The spoOA gene of Bacilus subtilis is critical for the initial stages in the developmental cycle leading to the formation of an endospore. We show that one function of the SpoOA protein is to negatively regulate another regulatory locus, abrB, which controls the expression of many genes associated with the onset of sporulation. Purified SpoOA protein binds to a specific region of the abrB promoter and functions as a repressor of transcription in an in vitro assay. The binding of the SpoOA protein is independent of the binding of the AbrB protein, which is known to autoregulate its expression. This independence mirrors the temporal sequence of events in abrB control.The products of the Bacillus subtilis spoO genes are required for the initiation of sporulation in this organism (1, 2). Mutations of spoO in B. subtilis block sporulation at its earliest stage and exhibit pleiotropic effects on the expression of a wide variety of gene products associated with the end of exponential growth and the onset of sporulation: the transition stage (1, 3). The most pleiotropic of the spoO mutations occur at the spoOA locus.It is believed that SpoOA is the main component through which the environmental signals for sporulation are relayed to the transcriptional machinery of the cell (4, 5). The spoOA gene codes for a protein of 29,700 Da (6), which exhibits amino acid sequence homology with the regulator class of molecule (e.g., OmpR, NtrC, CheB, CheY, etc.) in prokaryotic two-component regulatory systems (7-10). The SpoOA protein regulates the onset of stationary phase and sporulation, in part, by controlling the expression of the abrB gene. The product of the abrB gene is a repressor that functions to prevent the expression of transition stage-specific genes during vegetative growth (11,12). Among the genes regulated by abrB are some that are necessary for normal sporulation: spoOE (5), spoOH (13), and spoVG (14). During the transition stage, production of the AbrB protein decreases, thus allowing for the derepression of promoters regulated by AbrB (11). The modulation of AbrB synthesis depends upon the SpoOA protein (11,15).Although a large body of genetic evidence implicated the SpoOA protein in regulatory phenomena occurring at the onset of sporulation, the actual biochemical function of the SpoOA protein was unknown. In this communication we show that the SpoOA protein binds to a specific region of the abrB gene located downstream from the transcriptional start sites and functions as a repressor of transcription in vitro. MATERIALS AND METHODSDNase and Methylation Protection Assays. The 800-base-pair (bp) BamHI-HindIII fragment of pJM5134 (11) containing the abrB promoter and upstream region was labeled at the BamHI end by using either [a-32P] (12) with labeled DNA at 1-3 x 10-9 M final concentration and highly purified SpoOA protein (see below). The method used for determining which purine (both adenine and guanine) residues were protected from methylation by dimethyl sulfate due to binding of SpoOA was as follow...

The ability of bacteria to acquire and disseminate heterologous genes has been a major factor in the development of multiple drug resistance. A gene, intI4, was identified that encodes a previously unknown integrase that is associated with a "gene-VCR" organization (VCRs are Vibrio cholerae repeated sequences), similar to that of the well-characterized antibiotic resistance integrons. The similarity was confirmed by IntI1-mediated recombination of a gene-VCR cassette into a class 1 integron. VCR cassettes are found in a number of Vibrio species including a strain of V. metschnikovii isolated in 1888, suggesting that this mechanism of heterologous gene acquisition predated the antibiotic era.

The 72 Escherichia coli strains of the ECOR collection were examined for resistance to 10 different antimicrobial agents including ampicillin, tetracycline, mercury, trimethoprim, and sulfonamides. Eighteen strains were resistant to at least one of the antibiotics tested, and nearly 20% (14 of 72) were resistant to two or more. Several of the resistance determinants were shown to be carried on conjugative elements. The collection was screened for the presence of the three classes of integrons and for the sul1 gene, which is generally associated with class 1 integrons. The four strains found to carry a class 1 integron also had Tn21-encoded mercury resistance. One of the integrons encoded a novel streptomycin resistance gene, aadA7, with an attC site (or 59-base element) nearly identical to the attC site associated with the qacF gene cassette found in In40 (M. -

Fluorescence measurements and polymerase chain reaction amplification of streptomycete 16S ribosomal DNA sequences were used to show that a number of antibiotic preparations employed for human and animal use are contaminated with chromosomal DNA of the antibiotic-producing organism. The DNA contains identifiable antibiotic resistance gene sequences; the uptake of this DNA by bacteria and its functional incorporation into bacterial replicons would lead to the generation of antibiotic resistance determinants. We propose that the presence of DNA encoding drug resistance in antibiotic preparations has been a factor in the rapid development of multiple antibiotic resistance in bacteria.