Table I
Oligonucleotides used in this study.
| Primer | 5′–3′ primer sequence | Purpose |
|---|---|---|
| XRQFw | TGGAAGCCATGCATAAACTG | Internal pair for cpxR |
| XRQRv | TACGCTGCTGATGTTTCTGG | |
| 16SQFw | GAGCAAGCGGACCTCATAAAG | Internal pair for 16S |
| 16SQRv | TGCGGTTGGATTACCTCCT | |
| ERQFw | GGATTGGAAAACGTCAGCATG | Internal pair for eepR |
| ERQRv | GCCACGAAAAAGATGGCATC | |
| HSQFw | CTTCCAGCAGATCGACCATC | Internal pair for hexS |
| HSQRv | AGATCCTGCGCTTTAACGAC | |
| SDQFw | CGCGATCCAAAAATTGTACG | Internal pair for slpD |
| SDQRv | TCGTTCAGGTTGATCATCTG | |
| PSQFw | GACCTGGTACAACGTCAAC | Internal pair for prtS |
| PSQRv | GTAGCTCATCAGGCTGAAC | |
| PREcoF | CCCTGAATTCCGTTTTTATTTGCGGCTG | Pair for eepR promoter cloning into pSEVA246 |
| PRXbaR | TGGGTCTAGATTGTTATCCATTTGTTCCTTCG | |
| scSEVAF | AGCGGATAACAATTTCACACAGGA | Pair for pSEVA-based constructions screening |
| scSEVAR | CTTTCGGGAAAGATTTCAACCTGG | |
| scETFw | CCCTCAAGACCCGTTTAGAG | Pair for pET-based constructions screening |
| scETRv | CTCTTCCGAGGTGAAAACCG | |
| xRNdeF | CGCGCATATGAACAAGATTCTGTTAG | Pair for cpxR amplification |
| xRBamR | TTTGGATCCAAACTGTTGATCATGTTGC | |
| CRPFw | CCTGGTGCCGCGCGGCAGCCATGTTCTCGGCAAACCGCAAAC | Pair for crp amplification |
| CRPRv | CTGTCCACCAGTCATGCTAGCCATTAACGGGTGCCGTAGACG | |
| HexSFw | CCTGGTGCCGCGCGGCAGCCACATGACAACTGCAAATCGTCC | Pair for hexS amplification |
| HexSRv | CTGTCCACCAGTCATGCTAGCCACGTTATTCTTCTTCGTCCAC | |
| PeRFw1 | CAATAAAAAACCGGGACCC | Forward for eepR promoter (−358 nt from eepR start) |
| PeRFw2 | GCAGTCCRAGCGATGTG | Forward for eepR promoter (−271 nt from eepR start) |
| PeRRv2 | TTTCYGCTGAAAAAGCCAC | Reverse primer for eepR promoter (+45 nt from eepR start) |
| PeRRv1 | TTGTTATCCATTTGTTCCTTCG | Reverse primer for eepR promoter (−11 nt from eepR start) |
| recAFw | CAAGGCGAATGCCTGTAACT | Pair for EMSA negative control (internal for recA) |
| recARv | GAGGATAGGCGCCACATAAA | |
| UPxRFw | GGGTTTTTTCGCTGATCACGTACGATGCGCTGCTGATGTTTCTGG | Pair for cpxR upstream region |
| UPxRRv | GTTGCGCCAGCAGATACAGCAGCGAGGTCAACTCGCGGTC | |
| DWxRFw | GACCGCGAGTTGACCTCGCTGCTGTATCTGCTGGCGCAAC | Pair for cpxR downstream region |
| DWxRRv | GTACACCATGTGCACCGGTTCGAAGATGGTGACGATCAGCAGCAG | |
| scTOXF | CGCGACGGTTTCTTACAGTG | Pair for pTOX3-based construction screening |
| scTOXR | GCTTCCCGGTATCAACAGAG | |
| XRscFw | CCAGAAATTTGTTGCTCCATC | Pair for cpxR deletion strain screening |
| XRscRv | GGTCGGAACATCAGGTTGAT |
Fig. 1.
Zymography and proteolytic activity of Serratia marcescens strains.
A) SDS-PAGE of extracellular proteins precipitated by ammonium sulfate from bacterial cultures at 37°C (Coomassie stain). Identified proteins by mass spectrometry are indicated at the left; B) Zymography following SDS-PAGE using gelatin as substrate from supernatant cultures grown at 37°C. Arrowheads indicate gelatin degradation areas; C) Digestion of azocasein by normalized filtered supernatants.
Graphs represent the mean ± SEM from three independent experiments (**p < 0.01, ***p < 0.001).
Fig. 2.
Transcriptional analysis of prtS and regulator genes in Serratia marcescens HU1848, SmUNAM836 and Db10.
qRT-PCR analysis of gene expression of A) prtS, B) slpD, C) eepR D) cpxR, and E) hexS. RNA was extracted from bacterial cultures at 37°C. The mRNA levels were normalized to the 16S rRNA gene. Relative expression was calculated by 2−ΔCT method
Means ± SEM from three independent experiments are shown (*p < 0.05).
Fig. 3.
Differential eepR promoter expression and direct regulation of eepR by CpxR.
A) Scheme representation of the Serratia marcescens region encoding eepRS. DNA fragments employed in transcriptional fusion or in EMSA evaluations are depicted. White rectangles with asterisk indicate the two predicted CpxR binding sites. All nt positions are referred to eepR start codon; B) eepR promoter activity expressed as RLU during bacterial culture grown at 37°C. Filled and open triangles HU1848 carrying pPeepRHU1848 or pSEVA26, respectively. Filled and open circles SmUNAM836 carrying pPeepRSmUNAM or pSEVA26, respectively; C) EMSA using CRP (0, 0.05, 0.1, 0.2, 0.4 μM) and DNA region E1 amplified from SmUNAM836 or HU1848; D) EMSA using CpxR (0, 0.5, 1, 2, 3 μM) and the four DNA fragments (E1-E4) containing eepR upstream sequence of strain HU1848, reactions pre-incubated with acetyl phosphate are indicated (AcP).
E) qRT-PCR analysis of eepR expression from the S. marcescens strain HU1848 (WT), ΔcpxR strain and ΔcpxR carrying pPBADcpxR plasmid. Relative expression was calculated by 2–ΔCT method. Means ± SEM from three independent experiments are shown (**p < 0.01, *p < 0.05).