Unveiling the Coordinated Action of DesK/DesR and YvfT/YvfU to Control the Expression of an ABC Transporter in Bacillus subtilis.

Two-component systems (TCSs) are vital signal transduction pathways ubiquitous among bacteria, facilitating their responses to diverse environmental stimuli. In Bacillus subtilis, the DesK histidine kinase thermosensor, together with the response regulator DesR, constitute a TCS dedicated to membrane lipid homeostasis maintenance. This TCS orchestrates the transcriptional regulation of the des gene, encoding the sole desaturase in these bacteria, Δ5-Des. Additionally, B. subtilis possesses a paralog TCS, YvfT/YvfU, with unknown target gene(s). In this work, we show that YvfT/YvfU controls the expression of the yvfRS operon that codes for an ABC transporter. Interestingly, we found that this regulation also involves the action of DesK/DesR. Notably, opposite to des, yvfRS transcription is induced at 37°C and not at 25°C. Our in vivo and in vitro experiments demonstrate that both YvfU and DesR directly bind to the operon promoter region, with DesR exerting its control over yvfRS expression in its unphosphorylated state. Our study uncovers an intriguing case of cross-regulation where two homologous TCSs interact closely to finely tune gene expression in response to environmental cues. These findings shed light on the complexity of bacterial signal transduction systems and their critical role in bacterial adaptability.

An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

The oxidative stress response of Streptococcus pneumoniae: its contribution to both extracellular and intracellular survival.

Streptococcus pneumoniae is a gram-positive, aerotolerant bacterium that naturally colonizes the human nasopharynx, but also causes invasive infections and is a major cause of morbidity and mortality worldwide. This pathogen produces high levels of H2O2 to eliminate other microorganisms that belong to the microbiota of the respiratory tract. However, it also induces an oxidative stress response to survive under this stressful condition. Furthermore, this self-defense mechanism is advantageous in tolerating oxidative stress imposed by the host's immune response. This review provides a comprehensive overview of the strategies employed by the pneumococcus to survive oxidative stress. These strategies encompass the utilization of H2O2 scavengers and thioredoxins, the adaptive response to antimicrobial host oxidants, the regulation of manganese and iron homeostasis, and the intricate regulatory networks that control the stress response. Here, we have also summarized less explored aspects such as the involvement of reparation systems and polyamine metabolism. A particular emphasis is put on the role of the oxidative stress response during the transient intracellular life of Streptococcus pneumoniae, including coinfection with influenza A and the induction of antibiotic persistence in host cells.

Two-Component System Sensor Kinases from Asgardian Archaea May Be Witnesses to Eukaryotic Cell Evolution.

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.

Trends in the two-component system's role in the synthesis of antibiotics by Streptomyces.

Despite the advances in understanding the regulatory networks for secondary metabolite production in Streptomyces, the participation of the two-component systems (TCS) in this process still requires better characterization. These sensing systems and their responses to environmental stimuli have been described by evaluating mutant strains with techniques that allow in-depth regulatory responses. However, defining the stimulus that triggers their activation is still a task. The transmembrane nature of the sensor kinases and the high content of GC in the streptomycetes represent significant challenges in their study. In some examples, adding elements to the assay medium has determined the respective ligand. However, a complete TCS description and characterization requires specific amounts of the involved proteins that are most difficult to obtain. The availability of enough sensor histidine kinase concentrations could facilitate the identification of the ligand-protein interaction, and besides would allow the establishment of its phosphorylation mechanisms and determine their tridimensional structure. Similarly, the advances in the development of bioinformatics tools and novel experimental techniques also promise to accelerate the TCSs description and provide knowledge on their participation in the regulation processes of secondary metabolite formation. This review aims to summarize the recent advances in the study of TCSs involved in antibiotic biosynthesis and to discuss alternatives to continue their characterization. KEY POINTS: • TCSs are the environmental signal transducers more abundant in nature. • The Streptomyces have some of the highest number of TCSs found in bacteria. • The study of signal transduction between SHKs and RRs domains is a big challenge.

The Histidine Kinase CckA Is Directly Inhibited by a Response Regulator-like Protein in a Negative Feedback Loop.

In alphaproteobacteria, the two-component system (TCS) formed by the hybrid histidine kinase CckA, the phosphotransfer protein ChpT, and the response regulator CtrA is widely distributed. In these microorganisms, this system controls diverse functions such as motility, DNA repair, and cell division. In Caulobacterales and Rhizobiales, CckA is regulated by the pseudo- histidine kinase DivL, and the response regulator DivK. However, this regulatory circuit differs for other bacterial groups. For instance, in Rhodobacterales, DivK is absent and DivL consists of only the regulatory PAS domain. In this study, we report that, in Rhodobacter sphaeroides, the kinase activity of CckA is inhibited by Osp, a single domain response regulator (SDRR) protein that directly interacts with the transmitter domain of CckA. In vitro, the kinase activity of CckA was severely inhibited with an equimolar amount of Osp, whereas the phosphatase activity of CckA was not affected. We also found that the expression of osp is activated by CtrA creating a negative feedback loop. However, under growth conditions known to activate the TCS, the increased expression of osp does not parallel Osp accumulation, indicating a complex regulation. Phylogenetic analysis of selected species of Rhodobacterales revealed that Osp is widely distributed in several genera. For most of these species, we found a sequence highly similar to the CtrA-binding site in the control region of osp, suggesting that the TCS CckA/ChpT/CtrA is controlled by a novel regulatory circuit that includes Osp in these bacteria. IMPORTANCE The two-component systems (TCS) in bacteria in its simplest architecture consist of a histidine kinase (HK) and a response regulator (RR). In response to a specific stimulus, the HK is activated and drives phosphorylation of the RR, which is responsible of generating an adaptive response. These systems are ubiquitous among bacteria and are frequently controlled by accessory proteins. In alphaproteobacteria, the TCS formed by the HK CckA, the phosphotransferase ChpT, and the RR CtrA is widely distributed. Currently, most of the information of this system and its regulatory proteins comes from findings carried out in microorganisms where it is essential. However, this is not the case in many species, and studies of this TCS and its regulatory proteins are lacking. In this study, we found that Osp, a RR-like protein, inhibits the kinase activity of CckA in a negative feedback loop since osp expression is activated by CtrA. The inhibitory role of Osp and the similar action of the previously reported FixT protein, suggests the existence of a new group of RR-like proteins whose main function is to interact with the HK and prevent its phosphorylation.

RdsA Is a Global Regulator That Controls Cell Shape and Division in Rhizobium etli.

Unlike other bacteria, cell growth in rhizobiales is unipolar and asymmetric. The regulation of cell division, and its coordination with metabolic processes is an active field of research. In Rhizobium etli, gene RHE_PE00024, located in a secondary chromosome, is essential for growth. This gene encodes a predicted hybrid histidine kinase sensor protein, participating in a, as yet undescribed, two-component signaling system. In this work, we show that a conditional knockdown mutant (cKD24) in RHE_PE00024 (hereby referred as rdsA, after rhizobium division and shape) generates a striking phenotype, where nearly 64% of the cells present a round shape, with stochastic and uncoordinated cell division. For rod-shaped cells, a large fraction (12 to 29%, depending on their origin) present growth from the old pole, a sector that is normally inactive for growth in a wild-type cell. A fraction of the cells (1 to 3%) showed also multiple ectopic polar growths. Homodimerization of RdsA appears to be required for normal function. RNAseq analysis of mutant cKD24 reveals global changes, with downregulated genes in at least five biological processes: cell division, wall biogenesis, respiration, translation, and motility. These modifications may affect proper structuring of the divisome, as well as peptidoglycan synthesis. Together, these results indicate that the hybrid histidine kinase RdsA is an essential global regulator influencing cell division and cell shape in R. etli.

A new mutation in mgrb mediating polymyxin resistance in Klebsiella variicola.

Polymyxin resistance is a public health concern - present in humans, animals and the environment - caused by chromosomal-encoding or plasmid-encoding mechanisms. Chromosomal alterations in MgrB are frequently detected in Klebsiella spp., but not yet reported and characterised in Klebsiella variicola (K. variicola). This study performed microbiological and genomic characterisation of three polymyxin-resistant K. variicola isolates (M14, M15 and M50) recovered from the microbiota of migratory birds in Brazil. The isolates were submitted to SpeI-PFGE, broth microdilution and whole genome sequencing using Illumina MiSeq for analysis of genetic relatedness, sequence typing and detection of antimicrobial-resistance genes. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance only for polymyxins. Sequences of chromosomal two-component systems (PmrAB, PhoPQ, RstAB, CrrAB) and MgrB were evaluated by blastN and blastP against a polymyxin-susceptible K. variicola (A58243), and mutations with biological effect were checked by the PROVEAN tool. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance for polymyxins. In M14 and M15, phoQ deleterious mutations (D90N, I122S and G385S) were identified, while an mgrB variant containing a single deletion (C deletion on position 93) leading to the production of a non-functional protein was detected in M50. mgrB complementation studies showed restoration of polymyxin susceptibility (64 to ≤ 0.25 mg/L) as a wild-type mgrB was inserted into the mgrB-deficient M50. This study confirmed the role of a non-functional mgrB variant in conferring polymyxin resistance, stressing the role of this regulator in K. variicola and drawing attention to novel polymyxin resistance mechanisms emerging in wildlife.

The role of two-component regulatory systems in environmental sensing and virulence in Salmonella.

Adaptation to environments with constant fluctuations imposes challenges that are only overcome with sophisticated strategies that allow bacteria to perceive environmental conditions and develop an appropriate response. The gastrointestinal environment is a complex ecosystem that is home to trillions of microorganisms. Termed microbiota, this microbial ensemble plays important roles in host health and provides colonization resistance against pathogens, although pathogens have evolved strategies to circumvent this barrier. Among the strategies used by bacteria to monitor their environment, one of the most important are the sensing and signalling machineries of two-component systems (TCSs), which play relevant roles in the behaviour of all bacteria. Salmonella enterica is no exception, and here we present our current understanding of how this important human pathogen uses TCSs as an integral part of its lifestyle. We describe important aspects of these systems, such as the stimuli and responses involved, the processes regulated, and their roles in virulence. We also dissect the genomic organization of histidine kinases and response regulators, as well as the input and output domains for each TCS. Lastly, we explore how these systems may be promising targets for the development of antivirulence therapeutics to combat antibiotic-resistant infections.

Allostery and protein plasticity: the keystones for bacterial signaling and regulation.

Bacteria sense intracellular and environmental signals using an array of proteins as antennas. The information is transmitted from such sensory modules to other protein domains that act as output effectors. Sensor and effector can be part of the same polypeptide or instead be separate diffusible proteins that interact specifically. The output effector modules regulate physiologic responses, allowing the cells to adapt to the varying conditions. These biological machineries are known as signal transduction systems (STSs). Despite the captivating architectural diversity exhibited by STS proteins, a universal feature is their allosteric regulation: signal binding at one site modifies the activity at a physically distant site. Allostery requires protein plasticity, precisely encoded within their 3D structures, and implicating programmed molecular motions. This review summarizes how STS proteins connect stimuli to specific responses by exploiting allostery and protein plasticity. Illustrative examples spanning a wide variety of protein folds will focus on one- and two-component systems (TCSs). The former encompass the entire transmission route within a single polypeptide, whereas TCSs have evolved as separate diffusible proteins that interact specifically, sometimes including additional intermediary proteins in the pathway. Irrespective of their structural diversity, STS proteins are able to modulate their own molecular motions, which can be relatively slow, rigid-body movements, all the way to fast fluctuations in the form of macromolecular flexibility, thus spanning a continuous protein dynamics spectrum. In sum, STSs rely on allostery to steer information transmission, going from simple two-state switching to rich multi-state conformational order/disorder transitions.

Genomic Analysis of Carbapenem-Resistant Acinetobacter baumannii Isolates Belonging to Major Endemic Clones in South America.

Carbapenem-resistant Acinetobacter baumannii (CRAB) are emerging worldwide. In South America, clinical isolates presenting such a phenotype usually do not belong to the globally distributed international clone 2 (IC2). The majority of these isolates are also resistant to multiple other antimicrobials and are often designated extremely drug-resistant (XDR). The aim of this study was to characterize the resistance mechanisms presented by 18 carbapenem-resistant A. baumannii isolates from five different Brazilian hospitals. Species identification was determined by rpoB sequencing, and antimicrobial susceptibility was determined by broth microdilution. Isolates were submitted to whole genome sequencing using Illumina platform and genetic similarity was determined by PFGE, MLST, and cgMLST. Genome analysis was used to identify intrinsic and acquired resistance determinants, including mutations in the AdeRSABC efflux system and in outer membrane proteins (OMPs). All isolates were identified as A. baumannii and grouped into 4 pulsotypes by PFGE, which belonged to clonal complexes (CC) 15 Pas /103 Ox (n = 4) and 79 Pas /113 Ox (n = 14), corresponding to IC4 and IC5, respectively. High MIC values to carbapenems, broad-spectrum cephalosporins, amikacin, and ciprofloxacin were observed in all isolates, while MICs of ampicillin/sulbactam, gentamicin, and tigecycline varied among the isolates. Minocycline was the most active antimicrobial agent tested. Moreover, 12 isolates (66.7%) were considered resistant to polymyxins. Besides intrinsic OXA-51 and ADC variants, all isolates harbored an acquired carbapenem-hydrolyzing class D ß-lactamase (CHDL) encoding gene, either bla OXA- 23 or bla OXA- 72. A diversity of aminoglycoside modifying enzymes and resistance determinants to other antimicrobial classes were found, as well as mutations in gyrA and parC. Non-synonymous mutations have also been identified in the AdeRSABC efflux system and in most OMPs, but they were considered natural polymorphisms. Moreover, resistance to polymyxins among isolates belonging to IC5 were associated to non-synonymous mutations in pmrB, but no known polymyxin resistance mechanism was identified in isolates belonging to IC4. In conclusion, A. baumannii clinical isolates belonging to South America's major clones present a myriad of antimicrobial resistance determinants. Special attention should be paid to natural polymorphisms observed in each clonal lineage, especially regarding non-synonymous mutations in constitutive genes associated with distinct resistance phenotypes.

Identification of Z nucleotides as an ancient signal for two-component system activation in bacteria.

Two-component systems (TCSs) in bacteria are molecular circuits that allow the perception of and response to diverse stimuli. These signaling circuits rely on phosphoryl-group transfers between transmitter and receiver domains of sensor kinase and response regulator proteins, and regulate several cellular processes in response to internal or external cues. Phosphorylation, and thereby activation, of response regulators has been demonstrated to occur by their cognate histidine kinases but also by low molecular weight phosphodonors such as acetyl phosphate and carbamoyl phosphate. Here, we present data indicating that the intermediates of the de novo syntheses of purines and histidine, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) and/or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-triphosphate (ZTP), activate the response regulator UvrY, by promoting its autophosphorylation at the conserved aspartate at position 54. Moreover, these Z nucleotides are shown to also activate the nonrelated response regulators ArcA, CpxR, RcsB, and PhoQ. We propose that ZMP and/or ZTP act as alarmones for a wide range of response regulators in vivo, providing a novel mechanism by which they could impact gene expression in response to metabolic cues.

Diverse and emerging molecular mechanisms award polymyxins resistance to Enterobacteriaceae clinical isolates from a tertiary hospital of Recife, Brazil.

OBJECTIVE: To describe the molecular mechanisms of polymyxins resistance in five Enterobacteriaceae clinical isolates from a tertiary hospital of Recife, Brazil. METHODS: The species identification and the susceptibility to antimicrobials were firstly performed by automatized methods and polymyxin resistance was confirmed by broth microdilution methods. The genetic basis of resistance was characterized with WGS analyses to study their resistome, plasmidome and mobilome, by BLAST searches on reference databases. RESULTS: Five (5%) Enterobacteriaceae isolates, comprising Escherichia coli (n = 2), Klebsiella pneumoniae (n = 2) and Citrobacter freundii (n = 1) species, exhibited polymyxin resistance. The mcr-1.1 gene was found in identical IncX4-plasmids harbored by both K. pneumoniae C119 (PolB MIC = 512 mg/L) and E. coli C153 (PolB MIC = 8 mg/L). The remaining E. coli strain C027 harbored the mcr-5.1 gene on an undefined Inc-plasmid (PolB MIC 256 mg/L). Some amino acid substitutions in PmrA (S29G, G144S), PmrB (S202P; D283G, W350*, Y258N) and PhoP (I44L) was detected among the E. coli clinical isolates, however they were also found in colistin-susceptible strains and predicted as neutral alterations. The mgrB of the ST54 KPC-2-producing K. pneumoniae C151 (PolB MIC = 32 g/mL) was interrupted at 69 nt by the IS903 element. The ST117 C. freundii C156 (PolB MIC = 256 mg/L) showed the A91T substitution on HAMP domain of the histidine kinase sensor CrrB, predicted as deleterious and deemed the remarkable determinant to polymyxins resistance in this strain. CONCLUSIONS: Diverse mechanisms of polymyxins resistance were identified among clinical Enterobacteriaceae from a tertiary hospital of Recife, Brazil, such as plasmid-mediated MCR-1 and MCR-5; IS903-interruption of mgrB and mutation in CrrAB regulatory system. These findings highlight the involvement of the identified plasmids on mcr dissemination among Enterobacteriaceae; warn about co-selection of the polymyxin-resistant and KPC-producer K. pneumoniae ΔmgrB lineage by carbapenems usage; and demonstrate potential role of CrrAB on emerging of polymyxin resistance among Enterobacteriaceae, besides Klebsiella species.

A Novel Two-Component System, Encoded by the sco5282/sco5283 Genes, Affects Streptomyces coelicolor Morphology in Liquid Culture.

Streptomyces are mycelial bacteria adapted to grow in soil. They have become important producers of biomolecules with medical applications, but their growth in industrial fermenters is challenged by their peculiar morphology in liquid culture: the hyphae tend to clump and grow as large pellets, which are oxygen- and nutrient-limited, grow slowly and present diminished protein production. Here, by implementing an experimental evolution strategy, a S. coelicolor strain, 2L12, with dispersed morphology and reduced pellet size in liquid culture and no defects in either differentiation or secondary metabolism was selected. Genome sequencing revealed a single amino acid substitution in a sensor kinase, Sco5282, of unknown function to be responsible for the morphological changes. Moreover, genetic and biochemical scrutiny identified Sco5283 as the cognate response regulator and demonstrated that the acquired mutation activates this two-component system. Finally, transcriptomic analysis of the mutant strain revealed changes in expression of genes involved in central processes such as glycolysis, gluconeogenesis, stress-signaling pathways, proteins secretion and cell envelope metabolism. Thus a novel two-component system is proposed to play a key role in the control of Streptomyces extracellular metabolism.

Caracterização funcional de sistemas de dois componentes em Xanthomonas citri / Functional characterization of two-component systems in Xanthomonas citri

Xanthomonas citri subsp. citri, é uma bactéria pertencente à classe das Gamaproteobactérias, fitopatogênica, que exibe uma especificidade patógeno-hospedeiro extremamente alta. X. citri infecta plantas do gênero Citrus, causando o cancro cítrico, uma doença destrutiva encontrada em cultivos ao redor do mundo. Esta bactéria apresenta em seu genoma 34 genes que codificam proteínas relacionadas com o metabolismo do segundo mensageiro c-di-GMP. Em geral, níveis elevados de c-di-GMP favorecem a sessilidade e a produção de exopolissacarídeos, enquanto níveis mais baixos resultam em maior motilidade e aumento na dispersão do biofilme. Com o intuito inicial de buscar novos alvos de X. citri que dependessem dos níveis intracelulares desse segundo mensageiro, foram analisados os proteomas de linhagens mutantes em diguanilato ciclases específicas. Nas análises proteômicas por eletroforese bidimensional foram identificadas 15 proteínas diferencialmente expressas presentes em mais de um dos proteomas dos mutantes analisados. Entre estas, duas proteínas reguladoras de resposta e preditas de participar de sistemas de dois componentes, XAC0834 e XAC3443, foram encontradas sendo mais expressas em mutantes que apresentavam fenótipo de alto c-di-GMP; enquanto uma proteína hipotética provavelmente presente na membrana, XAC3657, estava mais expressa em linhagens com fenótipos relacionados a baixos níveis de c-di-GMP. Por meio de uma análise por qRT-PCR foi verificado que os níveis de mRNA para XAC0834 e XAC3443 não variam entre as linhagens e, portanto, a diferença nos níveis de expressão destas proteínas deve ocorrer póstranscricionalmente. Como os sistemas de dois componentes e proteínas de membrana são importantes para a adaptação das bactérias a diferentes condições ambientais, o objetivo do presente trabalho foi a caracterização funcional de XAC0834, XAC3433 e XAC3657, com maiorênfase em XAC0834 e na provável proteína sensora cognata, XAC0835, de forma a contribuir para a melhor compreensão dos processos de regulação da virulência de bactérias. Na análise da organização gênica dos genes que codificam estas proteínas, foi verificado que os genes XAC0834 e XAC0835 formam um operon, juntamente com a tioesterase XAC0833 e, portanto, o nível transcricional destes genes ocorre pelos mesmos reguladores, apoiando a hipótese de se tratarem de um sistema de dois componentes; assim como os genes XAC3442 e XAC3443. Utilizando uma linhagem mutante em XAC0834, mostramos que esta proteína impacta positivamente a motilidade sliding e a formação de biofilme, e tem efeito contrário no crescimento de X. citri em meio rico 2xTY e na motilidade twitching. Como estes fenótipos são modulados por c-di-GMP, é possível que a deleção deste gene altere significativamente os níveis de c-di-GMP nas células. Além disto, foi verificado que as proteínas XAC0835, XAC3443 e XAC3657 não afetam a motilidade sliding, mas, individualmente, XAC0835 é importante para a formação de biofilme; XAC3657 afeta negativamente o crescimento de X. citri em meio rico 2xTY; e XAC3443 afeta negativamente a motilidade twitching. Na análise do transcritoma da superexpressão de XAC0834, foi observado que havia aumento na expressão de genes relacionados ao sistema de secreção do tipo IV e na montagem do pilus do tipo IV, em comparação com a linhagem selvagem, o que pode estar relacionado aos fenótipos observados. Este trabalho forneceu subsídios importantes para a compreensão do papel fisiológico do sistema de dois componentes XAC0834/XAC0835, assim como do regulador de resposta XAC3443 e da proteína hipotética, XAC3657, em X. citri, o que pode contribuir para o entendimento da relação de c-di-GMP com os sistemas de dois componentes

Xanthomonas citri subsp. citri, is a phytopathogenic Gammaproteobacteria, with extremely high pathogen-host specificity. X. citri infects plants of the genus Citrus, causing citrus canker, a destructive disease found in crops around the world. The genome of X. citri pv. citri 306 (XAC 306) contains 34 genes encoding proteins related to the second messenger c-di-GMP metabolism. In general, high levels of c-di-GMP favor the sessility and exopolysaccharide production, whereas lower levels result in greater motility and increased biofilm dispersion. In order to initially search for new X. citri targets that depend on the intracellular levels of this second messenger, the proteomes of specific diguanylate cyclase mutant strains were analyzed by two-dimensional electrophoresis. Fifteen differentially expressed proteins present in more than one of the mutant proteomes compared to wild type were identified. Among these, two proteins predicted to participate as response regulators in two-component systems, XAC0834 and XAC3443, were found to be more expressed in mutants with high c-di-GMP phenotypes; whereas a hypothetical membrane protein, XAC3657, was more expressed in strains with low cdi-GMP-related phenotypes. Relative mRNA levels for XAC0834 and XAC3443, as determined by qRT-PCR, do not vary among the analyzed strains, suggesting post-transcriptional regulation. Because two-component systems and membrane proteins are important for the adaptation of bacteria to different environmental conditions, the aim of this work was the functional characterization of XAC0834, XAC3433 and XAC3657, with greater emphasis on XAC0834 and its probable cognate sensor protein, XAC0835, contributing to a better understanding of the processes of bacterial virulence regulation. Genes XAC0834 and XAC0835 form an operon, together with the XAC0833 coding for a thioesterase, suggesting that they are co-regulated, aswell as the XAC3442 and XAC3443 genes. Using a mutant strain in XAC0834, we show that this protein positively impacts sliding motility and biofilm formation and has the opposite effect on X. citri growth in rich medium and twitching motility. Because these phenotypes are modulated by c-di-GMP, deletion of this gene may alter cellular c-di-GMP levels. In addition, we found that XAC0835, XAC3443 and XAC3657 proteins do not affect sliding motility, but XAC0835 is important for biofilm formation; XAC3657 negatively affects X. citri growth in rich medium; and XAC3443 negatively affects twitching motility. The RNA-seq transcriptome of X. citri overexpressing XAC0834 was compared to the control strain, and there was an increase in the expression of genes for the type IV secretion system and the assembly of the type IV pilus, which may be related to the observed phenotypes. This work provided important insights for understanding the physiological role of the XAC0834/XAC0835 two-component system as well as the XAC3443 response regulator and the hypothetical protein XAC3657, in X. citri which may contribute to the understanding of the relationship of c- di-GMP with two-component systems

The variome of pneumococcal virulence factors and regulators.

BACKGROUND: In recent years, the idea of a highly immunogenic protein-based vaccine to combat Streptococcus pneumoniae and its severe invasive infectious diseases has gained considerable interest. However, the target proteins to be included in a vaccine formulation have to accomplish several genetic and immunological characteristics, (such as conservation, distribution, immunogenicity and protective effect), in order to ensure its suitability and effectiveness. This study aimed to get comprehensive insights into the genomic organization, population distribution and genetic conservation of all pneumococcal surface-exposed proteins, genetic regulators and other virulence factors, whose important function and role in pathogenesis has been demonstrated or hypothesized. RESULTS: After retrieving the complete set of DNA and protein sequences reported in the databases GenBank, KEGG, VFDB, P2CS and Uniprot for pneumococcal strains whose genomes have been fully sequenced and annotated, a comprehensive bioinformatic analysis and systematic comparison has been performed for each virulence factor, stand-alone regulator and two-component regulatory system (TCS) encoded in the pan-genome of S. pneumoniae. A total of 25 S. pneumoniae strains, representing different pneumococcal phylogenetic lineages and serotypes, were considered. A set of 92 different genes and proteins were identified, classified and studied to construct a pan-genomic variability map (variome) for S. pneumoniae. Both, pneumococcal virulence factors and regulatory genes, were well-distributed in the pneumococcal genome and exhibited a conserved feature of genome organization, where replication and transcription are co-oriented. The analysis of the population distribution for each gene and protein showed that 49 of them are part of the core genome in pneumococci, while 43 belong to the accessory-genome. Estimating the genetic variability revealed that pneumolysin, enolase and Usp45 (SP_2216 in S. p. TIGR4) are the pneumococcal virulence factors with the highest conservation, while TCS08, TCS05, and TCS02 represent the most conserved pneumococcal genetic regulators. CONCLUSIONS: The results identified well-distributed and highly conserved pneumococcal virulence factors as well as regulators, representing promising candidates for a new generation of serotype-independent protein-based vaccine(s) to combat pneumococcal infections.

Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action.

Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring.

An overview on transcriptional regulators in Streptomyces.

Streptomyces are Gram-positive microorganisms able to adapt and respond to different environmental conditions. It is the largest genus of Actinobacteria comprising over 900 species. During their lifetime, these microorganisms are able to differentiate, produce aerial mycelia and secondary metabolites. All of these processes are controlled by subtle and precise regulatory systems. Regulation at the transcriptional initiation level is probably the most common for metabolic adaptation in bacteria. In this mechanism, the major players are proteins named transcription factors (TFs), capable of binding DNA in order to repress or activate the transcription of specific genes. Some of the TFs exert their action just like activators or repressors, whereas others can function in both manners, depending on the target promoter. Generally, TFs achieve their effects by using one- or two-component systems, linking a specific type of environmental stimulus to a transcriptional response. After DNA sequencing, many streptomycetes have been found to have chromosomes ranging between 6 and 12Mb in size, with high GC content (around 70%). They encode for approximately 7000 to 10,000 genes, 50 to 100 pseudogenes and a large set (around 12% of the total chromosome) of regulatory genes, organized in networks, controlling gene expression in these bacteria. Among the sequenced streptomycetes reported up to now, the number of transcription factors ranges from 471 to 1101. Among these, 315 to 691 correspond to transcriptional regulators and 31 to 76 are sigma factors. The aim of this work is to give a state of the art overview on transcription factors in the genus Streptomyces.

Stress-triggered signaling affecting survival or suicide of Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen that can survive to stress conditions, such as the acidic environment of inflammatory foci, and tolerates lethal pH through a mechanism known as the acid tolerance response. We previously described that S. pneumoniae activates acidic-stress induced lysis in response to acidified environments, favoring the release of cell wall compounds, DNA and virulence factors. Here, we demonstrate that F(0)F(1)-ATPase is involved in the response to acidic stress. Chemical inhibitors (DCCD, optochin) of this proton pump repressed the ATR induction, but caused an increased ASIL. Confirming these findings, mutants of the subunit c of this enzyme showed the same phenotypes as inhibitors. Importantly, we demonstrated that F(0)F(1)-ATPase and ATR are necessary for the intracellular survival of the pneumococcus in macrophages. Alternatively, a screening of two-component system (TCS) mutants showed that ATR and survival in pneumocytes were controlled in contrasting ways by ComDE and CiaRH, which had been involved in the ASIL mechanism. Briefly, CiaRH was essential for ATR (ComE represses activation) whereas ComE was necessary for ASIL (CiaRH protects against induction). They did not regulate F0F1-ATPase expression, but control LytA expression on the pneumococcal surface. These results suggest that both TCSs and F(0)F(1)-ATPase control a stress response and decide between a survival or a suicide mechanism by independent pathways, either in vitro or in pneumocyte cultures. This biological model contributes to the current knowledge about bacterial response under stress conditions in host tissues, where pathogens need to survive in order to establish infections.