Fig 1.
Genetic maps of ICEBs1, Tn916, and hybrid elements.
Maps are shown of the ICEs used in these experiments: A) Tn916; B) ICEBs1; C) (ICEBs1-Tn916)-H1 D) (ICEBs1-Tn916)-H2. Attachment sites attL and attR are indicated by black boxes (ICEBs1, (ICEBs1-Tn916)-H1, and (ICEBs1-Tn916)-H2 are all integrated at trnS-leu2; Tn916 is integrated between yufK and yufL in donor cells. Open reading frames are indicated by horizontal arrows, pointing in the direction of transcription (gray for Tn916, black for ICEBs1). Gene names are located below the depicted open reading frame. Tn916 gene names are abbreviated to include only the number designation from the gene name (i.e., “orf23” is written as “23”), and the corresponding ICEBs1 homolog gene name is written in parentheses below, when appropriate. ardA of Tn916 encodes an anti-restriction protein [71]. Confirmed and putative promoters are indicated by bent arrows, putative transcription terminators in Tn916 are indicated by “T” shapes. The current model of transcriptional regulation of Tn916 (A) is adapted from [4]. Previously determined origins of transfer (oriT) and single strand origins of replication (sso) are indicated by a “-”above the genetic map [14,21,50,93].
Table 1.
Excision frequencies and conjugation efficiencies of ICEs.
Table 2.
Coupling proteins can recognize non-cognate relaxosome.
Fig 2.
Element conjugation efficiencies are dependent on recipient species.
Bacillus subtilis donors contained Tn916 (ELC1566); ICEBs1 (ELC1795); (ICEBs1-Tn916)-H1 (ELC1722); or (ICEBs1-Tn916)-H2 (ELC1725). Donors were D-alanine auxotrophs (alr::cat) for counter-selection of transconjugants during mating assays. ICEBs1, H1, and H2 donors also contained amyE::[(Pspank(hy)-rapI) spc] for IPTG-inducible overproduction of RapI to de-repress element gene expression that leads to excision (activation). Donor cells were grown to exponential phase in LB medium; IPTG or tetracycline was added as appropriate to stimulate element activation. Donors were mixed with recipients that had been grown to exponential phase in LB (B. subtilis: CAL419) or BHI (E. faecalis, E. caccae, and E. durans). Mixed cells were filtered and placed on a solid surface for one hour. Conjugation efficiencies were calculated as the number of transconjugants (tetR, D-alanine prototrophs) produced, normalized to the number of donors. Bars indicated the mean of 3 independent mating assays ± SEM.
Table 3.
Bacillus subtilis strains.