A family of shuttle plasmids was constructed for genetic transformation of <i>Escherichia coli</i> and of ruminal <i>Bacteroides </i>strains AR20 and AR29. Plasmids were based on the replicon from <i>Bacteroides</i> plasmid pBI191 and were designed for studies of chromosomal integration (pBA), for the identification and study of <i>Bacteroides</i> gene promoters (pPPR) and for the expression of heterologous genes in <i>Bacteroides</i> (pBAC). Electroporation efficiency of <i>Bacteroides</i> was up to 10<sup>5</sup> transformants/µg plasmid, depending on the source of the DNA. The largest plasmid, pBA, was maintained at approximately 8 copies per cell in AR20 and did not measurably alter in vitro growth of transformed cells. In the current work, pBA did not integrate into the chromosomes of AR20 or AR29. The ability of plasmid pPPR to select promoter sequences was demonstrated by removal and replacement of promoters that activate the clindamycin resistance gene. The suitability of pBAC for expression of heterologous genes was demonstrated by expression of the <i>Moraxella species</i> fluoroacetate dehalogenase gene H1 to give intracellular activity of 7 nmol fluoride released/min/mg soluble protein in AR20 and 4 nmol/min/mg in AR29. Spontaneous loss of pBAC under non-selective conditions was 0.11–0.165% per generation, significantly less than loss of the native <i>Bacteroides</i> plasmid pBI191, which was lost at 0.53% per generation.
