A toxin-antitoxin system promotes the maintenance of an integrative conjugative element - PubMed (original) (raw)

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

Rachel A F Wozniak et al. PLoS Genet. 2009 Mar.

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.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. 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.

Figure 2

Figure 2. Influence of genetic factors on the frequency of SXT loss.

The frequency of SXT loss was calculated as the ratio of SpecRCmS cfu / total cfu after 15 hour of growth. A) Factors that influence the frequency of SXT excision alter the frequency of SXT loss. The black diamonds indicate that the result is statistically different (p<0.05) than the result shown in the first column for the wild type reporter. The * signifies that the result was below the limit of detection which was ∼1×10−8. pXis and pXis-R harbor arabinose-inducible xis or its reverse complement respectively. B) SXT loss is not accompanied by heritable changes that influence maintenance of SXT. The ‘current SXT genotype’ refers to the ICE introduced into the host that lost the ICE designated as ‘previous SXT genotype’.

Figure 3

Figure 3. 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.

Figure 4

Figure 4. MosT inhibits E. coli growth and its toxicity can be neutralized by MosA.

Growth kinetics of E. coli strains CAG18439 (A and B), CAG18439 containing mosAT SXT (C) and CAG18439 containing wild type SXT (D). These strains, which harbored arabinose-inducible mosT and mosA, (pMosT or pMosA respectively) or control vectors, (pBAD33 and pBAD18), were grown in either 0.2% glucose (solid lines) or 0.02% arabinose (dashed lines).

Figure 5

Figure 5. 5′RACE analysis of mosA transcription start site.

A) Schematic of mosA transcription start site based on the 5′ RACE results shown in B). The +1 refers to the start of transcription as defined by the DNA sequence of the 5′RACE product obtained using primer A (small black arrow). The length of the PCR product is indicated. Black arrows represent ORFs as predicted using bioinformatics; the previously annotated s052 start codon is indicated. B) Shows a 1% agarose gel of the product of 5′RACE reaction using primer A.

Figure 6

Figure 6. Influence of Xis and SetCD on mosA expression.

A) Schematic of chromosomal mosA:: lacZ transcription reporter within the mosAT locus. Thick black arrows represent ORFs as predicted by bioinformatics and the thin arrow represents the predicted location of the mosA promoter. B, D) β-galactosidase activities of the chromosomal mosA::lacZ fusion in CAG18439 containing wt SXT (B) or in CAG18439 containing Δint SXT (D) along with the indicating expression vectors. C) β-galactasidase activities from a plasmid-borne mosA-lacZ fusion (in pPmosA) in the indicated strains. All β-galactasidase measurements were conducted on 15 hr cultures and the results shown are the means and standard deviation from at least 9 independent cultures.

Similar articles

Cited by

References

    1. Burrus V, Waldor MK. Shaping bacterial genomes with integrative and conjugative elements. Res Microbiol. 2004;155:376–386. - PubMed
    1. Churchward G. Back to the future: the new ICE age. Mol Microbiol. 2008;70:554–556. - PubMed
    1. Qiu X, Gurkar AU, Lory S. Interstrain transfer of the large pathogenicity island (PAPI-1) of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2006;103:19830–19835. - PMC - PubMed
    1. Auchtung JM, Lee CA, Monson RE, Lehman AP, Grossman AD. Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response. Proc Natl Acad Sci U S A. 2005;102:12554–12559. - PMC - PubMed
    1. Pavlovic G, Burrus V, Gintz B, Decaris B, Guedon G. Evolution of genomic islands by deletion and tandem accretion by site-specific recombination: ICESt1-related elements from Streptococcus thermophilus. Microbiology. 2004;150:759–774. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources