Dataset Information

Active segregation by the Bacillus subtilis partitioning system in Escherichia coli.

ABSTRACT: Bacterial genes required for proper partitioning consist of two transacting genes that encode proteins and a cis-acting gene that functions like a centromere. Plasmids actively partitioning by means of these genes migrate from midcell to the cell quarters and are tethered to these sites until the cells divide. Previously the partitioning genes were mainly found on plasmids and phages in Escherichia coli. However, progress in genome sequencing reveals that partitioning genes are ubiquitous in many bacterial plasmids and chromosomes. Each homologue of the two transacting genes belongs to a family, ParA or ParB. Moreover, phylogenic analysis of members of the ParA and ParB families indicates that each member falls into a chromosomal group or an extrachromosomal group. It is known that the parAB genes in the chromosomal group are located on relatively conserved chromosomal regions in several bacterial species. This suggests that the parAB genes were transferred from a chromosome to plasmids and phages, so the genes have diverged among bacterial species. To support this possibility, we show that the Bacillus subtilis Soj and Spo0J members of the ParAB families are responsible for the specific localization of plasmids at cell quarters in E. coli and can function as partition proteins. Host factors to tether actively partitioning plasmids at subcellular sites may be conserved in Gram-negative and Gram-positive bacteria so that phages and plasmids with the ParAB partitioning system can be stably inherited in host cells across bacterial species.

SUBMITTER: Yamaichi Y

PROVIDER: S-EPMC18974 | biostudies-literature | 2000 Dec

REPOSITORIES: biostudies-literature

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Active segregation by the Bacillus subtilis partitioning system in Escherichia coli.

Yamaichi Y Y Niki H H

Proceedings of the National Academy of Sciences of the United States of America 20001201 26

Bacterial genes required for proper partitioning consist of two transacting genes that encode proteins and a cis-acting gene that functions like a centromere. Plasmids actively partitioning by means of these genes migrate from midcell to the cell quarters and are tethered to these sites until the cells divide. Previously the partitioning genes were mainly found on plasmids and phages in Escherichia coli. However, progress in genome sequencing reveals that partitioning genes are ubiquitous in man ...[more]

PMID: 11121066

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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

Active segregation by the Bacillus subtilis partitioning system in Escherichia coli.

Publications

Active segregation by the Bacillus subtilis partitioning system in Escherichia coli.

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