Fig 1.
Schematic organization and operon verification of the creBCD-hp136 cluster of S. maltophilia KJ.
(A) Genomic organization of the creBCD-hp136 cluster and the structure of chromosomal xylE-transcription fusion constructs, mutants, and recombinant plasmids. The creBC operon forms a two-component regulatory system. CreD encodes a putative inner membrane protein. The small arrows indicate the primer HP136-C and CreD-C for the reverse transcription. The gray bar indicates the PCR amplicons using the CreDQ-F/R and CreCQ-F/R as the primer sets. The orientation of gene is indicated by the arrow. The white box indicates the deleted region for each deletion mutant construct. The crosshatched arrows represent the xylE cassette. (B) The 81-bp DNA sequence of intergenic region between creC and creD. A homologue of so-called cre/blr tag sequence (TTCACnnnnnnTTCAA) is marked in gray, at around -80 to -65 bp relative to the start codon of creD. (C) Agarose gel electrophoresis of reverse transcriptase-PCR (RT-PCR). The cDNAs of S. maltophilia KJ were obtained by RT-PCR using the primers HP136-C and CreD-C respectively, and then PCR was performed using different primer sets. The S. maltophilia KJ chromosome DNA was used as a control for the primers reliability. Lane 1, primers CreDQ-F and CreDQ-R; Lane 2, primers CreCQ-F and CreCQ-R; Lane 3, primers SmeXQ-F and SmeXQ-R. SmeX gene, which is intrinsically unexpressed in strain KJ, is used as a control for the DNA contamination during cDNA preparation.
Fig 2.
The C23O activity expressed by the chromosomal xylE-transcription fusion constructs of S. maltophilia KJ.
Overnight cultures of S. maltophilia strains assayed were inoculated into the fresh LB to the A450 of 0.15. Cells were grown aerobically, and the A450 and C23O activity were measured every 3 h.
Fig 3.
The promoter activity of creD gene was regulated by the bacterial culture density.
(A) The impact of plasmid and tetracycline on the bacterial growth and C23O expression of strain KJCreD23. Plasmid pRK415 was transported into KJCreD23 by conjugation. The bacterial growth (by recording the OD450nm) and C23O activity expressed from KJCreD23, KJCreD23(pRK415), and KJCreD23(pRK415) with 30 μg/ml tetracycline were monitored every 3 h. (B) The impact of kanamycin (Km) on the bacterial growth and C23O expression of strain KJΔBCCreD23. The bacterial growth (by recording the OD450nm) and C23O activity of KJΔBCCreD23 in the absence and presence of kanamycin (1 or 5 μg/ml) were monitored every 3 h. (C) The impact of menadione (K3) on the bacterial growth and C23O expression of strain KJΔBCCreD23. The bacterial growth (by recording the OD450nm) and C23O activity of KJΔBCCreD23 in the absence and presence of K3 (2 or 30 μg/ml) were monitored every 3 h. (D) The impact of benzalkonium chloride (BC) on the bacterial growth and C23O expression of strain KJΔBCCreD23. The bacterial growth (by recording the OD450nm) and C23O activity of KJΔBCCreD23 in the absence and presence of BC (1 or 5 μg/ml) were monitored every 3 h.
Fig 4.
The impact of phosphor-mimic variant of CreB, CreB(D55E), on the promoter activity of creD gene.
Overnight culture of strain KJCreD23Fua::CreB(D55E) was inoculated into the fresh medium to the OD450 of 0.15 in the absence and presence of fusaric acid (30 μg/ml). Cells were grown aerobically and the C23O activity were measured every 3 h.
Fig 5.
The impact of β-lactam on the promoter activity of creD gene.
Overnight culture of KJΔBCCreD23 was inoculated into the fresh medium to the OD450 of 0.15. After 30-min culture, the β-lactam as indicated was added and the culture was further incubated for 3 h. The OD450 and C23O activity were measured.
Fig 6.
The impact of CreD on bacterial morphology.
(A) Bacterial cells were stained with Gram stain and examined by light microscopy. (B) Scanning electron microscopy was performed as described in Materials and Methods. Images are representative of different fields of bacteria from exponentially growing cultures at 37°C.
Fig 7.
The impact of CreD on cell envelope integrity.
Each bar represents the mean of three independent experiments. Error bars indicate the average deviation. *, p≤0.05 significance calculated by s Student’s t-test. (A) Sodium dodecyl sulfate (SDS) survival analysis. The survival of KJ, KJΔCreD, and KJΔCreD(pCreD) in LB broth without or with 0.01% SDS was determined by colony forming units (CFUs) counting. The percentage of survival was defined as the CFUs ratio of the SDS-additive group to the SDS-free counterpart. (B) N-phenylanphthylamine (NPN) uptake assay. Each microtiter well was inoculated with 100 μl of the OD450 0.5 bacterial culture and 15 μM NPN, and incubated for 5 min. Fluorescence was monitored by fluorescence spectrophotometer at excitation and emission wavelengths of 355 nm and 402 nm, respectively.
Fig 8.
The linkage between creD inactivation and σE-mediated envelope stress response.
The promoter transcriptional fusion constructs of rpoE and smeI genes, pRpoExylE and pSmeIxylE, were transferred into KJ, KJΔCreD, and KJΔCreDΔRpoE cells and the expressed C23O activities were determined, respectively. Each bar represents the mean of three independent experiments. Error bars indicate the average deviation. *, p≤0.01 significance calculated by s Student’s t-test.