An operon of Bacillus subtilis motility genes transcribed by the sigma D form of RNA polymerase.

Two genes controlling motility functions in Bacillus subtilis were identified by DNA sequence analysis of a chromosomal fragment containing a strong promoter for sigma D RNA polymerase. Previous studies had shown that this sigma D-dependent promoter controls synthesis of a 1.6-kb transcript in vivo and in vitro. Sequence analysis revealed that the 1.6-kb transcript contains two open reading frames coding for protein sequences homologous to the Escherichia coli motA and motB gene products, respectively, and ends in a rho-independent termination site. Direct evidence linking these genes to motility functions in B. subtilis was obtained by precise localization by polymerase chain reaction of Tn917 transposon insertion mutations of Mot- strains, isolated by Zuberi et al. (A. R. Zuberi, C. Ying, H. M. Parker, and G. W. Ordal, J. Bacteriol. 172:6841-6848, 1990), to within this mot. operon. Replacement of each wild-type gene by in-frame deletion mutations yielded strains possessing paralyzed flagella and confirmed that both motA and motB are required for the motility of B. subtilis. These current findings support our earlier suggestions that sigma D in B. subtilis plays a central role in the control of gene expression for flagellar assembly, chemotaxis, and motility functions. Sigma F, the enteric homolog of sigma D, controls similar functions in E. coli and Salmonella typhimurium, and these factors appear to be representative of a family of factors implicated in flagellar synthesis in many bacterial species, which we propose to designate the sigma 28 family.

Direct sequencing of terminal regions of genomic P1 clones. A general strategy for the design of sequence-tagged site markers.

A method for the preparation of P1 DNA is presented, which allows the direct sequencing of ends of inserts in genomic P1 clones using the Applied Biosystems 373A DNA Sequencer and the Dye Terminator sequencing methodology. We surveyed several common methods of DNA preparation including alkaline lysis, Triton-lysozyme lysis, CsCl density-gradient purification, and a commercial column matrix DNA purification kit manufactured by Qiagen. We found that a modified alkaline lysis preparation of P1 DNA was most successful for generating P1 DNA that could be sequenced directly. We also noted that the host bacterial strain from which the P1 DNA was purified dramatically affected the quality of sequencing templates. The bacterial strains NS3145 and NS3529, in which the Drosophila melanogaster and human P1 genomic libraries are harbored, routinely yielded poor-quality sequencing templates. However, the bacterial strain DH10B routinely yielded P1 DNA that was sequenced successfully. A bacterial mating scheme is presented that exploits gamma delta transposition events to allow the transfer of P1 clones from the library host strain to DH10B. Using either an SP6 or a T7 primer, an average of 350 base pairs of DNA sequence was obtained with an uncalled base frequency of approximately 2%. About 4% of P1 end sequences generated corresponded to unique Drosophila loci present in the Genbank database. These single-pass DNA sequences were used to design sequence-tagged site markers for physical mapping studies in both humans and Drosophila.