Abstract
Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely.
Highlights
Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds.Spo0A is the key regulator for sporulation [1,2]
We found a clear signal of the size expected for full length Spo0A (,31 kDa) as early as 3 hours post inoculation, through transition phase (8 h) as well as 24 and 48 hours post inoculation (Figure 1B; 630Derm)
The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant (Fig. 1B, Clostron-generated spo0A mutant (CT)::spo0A)
Summary
Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds.Spo0A is the key regulator for sporulation [1,2]. Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA [3,4,5]. The signaling cascade allows for the integration of environmental signals into the regulation of Spo0A dependent processes, including sporulation. Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization [8,9], resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation [10]. The crystal structure of the DNA binding domain confirms specific and non-specific contacts between the protein and the consensus sequence [12,13]. It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis [14,15,16], virulence factors and stress responses in for instance B. anthracis and B. thuringiensis [17,18,19,20,21], and solvent production in Clostridium acetobutylicum [22,23]
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