Increased Proteolytic Activity of Serratia marcescens Clinical Isolate HU1848 Is Associated with Higher eepR Expression

De Anda-Mora, Karla L.; Tavares-Carreón, Faviola; Alvarez, Carlos; Barahona, Samantha; Becerril-García, Miguel A.; Treviño-Rangel, Rogelio J.; García-Contreras, Rodolfo; Andrade, Angel

doi:10.33073/pjm-2024-002

Figures & Tables

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). — 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).

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). — 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).

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). — 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).

Oligonucleotides used in this study_

Primer	5′–3′ primer sequence	Purpose
XRQFw	TGGAAGCCATGCATAAACTG	Internal pair for cpxR
XRQRv	TACGCTGCTGATGTTTCTGG	Internal pair for cpxR
16SQFw	GAGCAAGCGGACCTCATAAAG	Internal pair for 16S
16SQRv	TGCGGTTGGATTACCTCCT	Internal pair for 16S
ERQFw	GGATTGGAAAACGTCAGCATG	Internal pair for eepR
ERQRv	GCCACGAAAAAGATGGCATC	Internal pair for eepR
HSQFw	CTTCCAGCAGATCGACCATC	Internal pair for hexS
HSQRv	AGATCCTGCGCTTTAACGAC	Internal pair for hexS
SDQFw	CGCGATCCAAAAATTGTACG	Internal pair for slpD
SDQRv	TCGTTCAGGTTGATCATCTG	Internal pair for slpD
PSQFw	GACCTGGTACAACGTCAAC	Internal pair for prtS
PSQRv	GTAGCTCATCAGGCTGAAC	Internal pair for prtS
PREcoF	CCCTGAATTCCGTTTTTATTTGCGGCTG	Pair for eepR promoter cloning into pSEVA246
PRXbaR	TGGGTCTAGATTGTTATCCATTTGTTCCTTCG	Pair for eepR promoter cloning into pSEVA246
scSEVAF	AGCGGATAACAATTTCACACAGGA	Pair for pSEVA-based constructions screening
scSEVAR	CTTTCGGGAAAGATTTCAACCTGG	Pair for pSEVA-based constructions screening
scETFw	CCCTCAAGACCCGTTTAGAG	Pair for pET-based constructions screening
scETRv	CTCTTCCGAGGTGAAAACCG	Pair for pET-based constructions screening
xRNdeF	CGCGCATATGAACAAGATTCTGTTAG	Pair for cpxR amplification
xRBamR	TTTGGATCCAAACTGTTGATCATGTTGC	Pair for cpxR amplification
CRPFw	CCTGGTGCCGCGCGGCAGCCATGTTCTCGGCAAACCGCAAAC	Pair for crp amplification
CRPRv	CTGTCCACCAGTCATGCTAGCCATTAACGGGTGCCGTAGACG	Pair for crp amplification
HexSFw	CCTGGTGCCGCGCGGCAGCCACATGACAACTGCAAATCGTCC	Pair for hexS amplification
HexSRv	CTGTCCACCAGTCATGCTAGCCACGTTATTCTTCTTCGTCCAC	Pair for hexS amplification
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	Pair for EMSA negative control (internal for recA)
UPxRFw	GGGTTTTTTCGCTGATCACGTACGATGCGCTGCTGATGTTTCTGG	Pair for cpxR upstream region
UPxRRv	GTTGCGCCAGCAGATACAGCAGCGAGGTCAACTCGCGGTC	Pair for cpxR upstream region
DWxRFw	GACCGCGAGTTGACCTCGCTGCTGTATCTGCTGGCGCAAC	Pair for cpxR downstream region
DWxRRv	GTACACCATGTGCACCGGTTCGAAGATGGTGACGATCAGCAGCAG	Pair for cpxR downstream region
scTOXF	CGCGACGGTTTCTTACAGTG	Pair for pTOX3-based construction screening
scTOXR	GCTTCCCGGTATCAACAGAG	Pair for pTOX3-based construction screening
XRscFw	CCAGAAATTTGTTGCTCCATC	Pair for cpxR deletion strain screening
XRscRv	GGTCGGAACATCAGGTTGAT	Pair for cpxR deletion strain screening

Increased Proteolytic Activity of Serratia marcescens Clinical Isolate HU1848 Is Associated with Higher eepR Expression

Figures & Tables

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Oligonucleotides used in this study_

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