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A toxin-antitoxin system promotes the maintenance of an integrative conjugative element.

Wozniak RA, Waldor MK - PLoS Genet. (2009)

Bottom Line: We screened the entire 100 kb SXT genome and identified two genes within SXT, now designated mosA and mosT (for maintenance of SXT Antitoxin and Toxin), that promote SXT stability.These two genes, which lack similarity to any previously characterized genes, encode a novel toxin-antitoxin pair; expression of mosT greatly impaired cell growth and mosA expression ameliorated MosT toxicity.Thus, when the element is extrachromosomal and vulnerable to loss, SXT activates a TA module to minimize the formation of SXT-free cells.

View Article: PubMed Central - PubMed

Affiliation: Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
SXT is an integrative and conjugative element (ICE) that confers resistance to multiple antibiotics upon many clinical isolates of Vibrio cholerae. In most cells, this approximately 100 Kb element is integrated into the host genome in a site-specific fashion; however, SXT can excise to form an extrachromosomal circle that is thought to be the substrate for conjugative transfer. Daughter cells lacking SXT can theoretically arise if cell division occurs prior to the element's reintegration. Even though approximately 2% of SXT-bearing cells contain the excised form of the ICE, cells that have lost the element have not been detected. Here, using a positive selection-based system, SXT loss was detected rarely at a frequency of approximately 1 x 10(-7). As expected, excision appears necessary for loss, and factors influencing the frequency of excision altered the frequency of SXT loss. We screened the entire 100 kb SXT genome and identified two genes within SXT, now designated mosA and mosT (for maintenance of SXT Antitoxin and Toxin), that promote SXT stability. These two genes, which lack similarity to any previously characterized genes, encode a novel toxin-antitoxin pair; expression of mosT greatly impaired cell growth and mosA expression ameliorated MosT toxicity. Factors that promote SXT excision upregulate mosAT expression. Thus, when the element is extrachromosomal and vulnerable to loss, SXT activates a TA module to minimize the formation of SXT-free cells.

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Schematic of a positively selectable reporter of SXT loss.SXT containing lacIQ was introduced into a lacI E. coli host containing Plac-aad7 (a spectinomycin-resistance gene). If SXT (and lacIQ) is lost, the cells become SpecR. The black diamond represents the site of insertion for a fragment containing lacIQ (pFRTIq), thick black arrows represent ORFs, and the thin black line indicates the approximate location of the Δ9 deletion. Abbreviations: tet = tetracycline resistance gene, kan = kanamycin resistance gene, int = integrase, prfC = site of SXT insertion. Figure not to scale.
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pgen-1000439-g001: Schematic of a positively selectable reporter of SXT loss.SXT containing lacIQ was introduced into a lacI E. coli host containing Plac-aad7 (a spectinomycin-resistance gene). If SXT (and lacIQ) is lost, the cells become SpecR. The black diamond represents the site of insertion for a fragment containing lacIQ (pFRTIq), thick black arrows represent ORFs, and the thin black line indicates the approximate location of the Δ9 deletion. Abbreviations: tet = tetracycline resistance gene, kan = kanamycin resistance gene, int = integrase, prfC = site of SXT insertion. Figure not to scale.

Mentions: During serial passage of SXT-bearing strains without selection for SXT-encoded markers, we never detected cells that have lost this ICE, suggesting that SXT is stably maintained in the host population. Given the apparently low frequency of SXT loss, an E. coli reporter strain was designed to allow positive selection of rare cells that have lost SXT (Figure 1). To construct this strain, the Lac repressor, lacIQ, was introduced into an innocuous location in SXT (between traG and s079) while the native lacI was deleted from the chromosome. Additionally, a spectinomycin (Spec) resistance cassette under the control of the lac promoter was introduced into the gal locus in the chromosome (Figure 1). Therefore, in the presence of SXT (and therefore lacIQ), cells are sensitive to spectinomycin. If SXT (and lacIQ) are lost, cells become resistant to spectinomycin. As a result, even very rare cells lacking SXT can be detected as Spec-resistant colony forming units (cfu). Once Spec-resistant cfu are identified, they can subsequently be screened for the presence of SXT-encoded antibiotic resistances, such as chloramphenicol, to confirm SXT loss.


A toxin-antitoxin system promotes the maintenance of an integrative conjugative element.

Wozniak RA, Waldor MK - PLoS Genet. (2009)

Schematic of a positively selectable reporter of SXT loss.SXT containing lacIQ was introduced into a lacI E. coli host containing Plac-aad7 (a spectinomycin-resistance gene). If SXT (and lacIQ) is lost, the cells become SpecR. The black diamond represents the site of insertion for a fragment containing lacIQ (pFRTIq), thick black arrows represent ORFs, and the thin black line indicates the approximate location of the Δ9 deletion. Abbreviations: tet = tetracycline resistance gene, kan = kanamycin resistance gene, int = integrase, prfC = site of SXT insertion. Figure not to scale.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2654960&req=5

pgen-1000439-g001: Schematic of a positively selectable reporter of SXT loss.SXT containing lacIQ was introduced into a lacI E. coli host containing Plac-aad7 (a spectinomycin-resistance gene). If SXT (and lacIQ) is lost, the cells become SpecR. The black diamond represents the site of insertion for a fragment containing lacIQ (pFRTIq), thick black arrows represent ORFs, and the thin black line indicates the approximate location of the Δ9 deletion. Abbreviations: tet = tetracycline resistance gene, kan = kanamycin resistance gene, int = integrase, prfC = site of SXT insertion. Figure not to scale.
Mentions: During serial passage of SXT-bearing strains without selection for SXT-encoded markers, we never detected cells that have lost this ICE, suggesting that SXT is stably maintained in the host population. Given the apparently low frequency of SXT loss, an E. coli reporter strain was designed to allow positive selection of rare cells that have lost SXT (Figure 1). To construct this strain, the Lac repressor, lacIQ, was introduced into an innocuous location in SXT (between traG and s079) while the native lacI was deleted from the chromosome. Additionally, a spectinomycin (Spec) resistance cassette under the control of the lac promoter was introduced into the gal locus in the chromosome (Figure 1). Therefore, in the presence of SXT (and therefore lacIQ), cells are sensitive to spectinomycin. If SXT (and lacIQ) are lost, cells become resistant to spectinomycin. As a result, even very rare cells lacking SXT can be detected as Spec-resistant colony forming units (cfu). Once Spec-resistant cfu are identified, they can subsequently be screened for the presence of SXT-encoded antibiotic resistances, such as chloramphenicol, to confirm SXT loss.

Bottom Line: We screened the entire 100 kb SXT genome and identified two genes within SXT, now designated mosA and mosT (for maintenance of SXT Antitoxin and Toxin), that promote SXT stability.These two genes, which lack similarity to any previously characterized genes, encode a novel toxin-antitoxin pair; expression of mosT greatly impaired cell growth and mosA expression ameliorated MosT toxicity.Thus, when the element is extrachromosomal and vulnerable to loss, SXT activates a TA module to minimize the formation of SXT-free cells.

View Article: PubMed Central - PubMed

Affiliation: Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
SXT is an integrative and conjugative element (ICE) that confers resistance to multiple antibiotics upon many clinical isolates of Vibrio cholerae. In most cells, this approximately 100 Kb element is integrated into the host genome in a site-specific fashion; however, SXT can excise to form an extrachromosomal circle that is thought to be the substrate for conjugative transfer. Daughter cells lacking SXT can theoretically arise if cell division occurs prior to the element's reintegration. Even though approximately 2% of SXT-bearing cells contain the excised form of the ICE, cells that have lost the element have not been detected. Here, using a positive selection-based system, SXT loss was detected rarely at a frequency of approximately 1 x 10(-7). As expected, excision appears necessary for loss, and factors influencing the frequency of excision altered the frequency of SXT loss. We screened the entire 100 kb SXT genome and identified two genes within SXT, now designated mosA and mosT (for maintenance of SXT Antitoxin and Toxin), that promote SXT stability. These two genes, which lack similarity to any previously characterized genes, encode a novel toxin-antitoxin pair; expression of mosT greatly impaired cell growth and mosA expression ameliorated MosT toxicity. Factors that promote SXT excision upregulate mosAT expression. Thus, when the element is extrachromosomal and vulnerable to loss, SXT activates a TA module to minimize the formation of SXT-free cells.

Show MeSH
Related in: MedlinePlus