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Similar organization of the nusA-infB operon in Bacillus subtilis and Escherichia coli.

ABSTRACT: We reported previously the cloning and sequence of the Bacillus subtilis infB gene which encodes the essential IF2 factor required for initiation of translation (K. Shazand, J. Tucker, R. Chiang, K. Stansmore, H. U. Sperling-Petersen, M. Grunberg-Manago, J. C. Rabinowitz, and T. Leighton, J. Bacteriol. 172:2675-2687, 1990). The location of the 5' border of the infB operon was investigated by using integrative plasmids carrying various DNA fragments from the region upstream of the infB gene. The lethal effect of disruption of the infB transcriptional unit could be suppressed when the integrated plasmid introduced the spac promoter upstream of the infB operon and transformants were selected in conditions of induction of spac expression. Such an integrated plasmid was used as a starting point to clone the promoter of the infB operon. Primer extension mapping suggests that a single sigma A-type promoter controls transcription of the infB operon. The sequence of a 5,760-bp region encompassing the infB gene was determined. The infB operon is located immediately downstream of the polC gene and comprises seven open reading frames, four of which appear to be the homologs of genes present in the same order in the Escherichia coli infB operon, including nusA. The striking similarity between the E. coli and B. subtilis infB operons suggests that the function of each gene pair is conserved and that the B. subtilis NusA homolog, which is 124 residues shorter than its E. coli counterpart, could play a role similar to its role in E. coli.

SUBMITTER: Shazand K

PROVIDER: S-EPMC204605 | biostudies-other | 1993 May

REPOSITORIES: biostudies-other

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Similar organization of the nusA-infB operon in Bacillus subtilis and Escherichia coli.

Shazand K K Tucker J J Grunberg-Manago M M Rabinowitz J C JC Leighton T T

Journal of bacteriology 19930501 10

We reported previously the cloning and sequence of the Bacillus subtilis infB gene which encodes the essential IF2 factor required for initiation of translation (K. Shazand, J. Tucker, R. Chiang, K. Stansmore, H. U. Sperling-Petersen, M. Grunberg-Manago, J. C. Rabinowitz, and T. Leighton, J. Bacteriol. 172:2675-2687, 1990). The location of the 5' border of the infB operon was investigated by using integrative plasmids carrying various DNA fragments from the region upstream of the infB gene. The ...[more]

PMID: 8491709

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Project description:RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli, we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli, including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His6-MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His6-MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL.IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage in vitro We applied these two approaches to the B. subtilis enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in E. coli, providing evidence that both substrates are cleaved by RasP in vivo but, surprisingly, that only RsiW was cleaved in vitro, suggesting that FtsL has an additional requirement.

Dataset Information

Similar organization of the nusA-infB operon in Bacillus subtilis and Escherichia coli.

Publications

Similar organization of the nusA-infB operon in Bacillus subtilis and Escherichia coli.

Similar Datasets

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