comK is a positive autoregulatory gene occupying a central position in the competence-signal-transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late competence genes, which specify morphogenetic and structural proteins necessary for construction of the DNA-binding and uptake apparatus. In this report we demonstrate that ComK specifically binds to DNA fragments containing promoter and upstream sequences of the genes it affects (comC, comE, comF, comG and comK). Using portions of the region upstream of comC we show that the ComK-binding sequences are essential for the expression of competence. Moreover, we demonstrate that the presence of ComK stimulates the expression of comF-lacZ and comG-lacZ translational fusions in vivo in Escherichia coli. These results indicate that the gene product of comK is identical to the previously inferred competence transcription factor (CTF).

comK, which encodes the competence transcription factor, is itself transcriptionally activated at the transition from exponential growth to stationary phase in Bacillus subtilis. MecA, a negative regulator of competence, also inhibits comK transcription when overexpressed, and a mec4 null mutation results in comK overexpression. Although null mutations in mecA, as well as in another gene, mecB, are known to bypass the requirements for nearly all of the competence regulatory genes, the comK requirement is not suppressed by mecA inactivation. Various competence reulatory genes (comA srfA, degU, abrB, sin, and spoOA) are shown to be required for the expression of comK srfA transcription is shown to occur equally in cells destined for competence and those destined not to become competent. In contrast, comK transcription is restricted to the presumptive competent cells. These and other results are combined to describe a regulatory pathway for competence.

The expression of competence genes in Bacillus subt~is is controlled by a signal transduction cascade which increases the expression of a competence transcription factor (CTF, encoded by comK) during the transition from exponential growth to stationary phase. ComK (2, 3) is known to be a positively acting transcription factor, previously named CTF (competence transcription factor) (ref.

The development of competence in Bacillus subtilis is regulated by growth conditions and several regulatory genes. In complex media competence development is poor, and there is little or no expression of late competence genes. mec mutations permit competence development and late competence gene expression in complex media, and bypass the requirements for many of the competence regulatory genes. In this paper we describe the cloning and characterization of mecA. The mecA gene product acts negatively in the development of competence. Null mutations in mecA allowed expression of a late competence gene comG, under conditions where it is not normally expressed, including in complex media and in cells mutant for several competence regulatory genes. Overexpression of MecA from a multicopy plasmid resulted in inhibition of comG transcription. The DNA sequence of mecA was determined and the predicted gene product showed no significant similarity to any protein in the database. Expression of a mecA-lacZ translational fusion was constitutive during growth and did not vary significantly in the different media tested. The role of mecA in competence development and other stationary phase phenomena is discussed.

Objective: The effects of mesenchymal stem cell (MSC)-derived exosomes on brain microvascular endothelial cells under oxygen-glucose deprivation (OGD), which mimics cells in deep hypothermic circulatory arrest (DHCA) in vitro, are yet to be studied. Methods: MSCs were co-cultured with primary rat brain endothelial cells, which were then exposed to OGD. Cell viability, apoptosis, the inflammatory factors (IL-1β, IL-6, and TNF-α), and the activation of inflammation-associated TLR4-mediated pyroptosis and the NF-κB signaling pathway were determined. Furthermore, exosomes derived from MSCs were isolated and incubated with endothelial cells to investigate whether the effect of MSCs is associated with MSC-derived exosomes. Apoptosis, cell viability, and the inflammatory response were also analyzed in OGD-induced endothelial cells incubated with MSC-derived exosomes. Results: OGD treatment promoted endothelial cell apoptosis, induced the release of inflammatory factors IL-1β, IL-6, and TNF-α, and inhibited cell viability. Western blot analysis showed that OGD treatment induced TLR4, and NF-κB p65 subunit phosphorylation and caspase-1 upregulation, while co-culture with MSCs could reduce the effect of OGD treatment on endothelial cells. As expected, the effect of MSC-derived exosomes on OGD-treated endothelial cells was similar to that of MSCs. MSC-derived exosomes alleviated the OGD-induced decrease in the viability of endothelial cells, and increased levels of apoptosis, inflammatory factors, and the activation of inflammatory and inflammatory focal pathways. Conclusion: Both MSCs and MSC-derived exosomes attenuated OGD-induced rat primary brain endothelial cell injury. These findings suggest that at least some of the protective effects of MSCs on endothelial cells are mediated by MSC-derived exosomes.