Abstract
The Gram-positive pathogen Bacillus cereus is able to grow in chains of rod-shaped cells, but the regulation of chaining remains largely unknown. Here, we observe that glucose-grown cells of B. cereus ATCC 14579 form longer chains than those grown in the absence of glucose during the late exponential and transition growth phases, and identify that the clhAB2 operon is required for this chain lengthening phenotype. The clhAB2 operon is specific to the B. cereus group (i.e., B. thuringiensis, B. anthracis and B. cereus) and encodes two membrane proteins of unknown function, which are homologous to the Staphylococcus aureus CidA and CidB proteins involved in cell death control within glucose-grown cells. A deletion mutant (ΔclhAB2) was constructed and our quantitative image analyses show that ΔclhAB2 cells formed abnormal short chains regardless of the presence of glucose. We also found that glucose-grown cells of ΔclhAB2 were significantly wider than wild-type cells (1.47 μm ±CI95% 0.04 vs 1.19 μm ±CI95% 0.03, respectively), suggesting an alteration of the bacterial cell wall. Remarkably, ΔclhAB2 cells showed accelerated autolysis under autolysis-inducing conditions, compared to wild-type cells. Overall, our data suggest that the B. cereus clhAB2 operon modulates peptidoglycan hydrolase activity, which is required for proper cell shape and chain length during cell growth, and down-regulates autolysin activity. Lastly, we studied the transcription of clhAB2 using a lacZ transcriptional reporter in wild-type, ccpA and codY deletion-mutant strains. We found that the global transcriptional regulatory protein CodY is required for the basal level of clhAB2 expression under all conditions tested, including the transition growth phase while CcpA, the major global carbon regulator, is needed for the high-level expression of clhAB2 in glucose-grown cells.
Highlights
In Staphylococcus aureus, the CidR regulon is necessary for optimal survival in late stationary phase cultures and during biofilm development in the presence of excess glucose (~0.6%) [1,2,3]
We first investigated how expression of the clhAB2 operon responded to the presence of glucose during growth of B. cereus ATCC 14579 wild-type strain (Bc)
We observed that clhAB2 transcription remained at a constant low level in LB medium, but in the LB supplemented with 0.35% glucose (LBG) medium, clhAB2 expression gradually increased from one hour before t0 to five hours after (Fig 1B)
Summary
In Staphylococcus aureus, the CidR regulon is necessary for optimal survival in late stationary phase cultures and during biofilm development in the presence of excess glucose (~0.6%) [1,2,3] Under these growth conditions, the CidR regulon is involved in the down-regulation of acetate production, which reduces cytoplasmic acidification and limits cell death and lysis [1,2]. It was revealed that CidA and CidB modulate cell death through the direct control of these overflow metabolic enzymes: CidC (pyruvate:menaquinone oxidoreductase) involved in acetate production and AlS (α-acetolactate synthetase) and AlD (α-acetolactate decarboxylase) involved in acetoin production [3] This was a surprising conclusion, because Bayles and collaborators had previously hypothesized that CidA and a structural homolog named LrgA were functionally similar to bacteriophage holin/anti-holin proteins [6]. According to Bayles, much about the Cid/Lrg family of cell-death modulators largely present in Gram positive and in Gram negative bacteria remains unknown; the identification and characterization of PHs involved in this cell death phenomena remain largely unknown [7]
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