Escherichia coil azi mutants, whose growth is resistant to millimolar concentrations of sodium azide, were among the earliest E. coil mutants isolated. Genetic complementation, mapping, and DNA sequence analysis now show that these mutations are alleles of the secA gene, which is essential for protein export across the E. coil plasma membrane. We have found that sodium azide is an extremely rapid and potent inhibitor of protein export in vivo and that azi mutants are more resistant to such inhibition. Furthermore, SecA-dependent in vitro protein translocation and ATPase activities are inhibited by sodium azide, and SecA protein prepared from an azi mutant strain is more resistant to such inhibition. These studies point to the utility ofspecific inhibitors of protein export, such as sodium azide, in facilitating the dissection of the function of individual components of the protein export machinery.

LuxR, the Vibno fischeri luminescence gene (lux) activator, is the best-studied member ofa family of bacterial transcription factors required for cell density-dependent expression of specific genes involved in associations with eukaryotic hosts. Neither LuxR nor any other LuxR homolog has been shown to bind DNA directly. We have purified the LuxR C-terminal transcriptional activator domain from extracts of recombinant Escherichia coli in which this polypeptide was expressed. The purified polypeptide by itself binds to lux regulatory DNA upstream of the lux box, a 20-bp palindrome that is required for LuxR activity in vivo, but it does not bind to the lux box. However, the LuxR C-terminal domain together with RNA polymerase protects a region including the lux box and the lux operon promoter from DNase I cleavage. There is very little protection of the lux operon promoter region from DNase I digestion in the presence of RNA polymerase alone. Apparently, there is a synergistic binding of the LuxR C-terminal domain and RNA polymerase to the promoter region LuxR-facilitated autoinduction controls transcription of luminescence genes in Vibriofischeri. LuxR homologs occur in a number of different Gram-negative -bacteria, and these transcription factors are involved in a phenomenon termed quorum sensing and response (for recent reviews, see refs.

In Escherichia coli, transcription of the inducible Vibrio fischeri luminescence operon, luxICDABE, has been reported to require sigma 32, the product of rpoH. Consistent with previous studies, we report that an E. coli delta rpoH mutant, KY1601 containing luxICDABE and luxR, which codes for the activator of luxICDABE transcription on a plasmid (pJE202), was weakly luminescent. Transformation of this E. coli strain with a plasmid containing rpoH under the control of the tac promoter resulted in high levels of cellular luminescence. However, the level of expression of the pJE202 luxICDABE was also high in E. coli 1603, a delta rpoH mutant with a second-site mutation that resulted in sigma 32-independent overexpression of the groE operon. Apparently, sigma 32 is not directly required for the transcription of luxICDABE in E. coli but is required for sufficient expression of groE, which is in turn required for the transcription of luxICDABE. This conclusion is supported by the finding that E. coli groE mutants containing pJE202 were weakly luminescent. In the E. coli delta rpoH mutant KY1601, the sigma 32 requirement for the transcription of luxICDABE was partially compensated for by the addition of saturating concentrations of the inducer to the culture medium and largely compensated for when cells were transformed with a luxR overexpression vector. These data support the hypothesis that sigma 32 is not required for transcription of luxICDABE. Rather, it appears that the products of groE are required for the folding of LuxR into an active protein, like they are for the folding of several other proteins.

Genetic and biochemical studies have shown that the product of the Escherichia coli secY gene is an integral membrane protein with a central role in protein secretion. We found the Bacillus subtilis secY homologue within the spc-alpha ribosomal protein operon at the same position occupied by E. coli secY. B. subtilis secY coded for a hypothetical product 41% identical to E. coli SecY, a protein thought to contain 10 membrane-spanning segments and 11 hydrophilic regions, six of which are exposed to the cytoplasm and five to the periplasm. We predicted similar segments in B. subtilis SecY, and the primary sequences of the second and third cytoplasmic regions and the first, second, fourth, fifth, seventh, and tenth membrane segments were particularly conserved, sharing greater than 50% identity with E. coli SecY. We propose that the conserved cytoplasmic regions interact with similar cytoplasmic secretion factors in both organisms and that the conserved membrane-spanning segments actively participate in protein export. Our results suggest that despite the evolutionary differences reflected in cell wall architecture, Gram-negative and Gram-positive bacteria possess a similar protein export apparatus.