Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed.

At specific times during bacterial growth, the transcription factor DksA and the unusual nucleotide regulator ppGpp work synergistically to inhibit some Escherichia coli promoters (e.g. rRNA promoters) and to stimulate others (e.g. promoters for amino‐acid synthesis and transport). However, the mechanism of DksA action remains uncertain, in part because DksA does not function like conventional transcription factors. To gain insights into DksA function, we identified mutations in dksA that bypassed the requirement for ppGpp by selecting for growth of cells lacking ppGpp on minimal medium without amino acids. We show here that two substitutions in DksA, L15F and N88I, result in higher DksA activity both in vivo and in vitro, primarily by increasing the apparent affinity of DksA for RNA polymerase (RNAP). The mutant DksA proteins suggest potential roles for ppGpp in DksA function, identify potential surfaces on DksA crucial for RNAP binding, and provide tools for future studies to elucidate the mechanism of DksA action.

σ is an alternative sigma factor, encoded by the gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σ general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σ levels transcriptionally, translationally, and post-translationally in It was shown previously that the transcription factor DksA and its cofactor ppGpp are among the many factors governing σ synthesis, thus playing an important role in activation of the σ stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully. We describe here how DksA and ppGpp directly activate the promoters for the anti-adaptor protein IraP and the small regulatory RNA DsrA, thereby indirectly influencing σ levels. In addition, based on effects of DksA, a previously identified DksA variant with increased affinity for RNAP, we show that DksA can increase σ activity by another indirect mechanism. We propose that by reducing rRNA transcription, DksA and ppGpp increase the availability of core RNAP for binding to σ and also increase transcription from other promoters, including P and P By improving the translation and stabilization of σ, as well as the ability of other promoters to compete for RNAP, DksA and ppGpp contribute to the switch in the transcription program needed for stress adaptation. Bacteria spend relatively little time in log phase outside of the optimized environment found in a laboratory. They have evolved to make the most of alternating feast and famine conditions by seamlessly transitioning between rapid growth and stationary phase, a lower metabolic mode that is crucial for long-term survival. One of the key regulators of the switch in gene expression that characterizes stationary phase is the alternative sigma factor σ Understanding the factors governing σ activity is central to unraveling the complexities of growth, adaptation to stress, and pathogenesis. Here we describe three mechanisms by which the RNA polymerase binding factor DksA and the second messenger ppGpp regulate σ levels.

The microbiome is important to all animals, including poultry, playing a critical role in health and performance. Low-dose antibiotics have historically been used to modulate food production animals and their microbiome. Identifying alternatives to antibiotics conferring similar modulatory properties has been elusive. The purpose of this study was to determine if a host-tailored probiotic could recapitulate effects of a low-dose antibiotic on host response and the developing microbiome. Over 13 days of life, turkey poults were supplemented continuously with a low-dose antibiotic or oral supplementation of a prebiotic with or without two different probiotics (8 cage units, n = 80 per group). Gastrointestinal bacterial and fungal communities of poults were characterized by 16S rRNA gene and ITS2 amplicon sequencing. Localized and systemic host gene expression was assessed using transcriptome sequencing (RNA-Seq), kinase activity was assessed by avian-specific kinome peptide arrays, and performance parameters were assessed. We found that development of the early-life microbiome of turkey poults was tightly ordered in a tissue- and time-specific manner. Low-dose antibiotic and turkey-tailored probiotic supplementation, but not nontailored probiotic supplementation, elicited similar shifts in overall microbiome composition during development compared to controls. Treatment-induced bacterial changes were accompanied by parallel shifts in the fungal community and host gene expression and enhanced performance metrics. These results were validated in pen trials that identified further additive effects of the turkey-tailored probiotic combined with different prebiotics. Alternative approaches to low-dose antibiotic use in poultry are feasible and can be optimized utilizing the indigenous poultry microbiome. Similar approaches may also be beneficial for humans. IMPORTANCE Alternative approaches are greatly needed to reduce the need for antibiotic use in food animal production. This study utilized a pipeline for the development of a host-tailored probiotic to enhance performance in commercial turkeys and modulate their microbiota, similar to the effects of low-dose antibiotic administration. We determined that a host-tailored probiotic, developed in the context of the commercial turkey gut microbiome, was more effective at modulating these parameters than a nontailored probiotic cocktail. Furthermore, the host-tailored probiotic mimicked many of the effects of a low-dose antibiotic growth promoter. Surprisingly, the effects of the antibiotic growth promoter and host-tailored probiotic were observed across kingdoms, illustrating the coordinated interkingdom effects of these approaches. This work suggests that tailored approaches to probiotic development hold promise for modulating the avian host and its microbiota.

cHorizontal gene transfer by conjugation plays a major role in bacterial evolution, allowing the acquisition of new traits, such as virulence and resistance to antibacterial agents. With the increased antibiotic resistance in bacterial pathogens, a better understanding of how bacteria modulate conjugation under changing environments and the genetic factors involved is needed. Despite the evolutionary advantages conjugation may confer, the process can be quite stressful for the donor cell. Here, we characterize the ability of TraR, encoded on the episomal F= plasmid, to upregulate the E extracytoplasmic stress pathway in Escherichia coli. TraR, a DksA homolog, modulates transcription initiation through the secondary channel of RNA polymerase. We show here that TraR activates transcription directly; however, unlike DksA, it does so without using ppGpp as a cofactor. TraR expression can stimulate the E extracytoplasmic stress response independently of the DegS/RseA signal transduction cascade. In the absence of TraR, bacteria carrying conjugative plasmids become more susceptible to external stress. We propose that TraR increases the concentrations of periplasmic chaperones and proteases by directly activating the transcription of E -dependent promoters; this increased protein folding capacity may prepare the bacterium to endure the periplasmic stress of sex pilus biosynthesis during mating.