KdpD is a tandem serine histidine kinase that controls K+ pump KdpFABC transcriptionally and post-translationally.

Two-component systems, consisting of a histidine kinase and a response regulator, serve signal transduction in bacteria, often regulating transcription in response to environmental stimuli. Here, we identify a tandem serine histidine kinase function for KdpD, previously described as a histidine kinase of the KdpDE two-component system, which controls production of the potassium pump KdpFABC. We show that KdpD additionally mediates an inhibitory serine phosphorylation of KdpFABC at high potassium levels, using not its C-terminal histidine kinase domain but an N-terminal atypical serine kinase domain. Sequence analysis of KdpDs from different species highlights that some KdpDs are much shorter than others. We show that, while Escherichia coli KdpD's atypical serine kinase domain responds directly to potassium levels, a shorter version from Deinococcus geothermalis is controlled by second messenger cyclic di-AMP. Our findings add to the growing functional diversity of sensor kinases while simultaneously expanding the framework for regulatory mechanisms in bacterial potassium homeostasis.

Structural insights into the regulation of protein-arginine kinase McsB by McsA.

In gram-positive bacteria, phosphorylated arginine functions as a protein degradation signal in a similar manner as ubiquitin in eukaryotes. The protein-arginine phosphorylation is mediated by the McsAB complex, where McsB possesses kinase activity and McsA modulates McsB activity. Although mcsA and mcsB are regulated within the same operon, the role of McsA in kinase activity has not yet been clarified. In this study, we determined the molecular mechanism by which McsA regulates kinase activity. The crystal structure of the McsAB complex shows that McsA binds to the McsB kinase domain through a second zinc-coordination domain and the subsequent loop region. This binding activates McsB kinase activity by rearranging the catalytic site, preventing McsB self-assembly, and enhancing stoichiometric substrate binding. The first zinc-coordination and coiled-coil domains of McsA further activate McsB by reassembling the McsAB oligomer. These results demonstrate that McsA is the regulatory subunit for the reconstitution of the protein-arginine kinase holoenzyme. This study provides structural insight into how protein-arginine kinase directs the cellular protein degradation system.

Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.

Biomimetic NIR-II fluorescent proteins created from chemogenic protein-seeking dyes for multicolor deep-tissue bioimaging.

Near-infrared-I/II fluorescent proteins (NIR-I/II FPs) are crucial for in vivo imaging, yet the current NIR-I/II FPs face challenges including scarcity, the requirement for chromophore maturation, and limited emission wavelengths (typically < 800 nm). Here, we utilize synthetic protein-seeking NIR-II dyes as chromophores, which covalently bind to tag proteins (e.g., human serum albumin, HSA) through a site-specific nucleophilic substitution reaction, thereby creating proof-of-concept biomimetic NIR-II FPs. This chemogenic protein-seeking strategy can be accomplished under gentle physiological conditions without catalysis. Proteomics analysis identifies specific binding site (Cys 477 on DIII). NIR-II FPs significantly enhance chromophore brightness and photostability, while improving biocompatibility, allowing for high-performance NIR-II lymphography and angiography. This strategy is universal and applicable in creating a wide range of spectrally separated NIR-I/II FPs for real-time visualization of multiple biological events. Overall, this straightforward biomimetic approach holds the potential to transform fluorescent protein-based bioimaging and enables in-situ albumin targeting to create NIR-I/II FPs for deep-tissue imaging in live organisms.

Patchy and widespread distribution of bacterial translation arrest peptides associated with the protein localization machinery.

Regulatory arrest peptides interact with specific residues on bacterial ribosomes and arrest their own translation. Here, we analyse over 30,000 bacterial genome sequences to identify additional Sec/YidC-related arrest peptides, followed by in vivo and in vitro analyses. We find that Sec/YidC-related arrest peptides show patchy, but widespread, phylogenetic distribution throughout the bacterial domain. Several of the identified peptides contain distinct conserved sequences near the C-termini, but are still able to efficiently stall bacterial ribosomes in vitro and in vivo. In addition, we identify many arrest peptides that share an R-A-P-P-like sequence, suggesting that this sequence might serve as a common evolutionary seed to overcome ribosomal structural differences across species.

Characterization of a pleiotropic regulator MtrA in Streptomyces avermitilis controlling avermectin production and morphological differentiation.

BACKGROUND: The macrolide antibiotic avermectin, a natural product derived from Streptomyces avermitilis, finds extensive applications in agriculture, animal husbandry and medicine. The mtrA (sav_5063) gene functions as a transcriptional regulator belonging to the OmpR family. As a pleiotropic regulator, mtrA not only influences the growth, development, and morphological differentiation of strains but also modulates genes associated with primary metabolism. However, the regulatory role of MtrA in avermectin biosynthesis remains to be elucidated. RESULTS: In this study, we demonstrated that MtrA, a novel OmpR-family transcriptional regulator in S. avermitilis, exerts global regulator effects by negatively regulating avermectin biosynthesis and cell growth while positively controlling morphological differentiation. The deletion of the mtrA gene resulted in an increase in avermectin production, accompanied by a reduction in biomass and a delay in the formation of aerial hyphae and spores. The Electrophoretic Mobility Shift Assay (EMSA) revealed that MtrA exhibited binding affinity towards the upstream region of aveR, the intergenic region between aveA1 and aveA2 genes, as well as the upstream region of aveBVIII in vitro. These findings suggest that MtrA exerts a negative regulatory effect on avermectin biosynthesis by modulating the expression of avermectin biosynthesis cluster genes. Transcriptome sequencing and fluorescence quantitative PCR analysis showed that mtrA deletion increased the transcript levels of the cluster genes aveR, aveA1, aveA2, aveC, aveE, aveA4 and orf-1, which explains the observed increase in avermectin production in the knockout strain. Furthermore, our findings demonstrate that MtrA positively regulates the cell division and differentiation genes bldM and ssgC, while exerting a negative regulatory effect on bldD, thereby modulating the primary metabolic processes associated with cell division, differentiation and growth in S. avermitilis, consequently impacting avermectin biosynthesis. CONCLUSIONS: In this study, we investigated the negative regulatory effect of the global regulator MtrA on avermectin biosynthesis and its effects on morphological differentiation and cell growth, and elucidated its transcriptional regulatory mechanism. Our findings indicate that MtrA plays crucial roles not only in the biosynthesis of avermectin but also in coordinating intricate physiological processes in S. avermitilis. These findings provide insights into the synthesis of avermectin and shed light on the primary and secondary metabolism of S. avermitilis mediated by OmpR-family regulators.

Quantitative proteomics analysis reveals an important role of the transcriptional regulator UidR in the bacterial biofilm formation of Aeromonas hydrophila.

Introduction: Bacterial biofilm is a well-known characteristic that plays important roles in diverse physiological functions, whereas the current intrinsic regulatory mechanism of its formation is still largely unknown. Methods: In the present study, a label-free based quantitative proteomics technology was conducted to compare the differentially expressed proteins (DEPs) between ΔuidR and the wild-type strain in the biofilm state. Results: The results showed that the deletion of gene uidR encoding a TetR transcriptional regulator significantly increased the biofilm formation in Aeromonas hydrophila. And there was a total of 220 DEPs, including 120 up-regulated proteins and 100 down-regulated proteins between ΔuidR and the wild-type strain based on the quantitative proteomics. Bioinformatics analysis suggested that uidR may affect bacterial biofilm formation by regulating some related proteins in glyoxylic acid and dicarboxylic acid pathway. The expressions of selected proteins involved in this pathway were further confirmed by q-PCR assay, and the results was in accordance with the quantitative proteomics data. Moreover, the deletion of four genes (AHA_3063, AHA_3062, AHA_4140 and aceB) related to the glyoxylic acid and dicarboxylic acid pathway lead to a significant decrease in the biofilm formation. Discussion: Thus, the results indicated that uidR involved in the regulatory of bacterial biofilm formation, and it may provide a potential target for the drug development and a new clue for the prevention of pathogenic A. hydrophila in the future.

Genetic dissection of the tissue-specific roles of type III effectors and phytotoxins in the pathogenicity of Pseudomonas syringae pv. syringae to cherry.

When compared with other phylogroups (PGs) of the Pseudomonas syringae species complex, P. syringae pv. syringae (Pss) strains within PG2 have a reduced repertoire of type III effectors (T3Es) but produce several phytotoxins. Effectors within the cherry pathogen Pss 9644 were grouped based on their frequency in strains from Prunus as the conserved effector locus (CEL) common to most P. syringae pathogens; a core of effectors common to PG2; a set of PRUNUS effectors common to cherry pathogens; and a FLEXIBLE set of T3Es. Pss 9644 also contains gene clusters for biosynthesis of toxins syringomycin, syringopeptin and syringolin A. After confirmation of virulence gene expression, mutants with a sequential series of T3E and toxin deletions were pathogenicity tested on wood, leaves and fruits of sweet cherry (Prunus avium) and leaves of ornamental cherry (Prunus incisa). The toxins had a key role in disease development in fruits but were less important in leaves and wood. An effectorless mutant retained some pathogenicity to fruit but not wood or leaves. Striking redundancy was observed amongst effector groups. The CEL effectors have important roles during the early stages of leaf infection and possibly acted synergistically with toxins in all tissues. Deletion of separate groups of T3Es had more effect in P. incisa than in P. avium. Mixed inocula were used to complement the toxin mutations in trans and indicated that strain mixtures may be important in the field. Our results highlight the niche-specific role of toxins in P. avium tissues and the complexity of effector redundancy in the pathogen Pss 9644.

Revisiting old friends: updates on the role of two-component signaling systems in Listeria monocytogenes survival and pathogenesis.

Listeria monocytogenes is well recognized for both its broad resistance to stress conditions and its ability to transition from a soil bacterium to an intracellular pathogen of mammalian hosts. The bacterium's impressive ability to adapt to changing environments and conditions requires the rapid sensing of environmental cues and the coordinated response of gene products that enable bacterial growth and survival. Two-component signaling systems (TCSs) have been long recognized for their ability to detect environmental stimuli and transmit those signals into transcriptional responses; however, often the precise nature of the stimulus triggering TCS responses can be challenging to define. L. monocytogenes has up to 16 TCSs that have been recognized based on homology and included in this list are several whose functions remain poorly described. This review highlights the current understanding of the breadth and scope of L. monocytogenes TCS as relates to stress resistance and pathogenesis. Precise signals still often remain elusive, but the gene networks associated with TCSs are providing clues into possible functions.

The twin-arginine translocation (Tat) system.

In this Quick guide, Palmer and Berks introduce the twin-arginine translocation (Tat) systems. Tats are found in a variety of microbes and microbe-derived organelles, and are known to translocate folded substrate proteins across biological membranes.

HemU and TonB1 contribute to hemin acquisition in Stenotrophomonas maltophilia.

Introduction: The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins to shuttle hemin, an inner membrane transporter that transports hemin into the cytoplasm, and cytoplasmic heme oxygenase to release iron. Fur and HemP are two known regulators involved in the regulation of hemin acquisition. The hemin acquisition system of Stenotrophomonas maltophilia is poorly understood, with the exception of HemA as a TonB-dependent transporter for hemin uptake. Methods: Putative candidates responsible for hemin acquisition were selected via a homolog search and a whole-genome survey of S. maltophilia. Operon verification was performed by reverse transcription-polymerase chain reaction. The involvement of candidate genes in hemin acquisition was assessed using an in-frame deletion mutant construct and iron utilization assays. The transcript levels of candidate genes were determined using quantitative polymerase chain reaction. Results: Smlt3896-hemU-exbB2-exbD2-tonB2 and tonB1-exbB1-exbD1a-exbD1b operons were selected as candidates for hemin acquisition. Compared with the parental strain, hemU and tonB1 mutants displayed a defect in their ability to use hemin as the sole iron source for growth. However, hemin utilization by the Smlt3896 and tonB2 mutants was comparable to that of the parental strain. HemA expression was repressed by Fur in iron-replete conditions and derepressed in iron-depleted conditions. HemP negatively regulated hemA expression. Like hemA, hemU was repressed by Fur in iron-replete conditions; however, hemU was moderately derepressed in response to iron-depleted stress and fully derepressed when hemin was present. Unlike hemA and hemU, the TonB1-exbB1-exbD1a-exbD1b operon was constitutively expressed, regardless of the iron level or the presence of hemin, and Fur and HemP had no influence on its expression. Conclusion: HemA, HemU, and TonB1 contribute to hemin acquisition in S. maltophilia. Fur represses the expression of hemA and hemU in iron-replete conditions. HemA expression is regulated by low iron levels, and HemP acts as a negative regulator of this regulatory circuit. HemU expression is regulated by low iron and hemin levels in a hemP-dependent manner.

Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains.

BACKGROUND: The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS. RESULTS: In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27-45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84-224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively. CONCLUSION: While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Staphylococcus aureus foldase PrsA contributes to the folding and secretion of protein A.

BACKGROUND: Staphylococcus aureus secretes a variety of proteins including virulence factors that cause diseases. PrsA, encoded by many Gram-positive bacteria, is a membrane-anchored lipoprotein that functions as a foldase to assist in post-translocational folding and helps maintain the stability of secreted proteins. Our earlier proteomic studies found that PrsA is required for the secretion of protein A, an immunoglobulin-binding protein that contributes to host immune evasion. This study aims to investigate how PrsA influences protein A secretion. RESULTS: We found that in comparison with the parental strain HG001, the prsA-deletion mutant HG001ΔprsA secreted less protein A. Deleting prsA also decreased the stability of exported protein A. Pulldown assays indicated that PrsA interacts with protein A in vivo. The domains in PrsA that interact with protein A are mapped to both the N- and C-terminal regions (NC domains). Additionally, the NC domains are essential for promoting PrsA dimerization. Furthermore, an immunoglobulin-binding assay revealed that, compared to the parental strain HG001, fewer immunoglobulins bound to the surface of the mutant strain HG001ΔprsA. CONCLUSIONS: This study demonstrates that PrsA is critical for the folding and secretion of protein A. The information derived from this study provides a better understanding of virulent protein export pathways that are crucial to the pathogenicity of S. aureus.

Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood-brain barrier damage in ducks during Riemerella anatipestifer infection.

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

PySupercharge: a python algorithm for enabling ABC transporter bacterial secretion of all proteins through amino acid mutation.

BACKGROUND: The process of producing proteins in bacterial systems and secreting them through ATP-binding cassette (ABC) transporters is an area that has been actively researched and used due to its high protein production capacity and efficiency. However, some proteins are unable to pass through the ABC transporter after synthesis, a phenomenon we previously determined to be caused by an excessive positive charge in certain regions of their amino acid sequence. If such an excessive charge is removed, the secretion of any protein through ABC transporters becomes possible. RESULTS: In this study, we introduce 'linear charge density' as the criteria for possibility of protein secretion through ABC transporters and confirm that this criterion can be applied to various non-secretable proteins, such as SARS-CoV-2 spike proteins, botulinum toxin light chain, and human growth factors. Additionally, we develop a new algorithm, PySupercharge, that enables the secretion of proteins containing regions with high linear charge density. It selectively converts positively charged amino acids into negatively charged or neutral amino acids after linear charge density analysis to enable protein secretion through ABC transporters. CONCLUSIONS: PySupercharge, which also minimizes functional/structural stability loss of the pre-mutation proteins through the use of sequence conservation data, is currently being operated on an accessible web server. We verified the efficacy of PySupercharge-driven protein supercharging by secreting various previously non-secretable proteins commonly used in research, and so suggest this tool for use in future research requiring effective protein production.

YabJ from Staphylococcus aureus entraps chlorides within its pocket.

Chlorination is a potent disinfectant against various microorganisms, including bacteria and viruses, by inducing protein modifications and functional changes. Chlorine, in the form of sodium hypochlorite, stands out as the predominant sanitizer choice due to its cost-effectiveness and powerful antimicrobial properties. Upon exposure to chlorination, proteins undergo modifications, with amino acids experiencing alterations through the attachment of chloride or oxygen atoms. These modifications lead to shifts in protein function and the modulation of downstream signaling pathways, ultimately resulting in a bactericidal effect. However, certain survival proteins, such as chaperones or transcription factors, aid organisms in overcoming harsh chlorination conditions. The expression of YabJ, a highly conserved protein from Staphylococcus aureus, is regulated by a stress-activated sigma factor called sigma B (σB). This research revealed that S. aureus YabJ maintains its structural integrity even under intense chlorination conditions and harbors sodium hypochlorite molecules within its surface pocket. Notably, the pocket of S. aureus YabJ is primarily composed of amino acids less susceptible to chlorination-induced damage, rendering it resistant to such effects. This study elucidates how S. aureus YabJ evades the detrimental effects of chlorination and highlights its role in sequestering sodium hypochlorite within its structure. Consequently, this process enhances resilience and facilitates adaptation to challenging environmental conditions.

Reduced OxyR positively regulates the prodigiosin biosynthesis in Serratia marcescens FS14.

OxyR, a LysR family transcriptional regulator, plays vital roles in bacterial oxidative stress response. In this study, we found that the deletion of oxyR not only inhibited the antioxidant capacity of S. marcescens FS14, but also decreased the production of prodigiosin. Further study revealed that OxyR activated the prodigiosin biosynthesis at the transcriptional level. Complementary results showed that not only the wild-type OxyR but also the reduced form OxyRC199S could activate the prodigiosin biosynthesis. We further demonstrated that reduced form of wild type OxyR could bind to the promoter of pig gene cluster, and identified the binding sites which is different from oxidized OxyR binding sites in E. coli. Our results demonstrated that OxyR in FS14 uses oxidized form to regulate the expression of the antioxidant related genes and utilizes reduced form to activate prodigiosin production. Further in silico analysis suggested that the activation of prodigiosin biosynthesis by reduced OxyR should be general in S. marcesencs. To our knowledge, this is the first report to show that OxyR uses the reduced form to activate the gene's expression, therefore, our results provide a novel regulation mechanism of OxyR.

DNA binding reveals hidden interdomain allostery of a MazE antitoxin from Mycobacterium tuberculosis.

Type II toxin-antitoxin (TA) systems are ubiquitously distributed genetic elements in prokaryotes and are crucial for cell maintenance and survival under environmental stresses. The antitoxin is a modular protein consisting of the disordered C-terminal region that physically contacts and neutralizes the cognate toxin and the well-folded N-terminal DNA binding domain responsible for autorepression of TA transcription. However, how the two functional domains communicate is largely unknown. Herein, we determined the crystal structure of the N-terminal domain of the type II antitoxin MazE-mt10 from Mycobacterium tuberculosis, revealing a homodimer of the ribbon-helix-helix (RHH) fold with distinct DNA binding specificity. NMR studies demonstrated that full-length MazE-mt10 forms the helical and coiled states in equilibrium within the C-terminal region, and that helical propensity is allosterically enhanced by the N-terminal binding to the cognate operator DNA. This coil-to-helix transition may promote toxin binding/neutralization of MazE-mt10 and further stabilize the TA-DNA transcription repressor. This is supported by many crystal structures of type II TA complexes in which antitoxins form an α-helical structure at the TA interface. The hidden helical state of free MazE-mt10 in solution, favored by DNA binding, adds a new dimension to the regulatory mechanism of type II TA systems. Furthermore, complementary approaches using X-ray crystallography and NMR allow us to study the allosteric interdomain interplay of many other full-length antitoxins of type II TA systems.