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Title:
Development of an Adhesion Assay and Characterization of an Adhesion-Deficient Mutant of Pseudomonas fluorescens

RESULTS Bacterial adhesion to sand columns. In the initial column assays with soil-and-sand mixtures, P. fluorescens PfO-1 and E. coli SLH25 had the highest and lowest adhesion values, respectively. Therefore, the sand column assay was optimized to yield the greatest difference in percent adhesion for these two strains. PfO-1 bacteria grown in minimal medium to the logarithmic phase were washed and suspended in either minimal medium or buffered saline and added to three columns each at identical titers. The cells that were added in minimal medium adhered at percentages ranging from 46 to 82%. Cells suspended in buffered saline, however, adhered at 93 to 95% of the total introduced. Growth of PfO-1 in L broth, as opposed to minimal medium, with subsequent washing in buffered saline, yielded lower adhesion values (x = 77%, n = 5). Adding PfO-1 to the column in buffered saline with two and three times the NaCl content only slightly decreased the percentage that adhered to the column. The effect of the growth phase on adhesion of the inoculum was tested by comparing PfO-1 in the logarithmic phase, the stationary phase (18-, 24-, and 30-h cultures), and a starvation state (i.e., suspension in buffered saline for 48 h). All cultures were prepared by washing and suspending the cells in buffered saline before addition to the column. While logarithmic-phase cells consistently adhered at >90% (93 + 3%, n = 6), starved cells adhered less well (73 + 5%, n = 6) and stationary-phase cultures adhered the least (52 + 16%, n = 6). Column parameters affecting adhesion. PfO-1 bacteria (107) added to a 12-g sand column adhered at >90% whether the sand was contained in a 20- or 50-ml syringe, indicating that the depth of the sand in the column was not a determining factor. Increasing the attachment over a period of 1 to 4 h, with samples taken every hour, also did not affect the total number of cells that adhered (92 + 1.6%). The number and salinity of the washes applied to each column were also tested. Columns containing either P. fluorescens PfO-1 or E. coli SLH25 were washed with six 3-ml samples of buffered saline, and each wash was counted separately. For the PfO-1 columns, all nonadhering cells came off in the first three washes (6% of the total cells were removed). With SLH25, 88% of the cells were removed in the first three washes. The greatest difference in adhesion between the two strains occurred after four washes, with PfO-1 adhering at 94% andSLH25 adhering at 10%. Doubling and tripling the amount of sodium chloride contained in the buffered saline used to wash the columns had little effect on the amount of strain Pf0-1 bacteria that adhered to the sand column. After four 3-ml washes, the column washed with buffered saline had 94% adhesion of PfO-1, while the columns treated with the higher-salt washes both had adhesion values of 92%. PfO-1 and SLH25 were also tested together in the same column. In six separate experiments, PfO-1 adhered at 93 ± 4% while SLH25 adhered at only 9 ± 7%. Under conditions yielding the greatest difference in adhesion between P. fluorescens PfO-1 and E. coli SLH25, the percent adhesion values of different strains were determined (Table 2). Other pseudomonads adhered at 40 to 60%, whereas E. coli HB101 adhered at about 20%. Isolation of adhesion-deficient mutants. From a total of 2,500 PfO-i::TnS mutants tested in the sand column assay, we isolated two adhesion-deficient mutants (NO-5 and NO- 10) which consistently adhered at <50%. The position of the TnS insertion in chromosomal digests of PfO-5 was different from that of TnS in digests of PfO-10 and NO-6, a TnS insertion mutant that adhered at >90% (Fig. 1). We selected PfO-5 for further study. Comparison of outer membrane proteins in mutant and wild-type strains. The outer membrane proteins of strains PfO-1 (wild type), NO-6, and PfO-5 were compared. PfO-1 and PfO-6 showed indistinguishable membrane protein profiles. Both contained a major protein at -34 kilodaltons (kDa) that was not present among the outer membrane proteins of PfO-5, the adhesion-deficient mutant (Fig. 2). The outer membrane proteins of PfO-1 cultures in logarithmic, stationary, and starved conditions were identical (data not shown). Transmission electron microscopy. Electron microscopic studies of strain NO-1 revealed long, thin (-10 nm), multitrichous polar flagella extending from the cell. Strain PfO-5, however, lacked these cell surface structures (Fig. 3). Purified flagellin from PfO-i migrated as a 34-kDA protein in sodium dodecyl sulfate-polyacrylamide gels (data not shown), indicating that the membrane protein missing in PfO-5 is attributable to the absence of flagella in the mutant. Motility studies. Growth of PfO-1 in the motility test agar extended throughout the tube, while PfO-5 grew only along the stab line. This result was confirmed by microscopic observation of these cells, which indicated that NO-1 was motile while PfO-5 was nonmotile. Growth of mutant in media and sterile soil. Growth of PfO-i and PfO-5 was compared in liquid culture. In L broth, both doubled in 40 min; in minimal medium, however, PfO-5 doubled in only 65 min, in contrast to the 85-min doubling time of NO-1 (Fig. 4). In previous studies (10; B. J. Al-Achi, E. Platsouka, and S. B. Levy, submitted for publication), sterile-soil assays have been used to assess the competitive fitness of mutants compared with the parent strain. When PfN-1 and the adheFIG. 1. (A) Sall digestion of chromosomal DNAs. Lanes: 1, PfO-1; 2, PfO-6; 3, PfO-10; 4, PfO-5. (B) Autoradiogram resulting from hybridization with the 32P-labeled TthlllI-SalI fragment of Tn5. sion-deficient mutant PfO-5 were added individually to sterile soil, both reached titers of 4 x 108 CFU/g of soil after 48 h and maintained that level over a period of 5 days (data not shown). When added together into sterile soil, both reached titers of -1 x 108 to 2 x 108 CFU/g of soil after 48 h and maintained that level over the course of the experiment (5 to 7 days; data not shown). Adhesion of mutant to soil columns. Strain PfO-1 and PfO-5 bacteria were added in equal numbers to sterile soil-sand columns and watered for a period of 5 days. Subsequent analysis showed that the top layers of soil and sand contained 45 to 71% (58 + 12%, n = 4) less PfO-5 than PfO-1, with the greatest differences between the two strains found in the 6-g soil-sand columns. The bottom sand layers had 68 to 84% (75 + 7%, n = 4) less PfO-5 than PfO-1. Site identified by TnS. Two plasmids of different sizes (pMFD1 and pMFD2) were selected from the kanamycinand tetracycline-resistant E. coli BD1527 that resulted from transfection with cosmid-containing phage X. These two plasmids were mated into wild-type PfO-1 for subsequent recombination of the TnS-containing insert into the chromosome. Thirty-nine percent of the Kmr Tcs Gmr colonies grown for >100 generations lost pPHlJI (loss of Gmr). The two different recombinant PfO-1/5a and PfO-1/5b (Kmr Tcs Gms) strains tested in the adhesion assay adhered at 48 and 32%, respectively. Outer membrane protein preparations of these two recombinants revealed a profile identical to that of PfO-5 (Fig. 5). Probing SalI chromosomal digests with the TthlllI-SalI fragment of TnS indicated that TnS was in the same position in the recombinant Pf0-1 strains as in PfO-5 (Fig. 6)

what would be a good 4-5 sentence summary for these results?

Answer 1

The adhesion value of Pf0-1 was lower in broth as compared to minimal media. Similarly, logarithmic-phase cells have adhered more than starved cells and stationary-phase cells. Studies on column parameters such as depth of the sand in the column, column retention time, sodium chloride concentration have not showed any impact on adhesion value of Pf0-1. Only number of column washes have showed difference with PfO-1 adhering at 94% and SLH25 adhering at 10%occurred after four washes. From 2,500 Pf0-l::TnS mutants examined in the sand column assay, two adhesion-deficient Pf0-1 mutants showing <50% adhesion were isolated. Linkage between the Tn5 insertion and the adhesion deficient phenotype of PfO-5 was demonstrated by the marker exchange technique. Placement of TnS into the same chromosomal site in unmutated strain PfO-1 caused the adhesion deficiency phenotype of Pf0-5.