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Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons.

Cambray G, Sanchez-Alberola N, Campoy S, Guerin E, Da Re S, González-Zorn B, Ploy MC, Barbé J, Mazel D, Erill I - Mob DNA (2011)

Bottom Line: Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens.In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons.Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, Unité Plasticité du Génome Bactérien, CNRS URA 2171, 75015 Paris, France. mazel@pasteur.fr.

ABSTRACT

Background: Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens. The SOS response is a regulatory network under control of the repressor protein LexA targeted at addressing DNA damage, thus promoting genetic variation in times of stress. We recently reported a direct link between the SOS response and the expression of integron integrases in Vibrio cholerae and a plasmid-borne class 1 mobile integron. SOS regulation enhances cassette swapping and capture in stressful conditions, while freezing the integron in steady environments. We conducted a systematic study of available integron integrase promoter sequences to analyze the extent of this relationship across the Bacteria domain.

Results: Our results showed that LexA controls the expression of a large fraction of integron integrases by binding to Escherichia coli-like LexA binding sites. In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons. There was a significant correlation between lack of LexA control and predicted inactivation of integrase genes, even though experimental evidence also indicates that LexA regulation may be lost to enhance expression of integron cassettes.

Conclusions: Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA. The data also indicated that SOS regulation has been actively preserved in mobile integrons and large chromosomal integrons, suggesting that unregulated integrase activity is selected against. Nonetheless, additional adaptations have probably arisen to cope with unregulated integrase activity. Identifying them may be fundamental in deciphering the uneven distribution of integrons in the Bacteria domain.

No MeSH data available.


Related in: MedlinePlus

Electrophoretic mobility-shift assay (EMSA) and quantitative real-time reverse transcription PCR on different intI genes and their respective promoters. (A) Vibrio parahaemolyticus integron. EMSA of V. parahaemolyticus intIA promoter with purified V. parahaemolyticus LexA protein. Competitive assays using either non-specific or Pint non-labeled DNA are also shown. The intA expression factor was calculated as the ratio of the relative intA mRNA concentration in the V. parahaemolyticus lexA mutant strain with respect to that in the wild type. (B) E. coli pMUR050 integrons. EMSA of pMUR050 intI1-and intI1+(containing GGG insertion) promoters with purified E. coli LexA protein. The expression factor for both intI genes was calculated as the ratio of each relative intI mRNA concentration in the E. coli lexA-sulA mutant strain with respect to that in the wild type. In all cases, the expression factor of recA is shown as a control, and all expression factors are the mean value from three independent experiments (each in triplicate).
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Figure 3: Electrophoretic mobility-shift assay (EMSA) and quantitative real-time reverse transcription PCR on different intI genes and their respective promoters. (A) Vibrio parahaemolyticus integron. EMSA of V. parahaemolyticus intIA promoter with purified V. parahaemolyticus LexA protein. Competitive assays using either non-specific or Pint non-labeled DNA are also shown. The intA expression factor was calculated as the ratio of the relative intA mRNA concentration in the V. parahaemolyticus lexA mutant strain with respect to that in the wild type. (B) E. coli pMUR050 integrons. EMSA of pMUR050 intI1-and intI1+(containing GGG insertion) promoters with purified E. coli LexA protein. The expression factor for both intI genes was calculated as the ratio of each relative intI mRNA concentration in the E. coli lexA-sulA mutant strain with respect to that in the wild type. In all cases, the expression factor of recA is shown as a control, and all expression factors are the mean value from three independent experiments (each in triplicate).

Mentions: We have previously shown that LexA regulates the expression of intI in V. cholerae, and our in silico search identified LexA binding sites in the promoter region of intI for all sequenced Vibrio species (see Additional file 1). To further assess the overall functionality of the in silico predicted LexA binding sites, we evaluated integrase LexA regulation in Vibrio parahaemolyticus strain ATCC 17802, which harbors a LexA binding site upstream of its intIA gene in a genomic context that is substantially different from that of V. cholerae (Figure 2A). Using quantitative reverse transcriptase (RT)-PCR, we determined the intIA expression level in both the wild-type strain and its lexA(Def) derivative (lacking a functional lexA gene). We found an expression ratio of 9.28, revealing a strong LexA regulation of the intIA gene expression (Figure 3A). Furthermore, electrophoretic mobility-shift assays (EMSA) with purified V. parahaemolyticus LexA protein showed that the observed upregulation of intIA expression was directly mediated by LexA in this organism (Figure 3A).


Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons.

Cambray G, Sanchez-Alberola N, Campoy S, Guerin E, Da Re S, González-Zorn B, Ploy MC, Barbé J, Mazel D, Erill I - Mob DNA (2011)

Electrophoretic mobility-shift assay (EMSA) and quantitative real-time reverse transcription PCR on different intI genes and their respective promoters. (A) Vibrio parahaemolyticus integron. EMSA of V. parahaemolyticus intIA promoter with purified V. parahaemolyticus LexA protein. Competitive assays using either non-specific or Pint non-labeled DNA are also shown. The intA expression factor was calculated as the ratio of the relative intA mRNA concentration in the V. parahaemolyticus lexA mutant strain with respect to that in the wild type. (B) E. coli pMUR050 integrons. EMSA of pMUR050 intI1-and intI1+(containing GGG insertion) promoters with purified E. coli LexA protein. The expression factor for both intI genes was calculated as the ratio of each relative intI mRNA concentration in the E. coli lexA-sulA mutant strain with respect to that in the wild type. In all cases, the expression factor of recA is shown as a control, and all expression factors are the mean value from three independent experiments (each in triplicate).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108266&req=5

Figure 3: Electrophoretic mobility-shift assay (EMSA) and quantitative real-time reverse transcription PCR on different intI genes and their respective promoters. (A) Vibrio parahaemolyticus integron. EMSA of V. parahaemolyticus intIA promoter with purified V. parahaemolyticus LexA protein. Competitive assays using either non-specific or Pint non-labeled DNA are also shown. The intA expression factor was calculated as the ratio of the relative intA mRNA concentration in the V. parahaemolyticus lexA mutant strain with respect to that in the wild type. (B) E. coli pMUR050 integrons. EMSA of pMUR050 intI1-and intI1+(containing GGG insertion) promoters with purified E. coli LexA protein. The expression factor for both intI genes was calculated as the ratio of each relative intI mRNA concentration in the E. coli lexA-sulA mutant strain with respect to that in the wild type. In all cases, the expression factor of recA is shown as a control, and all expression factors are the mean value from three independent experiments (each in triplicate).
Mentions: We have previously shown that LexA regulates the expression of intI in V. cholerae, and our in silico search identified LexA binding sites in the promoter region of intI for all sequenced Vibrio species (see Additional file 1). To further assess the overall functionality of the in silico predicted LexA binding sites, we evaluated integrase LexA regulation in Vibrio parahaemolyticus strain ATCC 17802, which harbors a LexA binding site upstream of its intIA gene in a genomic context that is substantially different from that of V. cholerae (Figure 2A). Using quantitative reverse transcriptase (RT)-PCR, we determined the intIA expression level in both the wild-type strain and its lexA(Def) derivative (lacking a functional lexA gene). We found an expression ratio of 9.28, revealing a strong LexA regulation of the intIA gene expression (Figure 3A). Furthermore, electrophoretic mobility-shift assays (EMSA) with purified V. parahaemolyticus LexA protein showed that the observed upregulation of intIA expression was directly mediated by LexA in this organism (Figure 3A).

Bottom Line: Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens.In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons.Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, Unité Plasticité du Génome Bactérien, CNRS URA 2171, 75015 Paris, France. mazel@pasteur.fr.

ABSTRACT

Background: Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens. The SOS response is a regulatory network under control of the repressor protein LexA targeted at addressing DNA damage, thus promoting genetic variation in times of stress. We recently reported a direct link between the SOS response and the expression of integron integrases in Vibrio cholerae and a plasmid-borne class 1 mobile integron. SOS regulation enhances cassette swapping and capture in stressful conditions, while freezing the integron in steady environments. We conducted a systematic study of available integron integrase promoter sequences to analyze the extent of this relationship across the Bacteria domain.

Results: Our results showed that LexA controls the expression of a large fraction of integron integrases by binding to Escherichia coli-like LexA binding sites. In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons. There was a significant correlation between lack of LexA control and predicted inactivation of integrase genes, even though experimental evidence also indicates that LexA regulation may be lost to enhance expression of integron cassettes.

Conclusions: Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA. The data also indicated that SOS regulation has been actively preserved in mobile integrons and large chromosomal integrons, suggesting that unregulated integrase activity is selected against. Nonetheless, additional adaptations have probably arisen to cope with unregulated integrase activity. Identifying them may be fundamental in deciphering the uneven distribution of integrons in the Bacteria domain.

No MeSH data available.


Related in: MedlinePlus