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Addiction toxin Fst has unique effects on chromosome segregation and cell division in Enterococcus faecalis and Bacillus subtilis.

ABSTRACT: The Fst toxin of the Enterococcus faecalis pAD1-encoded par addiction module functions intracellularly to kill plasmid-free segregants. Previous results had shown that Fst induction results in membrane permeabilization and cessation of macromolecular synthesis, but only after 45 min. Electron micrographs of toxin-induced cells showed no obvious membrane abnormalities but did reveal defects in nucleoid segregation and cell division, begging the question of which is the primary effect of Fst. To distinguish the possibilities, division septae and nucleoids were visualized simultaneously with fluorescent vancomycin and a variety of DNA stains. Results showed that division and segregation defects occurred in some cells within 15 min after induction. At these early time points, affected cells remained resistant to membrane-impermeant DNA stains, suggesting that loss of membrane integrity is a secondary effect caused by ongoing division and/or segregation defects. Fst-resistant mutants showed greater variability in cell length and formed multiple septal rings even in the absence of Fst. Fst induction was also toxic to Bacillus subtilis. In this species, Fst induction caused only minor division abnormalities, but all cells showed a condensation of the nucleoid, suggesting that effects on the structure of the chromosomal DNA might be paramount.

SUBMITTER: Patel S

PROVIDER: S-EPMC1540048 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Addiction toxin Fst has unique effects on chromosome segregation and cell division in Enterococcus faecalis and Bacillus subtilis.

Patel S S Weaver K E KE

Journal of bacteriology 20060801 15

The Fst toxin of the Enterococcus faecalis pAD1-encoded par addiction module functions intracellularly to kill plasmid-free segregants. Previous results had shown that Fst induction results in membrane permeabilization and cessation of macromolecular synthesis, but only after 45 min. Electron micrographs of toxin-induced cells showed no obvious membrane abnormalities but did reveal defects in nucleoid segregation and cell division, begging the question of which is the primary effect of Fst. To d ...[more]

PMID: 16855226

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Project description:By chance, we discovered a window of extracellular magnesium (Mg2+) availability that modulates the division frequency of Bacillus subtilis without affecting its growth rate. In this window, cells grown with excess Mg2+ produce shorter cells than do those grown in unsupplemented medium. The Mg2+-responsive adjustment in cell length occurs in both rich and minimal media as well as in domesticated and undomesticated strains. Of other divalent cations tested, manganese (Mn2+) and zinc (Zn2+) also resulted in cell shortening, but this occurred only at concentrations that affected growth. Cell length decreased proportionally with increasing Mg2+ from 0.2 mM to 4.0 mM, with little or no detectable change being observed in labile, intracellular Mg2+, based on a riboswitch reporter. Cells grown in excess Mg2+ had fewer nucleoids and possessed more FtsZ-rings per unit cell length, consistent with the increased division frequency. Remarkably, when shifting cells from unsupplemented to supplemented medium, more than half of the cell length decrease occurred in the first 10 min, consistent with rapid division onset. Relative to unsupplemented cells, cells growing at steady-state with excess Mg2+ showed an enhanced expression of a large number of SigB-regulated genes and the activation of the Fur, MntR, and Zur regulons. Thus, by manipulating the availability of one nutrient, we were able to uncouple the growth rate from the division frequency and identify transcriptional changes that suggest that cell division is accompanied by the general stress response and an enhanced demand to sequester and/or increase the uptake of iron, Mn2+, and Zn2+. IMPORTANCE The signals that cells use to trigger cell division are unknown. Although division is often considered intrinsic to the cell cycle, microorganisms can continue to grow and repeat rounds of DNA replication without dividing, indicating that cycles of division can be skipped. Here, we show that by manipulating a single nutrient, namely, Mg2+, cell division can be uncoupled from the growth rate. This finding can be applied to investigate the nature of the cell division signal(s).

Project description:The enterococci, which are among the leading causes of multidrug-resistant (MDR) hospital infection, are notable for their environmental ruggedness, which extends to intrinsic antibiotic resistance. To identify genes that confer this unique property, we used Tn-seq to comprehensively explore the genome of MDR Enterococcus faecalis strain MMH594 for genes important for growth in nutrient-containing medium and with low-level antibiotic challenge. As expected, a large core of genes for DNA replication, expression, and central metabolism, shared with other bacteria, are intolerant to transposon disruption. However, genes were identified that are important to E. faecalis that are either absent from or unimportant for Staphylococcus aureus and Streptococcus pneumoniae fitness when similarly tested. Further, 217 genes were identified that when challenged by sub-MIC antibiotic levels exhibited reduced tolerance to transposon disruption, including those previously shown to contribute to intrinsic resistance, and others not previously ascribed this role. E. faecalis is one of the few Gram-positive bacteria experimentally shown to possess a functional Entner-Doudoroff pathway for carbon metabolism, a pathway that contributes to stress tolerance in other microbes. Through functional genomics and network analysis we defined the unusual structure of this pathway in E. faecalis and assessed its importance. These approaches also identified toxin-antitoxin and related systems that are unique and active in E. faecalis Finally, we identified genes that are absent in the closest nonenterococcal relatives, the vagococci, and that contribute importantly to fitness with and without antibiotic selection, advancing an understanding of the unique biology of enterococci.IMPORTANCE Enterococci are leading causes of antibiotic-resistant infection transmitted in hospitals. The intrinsic hardiness of these organisms allows them to survive disinfection practices and then proliferate in the gastrointestinal tracts of antibiotic-treated patients. The objective of this study was to identify the underlying genetic basis for its unusual hardiness. Using a functional genomic approach, we identified traits and pathways of general importance for enterococcal survival and growth that distinguish them from closely related pathogens as well as ancestrally related species. We further identified unique traits that enable them to survive antibiotic challenge, revealing a large set of genes that contribute to intrinsic antibiotic resistance and a smaller set of uniquely important genes that are rare outside enterococci.

Dataset Information

Addiction toxin Fst has unique effects on chromosome segregation and cell division in Enterococcus faecalis and Bacillus subtilis.

Publications

Addiction toxin Fst has unique effects on chromosome segregation and cell division in Enterococcus faecalis and Bacillus subtilis.

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