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Figur e Legends
Fig. 1. The physical map and sequence characterization of the flhDCSm genetic locus.
(a) The physical map of S. marcescens flhDCSm genetic locus. PI, PstI; EI, EcoRI; SI, SalI; XI, XhoI; SpI, SphI.
(b) DNA and predicted protein sequences of the flhDCSmlocus. A potential Shine-Dalgarno (S.D.) sequence was indicated. A sequence TAAAAT, similar to the E. coli consensus –10 box, is found between the nucleotide sequence 413~418. The sequence, TTGCGC, similar to the E. coli consensus – 35 box, is found between positions 387~392 (The shaded area). Aσ28 consensus sequence was found between 182~208, and a putative consensus CRP binding site was found between 311~331, with the conserved sequence being boxed.
Fig. 2. Confirmation of inactivation of flhDSm by streptomycin (SM) through homologous recombination.
(a) A pair of primers (5’CATCCATACACGTTGGTTTACGCT-3’ and 5’-TGCTCGTCCAGCAGTTGAGGAATA-3’) were used in the PCR for confirmation of insertion mutagenesis. S. marcescens CH-1 chromosomal DNA (lane 1), pJH04 (lane 2), pCR2.1flhDCSm::Sm (lane 3), S. marcescens F-1 chromosomal DNA (lane 4), and S. marcescens S-2 chromosomal DNA (lane 5) were used as the template. The arrows indicate the DNA size markers.
(b) Southern blot hybridization using the PCR amplified 1.1 Kb DNA fragment (the same as a) as the probe. Chromosomal DNA of S. marcescens CH-1 (lane 1, 4, 7), S. marcescens F-1 (lane 2, 5, 8) and S. marcescens S-2 (3, 6, 9) were digested by SalI, EcoRI/HindIII, and PstI separately as indicated.
Arrows indicate the DNA size markers.
Fig. 3. The flhDSmmutant phenotypes.
(a) The swimming of F-1 (A) and CH-1 (B), and swarming of F-1(C) and CH-1 (D) in LB plates at 30℃. A and B, 0.4 % agar; C and D, 0.8 % agar.
(b) The growth curve [CH-1 (O); F-1 ( )] and mean cell length units [CH-1 (filled column); F-1 (empty column)] of S. marcescens CH-1 and F-1 following the growth process in LB broth culture at 30℃. Results are the means of at least 3 independent experiments (SEM < 5 %).
Fig. 4. Effect of flhDCSmon the phospholipase activity and nucASmexpression.
(a) The phospholipase–negative cells (upper arrow) were observed in flhD mutant (S. marcescens F-1) after O/N culture on egg-yolk agar plate (Koneman et al., 1997). The lower arrow indicates the wild-type control cells.
(b) Expression of nucASmwas activated by flhDCSm. Following the growth process in LB broth culture at 30℃, the light emission of S. marcescens 1/ pBCSK+ ( ), 2/ pBCSK+ (O), and
N-2/pJH05 (Ž) were measured and the specific activity (spe. act.) was expressed as (R.L.U./O.D.).
Results are the means of three independent experiments (SEM < 6 %).
Fig. 5. Cells with differentiated characteristics were observed in S. marcescens CH-1/pJH05 in MGM broth culture.
S. marcescens CH-1/pJH05 was inoculated in MGM broth culture at 30℃. Following the growth process, cells were harvested for observation of cellular morphology. The cells shown in the left (A) were taken from early log phase (O.D. 0.2). The cells shown in the right (B) was taken from S.
marcescens CH-1/ pBCSK+ control cells (O.D. 0.2).
Fig. 6. PflhDCSm was self-regulated.
Following the growth process in LB broth culture at 30℃, the light emissions of S. marcescens F-3
( ) and F-4 (O) were measured and expressed as specific activity (spe. act., R.L.U./O. D.). Results are the means of three independent experiments (SEM < 6 %).
Fig. 7. Effect of temperature, osmolarity and glucose concentration on the expression of PflhDCSm. (a)S. marcescens F-3 (PflhDCSm::luxAB) was cultured in LB broth at either 30 (O) or 37℃ ( ). The
light emission was measured following the growth process. For comparison, S. marcescens S-1 (PhagSm::luxAB) was also assayed at 30 (Ž) or 37℃ ( ).
(b) S. marcescens F-3 was cultured at increasing NaCl concentration [8.5 (O), 100 ( ), 250 (Ž) and
500 ( ) mM] in LB broth, followed by measuring the light emission. (c) S. marcescens F-3 was
cultured at increasing glucose concentration [0.4 % (O), 0.8 % (Ž), 1.2 % ( ) and 2.0 % ( )] in MGM broth culture. Results are the means of three independent experiments (SEM < 5 %). spe. act., specific activity (R. L. U./O. D.).