BackgroundSoxR and SoxS constitute an intracellular signal response system that rapidly detects changes in superoxide levels and modulates gene expression in E. coli. A time series microarray design was used to identify co-regulated SoxRS-dependent and independent genes modulated by superoxide minutes after exposure to stress.Methodology/Principal Findings soxS mRNA levels surged to near maximal levels within the first few minutes of exposure to paraquat, a superoxide-producing compound, followed by a rise in mRNA levels of known SoxS-regulated genes. Based on a new method for determining the biological significance of clustering results, a total of 138 genic regions, including several transcription factors and putative sRNAs were identified as being regulated through the SoxRS signaling pathway within 10 minutes of paraquat treatment. A statistically significant two-block SoxS motif was identified through analysis of the SoxS-regulated genes. The SoxRS-independent response included members of the OxyR, CysB, IscR, BirA and Fur regulons. Finally, the relative sensitivity to superoxide was measured in 94 strains carrying deletions in individual, superoxide-regulated genes.Conclusions/SignificanceBy integrating our microarray time series results with other microarray data, E. coli databases and the primary literature, we propose a model of the primary transcriptional response containing 226 protein-coding and sRNA sequences. From the SoxS dependent network the first statistically significant SoxS-related motif was identified.

Human viruses possess very complex supramolecular structures. Both icosahedral and enveloped viruses typically display an array of viral-encoded protein antigens at varied spatial densities on the viral particle surface. The viral nucleic acid genome, on the other hand, is encapsulated inside the viral particle. Although both the surface antigen and the interior nucleic acids could independently produce immunological responses, how B cells integrate these two types of signals and respond to a typical virus particle to initiate activation is not well understood at a molecular level. The study of these fundamental biological processes would benefit from the development of viral structural mimics that are well constructed to incorporate both quantitative and qualitative viral features for presentation to B cells. These novel tools would enable researchers to systematically dissect the underlying processes. Here we report the development of such particulate antigens based on liposomes engineered to display a model protein antigen, hen egg lysozyme (HEL). We developed methods to overexpress and purify various affinity mutants of HEL from E. coli. We conjugated the purified recombinant HEL proteins onto the surface of a virion-sized liposome in an orientation-specific manner at defined spatial densities and also encapsulated nucleic acid molecules into the interior of the liposome. Both the chemical conjugation of the HEL antigen on liposome surfaces and the encapsulation of nucleic acids were stable under physiologically relevant conditions. These liposomes elicited antigen-specific B-cell responses in vitro, which validate these supramolecular structures as a novel and effective approach to mimic and systematically isolate the role of essential viral features in directing the B-cell response to particulate antigens.

SignificanceThe opportunistic pathogen Streptococcus pneumoniae (pneumococcus) participates in horizontal gene transfer through genetic competence and produces antimicrobial peptides called “bacteriocins.” Here, we show that the competence and bacteriocin-related ABC transporters ComAB and BlpAB share the same substrate pool, resulting in bidirectional crosstalk between competence and bacteriocin regulation. We also clarify the role of each transporter in bacteriocin secretion and show that, based on their transporter content, pneumococcal strains can be separated into a majority opportunist group that uses bacteriocins only to support competence and a minority aggressor group that uses bacteriocins in broader contexts. Our findings will impact how bacteriocin regulation and production is modeled in the many other bacterial species that use ComAB/BlpAB-type transporters.

Maintaining cellular iron homeostasis is critical for organismal survival. Whereas iron depletion negatively affects the many metabolic pathways that depend on the activity of iron-containing enzymes, any excess of iron can cause the rapid formation of highly toxic reactive oxygen species (ROS) through Fenton chemistry. Although several cellular iron chelators have been identified, little is known about if and how organisms can prevent the Fenton reaction. By studying the effects of cisplatin, a commonly used anticancer drug and effective antimicrobial, we discovered that cisplatin elicits severe iron stress and oxidative DNA damage in bacteria. We found that both of these effects are successfully prevented by polyphosphate (polyP), an abundant polymer consisting solely of covalently linked inorganic phosphates. Subsequent in vitro and in vivo studies revealed that polyP provides a crucial iron reservoir under nonstress conditions and effectively complexes free iron and blocks ROS formation during iron stress. These results demonstrate that polyP, a universally conserved biomolecule, plays a hitherto unrecognized role as an iron chelator and an inhibitor of the Fenton reaction. IMPORTANCE How do organisms deal with free iron? On the one hand, iron is an essential metal that plays crucial structural and functional roles in many organisms. On the other hand, free iron is extremely toxic, particularly under aerobic conditions, where iron rapidly undergoes the Fenton reaction and produces highly reactive hydroxyl radicals. Our study now demonstrates that we have discovered one of the first physiologically relevant nonproteinaceous iron chelators and Fenton inhibitors. We found that polyphosphate, a highly conserved and ubiquitous inorganic polyanion, chelates iron and, through its multivalency, prevents the interaction of iron with peroxide and therefore the formation of hydroxyl radicals. We show that polyP provides a crucial iron reservoir for metalloproteins under nonstress conditions and effectively chelates free iron during iron stress. Importantly, polyP is present in all cells and organisms and hence is likely to take on this crucial function in both prokaryotic and eukaryotic cells.

Epitope density has a profound impact on B cell responses to particulate Ags, the molecular mechanisms of which remain to be explored. To dissect the role of epitope density in this process, we have synthesized a series of liposomal particles, similar to the size of viruses, that display a model self-antigen peptide at defined surface densities. Immunization of C57BL/6J mice using these particles elicited both IgM and class-switched IgG1, IgG2b, and IgG3 autoreactive Abs that depended on the epitope density. In C57BL/6 gene knockout mice lacking either functional TCRs or MHC class II molecules on B cells, the liposomal particles also elicited IgM, IgG1, IgG2b, and IgG3 responses that were comparable in magnitudes to wild-type mice, suggesting that this B cell response was independent of cognate T cell help. Notably, the titer of the IgG in wild-type animals could be increased by more than 200-fold upon replacement of liposomes with bacteriophage Qb virus-like particles that displayed the same self-antigen peptide at comparable epitope densities. This enhancement was lost almost completely in gene knockout mice lacking either TCRs or MHC class II molecules on B cells. In conclusion, epitope density above a threshold on particulate Ags can serve as a stand-alone signal to trigger secretion of autoreactive and class-switched IgG in vivo in the absence of cognate T cell help or any adjuvants. The extraordinary immunogenicity of Qb viral-like particles relies, in large part, on their ability to effectively recruit T cell help after B cell activation.