The toxicity of the amino acids glycine and L-serine at high concentrations in bacteria was discovered decades ago. In this work, we used transposon insertion sequencing (Tn-seq) experiments to determine the genes necessary to tolerate excess L-serine, diglycine, or glycine in the human pathogen Staphylococcus aureus. Our results indicate that intracellular accumulation of specific counterbalancing amino acids-such as alanine in the presence of excess glycine-is the primary mechanism of resistance to amino acid toxicity. Consistent with this model, specific amino acid and peptide uptake transporters were required for fitness in each treatment; the peptide transporter DtpT was crucial for fitness in excess L-serine or glycine, and the alanine transporter AapA was necessary for fitness in diglycine. Tn-seq results also identified the cystine/cysteine uptake transporter TcyABC as necessary in excess L-serine, suggesting that both peptide and cysteine uptake contribute to L-serine tolerance. In addition to uptake mechanisms, glycine and diglycine toxicity is neutralized by D-alanine aminotransferase (Dat), which is required for D-alanine synthesis. The requirement for Dat and DtpT function-but not AapA-in excess glycine is explained by excess glycine inhibiting alanine uptake. Building on this finding, we found that combined treatment with glycine and the alanine analog antibiotic D-cycloserine was strongly synergistic in inhibiting S. aureus growth. Overall, our findings identify targetable mechanisms underlying excess amino acid tolerance in S. aureus, with implications for developing novel combination treatments using the accessible and biocompatible amino acids glycine and L-serine.

The multilayered cell envelope of Acinetobacter baumannii is an essential structure that maintains cellular integrity and protects the bacterial cell against external stresses and antibiotics. It consists of an inner membrane, a thin peptidoglycan (PG) layer, and an asymmetric outer membrane (OM) enriched in lipooligosaccharide (LOS), whose lipid A moiety is the target of colistin, a last-resort antibiotic. Although lipid A is essential in most Gram-negative bacteria, A. baumannii can survive without LOS through envelope remodeling, particularly in strains producing low levels of the bifunctional penicillin-binding protein PBP1A (encoded by mrcA) or in ΔmrcA mutants. Here, we identify a genetic and functional interplay between the LD-transpeptidase LdtJ, which generates 3-3 cross-links, and PBP1A, which catalyzes 4-3 transpeptidation during PG synthesis. We show that simultaneous inactivation of both enzymes severely affects growth, viable counts, morphology, and OM homeostasis. PG analyses revealed that the ΔldtJ ΔmrcA mutants display reduced overall cross-linking and shorter glycan chains, consistent with a weakened sacculus. Co-immunoprecipitation demonstrated that PBP1A associates with LdtJ, suggesting that the two proteins may act in close proximity during PG synthesis. Notably, ΔldtJ ΔmrcA mutants exhibited the highest recovery frequency of colistin-resistant, LOS-deficient variants compared with wild type or single mutants. Together, our findings indicate that 4-3 and 3-3 transpeptidase activities play complementary roles in PG synthesis and remodeling, contributing to envelope stability in A. baumannii, and that disruption of these activities favors the emergence of colistin resistance. This work further highlights the importance of PG remodeling in maintaining envelope homeostasis and suggests that proper PG cross-linking acts as a physiological constraint to limit the emergence of LOS-deficient colistin-resistant variants in A. baumannii, providing new insight into mechanisms that contribute to envelope destabilization.IMPORTANCEThe global rise of multidrug-resistant Acinetobacter baumannii represents an urgent clinical threat, driven by its capacity to withstand cell envelope damage and escape last-resort antibiotics such as colistin. Although peptidoglycan synthesis and remodeling are known to influence outer membrane stability, how these processes interact in A. baumannii remains unclear. Here, we identify genetic and functional interactions between two major peptidoglycan biosynthetic and remodeling activities, 4-3 and 3-3 transpeptidation, mediated by the bifunctional PBP1A and the LD-transpeptidase LdtJ, respectively. Disrupting these activities weakens the peptidoglycan, destabilizes the outer membrane, and increases the emergence of lipooligosaccharide-deficient, colistin-resistant variants. These findings highlight the importance of proper peptidoglycan cross-linking for envelope homeostasis in A. baumannii and provide insight into how disruptions in peptidoglycan architecture influence antibiotic resistance.

Isophthalate serves as a carbon and energy source for several microorganisms through a dedicated catabolic pathway, in which isophthalate dihydrodiol dehydrogenase (IphB) mediates the NAD(P)[+]-dependent oxidation of 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD) to protocatechuate. Here, we present the structural and biochemical characterization of isophthalate dihydrodiol dehydrogenase (IphBKF1) from Comamonas testosteroni KF1. Enzyme kinetics revealed IphBKF1 catalyzes the dehydrogenation of 1,5-DCD substrate with both NAD[+] and NADP[+], exhibiting a ~4 fold higher catalytic efficiency (kcat/KM) with NADP[+]. Crystal structures of IphBKF1 were determined in the apo form, in complex with NADP[+], and as a ternary complex with NADP[+] and the product 3,4-dihydroxybenzoic acid (3,4-DHB; protocatechuate) at resolutions of 1.5, 1.8, and 1.5 Å, respectively. Structural analysis reveals an NADP[+]-induced conformational change that facilitates cofactor accommodation and identifies key residues involved in NADP[+] binding. Sequence and structural analyses highlight two conserved motifs, GXXXGXG and YXXXK, characteristic of the short-chain dehydrogenase/reductase (SDR) superfamily. Ternary complex of IphBKF1 (NADP[+] and 3,4-DHB) and molecular docking with 1,5-DCD suggests that specific active-site residues contribute to substrate recognition and underpin the distinct substrate specificity of IphBKF1 compared to other SDR family members. Based on structural insights and comparison with homologous enzymes, we propose a catalytic mechanism for isophthalate oxidation. Together, these findings provide a comprehensive framework for understanding substrate specificity and catalysis in isophthalate dihydrodiol dehydrogenases.IMPORTANCEIsophthalate is a recalcitrant aromatic compound linked to the environmental release of phthalate-based plastics and plasticizers. However, the microbial degradation of isophthalate is relatively less known in comparison to other phthalate isomers. Efficient bacterial utilization of isophthalate requires a coordinated pathway in which the cis-dihydrodiol dehydrogenase IphB converts intermediates into protocatechuate, a central carbon metabolism intermediate. Despite its importance, the structural basis of this transition has remained unclear. Here, we provide a comprehensive structural and biochemical investigation of isophthalate dihydrodiol dehydrogenase (IphBKF1), revealing the structural basis for cofactor binding, active-site architecture, and conformational dynamics leading to the selective processing of isophthalate-derived substrates. These findings provide a framework for the design of enzymes with improved activity and selectivity for pollutant removal.

Heterocyst-forming cyanobacteria (order Nostocales) are a monophyletic group of organisms that grow as filaments usually made of dozens to hundreds of cells (termed vegetative cells) that can perform oxygenic photosynthesis. Under combined nitrogen deficiency, some cells in the filament differentiate into N2-fixing heterocysts, which do not produce oxygen and, indeed, keep a microoxic environment appropriate for the expression and functioning of the N2-fixing enzyme, nitrogenase. Thus, in the diazotrophic filament, oxygenic photosynthesis and N2 fixation are segregated into two different cell types, vegetative cells and heterocysts, which require intercellular molecular exchange to feed each other with fixed nitrogen and reduced carbon, respectively. The current understanding of heterocyst biology, including regulation of heterocyst differentiation, which also involves intercellular signaling, is the result of studying mainly a few strains of the genus Anabaena. Here, we review the classical studies that established Anabaena as a model for the Nostocales and clarified the basics of N2 fixation in these organisms. Then, we show how molecular genetics came into the scene and how it was complemented by genomic analysis. This, together with the introduction of modern cell biology approaches, led to a renewed understanding of cellular differentiation and unraveled a system for intercellular communication that is unique in the biological world. In turn, the new accumulated knowledge on Anabaena physiology and genetics makes these organisms an excellent system for the development of new synthetic biology tools that will be crucial for their use in biotechnology.

A hallmark of Mycobacterium tuberculosis survival and proliferation within the host is attributable to the effective stress response of the pathogen against hostile environmental conditions. Although controlled iron (Fe) uptake and maintenance of Fe homeostasis are closely associated with intracellular survival of mycobacteria, the underlying mechanism that accounts for the interdependence of mycobacterial Fe regulation and oxidative stress response remains unknown. In this study, we discovered that Fe accumulation connects intra-bacterial redox environment via the phoP locus. Gene expression analyses coupled with genetic, biochemical, and transcriptomic studies uncover a distinct mechanism that utilizes Lsr2 and the virulence regulator PhoP to modulate transcriptional control of the major Fe storage protein-encoding gene bfrB. Remarkably, regulated expression of bfrB determines bacterial tolerance to oxidative stress and its intracellular survival. Although both regulators could independently influence bfrB expression, the most fundamental insights are derived from the inhibition of Lsr2 binding at the bfrB regulatory region of the phoP-KO mutant strain owing to Lsr2-PhoP protein-protein interactions. Building on these results, we proposed a model suggesting how Lsr2-PhoP interaction, or lack thereof, contributes to stress-specific regulation of bfrB expression. Collectively, these results uncover a key mechanism integrating Fe accumulation to oxidative stress response, an essential pathogenic determinant of M. tuberculosis.IMPORTANCEWhile the availability of Fe remains essential for Mycobacterium tuberculosis infection and replication of the pathogen in human macrophages, Fe may be harmful as it catalyzes the generation of toxic reactive oxygen species from products of aerobic respiration to induce oxidative stress. Although interdependence of mycobacterial Fe regulation and oxidative stress response has been reported, the mechanism connecting Fe acquisition and oxidative stress response is yet to be fully understood. Results reported in this study identify and elaborate on a new mechanism of regulation of expression of the major mycobacterial Fe storage protein, which determines bacterial tolerance to oxidative stress and impacts intracellular survival of the pathogen. We demonstrate that the virulence-associated phoP locus controls Fe accumulation and contributes to the intra-bacterial redox environment. The results uncover a distinctly different transcriptional control mechanism of the major Fe storage protein requiring a co-repressor-dependent regulatory circuit, including nucleoid-associated protein Lsr2 and the virulence regulator PhoP. Together, these results unveil a unique mechanism of stress-specific gene expression, which integrates Fe homeostasis to oxidative stress response, an essential pathogenic determinant of M. tuberculosis.

Biofilm and colony growth create microenvironments that require coordinated localization and function of cell envelope components. However, the mechanisms by which membrane organization contributes to cell envelope organization during multicellular growth remain poorly understood. Mycobacterium smegmatis contains an inner membrane domain (IMD) at the subpolar regions of the cell that contributes to cell envelope synthesis. Prior research identified PonA2 as important for de novo formation of distinct plasma membrane domains during planktonic growth. PonA2 is a penicillin-binding protein with transglycosylase (TG) and transpeptidase (TP) activities involved in peptidoglycan synthesis. To investigate the role of PonA2 during colony and pellicle biofilm growth, wild-type, ΔponA2, the complement strain (cponA2), and catalytically inactive PonA2 variants (TG-, TP-, and TG-/TP-) were analyzed. Subpolar IMD localization was maintained in wild-type cells during both colony and pellicle biofilm growth, demonstrating that IMD localization is not exclusive to planktonic growth. Loss of ponA2 resulted in smooth colony and early pellicle biofilm architecture and was associated with reduced abundance of trehalose monomycolate, trehalose dimycolate, and glycopeptidolipids. ΔponA2 cells in colony growth exhibited shortened cell morphology and lacked detectable IMD localization, whereas ΔponA2 cells in pellicle biofilm growth retained bacillary morphology and subpolar IMD localization. In contrast, the catalytically inactive PonA2 variants maintained rugose multicellular architecture, bacillary morphology, and subpolar IMD localization similar to that of wild-type. Together, these findings identify a previously unrecognized noncatalytic role for PonA2 in maintaining normal colony and pellicle biofilm development in M. smegmatis.IMPORTANCEPlanktonic, pellicle biofilm, and colony growth expose mycobacteria to distinct environmental conditions that can affect cell-envelope organization and survival. Prior research has shown that mycobacteria form subpolar plasma membrane domains that support polar cell elongation in planktonic growth, but it remains unclear whether this organization is conserved in multicellular biofilm and colony growth, where cells experience nutrient and oxygen gradients and altered cell-to-cell interactions. Our current study analyzed the plasma membrane domain formation across the two growth states and showed that membrane domain localization is conserved, while the mechanisms required to maintain these domains differ depending on growth conditions. These findings suggest that mycobacteria use growth state-specific mechanisms to coordinate membrane organization.

Vibrio cholerae is the causative agent of the human diarrheal disease cholera. Previous studies identified "hyperswarming" (HS) mutant V. cholerae with enhanced motility and decreased virulence factor expression, but the genetic basis of HS was unknown. Through whole-genome sequencing, we have identified point mutations within toxT, which encodes the master virulence regulator, as being responsible for the HS phenotype. These mutations are loss of function, and they eliminate transcription of cholera toxin and toxin-coregulated pilus genes. Using single-cell video tracking, these HS strains were also observed to exhibit enhanced motility/chemotaxis (increased speed and decreased directional change). Two methyl-accepting chemoreceptors (methyl-accepting chemotaxis proteins), AcfB and TcpI, are positively regulated by ToxT, and expression of both AcfB and TcpI within HS mutants restores wild-type motility. Moreover, a V. cholerae strain with a hyperactive ToxT (N106A) exhibits reduced motility (decreased speed and increased directional change), and knocking out acfB and tcpI in this strain restores wild-type motility. AcfB and TcpI localize to the bacterial cell poles, suggesting they incorporate into the chemotaxis arrays to modulate swimming behavior. Our findings demonstrate that the absence of the ToxT-dependent MCPs AcfB and TcpI is responsible for the HS phenotype, demonstrating a direct role of ToxT in the swimming behavior of V. cholerae.IMPORTANCEVibrio cholerae causes the human diarrheal disease cholera, and the master virulence regulator ToxT is responsible for the expression of the most important virulence factors, cholera toxin and the toxin-coregulated pilus, necessary for disease. Motility is also important for the bacteria to cause disease, and in this study, we provide evidence that ToxT also modulates motility, providing a regulatory link between motility and the other virulence factors required for the pathogenic lifestyle of V. cholerae.

The genus Yersinia includes three human pathogens-Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica-where biofilm formation plays distinct roles in transmission and environmental persistence. All three species share the core biofilm machinery-the hmsHFRS operon, encoding enzymes for poly-β-1,6-N-acetylglucosamine (PNAG) exopolysaccharide production, and the c-di-GMP signaling controlling this production-but their regulatory networks and biofilm outputs differ. Transcriptional control of hms involves an integrated network that includes the Rcs phosphorelay (reshaped by rcsA pseudogenization), PhoP/PhoQ, EnvZ/OmpR, BarA/UvrY, Fur/Zur, CRP-cAMP, RovM/RovA, and multiple sigma factors. Post-transcriptionally, the Hfq-sRNA and CsrA-CsrB/CsrC systems modulate gene expression; the latter is directly validated in Y. pestis and Y. pseudotuberculosis, whereas its role in Y. enterocolitica is inferred from genomic conservation. At the translational and enzymatic levels, polyamines sustain Hms protein production, while redox signals govern the HmsC-HmsD disulfide switch, controlling diguanylate cyclase activity. This multi-layer architecture ensures that biofilm output is tuned to species-specific ecological niches. In host adaptation, Y. pestis transitioned from a foodborne enteric pathogen to a flea-borne lifestyle through key genetic events-acquisition of ymt, pseudogenization of rcsA, inactivation of y2909/y3389, and ureD-which are not shared with Y. pseudotuberculosis or Y. enterocolitica, thereby explaining their different biofilm-dependent transmission strategies. Studies in Caenorhabditis elegans have identified host O-glycosylation modifications as receptors for biofilm attachment. This review summarizes progress on the molecular mechanisms, regulatory networks, and host-adaptive functions of Yersinia biofilm formation and proposes future research directions to address knowledge gaps. Future priorities include the identification of c-di-GMP effectors, flea-specific signals, 3D biofilm imaging, and CRISPR-based detection.

Brucella species are intracellular bacterial pathogens that cause brucellosis, one of the most common zoonoses worldwide. This review traces how Brucella, first discovered in the 19th century through investigations of human and bovine disease, became a modern experimental model system. Work on Brucella has illuminated how intracellular bacteria evade and adapt to host defenses and how they remodel host cell biology to establish a replicative niche. It has also yielded insights that extend beyond pathogenesis into bacterial cell envelope architecture and development. Though long regarded as a small, homogeneous group of mammalian pathogens, the genus has expanded in recent years as "non-classical" Brucella have been isolated from unexpected animal hosts, opening new opportunities to understand the fascinating host preferences and evolutionary history of these closely related Alphaproteobacteria. More than a century after their discovery, Brucella species remain versatile models for the study of intracellular infection, bacterial cell biology, and physiology, and the evolution of host association.

Mycobacterium abscessus is a non-tuberculous Mycobacterium (NTM) that is often associated with respiratory infections in persons with cystic fibrosis. High levels of antibiotic resistance make M. abscessus infections difficult to treat, highlighting the urgent need for alternative therapeutic strategies. Mycobacteriophage therapy-treatment of infections with viruses that infect Mycobacterium hosts-shows considerable promise, but the narrow host specificity of the current mycobacteriophage repertoire limits this approach to personalized therapies. Here, we characterize the anti-M. abscessus activity of mycobacteriophage-encoded Lysin B enzymes (lysterases). Lysterases are serine-esterases that cleave the ester bonds linking mycolic acids to either arabinogalactan or trehalose in the mycobacterial cell envelope to complete the lysis process. In this study, we explored their ability to kill a spectrum of clinically isolated M. abscessus strains when administered exogenously. Six phylogenetically distinct lysterases were purified, characterized on the surrogate M. smegmatis, and their activity against a diverse panel of 24 M. abscessus clinical strains was assessed. The enzymes exhibit a range of activities against these strains, but most have broad-spectrum activity with only minor variations among the M. abscessus isolates. However, the activities can be quite potent, resulting in up to six orders of magnitude of reduction in viability within 4 h with one activity unit of the enzymes, and act synergistically with amikacin. Overall, mycobacteriophage-encoded lysterases show considerable potential for antimycobacterial control, especially for topical applications.IMPORTANCEMycobacterium infections are clinically challenging, and there is a high failure of resolving Mycobacterium abscessus infections using antibiotics. Mycobacteriophages show promise in treating M. abscessus infections, but there is substantial variation among clinical isolates in their phage infection profiles. Mycobacteriophages are unusual in coding for lysis enzymes-lysterases-that target cell wall exposed constituents, and these can have potential antibacterial activity. Exploring the vast sequence diversity of thousands of phage-encoded lysterases identifies a subset that was expressed, purified, and tested for their ability to kill a broad range of M. abscessus clinical strains. Lysterases with the broadest and greatest activity offer substantial potential for treatment of M. abscessus infection, especially where they display synergism with current antibiotics.

The gram-negative cell envelope requires coordinated remodeling of the peptidoglycan layer and outer membrane to preserve envelope integrity during growth and division. The conserved Tol-Pal system contributes to envelope maintenance, although the physiological consequences of its loss vary across environmental contexts and among gram-negative bacteria. Here, we examined the contribution of Tol-Pal across diverse growth environments in Acinetobacter baumannii. Loss of Tol-Pal did not cause a substantial population growth defect, and septal peptidoglycan incorporation remained largely preserved under standard laboratory conditions. In contrast, nutrient-rich media, conditions that alter osmotic balance, and a host-like environment exposed defects in cell division organization and cell morphology. Tol-Pal-deficient cells also exhibited modest but reproducible reductions in outer membrane barrier robustness, and disruption of the Mla phospholipid transport system further exacerbated these defects. Consistent with these phenotypes, loss of Tol-Pal reduced bacterial fitness under environmental stress and host-associated models. Together, these findings indicate that Tol-Pal contributes to outer membrane barrier robustness and that additional consequences of its loss become apparent under conditions that challenge the cell envelope.IMPORTANCEThe physiological consequences of disrupting conserved envelope systems can vary with the conditions in which bacteria grow. In Acinetobacter baumannii, loss of Tol-Pal has little effect on population growth under routine laboratory conditions but modestly compromises outer membrane barrier robustness, whereas specific environmental and host-associated conditions reveal additional defects in cell division organization and bacterial fitness. These findings highlight how environmental context shapes the physiological consequences of Tol-Pal deficiency and provide a framework for understanding why the contribution of this conserved envelope system differs among gram-negative bacteria.

The nucleoside diphosphates linked to moiety-X (Nudix) hydrolases are a superfamily of proteins defined by the characteristic Nudix box sequence, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. The Nudix hydrolase associated with CRISPR-Cas-associated adenosine deaminases (CAAD) from Limisphaera ngatamarikiensis (LngNH) mitigates CAAD-induced toxicity by hydrolyzing ITP to IMP and pyrophosphate. Here, we determined crystal structures of LngNH in the apo form and in complex with ITP using catalytic mutants and validated key interactions by site-directed mutagenesis. The hypoxanthine of ITP is deeply buried in a hydrophobic pocket and engages a distinct hydrogen-bond network, whereas the triphosphate tail is coordinated by conserved residues and divalent metal ions. Together, these findings provide a structural framework for CAAD-linked nucleotide sanitization and suggest that CRISPR-associated Nudix hydrolases function as local self-protection modules that limit toxicity from noncanonical inosine nucleotides.IMPORTANCENucleotide quality-control enzymes protect cells from harmful metabolic by-products. Here, we characterize a CRISPR-associated nucleoside diphosphates linked to moiety-X (Nudix) hydrolase that removes ITP, a toxic noncanonical nucleotide generated during antiviral defense. Biochemical and structural analyses show how this enzyme specifically recognizes and hydrolyzes ITP while avoiding related cellular nucleotides. Our findings suggest that CRISPR-associated nucleotide deaminase systems require "self-protection" mechanisms to prevent damage from their own antiviral activities. These results expand our understanding of how bacteria maintain nucleotide homeostasis during immune responses and reveal an additional regulatory layer within CRISPR-associated defense systems. The structural framework reported here also provides a foundation for future studies of nucleotide sanitization enzymes in microbial physiology and antiviral immunity.

Clostridioides difficile is an urgent threat to human health. Current treatments for C. difficile infections (CDIs) are antibiotics and microbiome restoration therapy (MRT) for recurrent cases. However, antibiotics contribute to antibiotic resistance and recurrent CDIs, and the long-term sustainability and accessibility of MRTs remain to be determined. Since a dysbiotic gut microbiome is the primary risk factor for CDI, a better understanding of the interactions between C. difficile, the microbiome, and the host will aid development of treatments with improved precision. Emerging evidence supports that butyrate, a prominent end product of gut microbiome metabolism, is a key determinant of C. difficile pathogenesis. Notably, C. difficile releases more of its toxins (TcdA and TcdB) in butyrate-rich environments. Here, we demonstrate that butyrate-dependent toxin release is not driven by two previously characterized modes of toxin release (e.g., TcdE-dependent secretion or Cwp19-dependent autolysis). Instead, butyrate enhances the expression of a broadly conserved endolysin (EndD), which is responsible for butyrate-dependent toxin release. We additionally demonstrate that endD-dependent toxin release does not universally occur under all growth conditions, that its expression is dependent on the late-stage sporulation sigma factor SigK, and that endD enhances butyrate-dependent sporulation. Overall, our findings provide deeper insight into butyrate-dependent effects on C. difficile pathogenesis and set the stage for future work to better understand the molecular and genetic underpinnings of endD regulation.IMPORTANCEClostridioides difficile is a leading cause of infectious diarrhea. The gut microbiome-derived short-chain fatty acid butyrate triggers C. difficile to release its major virulence factors, toxins TcdA and TcdB. Here, we show that butyrate-dependent toxin release is independent of classical secretion or autolytic pathways and is instead mediated by EndD, an endolysin broadly conserved among C. difficile clinical isolates. We also demonstrate that endD expression is governed by the sporulation factor SigK and enhances sporulation, directly linking transmission and virulence. Uncovering how C. difficile couples metabolic sensing to EndD-mediated toxin release and sporulation advances our understanding of pathogen-microbiome interactions and highlights potential targets for non-antibiotic therapeutics.