Fig 1.
Role of CreBC TCS in bacterial and flagella morphologies.
The overnight-cultured bacteria were adjusted to an initial OD450 of 0.15. After a 5-h incubation at 37°C, the logarithmic-phase cells were harvested for morphology observation. (A) Bacterial morphology. The samples for bacterial morphology observation were processed by glutaraldehyde-osmium tetraoxide (OsO4)-ethanol method and examined suing SEM. (B) Flagella morphology. The flagella were negatively strained with 1% phosphotungstic acid (pH 7.4) and observed by TEM.
Fig 2.
The role of CreBC in the secreted protease activity.
Forty microliters of bacterial cell suspension was dipped onto LB agar containing 1% skim milk. After incubation at 37°C for 72 hour, the proteolytic activity of bacteria was assessed by measuring the transparent zones around the bacteria. Data represent the means from 3 independent experiments. Error bars represent the standard deviations for three triplicate samples. *, p < 0.05; ***, p < 0.001. (A) The secreted protease activities of creB, creC, and creBC mutants. (B) The secreted protease activities of creBC mutant and its derived complementation strains. Tetracycline (30 μg/ml) was added for the plasmid maintenance.
Fig 3.
The role of CreBC TCS in the swimming motility.
Two microliters of bacterial cell suspension was inoculated onto the swimming agar (1% tryptone, 0.5% NaCl, and 0.15% agar). Results were expressed as diameters (millimetres) of swimming zones after 48 h of incubation at 37°C. Data represent the means from 3 independent experiments. Error bars represent the standard deviations for three triplicate samples. *, p < 0.01; **, p < 0.001. (A) The swimming motility of creB, creC, creBC, and creBCD mutants. (B) The swimming motility of creBC mutant and its derived complementation strains. Tetracycline of 30 μg/ml was added for the plasmid maintenance.
Fig 4.
The transcript changes (folds) of flagella-related genes between KJ and KJΔBC cells by transcriptome analysis.
Total mRNA was extracted from KJ and KJΔBC logarithmic-phase cultures. The ribosomal RNA (rRNA) depletion, adapter-ligated cDNA library construction and enrichment, and cDNA sequencing were performed as described in Materials and Methods. Transcript changes (folds) of a gene is expressed as the transcript in KJΔBC relative to the transcript in wild-type KJ (KJΔBC/KJ). Navy blue color indicates the transcript change of the gene is greater than or equal to 3. Blue color indicates the transcript change of the gene is less than 3 and greater than 2. Light blue color indicates the transcript change of the gene is less than 2 and greater than 1. (A) The genomic organizations of the flagella-related genes. Based on the transcriptome assay, the flagella-related genes, upregulated in case of creBC inactivation, are located in three clusters, Smlt0561-0562, Smlt2265-2290, and Smlt2302-2321. The orientation of gene is indicated by the arrow. (B) Schematic diagram of bacterial flagellum. The flagellum consists of the basal body, the hook, and the filament. The composition proteins are labelled. OM, outer membrane; PG, peptidoglycan layer; IM, inner membrane.
Table 1.
Flagella-associated genes differently expressed in S. maltophilia KJ and KJΔBC cells.
Fig 5.
The transcript changes (folds) of selected flagella-related genes between KJ and KJΔBC cells by qRT-PCR.
Total mRNA was extracted from KJ and KJΔBC logarithmic-phase cultures. cDNA was prepared by RT-PCR and used as the template for qRT-PCR. The expression of target gene transcripts in qRT-PCR were normalized to the level of expression of the 16S rRNA gene by using the ΔΔCT method. Data are the means from three independent experiments. Error bars represent the standard deviations for three triplicate samples. *, p < 0.05; **, p < 0.005.
Fig 6.
The roles of creBC and creD in membrane susceptibility to Triton X-100.
The overnight-cultured bacteria were inoculated into fresh LB broth containing Triton X-100 of 200 μg/ml at the initial OD450 of 0.15. The bacterial growth was monitored by recording the OD450nm. Data are the means from three independent experiments. Error bars indicate the standard deviations for three triplicate samples.
Table 2.
The CCCP50 values of S. maltophilia KJ, its isogenic creBC mutant (KJΔBC), and the complementary strains.