Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Abstract. RPTPIx is a transmembrane protein tyrosine phosphatase with an adhesion molecule-like ectodomain. It has recently been shown that RPTPIx mediates homophilic interactions when expressed in insect cells. In this study, we have examined how RPTPIx may function as a cell contact receptor in mink lung epithelial cells, which express RPTPIx endogenously, as well as in transfected 3T3 cells. We find that RPTPI x has a relatively short half-life (3--4 hours) and undergoes posttranslational cleavage into two noncovalently associated subunits, with both cleaved and uncleaved molecules being present on the cell surface (roughly at a 1:1 ratio); shedding of the ectodomain subunit is observed in exponentially growing cells. Immunofluorescence analysis reveals that surface expression of RPTPIx is restricted to regions of tight cell-cell contact.RPTPIx surface expression increases significantly with increasing cell density. This density-induced upregulation of RPTPIx is independent of its catalytic activity and is also observed when transcription is driven by a constitutive promoter, indicating that modulation of RPTPIx surface expression occurs posttranscriptionally. Based on our results, we propose the following model of RPTPIx function: In the absence of cell--cell contact, newly synthesized RPTPIx molecules are rapidly cleared from the cell surface. Cell-cell contact causes RPTPIx to be trapped at the surface through homophilic binding, resulting in accumulation of RPTPIx at intercellular contact regions. This contact-induced clustering of RPTPIx may then lead to tyrosine dephosphorylation of intracellular substrates at cell-cell contacts.ECZl'ToR-like protein tyrosine phosphatases (receptor PTPs) 1 represent a new family of transmembrane proteins that are thought to transduce external signals by dephosphorylating phosphotyrosine residues on intracellular substrates (for review see Walton and Dixon, 1993). Although the receptor PTPs are growing in number and diversity, little is known about the nature of their extracellular ligands and their normal intracellular activities. It is also unknown where at the cell surface receptor PTPs are localized and how delivery and localization to the cell surface is regulated.We have recently cloned and characterized a mammalian receptor PTP, termed RPTPIx, which has an ectodomain similar to both the Ig-like and fibronectin type III-like domains of cell adhesion molecules (Gebbink et al., 1991).

The receptor-like protein tyrosine phosphatases (RPTP) mu and RPTP kappa have a modular ectodomain consisting of four fibronectin type III-like repeats, a single Ig-like domain, and a newly identified N-terminal MAM domain. The function of the latter module, which comprises about 160 amino acids and is found in diverse transmembrane proteins, is not known. We previously reported that both RPTP mu and RPTP kappa can mediate homophilic cell interactions when expressed in insect cells. Here we show that despite their striking structural similarity, RPTP mu and RPTP kappa fail to interact in a heterophilic manner. To examine the role of the MAM domain in homophilic binding, we expressed a mutant RPTP mu lacking the MAM domain in insect Sf9 cells. Truncated RPTP mu is properly expressed at the cell surface but fails to promote cell-cell adhesion. Homophilic cell adhesion is fully restored in a chimeric RPTP mu molecule containing the MAM domain of RPTP kappa. However, this chimeric RPTP mu does not interact with either RPTP mu or RPTP kappa. These results indicate that the MAM domain of RPTP mu and RPTP kappa is essential for homophilic cell-cell interaction and helps determine the specificity of these interactions.

Spheroplasts were used to study the early interactions of newly synthesized outer membrane protein PhoE with periplasmic proteins employing a protein cross-linking approach. Newly translocated PhoE protein could be cross-linked to the periplasmic chaperone Skp at the periplasmic side of the inner membrane. To study the timing of this interaction, a PhoE-dihydrofolate reductase hybrid protein was constructed that formed translocation intermediates, which had the PhoE moiety present in the periplasm and the dihydrofolate reductase moiety tightly folded in the cytoplasm.

The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastid-localized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific lambda gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.

Background:The FtsQBL complex plays a key role in bacterial cell division. Results: Periplasmic domains of FtsQ, FtsB, and FtsL form a trimeric complex with submicromolar affinity. Interactions are focused at the C termini of the subunits. Conclusion: FtsQ, FtsB, and FtsL form a complex with 1:1:1 stoichiometry. Significance: Insight into FtsQBL complex formation will facilitate drug design.

YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Secassociated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth.

Eeyarestatin 1 (ES1) is an inhibitor of endoplasmic reticulum (ER) associated protein degradation, Sec61‐dependent Ca2+ homeostasis and protein translocation into the ER. Recently, evidence was presented showing that a smaller analog of ES1, ES24, targets the Sec61‐translocon, and captures it in an open conformation that is translocation‐incompetent. We now show that ES24 impairs protein secretion and membrane protein insertion in Escherichia coli via the homologous SecYEG‐translocon. Transcriptomic analysis suggested that ES24 has a complex mode of action, probably involving multiple targets. Interestingly, ES24 shows antibacterial activity toward clinically relevant strains. Furthermore, the antibacterial activity of ES24 is equivalent to or better than that of nitrofurantoin, a known antibiotic that, although structurally similar to ES24, does not interfere with SecYEG‐dependent protein trafficking. Like nitrofurantoin, we find that ES24 requires activation by the NfsA and NfsB nitroreductases, suggesting that the formation of highly reactive nitroso intermediates is essential for target inactivation in vivo.