In prsA (protein secretion) mutants of Bacillus subtilis, decreased levels of exoproteins, including alpha-amylase and subtilisins, are found extracellularly. The effect of prsA on subtilisin secretion is elaborated here. Extracytoplasmic folding and secretion of active subtilisin is assisted by the N-terminal pro-sequence of its precursor. In this paper we present evidence that the product of the prsA gene is additionally required for these processes in vivo. We examined inducible expression of different subtilisin-alkaline phosphatase fusion genes in the prsA3 mutant. We found massive degradation of the fusion proteins, and a lack of enzymatic activity in the protein secreted. We suggest that PrsA is a novel chaperone with a predicted extracytoplasmic location, and is important in vivo for the proper conformation of various exoproteins, including those with pro-sequence (like subtilisin) and those without (like alpha-amylase).

The gene encoding subtilisin Carlsberg from Bacillus licheniformis has been isolated by molecular cloning using a mixture of synthetic oligonucleotides. The entire nucleotide sequence of the coding sequence as well as 5' and 3' flanking sequences have been determined. The deduced amino acid sequence reveals an N-terminal signal peptide consisting of 29 residues, a pro-peptide of 76 residues followed by the mature protein comprising 274 residues. The ATG initiator codon is preceded by two putative overlapping ribosomal binding sequences. A palindromic sequence typical for transcription termination is found downstream from the TAA stop codon. Structural comparisons between different known subtilisin genes reveal extensive homology, particularly in the parts coding for the pro-region and the mature protein. Expression studies in Bacillus subtilis show that the cloned fragment produces a functional enzyme when inserted after a B. subtilis promoter.

Protective antigen (PA) is a component of the Bacillus anthracis lethal and edema toxins and the basis of the current anthrax vaccine. In its heptameric form, PA targets host cells and internalizes the enzymatically active components of the toxins, namely lethal and edema factors. PA and other toxin components are secreted from B. anthracis using the Sec-dependent secretion pathway. This requires them to be translocated across the cytoplasmic membrane in an unfolded state and then to be folded into their native configurations on the trans side of the membrane, prior to their release from the environment of the cell wall. In this study we show that recombinant PA (rPA) requires the extracellular chaperone PrsA for efficient folding when produced in the heterologous host, B. subtilis; increasing the concentration of PrsA leads to an increase in rPA production. To determine the likelihood of PrsA being required for PA production in its native host, we have analyzed the B. anthracis genome sequence for the presence of genes encoding homologues of B. subtilis PrsA. We identified three putative B. anthracis PrsA proteins (PrsAA, PrsAB, and PrsAC) that are able to complement the activity of B. subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be PrsA-dependent.

Summaryecs is a three-cistron operon of Bacillus subtilis, encoding proteins with similarity to the ATPase (EcsA) and hydrophobic components (EcsB) of ABC transporters. The ecsA26 point mutation was shown to cause a strong processing defect of a secreted ␣-amylase precursor (preAmyQ) and of three other exoproteins. Northern analysis of the level of amyQ mRNA showed that ecsA26 also decreases amyQ transcription. This effect too was pleiotropic, as judged by a drastic decrease in the expression from an exoprotease promoter of a reporter protein. A knockout mutation of the ecsB cistron caused a processing defect similar to ecsA26 but, unlike ecsA26, did not affect amyQ transcription. There was also no defect in transcription in the ecsA ecsB double mutant. Thus, an intact ecsB product was required for the downregulation of amyQ by the mutant ecsA. These results suggest a dual regulatory function for Ecs, in which Ecs, possibly as part of a signal transduction mechanism, regulates some component(s) of the protein secretion apparatus as well as secretory protein transcription in a co-ordinated fashion.

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Bioluminescence levels comparable to those achievable in Escherichia coli have yet to be obtained from luxAB expression in gram-positive bacteria. In this communication we describe the gene engineering required to generate a highly bioluminescent derivative of Bacillus subtilis. The combination of a powerful promoter, Pxyn, a fusion derivative of luxAB from Vibrio harveyi and translational coupling have overcome the previously reported limitations in luxAB expression. The implications for highly bioluminescent gram-positive organisms are discussed.

Inefficient translational initiation is often the cause of poor foreign gene expression in gram-positive organisms. The expression of bacterial luciferase (lux) genes in Streptococcus thermophilus (bioluminescence) was improved by addressing this problem in two ways; by ribosome-binding site (RBS) replacement, and by enhancing lux RBS access by polymerase chain reaction modification either alone or combined with translational coupling to a truncated upstream open- reading frame (orf') having its own RBS. Lactococcal expression signals were employed for plasmid-based lux expression. The same constructs were used to monitor bioluminescence in Lactococcus lactis, as well as two non-lactic bacterial strains, for comparison. High lux expression was achieved in all four organisms with a heterodimeric thermostable enzyme. Surprisingly, where ready access to the lux RBS was predicted, translational coupling to the lactococcal orf remained a prerequisite for detectable lux expression in L. lactis. In contrast, high bioluminescence in S. thermophilus was independent of coupling. Consistent with these observations, inspection of published gene sequences suggests that RBS "strength" may be a more important factor in translation in S. thermophilus than in L. lactis. Using reduced light production in highly bioluminescent S. thermophilus as an indicator of biocide presence in milk, test times could be significantly shortened compared with a commercial test utilizing the related non-bioluminescent strain. lux genes appear to be sensitive, exponential-phase reporters of gene activity in S. thermophilus, an organism with molecular biology and genetics that remain largely unstudied.