A new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms - PubMed (original) (raw)

A new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms

Marc Marenda et al. J Bacteriol. 2006 Jun.

Abstract

An integrative conjugative element, ICEA, was characterized in Mycoplasma agalactiae strain 5632, in which it occurs as multiple chromosomal copies and as a free circular form. The distribution of ICEA sequences in M. agalactiae strains and their occurrence in Mycoplasma bovis suggest the spreading of the element within or between species.

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Figures

FIG. 1.

FIG. 1.

Genetic organization of ICEs characterized in M. agalactiae and M. fermentans. (A) Restriction map and genetic organization of the ICEA5632-I locus of M. agalactiae strain 5632. E, EcoRI; V, EcoRV; X, XbaI; H, HindIII. Diamonds indicate the ICE termini; gray arrows, ORFs found in both ICEA5632-I and ICEF; black arrows, CDSs found only in ICEA5632-I. The locations of the specific probes (horizontal bars) and of the PCR primers (filled or open arrowheads) used in this study are indicated below the ICEA5632-I locus representation. (B) ICEF versions previously described for M. fermentans strain PG18. White arrows, ICEF-specific CDSs; hatched arrows, locus-variable ICEF-specific CDSs.

FIG. 2.

FIG. 2.

Occurrence of M. agalactiae ICEA5632-I as an integrated or free circular form. (A) The integrated ICEA5632-I (solid line) and its flanking chromosomal regions (double gray line) are represented along with relevant CDSs (arrows) and PCR primers (arrowheads). Filled and open diamonds, ICEA5632-I left and right termini, respectively. Inverted-repeat (boldfaced letters) and direct-repeat sequences of motifs I, II, and III are given above the diagram, with the numbers of nucleotides separating the repeats given in parentheses. (B) Proposed configuration of the ICEA5632-I circular form. The black-and-white diamond represents the coupling sequence resulting from the reassociation of the left and right repeats of motif II. (C) ICEA5632-I-specific PCR assays analyzed on a 1% agarose gel. MW, molecular weight marker. C, negative control. Primer pairs: Right2/Left1 (lane 1), Right2/5′-1-R (lane 2), 3′-22-F/Left1 (lane 3). (D) Proposed configuration of the chromosomal locus following excision of ICEA5632-I. The sequence given above the diagram corresponds to part of the sequence of a PCR product using OL-1 and OR2 as primers and 5632 total DNA as the template. Asterisk indicates the motif II unique sequence resulting from the excision.

FIG. 3.

FIG. 3.

Southern blot analyses showing the presence of several ICEA5632 chromosomal copies in M. agalactiae strain 5632. The enzymes used to digest the genomic DNA are indicated on top of each panel: E, EcoRI; V, EcoRV; X, XbaI; H, HindIII. MW, Dig-labeled lambda HindIII DNA. Membranes were hybridized with either a CDS1-specific probe (A) or a CDS22-specific probe (B); the membrane from panel B was stripped and rehybridized with the RFR probe (C). Stars indicate the EcoRV DNA fragments that reacted with both the CDS22 and RFR probes. Dots indicate the fragment displaying the expected size and corresponding to the ICEA5632-I locus sequenced in this study.

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