Changes in the Spore Proteome of Bacillus cereus in Response to Introduction of Plasmids

Abstract

Fluorescent fusion proteins were expressed in Bacillus cereus to visualize the germinosome by introducing a plasmid that carries fluorescent fusion proteins of germinant receptor GerR subunits or germinosome scaffold protein GerD. The effects of plasmid insertion and recombinant protein expression on the spore proteome were investigated. Proteomic analysis showed that overexpression of the target proteins had negligible effects on the spore proteome. However, plasmid-bearing spores displayed dramatic abundance changes in spore proteins involved in signaling and metabolism. Our findings indicate that the introduction of a plasmid alone alters the spore protein composition dramatically, with 993 proteins significantly down-regulated and 415 proteins significantly up-regulated among 3323 identified proteins. This shows that empty vector controls are more appropriate to compare proteome changes due to plasmid-encoded genes than is the wild-type strain, when using plasmid-based genetic tools. Therefore, researchers should keep in mind that molecular cloning techniques can alter more than their intended targets in a biological system, and interpret results with this in mind.

Keywords: effects of plasmids; fluorescent proteins; germinant receptor; proteomics.

Figure 1

(A): The number of identified spore proteins in each strain. (B): Coefficient of variation between replicates of each strain.

Figure 2

Volcano plots of t-test proteomes between spores of different strains. * denotes control group. The orange dots represent significantly up-regulated proteins, and blue dots indicate significantly down-regulated proteins compared with the control group. The vertical dotted lines indicate two-fold change. The horizontal dotted line represents p = 0.01.

Figure 3

The KEGG functional enrichment of differentially expressed proteins in spores of strains cotE and M001 compared with WT spores.

Figure 4

The abundances of three proteins encoded by the gerR operon (GerRA, GerRC and GerRB), GerD and CotE in spores of various strains. The z-scores of the LFQ intensities are shown to compare relative expression levels between different strains of a given protein; note that the quantitative data for GerRB are based on only a single peptide.

Figure 5

The heatmap of proteins associated with spore germination in spores of various strains (A). Color represents z-score of LFQ values; a cross means not detected. The log2 (fold changes) of SASPs (D), coat proteins (C) and heat shock proteins (B) are shown in a colored heatmap. Red represents elevated protein abundance and blue represents decreased LFQ intensities compared to WT spores.

Figure 6

Enzyme levels (log2 (fold changes)) in central metabolic pathways of B. cereus strain M001 spores compared to levels in WT spores. Enzyme levels in blue are reduced, while those in red are increased. The multiple boxes next to some enzymes correspond to isoforms of the same enzyme. The regulation of the relevant genes and the enzymes for which they are coding are presented in Supplementary Table S3.