Oscillating behavior of Clostridium difficile Min proteins in Bacillus subtilis

Abstract

In rod-shaped bacteria, the proper placement of the division septum at the midcell relies, at least partially, on the proteins of the Min system as an inhibitor of cell division. The main principle of Min system function involves the formation of an inhibitor gradient along the cell axis; however, the establishment of this gradient differs between two well-studied gram-negative and gram-positive bacteria. While in gram-negative Escherichia coli, the Min system undergoes pole-to-pole oscillation, in gram-positive Bacillus subtilis, proper spatial inhibition is achieved by the preferential attraction of the Min proteins to the cell poles. Nevertheless, when E.coli Min proteins are inserted into B.subtilis cells, they still oscillate, which negatively affects asymmetric septation during sporulation in this organism. Interestingly, homologs of both Min systems were found to be present in various combinations in the genomes of anaerobic and endospore-forming Clostridia, including the pathogenic Clostridium difficile. Here, we have investigated the localization and behavior of C.difficile Min protein homologs and showed that MinDE proteins of C.difficile can oscillate when expressed together in B.subtilis cells. We have also investigated the effects of this oscillation on B.subtilis sporulation, and observed decreased sporulation efficiency in strains harboring the MinDE genes. Additionally, we have evaluated the effects of C.difficile Min protein expression on vegetative division in this heterologous host.

Keywords: Bacillus subtilis; Clostridium difficile; Min system oscillation; bacterial cell division; sporulation.

Figure 1

Cell length histograms. Left column: effects of overexpression of C. difficile Min proteins in wild‐type background. Expression was induced using 0.1 mM IPTG and/or 0.02% xylose, except for strain expressing MinC_{C d} (IB1549), in which 0.3% xylose was used. Right column: complementation of Min_Bs proteins absence by Min_Cd proteins. Shown are induction conditions exhibiting the most notable complementation, that is 0.1 mM IPTG and/or 0.02% xylose except for strain ∆min D_{B s} MinDE_{C d} (IB1418), in which higher concentrations were used (0.5 mM IPTG and 0.3% xylose). Parental strains are in gray. Summary of all measurements can be found in Table S3.

Figure 2

Localization and oscillation of C.difficile Min proteins. (A)–(C) Localization of YFP‐tagged MinD_{C d} expressed from P_hyperspank in wild‐type and mutant B. subtilis backgrounds. (A) wt (IB1415), (B) ∆min D_{B s} (IB1416), (C) ∆min J_{B s} (IB1553). Full arrows point to examples of cells where the fine‐structure signal resembles the localization pattern of the native MinD_{B s} along lipid spirals; the empty arrow indicates an example of localization to the vegetative septum, and the asterisk, to the asymmetric septum. Expression was induced with 0.1 mM IPTG; the scale bar represents 5 μm. (D) Time‐lapse images recorded over a period of 6 min showing oscillation of MinD_{C d}MinE_{C d} in a B.subtilis ∆min D_{B s} background (IB1418), compared to oscillation of E.coli proteins in a B.subtilis ∆min D_{B s} background (Jamroškovič et al. 2012). Expression of MinD_{C d} was induced with 0.1 mM IPTG and MinE_{C d} with 0.02% xylose; scale bar represents 1 μm. Available also as Video S1 in Supporting Information.

Figure 3

Sporulation efficiency of B.subtilis strains. Sporulation efficiency is given as the mean ± SD of at least three independent assays, each normalized against a wild‐type control. Sporulating colonies develop brown color, while nonsporulating light are brown to translucent, as seen in the ∆spo0A negative control (IB220).

Figure 4

Protein–protein interactions between MinD_{C d} and the B.subtilis Min proteins compared alongside interactions among the B.subtilis Min proteins as detected by bacterial two‐hybrid system BACTH. Interactions were quantified using a β‐galactosidase assay and are expressed in Miller units as mean values ± SD of at least three independent measurements. The color intensity corresponds to the strength of the interaction; red boxes highlight strong positive interactions between heterologous proteins. Negative controls were all below 80 MU.

Figure 5

Bioinformatic analysis of Min_Cd proteins. (A) Percent sequence identity (same residues)/similarity (same residues + positive substitutions) between the Min proteins of B.subtilis (Bs), C.difficile (Cd) and E.coli (Ec) based on data from BLAST search (Altschul et al. 1997). (B) Model of the attachment of a MinD_{E c} dimer to the membrane interface through its amphipathic helix. This model is based on the MinD_{E c} crystal structure (PDB ID: 3Q9L), which contains residues 1–260, and thus lacks some of the residues involved in helix formation. Below: Alignment of the C‐terminal region of MinD containing the amphipathic helix. The consensus region is boxed, identical residues are violet, and conservation is indicated by color intensity.