Bacteria either swarming or non-swarming were seeded on plate conditions mentioned in the text. Extraction of cellular fatty acids was performed followed the standard procedure (MIDI, USA). Samples were prepared for analysis in MIDI GC-FAME analysis system (Microbe Inotech Laboratories, USA).
Acknowledgements:
DOEA project…..
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Figure Legends:
Fig. 1. Swarming of S. marcescens CH-1, S. marcescens CH-1∆A and S. marcescens
CH-1∆B. Swarming assay of the three strains on 0.8% LB swarming plates and comparison of swarming velocity (Gygi et al., 1995) were performed at both 30 and 37°C and were shown in (A) and (C) respectively. Cells were grown overnight in LB broth, washed once in 1 × PBS, and a 5-µl spot of inoculum was added to the center of the agar. The plates were incubated and observed after 12 h. Swarming assay of the three strains on 1.2% LB agar plates at 30°C after 10 h incubation was shown in (B).Symbols: ( )CH-1, 30°C; ( )CH-1, 37°C; (S)CH-1∆A, 30°C; (∆)CH-1∆A, 37°C;
(•)CH-1∆B, 30°C; (ο)CH-1∆B, 37°C. Error bars, standard errors of the means (n = 4).
Fig. 2. Location of the Tn5 insertion on the S. marcescens chromosome and analysis
of the RssA protein sequence. The insertion point of the mini-Tn5-Km transposon in 5’region of rssA was shown in (A). The nucleotide sequence containing rssA and rssB has been submitted to the DDBJ/EMBL/GenBank databases under accession no.AF465237. A hydropathy profile of RssA constructed by DAS (Cserzo et al., 1997) was shown in (B). The solid and dashed lines represent the strict and loose cutoffs, respectively. Prediction of membrane topology (Mitaku and Hirokawa 1999) was shown in (C). Alignment of RssA domains with those of related bacterial sensor proteins (Hoch and Silhavy 1995) was shown in (D). Conserved sequences are highlighted.
Fig. 3 S. marcescens CH-1∆A initiates swarming at a lower cell density.
The density-dependent initiation of swarming behavior of CH-1∆A(pPC300) was assayed on 0.8% LB swarming plates followed by hourly light microscopy observation (Ai) and X-ray film exposure (Aii) for monitoring the light emission pattern which is a reporter for flhDCSm expression (Liu et al., 2000) and bacterial swarming. Number means the time (hr) of incubation. The density-dependent initiation of swarming behavior in CH-1 and CH-1∆A cells was compared. The initial inoculum concentration was varied in 10-fold increments from 1 × 106 to 1 × 102 cells (CFU) delivered in 5-µl-aliquot droplets to the LB swarming plates. The onset time of swarming behavior and the lowest cell number (CFU) needed for swarming initiation were then recorded (B). Bacterial cell number shown was the mean of 4 independent experiments.
Fig. 4 The flagellar swimming motility and production of surfactant were not affected
in CH-1∆A and CH-1∆B mutants. Swimming assay of CH-1, CH-1∆A and CH-1∆Bwas performed on 0.35% LB plates at both 30 and 37°C for over night (A). The swimming velocity measured at 30 and 37°C (B) and flhDCSm promoter activity assayed at 37°C (C) for the 3 strains were recorded. Drop collapsing assay (Horng et al., 2002) (D) and TLC assay (Horng et al., 2002) (E) were performed to qualitatively and semi-quantitatively determine serrawettin production in CH-1 and CH-1∆A at 30 and 37°C. f, TLC front; p, prodigiosin pigment; w, serrawettin; o, TLC origin.
Symbols B: ( )CH-1, 30°C; ( )CH-1, 37°C; (S)CH-1∆A, 30°C; (∆)CH-1∆A, 37°C;
(•)CH-1∆B, 30°C; (ο)CH-1∆B, 37°C. Symbols C: ( )CH-1(pPC300);
(S)CH-1∆A(pPC300) and (•)CH-1∆B(pPC300).
Fig. 5 S. marcescens CH-1∆A and CH-1∆B are defective in biofilm formation and
show increased haemolysin activity at 30 and 37°C. Microtiter well assay (O’Tolle and Kolter 1998) was performed to monitor the biofilm formation activity of CH-1, CH-1∆A and CH-1∆B (A). Intensity of crystal violet measured at 550nm (OD550) was shown in (B), where blank column means test performed at 30°C and black column means at 37°C. The specific haemolysin activity (Koronakis et al., 1987) and transcriptional activity of the haemolysin gene shlA expressed as specific bioluminescence activity of CH-1(pSA400), CH-1∆A(pSA400) and CH-1∆B(pSA400) at 30 and 37°C were shown in (C) and (D), respectively. Symbols C: ( )CH-1, 30°C;( )CH-1, 37°C; (S)CH-1∆A, 30°C; (∆)CH-1∆A, 37°C; (•)CH-1∆B, 30°C;
(ο)CH-1∆B, 37°C. Symbols D: ( )CH-1(pSA400), 30°C; ( )CH-1(pSA400), 37°C;
(S)CH-1∆A(pSA400), 30°C; (∆)CH-1∆A(pSA400), 37°C; (•)CH-1∆B(pSA400), 30°C; (ο)CH-1∆B(pSA400), 37°C.
Fig. 6 S. marcescens CH-1 swarming phenotype is inhibited by saturated fatty acid in
a dose-dependent way, and shows close correlation to cellular fatty acid profiles.Swarming assay of CH-1 and CH-1∆A was performed on 0.8% MGM plates either without or with additives at 30 and 37°C (A). Effect of myristic acid (C14) on swarming was further assayed on 0.8% MGM plates containing 2x serially diluted myristic acid from 0.01% to 0.00125% at 37°C for 12 hr (B). The cellular fatty acid profiles of CH-1, CH-1∆A and CH-1∆B inoculated under different growth conditions leading to either swarming or non-swarming phenotypes were analysed by Gas Chromatography. The average percentage of fatty acids between swarming (white bars) and non-swarming cells (black bars) was shown in (C). Results were the mean of 3 independent experiments.
Fig. 7 Effect of myristic acid on the activation of fabG
Sm in S. marcescens CH-1 was down-regulated by RssA-RssB. CH-1(pSA401), CH-1∆A(pSA401) andCH-1∆B(pSA401) grown in LB broth containing myristic acid at the concentration of 0.01% (w/v)were tested for fabGSm promoter activity using luxCDABE as the reporter following the growth at 30°C and was shown in (A). Symbols: (S)CH-1(pSA401);
(◆ )CH-1∆A(pSA401); ( )CH-1∆B(pSA401). Northern blot hybridization using partial fabGSm as the probe detecting fabGSm transcriptional level in CH-1, CH-1∆A and CH-1∆B was shown in (B).