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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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Genetic analysis of loss of mosT SXT.A) Schematic of the region deleted from Δ9 SXT. Black arrows represent ORFs, thin black arrows represent the deletions studied below. B) Frequency of loss of the indicated mutant SXT. C) Influence of mosT expression in trans on the stability of mosT SXT. All cultures were grown in the presence of 0.02% arabinose for 15 hr. Black diamond represents a statistically significant (p<.05) result compared to loss in the presence of pBAD33. D) Loss of mosT SXT is influenced by factors affecting SXT excision. All cultures were grown for 15 hr except as noted. Black diamonds represent a statistically significant (p<0.05) result compared to the mosT SXT grown for 15 hr. The * signifies that the result was below the limit of detection which was ∼1×10−8. aLoss was calculated following 3 hours of growth in an early log phase culture.
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pgen-1000439-g003: Genetic analysis of loss of mosT SXT.A) Schematic of the region deleted from Δ9 SXT. Black arrows represent ORFs, thin black arrows represent the deletions studied below. B) Frequency of loss of the indicated mutant SXT. C) Influence of mosT expression in trans on the stability of mosT SXT. All cultures were grown in the presence of 0.02% arabinose for 15 hr. Black diamond represents a statistically significant (p<.05) result compared to loss in the presence of pBAD33. D) Loss of mosT SXT is influenced by factors affecting SXT excision. All cultures were grown for 15 hr except as noted. Black diamonds represent a statistically significant (p<0.05) result compared to the mosT SXT grown for 15 hr. The * signifies that the result was below the limit of detection which was ∼1×10−8. aLoss was calculated following 3 hours of growth in an early log phase culture.

Mentions: However, deletion of the 8 genes in the Δ9 mutant, which spans the region from s052 to traN, resulted in growth of markedly more colonies that lacked SXT (∼1×10−4), than observed to lack wt SXT (∼1×10−7) (Table 3 and Figure 3B). Five of the eight genes (see Figure 3A) in this deletion (traC, trsF, traW, traU, traN) are important for conjugative transfer of SXT. s054 encodes a homologue of a disulfide bond isomerase-related protein and s052 and s053 are currently classified as hypothetical genes with no known function, characterized homologues, or recognizable motifs. We constructed smaller deletions within the s052-traN region to pinpoint those gene(s) that account for the increased loss observed for the Δ9 mutant. As expected, ΔA, which only removed genes implicated in conjugative transfer, did not alter SXT stability (Figure 3B); deletion of s054 did not influence SXT stability either. However ΔB, which contains s052 and s053, resulted in a similar frequency of loss as the Δ9 mutant. Finally, deletion of s053 alone was sufficient to recapitulate the Δ9 phenotype (Figure 3B). It is important to note that while s052 and s053 could be deleted together, s052 could not be deleted in the presence of s053. From this point on, for reasons that will become clear below, s052 and s053 are referred to as mosA and mosT, respectively, for maintenance of SXT (Antitoxin, Toxin).


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

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

Genetic analysis of loss of mosT SXT.A) Schematic of the region deleted from Δ9 SXT. Black arrows represent ORFs, thin black arrows represent the deletions studied below. B) Frequency of loss of the indicated mutant SXT. C) Influence of mosT expression in trans on the stability of mosT SXT. All cultures were grown in the presence of 0.02% arabinose for 15 hr. Black diamond represents a statistically significant (p<.05) result compared to loss in the presence of pBAD33. D) Loss of mosT SXT is influenced by factors affecting SXT excision. All cultures were grown for 15 hr except as noted. Black diamonds represent a statistically significant (p<0.05) result compared to the mosT SXT grown for 15 hr. The * signifies that the result was below the limit of detection which was ∼1×10−8. aLoss was calculated following 3 hours of growth in an early log phase culture.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000439-g003: Genetic analysis of loss of mosT SXT.A) Schematic of the region deleted from Δ9 SXT. Black arrows represent ORFs, thin black arrows represent the deletions studied below. B) Frequency of loss of the indicated mutant SXT. C) Influence of mosT expression in trans on the stability of mosT SXT. All cultures were grown in the presence of 0.02% arabinose for 15 hr. Black diamond represents a statistically significant (p<.05) result compared to loss in the presence of pBAD33. D) Loss of mosT SXT is influenced by factors affecting SXT excision. All cultures were grown for 15 hr except as noted. Black diamonds represent a statistically significant (p<0.05) result compared to the mosT SXT grown for 15 hr. The * signifies that the result was below the limit of detection which was ∼1×10−8. aLoss was calculated following 3 hours of growth in an early log phase culture.
Mentions: However, deletion of the 8 genes in the Δ9 mutant, which spans the region from s052 to traN, resulted in growth of markedly more colonies that lacked SXT (∼1×10−4), than observed to lack wt SXT (∼1×10−7) (Table 3 and Figure 3B). Five of the eight genes (see Figure 3A) in this deletion (traC, trsF, traW, traU, traN) are important for conjugative transfer of SXT. s054 encodes a homologue of a disulfide bond isomerase-related protein and s052 and s053 are currently classified as hypothetical genes with no known function, characterized homologues, or recognizable motifs. We constructed smaller deletions within the s052-traN region to pinpoint those gene(s) that account for the increased loss observed for the Δ9 mutant. As expected, ΔA, which only removed genes implicated in conjugative transfer, did not alter SXT stability (Figure 3B); deletion of s054 did not influence SXT stability either. However ΔB, which contains s052 and s053, resulted in a similar frequency of loss as the Δ9 mutant. Finally, deletion of s053 alone was sufficient to recapitulate the Δ9 phenotype (Figure 3B). It is important to note that while s052 and s053 could be deleted together, s052 could not be deleted in the presence of s053. From this point on, for reasons that will become clear below, s052 and s053 are referred to as mosA and mosT, respectively, for maintenance of SXT (Antitoxin, Toxin).

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