Robert B. Bourret

Bacterial motility and biofilm formation are essential for the adaptation and survival of Vibrio cholerae , the causative agent of cholera. In many bacterial species, the second messenger c-di-GMP regulates these processes through PilZ domain proteins, however the downstream mechanisms have remained poorly defined. Here, we identify the PilZ domain protein PlzC as a key positive regulator of motility and biofilm formation through distinct mechanisms. A suppressor screen for mutants restoring Δ plzC migration identified downstream regulators, including CheX, a CheY-3 phosphatase. Genetic and phenotypic analyses revealed that PlzC promotes motility by modulating CheX, thereby influencing the frequency of direction changes during swimming. Notably, the role of PlzC in motility regulation is independent of CheZ, another CheY-3 phosphatase, demonstrating that among the two CheY-3 phosphatases, only CheX is under PlzC control. This distinction suggests that CheY-3-P levels are regulated by at least two separate signaling pathways, one of which operates through PlzC. Despite its function in motility, CheX is not required for PlzC-mediated biofilm regulation, indicating pathway specificity. PlzC regulates biofilm formation through a mechanism that involves c-di-GMP binding, separating it from its CheX-dependent motility regulation. Together, our findings establish PlzC as a central regulator linking CheX-mediated motility and c-di-GMP signaling, thereby impacting motility and biofilm formation in V. cholerae .

Two-component regulatory systems typically consist of a sensor kinase and a response regulator. Phosphorylation of the receiver domain controls response regulator activity. Pseudo-receivers (PsRs) are identified computationally as receivers but lack key residues to catalyze phosphotransfer reactions. Although PsRs are common, molecular mechanisms that activate and inactivate bacterial PsRs remain a mystery. We untangled four potentially related but distinct concepts: bacterial PsRs, receivers with regulatory mechanisms in addition to phosphorylation, receivers that are active without phosphorylation, and orphan receivers without an obvious partner sensor kinase. We also analyzed bacterial PsR sequences and structures to identify regions of likely functional significance.

Two-component regulatory systems typically consist of a sensor kinase and a response regulator. All response regulators contain a receiver domain; most also contain an output domain. Response regulator activity is controlled by the phosphorylation state of the receiver. Receivers contain five conserved active site residues that catalyze phosphorylation and dephosphorylation reactions. Some protein domains identified computationally as receivers (PF00072) lack one or more of the five key conserved residues and are termed pseudoreceivers (PsRs). Because receivers are among the most abundant protein domains in nature, PsRs are also common. PsRs are especially common in plants, where they control circadian rhythms. Little is known about PsRs in fungi and archaea. Here, we focused on bacterial PsRs. We created representative datasets of 9,153 PsR and 143,116 true receiver domain sequences from bacteria. Comparison of amino acid composition of PsR and true receiver domains at each position showed (i) many differences at positions known to be important for phosphorylation-mediated signaling in true receiver domains, consistent with diminished importance in PsRs; and (ii) greatest differences between PsR and true receiver domains in the β3α3 and β4α4 loops, potentially highlighting functionally important regions of PsRs. We also performed covariation analyses of PsR and true receiver domains, which suggested six networks of linked residues that may be important for PsR function. Our analyses lay the foundation for rational experimental approaches to investigate molecular mechanisms of signaling by bacterial PsRs.

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.

The rapid increase of ‘-omics' data warrants the reconsideration of experimental strategies to investigate general protein function. Studying individual members of a protein family is likely insufficient to provide a complete mechanistic understanding of family functions, especially for diverse families with thousands of known members. Strategies that exploit large amounts of available amino acid sequence data can inspire and guide biochemical experiments, generating broadly applicable insights into a given family. Here we review several methods that utilize abundant sequence data to focus experimental efforts and identify features truly representative of a protein family or domain. First, coevolutionary relationships between residues within primary sequences can be successfully exploited to identify structurally and/or functionally important positions for experimental investigation. Second, functionally important variable residue positions typically occupy a limited sequence space, a property useful for guiding biochemical characterization of the effects of the most physiologically and evolutionarily relevant amino acids. Third, amino acid sequence variation within domains shared between different protein families can be used to sort a particular domain into multiple subtypes, inspiring further experimental designs. Although generally applicable to any kind of protein domain because they depend solely on amino acid sequences, the second and third approaches are reviewed in detail because they appear to have been used infrequently and offer immediate opportunities for new advances. Finally, we speculate that future technologies capable of analyzing and manipulating conserved and variable aspects of the three-dimensional structures of a protein family could lead to broad insights not attainable by current methods.

2 SUMMARY: Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against the pertussis toxin but does not prevent transmission or colonization. Cases of B. pertussis infections are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past three days in the lung, suggesting PlrSR works in a phosphorylation dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 and Glu522 were essential for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS can both phosphotransfer to and exert phosphatase activity towards PlrR. Unexpectedly, PlrR forms a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics. 10 GRAPHICAL

The ability to control locomotion in a dynamic environment provides a competitive advantage for microorganisms, thus driving the evolution of sophisticated regulatory systems. In total, 19 known categories of chemotaxis systems control motility mediated by flagella or Type IV pili, plus other cellular functions. A key feature that distinguishes chemotaxis systems from generic two‐component regulatory systems is separation of receptor and kinase functions into distinct proteins, linked by CheW scaffold proteins. This arrangement allows for formation of varied arrays with remarkable signaling properties. We recently analyzed sequences of CheW‐like domains found in CheA kinases and CheW and CheV scaffold proteins. In total, 16 Architectures of CheA, CheW, and CheV proteins contain ~94% of all CheW‐like domains and form six Classes with likely functional specializations. We surveyed chemotaxis system categories and proteins containing CheW‐like domains in ~1900 prokaryotic species, the most comprehensive analysis to date, revealing new insights. Co‐occurrence analyses suggested that many chemotaxis systems occur in non‐random combinations within species, implying synergy or antagonism. Furthermore, many Architectures of proteins containing CheW‐like domains occurred predominantly with specific categories of chemotaxis systems, suggesting specialized functional interactions. We propose Class 1 (~80%) and Class 6 (~20%) CheW proteins exhibit preferences for distinct chemoreceptor structures. Furthermore, rare (~1%) Class 2 CheW proteins frequently co‐occurred with methyl‐accepting coiled coil proteins, which contain both receptor and kinase functions and so do not require connection via a CheW scaffold but may benefit from arrays. Last, rare multidomain CheW proteins may interact with different receptors than single‐domain CheW proteins.

Domains are the three-dimensional building blocks of proteins. An individual domain can occur in a variety of domain architectures that perform unique functions and are subject to different evolutionary selective pressures. We describe an approach to evaluate the variability in amino add sequences of a single domain across architectural contexts. The ability to distinguish different evolutionary outcomes of one protein domain can help determine whether existing knowledge about a specific domain will apply to an uncharacterized protein, lead to insights and hypotheses about function, and guide experimental priorities. We developed and tested our approach on CheW-like domains (PF01584), which mediate protein/protein interactions and are difficult to compare experimentally. CheW-like domains occur in CheW scaffolding proteins, CheA kinases, and CheV proteins that regulate bacterial chemotaxis. We analyzed 16 domain architectures that included 94% of all CheW-like domains found in nature. We identified six Classes of CheW-like domains with presumed functional differences. CheV and most CheW proteins contained Class 1 domains, whereas some CheW proteins contained Class 6 (similar to 20%) or Class 2 (similar to 1%) domains instead. Most CheA proteins contained Class 3 domains. CheA proteins with multiple Hpt domains contained Class 4 domains. CheA proteins with two CheW-like domains contained one Class 3 and one Class 5. We also created SimpLogo, an innovative method for visualizing amino acid composition across large sets of multiple sequence alignments of arbitrary length. SimpLogo offers substantial advantages over standard sequence logos for comparison and analysis of related protein sequences. The R package for SimpLogo is freely available.

Domains are the three-dimensional building blocks of proteins. An individual domain can occur in a variety of domain architectures that perform unique functions and are subject to different evolutionary selective pressures. We describe an approach to evaluate the variability in amino acid sequences of a single domain across architectural contexts. The ability to distinguish different evolutionary outcomes of one protein domain can help determine whether existing knowledge about a specific domain will apply to an uncharacterized protein, lead to insights and hypotheses about function, and guide experimental priorities. We developed and tested our approach on CheW-like domains (PF01584), which mediate protein/protein interactions and are difficult to compare experimentally. CheW-like domains occur in CheW scaffolding proteins, CheA kinases, and CheV proteins that regulate bacterial chemotaxis. We analyzed 16 domain architectures that included 94% of all CheW-like domains found in nature. We identified six Classes of CheW-like domains with presumed functional differences. CheV and most CheW proteins contained Class 1 domains, whereas some CheW proteins contained Class 6 (∼20%) or Class 2 (∼1%) domains instead. Most CheA proteins contained Class 3 domains. CheA proteins with multiple Hpt domains contained Class 4 domains. CheA proteins with two CheW-like domains contained one Class 3 and one Class 5. We also created SimpLogo, an innovative method for visualizing amino acid composition across large sets of multiple sequence alignments of arbitrary length. SimpLogo offers substantial advantages over standard sequence logos for comparison and analysis of related protein sequences. The R package for SimpLogo is freely available.

The ability to control locomotion in a dynamic environment provides a competitive advantage for microorganisms, thus driving the evolution of sophisticated regulatory systems. Nineteen known categories of chemotaxis systems control motility mediated by flagella or Type IV pili, plus other cellular functions. A key feature that distinguishes chemotaxis systems from generic two-component regulatory systems is separation of receptor and kinase functions into distinct proteins, linked by CheW scaffold proteins. This arrangement allows for formation of varied arrays with remarkable signaling properties. We recently analyzed sequences of CheW-like domains found in CheA kinases and CheW and CheV scaffold proteins. Sixteen Architectures of CheA, CheW, and CheV proteins contain ∼94% of all CheW-like domains and form six Classes with likely functional specializations. We surveyed chemotaxis system categories and proteins containing CheW-like domains in ∼1900 prokaryotic species, the most comprehensive analysis to date, revealing new insights. Co-occurrence analyses suggested that many chemotaxis systems occur in non-random combinations within species, implying synergy or antagonism. Furthermore, many Architectures of proteins containing CheW-like domains occurred predominantly with specific categories of chemotaxis systems, suggesting specialized functional interactions. We propose Class 1 (∼80%) and Class 2 (∼20%) CheW proteins exhibit preferences for distinct chemoreceptor structures. Furthermore, rare (∼1%) Class 6 CheW proteins frequently co-occurred with methyl-accepting coiled coil (MAC) proteins, which contain both receptor and kinase functions and so do not require connection via a CheW scaffold but may benefit from arrays. Lastly, rare multi-domain CheW proteins may interact with different receptors than single domain CheW proteins.