Yersinia enterocolitica produces compounds capable of transcriptionally activating the Photobacterium fischeri bioluminescence (lux) operon. Using high-performance liquid chromatography, high resolution tandem mass spectrometry in conjunction with chemical synthesis, two signal molecules were identified and shown to be N-hexanoyl-L-homoserine lactone (HHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). A gene (yenI) was isolated from Y. enterocolitica and demonstrated to direct the synthesis of both HHL and OHHL. DNA sequence analysis revealed an open reading frame (ORF) of 642 bp encoding a protein (YenI) of 24.6 kDa with approximately 20% identity to the LuxI family of proteins. Northern blot analysis of yenI expression indicated yenI is transcribed as a single gene and 5' transcript mapping of yenI identified a transcriptional start site 89 bp upstream of the ORF. DNA sequence analysis of the region downstream of yenI located a second ORF, termed yenR, with significant homology to the LuxR family of transcriptional activators. An insertion mutation of yenI abolishes HHL and OHHL production, indicating its central role in N-acylhomoserine lactone synthesis in Y. enterocolitica. Transcriptional analysis using a chromosomal yenI::luxAB fusion has demonstrated that yenI is not subject to autoinduction but is expressed constitutively. Whilst production of the Yop proteins in the wild type and in yenI mutants is indistinguishable, two-dimensional SDS-PAGE analysis of total cell proteins indicated that a number of proteins lack the yenI mutant.

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Proteolytic enzymes require the presence of their pro-regions for correct folding. Of the four proteolytic enzymes from Carica papaya, papain and papaya proteinase IV (PPIV) have 68% sequence identity. We find that their pro-regions are even more similar, exhibiting 73.6% identity. cDNAs encoding the pro-regions of these two proteinases have been expressed in Escherichia coli independently from their mature enzymes. The recombinant pro-regions of papain and PPIV have been shown to be high affinity inhibitors of all four of the mature native papaya cysteine proteinases. Their inhibition constants are in the range 10(-6) - 10(-9) M. PPIV was inhibited two to three orders of magnitude less effectively than papain, chymopapain and caricain. The pro-region of PPIV, however, inhibited its own mature enzyme more effectively than did the pro-region of papain. Alignment of the sequences of the four papaya enzymes shows that there is a highly variable section towards the C-terminal of the pro-region. This region may therefore confer selectivity to the pro-regions for the individual proteolytic enzymes.

bMuch of our knowledge of the initiation of DNA replication comes from studies in the Gram-negative model organism Escherichia coli. However, the location and structure of the origin of replication within the E. coli genome and the identification and study of the proteins which constitute the E. coli initiation complex suggest that it might not be as universal as once thought. The archetypal low-G؉C-content Gram-positive Firmicutes initiate DNA replication via a unique primosomal machinery, quite distinct from that seen in E. coli, and an examination of oriC in the Firmicutes species Bacillus subtilis indicates that it might provide a better model for the ancestral bacterial origin of replication. Therefore, the study of replication initiation in organisms other than E. coli, such as B. subtilis, will greatly advance our knowledge and understanding of these processes as a whole. In this minireview, we highlight the structure-function relationships of the Firmicutes primosomal proteins, discuss the significance of their oriC architecture, and present a model for replication initiation at oriC. The Firmicutes are Gram-positive bacteria encompassing three major classes, Bacilli, Clostridia, and Mollicutes. They have a relatively low GϩC content in their genomes and are morphologically and physiologically diverse. Rod-shaped bacilli, spherical cocci, and aerobic, anaerobic spore-forming, and non-sporeforming bacteria are found in this group. They likely represent the most ancestral phylum of prokaryotes, with high-GϩC-content Gram-positive and Gram-negative bacteria having diverged from the Firmicutes at a later stage in evolution (13,70). Bacillus subtilis is the best studied of the Firmicutes and is widely considered the Gram-positive model bacterium, but other bacilli, streptococci, staphylococci, and clostridia have been extensively studied because of their medical and industrial importance.In addition to the universally conserved replication initiation protein DnaA (32, 65) and the replication restart protein PriA (17, 39), the low-GϩC-content Firmicutes generally have two unique essential genes, dnaD and dnaB, coding for the replication initiation proteins DnaD and DnaB, respectively ( Table 1) (note that DnaB in B. subtilis is unrelated to DnaB in Escherichia coli). In some cases-for example, in several Mollicutes-there are no distinct dnaD and dnaB genes, but there is, instead, a single gene annotated as dnaD-like which may combine both functions. No homologous proteins are found outside the Firmicutes, suggesting a replication initiation machinery distinctly different from those of other bacteria (31). In addition, there are a number of regulatory proteins which are not found in the Gram-negative model organism Escherichia coli, including YabA, Soj, SirA, and Spo0A, while other regulatory proteins are found in E. coli but not B. subtilis (these regulatory proteins have been subject to a recent review [29]). DnaA, DnaD, and DnaB, together with the helicase loader DnaI (called DnaC in E. coli), the replicativ...