Jonathan W. Willett

Cell growth is determined by substrate availability and the cell’s metabolic capacity to assimilate substrates into building blocks. Metabolic genes that determine growth rate may interact synergistically or antagonistically, and can accelerate or slow growth, depending on genetic background and environmental conditions. We evolved a diverse set of Escherichia coli single-gene deletion mutants with a spectrum of growth rates and identified mutations that generally increase growth rate. Despite the metabolic differences between parent strains, mutations that enhanced growth largely mapped to core transcription machinery, including the β and β’ subunits of RNA polymerase (RNAP) and the transcription elongation factor, NusA. The structural segments of RNAP that determine enhanced growth have been previously implicated in antibiotic resistance and in the control of transcription elongation and pausing. We further developed a computational framework to characterize how the transcriptional changes that occur upon acquisition of these mutations affect growth rate across strains. Our experimental and computational results provide evidence for cases in which RNAP mutations shift the competitive balance between active transcription and gene silencing. This study demonstrates that mutations in specific regions of RNAP are a convergent adaptive solution that can enhance the growth rate of cells from distinct metabolic states. The loss of a metabolic function caused by gene deletion can be compensated, in certain cases, by the concurrent mutation of a second gene. Whether such gene pairs share a local chemical or regulatory relationship or interact via a non-local mechanism has implications for the co-evolution of genetic changes, development of alternatives to gene therapy, and the design of combination antimicrobial therapies that select against resistance. Yet, we lack a comprehensive knowledge of adaptive responses to metabolic mutations, and our understanding of the mechanisms underlying genetic rescue remains limited. We present results of a laboratory evolution approach that has the potential to address both challenges, showing that mutations in specific regions of RNA polymerase enhance growth rates of distinct mutant strains of Escherichia coli with a spectrum of growth defects. Several of these adaptive mutations are deleterious when engineered directly into the original wild-type strain under alternative cultivation conditions, and thus have epistatic rescue properties when paired with the corresponding primary metabolic gene deletions. Our combination of adaptive evolution, directed genetic engineering, and mathematical analysis of transcription and growth rate distinguishes between rescue interactions that are specific or non-specific to a particular deletion. Our study further supports a model for RNA polymerase as a locus of convergent adaptive evolution from different sub-optimal metabolic starting points.

Summary: The environment of a cell has a profound influence on its physiology, development and evolution. Accordingly, the capacity to sense and respond to physical and chemical signals in the environment is an important feature of cellular biology. In bacteria, environmental sensory perception is often regulated by two‐component signal transduction systems (TCSTs). Canonical TCST entails signal‐induced autophosphorylation of a sensor histidine kinase (HK) followed by phosphoryl transfer to a cognate response regulator (RR) protein, which may affect gene expression at multiple levels. Recent studies provide evidence for systems that do not adhere to this archetypal TCST signaling model. We present selected examples of atypical modes of signal transduction including inactivation of HK activity via homo‐ and hetero oligomerization, and cross‐phosphorylation between HKs. These examples highlight mechanisms bacteria use to integrate environmental signals to control complex adaptive processes.

The general stress response (GSR) system of the intracellular pathogenBrucella abortuscontrols the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required forB. abortussurvival under nonoptimal growth conditionsin vitroand for maintenance of chronic infection in anin vivomouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined.bab1_1070is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditionsin vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However,B. abortus WrbA-relatedprotein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductasein vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion ofwrpA(ΔwrpA) does not compromise cell survival under acute oxidative stressin vitroor attenuate infection in cell-based or mouse models. However, a ΔwrpAstrain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulatesB. abortusinteraction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose thatB. abortusWrpA represents a functionally distinct member of the diverse flavodoxin family.IMPORTANCEBrucella abortusis an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system ofB. abortuscontrols the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We presentin vitroandin vivofunctional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activityin vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.

The Alphaproteobacteria uniquely integrate features of two-component signal transduction and alternative σ factor regulation to control transcription of genes that ensure growth and survival across a range of stress conditions. Research over the past decade has led to the discovery of the key molecular players of this general stress response (GSR) system, including the sigma factor σ^EcfG, its anti-σ factor NepR, and the anti-anti-σ factor PhyR. The central molecular event of GSR activation entails aspartyl phosphorylation of PhyR, which promotes its binding to NepR and thereby releases σ^EcfG to associate with RNAP and direct transcription. Recent studies are providing a new understanding of complex, multilayered sensory networks that activate and repress this central protein partner switch. This review synthesizes our structural and functional understanding of the core GSR regulatory proteins and highlights emerging data that are defining the systems that regulate GSR transcription in a variety of species.

The anti-σ factor NepR plays a central role in regulation of the general stress response (GSR) in alphaproteobacteria. This small protein has two known interaction partners: its cognate extracytoplasmic function (ECF) σ factor and the anti-anti-σ factor, PhyR. Stress-dependent phosphorylation of PhyR initiates a protein partner switch that promotes phospho-PhyR binding to NepR, which frees ECF σ to activate transcription of genes required for cell survival under adverse or fluctuating conditions. We have defined key functional roles for structured and intrinsically disordered domains of Caulobacter crescentus NepR in partner binding and activation of GSR transcription. We further demonstrate that NepR strongly stimulates the rate of PhyR phosphorylation in vitro and that this effect requires the structured and disordered domains of NepR. This result provides evidence for an additional layer of GSR regulation in which NepR directly influences activation of its binding partner, PhyR, as an anti-anti-σ factor. We conclude that structured and intrinsically disordered domains of NepR coordinately control multiple functions in the GSR signaling pathway, including core protein partner switch interactions and pathway activation by phosphorylation. IMPORTANCE Anti-σ factors are key molecular participants in a range of adaptive responses in bacteria. The anti-σ factor NepR plays a vital role in a multiprotein partner switch that governs general stress response (GSR) transcription in alphaproteobacteria. We have defined conserved and unconserved features of NepR structure that determine its function as an anti-σ factor and uncovered a functional role for intrinsically disordered regions of NepR in partner binding events required for GSR activation. We further demonstrate a novel function for NepR as an enhancer of PhyR phosphorylation; this activity also requires the disordered domains of NepR. Our results provide evidence for a new layer of GSR regulatory control in which NepR directly modulates PhyR phosphorylation and, hence, activation of the GSR.

Significance: Brucella abortus is an intracellular bacterial pathogen that inflicts a significant health burden on both humans and their livestock on a global scale. We demonstrate that an essential regulatory system controls the growth and morphology of B. abortus , and that this system is required for survival inside mammalian host cells. Using experimental and computational tools of structural biology, we further define how the protein components of this regulatory pathway interact at the atomic scale. Our results provide evidence for multiple, asymmetric modes of binding between essential pathway proteins that control transcription. The multimodal molecular interactions we observe provide evidence for new layers of allosteric control of this conserved gene regulatory system.

Summary: In the intracellular pathogen Brucella abortus, the general stress response (GSR) signalling system determines survival under acute stress conditions in vitro, and is required for long‐term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signalling have remained undefined. We demonstrate that the flavin‐binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK rapidly and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long‐term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N‐terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signalling pathway that regulates B. abortus stress physiology and infection biology.

Two-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In general, these reactions appear to be highly specific and require an appropriate association between the HK and RR proteins. The Myxococcus xanthus genome encodes one of the largest repertoires of signaling proteins in bacteria (685 open reading frames [ORFs]), including at least 127 HKs and at least 143 RRs. Of these, 27 are bona fide NtrC-family response regulators, 21 of which are encoded adjacent to their predicted cognate kinases. Using system-wide profiling methods, we determined that the HK-NtrC RR pairs display a kinetic preference during both phosphotransfer and phosphatase functions, thereby defining cognate signaling systems in M. xanthus . Isothermal titration calorimetry measurements indicated that cognate HK-RR pairs interact with dissociation constants ( K _d ) of approximately 1 µM, while noncognate pairs had no measurable binding. Lastly, a chimera generated between the histidine kinase, CrdS, and HK1190 revealed that residues conferring phosphotransfer and phosphatase specificity dictate binding affinity, thereby establishing discrete protein-protein interactions which prevent cross talk. The data indicate that binding affinity is a critical parameter governing system-wide signaling fidelity for bacterial signal transduction proteins. IMPORTANCE Using in vitro phosphotransfer and phosphatase profiling assays and isothermal titration calorimetry, we have taken a system-wide approach to demonstrate specificity for a family of two-component signaling proteins in Myxococcus xanthus . Our results demonstrate that previously identified specificity residues dictate binding affinity and that phosphatase specificity follows phosphotransfer specificity for cognate HK-RR pairs. The data indicate that preferential binding affinity is the basis for signaling fidelity in bacterial two-component systems.

Myxococcus xanthus is a member of the Myxococcales order within the deltaproteobacterial subdivision. Here, we report the whole-genome shotgun sequence of the type IV pilus (T4P) defective strain DZF1, which includes many genes found in strain DZ2 but absent from strain DK1622.

Myxococcus xanthus is a member of the Myxococcales order within the Deltaproteobacteria subdivision. The myxobacteria reside in soil, have relatively large genomes, and display complex life cycles. Here, we report the whole-genome shotgun sequence of strain DZ2, which includes unique genes not found previously in strain DK1622.