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Journal of Bacteriology, December 2008, p. 7739-7753, Vol. 190, No. 23
0021-9193/08/$08.00+0     doi:10.1128/JB.00361-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetic Analysis of the Enterococcus Vancomycin Resistance Conjugative Plasmid pHTβ: Identification of the Region Involved in Cell Aggregation and traB, a Key Regulator Gene for Plasmid Transfer and Cell Aggregation ^,

Haruyoshi Tomita^1* and Yasuyoshi Ike^1,2

Department of Bacteriology, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan,¹ Laboratory of Bacterial Drug Resistance, Gunma University Graduates School of Medicine, Maebashi, Gunma 371-8511, Japan²

Received 12 March 2008/ Accepted 22 September 2008

The Enterococcus plasmid pHTβ (63.7 kbp) is a pheromone-independent, highly conjugative pMG1-like plasmid that carries a Tn1546-like transposon encoding vancomycin resistance. The transfer-related regions (Tra I, Tra II, and Tra III) containing oriT and a putative nickase gene (traI) have previously been identified in pHTβ, and in this study, we found that the plasmid conferred the ability to self-aggregate on the host strain Enterococcus faecalis FA2-2. A region where mutation resulted in the impairment of aggregation was identified and mapped to a point upstream of the transfer-related Tra I region. This region consisted of an approximately 6-kbp segment that contained the five open reading frames (ORFs) ORF9 to ORF13. These ORFs are considered to encode the aggregation function, although the precise mode of action of each ORF has not yet been elucidated. An in-frame deletion mutant of ORF10 resulted in reduced aggregation and decreased transfer frequency in broth mating. Transcription analysis of the aggregation region showed that the five ORFs from ORF9 to ORF13 form an operon structure, and a long transcript that started from a promoter region located upstream of ORF9 was identified. Tra II spans a 1.7-kbp region containing ORF56 and ORF57. Tn917-lac insertions into or an in-frame deletion mutant of ORF56 (187 amino acids) resulted in impaired transfer and aggregation. The cloned ORF56 complemented these functions in trans. The transcription levels of ORF10 and ORF13 were reduced in the in-frame mutants of ORF56, but this reduction was complemented by a cloned ORF56 in trans. The results indicated that ORF56 positively regulated the aggregation and plasmid transfer in the host strain, and ORF56 was designated traB.

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* Corresponding author. Mailing address: Department of Bacteriology, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan. Phone: (81) 27 220 7992. Fax: (81) 27 220 7996. E-mail: tomitaha{at}med.gunma-u.ac.jp

Published ahead of print on 3 October 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, December 2008, p. 7739-7753, Vol. 190, No. 23
0021-9193/08/$08.00+0     doi:10.1128/JB.00361-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.