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Toxin-antitoxin systems are widely distributed genetic modules that regulate growth and persistence in bacteria. Many systems, including E. coli MazEF, include toxins that are endoribonucleases, but the full set of targets for these toxins remains poorly defined. Previous studies on a limited set of transcripts suggested that MazF creates a pool of leaderless mRNAs that are preferentially translated by specialized ribosomes created through MazF cleavage of mature 16S rRNA. Here, using paired-end RNA sequencing (RNA-seq) and ribosome profiling, we provide a comprehensive, global analysis of MazF cleavage specificity and its targets. We find that MazF cleaves most transcripts at multiple sites within their coding regions, with very few full-length, leaderless mRNAs created. Additionally, our results demonstrate that MazF does not create a large pool of specialized ribosomes but instead rapidly disrupts ribosome biogenesis by targeting both ribosomal protein transcripts and rRNA precursors, helping to inhibit cell growth., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Canonical bacterial transcription activators bind to non-transcribed promoter elements to increase transcription of their target genes. Here we report crystal structures of binary complexes comprising domains of Caulobacter crescentus GcrA, a noncanonical bacterial transcription factor, that support a novel mechanism for transcription activation through the preferential binding of methylated cis-regulatory elements and the promotion of open complex formation through an interaction with region 2 of the principal σ factor, σ70. We present crystal structures of the C-terminal, σ factor-interacting domain (GcrA-SID) in complex with domain 2 of σ70 (σ702), and the N-terminal, DNA-binding domain (GcrA-DBD) in complex with methylated double-stranded DNA (dsDNA). The structures reveal interactions essential for transcription activation and DNA recognition by GcrA. These structures, along with mutational analyses, support a mechanism of transcription activation in which GcrA associates with RNA polymerase (RNAP) prior to promoter binding through GcrA-SID, arming RNAP with a flexible GcrA-DBD. The RNAP-GcrA complex then binds and activates target promoters harboring a methylated GcrA binding site either upstream or downstream of the transcription start site.

The structural maintenance of chromosomes (SMC) complex plays an important role in chromosome organization and segregation in most living organisms. In Caulobacter crescentus, SMC is required to align the left and the right arms of the chromosome that run in parallel down the long axis of the cell. However, the mechanism of SMC-mediated alignment of chromosomal arms remains elusive. Here, using genome-wide methods and microscopy of single cells, we show that Caulobacter SMC is recruited to the centromeric parS site and that SMC-mediated arm alignment depends on the chromosome-partitioning protein ParB. We provide evidence that SMC likely tethers the parS-proximal regions of the chromosomal arms together, promoting arm alignment. Furthermore, we show that highly transcribed genes near parS that are oriented against SMC translocation disrupt arm alignment, suggesting that head-on transcription interferes with SMC translocation. Our results demonstrate a tight interdependence of bacterial chromosome organization and global patterns of transcription., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cross-experiment comparisons in public data compendia are challenged by unmatched conditions and technical noise. The ADAGE method, which performs unsupervised integration with denoising autoencoder neural networks, can identify biological patterns, but because ADAGE models, like many neural networks, are over-parameterized, different ADAGE models perform equally well. To enhance model robustness and better build signatures consistent with biological pathways, we developed an ensemble ADAGE (eADAGE) that integrated stable signatures across models. We applied eADAGE to a compendium of Pseudomonas aeruginosa gene expression profiling experiments performed in 78 media. eADAGE revealed a phosphate starvation response controlled by PhoB in media with moderate phosphate and predicted that a second stimulus provided by the sensor kinase, KinB, is required for this PhoB activation. We validated this relationship using both targeted and unbiased genetic approaches. eADAGE, which captures stable biological patterns, enables cross-experiment comparisons that can highlight measured but undiscovered relationships., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair-the attenuation of DNA-end processing and the initiation of homology search by RecA-thereby helping to ensure that genomic integrity is maintained during DSB repair.

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common.

Structural maintenance of chromosomes (SMC) complexes play critical roles in chromosome dynamics in virtually all organisms, but how they function remains poorly understood. In the bacterium Bacillus subtilis, SMC-condensin complexes are topologically loaded at centromeric sites adjacent to the replication origin. Here we provide evidence that these ring-shaped assemblies tether the left and right chromosome arms together while traveling from the origin to the terminus (>2 megabases) at rates >50 kilobases per minute. Condensin movement scales linearly with time, providing evidence for an active transport mechanism. These data support a model in which SMC complexes function by processively enlarging DNA loops. Loop formation followed by processive enlargement provides a mechanism by which condensin complexes compact and resolve sister chromatids in mitosis and by which cohesin generates topologically associating domains during interphase., (Copyright © 2017, American Association for the Advancement of Science.)

The Clp family of proteases is responsible for controlling both stress responses and normal growth. In Caulobacter crescentus, the ClpXP protease is essential and drives cell cycle progression through adaptor-mediated degradation. By contrast, the physiological role for the ClpAP protease is less well understood with only minor growth defects previously reported for ΔclpA cells. Here, we show that ClpAP plays an important role in controlling chromosome content and cell fitness during extended growth. Cells lacking ClpA accumulate aberrant numbers of chromosomes upon prolonged growth suggesting a defect in replication control. Levels of the replication initiator DnaA are elevated in ΔclpA cells and degradation of DnaA is more rapid in cells lacking the ClpA inhibitor ClpS. Consistent with this observation, ClpAP degrades DnaA in vitro while ClpS inhibits this degradation. In cells lacking Lon, the protease previously shown to degrade DnaA in Caulobacter, ClpA overexpression rescues defects in fitness and restores degradation of DnaA. Finally, we show that cells lacking ClpA are particularly sensitive to inappropriate increases in DnaA activity. Our work demonstrates an unexpected effect of ClpAP in directly regulating replication through degradation of DnaA and expands the functional role of ClpAP in Caulobacter., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (© 2016 John Wiley & Sons Ltd.)

In Escherichia coli and other γ-proteobacteria, the PhoQ-PhoP two-component signaling system responds to low extracellular Mg ++ and cationic antimicrobial peptides. On transition to inducing conditions, the expression of PhoP-dependent genes increases rapidly, but then decays to a new, intermediate steady-state level, a phenomenon often referred to as partial adaptation. The molecular basis for this partial adaptation has been unclear. Here, using time-lapse fluorescence microscopy to examine PhoP-dependent gene expression in individual E. coli cells we show that partial adaptation arises through a negative feedback loop involving the small protein MgrB. When E. coli cells are shifted to low Mg ++ , PhoQ engages in multiple rounds of autophosphorylation and phosphotransfer to PhoP, which, in turn, drives the expression of mgrB. MgrB then feeds back to inhibit the kinase activity of PhoQ. PhoQ is bifunctional such that, when not active as a kinase, it can stimulate the dephosphorylation of PhoP. Thus, MgrB drives the inactivation of PhoP and the observed adaptation in PhoP-dependent gene expression. Our results clarify the source of feedback inhibition in the E. coli PhoQ-PhoP system and reveal how exogenous factors, such as MgrB, can combine with a canonical two-component signaling pathway to produce complex temporal dynamics in target gene expression., (© 2016 John Wiley & Sons Ltd.)

As we become increasingly dependent on electronic information-processing systems at home and work, it's easy to lose sight of the fact that our very survival depends on highly complex biological information-processing systems. Each of the trillions of cells that form the human body has the ability to detect and respond to a wide range of stimuli and inputs, using an extraordinary set of signaling proteins to process this information and make decisions accordingly. Indeed, cells in all organisms rely on these signaling proteins to survive and proliferate in unpredictable and sometimes rapidly changing environments. But how exactly do these proteins relay information within cells, and how do they keep a multitude of incoming signals straight? Here, I describe recent efforts to understand the fidelity of information flow inside cells. This work is providing fundamental insight into how cells function. Additionally, it may lead to the design of novel antibiotics that disrupt the signaling of pathogenic bacteria or it could help to guide the treatment of cancer, which often involves information-processing gone awry inside human cells.

Chromosomes in all organisms are highly organized and divided into multiple chromosomal interaction domains, or topological domains. Regions of active, high transcription help establish and maintain domain boundaries, but precisely how this occurs remains unclear. Here, using fluorescence microscopy and chromosome conformation capture in conjunction with deep sequencing (Hi-C), we show that in Caulobacter crescentus, both transcription rate and transcript length, independent of concurrent translation, drive the formation of domain boundaries. We find that long, highly expressed genes do not form topological boundaries simply through the inhibition of supercoil diffusion. Instead, our results support a model in which long, active regions of transcription drive local decompaction of the chromosome, with these more open regions of the chromosome forming spatial gaps in vivo that diminish contacts between DNA in neighboring domains. These insights into the molecular forces responsible for domain formation in Caulobacter likely generalize to other bacteria and possibly eukaryotes., (© 2016 The Authors.)

Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ(70)-dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ(70) interact with domain 4, GcrA interfaces with domain 2, the region that binds the -10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼ 200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively., (© 2015 Haakonsen et al.; Published by Cold Spring Harbor Laboratory Press.)

Interacting proteins typically coevolve, and the identification of coevolving amino acids can pinpoint residues required for interaction specificity. This approach often assumes that an interface-disrupting mutation in one protein drives selection of a compensatory mutation in its partner during evolution. However, this model requires a non-functional intermediate state prior to the compensatory change. Alternatively, a mutation in one protein could first broaden its specificity, allowing changes in its partner, followed by a specificity-restricting mutation. Using bacterial toxin-antitoxin systems, we demonstrate the plausibility of this second, promiscuity-based model. By screening large libraries of interface mutants, we show that toxins and antitoxins with high specificity are frequently connected in sequence space to more promiscuous variants that can serve as intermediates during a reprogramming of interaction specificity. We propose that the abundance of promiscuous variants promotes the expansion and diversification of toxin-antitoxin systems and other paralogous protein families during evolution., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: An ability to sense and respond to changes in extracellular phosphate is critical for the survival of most bacteria. For Caulobacter crescentus, which typically lives in phosphate-limited environments, this process is especially crucial. Like many bacteria, Caulobacter responds to phosphate limitation through a conserved two-component signaling pathway called PhoR-PhoB, but the direct regulon of PhoB in this organism is unknown. Here we used chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) to map the global binding patterns of the phosphate-responsive transcriptional regulator PhoB under phosphate-limited and -replete conditions. Combined with genome-wide expression profiling, our work demonstrates that PhoB is induced to regulate nearly 50 genes under phosphate-starved conditions. The PhoB regulon is comprised primarily of genes known or predicted to help Caulobacter scavenge for and import inorganic phosphate, including 15 different membrane transporters. We also investigated the regulatory role of PhoU, a widely conserved protein proposed to coordinate phosphate import with expression of the PhoB regulon by directly modulating the histidine kinase PhoR. However, our studies show that it likely does not play such a role in Caulobacter, as PhoU depletion has no significant effect on PhoB-dependent gene expression. Instead, cells lacking PhoU exhibit striking accumulation of large polyphosphate granules, suggesting that PhoU participates in controlling intracellular phosphate metabolism., Importance: The transcription factor PhoB is widely conserved throughout the bacterial kingdom, where it helps organisms respond to phosphate limitation by driving the expression of a battery of genes. Most of what is known about PhoB and its target genes is derived from studies of Escherichia coli. Our work documents the PhoB regulon in Caulobacter crescentus, and comparison to the regulon in E. coli reveals significant differences, highlighting the evolutionary plasticity of transcriptional responses driven by highly conserved transcription factors. We also demonstrated that the conserved protein PhoU, which is implicated in bacterial persistence, does not regulate PhoB activity, as previously suggested. Instead, our results favor a model in which PhoU affects intracellular phosphate accumulation, possibly through the high-affinity phosphate transporter., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Although DSB repair via homologous recombination has been well characterized, the spatial organization of this process inside cells remains poorly understood, and the mechanisms used for chromosome resegregation after repair are unclear. In this paper, we introduced site-specific DSBs in Caulobacter crescentus and then used time-lapse microscopy to visualize the ensuing chromosome dynamics. Damaged loci rapidly mobilized after a DSB, pairing with their homologous partner to enable repair, before being resegregated to their original cellular locations, independent of DNA replication. Origin-proximal regions were resegregated by the ParABS system with the ParA structure needed for resegregation assembling dynamically in response to the DSB-induced movement of an origin-associated ParB away from one cell pole. Origin-distal regions were resegregated in a ParABS-independent manner and instead likely rely on a physical, spring-like force to segregate repaired loci. Collectively, our results provide a mechanistic basis for the resegregation of chromosomes after a DSB., (© 2015 Badrinarayanan et al.)

SMC condensin complexes play a central role in compacting and resolving replicated chromosomes in virtually all organisms, yet how they accomplish this remains elusive. In Bacillus subtilis, condensin is loaded at centromeric parS sites, where it encircles DNA and individualizes newly replicated origins. Using chromosome conformation capture and cytological assays, we show that condensin recruitment to origin-proximal parS sites is required for the juxtaposition of the two chromosome arms. Recruitment to ectopic parS sites promotes alignment of large tracks of DNA flanking these sites. Importantly, insertion of parS sites on opposing arms indicates that these "zip-up" interactions only occur between adjacent DNA segments. Collectively, our data suggest that condensin resolves replicated origins by promoting the juxtaposition of DNA flanking parS sites, drawing sister origins in on themselves and away from each other. These results are consistent with a model in which condensin encircles the DNA flanking its loading site and then slides down, tethering the two arms together. Lengthwise condensation via loop extrusion could provide a generalizable mechanism by which condensin complexes act dynamically to individualize origins in B. subtilis and, when loaded along eukaryotic chromosomes, resolve them during mitosis., (© 2015 Wang et al.; Published by Cold Spring Harbor Laboratory Press.)

Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

Bacteria sense and respond to numerous environmental signals through two-component signaling pathways. Typically, a given stimulus will activate a sensor histidine kinase to autophosphorylate and then phosphotransfer to a cognate response regulator, which can mount an appropriate response. Although these signaling pathways often appear to be simple switches, they can also orchestrate surprisingly sophisticated and complex responses. These temporal dynamics arise from several key regulatory features, including the bifunctionality of histidine kinases as well as positive and negative feedback loops. Two-component signaling pathways are also dynamic on evolutionary time-scales, expanding dramatically in many species through gene duplication and divergence. Here, we review recent work probing the temporal and evolutionary dynamics of two-component signaling systems., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mapping protein sequence space is a difficult problem that necessitates the analysis of 20(N) combinations for sequences of length N. We systematically mapped the sequence space of four key residues in the Escherichia coli protein kinase PhoQ that drive recognition of its substrate PhoP. We generated a library containing all 160,000 variants of PhoQ at these positions and used a two-step selection coupled to next-generation sequencing to identify 1659 functional variants. Our results reveal extensive degeneracy in the PhoQ-PhoP interface and epistasis, with the effect of individual substitutions often highly dependent on context. Together, epistasis and the genetic code create a pattern of connectivity of functional variants in sequence space that likely constrains PhoQ evolution. Consequently, the diversity of PhoQ orthologs is substantially lower than that of functional PhoQ variants., (Copyright © 2015, American Association for the Advancement of Science.)

If fully stretched out, a typical bacterial chromosome would be nearly 1 mm long, approximately 1,000 times the length of a cell. Not only must cells massively compact their genetic material, but they must also organize their DNA in a manner that is compatible with a range of cellular processes, including DNA replication, DNA repair, homologous recombination, and horizontal gene transfer. Recent work, driven in part by technological advances, has begun to reveal the general principles of chromosome organization in bacteria. Here, drawing on studies of many different organisms, we review the emerging picture of how bacterial chromosomes are structured at multiple length scales, highlighting the functions of various DNA-binding proteins and the impact of physical forces. Additionally, we discuss the spatial dynamics of chromosomes, particularly during their segregation to daughter cells. Although there has been tremendous progress, we also highlight gaps that remain in understanding chromosome organization and segregation.

In all organisms, chromosomal DNA must be compacted nearly three orders of magnitude to fit within the limited volume of a cell. However, chromosomes cannot be haphazardly packed, and instead must adopt structures compatible with numerous cellular processes, including DNA replication, chromosome segregation, recombination, and gene expression. Recent technical advances have dramatically enhanced our understanding of how chromosomes are organized in vivo and have begun to reveal the mechanisms and forces responsible. Here, we review the current arsenal of techniques used to query chromosome structure, focusing first on single-cell fluorescence microscopy approaches that directly examine chromosome structure and then on population-averaged biochemical methods that infer chromosome structure based on the interaction frequencies of different loci. We describe the power of these techniques, highlighting the major advances they have produced while also discussing their limitations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Genetic memory enables the recording of information in the DNA of living cells. Memory can record a transient environmental signal or cell state that is then recalled at a later time. Permanent memory is implemented using irreversible recombinases that invert the orientation of a unit of DNA, corresponding to the [0,1] state of a bit. To expand the memory capacity, we have applied bioinformatics to identify 34 phage integrases (and their cognate attB and attP recognition sites), from which we build 11 memory switches that are perfectly orthogonal to each other and the FimE and HbiF bacterial invertases. Using these switches, a memory array is constructed in Escherichia coli that can record 1.375 bytes of information. It is demonstrated that the recombinases can be layered and used to permanently record the transient state of a transcriptional logic gate.

Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage., Competing Interests: The authors have declared that no competing interests exist.

Toxin-antitoxin (TA) systems are ubiquitous on bacterial chromosomes, yet the mechanisms regulating their activity and the molecular targets of toxins remain incompletely defined. Here, we identify SocAB, an atypical TA system in Caulobacter crescentus. Unlike canonical TA systems, the toxin SocB is unstable and constitutively degraded by the protease ClpXP; this degradation requires the antitoxin, SocA, as a proteolytic adaptor. We find that the toxin, SocB, blocks replication elongation through an interaction with the sliding clamp, driving replication fork collapse. Mutations that suppress SocB toxicity map to either the hydrophobic cleft on the clamp that binds DNA polymerase III or a clamp-binding motif in SocB. Our findings suggest that SocB disrupts replication by outcompeting other clamp-binding proteins. Collectively, our results expand the diversity of mechanisms employed by TA systems to regulate toxin activity and inhibit bacterial growth, and they suggest that inhibiting clamp function may be a generalizable antibacterial strategy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chromosomes must be highly compacted and organized within cells, but how this is achieved in vivo remains poorly understood. We report the use of chromosome conformation capture coupled with deep sequencing (Hi-C) to map the structure of bacterial chromosomes. Analysis of Hi-C data and polymer modeling indicates that the Caulobacter crescentus chromosome consists of multiple, largely independent spatial domains that are probably composed of supercoiled plectonemes arrayed into a bottle brush-like fiber. These domains are stable throughout the cell cycle and are reestablished concomitantly with DNA replication. We provide evidence that domain boundaries are established by highly expressed genes and the formation of plectoneme-free regions, whereas the histone-like protein HU and SMC (structural maintenance of chromosomes) promote short-range compaction and the colinearity of chromosomal arms, respectively. Collectively, our results reveal general principles for the organization and structure of chromosomes in vivo.

Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, Escherichia coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes and a systematic mutagenesis study reveal how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions and protein evolution and for the design of novel protein interfaces., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The decision to initiate DNA replication is a critical step in the cell cycle of all organisms. Cells often delay replication in the face of stressful conditions, but the underlying mechanisms remain incompletely defined. Here, we demonstrate in Caulobacter crescentus that proteotoxic stress induces a cell-cycle arrest by triggering the degradation of DnaA, the conserved replication initiator. A depletion of available Hsp70 chaperone, DnaK, either through genetic manipulation or heat shock, induces synthesis of the Lon protease, which can directly degrade DnaA. Unexpectedly, we find that unfolded proteins, which accumulate following a loss of DnaK, also allosterically activate Lon to degrade DnaA, thereby ensuring a cell-cycle arrest. Our work reveals a mechanism for regulating DNA replication under adverse growth conditions. Additionally, our data indicate that unfolded proteins can actively and directly alter substrate recognition by cellular proteases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Bacteria frequently use two-component signal transduction pathways to sense and respond to environmental and intracellular stimuli. Upon receipt of a stimulus, a homodimeric sensor histidine kinase autophosphorylates and then transfers its phosphoryl group to a cognate response regulator. The autophosphorylation of histidine kinases has been reported to occur both in cis and in trans, but the molecular determinants dictating which mechanism is employed are unknown. Based on structural considerations, one model posits that the handedness of a loop at the base of the helical dimerization domain plays a critical role. Here, we tested this model by replacing the loop from Escherichia coli EnvZ, which autophosphorylates in trans, with the loop from three PhoR orthologs that autophosphorylate in cis. These chimeric kinases autophosphorylated in cis, indicating that this small loop is sufficient to determine autophosphorylation mechanism. Further, we report that the mechanism of autophosphorylation is conserved in orthologous sets of histidine kinases despite highly dissimilar loop sequences. These findings suggest that histidine kinases are under selective pressure to maintain their mode of autophosphorylation, but they can do so with a wide range of sequences., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Maintaining the faithful flow of information through signal transduction pathways is critical to the survival and proliferation of organisms. This problem is particularly challenging as many signaling proteins are part of large, paralogous families that are highly similar at the sequence and structural levels, increasing the risk of unwanted cross-talk. To detect environmental signals and process information, bacteria rely heavily on two-component signaling systems comprised of sensor histidine kinases and their cognate response regulators. Although most species encode dozens of these signaling pathways, there is relatively little cross-talk, indicating that individual pathways are well insulated and highly specific. Here, we review the molecular mechanisms that enforce this specificity. Further, we highlight recent studies that have revealed how these mechanisms evolve to accommodate the introduction of new pathways by gene duplication., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1-S transition involves the degradation of an essential DNA-binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1-S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1-S transition so that cells can properly activate CtrA-dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division., (© 2013 Blackwell Publishing Ltd.)

Determining which residues of a protein contribute to a specific function is a difficult problem. Analyses of amino acid covariation within a protein family can serve as a useful guide by identifying residues that are functionally coupled. Covariation analyses have been successfully used on several different protein families to identify residues that work together to promote folding, enable protein-protein interactions, or contribute to an enzymatic activity. Covariation is a statistical signal that can be measured in a multiple sequence alignment of homologous proteins. As sequence databases have expanded dramatically, covariation analyses have become easier and more powerful. In this chapter, we describe how functional covariation arises during the evolution of proteins and how this signal can be distinguished from various background signals. We discuss the basic methodology for performing amino acid covariation analysis, using bacterial two-component signal transduction proteins as an example. We provide practical suggestions for each step of the process including assembly of protein sequences, construction of a multiple sequence alignment, measurement of covariation, and analysis of results., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The production of asymmetric daughter cells is a hallmark of metazoan development and critical to the life cycle of many microbes, including the α-proteobacterium Caulobacter crescentus. For Caulobacter, every cell division is asymmetric, yielding daughter cells with different morphologies and replicative potentials. This asymmetry in daughter cell fate is governed by the response regulator CtrA, a transcription factor that can also bind and silence the origin of replication. CtrA activity is controlled by a complex regulatory circuit that includes several polarly localized histidine kinases. This circuit ensures differential activation of CtrA in daughter cells, leading to their asymmetric replicative potentials. Here, we review progress in elucidating the molecular mechanisms regulating CtrA and the role of cellular polarity in this process., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Signal transduction proteins are often multi-domain proteins that arose through the fusion of previously independent proteins. How such a change in the spatial arrangement of proteins impacts their evolution and the selective pressures acting on individual residues is largely unknown. We explored this problem in the context of bacterial two-component signalling pathways, which typically involve a sensor histidine kinase that specifically phosphorylates a single cognate response regulator. Although usually found as separate proteins, these proteins are sometimes fused into a so-called hybrid histidine kinase. Here, we demonstrate that the isolated kinase domains of hybrid kinases exhibit a dramatic reduction in phosphotransfer specificity in vitro relative to canonical histidine kinases. However, hybrid kinases phosphotransfer almost exclusively to their covalently attached response regulator domain, whose effective concentration exceeds that of all soluble response regulators. These findings indicate that the fused response regulator in a hybrid kinase normally prevents detrimental cross-talk between pathways. More generally, our results shed light on how the spatial properties of signalling pathways can significantly affect their evolution, with additional implications for the design of synthetic signalling systems., (© 2012 Blackwell Publishing Ltd.)

Orthologous proteins often harbor numerous substitutions, but whether these differences result from neutral or adaptive processes is usually unclear. To tackle this challenge, we examined the divergent evolution of a model bacterial signaling pathway comprising the kinase PhoR and its cognate substrate PhoB. We show that the specificity-determining residues of these proteins are typically under purifying selection but have, in α-proteobacteria, undergone a burst of diversification followed by extended stasis. By reversing mutations that accumulated in an α-proteobacterial PhoR, we demonstrate that these substitutions were adaptive, enabling PhoR to avoid crosstalk with a paralogous pathway that arose specifically in α-proteobacteria. Our findings demonstrate that duplication and the subsequent need to avoid crosstalk strongly influence signaling protein evolution. These results provide a concrete example of how system-wide insulation can be achieved postduplication through a surprisingly limited number of mutations. Our work may help explain the apparent ease with which paralogous protein families expanded in all organisms., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Heterogeneity within a clonal population of cells can increase survival in the face of environmental stress. In a recent issue of Science, Aldridge et al. (2012) demonstrate that cell division in mycobacteria is asymmetric, producing daughter cells that differ in size, growth rate, and susceptibility to antibiotics., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To exist in a wide range of environmental niches, bacteria must sense and respond to a variety of external signals. A primary means by which this occurs is through two-component signal transduction pathways, typically composed of a sensor histidine kinase that receives the input stimuli and then phosphorylates a response regulator that effects an appropriate change in cellular physiology. Histidine kinases and response regulators have an intrinsic modularity that separates signal input, phosphotransfer, and output response; this modularity has allowed bacteria to dramatically expand and diversify their signaling capabilities. Recent work has begun to reveal the molecular basis by which two-component proteins evolve. How and why do orthologous signaling proteins diverge? How do cells gain new pathways and recognize new signals? What changes are needed to insulate a new pathway from existing pathways? What constraints are there on gene duplication and lateral gene transfer? Here, we review progress made in answering these questions, highlighting how the integration of genome sequence data with experimental studies is providing major new insights.

Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In Caulobacter crescentus, phosphorylation of key regulators is coordinated with the second messenger cyclic di-GMP to drive cell-cycle progression and differentiation. The diguanylate cyclase PleD directs pole morphogenesis, while the c-di-GMP effector PopA initiates degradation of the replication inhibitor CtrA by the AAA+ protease ClpXP to license S phase entry. Here, we establish a direct link between PleD and PopA reliant on the phosphodiesterase PdeA and the diguanylate cyclase DgcB. PdeA antagonizes DgcB activity until the G1-S transition, when PdeA is degraded by the ClpXP protease. The unopposed DgcB activity, together with PleD activation, upshifts c-di-GMP to drive PopA-dependent CtrA degradation and S phase entry. PdeA degradation requires CpdR, a response regulator that delivers PdeA to the ClpXP protease in a phosphorylation-dependent manner. Thus, CpdR serves as a crucial link between phosphorylation pathways and c-di-GMP metabolism to mediate protein degradation events that irreversibly and coordinately drive bacterial cell-cycle progression and development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Complex regulatory circuits in biology are often built of simpler subcircuits or modules. In most cases, the functional consequences and evolutionary origins of modularity remain poorly defined., Results: Here, by combining single-cell microscopy with genetic approaches, we demonstrate that two separable modules independently govern the temporal and spatial control of DNA replication in the asymmetrically dividing bacterium Caulobacter crescentus. DNA replication control involves DnaA, which promotes initiation, and CtrA, which silences initiation. We show that oscillations in DnaA activity dictate the periodicity of replication while CtrA governs the asymmetric replicative fates of daughter cells. Importantly, we demonstrate that DnaA activity oscillates independently of CtrA., Conclusions: The genetic separability of spatial and temporal control modules in Caulobacter reflects their evolutionary history. DnaA is the central component of an ancient and phylogenetically widespread circuit that governs replication periodicity in Caulobacter and most other bacteria. By contrast, CtrA, which is found only in the asymmetrically dividing α-proteobacteria, was integrated later in evolution to enforce replicative asymmetry on daughter cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Following DNA damage, cells typically delay cell cycle progression and inhibit cell division until their chromosomes have been repaired. The bacterial checkpoint systems responsible for these DNA damage responses are incompletely understood. Here, we show that Caulobacter crescentus responds to DNA damage by coordinately inducing an SOS regulon and inhibiting the master regulator CtrA. Included in the SOS regulon is sidA (SOS-induced inhibitor of cell division A), a membrane protein of only 29 amino acids that helps to delay cell division following DNA damage, but is dispensable in undamaged cells. SidA is sufficient, when overproduced, to block cell division. However, unlike many other regulators of bacterial cell division, SidA does not directly disrupt the assembly or stability of the cytokinetic ring protein FtsZ, nor does it affect the recruitment of other components of the cell division machinery. Instead, we provide evidence that SidA inhibits division by binding directly to FtsW to prevent the final constriction of the cytokinetic ring.

Cellular asymmetry is critical to metazoan development and the life cycle of many microbes. In Caulobacter, cell cycle progression and the formation of asymmetric daughter cells depend on the polarly-localized histidine kinase CckA. How CckA is regulated and why activity depends on localization are unknown. Here, we demonstrate that the unorthodox kinase DivL promotes CckA activity and that the phosphorylated regulator DivK inhibits CckA by binding to DivL. Early in the cell cycle, CckA is activated by the dephosphorylation of DivK throughout the cell. However, in later stages, when phosphorylated DivK levels are high, CckA activation relies on polar localization with a DivK phosphatase. Localization thus creates a protected zone for CckA within the cell, without the use of membrane-enclosed compartments. Our results reveal the mechanisms by which CckA is regulated in a cell-type-dependent manner. More generally, our findings reveal how cells exploit subcellular localization to orchestrate sophisticated regulatory processes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Spatial asymmetry is crucial to development. One mechanism for generating asymmetry involves the localized synthesis of a key regulatory protein that diffuses away from its source, forming a spatial gradient. Although gradients are prevalent in eukaryotes, at both the tissue and intracellular levels, it is unclear whether gradients of freely diffusible proteins can form within bacterial cells given their small size and the speed of diffusion. Here, we show that the bacterium Caulobacter crescentus generates a gradient of the active, phosphorylated form of the master regulator CtrA, which directly regulates DNA replication. Using a combination of mathematical modeling, single-cell microscopy, and genetic manipulation, we demonstrate that this gradient is produced by the polarly localized phosphorylation and dephosphorylation of CtrA. Our data indicate that cells robustly establish the asymmetric fates of daughter cells before cell division causes physical compartmentalization. More generally, our results demonstrate that uniform protein abundance may belie gradients and other sophisticated spatial patterns of protein activity in bacterial cells.

We have evolved a robust two-component signal transduction pathway from a sensor kinase (SK) and non-partner response regulator (RR) that show weak cross-talk in vitro and no detectable cross-talk in vivo in wild-type strains. The SK, CpxA, is bifunctional, with both kinase and phosphatase activities for its partner RR. We show that by combining a small number of mutations in CpxA that individually increase phosphorylation of the non-partner RR OmpR, phosphatase activity against phospho-OmpR emerges. The resulting circuit also becomes responsive to input signal to CpxA. The effects of combining these mutations in CpxA appear to reflect complex intragenic interactions between multiple sites in the protein. However, by analyzing a simple model of two-component signaling, we show that the behavior can be explained by a monotonic change in a single parameter controlling protein-protein interaction strength. The results suggest one possible mode of evolution for two-component systems with bifunctional SKs whereby the remarkable properties and competing reactions that characterize these systems can emerge by combining mutations of the same effect.

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk., Competing Interests: The authors have declared that no competing interests exist.

Progression through the Caulobacter cell cycle is driven by the master regulator CtrA, an essential two-component signaling protein that regulates the expression of nearly 100 genes. CtrA is abundant throughout the cell cycle except immediately prior to DNA replication. However, the expression of CtrA-activated genes is generally restricted to S phase. We identify the conserved protein SciP (small CtrA inhibitory protein) and show that it accumulates during G1, where it inhibits CtrA from activating target genes. The depletion of SciP from G1 cells leads to the inappropriate induction of CtrA-activated genes and, consequently, a disruption of the cell cycle. Conversely, the ectopic synthesis of SciP is sufficient to inhibit CtrA-dependent transcription, also disrupting the cell cycle. SciP binds directly to CtrA without affecting stability or phosphorylation; instead, SciP likely prevents CtrA from recruiting RNA polymerase. CtrA is thus tightly regulated by a protein-protein interaction which is critical to cell-cycle progression., (Copyright 2010 Elsevier Inc. All rights reserved.)

In a developmental strategy designed to efficiently exploit and colonize sparse oligotrophic environments, Caulobacter crescentus cells divide asymmetrically, yielding a motile swarmer cell and a sessile stalked cell. After a relatively fixed time period under typical culture conditions, the swarmer cell differentiates into a replicative stalked cell. Since differentiation into the stalked cell type is irreversible, it is likely that environmental factors such as the availability of essential nutrients would influence the timing of the decision to abandon motility and adopt a sessile lifestyle. We measured two different parameters in nutrient-limited chemostat cultures, biomass concentration and the ratio of nonstalked to stalked cells, over a range of flow rates and found that nitrogen limitation significantly extended the swarmer cell life span. The transcriptional profiling experiments described here generate the first comprehensive picture of the global regulatory strategies used by an oligotroph when confronted with an environment where key macronutrients are sparse. The pattern of regulated gene expression in nitrogen- and carbon-limited cells shares some features in common with most copiotrophic organisms, but critical differences suggest that Caulobacter, and perhaps other oligotrophs, have evolved regulatory strategies to deal distinctly with their natural environments. We hypothesize that nitrogen limitation extends the swarmer cell lifetime by delaying the onset of a sequence of differentiation events, which when initiated by the correct combination of external environmental cues, sets the swarmer cell on a path to differentiate into a stalked cell within a fixed time period.

In Caulobacter crescentus, progression through the cell cycle is governed by the periodic activation and inactivation of the master regulator CtrA. Two phosphorelays, each initiating with the histidine kinase CckA, promote CtrA activation by driving its phosphorylation and by inactivating its proteolysis. Here, we examined whether the CckA phosphorelays also influence the downregulation of CtrA. We demonstrate that CckA is bifunctional, capable of acting as either a kinase or phosphatase to drive the activation or inactivation, respectively, of CtrA. By identifying mutations that uncouple these two activities, we show that CckA's phosphatase activity is important for downregulating CtrA prior to DNA replication initiation in vivo but that other phosphatases may exist. Our results demonstrate that cell cycle transitions in Caulobacter require and are likely driven by the toggling of CckA between its kinase and phosphatase states. More generally, our results emphasize how the bifunctional nature of histidine kinases can help switch cells between mutually exclusive states.

Vibrio cholerae causes the disease cholera and inhabits aquatic environments. One key factor in the environmental survival of V. cholerae is its ability to form matrix-enclosed, surface-associated microbial communities known as biofilms. Mature biofilms rely on Vibrio polysaccharide to connect cells to each other and to a surface. We previously described a core regulatory network, which consists of two positive transcriptional regulators, VpsR and VpsT, and a negative transcriptional regulator HapR, that controls biofilm formation by regulating the expression of vps genes. In this study, we report the identification of a sensor histidine kinase, VpsS, which can control biofilm formation and activates the expression of vps genes. VpsS required the response regulator VpsR to activate vps expression. VpsS is a hybrid sensor histidine kinase that is predicted to contain both histidine kinase and response regulator domains, but it lacks a histidine phosphotransferase (HPT) domain. We determined that VpsS acts through the HPT protein LuxU, which is involved in a quorum-sensing signal transduction network in V. cholerae. In vitro analysis of phosphotransfer relationships revealed that LuxU can specifically reverse phosphotransfer to CqsS, LuxQ, and VpsS. Furthermore, mutational and phenotypic analyses revealed that VpsS requires the response regulator LuxO to activate vps expression, and LuxO positively regulates the transcription of vpsR and vpsT. The induction of vps expression via VpsS was also shown to occur independent of HapR. Thus, VpsS utilizes components of the quorum-sensing pathway to modulate biofilm formation in V. cholerae.

Two-component signal transduction systems are the predominant means by which bacteria sense and respond to environmental stimuli. Bacteria often employ tens or hundreds of these paralogous signaling systems, comprised of histidine kinases (HKs) and their cognate response regulators (RRs). Faithful transmission of information through these signaling pathways and avoidance of detrimental crosstalk demand exquisite specificity of HK-RR interactions. To identify the determinants of two-component signaling specificity, we examined patterns of amino acid coevolution in large, multiple sequence alignments of cognate kinase-regulator pairs. Guided by these results, we demonstrate that a subset of the coevolving residues is sufficient, when mutated, to completely switch the substrate specificity of the kinase EnvZ. Our results shed light on the basis of molecular discrimination in two-component signaling pathways, provide a general approach for the rational rewiring of these pathways, and suggest that analyses of coevolution may facilitate the reprogramming of other signaling systems and protein-protein interactions.

The two-component phosphorylation network is of critical importance for bacterial growth and physiology. Here, we address plasticity and interconnection of distinct signal transduction pathways within this network. In Caulobacter crescentus antagonistic activities of the PleC phosphatase and DivJ kinase localized at opposite cell poles control the phosphorylation state and subcellular localization of the cell fate determinator protein DivK. We show that DivK functions as an allosteric regulator that switches PleC from a phosphatase into an autokinase state and thereby mediates a cyclic di-GMP-dependent morphogenetic program. Through allosteric activation of the DivJ autokinase, DivK also stimulates its own phosphorylation and polar localization. These data suggest that DivK is the central effector of an integrated circuit that operates via spatially organized feedback loops to control asymmetry and cell fate determination in C. crescentus. Thus, single domain response regulators can facilitate crosstalk, feedback control, and long-range communication among members of the two-component network.