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Protein phosphorylation is a fundamental cellular regulatory mechanism that governs the activation and deactivation of numerous proteins. In two-component signaling transduction pathways, the phosphorylation of response regulator proteins and their subsequent diffusion play pivotal roles in signal transmission. However, the impact of protein phosphorylation on their dispersion properties remains elusive. In this study, using the response regulator CheY in bacterial chemotaxis as a model, we performed comprehensive measurements of the spatial distributions and diffusion characteristics of CheY and phosphorylated CheY through single-molecule tracking within live cells. We discovered that phosphorylation significantly enhances diffusion and mitigates the constraining influence of the cell membrane on these proteins. Moreover, we observed that ATP-dependent fluctuations also promote protein diffusion and reduce the restraining effect of the cell membrane. These findings highlight important effects of phosphorylation beyond protein activation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Tigecycline (Tgc), a third-generation tetracycline is found as the last line of defense against multi-drug resistant bacteria. Recent increased rate of resistance to tgc, a human-restricted agent among animal bacteria poses a significant global health challenge. Overuse of first generation tetracyclines (Tet) and phenicols in animals have been suggested to be associated with Tgc resistance development. In the current study we aimed to determine the effect of tetracycline (Tet) and chloramphenicol (Chl) overexposure on Tgc susceptibility. A Tet and Chl-susceptible isolate of K. pneumoniae and E. coli were exposed to successively increasing concentrations of tetracycline and chloramphenicol separately until a ≥4 times increase in Tet and Chl MICs was observed. Susceptibility changes to several antimicrobial agents were tested using disk diffusion and broth dilution methods. The genetic alterations of genes coding for major AcrAB regulators including acrR (repressor of acrAB), ramR (repressor of ramA), soxR (repressor of soxS) in K. pneumoniae and lon (proteolytic degradation of MarA), marR (repressor of marA), acrR and soxR in E. coli were investigated. The expression level of acrB was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method. The excessive exposure (15 to 40 selection cycles) of studied bacteria to both antibiotics significantly decreased susceptibility of Tet-resistant (R) and Chl-R variants of E. coli (n = 6) and K. pneumoniae (n = 6) to several groups of antibiotics including tigecycline (4-16 and 8-64 times respectively) and quinolones. About 58% of variants (n = 7) carried genetic alterations in AcrAB regulators including ramR (frameshift mutations/locus deletion), MarR (L33R, A70T, G15S amino acid substitutions) and Lon (L630F change, frameshift mutation) which were associated with acrB upregulation. Our study demonstrated the capacity of chloramphenicol and tetracycline exposure for selection of mutants which revealed tigecycline resistance/decreased susceptibility mostly mediated by active efflux mechanism. Unaltered acrB expression level in some strains indicates possible contribution of other efflux pumps or non-efflux-based mechanisms in the development of multiple- antibiotic resistance phenotype., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Nasralddin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cell cycle adaptability assists bacteria in response to adverse stress. The effect of oxidative stress on replication initiation in Escherichia coli remains unclear. This work examined the impact of exogenous oxidant and genetic mutation-mediated oxidative stress on replication initiation. We found that 0-0.5 mM H 2 O 2 suppresses E. coli replication initiation in a concentration-dependent manner but does not lead to cell death. Deletion of antioxidant enzymes SodA-SodB, KatE, or AhpC results in delayed replication initiation. The antioxidant N-acetylcysteine (NAC) promotes replication initiation in ΔkatE and ΔsodAΔsodB mutants. We then explored the factors that mediate the inhibition of replication initiation by oxidative stress. MutY, a base excision repair DNA glycosylase, resists inhibition of replication initiation by H 2 O 2 . Lon protease deficiency eliminates inhibition of replication initiation mediated by exogenous H 2 O 2 exposure but not by katE or sodA-sodB deletion. The absence of clpP and hslV further delays replication initiation in the ΔktaE mutant, whereas hflK deletion promotes replication initiation in the ΔkatE and ΔsodAΔsodB mutants. In conclusion, non-lethal oxidative stress inhibits replication initiation, and AAA+ proteases are involved and show flexible regulation in E. coli., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Oscillation of the active form of the initiator protein DnaA (ATP-DnaA) allows for the timely regulation for chromosome replication. After initiation, DnaA-bound ATP is hydrolyzed, producing inactive ADP-DnaA. For the next round of initiation, ADP-DnaA interacts with the chromosomal locus DARS2 bearing binding sites for DnaA, a DNA-bending protein IHF, and a transcription activator Fis. The IHF binding site is about equidistant between the DnaA and Fis binding sites within DARS2. The DARS2-IHF-Fis complex promotes ADP dissociation from DnaA and furnishes ATP-DnaA at the pre-initiation stage, which dissociates Fis in a negative-feedback manner. However, regulation for IHF binding as well as mechanistic roles of Fis and specific DNA structure at DARS2 remain largely unknown. We have discovered that negative DNA supercoiling of DARS2 is required for stimulating IHF binding and ADP dissociation from DnaA in vitro. Consistent with these, novobiocin, a DNA gyrase inhibitor, inhibits DARS2 function in vivo. Fis Gln68, an RNA polymerase-interaction site, is suggested to be required for interaction with DnaA and full DARS2 activation. Based on these and other results, we propose that DNA supercoiling activates DARS2 function by stimulating stable IHF binding and DNA loop formation, thereby directing specific Fis-DnaA interaction., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phenotypic variability in isogenic bacterial populations is a remarkable feature that helps them cope with external stresses, yet it is incompletely understood. This variability can stem from gene expression noise and/or the unequal partitioning of low-copy-number freely diffusing proteins during cell division. Some high-copy-number components are transiently associated with almost immobile large assemblies (hyperstructures) and may be unequally distributed, contributing to bacterial phenotypic variability. We focus on the nucleoid hyperstructure containing numerous DNA-associated proteins, including the replication initiator DnaA. Previously, we found an increasing asynchrony in the nucleoid segregation dynamics in growing E. coli cell lineages and suggested that variable replication initiation timing may be the main cause of this phenomenon. Here, we support this hypothesis revealing that DnaA/DNA variability represents a key factor leading to the enhanced asynchrony in E. coli . We followed the intra- and intercellular distribution of fluorescently tagged DnaA and histone-like HU chromosomally encoded under their native promoters. The diffusion rate of DnaA is low, corresponding to a diffusion-binding mode of mobility, but still one order faster than that of HU. The intracellular distribution of DnaA concentration is homogeneous in contrast to the significant asymmetry in the distribution of HU to the cell halves, leading to the unequal DNA content of nucleoids and DnaA/DNA ratios in future daughter compartments. Accordingly, the intercellular variabilities in HU concentration (CV = 26%) and DnaA/DNA ratio (CV = 18%) are high. The variable DnaA/DNA may cause a variable replication initiation time (initiation noise). Asynchronous initiation at different replication origins may, in turn, be the mechanism leading to the observed asymmetric intracellular DNA distribution. Our findings indicate that the feature determining the variability of the initiation time in E. coli is the DnaA/DNA ratio, rather than each of them separately. We provide a likely mechanism for the 'loss of segregation synchrony' phenomenon.

GntR/FadR family featuring an N-terminal winged helix-turn-helix DNA-binding domain and a C-terminal α-helical effector-binding and oligomerization domain constitutes one of the largest families of transcriptional regulators. Several GntR/FadR regulators govern the metabolism of sugar acids, carbon sources implicated in bacterial-host interactions. Although effectors are known for a few sugar acid regulators, the unavailability of relevant structures has left their allosteric mechanism unexplored. Here, using DgoR, a transcriptional repressor of d-galactonate metabolism in Escherichia coli, as a model, and its superrepressor alleles, we probed allostery in a GntR/FadR family sugar acid regulator. Genetic and biochemical studies established compromised response to d-galactonate as the reason for the superrepressor behavior of the mutants: T180I does not bind d-galactonate, and while A97V, S171L and M188I bind d-galactonate, effector binding does not induce a conformational change required for derepression, suggesting altered allostery. For mechanistic insights into allosteric communication, we performed simulations of the modeled DgoR structure in different allosteric states for both the wild-type and mutant proteins. We found that each mutant exhibits unique dynamics disrupting the intrinsic allosteric communication pathways, thereby impacting DgoR function. We finally validated the allosteric communication model by testing in silico predictions with experimental data., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

H-NS, a nucleoid-associated protein (NAP) from enterobacteria, regulates gene expression by dynamically transducing environmental cues to conformational assembly and DNA binding. In this work, we show that H-NS from Escherichia coli, which can assemble into octameric and tetrameric oligomerization states, forms spontaneous micron-sized liquid-like condensates with DNA at sub-physiological concentrations in vitro. The heterotypic condensates are metastable at 298 K, partially solubilizing with time, while still retaining their liquid-like properties. The condensates display UCST-like phase behavior solubilizing at higher temperatures, but with a large decrease in droplet-assembly propensities at 310 K and at higher ionic strength. Condensate formation can be tuned in a cyclic manner between 298 and 310 K with the extent of reversibility determined by the incubation time, highlighting strong hysteresis. An engineered phospho-mimetic variant of H-NS (Y61E), which is dimeric and only weakly binds DNA, is unable to form condensates. The Y61E mutant solubilizes pre-formed H-NS condensates with DNA in a few minutes with nearly an order of magnitude speed-up in droplet dissolution at 310 K relative to 298 K, demonstrating rapid molecular transport between dilute and condensed phases. Our results establish that the oligomerization of H-NS is intrinsically tied not only to DNA binding but also its phase-separation tendencies, while showcasing the regulatable and programmable nature of heterotypic condensates formed by an archetypal NAP via multiple cues and their lifetimes., (© 2024 The Protein Society.)

The reducing system of SoxR consists of a putative electron transfer system encoded by the rsxABCDGE operon, RseC encoded from the unlinked rpoE-rseABC operon, and ApbE. RseC is composed of two transmembrane helices, with both the N-terminal and C-terminal domains located in the cytoplasm. The N-terminal domain has a four-cysteine motif, CX 5 CX 2 CX 5 C, in the cytoplasm, with the latter three cysteines highly conserved in RseC homologs, allowing the SoxR reducer complex to function in Escherichia coli . These three cysteines can form an oxygen-sensitive Fe-S cluster when only the N-terminal domain is expressed in a truncated form. Without the C-terminal domain, RseC shows no significant difference in interaction with the SoxR reducer complex, but its ability to complement the function of an rseC mutant is greatly reduced. Additionally, the rseC mutant exhibits weak resistance to cumene hydrogen peroxide in the stationary phase and increased sensitivity to hydrogen peroxide in the exponential phase, independent of the SoxR regulon. This suggests that the full-length sequence of RseC is essential for its function and that it may have SoxR-independent additional roles.

Cold-shock proteins (Csps), of around 70 amino acids, share a protein fold for the cold-shock domain (CSD) that contains RNA-binding motifs, RNP1 and RNP2, and constitute one family of bacterial RNA-binding proteins. Despite similar amino acid composition, Csps have been shown to individually possess inherent specific functions. Here, we identify the molecular differences in Csps that allow selective recognition of RNA targets. Using chimeras and mutants of Escherichia coli CspD and CspA, we demonstrate that Lys43-Ala44 in an internal loop of CspD, and the N-terminal portion with Lys4 of CspA, are important for determining their target specificities. Pull-down assays suggest that these distinct specificities reflect differences in the ability to act on the target RNAs rather than differences in binding to the RNA targets. A phylogenetic tree constructed from 1,573 Csps reveals that the Csps containing Lys-Ala in the loop form a monophyletic clade, and the members in this clade are shown to have target specificities similar to E. coli CspD. The phylogenetic tree also finds a small cluster of Csps containing Lys-Glu in the loop, and these exhibit a different specificity than E. coli CspD. Examination of this difference suggests a role of the loop of CspD-type proteins in recognition of specific targets. Additionally, each identified type of Csp shows a different distribution pattern among bacteria. Our findings provide a basis for subclassification of Csps based on target RNA specificity, which will be useful for understanding the functional specialization of Csps., (© 2025 Hasegawa et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Transition metals like Zn are essential for all organisms including bacteria, but fluctuations of their concentrations in the cell can be lethal. Organisms have thus evolved complex mechanisms for cellular metal homeostasis. One mechanistic paradigm involves pairs of transcription regulators sensing intracellular metal concentrations to regulate metal uptake and efflux. Here we report that Zur and ZntR, a prototypical pair of regulators for Zn uptake and efflux in E. coli, respectively, can coordinate their regulation through DNA, besides sensing cellular Zn 2+ concentrations. Using a combination of live-cell single-molecule tracking and in vitro single-molecule FRET measurements, we show that unmetallated ZntR can enhance the unbinding kinetics of Zur from DNA by directly acting on Zur-DNA complexes, possibly through forming heteromeric ternary and quaternary complexes that involve both protein-DNA and protein-protein interactions. This 'through-DNA' mechanism may functionally facilitate the switching in Zn-uptake regulation when bacteria encounter changing Zn environments, such as facilitating derepression of Zn-uptake genes upon Zn depletion; it could also be relevant for regulating the uptake-vs.-efflux of various metals across different bacterial species and yeast., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Fluidity is an inherent property of biological membranes and its maintenance (homeoviscous adaptation) is important for optimal functioning of membrane-associated processes. The fluidity of bacterial cytoplasmic membrane increases with temperature or an increase in the proportion of unsaturated fatty acids and vice versa. We found that strains deficient in the synthesis of guanine nucleotide analogs (p)ppGpp and lacking FadR, a transcription factor involved in fatty acid metabolism exhibited a growth defect that was rescued by an increase in growth temperature or unsaturated fatty acid content. The strain lacking (p)ppGpp was sensitive to genetic or chemical perturbations that decrease the proportion of unsaturated fatty acids over saturated fatty acids. Microscopy showed that the growth defect was associated with cell filamentation and lysis and rescued by combined expression of cell division genes ftsQ, ftsA, and ftsZ from plasmid or the gain-of-function ftsA* allele but not over-expression of ftsN. The results implicate (p)ppGpp in positive regulation of cell division during membrane fluidity loss through enhancement of FtsZ proto-ring stability. To our knowledge, this is the first report of a (p)ppGpp-mediated regulation needed for adaptation to membrane fluidity loss in bacteria., (© 2024 John Wiley & Sons Ltd.)

A cell wall is required to control cell shape and size to maintain growth and division. However, some bacterial species maintain their morphology and size without a cell wall, calling into question the importance of the cell wall to maintain shape and size. It has been very difficult to examine the dispensability of cell wall synthesis in rod-shaped bacteria such as Escherichia coli for maintenance of their shape and size because they lyse without cell walls under normal culture conditions. Here, we show that wall-less E. coli L-form cells, which have a heterogeneous cell morphology, can be converted to a mostly uniform oval shape solely by FtsZ-dependent division, even in the absence of cylindrical cell wall synthesis. This FtsZ-dependent control of cell shape and size in the absence of a cell wall requires at least either the Min or nucleoid occlusion systems for positioning FtsZ at mid cell division sites., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

A critical cell cycle checkpoint for most bacteria is the onset of constriction when the septal peptidoglycan synthesis starts. According to the current understanding, the arrival of FtsN to midcell triggers this checkpoint in Escherichia coli. Recent structural and in vitro data suggests that recruitment of FtsN to the Z-ring leads to a conformational switch in actin-like FtsA, which links FtsZ protofilaments to the cell membrane and acts as a hub for the late divisome proteins. Here, we investigate this putative pathway using in vivo measurements and stochastic cell cycle modeling at moderately fast growth rates. Quantitatively upregulating protein concentrations and determining the resulting division timings shows that FtsN and FtsA numbers are not rate-limiting for the division in E. coli. However, at higher overexpression levels, they affect divisions: FtsN by accelerating and FtsA by inhibiting them. At the same time, we find that the FtsZ numbers in the cell are one of the rate-limiting factors for cell divisions in E. coli. Altogether, these findings suggest that instead of FtsN, accumulation of FtsZ in the Z-ring is one of the main drivers of the onset of constriction in E. coli at faster growth rates., Competing Interests: Competing interests The authors declare no competing interest., (© 2024. The Author(s).)

Iron-sulfur [Fe-S] clusters are essential protein cofactors allowing bacteria to perceive environmental redox modification and to adapt to iron limitation. Escherichia coli , which served as a bacterial model, contains two [Fe-S] cluster biogenesis systems, ISC and SUF, which ensure [Fe-S] cluster synthesis under balanced and stress conditions, respectively. However, our recent phylogenomic analyses revealed that most bacteria possess only one [Fe-S] cluster biogenesis system, most often SUF. The opportunist human pathogen Pseudomonas aeruginosa is atypical as it harbors only ISC. Here, we confirmed the essentiality of ISC in P. aeruginosa under both normal and stress conditions. Moreover, P. aeruginosa ISC restored viability, under balanced growth conditions, to an E. coli strain lacking both ISC and SUF. Reciprocally, the E. coli SUF system sustained growth and [Fe-S] cluster-dependent enzyme activities of ISC-deficient P. aeruginosa . Surprisingly, an ISC-deficient P. aeruginosa strain expressing E. coli SUF showed defects in resistance to H 2 O 2 stress and paraquat, a superoxide generator. Similarly, the P. aeruginosa ISC system did not confer stress resistance to a SUF-deficient E. coli mutant. A survey of 120 Pseudomonadales genomes confirmed that all but five species have selected ISC over SUF. While highlighting the great versatility of bacterial [Fe-S] cluster biogenesis systems, this study emphasizes that their contribution to cellular homeostasis must be assessed in the context of each species and its own repertoire of stress adaptation functions. As a matter of fact, despite having only one ISC system, P. aeruginosa shows higher fitness in the face of ROS and iron limitation than E. coli ., Importance: ISC and SUF molecular systems build and transfer Fe-S cluster to cellular apo protein clients. The model Escherichia coli has both ISC and SUF and study of the interplay between the two systems established that the ISC system is the house-keeping one and SUF the stress-responding one. Unexpectedly, our recent phylogenomic analysis revealed that in contrast to E. coli (and related enterobacteria such as Salmonella), most bacteria have only one system, and, in most cases, it is SUF. Pseudomonas aeruginosa fits the general rule of having only one system but stands against the rule by having ISC. This study aims at engineering P. aeruginosa harboring E. coli systems and vice versa. Comparison of the recombinants allowed to assess the functional versatility of each system while appreciating their contribution to cellular homeostasis in different species context., Competing Interests: The authors declare no conflict of interest.

The pathway for synthesis of proline in most forms of life produces a highly unstable intermediate, γ-L-glutamyl 5-phosphate (GP). For nearly 70 y, channeling of this intermediate from the active site of glutamate 5-kinase to the active site of GP reductase has been believed to protect GP from cyclization to a dead-end product. However, the evidence presented in support of this idea is not conclusive. We show that changes in the structures of the kinase or reductase that should preclude a protein-protein interaction do not compromise proline synthesis in Escherichia coli , demonstrating that channeling does not occur. We calculate that the half-life of GP is 320 ms. Although GP is indeed unstable, it should diffuse the length of an E. coli cell in less than 3 ms. Thus, most GP produced by glutamate 5-kinase should encounter the active site of GP reductase before cyclization occurs., Competing Interests: Competing interests statement:The authors declare no competing interest.

Antimicrobial resistance (AMR) is a significant global threat, and the presence of resistance-determinant genes is one of the major driving forces behind it. The bacterial rod complex is an essential set of proteins that is crucial for cell survival due to its role in cell wall biogenesis and shape maintenance. Therefore, these proteins offer excellent potential as drug targets; however, compensatory mutations in nontarget genes render this complex nonessential. The MreB protein of this complex is an actin homologue that rotates along the longitudinal axis of the cell to provide rod shape to the bacteria. In this study, using chemical-chemical interaction profiling and FtsZ suppression assay, we identified the MreB targeting activity of IITR07865, a previously discovered small molecule in our lab. Escherichia coli suppressors against IITR07865 revealed mutations in two cell division-associated genes, min C and pal , that have not been previously implicated in rod complex essentiality. IITR07865 resistant mutants were found to inactivate and render the rod complex nonessential, making the rod complex inhibitors ineffective. Further, through transcriptome analysis, we reveal the primary cause of resistance in suppressor strains to be the overexpression of an l, d-transpeptidase A enzyme, which is involved in peptidoglycan and Braun's lipoprotein cross-linking. Our results demonstrate a novel mechanism of resistance development in rod-shaped Gram-negative bacterial pathogen E. coli involved in UTIs where mecillinam, a clinically used antibiotic that targets rod complex, is a drug of choice.

Escherichia coli has many periplasmic hydrolases to degrade and modify peptidoglycan (PG). However, the redundancy of eight PG endopeptidases makes it challenging to define specific roles to individual enzymes. Therefore, the cellular role of PBP7 (encoded by pbpG) is not clearly defined. In this work, we show that PBP7 localizes in the lateral cell envelope and at midcell. The C-terminal α-helix of PBP7 is crucial for midcell localization but not for its activity, which is dispensable for this localization. Additionally, midcell localization of PBP7 relies on the assembly of FtsZ up to FtsN in the divisome, and on the activity of PBP3. PBP7 was found to affect the assembly timing of FtsZ and FtsN in the divisome. The absence of PBP7 slows down the assembly of FtsN at midcell. The ΔpbpG mutant exhibited a weaker incorporation of the fluorescent D-amino acid HADA, reporting on transpeptidase activity, compared to wild-type cells. This could indicate reduced PG synthesis at the septum of the ΔpbpG strain, explaining the slower accumulation of FtsN and suggesting that endopeptidase-mediated PG cleavage may be a rate-limiting step for septal PG synthesis., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

Lysine and arginine methylation is an important regulator of enzyme activity and transcription in eukaryotes. However, little is known about this covalent modification in bacteria. In this work, we investigated the role of methylation in bacteria. By reanalyzing a large phyloproteomics data set from 48 bacterial strains representing six phyla, we found that almost a quarter of the bacterial proteome is methylated. Many of these methylated proteins are conserved across diverse bacterial lineages, including those involved in central carbon metabolism and translation. Among the proteins with the most conserved methylation sites is ribosomal protein L11 (bL11). bL11 methylation has been a mystery for five decades, as the deletion of its methyltransferase PrmA causes no cell growth defects. Comparative proteomics analysis combined with inorganic polyphosphate and guanosine tetra/pentaphosphate assays of the ΔprmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. In the stationary phase, we found that the ΔprmA mutant has impaired guanosine tetra/pentaphosphate production. This leads to a reduction in inorganic polyphosphate levels, accumulation of RNA and ribosomal proteins, and an abnormal polysome profile. Overall, our investigation demonstrates that the evolutionarily conserved bL11 methylation is important for stringent response signaling and ribosomal activity regulation and turnover., Importance: Protein methylation in bacteria was first identified over 60 years ago. Since then, its functional role has been identified for only a few proteins. To better understand the functional role of methylation in bacteria, we analyzed a large phyloproteomics data set encompassing 48 diverse bacteria. Our analysis revealed that ribosomal proteins are often methylated at conserved residues, suggesting that methylation of these sites may have a functional role in translation. Further analysis revealed that methylation of ribosomal protein L11 is important for stringent response signaling and ribosomal homeostasis., Competing Interests: The authors declare no conflict of interest.

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E coli (EAEC)-specific virulence factors. Here, we identified an E coli O104:H4 isolate that carried a single-nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E coli O104:H4 Δstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and ΔrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E coli O104:H4 virulence, primarily through an AggR-dependent mechanism., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Many chemoreceptors contain a C-terminal pentapeptide at the end of a linker. In Escherichia coli, this pentapeptide forms a high-affinity binding site for CheR and phosphorylated CheB, and its removal interferes with chemoreceptor adaptation. Analysis of chemoreceptors revealed significant variation in their pentapeptide sequences, and bacteria often possess multiple chemoreceptors with differing pentapeptides. To assess whether this sequence variation alters CheR affinity and chemotaxis, we used Pectobacterium atrosepticum SCRI1043 as a model. SCRI1043 has 36 chemoreceptors, with 19 of them containing a C-terminal pentapeptide. We show that the affinity of CheR for the different pentapeptides varies up to 11-fold (K D 90 nM to 1 μM). Pentapeptides with the highest and lowest affinities differ only in a single amino acid. Deletion of the cheR gene abolishes chemotaxis. The replacement of the pentapeptide in the PacC chemoreceptor with those of the highest and lowest affinities significantly reduced chemotaxis to its cognate chemoeffector, L-Asp. Altering the PacC pentapeptide also reduced chemotaxis to L-Ser, but not to nitrate, which are responses mediated by the nontethered PacB and PacN chemoreceptors, respectively. Changes in the pentapeptide sequence thus modulate the response of the cognate receptor and that of another chemoreceptor., (© 2024 John Wiley & Sons Ltd.)

The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

During bacterial cytokinesis, polymers of the bacterial tubulin FtsZ coalesce into the Z ring to orchestrate divisome assembly and septal cell wall synthesis. Previous studies have found that Z ring condensation and stability is critical for successful cell division. However, how FtsZ filaments condense into a Z ring remains enigmatic and whether septal cell wall synthesis can feedback to the Z ring has not been investigated. Here, we show that FtsZ-associated proteins (Zaps) play important roles in Z ring condensation and stability, and discover septal cell wall synthesis as a novel player for Z ring condensation and stabilization in Escherichia coli and Caulobacter crescentus. Moreover, we find that the interaction between the Z ring membrane anchor, FtsA, and components of the septal cell wall synthetic complex are critical for septal cell wall synthesis-mediated Z ring condensation. Altogether, these findings suggest that the divisome is a self-enhancing machine in these two gram-negative bacteria, where the Z ring and the septal cell wall synthetic complex communicate with and reinforce each other to ensure robustness of cell division., (© 2024. The Author(s).)

The CRISPR-Cas system provides a versatile RNA-guided approach for a broad range of applications. Thanks to advances in RNA synthetic biology, the engineering of guide RNAs (gRNAs) has enabled the conditional control of the CRISPR-Cas system. However, achieving precise regulation of the CRISPR-Cas system for efficient modulation of internal metabolic processes remains challenging. In this work, we developed a robust dCas9 regulator with engineered conditional gRNAs to enable tight control of endogenous genes. Our conditional gRNAs in Escherichia coli can control gene expression upon specific interaction with trigger RNAs with a dynamic range as high as 130-fold, evaluating up to a three-input logic A OR (B AND C). The conditional gRNA-mediated targeting of endogenous metabolic genes, lacZ, malT and poxB, caused differential regulation of growth in Escherichia coli via metabolic flux control. Further, conditional gRNAs could regulate essential cytoskeleton genes, ftsZ and mreB, to control cell filamentation and division. Finally, three types of two-input logic gates could be applied for the conditional control of ftsZ regulation, resulting in morphological changes. The successful operation and application of conditional gRNAs based on programmable RNA interactions suggests that our system could be compatible with other Cas-effectors and implemented in other host organisms., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

All sulfur transfer pathways have generally a l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of numerous sulfur-containing biomolecules in the cell. In Escherichia coli , the housekeeping l-cysteine desulfurase IscS has several interaction partners, which bind at different sites of the protein. So far, the interaction sites of IscU, Fdx, CyaY, and IscX involved in iron-sulfur (Fe-S) cluster assembly have been mapped, in addition to TusA, which is required for molybdenum cofactor biosynthesis and mnm 5 s 2 U34 tRNA modifications, and ThiI, which is involved in thiamine biosynthesis and s 4 U8 tRNA modifications. Previous studies predicted that the sulfur acceptor proteins bind to IscS one at a time. E. coli TusA has, however, been suggested to be involved in Fe-S cluster assembly, as fewer Fe-S clusters were detected in a ∆ tusA mutant. The basis for this reduction in Fe-S cluster content is unknown. In this work, we investigated the role of TusA in iron-sulfur cluster assembly and iron homeostasis. We show that the absence of TusA reduces the translation of fur , thereby leading to pleiotropic cellular effects, which we dissect in detail in this study.IMPORTANCEIron-sulfur clusters are evolutionarily ancient prosthetic groups. The ferric uptake regulator plays a major role in controlling the expression of iron homeostasis genes in bacteria. We show that a ∆tusA mutant is impaired in the assembly of Fe-S clusters and accumulates iron. TusA, therefore, reduces fur mRNA translation leading to pleiotropic cellular effects., Competing Interests: The authors declare no conflict of interest.

Initiation of DNA replication in Escherichia coli is coupled to cell size via the DnaA protein, whose activity is dependent on its nucleotide-bound state. However, the oscillations in DnaA activity have never been observed at the single-cell level. By measuring the volume-specific production rate of a reporter protein under control of a DnaA-regulated promoter, we could distinguish two distinct cell-cycle oscillators. The first, driven by both DnaA activity and SeqA repression, shows a causal relationship with cell size and divisions, similarly to initiation events. The second one, a reporter of DnaA activity alone, loses the synchrony and causality properties. Our results show that transient inhibition of gene expression by SeqA keeps the oscillation of volume-sensing DnaA activity in phase with the subsequent division event and suggest that DnaA activity peaks do not correspond directly to initiation events.

In this issue of Molecular Cell, Roeselová et al. 1 provide insights into co-translational folding of a multidomain protein in bacteria, revealing how the chaperones Trigger Factor, DnaJ, and DnaK work together to facilitate the folding of nascent chains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In 1967, in this journal, Evelyn Witkin proposed the existence of a coordinated DNA damage response in Escherichia coli , which later came to be called the "SOS response." We revisited this response using the replication inhibitor azidothymidine (AZT) and RNA-Seq analysis and identified several features. We confirm the induction of classic Save our ship (SOS) loci and identify several genes, including many of the pyrimidine pathway, that have not been previously demonstrated to be DNA damage-inducible. Despite a strong dependence on LexA, these genes lack LexA boxes and their regulation by LexA is likely to be indirect via unknown factors. We show that the transcription factor "stringent starvation protein" SspA is as important as LexA in the regulation of AZT-induced genes and that the genes activated by SspA change dramatically after AZT exposure. Our experiments identify additional LexA-independent DNA damage inducible genes, including 22 small RNA genes, some of which appear to activated by SspA. Motility and chemotaxis genes are strongly down-regulated by AZT, possibly as a result of one of more of the small RNAs or other transcription factors such as AppY and GadE, whose expression is elevated by AZT. Genes controlling the iron siderophore, enterobactin, and iron homeostasis are also strongly induced, independent of LexA. We confirm that IraD antiadaptor protein is induced independent of LexA and that a second antiadaptor, IraM is likewise strongly AZT-inducible, independent of LexA, suggesting that RpoS stabilization via these antiadaptor proteins is an integral part of replication stress tolerance., Competing Interests: Competing interests statement:The authors declare no competing interest.

The rpoN operon, an important regulatory hub in Enterobacteriaceae, includes rpoN encoding sigma factor σ 54 , hpf involved in ribosome hibernation, rapZ regulating glucosamine-6-phosphate levels, and two genes encoding proteins of the nitrogen-related phosphotransferase system. Little is known about regulatory mechanisms controlling the abundance of these proteins. This study employs transposon mutagenesis and chemical screens to dissect the complex expression of the rpoN operon. We find that envelope stress conditions trigger read-through transcription into the rpoN operon from a promoter located upstream of the preceding lptA-lptB locus. This promoter is controlled by the envelope stress sigma factor E and response regulator PhoP is required for its full response to a subset of stress signals. σ E also stimulates ptsN-rapZ-npr expression using an element downstream of rpoN, presumably by interfering with mRNA processing by RNase E. Additionally, we identify a novel promoter in the 3' end of rpoN that directs transcription of the distal genes in response to ethanol. Finally, we show that translation of hpf and ptsN is individually regulated by the RNA chaperone Hfq, perhaps involving small RNAs. Collectively, our work demonstrates that the rpoN operon is subject to complex regulation, integrating signals related to envelope stress and carbon source quality., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

MutT proteins belong to the Nudix hydrolase superfamily that includes a diverse group of Mg 2+ requiring enzymes. These proteins use a generalized substrate, nucleoside diphosphate linked to a chemical group X (NDP-X), to produce nucleoside monophosphate (NMP) and the moiety X linked with phosphate (XP). E. coli MutT (EcoMutT) and mycobacterial MutT1 (MsmMutT1) belong to the Nudix hydrolase superfamily that utilize 8-oxo-(d)GTP (referring to both 8-oxo-GTP or 8-oxo-dGTP). However, predominant products of their activities are different. While EcoMutT produces 8-oxo-(d)GMP, MsmMutT1 gives rise to 8-oxo-(d)GDP. Here, we show that the altered cleavage specificities of the two proteins are largely a consequence of the variation at the equivalent of Gly37 (G37) in EcoMutT to Lys (K65) in the MsmMutT1. Remarkably, mutations of G37K (EcoMutT) and K65G (MsmMutT1) switch their cleavage specificities to produce 8-oxo-(d)GDP, and 8-oxo-(d)GMP, respectively. Further, a time course analysis using 8-oxo-GTP suggests that MsmMutT1(K65G) hydrolyses 8-oxo-(d)GTP to 8-oxo-(d)GMP in a two-step reaction via 8-oxo-(d)GDP intermediate. Expectedly, unlike EcoMutT (G37K) and MsmMutT1, EcoMutT and MsmMutT1 (K65G) rescue an E. coli ΔmutT strain, better by decreasing A to C mutations., Competing Interests: Declaration of Competing Interest None to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Escherichia coli O157:H7 ( E. coli O157:H7) and Campylobacter jejuni ( C. jejuni ) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2 , and eaeA . Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.

In recent years, new evidence has shown that the SOS response plays an important role in the response to antimicrobials, with involvement in the generation of clinical resistance. Here we evaluate the impact of heterogeneous expression of the SOS response in clinical isolates of Escherichia coli on response to the fluoroquinolone, ciprofloxacin. In silico analysis of whole genome sequencing data showed remarkable sequence conservation of the SOS response regulators, RecA and LexA. Despite the genetic homogeneity, our results revealed a marked differential heterogeneity in SOS response activation, both at population and single-cell level, among clinical isolates of E. coli in the presence of subinhibitory concentrations of ciprofloxacin. Four main stages of SOS response activation were identified and correlated with cell filamentation. Interestingly, there was a correlation between clinical isolates with higher expression of the SOS response and further progression to resistance. This heterogeneity in response to DNA damage repair (mediated by the SOS response) and induced by antimicrobial agents could be a new factor with implications for bacterial evolution and survival contributing to the generation of antimicrobial resistance., Competing Interests: Declaration of Competing Interest On behalf of all authors, I declare that we have no commercial interests related to the submitted manuscript., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after the completion of successful antibiotic treatment. Many clinical isolates, including the well-characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex integration host factor and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two-hybrid assays. We report direct interactions between the succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions may enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility., Importance: Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections (UTIs). Upon invasion into bladder epithelial cells, UPEC establish quiescent intracellular reservoirs that may lead to antibiotic tolerance and recurrent UTIs. Here, we demonstrate using an in vitro system that quiescent UPEC cells are tolerant to ampicillin and have decreased metabolism characterized by succinyl-CoA limitation. We identify the global regulator integration host factor complex and the cell division protein ZapE as critical modifiers of quiescence and antibiotic tolerance. Finally, we show that ZapE interacts with components of both the cell division machinery and the tricarboxylic acid cycle, and this interaction is conserved in non-pathogenic E. coli , establishing a novel link between cell division and metabolism., Competing Interests: The authors declare no conflict of interest.

The EnvZ-OmpR two-component system of Escherichia coli regulates the expression of the ompF and ompC porin genes in response to medium osmolarity. However, certain mutations in envZ confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel envZ and ompR pleiotropic alleles, envZT15P and ompRL19Q , among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike envZ , pleiotropic mutations in ompR have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (Δ degP ) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential β-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a Δ bamB mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic ompR / envZ alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity., Competing Interests: The authors declare no conflict of interest.

The Type VI secretion system (T6SS) functions as a protein transport nanoweapon in several stages of bacterial life. Even though bacterial competition is the primary function of T6SS, different bacteria exhibit significant variations. Particularly in Extraintestinal pathogenic Escherichia coli (ExPEC), research into T6SS remains relatively limited. This study identified the uncharacterized gene evfG within the T6SS cluster of ExPEC RS218. Through our experiments, we showed that evfG is involved in T6SS expression in ExPEC RS218. We also found evfG can modulate T6SS activity by competitively binding to c-di-GMP, leading to a reduction in the inhibitory effect. Furthermore, we found that evfG can recruit sodA to alleviate oxidative stress. The research shown evfG controls an array of traits, both directly and indirectly, through transcriptome and additional tests. These traits include cell adhesion, invasion, motility, drug resistance, and pathogenicity of microorganisms. Overall, we contend that evfG serves as a multi-functional regulator for the T6SS and several crucial activities. This forms the basis for the advancement of T6SS function research, as well as new opportunities for vaccine and medication development., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier GmbH.)

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E. coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In many organisms, stress responses to adverse environments can trigger secondary functions of certain proteins by altering protein levels, localization, activity, or interaction partners. Escherichia coli cells respond to the presence of specific cationic antimicrobial peptides by strongly activating the PhoQ/PhoP two-component signaling system, which regulates genes important for growth under this stress. As part of this pathway, a biosynthetic enzyme called QueE, which catalyzes a step in the formation of queuosine (Q) tRNA modification is upregulated. When cellular QueE levels are high, it co-localizes with the central cell division protein FtsZ at the septal site, blocking division and resulting in filamentous growth. Here we show that QueE affects cell size in a dose-dependent manner. Using alanine scanning mutagenesis of amino acids in the catalytic active site, we pinpoint residues in QueE that contribute distinctly to each of its functions-Q biosynthesis or regulation of cell division, establishing QueE as a moonlighting protein. We further show that QueE orthologs from enterobacteria like Salmonella typhimurium and Klebsiella pneumoniae also cause filamentation in these organisms, but the more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this ability. By comparative analysis of E. coli QueE with distant orthologs, we elucidate a unique region in this protein that is responsible for QueE's secondary function as a cell division regulator. A dual-function protein like QueE is an exception to the conventional model of "one gene, one enzyme, one function", which has divergent roles across a range of fundamental cellular processes including RNA modification and translation to cell division and stress response., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Srujana S. Yadavalli consults for and collaborates with Designs for Vision Inc. and Samuel A. Adeleye declared that no competing interests exist., (Copyright: © 2024 Adeleye, Yadavalli. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA∗ alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologs revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The bacterial chemotaxis system is a well-understood signaling pathway that promotes bacterial success. Chemotaxis systems comprise chemoreceptors and the CheA kinase, linked by CheW or CheV scaffold proteins. Scaffold proteins provide connections between chemoreceptors and CheA and also between chemoreceptors to create macromolecular arrays. Chemotaxis is required for host colonization by many microbes, including the stomach pathogen Helicobacter pylori . This bacterium builds chemoreceptor-CheA contacts with two distinct scaffold proteins, CheW and CheV1. H. pylori cheW or cheV1 deletion mutants both lose chemoreceptor array formation, but show differing semisolid agar chemotaxis assay behaviors: ∆ cheW mutants exhibit total migration failure, whereas ∆ cheV1::cat mutants display a 50% reduction. On investigating these varied responses, we found that both mutants initially struggle with migration. However, over time, ∆ cheV1::cat mutants develop a stable, enhanced migration capability, termed "migration-able" (Mig + ). Whole-genome sequencing analysis of four distinct ∆ cheV1::cat Mig + strains identified single-nucleotide polymorphisms (SNPs ) in hpg27_252 (hp0273 ) that were predicted to truncate the encoded protein. Computational analysis of the hpg27_252 -encoded protein revealed it encoded a hypothetical protein that was a remote homolog of the PilO Type IV filament membrane alignment complex protein. Although H. pylori lacks Type IV filaments, our analysis showed it retains an operon of genes for homologs of PilO, PilN, and PilM. Deleting hpg27_252 in the ∆ cheV1::cat or wild type strain resulted in enhanced migration in semisolid agar. Our study thus reveals that while cheV1 mutants initially have significant migration defects, they can recover the migration ability through genetic suppressors, highlighting a complex regulatory mechanism in bacterial migration., Importance: Chemotactic motility, present in over half of bacteria, depends on chemotaxis signaling systems comprising receptors, kinases, and scaffold proteins. In Helicobacter pylori , a stomach pathogen, chemotaxis is crucial for colonization, with CheV1 and CheW as key scaffold proteins. While both scaffolds are essential for building chemoreceptor complexes, their roles vary in other assays. Our research reexamines cheV1 mutants' behavior in semisolid agar, a standard chemotaxis test. Initially, cheV1 mutants exhibited defects similar to those of cheW mutants, but they evolved genetic suppressors that enhanced migration. These suppressors involve mutations in a previously uncharacterized gene, unknown in motility behavior. Our findings highlight the significant chemotaxis defects in cheV1 mutants and identify new elements influencing bacterial motility., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s).)

The stringent response exerted by (p)ppGpp and RNA-polymerase binding protein DksA regulates gene expression in diverse bacterial species. To control gene expression (p)ppGpp, synthesized by enzymes RelA and SpoT, interacts with two sites within the RNA polymerase; site 1, located in the interphase between subunits β' and ω (rpoZ), and site 2 located in the secondary channel that is dependent on DksA protein. In Escherichia coli, inactivation of dksA results in a reduced sigma factor RpoS expression. In Azotobacter vinelandii the synthesis of polyhydroxybutyrate (PHB) is under RpoS regulation. In this study, we found that the inactivation of relA or dksA, but not rpoZ, resulted in a negative effect on PHB synthesis. We also found that the dksA, but not the relA mutation reduced both rpoS transcription and RpoS protein levels, implying that (p)ppGpp and DksA control PHB synthesis through different mechanisms. Interestingly, despite expressing rpoS from a constitutive promoter in the dksA mutant, PHB synthesis was not restored to wild type levels. A transcriptomic analysis in the dksA mutant, revealed downregulation of genes encoding enzymes needed for the synthesis of acetyl-CoA, the precursor substrate for PHB synthesis. Together, these data indicate that DksA is required for optimal expression of RpoS which in turn activates transcription of genes for PHB synthesis. Additionally, DksA is required for optimal transcription of genes responsible for the synthesis of precursors for PHB synthesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ortiz-Vasco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s).)

It was generally postulated that when intracellular free iron content is elevated in bacteria, the ferric uptake regulator (Fur) binds its corepressor a mononuclear ferrous iron to regulate intracellular iron homeostasis. However, the proposed iron-bound Fur had not been identified in any bacteria. In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box. Here we find that the iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content. Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro. While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF. Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur and that regulation of intracellular iron homeostasis is closely coupled with iron-sulfur cluster biogenesis in E. coli., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The acid signal transduction system can sense the acidic environment and translate it into signals to regulate various acid tolerance mechanisms within bacteria, helping them to cope with the stress of the acidic environment and survive the acidic environments. This review describes several major acid signal transduction systems that play important roles in acid-tolerant bacteria: EvgS/EvgA, PhoQ/PhoP, ArsS/ArsR, and CadC. The structural components of these systems and their regulation of acid-tolerant systems were used to analyze how acid-tolerant bacteria transduce signal in an acid environment to activate the corresponding acid-tolerance mechanisms and cope with the acid stress. An in-depth understanding of the regulatory mechanisms of acid-tolerant systems can help the mining, optimal design and construction of multiple acid-tolerant parts to improve the growth and metabolism of target strains in acidic environments. It helps to better utilize engineered microorganisms with super acid-resistance for industrial production of valuable metabolites, bioremediation of pollution in acidic environments. Moreover, it also helps to provide novel targets for inhibiting the growth of acid-tolerant pathogenic bacteria.

RpoS is an alternative sigma factor needed for the induction of the general stress response in many gammaproteobacteria. Tight regulation of RpoS levels and activity is required for bacterial growth and survival under stress. In Escherichia coli, various stresses lead to higher levels of RpoS due to increased translation and decreased degradation. During non-stress conditions, RpoS is unstable, because the adaptor protein RssB delivers RpoS to the ClpXP protease. RpoS degradation is prevented during stress by the sequestration of RssB by anti-adaptors, each of which is induced in response to specific stresses. Here, we examined how the stabilization of RpoS is reversed during recovery of the cell from stress. We found that RpoS degradation quickly resumes after recovery from phosphate starvation, carbon starvation, and when transitioning from stationary phase back to exponential phase. This process is in part mediated by the anti-adaptor IraP, known to promote RpoS stabilization during phosphate starvation via the sequestration of adaptor RssB. The rapid recovery from phosphate starvation is dependent upon a feedback loop in which RpoS transcription of rssB, encoding the adaptor protein, plays a critical role. Crl, an activator of RpoS that specifically binds to and stabilizes the complex between the RNA polymerase and RpoS, is also required for the feedback loop to function efficiently, highlighting a critical role for Crl in restoring RpoS basal levels., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only ∼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging., Competing Interests: Declaration of interests A.N.K. is a co-founder and shareholder of Oxford Nanoimaging (ONI), a company that builds, sells, and supports miniaturized fluorescence microscopes for single-molecule imaging, including for single-particle tracking., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the P lac /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS)., (© 2024 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Bacteria express lytic enzymes such as glycosidases, which have potentially self-destructive peptidoglycan (PG)-degrading activity and, therefore, require careful regulation in bacteria. The PG glycosidase EtgA is regulated by localization to the assembling type III secretion system (T3SS), generating a hole in the PG layer for the T3SS to reach the outer membrane. The EtgA localization was found to be mediated via EtgA interacting with the T3SS inner rod protein EscI. To gain structural insights into the EtgA recognition of EscI, we determined the 2.01 Å resolution structure of an EscI (51-87)-linker-EtgA fusion protein designed based on AlphaFold2 predictions. The structure revealed EscI residues 72-87 forming an α-helix interacting with the backside of EtgA, distant from the active site. EscI residues 56-71 also were found to interact with EtgA, with these residues stretching across the EtgA surface. The ability of the EscI to interact with EtgA was also probed using an EscI peptide. The EscI peptide comprising residues 66-87, slightly larger than the observed EscI α-helix, was shown to bind to EtgA using microscale thermophoresis and thermal shift differential scanning fluorimetry. The EscI peptide also had a two-fold activity-enhancing effect on EtgA, whereas the EscI-EtgA fusion protein enhanced activity over four-fold compared to EtgA. Our studies suggest that EtgA regulation by EscI could be trifold involving protein localization, protein activation, and protein stabilization components. Analysis of the sequence conservation of the EscI EtgA interface residues suggested a possible conservation of such regulation for related proteins from different bacteria., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The bacterial divisome is a complex nanomachine that drives cell division and separation. The essentiality of these processes leads to the assumption that proteins with core roles will be strictly conserved across all bacterial genomes. However, recent studies in diverse proteobacteria have revealed considerable variation in the early divisome compared with Escherichia coli. While some proteins are highly conserved, their specific functions and interacting partners vary. Meanwhile, different subphyla use clade-specific proteins with analogous functions. Thus, instead of focusing on gene conservation, we must also explore how key functions are maintained during early division by diverging protein networks. An enhanced awareness of these complex genetic networks will clarify the physical and evolutionary constraints of bacterial division., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The DedA superfamily is a highly conserved family of membrane proteins. Deletion of Escherichia coli yqjA and yghB , encoding related DedA family proteins, results in sensitivity to elevated temperature, antibiotics, and alkaline pH. The human pathogen Klebsiella pneumoniae possesses genes encoding DedA family proteins with >90% amino acid identity to E. coli YqjA and YghB. We hypothesized that the deletion of K. pneumoniae yqjA and yghB will impact its physiology and may reduce its virulence. The K. pneumoniae Δ yqjA Δ yghB mutant (strain VT101) displayed a growth defect at 42°C and alkaline pH sensitivity, not unlike its E. coli counterpart. However, VT101 retained mostly wild-type resistance to antibiotics. We found VT101 was sensitive to the chelating agent EDTA, the anionic detergent SDS, and agents capable of alkalizing the bacterial cytoplasm such as bicarbonate or chloroquine. We could restore growth at alkaline pH and at elevated temperature by addition of 0.5-2 mM Ca 2+ or Mg 2+ to the culture media. VT101 displayed a slower uptake of calcium, which was dependent upon calcium channel activity. VT201, with similar deletions as VT101 but derived from a virulent K. pneumoniae strain, was highly susceptible to phagocytosis by alveolar macrophages and displayed a defect in the production of capsule. These findings suggest divalent cation homeostasis and virulence are interlinked by common functions of the DedA family.IMPORTANCE Klebsiella pneumoniae is a dangerous human pathogen. The DedA protein family is found in all bacteria and is a membrane transporter often required for virulence and antibiotic resistance. K. pneumoniae possesses homologs of E. coli YqjA and YghB, with 60% amino acid identity and redundant functions, which we have previously shown to be required for tolerance to biocides and alkaline pH. A K. pneumoniae strain lacking yqjA and yghB was found to be sensitive to alkaline pH, elevated temperature, and EDTA/SDS and displayed a defect in calcium uptake. Sensitivity to these conditions was reversed by addition of calcium or magnesium to the growth medium. Introduction of Δ yqjA and Δ yghB mutations into virulent K. pneumoniae resulted in the loss of capsule, increased phagocytosis by macrophages, and a partial loss of virulence. These results show that targeting the Klebsiella DedA family results in impaired divalent cation transport and, in turn, loss of virulence., Competing Interests: The authors declare no conflict of interest.

The expression of most molybdoenzymes in Escherichia coli has so far been revealed to be regulated by anaerobiosis and requires the presence of iron, based on the necessity of the transcription factor FNR to bind one [4Fe-4S] cluster. One exception is trimethylamine- N -oxide reductase encoded by the torCAD operon, which has been described to be expressed independently from FNR. In contrast to other alternative anaerobic respiratory systems, the expression of the torCAD operon was shown not to be completely repressed by the presence of dioxygen. To date, the basis for the O 2 -dependent expression of the torCAD operon has been related to the abundance of the transcriptional regulator IscR, which represses the transcription of torS and torT, and is more abundant under aerobic conditions than under anaerobic conditions. In this study, we reinvestigated the regulation of the torCAD operon and its dependence on the presence of iron and identified a novel regulation that depends on the presence of the bis-molybdopterin guanine dinucleotide (bis-MGD) molybdenum cofactor . We confirmed that the torCAD operon is directly regulated by the heme-containing protein TorC and is indirectly regulated by ArcA and by the availability of iron via active FNR and Fur, both regulatory proteins that influence the synthesis of the molybdenum cofactor. Furthermore, we identified a novel regulation mode of torCAD expression that is dependent on cellular levels of bis-MGD and is not used by other bis-MGD-containing enzymes like nitrate reductase.IMPORTANCEIn bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. FNR is a very important transcription factor that represents the master switch for the expression of target genes in response to anaerobiosis. Only Escherichia coli trimethylamine- N -oxide (TMAO) reductase escapes this regulation by FNR. We identified that the expression of TMAO reductase is regulated by the amount of bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor synthesized by the cell itself, representing a novel regulation pathway for the expression of an operon coding for a molybdoenzyme. Furthermore, TMAO reductase gene expression is indirectly regulated by the presence of iron, which is required for the production of the bis-MGD cofactor in the cell., Competing Interests: The authors declare no conflict of interest.