Bacteria identify and respond to DNA damage using the SOS response. LexA, a central transcription repressor in the response, has been implicated in the regulation of lysogeny in various temperate bacteriophages. During infection of Bacillus thuringiensis with GIL01 bacteriophage, LexA represses the SOS response and the phage lytic cycle by binding DNA, an interaction further stabilized upon binding of a viral protein, gp7. gp7 protein acts as corepressor and is needed for GIL01 to establish lysogeny. Here we report the crystallographic structure of phage-borne gp7 at 1.7-A resolution, and characterize the 4:2 stoichiometry and potential interaction with LexA using surface plasmon resonance, static light scattering, and small-angle X-ray scattering. These data indicate that gp7 stabilizes LexA binding to operator DNA via coordination of the N- and C-terminal domains of LexA. Furthermore, we have found that gp7 can interact with LexA from Staphylococcus aureus, a significant human pathogen. Our results provide first structural evidence as to how phage factors can directly associate with LexA to modulate the SOS response.
Cells employ specific and nonspecific mechanisms to protect their genome integrity against exogenous and endogenous factors. The clbS gene is part of the polyketide synthase machinery (pks genomic island) encoding colibactin, a genotoxin implicated in promoting colorectal cancer. The pks is found among the Enterobacteriaceae, in particular Escherichia coli strains of the B2 phylogenetic group. Several resistance mechanisms protect toxin producers against toxicity of their products. ClbS, a cyclopropane hydrolase, was shown to confer colibactin resistance by opening its electrophilic cyclopropane ring. Here we report that ClbS sustained viability and enabled growth also of E. coli expressing another genotoxin, the Usp nuclease. The recA::gfp reporter system showed that ClbS protects against Usp induced DNA damage. To elucidate the mechanism of ClbS mediated protection, we studied the DNA binding ability of the ClbS protein. We show that ClbS directly interacts with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), whereas ssDNA seems to be the preferred substrate. Thus, the ClbS DNA-binding characteristics may serve bacteria to protect their genomes against DNA degradation.
The GIL01 bacteriophage is a temperate phage that infects the insect pathogen Bacillus thuringiensis. Unlike most temperate phages, GIL01 lysogeny is not maintained by a dedicated phage repressor but rather by the host's regulator of the SOS response, LexA. Previously we showed that the lytic cycle was induced by DNA damage and that LexA, in conjunction with phage-encoded protein gp7, repressed lysogenic promoter. Here we examine the lytic/lysogenic switch in more detail and show that lytic promoter is also repressed by a LexA-gp7 complex, binding to tandem LexA boxes within the promoter. We also demonstrate that expression from lytic promoter is considerably delayed after DNA damage, requiring the phage-encoded DNA binding protein, gp6. We show that gp6 binds to the site overlapping the -35 promoter element and thus presumably acts as a type II activator that directly interacts with the sigma subunit of the RNA polymerase holoenzyme in the complex with the promoter. Surprisingly, gp6 is homologous to LexA itself and, thus, is a rare example of a LexA homologue directly activating transcription. This is the first study showing that the interplay between these two LexA family members, with opposing functions, ensures the timely expression of a certain genes - GIL01 phage late genes.
Aegerolysins are proteins produced by bacteria, fungi, plants and protozoa. The biological role of bacterial aegerolysins is unknown. Genome analyses show a particularly high frequency of aegerolysin genes in bacteria, including the pathogenic genera Pseudomonas and Vibrio; these are human pathogens of high clinical relevance and can thrive in a variety of other species. We show that Pseudomonas aeruginosa aegerolysin RahU interacts with ceramide phosphorylethanolamine (CPE) containing artificial membranes, and that RahU interacts with the insect cell line producing CPE but not with the membrane of the human cell line. We show that RahU localizes to the nucleus of the formaldehyde-treated human cell line, with or without the cystic fibrosis transmembrane conductance regulator (CFTR), a key factor of cystic fibrosis incidence. We report crystal structures of RahU alone and in complex with tris(hydroxymethyl)aminomethane (Tris), which, like the phosphorylethanolamine head group of CPE, contains a primary amine. The RahU structures suggest the importance of the ligand cavity between the loops and its proximity in RahU membrane and possible also DNA interaction inside nucleus of the host cell. Our results thus represent a starting point for a better understanding of the role of P. aeruginosa RahU, and possibly other bacterial aegerolysins, in bacterial interactions with other organisms.
Transcription in most bacteria is tightly regulated in order to facilitate bacterial adaptation to different environments, and transcription factors play a key role in this. Here we give a brief overview of the essential features of bacterial transcription factors and how they affect transcript initiation at target promoters. We focus on complex promoters that are regulated by combinations of activators and repressors, combinations of repressors only, or combinations of activators. At some promoters, transcript initiation is regulated by nucleoid-associated proteins, which often work together with transcription factors. We argue that the distinction between nucleoid-associated proteins and transcription factors is blurred and that they likely share common origins.