Browsing by Author "Scharf, Birgit"

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    Characterizing a Small Regulatory RNA in Brucella abortus Linked to Outer Membrane Stress Resistance
    Stoyanof, Stephen Tristan (Virginia Tech, 2023-12-14)
    Brucella abortus is a bacterial species that infects cattle, elk, and bison herds worldwide and is a causative agent of brucellosis. B. abortus is a common form of zoonosis, as incidental spillover into the human population results in millions of infections annually. Current treatment options are limited to culling infected animals and treating humans with a rigorous antibiotic regimen, which still results in up to a 30% relapse rate. Detection of the pathogen is difficult due to the replicative niche residing within the host's immune cells, specifically macrophages and dendritic cells. Numerous small regulatory RNAs (sRNAs) were found to be expressed by B. abortus, and it was hypothesized that they may be important for virulence. One sRNA, when deleted, was shown to be linked to outer membrane stress resistance and was named MssR (membrane sensitivity sRNA). When the ΔmssR strain was tested in both macrophage and mouse models of infection, there were no virulence defects. Additionally, proteomic and transcriptomic studies of the ΔmssR strain showed very few dysregulated targets. Expression of mssR was tested under numerous biologically relevant conditions, and it was shown to be expressed significantly more during exponential phase of growth, compared to stationary phase. Initial microscopical analysis of mutant cells after treatment with sodium dodecyl sulfate (SDS_ did not reveal any morphological differences. It is unknown what contributes to the observed phenotypes and additional experiments are required to determine what is causing the perturbations in the outer membrane of the ΔmssR strain.
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    Discovery and Characterization of a Novel Regulatory Small RNA, VcrS, Required for Virulence in Brucella abortus
    King, Kellie Alexandra (Virginia Tech, 2022-02-01)
    Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses, such as acidic conditions and reactive oxygen species, as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation through the use of small regulatory RNAs (sRNAs). sRNAs bind to messenger RNA (mRNA) targets via complementary base pairing. sRNAs are a class of regulatory molecules in bacteria that elicit rapid post-transcriptional regulation. sRNA-mRNA binding can positively or negatively influence gene expression. Positive regulation can occur through sRNA binding to protect the mRNA from RNases. sRNA binding can also alleviate the secondary structure and reveal the ribosomal binding site. Alternatively, sRNA-mRNA interactions can have negative consequences on gene expression through degradation via RNases or sRNA binding can occlude the ribosomal binding site. Although some sRNAs have been discovered in B. abortus, few have been characterized in regards to virulence. In this study, B. abortus was stressed in conditions relevant to the macrophage, including, including low pH, oxidative stress, and nutrient limitation. Transcriptomic analysis revealed high levels of transcripts in intergenic regions, a hallmark of sRNAs, which led to the discovery of VcrS for virulence and cell wall regulating sRNA. A ΔvcrS was engineered and this mutant was used to infect both naïve murine macrophages, as well as BALB/c mice. Both virulence studies demonstrated significantly decreased bacterial recovery of ΔvcrS compared to the wildtype strain. Quantitative proteomics revealed that one protein, BAB1_1454, is 30-fold over-produced in ΔvcrS compared to wildtype. This essential protein encodes MurF, which catalyzes the final cytoplasmic step of generating the mura-pentapeptide precursor for peptidoglycan synthesis. VcrS is hypothesized to interact with murF mRNA and interfere with translation initiation. Sequence data indicates a putative 6 nucleotide motif in VcrS that has complementarity to the ribosomal binding site of murF. Identification of the binding site and further characterization of VcrS will showcase the importance of sRNA regulation in the virulence of B. abortus.
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    Harnessing Systems Bioengineering Approaches to Study Microbe-Microbe and Host-Microbe Interactions in Health and Disease
    Datla, Udaya Sree (Virginia Tech, 2024-03-22)
    The core of the dissertation lies in developing two novel systems bioengineering approaches, a synthetic Escherichia coli killer-prey microecology, and a combined infection-inflammation NET-array system, to investigate the role of the mechanochemical complexity of the microenvironment in driving the microbe-microbe and host-microbe interactions, respectively. Herein, the first part of the dissertation includes designing and engineering a synthetic E. coli killer-prey microecological system where we quantified the quorum-sensing mediated interactions between the engineered killer and prey E. coli bacterial strains plated on nutrient-rich media. In this work, we developed the plate assay followed by plasmid sequencing and computational modeling that emphasizes the concept of the constant evolution of species or acquired resistance in the prey E. coli, in the vicinity of the killer strain. We designed the microecological system such that the killer cells (dotted at the center of the plate) constitutively produce and secrete AHL quorum-sensing molecules into the microenvironment. AHL then diffuses into the prey cells (spread throughout the plate) and upregulates the expression of a protein that lyses the prey. Through time-lapse imaging on petri plates automated using a scanner, we recorded the "kill wave" that originates outside the killer colony and travels outward as the prey dies. We found that the prey population density surrounding the killer decreased in comparison to other locations on the plate far from the killer. However, some of the prey colonies evolve to be resistant to the effects of AHL secreted by the killer. These prey colonies resistant to the killer were then selected and confirmed by plasmid sequencing. Using this empirical data, we developed the first ecological model emphasizing the concept of the constant evolution of species, where the survival of the prey species is dependent on the location (distance from the killer) or the evolution of resistance. The importance of this work lies in the context of the evolution of antibiotic-resistant bacterial strains and in understanding the communication between the microbial consortia, such as in the gut microbiome. Further, the second part of the dissertation includes quantifying the interactions between immune cells (primary healthy human neutrophils) and motile Pseudomonas aeruginosa bacteria in an inflammation-rich microenvironment. Neutrophils, being the first responding immune cells to infection, defend by deploying various defense mechanisms either by phagocytosing and killing the pathogen intracellularly or through a suicidal mechanism of releasing their DNA to the extracellular space in the form of Neutrophil Extracellular Traps (NETs) to trap the invading pathogens. Although the release of NETs is originally considered a protective mechanism, it is shown to increase the inflammation levels in the host if unchecked, ultimately resulting in end-organ damage (especially lung and kidney damage), as with the severe cases of sepsis and COVID-19. In our work, we developed a combined infection-inflammation NET-array system integrated with a live imaging assay to quantify the spatiotemporal dynamics of NET release in response to P. aeruginosa infection in an inflammatory milieu at a single-cell resolution. Importantly, we found increased NET release to P. aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but not leukotriene B4 (LTB4), compared to the infection alone. Our device platform is unique in that the nanoliter well-assisted individual neutrophil trapping enables us to quantify NET release with single-cell precision. Besides, incorporating confined side loops in the device helped us study the role of mechanical confinement on NET release, showing reduced NET release from neutrophils confined in the side loops compared to the relatively wider chambers of our microsystem. In summary, our work emphasizes the importance of studying the heterogeneity of NET release in host defense and inflammation. In the future, our system can be used for screening novel neutrophil-based immunotherapies and serve as a valuable research tool in precision medicine.
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    Vibrio cholerae Bacteremia: An Enigma in Cholera-Endemic African Countries
    Agyei, Foster K.; Scharf, Birgit; Duodu, Samuel (MDPI, 2024-05-02)
    Cholera is highly endemic in many sub-Saharan African countries. The bacterium Vibrio cholerae is responsible for this severe dehydrating diarrheal disease that accounts for over 100,000 deaths each year globally. In recent years, the pathogen has been found to invade intestinal layers and translocate into the bloodstream of humans. The non-toxigenic strains of V. cholerae (non-O1/O139), also known as NOVC, which do not cause epidemic or pandemic cases of cholera, are the major culprits of V. cholerae bacteremia. In non-cholera-endemic regions, clinical reports on NOVC infection have been noted over the past few decades, particularly in Europe and America. Although low–middle-income countries are most susceptible to cholera infections because of challenges with access to clean water and inappropriate sanitation issues, just a few cases of V. cholerae bloodstream infections have been reported. The lack of evidence-based research and surveillance of V. cholerae bacteremia in Africa may have significant clinical implications. This commentary summarizes the existing knowledge on the host risk factors, pathogenesis, and diagnostics of NOVC bacteremia.
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    The interplay between pathogenic bacteria and bacteriophage Chi: New directions in motility and phage-host interactions in Enterobacterales
    Esteves, Nathaniel Carlos (Virginia Tech, 2024-04-15)
    The bacterial flagellum is a rotary motor that propels motile bacteria through their surroundings via swimming motility, or on surfaces via swarming motility. The flagellum is a key virulence factor for motile pathogenic bacteria. Viruses that infect bacteria via this appendage are known as flagellotropic or flagellum-dependent bacteriophages. Much like other phages, flagellotropic phages are of interest for clinical applications as antibacterial agents, particularly against multidrug resistant (MDR) bacteria. Bacteriophage χ is a flagellotropic phage that infects multiple species of motile pathogens. In the projects described below, we characterized several aspects of the complex interactions between χ and two of its hosts: Salmonella enterica and Serratia marcescens. In Chapter I, we describe in detail the existing knowledge on flagellum-dependent bacteriophages, pathogenic bacteria, and the flagellar motility system. We also expand significantly on flagellotropic phage χ. In Chapter II, we describe our discovery of S. enterica cellular components other than motility that are crucial for bacteriophage χ infection, making the key discovery that the AcrABZ-TolC multi-drug efflux system is required for infection to proceed. We additionally found that the host molecular chaperone trigger factor is important for the χ phage lifecycle. In Chapter III, we outline our characterization of the initial binding interaction between χ and the flagellum, determining that of flagellin's seven domains, C-terminal domain D2 is the most important for χ adsorption. In Chapter IV, we expand on this by discussing our work that determined that the χ tail fiber protein is encoded by the gene CHI_31, purification of this recombinantly-expressed protein, and demonstration of its direct interaction with the flagellar filament. Lastly, in Chapter V, our findings indicate that S. marcescens is able to detect χ infection and lysis in the surroundings and alter gene expression, resulting in an increase in the production of the red pigment prodigiosin. Overall, our hypothetical model for χ infection is as follows: χ binds to the flagellum of its host using its single tail fiber, composed of monomers of the CHI_31 gene product gp31. This tail fiber interacts with CTD2 of flagellin, and the rotation of the flagellum brings the phage to the cell surface, where it interacts with AcrABZ-TolC to inject its genetic material into the host cytoplasm. At some point during the process of production of phage particles and subsequent cell lysis, the host molecular chaperone trigger factor likely assists with proper folding of χ proteins. After cell lysis, cells in the surroundings are capable of detecting lysis and responding accordingly, at least in the case of S. marcescens. This research is clinically relevant for a number of reasons. Phage therapy, the use of bacteriophages as antibacterial agents, requires knowledge of phage infection pathways for optimal implementation. The fact that the flagellum and a complex mediating MDR are both essential for χ infection leads to particular interest in χ for this application. Knowledge of the host-determining factors between χ and Salmonella may lead to the ability to alter the χ phage genome to target specific pathogenic Salmonella or Escherichia coli strains while avoiding disruption of beneficial bacterial communities.
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    The little engine that could: Characterization of noncanonical components in the speed-variable flagellar motor of the symbiotic soil bacterium Sinorhizobium meliloti
    Sobe, Richard Charles (Virginia Tech, 2022-06-07)
    The bacterial flagellum is a fascinating corkscrew-shaped macromolecular rotary machine used primarily to propel bacterial cells through their environment via the conversion of chemical potential energy into rotational power and thrust. Flagella are the principal targets of complex chemotaxis systems, which allow microbes to navigate their habitats to locate favorable conditions and avoid harmful ones by continuous sampling of environmental compounds and cues. Flagella serve as surface and temperature sensors, mediators of host cell adherence by bacterial pathogens and symbionts alike, and important virulence factors for disease-causing microbes. They play several essential roles in accelerating the foundational stages of biofilm formation, during which bacteria build highly intricate microbial communities with increased resistance to predation and environmental assaults. Flagellum-mediated chemotaxis has broad and impactful implications in fields of bioremediation, targeted drug delivery, bacterial-mediated cancer therapy and diagnostics, and cross-kingdom horizontal gene transfer. While the core structural and functional components of flagella have been well characterized in the closely related enteric bacteria, Escherichia coli and Salmonella typhimurium, major departures from this paradigm have been identified in other diverse species that merit further investigation. Many bacteria employ additional reinforcement modules to surround and stabilize their more powerful flagellar motors and provide increased contact points in the inner membrane, the peptidoglycan sacculus, and, in Gram-negative bacteria, the outer membrane. Additionally, the soil-dwelling bacterium Sinorhizobium meliloti exhibits marked distinctions in the regulation, structure, and function of its navigation systems. S. meliloti is a nitrogen-fixing symbiont of the agronomically valuable leguminous plant, Medicago sativa Lucerne, and uses its coupled chemotaxis and flagellar motility systems to search for host plant roots to colonize. Following root colonization, the bacterium converts to a nitrogen-fixing factory for the plant and the combined influences of this symbiosis can quadruple the yields of the host. This dissertation is aimed at delivering a thorough representative overview of the processes facilitating bacterial flagellum-mediated chemotaxis and motility. Chapter 1 describes the interplay between chemotaxis and flagellar motility pathways as well as the structure, function, and regulation of these systems in several model bacteria. Particular emphasis is placed on the comparison of flagellar systems from the soil-dwelling legume symbiont, Sinorhizobium meliloti with other model systems, and a brief introduction is provided for its primary counterpart, the agronomically valuable legume, Medicago sativa, more commonly referred to as alfalfa. Chapter 2 embodies the first report of a flagellar system to require two copies of a protein known as FliL for its function. FliL is found in all bacterial flagellar systems reported to date but is only essential for some to drive motility. The more conserved copy of the protein has retained the title of FliL and several experiments to assay the proficiency of flagellar motor function revealed that in the absence of FliL swimming is essentially abolished as is the presence of flagella on the cell body. Flagellar motor activity and swimming proficiency of mutants lacking the FliL-paralog MotF was nearly as abysmal as those without FliL but flagellation was essentially normal indicating distinct roles for the two proteins. FliL is implicated in initial stator recruitment to the motor while MotF was found to serve as a power or speed modulator. A model to accommodate and explain the roles of these proteins in the flagellar motor of S. meliloti is provided. Chapter 3 links a never-before characterized flagellar protein, currently named Orf23, to a role in promoting maximum swimming velocity and perhaps stator alignment with the rotor in a peptidoglycan-dependent manner. The loss of LdtR, a transcriptional regulator of peptidoglycan-modification genes, caused defects in swimming motility that are restored only by removal of Orf23 or by replacing a nonpolar glycine with a polar serine in the periphery of stator units. Bioinformatics analyses, immunoblotting, and membrane topology reporter assays revealed that Orf23 is likely embedded in the inner membrane and that the remainder of the protein extends into the periplasm. Building on findings from Chapter 2, Orf23 is anticipated to influence stator positioning through interactions with MotF, FliL, and/or stator units directly. The chapter is concluded with the description of future experiments aimed to more thoroughly characterize Orf23. Altogether, this work increases the depth and breadth of knowledge regarding the composition and function of the speed-variable bacterial flagellar motor. We have identified several components required for stator incorporation and function, as well as an accessory component that improves stator performance. A wise society will draw inspiration from these fascinating and powerful machines to inform new technologies to achieve modern goals including targeted drug delivery, bioremediation, and perhaps one day our own exploration.
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    Mathematical modeling of biological dynamics
    Li, Xiaochu (Virginia Tech, 2023-12-11)
    This dissertation unravels intricate biological dynamics in three distinct biological systems as the following. These studies combine mathematical models with experimental data to enhance our understanding of these complex processes. 1. Bipolar Spindle Assembly: Mitosis relies on the formation of a bipolar mitotic spindle, which ensures an even distribution of duplicated chromosomes to daughter cells. We address the issue of how the spindle can robustly recover bipolarity from the irregular forms caused by centrosome defects/perturbations. By developing a biophysical model based on experimental data, we uncover the mechanisms that guide the separation and/or clustering of centrosomes. Our model identifies key biophysical factors that play a critical role in achieving robust spindle bipolarization, when centrosomes initially organize a monopolar or multipolar spindle. These factors encompass force fluctuations between centrosomes, balance between repulsive and attractive inter-centrosomal forces, centrosome exclusion from the cell center, proper cell size and geometry, and limitation of the centrosome number. 2. Chromosome Oscillation: During mitotic metaphase, chromosomes align at the spindle equator in preparation for segregation, and form the metaphase plate. However, these chromosomes are not static; they exhibit continuous oscillations around the spindle equator. Notably, either increasing or decreasing centromeric stiffness in PtK1 cells can lead to prolonged metaphase chromosome oscillations. To understand this biphasic relationship, we employ a force-balance model to reveal how oscillation arises in the spindle, and how the amplitude and period of chromosome oscillations depend on the biological properties of spindle components, including centromeric stiffness. 3. Pattern Formation in Bacterial-Phage Systems: The coexistence of bacteriophages (phages) and their host bacteria is essential for maintaining microbial communities. In resource-limited environments, mobile bacteria actively move toward nutrient-rich areas, while phages, lacking mobility, infect these motile bacterial hosts and disperse spatially through them. We utilize a combination of experimental methods and mathematical modeling to explore the coexistence and co-propagation of lytic phages and their mobile host bacteria. Our mathematical model highlights the role of local nutrient depletion in shaping a sector-shaped lysis pattern in the 2D phage-bacteria system. Our model further shows that this pattern, characterized by straight radial boundaries, is a distinctive indicator of extended coexistence and co-propagation of bacteria and phages. Such patterns rely on a delicate balance among the intrinsic biological characteristics of phages and bacteria, which have likely arisen from the coevolution of cognate pairs of phages and bacteria.
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    Mathematical modeling of molecular mechanisms governing cell cycle progression in Caulobacter crescentus and differentiation of immune system progenitor cells
    Weston, Bronson Ray (Virginia Tech, 2021-02-01)
    Mathematical modeling of biological systems can be useful to reveal new insights into biological observations. Here we apply mathematical modeling to study the underlying molecular networks driving observed behaviors of two systems. First, we apply systems biology and dynamic systems theory techniques to reveal new insights into the process of hematopoiesis. More specifically, we search the literature to deduce the underlying molecular mechanism that drives cell fate determination in granulocyte-monocyte progenitor (GMP) cells that are exposed to various cytokines. By converting this molecular mechanism into a set of ordinary differential equations (ODEs), we acquired new insights into the behavior of differentiating GMP cells. Next, we explore the cell cycle of the model prokaryotic organism, Caulobacter crescentus. Caulobacter is a uniquely successful oligotrophic bacterium, found abundantly in freshwater systems. While it is not a pathogenic species, Caulobacter is extremely well studied due to its distinguishable asymmetrical morphology and the ability to synchronize populations by cell cycle stage. We built a detailed mathematical model of the molecular mechanism driving the cell cycle. This research suggests a previously unknown role for the unknown form of the master regulator, CtrA, in regulating the G1-S transition. Furthermore, we incorporate a nutrient signaling model into the cell cycle model to investigate how Caulobacter responds to nutrient deprivation. We find that regulation of DivK phosphorylation is an essential component of the nutrient signaling pathway and demonstrate how starvation signals work together in synergy to manifest in observed cell cycle response.
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    Similarities and variations of the enterobacterial chemotaxis paradigm in Sinorhizobium meliloti
    Agbekudzi, Alfred (Virginia Tech, 2023-12-21)
    Sinorhizobium meliloti is a nitrogen-fixing endosymbiont of the legume Medicago sativa commonly known as alfalfa. It uses flagellar rotation and chemotaxis to seek roots of host plants to inhabit. This symbiosis serves as a great model system for studying biological nitrogen fixation and plant-microbe interactions. Since alfalfa brings enormous economic value to the USA, investments into the knowledge of the chemotaxis process that initiates symbiosis have the ability to mitigate deterioration of the environment and significantly increase food supply. The chemotaxis system in the enteric bacteria Escherichia coli is well studied and has been a great resource to understanding the process in other bacterial systems including our model organism S. meliloti. This dissertation compares and contrasts the chemotaxis features in E. coli and S. meliloti and investigates their molecular functions. Based on the understanding gained so far, we attempt to offer plausible explanations for the underlying mechanisms of the S. meliloti chemotaxis pathway. Chapter 1 describes why biological nitrogen fixation is important for agriculture and the health of our environment. This chapter also sheds light on the symbiotic relationship between alfalfa and S. meliloti, which culminates in the formation of nitrogen fixing nodules. We expound on the chemotaxis systems in E. coli and other bacteria including S. meliloti and Bacillus subtilis. In chapter 2, we compare the distribution of C-terminal pentapeptide-bearing receptors and the adaptation proteins that they tether in E. coli and S. meliloti. The stoichiometry data show that the ratio of pentapeptide-bearing chemoreceptors to chemotaxis protein (Che)R and CheB molecules are approximately 500- and 160-fold higher in S. meliloti than in E. coli, respectively. Since not all chemoreceptors in chemotactic bacteria have and utilize the pentapeptide moiety, we investigated the S. meliloti system and observed a strong interaction between CheR, activated CheB and the isolated pentapeptides via in-vitro binding studies. On the contrary, unmodified CheB showed weak binding to the pentapeptide. Through in-vivo studies, we highlighted the physiological necessity of the pentapeptide for chemotaxis. S. meliloti strains with substitutions of the conserved tryptophan residue to alanine in one or all four pentapeptide-bearing Methyl-accepting Chemotaxis Proteins (MCPs) resulted in diminished or loss of chemotaxis to glycine betaine, lysine, and acetate, ligands sensed by pentapeptide-bearing McpX and pentapeptide-lacking McpU and McpV, respectively. The flexible linker connecting the pentapeptide to the MCPs together with the pentapeptide itself were shown to be functional on pentapeptide-lacking chemoreceptors and provided adaptational assistance to other chemoreceptors that lacked a functional pentapeptide. Based on these results, we concluded that S. meliloti employs a pentapeptide-dependent adaptation system with MCPs possessing a consensus pentapeptide motif (N/D)WE(E/N)F). Finally, we postulated that the higher abundance of CheR and CheB in S. meliloti compared to E. coli compensates for the lower number of pentapeptide-bearing chemoreceptors in the chemosensory array. In chapter 3, we explored the putative phosphatase function of a novel protein, CheT, on phosphorylated S. meliloti response regulators. The kinase CheA phosphorylates both the sink response regulator, CheY1, and the flagellar motor interacting response regulator, CheY2. CheY1 competes with CheY2 for these phosphate groups, but we have discovered another layer of complexity to the story. Sequence comparison of S. meliloti CheT and the E. coli phosphatase CheZ shows little sequence homology. However, both proteins share a DXXXQ phosphatase motif. Phosphorylation assays performed using radiolabeled [γ-32P]-ATP revealed that CheT acts as a phosphatase of CheY1~P and accelerates dephosphorylation of CheY1~P by at least two-fold. Interestingly, we also discovered that CheT interacts with CheR, but this interaction did not affect the enzymatic activity of either protein under the examined conditions. Unexpectedly, a cheT deletion strain and strains carrying mutations in the phosphatase motif exhibit an increased swimming speed, a phenotype that does not conform with the model that the absence of CheT or its activity results in increased CheY2~P levels and reduced swimming speed. We concluded that a revised S. meliloti signal termination pathway should include CheT enhancing dephosphorylation of CheY1~P and sensory adaptation involving the yet unknown function of CheT on CheR. While the adaptation system in S. meliloti is unexplored, this work provides first insights into fascinating deviations and similarities to the known paradigm. We have also delivered evidence that the S. meliloti signal termination system requires a dedicated phosphatase. The knowledge gained here takes us a step closer to enhance the S. meliloti chemotaxis pathway towards improved symbiosis with alfalfa and to reduce our dependence on environmentally deleterious synthetic fertilizers.
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    Structural Studies of the Bacterial Histidine Kinases RetS and GacS, Key Components of the Multikinase Network that Controls the Switch Between a Motile Invasive Lifestyle and a Sessile Biofilm Lifestyle in Pseudomonas aeruginosa
    Ryan, Kylie Meghan (Virginia Tech, 2021-11-15)
    Signal transduction networks enable organisms to respond to environmental stimuli. Bacteria utilize two-component systems (TCSs) and phosphorelays as their primary means of signal transduction. Histidine kinase (HK) and response regulator (RR) proteins comprise these TCSs and phosphorelays. Previously, signal transduction within TCSs and phosphorelays was thought to only occur through a linear series of phosphotransfers between HKs and RRs. Recently multikinase networks have been shown to be involved in TCS and phosphorelay signal transmission. A multikinase network that includes the HKs RetS and GacS controls the switch between the motile invasive lifestyle and the sessile biofilm lifestyle of the opportunistic human pathogen Pseudomonas aeruginosa. GacS promotes the sessile biofilm lifestyle, while RetS promotes the motile invasive lifestyle via the inhibition of GacS. This inhibition occurs through three distinct mechanisms. Two of the mechanisms are dephosphorylating mechanisms and the third mechanism is a direct interaction between RetS and GacS which results in the inhibition of GacS autophosphorylation. This study examines the direct binding interaction between RetS and GacS using structural biology. We observed a heterodimeric RetS-GacS complex in which the canonical homodimerization interface was replaced with a heterodimeric interface. Heterodimerization between bacterial HKs is currently a novel observation, but it is likely that other HKs heterodimerize. The RetS-GacS direct interaction can serve as a model for HK-HK binding in multikinase networks.