Regulatory interactions between daptomycin- and bacitracin-responsive pathways coordinate the cell envelope antibiotic resistance response of Enterococcus faecalis.

Enterococcal infections frequently show high levels of antibiotic resistance, including to cell envelope-acting antibiotics like daptomycin (DAP). While we have a good understanding of the resistance mechanisms, less is known about the control of such resistance genes in enterococci. Previous work unveiled a bacitracin resistance network, comprised of the sensory ABC transporter SapAB, the two-component system (TCS) SapRS and the resistance ABC transporter RapAB. Interestingly, components of this system have recently been implicated in DAP resistance, a role usually regulated by the TCS LiaFSR. To better understand the regulation of DAP resistance and how this relates to mutations observed in DAP-resistant clinical isolates of enterococci, we here explored the interplay between these two regulatory pathways. Our results show that SapR regulates an additional resistance operon, dltXABCD, a known DAP resistance determinant, and show that LiaFSR regulates the expression of sapRS. This regulatory structure places SapRS-target genes under dual control, where expression is directly controlled by SapRS, which itself is up-regulated through LiaFSR. The network structure described here shows how Enterococcus faecalis coordinates its response to cell envelope attack and can explain why clinical DAP resistance often emerges via mutations in regulatory components.

Cytokine Hemoadsorption During Cardiac Surgery Versus Standard Surgical Care for Infective Endocarditis (REMOVE): Results From a Multicenter Randomized Controlled Trial.

BACKGROUND: Cardiac surgery often represents the only treatment option in patients with infective endocarditis (IE). However, IE surgery may lead to a sudden release of inflammatory mediators, which is associated with postoperative organ dysfunction. We investigated the effect of hemoadsorption during IE surgery on postoperative organ dysfunction. METHODS: This multicenter, randomized, nonblinded, controlled trial assigned patients undergoing cardiac surgery for IE to hemoadsorption (integration of CytoSorb to cardiopulmonary bypass) or control. The primary outcome (change in sequential organ failure assessment score [ΔSOFA]) was defined as the difference between the mean total postoperative SOFA score, calculated maximally to the 9th postoperative day, and the basal SOFA score. The analysis was by modified intention to treat. A predefined intergroup comparison was performed using a linear mixed model for ΔSOFA including surgeon and baseline SOFA score as fixed effect covariates and with the surgical center as random effect. The SOFA score assesses dysfunction in 6 organ systems, each scored from 0 to 4. Higher scores indicate worsening dysfunction. Secondary outcomes were 30-day mortality, duration of mechanical ventilation, and vasopressor and renal replacement therapy. Cytokines were measured in the first 50 patients. RESULTS: Between January 17, 2018, and January 31, 2020, a total of 288 patients were randomly assigned to hemoadsorption (n=142) or control (n=146). Four patients in the hemoadsorption and 2 in the control group were excluded because they did not undergo surgery. The primary outcome, ΔSOFA, did not differ between the hemoadsorption and the control group (1.79±3.75 and 1.93±3.53, respectively; 95% CI, -1.30 to 0.83; P=0.6766). Mortality at 30 days (21% hemoadsorption versus 22% control; P=0.782), duration of mechanical ventilation, and vasopressor and renal replacement therapy did not differ between groups. Levels of interleukin-1ß and interleukin-18 at the end of integration of hemoadsorption to cardiopulmonary bypass were significantly lower in the hemoadsorption than in the control group. CONCLUSIONS: This randomized trial failed to demonstrate a reduction in postoperative organ dysfunction through intraoperative hemoadsorption in patients undergoing cardiac surgery for IE. Although hemoadsorption reduced plasma cytokines at the end of cardiopulmonary bypass, there was no difference in any of the clinically relevant outcome measures. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03266302.

Expansion and re-classification of the extracytoplasmic function (ECF) σ factor family.

Extracytoplasmic function σ factors (ECFs) represent one of the major bacterial signal transduction mechanisms in terms of abundance, diversity and importance, particularly in mediating stress responses. Here, we performed a comprehensive phylogenetic analysis of this protein family by scrutinizing all proteins in the NCBI database. As a result, we identified an average of ∼10 ECFs per bacterial genome and 157 phylogenetic ECF groups that feature a conserved genetic neighborhood and a similar regulation mechanism. Our analysis expands previous classification efforts ∼50-fold, enriches many original ECF groups with previously unclassified proteins and identifies 22 entirely new ECF groups. The ECF groups are hierarchically related to each other and are further composed of subgroups with closely related sequences. This two-tiered classification allows for the accurate prediction of common promoter motifs and the inference of putative regulatory mechanisms across subgroups composing an ECF group. This comprehensive, high-resolution description of the phylogenetic distribution of the ECF family, together with the massive expansion of classified ECF sequences and an openly accessible data repository called 'ECF Hub' (https://www.computational.bio.uni-giessen.de/ecfhub), will serve as a powerful hypothesis-generator to guide future research in the field.

Determinants of COVID-19 Disease Severity-Lessons from Primary and Secondary Immune Disorders including Cancer.

At the beginning of the COVID-19 pandemic, patients with primary and secondary immune disorders-including patients suffering from cancer-were generally regarded as a high-risk population in terms of COVID-19 disease severity and mortality. By now, scientific evidence indicates that there is substantial heterogeneity regarding the vulnerability towards COVID-19 in patients with immune disorders. In this review, we aimed to summarize the current knowledge about the effect of coexistent immune disorders on COVID-19 disease severity and vaccination response. In this context, we also regarded cancer as a secondary immune disorder. While patients with hematological malignancies displayed lower seroconversion rates after vaccination in some studies, a majority of cancer patients' risk factors for severe COVID-19 disease were either inherent (such as metastatic or progressive disease) or comparable to the general population (age, male gender and comorbidities such as kidney or liver disease). A deeper understanding is needed to better define patient subgroups at a higher risk for severe COVID-19 disease courses. At the same time, immune disorders as functional disease models offer further insights into the role of specific immune cells and cytokines when orchestrating the immune response towards SARS-CoV-2 infection. Longitudinal serological studies are urgently needed to determine the extent and the duration of SARS-CoV-2 immunity in the general population, as well as immune-compromised and oncological patients.

Symmetric activity of DNA polymerases at and recruitment of exonuclease ExoR and of PolA to the Bacillus subtilis replication forks.

DNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5' overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.

Toxic but tasty - temporal dynamics and network architecture of heme-responsive two-component signaling in Corynebacterium glutamicum.

Heme is an essential cofactor and alternative iron source for almost all bacterial species but may cause severe toxicity upon elevated levels and consequently, regulatory mechanisms coordinating heme homeostasis represent an important fitness trait. A remarkable scenario is found in several corynebacterial species, e.g. Corynebacterium glutamicum and Corynebacterium diphtheriae, which dedicate two paralogous, heme-responsive two-component systems, HrrSA and ChrSA, to cope with the Janus nature of heme. Here, we combined experimental reporter profiling with a quantitative mathematical model to understand how this particular regulatory network architecture shapes the dynamic response to heme. Our data revealed an instantaneous activation of the detoxification response (hrtBA) upon stimulus perception and we found that kinase activity of both kinases contribute to this fast onset. Furthermore, instant deactivation of the PhrtBA promoter is achieved by a strong ChrS phosphatase activity upon stimulus decline. While the activation of detoxification response is uncoupled from further factors, heme utilization is additionally governed by the global iron regulator DtxR integrating information on iron availability into the regulatory network. Altogether, our data provide comprehensive insights how TCS cross-regulation and network hierarchy shape the temporal dynamics of detoxification (hrtBA) and utilization (hmuO) as part of a global homeostatic response to heme.

Stringent response leads to continued cell division and a temporal restart of DNA replication after initial shutdown in Vibrio cholerae.

Vibrio cholerae is an aquatic bacterium with the potential to infect humans and cause the cholera disease. While most bacteria have single chromosomes, the V. cholerae genome is encoded on two replicons of different size. This study focuses on the DNA replication and cell division of this bi-chromosomal bacterium during the stringent response induced by starvation stress. V. cholerae cells were found to initially shut DNA replication initiation down upon stringent response induction by the serine analog serine hydroxamate. Surprisingly, cells temporarily restart their DNA replication before finally reaching a state with fully replicated single chromosome sets. This division-replication pattern is very different to that of the related single chromosome model bacterium Escherichia coli. Within the replication restart phase, both chromosomes of V. cholerae maintained their known order of replication timing to achieve termination synchrony. Using flow cytometry combined with mathematical modeling, we established that a phase of cellular regrowth be the reason for the observed restart of DNA replication after the initial shutdown. Our study shows that although the stringent response induction itself is widely conserved, bacteria developed different ways of how to react to the sensed nutrient limitation, potentially reflecting their individual lifestyle requirements.

The role of C-terminal extensions in controlling ECF σ factor activity in the widely conserved groups ECF41 and ECF42.

The activity of extracytoplasmic function σ-factors (ECFs) is typically regulated by anti-σ factors. In a number of highly abundant ECF groups, including ECF41 and ECF42, σ-factors contain fused C-terminal protein domains, which provide the necessary regulatory function instead. Here, we identified the contact interface between the C-terminal extension and the core σ-factor regions required for controlling ECF activity. We applied direct coupling analysis (DCA) to infer evolutionary covariation between contacting amino acid residues for groups ECF41 and ECF42. Mapping the predicted interactions to a recently solved ECF41 structure demonstrated that DCA faithfully identified an important contact interface between the SnoaL-like extension and the linker between the σ2 and σ4 domains. Systematic alanine substitutions of contacting residues support this model and suggest that this interface stabilizes a compact conformation of ECF41 with low transcriptional activity. For group ECF42, DCA supports a structural homology model for their C-terminal tetratricopeptide repeat (TPR) domains and predicts an intimate contact between the first TPR-helix and the σ4 domain. Mutational analyses demonstrate the essentiality of the predicted interactions for ECF42 activity. These results indicate that C-terminal extensions indeed bind and regulate the core ECF regions, illustrating the potential of DCA for discovering regulatory motifs in the ECF subfamily.

BceAB-Type Antibiotic Resistance Transporters Appear To Act by Target Protection of Cell Wall Synthesis.

Resistance against cell wall-active antimicrobial peptides in bacteria is often mediated by transporters. In low-GC-content Gram-positive bacteria, a common type of such transporters is BceAB-like systems, which frequently provide high-level resistance against peptide antibiotics that target intermediates of the lipid II cycle of cell wall synthesis. How a transporter can offer protection from drugs that are active on the cell surface, however, has presented researchers with a conundrum. Multiple theories have been discussed, ranging from removal of the peptides from the membrane and internalization of the drug for degradation to removal of the cellular target rather than the drug itself. To resolve this much-debated question, we here investigated the mode of action of the transporter BceAB of Bacillus subtilis We show that it does not inactivate or import its substrate antibiotic bacitracin. Moreover, we present evidence that the critical factor driving transport activity is not the drug itself but instead the concentration of drug-target complexes in the cell. Our results, together with previously reported findings, lead us to propose that BceAB-type transporters act by transiently freeing lipid II cycle intermediates from the inhibitory grip of antimicrobial peptides and thus provide resistance through target protection of cell wall synthesis. Target protection has so far only been reported for resistance against antibiotics with intracellular targets, such as the ribosome. However, this mechanism offers a plausible explanation for the use of transporters as resistance determinants against cell wall-active antibiotics in Gram-positive bacteria where cell wall synthesis lacks the additional protection of an outer membrane.

Vibrio natriegens: an ultrafast-growing marine bacterium as emerging synthetic biology chassis.

The marine bacterium Vibrio natriegens is the fastest-growing non-pathogenic bacterium known to date and is gaining more and more attention as an alternative chassis organism to Escherichia coli. A recent wave of synthetic biology efforts has focused on the establishment of molecular biology tools in this fascinating organism, now enabling exciting applications - from speeding up our everyday laboratory routines to increasing the pace of biotechnological production cycles. In this review, we seek to give a broad overview on the literature on V. natriegens, spanning all the way from its initial isolation to its latest applications. We discuss its natural ecological niche and interactions with other organisms, unveil some of its extraordinary traits, review its genomic organization and give insight into its diverse metabolism - key physiological insights required to further develop this organism into a synthetic biology chassis. By providing a comprehensive overview on the established genetic tools, methods and applications we highlight the current possibilities of this organism, but also identify some of the gaps that could drive future lines of research, hopefully stimulating the growth of the V. natriegens research community.

Engineering orthogonal synthetic timer circuits based on extracytoplasmic function σ factors.

The rational design of synthetic regulatory circuits critically hinges on the availability of orthogonal and well-characterized building blocks. Here, we focus on extracytoplasmic function (ECF) σ factors, which are the largest group of alternative σ factors and hold extensive potential as synthetic orthogonal regulators. By assembling multiple ECF σ factors into regulatory cascades of varying length, we benchmark the scalability of the approach, showing that these 'autonomous timer circuits' feature a tuneable time delay between inducer addition and target gene activation. The implementation of similar timers in Escherichia coli and Bacillus subtilis shows strikingly convergent circuit behavior, which can be rationalized by a computational model. These findings not only reveal ECF σ factors as powerful building blocks for a rational, multi-layered circuit design, but also suggest that ECF σ factors are universally applicable as orthogonal regulators in a variety of bacterial species.

Transporters as information processors in bacterial signalling pathways.

Transporters are essential players in bacterial growth and survival, since they are key for uptake of nutrients on the one hand, and for defence against endogenous and environmental stresses on the other hand. Remarkably, in addition to their primary role in substrate translocation, it has become clear that some transporters have acquired a secondary function as sensors and information processors in signalling pathways. In this review, we describe recent advances in our understanding of the role of transporters in such signalling cascades, and discuss some of the emergent dynamic behaviour found in hallmark examples. A particular focus is placed on new insights into mechanistic details of information transfer between transporters and regulatory proteins. Quantitative considerations reveal that these signalling complexes can implement a remarkable diversity of regulatory logic functions, where the transporter can act as activity switch, as positive or negative reporter of transport flux, or as a signalling hub for the integration of multiple inputs. Such a dual use of transport proteins not only enables efficient substrate translocation but is also an elegant strategy to integrate important information about the cell's external conditions with its current physiological state.

Single-Molecule Tracking of DNA Translocases in Bacillus subtilis Reveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA.

Like many bacteria, Bacillus subtilis possesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: an ftsA deletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzyme in vivo Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slow- and fast-moving molecules in live cells.IMPORTANCE DNA translocases couple the late events of chromosome segregation to cell division and thereby play an important role in the bacterial cell cycle. The proteins fall into one of two categories, integral membrane translocases or nonintegral translocases. We show that the membrane-bound translocase SpoIIIE moves slowly throughout the cell membrane in B. subtilis and does not show a clear association with the division septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell division across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the division septum as a component of the division machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the division machinery, but it is not the only factor that binds SftA. Our work represents a detailed in vivo study of DNA translocases at the single-molecule level.

Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo.

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory.

In-depth analysis of cost structure for electroconvulsive therapy in a performance-based hospital budget.

OBJECTIVE: New medical guideline recommendations for the treatment of major depressive disorders and regulative changes in the payment system of the German mental health care system warrant a revision of the framework in which electroconvulsive therapies (ECT) are offered. METHODS: A cost structure analysis of the clinical resources essential for the ECT procedure was conducted and economically validated, exemplified at a German inpatient ECT treatment center. RESULTS: The identification of directly attributable costs to the ECT intervention presupposes an accurate assessment of personnel engagement time and material consumption as well as an inclusion of overhead costs for the operational readiness of the hospital. CONCLUSION: The increasing importance of ECT in the clinical portfolio of therapy options demands an adequate refunding to support the expansion of this highly effective treatment. For the calculation of an appropriate reimbursement for ECT and ascertaining an acceptable contribution, a detailed knowledge of personnel costs and infrastructure settings of the respective hospitals is required.

Anatomy of the bacitracin resistance network in Bacillus subtilis.

Protection against antimicrobial peptides (AMPs) often involves the parallel production of multiple, well-characterized resistance determinants. So far, little is known about how these resistance modules interact and how they jointly protect the cell. Here, we studied the interdependence between different layers of the envelope stress response of Bacillus subtilis when challenged with the lipid II cycle-inhibiting AMP bacitracin. The underlying regulatory network orchestrates the production of the ABC transporter BceAB, the UPP phosphatase BcrC and the phage-shock proteins LiaIH. Our systems-level analysis reveals a clear hierarchy, allowing us to discriminate between primary (BceAB) and secondary (BcrC and LiaIH) layers of bacitracin resistance. Deleting the primary layer provokes an enhanced induction of the secondary layer to partially compensate for this loss. This study reveals a direct role of LiaIH in bacitracin resistance, provides novel insights into the feedback regulation of the Lia system, and demonstrates a pivotal role of BcrC in maintaining cell wall homeostasis. The compensatory regulation within the bacitracin network can also explain how gene expression noise propagates between resistance layers. We suggest that this active redundancy in the bacitracin resistance network of B. subtilis is a general principle to be found in many bacterial antibiotic resistance networks.

Single-cell characterization of metabolic switching in the sugar phosphotransferase system of Escherichia coli.

The utilization of several sugars in Escherichia coli is regulated by the Phosphotransferase System (PTS), in which diverse sugar utilization modules compete for phosphoryl flux from the general PTS proteins. Existing theoretical work predicts a winner-take-all outcome when this flux limits carbon uptake. To date, no experimental work has interrogated competing PTS uptake modules with single-cell resolution. Using time-lapse microscopy in perfused microchannels, we analyzed the competition between N-acetyl-glucosamine and sorbitol, as representative PTS sugars, by measuring both the expression of their utilization systems and the concomitant impact of sugar utilization on growth rates. We find two distinct regimes: hierarchical usage of the carbohydrates, and co-expression of the genes for both systems. Simulations of a mathematical model incorporating asymmetric sugar quality reproduce our metabolic phase diagram, indicating that under conditions of nonlimiting phosphate flux, co-expression is due to uncoupling of both sugar utilization systems. Our model reproduces hierarchical winner-take-all behaviour and stochastic co-expression, and predicts the switching between both strategies as a function of available phosphate flux. Hence, experiments and theory both suggest that PTS sugar utilization involves not only switching between the sugars utilized but also switching of utilization strategies to accommodate prevailing environmental conditions.

The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network.

The cell envelope stress response (CESR) encompasses all regulatory events that enable a cell to protect the integrity of its envelope, an essential structure of any bacterial cell. The underlying signaling network is particularly well understood in the Gram-positive model organism Bacillus subtilis. It consists of a number of two-component systems (2CS) and extracytoplasmic function σ factors that together regulate the production of both specific resistance determinants and general mechanisms to protect the envelope against antimicrobial peptides targeting the biogenesis of the cell wall. Here, we summarize the current picture of the B. subtilis CESR network, from the initial identification of the corresponding signaling devices to unraveling their interdependence and the underlying regulatory hierarchy within the network. In the course of detailed mechanistic studies, a number of novel signaling features could be described for the 2CSs involved in mediating CESR. This includes a novel class of so-called intramembrane-sensing histidine kinases (IM-HKs), which-instead of acting as stress sensors themselves-are activated via interprotein signal transfer. Some of these IM-HKs are involved in sensing the flux of antibiotic resistance transporters, a unique mechanism of responding to extracellular antibiotic challenge.

Cannibalism stress response in Bacillus subtilis.

When faced with carbon source limitation, the Gram-positive soil organism Bacillus subtilis initiates a survival strategy called sporulation, which leads to the formation of highly resistant endospores that allow B. subtilis to survive even long periods of starvation. In order to avoid commitment to this energy-demanding and irreversible process, B. subtilis employs another strategy called 'cannibalism' to delay sporulation as long as possible. Cannibalism involves the production and secretion of two cannibalism toxins, sporulation delaying protein (SDP) and sporulation killing factor (SKF), which are able to lyse sensitive siblings. The lysed cells are thought to then provide nutrients for the cannibals to slow down or even prevent them from entering sporulation. In this study, we uncovered the role of the cell envelope stress response (CESR), especially the Bce-like antimicrobial peptide detoxification modules, in the cannibalism stress response during the stationary phase. SDP and SKF specifically induce Bce-like systems and some extracytoplasmic function σ factors in stationary-phase cultures, but only the latter provide some degree of protection. A full Bce response is only triggered by mature toxins, and not by toxin precursors. Our study provides insights into the close relationship between stationary-phase survival and the CESR of B. subtilis.

Environmental Sensing in Actinobacteria: a Comprehensive Survey on the Signaling Capacity of This Phylum.

UNLABELLED: Signal transduction is an essential process that allows bacteria to sense their complex and ever-changing environment and adapt accordingly. Three distinct major types of signal-transducing proteins (STPs) can be distinguished: one-component systems (1CSs), two-component systems (2CSs), and extracytoplasmic-function σ factors (ECFs). Since Actinobacteria are particularly rich in STPs, we comprehensively investigated the abundance and diversity of STPs encoded in 119 actinobacterial genomes, based on the data stored in the Microbial Signal Transduction (MiST) database. Overall, we observed an approximately linear correlation between the genome size and the total number of encoded STPs. About half of all membrane-anchored 1CSs are protein kinases. For both 1CSs and 2CSs, a detailed analysis of the domain architectures identified novel proteins that are found only in actinobacterial genomes. Many actinobacterial genomes are particularly enriched for ECFs. As a result of this study, almost 500 previously unclassified ECFs could be classified into 18 new ECF groups. This comprehensive survey demonstrates that actinobacterial genomes encode previously unknown STPs, which may represent new mechanisms of signal transduction and regulation. This information not only expands our knowledge of the diversity of bacterial signal transduction but also provides clear and testable hypotheses about their mechanisms, which can serve as starting points for experimental studies. IMPORTANCE: In the wake of the genomic era, with its enormous increase in the amount of available sequence information, the challenge has now shifted toward making sense and use of this treasure chest. Such analyses are a prerequisite to provide meaningful information that can help guide subsequent experimental efforts, such as mechanistic studies on novel signaling strategies. This work provides a comprehensive analysis of signal transduction proteins from 119 actinobacterial genomes. We identify, classify, and describe numerous novel and conserved signaling devices. Hence, our work serves as an important resource for any researcher interested in signal transduction of this important bacterial phylum, which contains organisms of ecological, biotechnological, and medical relevance.