Identification and cloning of the conjugative transfer region of Staphylococcus aureus plasmid pGO1

Abstract

The conjugative transfer (tra) genes of a 52-kilobase (kb) staphylococcal plasmid, pGO1, were localized by deletion analysis and transposon insertional inactivation. All transfer-defective (Tra-) deletions and TnS51 or Tn917 transposon insertions occurred within a 14.5-kb BglII fragment. Deletions and insertions outside this fragment all left the plasmid transfer proficient (Tra'). The tra region was found to be flanked by directly repeated DNA sequences, approximately 900 base pairs in length, at either end. Clones containing the 14.5-kb BgllI fragment (pGO200) and subclones from this fragment were constructed in Escherichia coli on shuttle plasmids and introduced into Staphylococcus aureus protoplasts. Protoplasts could not be transformed with pGO200E (pGO200 on the staphylococcal replicon, pE194) or subclones containing DNA at one end of the tra fragment unless pGO1 or specific cloned tra DNA fragments were present in the recipient cell. However, once stabilized by sequences present on a second replicon, each tra fragment could be successfully introduced alone into other plasmid-free S. aureus recipients by conjugative mobilization or transduction. In this manner, two clones containing overlapping fragments comprising the entire 14.5-kb BglII fragment were shown to complement each other. The low-frequency transfer resulted in transconjugants containing one clone intact, deletions of that clone, and recombinants of the two clones. The resulting recombinant plasmid (pGO220), which regenerated the tra region intact on a single replicon, transferred at frequencies comparable to those of pGO1. Thus, all the genes necessary and sufficient for conjugative transfer of pGO1 are contained within a 14.5-kb region of DNA.Large, 40-to 60-kilobase (kb) conjugative plasmids have been found in both Staphylococcus aureus and S. epidermidis (3,11,24). Conjugative transfer of these plasmids has the following characteristics: transfer occurs at a low frequency The 52-kb conjugative plasmid pGO1, originally resident in a clinical S. aureus isolate at the Medical College of Virginia Hospital, has all of the transfer characteristics summarized above, encodes Gmr, Tpr, and Qamr, and has both restriction fragment similarity and DNA hybridization homology to conjugative plasmids isolated from our own hospital (2) as well as to plasmids from the University of * Corresponding author.Michigan (pAM899-1) and Creighton University (pCRG1600) (G. Archer, unpublished observations). Thus, this plasmid serves as a model for the study of conjugative staphylococcal plasmid tra genes. As a first step toward a detailed genetic and functional analysis of the tra region of these plasmids, in this report we describe a determination of the physical and functional boundary of the genes encoding transfer functions on pGO1. In addition, tra insertion and deletion mutants and subclones of the tra region on S. aureus-Escherichia coli shuttle vectors were constructed to aid in future studies of the molecular basis for the conjugative transfer of antimicrob...

SarT Influences sarS Expression in Staphylococcus aureus

“…The sarT mutant of S. aureus SH1000 was generated by transducing strain SH1000 with an 80␣ phage lysate of S. aureus ALC2313 as previously described (14,50). To confirm the genotype, chromosomal DNA from S. aureus cultures grown overnight was isolated by lysostaphin lysis and phenol extraction as described previously (14).…”

Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements

“…Exploiting the conditional function of this replicon, transposon mutants can be selected after inducing the loss of the delivery system. By this approach, transposon Tn917 and its derivatives have been successfully used in other gram-positive organisms, including species in the genera Lactococcus (23), Clostridium (1), Listeria (4,9), Staphylococcus (6,48), and Enterococcus (8) (in which it was first discovered [51]). However, success in the generation of mutants of S. mutans and other streptococcal species with pTV1Ts and derivatives have not been reported, indicating that the pE194 origin of replication may not promote efficient propagation and thus mutagenesis in this genus.…”

Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1

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