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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.

SUBMITTER: Gao X

PROVIDER: S-EPMC9503168 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Changes in the Spore Proteome of <i>Bacillus cereus</i> in Response to Introduction of Plasmids.

Gao Xiaowei X Swarge Bhagyashree N BN Roseboom Winfried W Wang Yan Y Dekker Henk L HL Setlow Peter P Brul Stanley S Kramer Gertjan G

Microorganisms 20220824 9

Fluorescent fusion proteins were expressed in <i>Bacillus cereus</i> 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 displa ...[more]

PMID: 36144297

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Project description:BackgroundBacillus cereus is a bacterial species which grows efficiently on a wide range of carbon sources and accumulates biopolymer poly-hydroxybutyrate (PHB) up to 80% cell dry weight. PHB is an aliphatic polymer produced and stored intracellularly as a reservoir of carbon and energy, its mobilization is a key biological process for sporulation in Bacillus spp. Previously, B. cereus tsu1 was isolated and cultured on rapeseed cake substrate (RCS), with maximum of PHB accumulation reached within 12 h, and depleted after 48 h. Fore-spore and spore structure were observed after 24 h culture.ResultsQuantitative proteomic analysis of B. cereus tsu1 identified 2952 quantifiable proteins, and 244 significantly changed proteins (SCPs) in the 24 h:12 h pair of samples, and 325 SCPs in the 48 h:12 h pair of samples. Based on gene ontology classification analysis, biological processes enriched only in the 24 h:12 h SCPs include purine nucleotide metabolism, protein folding, metal ion homeostasis, response to stress, carboxylic acid catabolism, and cellular amino acid catabolism. The 48 h:12 h SCPs were enriched into processes including carbohydrate metabolism, protein metabolism, oxidative phosphorylation, and formation of translation ternary structure. A key enzyme for PHB metabolism, poly(R)-hydroxyalkanoic acid synthase (PhaC, KGT44865) accumulated significantly higher in 12 h-culture. Sporulation related proteins SigF and SpoEII were significantly higher in 24 h-samples. Enzymes for nitrate respiration and fermentation accumulated to the highest abundance level in 48 h-culture.ConclusionsChanges in proteome of B. cereus tsu1 during PHB intracellular mobilization were characterized in this study. The key enzyme PhaC for PHB synthesis increased significantly after 12 h-culture which supports the highest PHB accumulation at this time point. The protein abundance level of SpoIIE and SigF also increased, correlating with sporulation in 24 h-culture. Enzymes for nitrate respiration and fermentation were significantly induced in 48 h-culture which indicates the depletion of oxygen at this stage and carbon flow towards fermentative growth. Results from this study provide insights into proteome profile changes during PHB accumulation and reuse, which can be applied to achieve a higher PHB yield and to improve bacterial growth performance and stress resistance.

Project description:To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCE Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus.

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

Publications

Changes in the Spore Proteome of <i>Bacillus cereus</i> in Response to Introduction of Plasmids.

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