Bacterial Physiology Research Articles

NorA and Tet38 efflux pumps enable Staphylococcus aureus survival in the cystic fibrosis airway environment, resistance to antibiotics, and coinfection with Pseudomonas aeruginosa.

Efflux pumps play multiple roles in bacterial physiology, environmental adaptation, and antibiotic resistance. Early cystic fibrosis (CF) airway infections start with Staphylococcus aureus (SA), often later followed by Pseudomonas aeruginosa (PA) infections. In this study, we have evaluated the role of SA pumps NorA and Tet38 in survival and interaction with PA in CF patients. Data showed a ≥4-log10CFU/mL growth deficit of SA mutants ΔnorA and Δtet38 in an artificial sputum medium (ASM), suggesting NorA and Tet38 contributed to SA growth in CF sputum. In ASM, mucin activated norA but inhibited tet38, while extracellular DNA activated tet38 but inhibited norA, demonstrating complementary roles of mucin and DNA in affecting NorA and Tet38 expression. Furthermore, exposure of SA wild type to PA-excreted molecules affected pump expression; 3,4-dihydroxy-2-heptylquinoline PQS caused an increase in tet38 but a decrease in norA, 4-hydroxy-2-heptylquinoline HHQ caused a decrease in norA and tet38, and pyocyanin PYO caused a modest increase in norA, demonstrating differing additional roles of PA-secreted molecules influencing NorA and Tet38 expression. Evaluation of 48 randomly selected unique CF-associated SA showed that 18.8% were NorA-overexpressors and 10.4% were Tet38-overexpressors. NorA-overexpressors showed a fourfold increase in pyocyanin MIC and ≥16-fold in ciprofloxacin MIC. Furthermore, 89% of NorA-overexpressors carried an insertion of CAAT/ACAA/CTAT at the (-10) motif of the norA promoter, and 62.5% of these were co-isolated with PA.These data showed that SA survives PA killing in CF sputum conditions using sputum key components and PA-specific signal molecules to regulate NorA and Tet38 expression.

The Characterization of a Gonococcal HicAB Toxin–Antitoxin System Capable of Causing Bacteriostatic Growth Arrest

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea. Preventative vaccines or novel treatments based on a better understanding of the molecular basis of N. gonorrhoeae infection are required as resistance to current antibiotics is widespread. Toxin–antitoxin (TA) systems modulate bacterial physiology by interfering with vital cellular processes; type II TA systems, where both toxin and antitoxin are proteins, are the best-studied. Bioinformatics analysis revealed genes encoding an uncharacterized type II HicAB TA system in the N. gonorrhoeae strain FA1090 chromosome, which were also present in >83% of the other gonococcal genome sequences examined. Gonococcal HicA overproduction inhibited bacterial growth in Escherichia coli, an effect that could be counteracted by the co-expression of HicB. Kill/rescue assays showed that this effect was bacteriostatic rather than bactericidal. The site-directed mutagenesis of key histidine and glycine residues (Gly22, His24, His29) abolished HicA-mediated growth arrest. N. gonorrhoeae FA1090∆hicAB and complemented derivatives that expressed IPTG-inducible hicA, hicB, or hicAB, respectively, grew as wild type, except for IPTG-induced FA1090∆hicAB::hicA. RT-PCR demonstrated that hicAB are transcribed in vitro under the culture conditions used. The deletion of hicAB had no effect on biofilm formation. Our study describes the first characterization of a HicAB TA system in N. gonorrhoeae.

Metabolomic Profiling and Anti-Helicobacter pylori Activity of Caulerpa lentillifera (Sea Grape) Extract

Helicobacter pylori is a gastric pathogen implicated in peptic ulcer disease and gastric cancer. The increasing prevalence of antibiotic-resistant strains underscores the urgent need for alternative therapeutic strategies. In this study, we investigated the chemical composition and antibacterial activity of an aqueous extract from Caulerpa lentillifera (sea grape), a farm-cultivated edible green seaweed collected from Krabi Province, Thailand. Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) revealed that the extract was enriched in bioactive nucleosides and phenolic compounds. In vitro assays demonstrated dose-dependent inhibition of H. pylori growth following exposure to sea grape extract. Furthermore, untargeted intracellular metabolomic profiling of H. pylori cells treated with the extract uncovered significant perturbations in central carbon and nitrogen metabolism, including pathways associated with the tricarboxylic acid (TCA) cycle, one-carbon metabolism, and alanine, aspartate, and glutamate metabolism. Pyrimidine biosynthesis was selectively upregulated, indicating a potential stress-induced shift toward nucleotide salvage and DNA repair. Of particular note, succinate levels were markedly reduced despite accumulation of other TCA intermediates, suggesting disruption of electron transport-linked respiration. These findings suggest that bioactive metabolites from C. lentillifera impair essential metabolic processes in H. pylori, highlighting its potential as a natural source of antimicrobial agents targeting bacterial physiology.

Synergistic effects of antibiotics and efflux pump inhibitors on multidrug-resistant Escherichia coli and Klebsiella pneumoniae.

The increasing prevalence of multidrug-resistant Escherichia coli and Klebsiella pneumoniae presents a critical threat to public health. As a first line of defence, efflux pumps promote antibiotic resistance among pathogens. This study aims to assess the potential of efflux pump inhibitors (EPIs) as adjuvants for enhancing antibiotic efficacy against resistant pathogens. This research investigated antibiotic resistance patterns, antibiotic resistance genes, and the effects and expression profiles of efflux pump genes. The study examined bacterial responses to sub-inhibitory levels of antibiotics, both individually and in conjunction with the efflux pump inhibitor. The qPCR demonstrated that antibiotic exposure led to an upregulation of efflux genes in E. coli and K. pneumoniae, with significant strain-specific differences. The co-administration of EPI resulted in downregulation of efflux genes, indicating successful inhibition of the pumps. Gene expression analyses suggested that PAβN influences the regulation of efflux genes and may trigger oxidative stress responses mediated by global regulators. This implies that EPIs could directly and indirectly affect bacterial physiology. The results highlight the therapeutic promise of EPIs in combating antibiotic resistance. This study underscores the complexity of efflux-mediated resistance and the need for integrated strategies to mitigate MDR in Gram-negative pathogens.

Design, development, and validation of new fluorescent strains for studying oral streptococci.

Bacterial strains that are genetically engineered to constitutively produce fluorescent proteins have aided our study of bacterial physiology, biofilm formation, and interspecies interactions. Here, we report on the construction and utilization of new strains that produce the blue fluorescent protein mTagBFP2, the green fluorescent protein sfGFP, and the red fluorescent protein mScarlet-I3 in species Streptococcus gordonii, Streptococcus mutans, and Streptococcus sanguinis. Gene fragments, developed to contain the constitutive promoter Pveg, the fluorescent gene of interest, as well as aad9, providing resistance to the antibiotic spectinomycin, were inserted into selected open reading frames on the chromosome that were both transcriptionally silent and whose loss caused no measurable changes in fitness. All strains, except for sfGFP in S. sanguinis, were validated to produce a detectable and specific fluorescent signal. Individual stains, along with extracellular polymeric substances (EPS) within biofilms, were visualized and quantified through either widefield or super-resolution confocal microscopy approaches. Finally, to validate the ability to perform single-cell-level analysis using the strains, we imaged and analyzed a triculture mixed-species biofilm of S. gordonii, S. mutans, and S. sanguinis grown with and without the addition of human saliva. Quantification of the loss in membrane integrity using a SYTOX dye revealed that all strains had increased loss of membrane integrity with water or human saliva added to the growth media, but the proportion of the population stained by the SYTOX dye varied by species. In all, these fluorescent strains will be a valuable resource for the continued study of oral microbial ecology.IMPORTANCEStreptococci are among the earliest colonizers of the soft and hard tissues of the oral cavity and are contributors to the oral health status of the host, with involvement in dental caries, endodontic infections, periodontal disease, and the development of oral cancer. Strains genetically modified to produce fluorescent proteins that can be either visualized through microscopy imaging or quantified by their specific fluorescent intensity signal are critical tools toward the study of individual or mixed-species cultures. Our report here details the development and testing of several new strains of fluorescent oral streptococci that can be utilized in the study of microbial ecology, increasing both the availability of tools and documenting experimental approaches toward in vitro assay applications such as the study of intermicrobial interactions.

Pairwise encounters boost bacterial motion by transient velocity spikes.

Pairwise encounters boost bacterial motion by transient velocity spikes.

Engineering of a monitorable expression system to characterize β-lactamase genes in Enterobacteriaceae.

Engineering of a monitorable expression system to characterize β-lactamase genes in Enterobacteriaceae.

Resource allocation to cell envelopes and the scaling of bacterial growth rate

Although various empirical studies have reported a positive correlation between the specific growth rate and cell size across bacteria, it is currently unclear what causes this relationship. We conjecture that such scaling occurs because smaller cells have a larger surface-to-volume ratio and thus have to allocate a greater fraction of the total resources to the production of the cell envelope, leaving fewer resources for other biosynthetic processes. To test this theory, we developed a coarse-grained model of bacterial physiology composed of the proteome that converts nutrients into biomass, with the cell envelope acting as a resource sink. Assuming resources are partitioned to maximize the growth rate, the model predicts that the growth rate and ribosomal mass fraction scale negatively, while the mass fraction of envelope-producing enzymes scales positively with surface-to-volume. These relationships are compatible with growth measurements and quantitative proteomics data reported in the literature.

Host glutathione is required for Rickettsia parkeri cell division and intracellular survival

Intracellular bacteria rely on eukaryotic metabolites for their fitness and pathogenesis. Yet, the mechanisms of how host metabolites promote bacterial physiology and immune evasion are often unclear. Here, we employed obligate cytosolic Rickettsia parkeri, which parasitizes over fifty host metabolites, to investigate bacterial utilization of host glutathione (GSH). We observed that GSH depletion impaired R. parkeri intracellular survival. Super-resolution microscopy revealed that GSH depletion caused bacterial chaining in the host cytosol, prohibiting proper actin-based motility and cell-to-cell spread. GSH was especially critical for bacterial survival in primary macrophages, where it enabled R. parkeri to evade ubiquitylation and antibacterial autophagy. Cell division and survival defects were restored by supplementing N-acetylcysteine, suggesting that GSH serves as a cysteine source for R. parkeri. Together, these data suggest that Rickettsia requires GSH as a nutrient source to promote cell division, actin-based motility, evasion of antibacterial autophagy, and intracellular survival. This knowledge contributes to the expanding paradigm that GSH plays diverse roles in the pathogenesis of intracellular bacteria and represents a potential target for host-acting therapeutics.

Estimating the relative rates of lipopolysaccharide synthesis in Escherichia coli K-12 by click chemistry-mediated labeling

Lipopolysaccharide (LPS), a critical glycolipid component of Gram-negative bacteria, plays a central role in bacterial membrane integrity and host immune interactions. Despite extensive studies on the regulation of LPS synthesis, methods to quantify its synthesis rate remain limited. Here, we present a novel approach to measure in vivo LPS synthesis rates in the E. coli K-12 strain MG1655 using click chemistry. This method involves the incorporation of an exogenous Kdo analog, 8-azido-3,8-dideoxy-D-manno-oct-2-ulosonic acid (Kdo-N3), into newly synthesized LPS, followed by a copper-free click reaction with a fluorescent alkyne. The labeled LPS is separated by SDS-PAGE and visualized via in-gel fluorescence. We compared two fluorescent dyes and found that AZDye 488 DBCO exhibited stronger sensitivity for labeling LPS during the log phase of bacterial growth. Our results further demonstrated that the amount of newly synthesized LPS correlates linearly with the pulse labeling time of Kdo-N3, validating this approach as a reliable method for estimating relative LPS synthesis rates during the exponential phase of E. coli MG1655 growth. This method offers a reliable, non-radioactive approach for measuring LPS synthesis in vivo, providing a powerful tool to investigate bacterial physiology and the regulation of LPS biogenesis.

Phage DisCo: targeted discovery of bacteriophages by co-culture

Phages interact with many components of bacterial physiology from the surface to the cytoplasm. Although there are methods to determine the receptors and intracellular systems a specified phage interacts with retroactively, finding a phage that interacts with a chosen piece of bacterial physiology a priori is very challenging. Variation in phage plaque morphology does not to reliably distinguish distinct phages, and therefore many potentially redundant phages may need to be isolated, purified, and individually characterized to find phages of interest. Here, we present a method in which multiple bacterial strains are co-cultured on the same screening plate to add an extra dimension to plaque morphology data. In this method, phage discovery by co-culture (Phage DisCo), strains are isogenic except for fluorescent tags and one perturbation expected to impact phage infection. Differential plaquing on the strains is easily detectable by fluorescent signal and implies that the perturbation made to the surviving strain in a plaque prevents phage infection. We validate the Phage DisCo method by showing that characterized phages have the expected plaque morphology on Phage DisCo plates and demonstrate the power of Phage DisCo for multiple targeted discovery applications, from receptors to phage defense systems.IMPORTANCEIn this work, we describe a targeted phage discovery method that allows immediate isolation of phages with specific traits. Currently, to find a phage with specific properties, huge libraries of phages must be collected and screened retroactively. This assay, Phage Discovery by Co-culture (Phage DisCo), works by co-culture of host strains that are identical except for one perturbation that may interfere with phage infection and a unique fluorescent marker. These strains are co-cultured with an environmental sample of interest in traditional plaque assay format, making phage characteristics easily identifiable by fluorescent signal after imaging of the screening plate. We validate that Phage DisCo can identify phages with specific properties, even when these phages are rare in samples. This approach allows rapid exploration of the diversity within phage samples with vastly streamlined processes, and we anticipate it will be widely adopted within the phage discovery field.

Structural basis of the catalytic and allosteric mechanism of bacterial acetyltransferase PatZ

GCN5-related N-acetyltransferases (GNATs) are essential for regulating bacterial metabolism by acetylating specific target proteins. Despite their importance in bacterial physiology, the mechanisms behind their enzymatic and regulatory functions remain poorly understood. In this study, we investigated the structures of Escherichia coli protein acetyltransferase Z (PatZ), a Type I GNAT, and examined its ligand interactions, catalytic mechanism, and allosteric regulation. PatZ functions as a homotetramer, with each subunit comprising a catalytic and a regulatory domain. Our results demonstrate that the regulatory domain is vital for acetyltransferase activity, as it triggers cooperative conformational changes in the catalytic domain and directly aids in the formation of substrate-binding pockets. Additionally, a protein structure-based evolutionary analysis of bacterial GNAT types revealed a distinct regulatory domain pattern across phyla, highlighting its crucial role in responding to cellular energy levels.

FapR regulates HssRS-mediated heme homeostasis in Bacillus anthracis

Understanding the mechanisms by which B. anthracis regulates heme homeostasis is crucial for developing new strategies to combat anthrax, a serious disease affecting both humans and animals. This study uncovers the role of the transcriptional regulator FapR in maintaining membrane integrity and facilitating the proper function of the HssRS two-component signaling system, which is essential for managing heme toxicity in B. anthracis, as well as other Gram-positive pathogens. By elucidating the connection between FapR and HssRS, our findings provide new insights into the molecular adaptation of bacteria to heme stress and expand our knowledge of bacterial physiology and pathogenicity. More importantly, targeting the regulatory pathways involved in heme sensing and homeostasis presents a promising approach for developing novel therapeutics against anthrax and potentially other bacterial infections that rely on similar mechanisms.

The Role of Bacteriophage-Derived Small RNA Molecules in Bacterial and Phage Interactions.

Small regulatory RNAs (sRNAs) play a critical role in bacterial gene expression, modulating various cellular processes, including stress responses, metabolism, virulence, and many others. While well-characterized in bacterial systems, an emerging class of phage-derived sRNAs has been identified, suggesting an underexplored regulatory network at phage-host interactions. These sRNAs, encoded within phage genomes, influence both bacterial and viral life cycles by modulating transcriptional and post-transcriptional gene expression processes. The interplay between phage-derived sRNAs and the host genome reveals a complex network of gene regulation, with an impact on bacterial fitness, pathogenesis, and horizontal gene transfer. This review explores the diverse functions of phage-encoded sRNAs, highlighting recent discoveries and their impact on bacterial physiology and phage-host interactions.

The Helicobacter pylori orphan ATTAAT-specific methyltransferase M.Hpy99XIX plays a central role in the coordinated regulation of genes involved in iron metabolism

Helicobacter pylori genomes contain a large and variable portfolio of methyltransferases (MTases), creating a highly diverse methylome. Here, we characterize a highly conserved ATTAAT-specific MTase, M.Hpy99XIX (H. pylori strain J99, alternative designations in other strains, M.HpyAVII and M.HpyPVII), the only H. pylori MTase never associated with an endonuclease ("orphan" MTase). Inactivation of M.Hpy99XIX resulted in a significant change in the transcription of >100 genes, despite the fact that only a small subset of their promoter regions contained an ATTAAT target motif. Patterns of transcriptional change showed significant correlations with changes reported for H. pylori mutants in the regulators involved in iron regulation. MTase inactivation also caused a higher susceptibility to diverse metal ions as well as iron chelation and oxidative stress. These phenotypes could be traced back to the methylation of single motifs in the promoter regions of iron transporters frpB1 and fecA1. Altogether, methylation of individual motifs in promoters can have a large downstream effect, causing major changes to metabolic pathways. These findings suggest that the methylome represents a universal and dynamic interface connecting genome diversity and transcriptional regulation. Very recently, a new ecospecies of H. pylori, Hardy, has been reported. M.Hpy99XIX is present in the majority of "normal" (Ubiquitous) H. pylori strains, whereas no single Hardy strain contained this gene, consistent with other reported differences between Hardy and Ubiquitous strains related to iron/metal homeostasis. ATTAAT methylation is intricately connected with the bacterial transcriptional network, highlighting the important role of bacterial epigenetic modifications in bacterial physiology and pathogenesis.IMPORTANCEHelicobacter pylori has one of the largest repertoires of methyltransferases. Methylation has been associated with multiple functions in H. pylori, including the defense against foreign DNA and transcriptional regulation. Regulation of gene expression by methylation has the potential to influence many distant genes across the genome via target motifs in proximity to transcription start sites. Here, we sought to understand the role of M.Hpy99XIX, an orphan methyltransferase targeting the ATTAAT motif that is highly conserved in H. pylori. We show that by directly regulating specific genes involved in iron uptake via methylated ATTAAT motifs, M.Hpy99XIX has a significant effect on iron homeostasis by triggering the canonical iron regulatory pathway. Furthermore, we show that M.Hpy99XIX appears to have been acquired after the split between the two ecospecies of H. pylori, suggesting that its role in the tuning of iron homeostasis might have contributed to this divergence.

Environmental and clinical impacts of antibiotics' sub-minimum inhibitory concentrations on the development of resistance in acinetobacter baumannii.

Environmental and clinical impacts of antibiotics' sub-minimum inhibitory concentrations on the development of resistance in acinetobacter baumannii.

Strain-dependent emergence of aminoglycoside resistance in Escherichia coli biofilms.

Strain-dependent emergence of aminoglycoside resistance in Escherichia coli biofilms.

A Methanolic Urea-enhanced Protein Extraction Enabling the Largest Bacterial Phosphorylation Resource.

A Methanolic Urea-enhanced Protein Extraction Enabling the Largest Bacterial Phosphorylation Resource.

Fatty acid synthesis and utilization in gram-positive bacteria: insights from Bacillus subtilis.

SUMMARYThe bacterial cytoplasmic membrane, consisting of roughly equal proportions of proteins and lipids, plays a crucial role in cellular growth, metabolism, and maintaining the cytoplasmic boundary. It is a dynamic, fluid matrix that separates intracellular compartments, where lipids and proteins coexist in a highly organized yet flexible arrangement. Membrane fluidity, defined as the inverse of viscosity, determines how rapidly molecules diffuse within the membrane at a given temperature. This property is vital for protein mobility and biomolecular interactions. Structurally, the membrane primarily comprises a lamellar lipid bilayer, with glycerophospholipids and fatty acids forming its core framework. In Bacillus subtilis, a key model organism for studying gram-positive bacterial physiology, major membrane lipids include phospholipids, glycolipids, and lipoteichoic acids, the latter anchored to diacylglycerol glycolipids. This review examines the synthesis and regulation of membrane lipids in B. subtilis, with a focus on fatty acid biosynthesis, its diversification, and post-synthetic modifications such as desaturation. It also explores the production of phosphatidic acid and the integration of fatty acid and phospholipid biosynthesis. We review the well-characterized pathway of cold-induced membrane lipid modification in B. subtilis, arguably the best-studied model system for temperature sensing. This pathway is tightly linked to transcriptional responses triggered by changes in bilayer viscosity, detected by a membrane-associated thermosensor. Finally, this review highlights the importance of fatty acid biosynthesis in B. subtilis differentiation and its contributions to the production of biotin and lipoic acid, two universal cofactors essential for fatty acid synthesis and intermediary metabolism.

Protein kinase G-a key regulator of pathogenesis in Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis (TB), remains a leading global health threat, exacerbated by rising drug resistance and the ability of this pathogen to persist within host macrophages. Central to the intracellular survival of M. tuberculosis is Protein Kinase G (PknG), a secreted, eukaryotic-like serine/threonine kinase that subverts host immune defenses and modulates bacterial physiology. This review provides a comprehensive overview of structural features and the multifaceted role of PknG in M. tuberculosis pathogenesis, including inhibition of phagosome-lysosome fusion, acid tolerance, metabolic reprogramming, autophagy suppression, and cell wall remodelling. Additionally, we discuss recent advancements in targeting PknG with small-molecule inhibitors, highlighting its promise as a therapeutic target. By delineating PknG's central role in host-pathogen interactions and stress adaptation, this review underscores its potential in shaping future anti-TB strategies, especially against drug-tolerant and latent infections..