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

Karla L. De Anda-Mora
Karla L. De Anda-Mora
, 
Faviola Tavares-Carreón
Faviola Tavares-Carreón
, 
Carlos Alvarez
Carlos Alvarez
, 
Samantha Barahona
Samantha Barahona
, 
Miguel A. Becerril-García
Miguel A. Becerril-García
, 
Rogelio J. Treviño-Rangel
Rogelio J. Treviño-Rangel
, 
Rodolfo García-Contreras
Rodolfo García-Contreras
 y 
Angel Andrade
Angel Andrade
   | 04 mar 2024
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Article Category: ORIGINAL PAPER
Publicado en línea: 04 mar 2024
Páginas: 11 - 20
Recibido: 22 sept 2023
Aceptado: 14 dic 2023
DOI: https://doi.org/10.33073/pjm-2024-002
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© 2024 Karla L. De Anda-Mora et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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).
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).
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).
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
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
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