coli. The restriction endonucleases, T4 DNA ligase and Pfu polymerase were provided by Promega (Promega Corporation, Madison, WI). Complementation of the gpsX mutant For complementation of the gpsX mutant 223 G4 (gpsX-), a 2,299-bp DNA fragment containing the intact open reading frame (ORF) of gpsX and 230 bp upstream of the 5′ end to 21 bp downstream of the 3′ end of the ORF, was amplified from the genomic DNA of Xac strain 306 using the primers C10-F (5′ -tcgaggtaccgttggtgtcgtcctcgaaat-3′) and C10-R (5′ – tcgtaagcttctcaccccgcaataaacaac-3′),

respectively Adriamycin nmr containing KpnI and HindIII restriction enzyme sites (underlined). The PCR product was digested with KpnI and HindIII and cloned into the complementary vector pUFR053 [33] to construct the recombinant plasmid pJU3110 (Table 2). The recombinant plasmid was transferred into the gpsX mutant 223 G4 (gpsX-) by triparental conjugation as described elsewhere [57], resulting in strain C233G4 (gpsX+) (Table 2). Quantitative determination of EPS production To estimate EPS production, strains were cultured in 100 ml NB or XVM2 liquid

medium containing 2% (wt/vol) various sugars (fructose, galactose, glucose, maltose, mannose, Selleckchem PU-H71 sucrose, and xylose) at 28°C with shaking at 200 rpm for 24 hours (in NB) or 48 hours (in XVM2). EPS was precipitated from the culture supernatant at different time point post inoculation with ethanol, dried, and weighed as described elsewhere [35]. Lipopolysaccharides (LPS) analysis Bacterial strains were cultured at 28°C in NB or XVM2 liquid medium with shaking (200 rpm). Five-milliliter samples acetylcholine of cultures at the exponential stage were collected and the LPS samples were isolated as described previously [23]. LPS was separated

by SDS-PAGE and visualized using silver staining following the manufacturer’s instructions (Bio-Rad Laboratories, Inc., Hercules, CA). Standard LPS from Salmonella entenica serovar Typhimurium was obtained from Sigma. The test was performed three times independently. Capsule assays Bacterial capsules were stained using a capsule-staining kit (Eng Scientific Inc., Clifton, NJ, USA) following the manufacturer’s instructions. The samples were photographed using a light microscope (Leica DMLB2; Leica Microsystems GmbH, Wetzlar, Germany) with a digital camera. The experiment was repeated three times. Biofilm formation assays Biofilm formation on polystyrene and glass surfaces were examined as described previously [23] with modifications. The average of four replicates was used for quantitative measurement. Assays of biofilm formation on leaf surfaces were conducted as described previously [58] with modifications. Briefly, 20 μl of each bacterial suspension (108 cfu/ml) was incubated on the abaxial surface of citrus leaves, and the leaves were kept at 28°C in a humidified chamber. After 24 h of incubation, biofilm formation on the leaf surface was visualized using crystal violet staining.