. D.2000; Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae – a pathogen, ice nucleus, and epiphyte. Microbiol Mol Biol Rev 64:624–653
HoangT. T.,
Karkhoff-SchweizerR. R.,
KutchmaA. J.,
SchweizerH. P.1998; A broad-host-range Flp- FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86
KeithL. M. W.,
BenderC. L.1999; AlgT ( σ 22) controls alginate production and tolerance to environmental stress in Pseudomonas syringae . J Bacteriol 181:7176–7184
KeithR. C.,
KeithL. M. W.,
Hernández-GuzmánG.,
UppalapatiS. R.,
BenderC. L.2003; Alginate gene expression by Pseudomonas syringae pv. tomato DC3000 in host and non-host plants. Microbiology 149:1127–1138
KidambiS. P.,
SundinG. W.,
PalmerD. A.,
ChakrabartyA. M.,
BenderC. L.1995; Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae. Appl Environ Microbiol 61:2172–2179
LamJ.,
ChanR.,
LamK.,
CostertonJ. W.1980; Production of mucoid microcolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis. Infect Immun 28:546–556
LindowS. E.,
AndersenG.,
BeattieG. A.1993; Characteristics of insertional mutants of Pseudomonas syringae with reduced epiphytic fitness. Appl Environ Microbiol 59:1593–1601
MartinD. W.,
SchurrM. J.,
YuH.,
DereticV.1994; Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa : relationship to sigma E and stress response. J Bacteriol 176:6688–6696
MatheeK.,
KharazamiA. A.,
HøibyN.2002; Role of exopolysaccharide in biofilm matrix formation: the alginate paradigm. In Molecular Ecology of Biofilms pp 23–55 Edited by
McLeanR. J. C.
Norfolk: Horizon;
OhmanD. E.,
MarheeK.,
McPhersonC. J.,
DeVriesC. A.,
MaS.,
WozniakD. J.,
FranklinM. J.1996; Regulation of the alginate ( algD ) operon in Pseudomonas aeruginosa . In Molecular Biology of Pseudomonads pp 472–483 Edited by
NakazawaT.,
FurukawaK.,
HaasD.,
SilverS.
Washington, DC: American Society for Microbiology;
PalmerD. A.,
BenderC. L.1993; Effects of environmental and nutritional factors on production of the polyketide phytotoxin coronatine by Pseudomonas syringae pv. glycinea. Appl Environ Microbiol 59:1619–1623
Peñaloza-VázquezA.,
FakhrM. K.,
BaileyA. M.,
BenderC. L.2004; AlgR functions in algC expression and virulence in Pseudomonas syringae pv. syringae. Microbiology 150:2727–2737
RudolphK. W. E.,
GroßM.,
Ebrahim-NesbatF.,
NöllenburgM.,
ZomorodianA.,
WydraK.,
NeugebauerM.,
HettwerU.,
El-ShounyW.other authors1994; The role of extracellular polysaccharides as virulence factors for phytopathogenic pseudomonads and xanthomonads. In Molecular Mechanisms for Bacterial Virulence pp 357–378 Edited by
KadoC. I.,
CrossJ. H.
Dordrecht: Kluwer;
SambrookJ.,
FritschE. F.,
ManiatisT.1989Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
SchenkA.,
BergerM.,
KeithL. M.,
BenderC. L.,
MuskhelishviliG.,
UllrichM. S.2006; The algT gene of Pseudomonas syringae pv. glycinea and new insights into the transcriptional organization of the algT - muc gene cluster. J Bacteriol 188:8013–8021
SchenkA.,
WeingartH.,
UllrichM. S.2008; Extraction of high quality bacterial and plant total RNA from infected leaf tissue for bacterial in planta gene expression analysis by multiplexed fluorescent Northern hybridization. Mol Plant Pathol in press
Schnider-KeelU.,
LejbølleK. B.,
BaehlerE.,
HaasD.,
KeelC.2001; The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0. Appl Environ Microbiol 67:5683–5693
SchurrM. J.,
DereticV.1997; Microbial pathogenesis in cystic fibrosis – co-ordinate regulation of heat-shock response and conversion to mucoidy in Pseudomonas aeruginosa . Mol Microbiol 24:411–420
TatnellP. J.,
RussellN. J.,
GacesaP.1994; GDP-mannose dehydrogenase is the key regulatory enzyme in alginate biosynthesis in Pseudomonas aeruginosa : evidence from metabolite studies. Microbiology 140:1745–1754
YuJ.,
Peñaloza-VázquezA.,
ChakrabartyA. M.,
BenderC. L.1999; Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae. Mol Microbiol 33:712–720
ZielinskiN. A.,
ChakrabartyA. M.,
BerryA.1991; Characterization and regulation of the Pseudomonas aeruginosa algC gene encoding phosphomannomutase. J Biol Chem 266:9754–9763