A study was conducted to determine the effect of one atmosphere uniform glow discharge plasma (OAUGDP) on inactivation of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on apples, cantaloupe, and lettuce, respectively. A five-strain mixture of cultured test organisms was washed, suspended in phosphate buffer, and spot inoculated onto produce (7 log CFU per sample). Samples were exposed inside a chamber affixed to the OAUGDP blower unit operated at a power of 9 kV and frequency of 6 kHz. This configuration allows the sample to be placed outside of the plasma generation unit while allowing airflow to carry the antimicrobial active species, including ozone and nitric oxide, onto the food sample. Cantaloupe and lettuce samples were exposed for 1, 3, and 5 min, while apple samples were exposed for 30 s, 1 min, and 2 min. After exposure, samples were pummeled in 0.1% peptone water-2% Tween 80, diluted, and plated in duplicate onto selective media and tryptic soy agar and incubated as follows: E. coli O157:H7 (modified eosin methylene blue) and Salmonella (xylose lysine tergitol-4) for 48 h at 37 degrees C, and L. monocytogenes (modified Oxford medium) at 48 h for 32 degrees C. E. coli O157:H7 populations were reduced by >1 log after 30-s and 1-min exposures and >2 log after a 2-min exposure. Salmonella populations were reduced by >2 log after 1 min. Three- and 5-min exposure times resulted in >3-log reduction. L. monocytogenes populations were reduced by 1 log after 1 min of exposure. Three- and 5-min exposure times resulted in >3- and >5-log reductions, respectively. This process has the capability of serving as a novel, nonthermal processing technology to be used for reducing microbial populations on produce surfaces.

(12)(13)(14)16) and pili (20,25), are posttranslationally modified. The biological significance of these modifications remains unknown. Although thought to be of little significance until recently, posttranslational phosphorylation in bacteria is now recognized as playing a vital role in individual-enzyme regulation (11, 21), as well as in global control mechanisms (5, 9, 23, 24).Tyrosine phosphorylation, although somewhat less common than modifications of serine and threonine, contributes to specific and highly important regulatory events in eukaryotic cells (4, 6). Tyrosine phosphorylation in prokaryotes remains controversial (10); however, evidence demonstrating the presence of tyrosine kinases and phosphorylation of several bacterial proteins at tyrosine has now accumulated (3,8,17).In an initial report, we demonstrated the presence of phosphotyrosine (P-Tyr) in b-type flagellin (15). In research presented here, we have extended these findings (i) to show that P-Tyr is present in a-type flagellins, (ii) to locate and compare distribution of phosphate in phosphopeptides of both flagellin types, and (iii) to substantiate the presence of unmodified tyrosine phosphate. Several lines of evidence presented support the conclusion that P. aeruginosa native flagellar filaments, as well as isolated flagellin, contain unmodified tyrosine phosphate.Isolation and purification of 32P-labeled a-and b-type flagellins. P. aeruginosa strains used in this study were common laboratory strains. The b-type strains used included M-2 and PAO1 (both containing seven tyrosines; Mr, * Corresponding author. 53,000). The a-type strains used were PAK (Mr 43,000; two Tyr), 170018 (Mr, 45,000; seven Tyr), 5940 (Mr 47,000; five Tyr), and 2993 (Mr, 50,000; two Tyr). Bacteria were grown in modified sodium succinate mineral salts medium (MSM) (1, 15). To achieve maximum radiolabeling, the total phosphate concentration was decreased from that in MSM to approximately twofold above growth-limiting levels (0.62 mM phosphate).[32P]phosphoric acid was added at a concentration of either 5 or 10 ,uCi/ml, and the cells were grown at 30°C for 22 h. Labeled flagellar preparations from the a-and b-type cells were isolated by mechanical shearing and differential centrifugation and were further purified by several steps of molecular sieving, as previously described (15). Since cells were not broken, flagellar purification procedures were designed primarily to remove excessive lipopolysaccharide (LPS). Labeling of flagellar protein was then demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of phosphate-labeled b-type flagellins has previously been shown (15). A purified preparation of a representative a-type flagellin (PAK) was separated by electrophoresis in 10% homogeneous gels and stained with Coomassie brilliant blue (Fig. 1A). As expected, the Mr of strain PAK (Fig. 1A, lane 1) was 43,000 to 45,000 (26). The autoradiogram shown in Fig. 1B showed radioactivity associated with a-type flagellin, as previously shown wi...

Previous evidence showed that b- and a-type flagellins of Pseudomonas aeruginosa are modified in vivo by phosphorylation at tyrosine. This research was designed to demonstrate phosphorylation of flagellin at tyrosine in vitro. Evidence presented showed that flagellin is labelled by [gamma-32P]-ATP, but not by [alpha-32P]-ATP, when incubated with cell envelope fractions. Results suggested that autophosphorylation of a 42 kDa membrane protein occurred. No activity was detected in cytoplasmic fractions. Flagellin protein was identified by flagella-specific monoclonal antibody (mAb) and was labelled with anti-phosphotyrosine mAb. Confirmation of tyrosine kinase activity was shown by labelling of synthetic poly(Glu:Tyr) as a substrate with [gamma-32P]-ATP. Labelling of poly(Glu:Tyr) was heat sensitive and time dependent. Labelled phosphotyrosine was observed in partial acid hydrolysates of substrates. Using poly(Glu:Tyr) as substrate, tyrosine kinase activity was shown to be inhibited by sulphydryl reagents. It appears that tyrosine kinase and flagellin phosphorylation occur in several Pseudomonas spp. Location of phosphotyrosine in a conserved region of flagellin may serve as a cell signal so that intact flagellin is appropriately exported.

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