Adaptive point mutation and amplification are induced responses to environmental stress, promoting genetic changes that can enhance survival. A specialized adaptive mutation mechanism has been documented in one Escherichia coli assay, but its enzymatic basis remained unclear. We report that the SOS-inducible, error-prone DNA polymerase (pol) IV, encoded by dinB, is required for adaptive point mutation in the E. coli lac operon. A nonpolar dinB mutation reduces adaptive mutation frequencies by 85% but does not affect adaptive amplification, growth-dependent mutation, or survival after oxidative or UV damage. We show that pol IV, together with the major replicase, pol III, can account for all adaptive point mutations at lac. The results identify a role for pol IV in inducible genetic change.

Mechanisms of DNA repair and mutagenesis are defined on the basis of relatively few proteins acting on DNA, yet the identities and functions of all proteins required are unknown. Here, we identify the network that underlies mutagenic repair of DNA breaks in stressed Escherichia coli and define functions for much of it. Using a comprehensive screen, we identified a network of ≥93 genes that function in mutation. Most operate upstream of activation of three required stress responses (RpoS, RpoE, and SOS, key network hubs), apparently sensing stress. The results reveal how a network integrates mutagenic repair into the biology of the cell, show specific pathways of environmental sensing, demonstrate the centrality of stress responses, and imply that these responses are attractive as potential drug targets for blocking the evolution of pathogens.

SER-109 successfully prevented CDI and had a favorable safety profile, supporting a novel microbiome-based intervention as a potential therapy for recurrent CDI.

Background Recurrent Clostridioides difficile infection (rCDI) is associated with loss of microbial diversity and microbe-derived secondary bile acids, which inhibit C. difficile germination and growth. SER-109, an investigational microbiome drug of donor-derived, purified spores, reduced recurrence in a dose-ranging, phase (P) 1 study in subjects with multiple rCDIs. Methods In a P2 double-blind trial, subjects with clinical resolution on standard-of-care antibiotics were stratified by age (< or ≥65 years) and randomized 2:1 to single-dose SER-109 or placebo. Subjects were diagnosed at study entry by PCR or toxin testing. Safety, C. difficile–positive diarrhea through week 8, SER-109 engraftment, and bile acid changes were assessed. Results 89 subjects enrolled (67% female; 80.9% diagnosed by PCR). rCDI rates were lower in the SER-109 arm than placebo (44.1% vs 53.3%) but did not meet statistical significance. In a preplanned analysis, rates were reduced among subjects ≥65 years (45.2% vs 80%, respectively; RR, 1.77; 95% CI, 1.11–2.81), while the <65 group showed no benefit. Early engraftment of SER-109 was associated with nonrecurrence (P < .05) and increased secondary bile acid concentrations (P < .0001). Whole-metagenomic sequencing from this study and the P1 study revealed previously unappreciated dose-dependent engraftment kinetics and confirmed an association between early engraftment and nonrecurrence. Engraftment kinetics suggest that P2 dosing was suboptimal. Adverse events were generally mild to moderate in severity. Conclusions Early SER-109 engraftment was associated with reduced CDI recurrence and favorable safety was observed. A higher dose of SER-109 and requirements for toxin testing were implemented in the current P3 trial. Clinical Trials Registration NCT02437487, https://clinicaltrials.gov/ct2/show/NCT02437487?term=SER-109&draw= 2&rank=4.

In vivo expression technology (IVET) has been widely used to study gene expression of human bacterial pathogens in animal models, but has heretofore not been used in humans to our knowledge. As part of ongoing efforts to understand Vibrio cholerae pathogenesis and develop improved V. cholerae vaccines, we have performed an IVET screen in humans for genes that are preferentially expressed by V. cholerae during infection. A library of 8,734 nontoxigenic V. cholerae strains carrying transcriptional fusions of genomic DNA to a resolvase gene was ingested by five healthy adult volunteers. Transcription of the fusion leads to resolvasedependent excision of a sacB-containing cassette and thus the selectable phenotype of sucrose resistance (Suc R ). A total of Ϸ20,000 Suc R isolates, those carrying putative in vivo-induced fusions, were recovered from volunteer stool samples. Analysis of the fusion junctions from >7,000 Suc R isolates from multiple samples from multiple volunteers identified 217 candidate genes for preferential expression during human infection. Of genes or operons induced in three or more volunteers, the majority of those tested (65%) were induced in an infant mouse model. VC0201 (fhuC), which encodes the ATPase of a ferrichrome ABC transporter, is one of the identified in vivo-induced genes and is required for virulence in the mouse model. gene expression ͉ genetics ͉ vaccinology ͉ virulence O ur understanding of the complex interactions between bacterial pathogens and humans relies heavily on the use of animal and tissue culture model systems that serve as surrogates of human infection. One useful genetic tool for discovering genes and pathways involved in virulence is in vivo expression technology (IVET), which was designed to identify genes of pathogens that are preferentially expressed during infection and has been extensively used in model systems (reviewed by refs. 1 and 2). IVET is a promoter-trapping strategy in which cells carrying a library of transcriptional fusions of genomic DNA to a reporter gene are used to infect a model host and those carrying fusions that are expressed in vivo can be identified by either a genetic screen or selection. This technique allows the identification of genes that may be expressed only under in vivo conditions [in vivo-induced genes (ivi genes)]. Such genes may be very difficult to identify during growth under laboratory conditions, but are likely to be important in the host for survival and virulence. One form of IVET, used in this work, is recombination-based IVET (3, 4) in which the reporter gene encodes a resolvase that effects a permanent genetic change, allowing a direct selection for cells that expressed the resolvase even transiently during infection.Here, we describe the use of recombination-based IVET to identify genes of Vibrio cholerae, the causative agent of the diarrheal disease cholera, that are expressed during human infection. This approach has the potential to identify important virulence genes that may not be expressed in vitro or during in...