Search Results (47)

Background/Objectives: The σ factor of bacterial RNA polymerase (RNAP) directs promoter recognition, recruits RNAP to initiate transcription, and is released from the elongation complex to participate in subsequent rounds of initiation. However, the dynamic recycling mechanism of the primary σ factor, σ⁷⁰ (RpoD), during transcription in Escherichia coli remains poorly understood. Methods: We employed in vivo and in vitro interaction assays to screen for σ⁷⁰-interacting partners under different growth conditions. Protein localization studies were performed using fluorescence microscopy. The transcriptomic profile of ΔclpB, ΔdnaK, ΔhtpG, or ΔyhgF mutant was assessed by RNA-seq. Results: The molecular chaperones ClpB, DnaK, HtpG, and the RNA-binding protein YhgF interacts with RpoD both in vivo and in vitro, and the interaction in vivo is growth medium-dependent (LB vs. ABTGcasa). During exponential growth, each of these proteins co-localizes with the nucleoid. The transcriptome profile in ΔclpB, ΔhtpG or ΔyhgF mutant is mutant-specific to some extent; differentially expressed genes (DEGs) associated with amino acid metabolism and lipopolysaccharide biosynthesis are down-regulated in ΔclpB, ΔhtpG or ΔyhgF mutant in a manner that is growth medium-dependent, in agreement with the medium-dependent interaction of RpoD with the chaperones and YhgF. In contrast, the absence of DnaK resulted in delays to initiation of replication with a slow growth, and decreases cell motility, accompanied by down-regulated flagellar assembly and up-regulated amino acid metabolism genes. In summary, ClpB, DnaK, HtpG, and YhgF may regulate transcription by directly interacting with σ⁷⁰. The σ factor recycling guides global transcription to select genes for transcription and subsequently allows cells to cope with the changing environments by responding to the nutrient level as a signal. Full article

Bioactive glass (BAG) S53P4 is a clinically approved bone substitute with antibacterial, osteoconductive and osteostimulatory properties. Its antibacterial effect is associated with ion release, local pH elevation and osmolality, but the precise biochemical and biophysical mode-of-action is unclear. This study investigates the antibacterial mechanism of BAG S53P4 eluates. BAG eluates, collected at 2, 4, 8, and 24 h, eradicated Staphylococcus aureus. Elemental analysis revealed an early increase in concentrations of Si and Na, a later rise in Ca, depletion of P over time and rapid loss of Mg. Membrane disturbances occurred within 5 min, evident by permeability for SYTOX, aligning with time-kill kinetics for S. aureus and Bacillus subtilis. In B. subtilis, 2h-BAG-eluate induced rapid delocalization of marker proteins for cell division and DNA repair, signaling membrane potential collapse and nucleoid condensation. Transcriptomics revealed early transcription remodeling reflecting ionic and energetic imbalance, including disruption of central metabolism, redox homeostasis, and translational stability. Scanning electron microscopy revealed severe cell surface damage and particulate deposits on S. aureus. Transmission electron microscopy showed cell envelop disruptions and cytoplasmic leakage. Energy dispersive X-ray analysis identified Si on bacterial cell surface at 4 h and intracellular accumulation in punctured, empty cells at 24 h. Overall, BAG ionic dissolution products kill bacteria through a stepwise mechanism involving membrane damage, protein delocalization and metabolic impairment, accompanied by Si deposition on bacterial surfaces and loss of Mg. This finally leads to cell wall degradation, cytoplasmic content leakage and further Si deposition on the cells and inside cell ghosts. Full article

H-NS (histone-like nucleoid-structuring protein) is a global regulator affecting diverse bacterial processes. This study aimed to elucidate the regulatory role of H-NS in the virulence of Klebsiella pneumoniae (K. pneumoniae), particularly in relation to capsule synthesis and anchoring. A clinically isolated ST11-KL64 strain of K. pneumoniae FK6741 with low virulence was used. The role of H-NS was evaluated using colony morphology, the string test, viscosity measurement, capsule quantification, transmission electron microscopy, growth curve, biofilm assay, a mouse infection model, transcriptomic analysis, and RT-qPCR. Deletion of hns converted FK6741 into a hypermucoid phenotype in the positive string test; capsule quantification and transmission electron microscopy (TEM) showed increased polysaccharide chains but a reduced and tightly bound capsule. The mutant was initially found to grow slowly but formed stronger biofilms. In vivo, it displayed reduced virulence but induced stronger inflammation. Molecular assays revealed upregulation of capsule synthesis genes (galF, wzi, wcaJ, and wzc) and downregulation of wabG, which is involved in capsule anchoring. H-NS represses capsule synthesis genes, limiting capsule formation in K. pneumoniae. In contrast, loss of H-NS downregulates wabG, a key gene involved in GalA-mediated capsule anchoring, resulting in unstable surface attachment and loss of capsular polysaccharides. Consequently, these unanchored polysaccharides fail to confer effective protection, resulting in reduced bacterial virulence. Full article

Ultrastructure changes in S. aureus and E. faecalis bacteria incubated with synthetic cationic amphiphile DL₄12 and its hybrids with the natural monoterpene carvacrol were studied. The hybrid compounds DL₄CAR-6, DL₅CAR-6, DLpCAR-6, and DLoCAR-6 contained two carvacrol molecules and differed in central linker structure. The study was conducted on ultrathin sections of bacteria fixed by the Ryter–Kellenberger method and on a Jem 1400 transmission electron microscope (Jeol, Tokyo, Japan). Ultrastructure changes in S. aureus and E. faecalis incubated with compound DL₄12 were species-specific. Destructive changes in S. aureus cells when exposed to DL₄12 compound and all DL₄12-carvacrol hybrids did not differ. DL₄12 and DL₄12-carvacrol hybrids in E. faecalis cells damaged all structures except the cell wall. Compound DL₄12 and its hybrids disrupted the ultrastructure of nucleoid and DNA strands in both bacterial species. Complete disorganization of ribosomes in cells of both bacteria occurred upon incubation with compound DL₄12 and its carvacrol-bearing analog DL₄CAR-6. Inclusions in bacterial cells exposed to all compounds had the same ultrastructure. The study showed that all compounds used possess multitarget properties; the structure of the central linker of hybrid compounds plays a significant role in the nature of their damaging effect on S. aureus and E. faecalis cells. Full article

Biomolecular condensates (BCs), formed through liquid–liquid phase separation (LLPS), are membraneless compartments that dynamically regulate key cellular processes. Beyond their canonical roles in energy metabolism and apoptosis, Mitochondria harbor distinct BCs, including mitochondrial RNA granules (MRGs), nucleoids, and degradasomes, that coordinate RNA processing, genome maintenance, and protein homeostasis. These structures rely heavily on proteins with intrinsically disordered regions (IDRs), which facilitate the transient and multivalent interactions necessary for LLPS. In this review, we explore the composition and function of mitochondrial BCs and their emerging involvement in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis, and Huntington’s disease. We provide computational evidence identifying IDR-containing proteins within the mitochondrial proteome and demonstrate their enrichment in BC-related functions. Many of these proteins are also implicated in mitochondrial stress responses, apoptosis, and pathways associated with neurodegeneration. Moreover, the evolutionary conservation of phase-separating proteins from bacteria to mitochondria underscores the ancient origin of LLPS-mediated compartmentalization. Comparative analysis reveals functional parallels between mitochondrial and prokaryotic IDPs, supporting the use of bacterial models to study mitochondrial condensates. Overall, this review underscores the critical role of mitochondrial BCs in health and disease and highlights the potential of targeting LLPS mechanisms in the development of therapeutic strategies. Full article

Amyloids are protein aggregates having a cross-β structure, and they reveal some unusual properties, like interactions with specific dyes and resistance to actions of detergents and proteases, as well as the capability to force some proteins to change their conformation from a soluble form to aggregates. The occurrence of amyloids is not restricted to humans and animals, as they also exist in microbial cells. However, contrary to animals, where amyloids are usually pathological molecules, bacterial amyloids are often functional, participating in various physiological processes. In this review, we focus on a specific property of bacterial amyloids, namely their ability to interact with nucleic acids and resultant regulatory mechanisms. Moreover, some of these interactions might play indirect roles in the pathomechanisms of human neurodegenerative and inflammatory diseases; these aspects are also summarized and discussed in this review. Full article

Microbial secondary metabolites (SMs) serve as the main source of natural antibiotics. Bioinformatics analyses reveal that multiple secondary metabolites biosynthetic gene clusters (BGCs) exist in the genomes of fungi and bacteria but the vast majority remains silent due to the control of intricate regulatory networks. An in-depth comprehension of these regulatory processes is required for the activation of cryptic gene clusters. Among them, the regulations at the proteomic level originating from epigenetic modifications and their correlations with secondary metabolite biosynthesis have gained increasing interest recently, especially the modifications on bacterial nucleoid-associated proteins. This article highlights the recent advances and important roles of bacterial nucleoid-associated proteins (NAPs) in the biosynthesis of SMs. Developing new tools around NAPs would be significant for the discovery of novel bioactive compounds in microbial resources. Full article

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. Full article

Phenol and its chlorinated derivatives are introduced into the environment with wastewater effluents from various industries, becoming toxic pollutants. Phenol-degrading bacteria are important objects of research; among them, representatives of the genus Rhodoccocus are often highlighted as promising. Strain 7Ba was isolated by enrichment culture. A new isolate was characterized using culturing, biochemistry, high-throughput sequencing, microscopy (including electron microscopy), and functional genome analysis. Rhodococcus erythropolis strain 7Ba is able to grow on phenol and chlorophenols without losing its properties during long-term storage. It was shown that strain 7Ba is able to form viable but nonculturable (VBNC) forms during long-term storage under nutrient limitation, preserving both cell viability and the ability to degrade phenols. The ultrastructural organization of the vegetative forms of cells and VBNC forms was characterized. The following distinctive features were found: modifications (thickening) of cell membranes, cell size reduction, nucleoid condensation. Functional analysis of the genome showed the presence of genes for the degradation of alkanes, and two branches of the β-ketoadipate pathway for the degradation of aromatic compounds. Also, the genome of strain 7Ba contains several copies of Rpf (resuscitation promoting factor) genes, a resuscitation factor of resting bacterial forms. The new isolate strain 7Ba is a promising biotechnological agent that can not only utilize toxic aromatic compounds but also remain viable during long-term storage. For this reason, its further application as an agent for bioremediation can be successful under changing conditions of climate and given the deficiency of nutrient compounds in nature. Minor biostimulation will allow the strain to recover its metabolic activity and effectively degrade pollution. Full article

Current antibiograms cannot discern the particular effect of a specific antibiotic when the bacteria are incubated with a mixture of antibiotics. To prove that this task is achievable, Escherichia coli strains were treated with ciprofloxacin for 45 min, immobilized on a slide and stained with SYBR Gold. In susceptible strains, the nucleoid relative surface started to decrease near the MIC, being progressively condensed as the dose increased. The shrinkage level correlated with the DNA fragmentation degree. Ciprofloxacin-resistant bacilli showed no change. Additionally, E. coli strains were incubated with ampicillin for 45 min and processed similarly. The ampicillin-susceptible strain revealed intercellular DNA fragments that increased with dose, unlike the resistant strain. Co-incubation with both antibiotics revealed that ampicillin did not modify the nucleoid condensation effect of ciprofloxacin, whereas the quinolone partially decreased the background of DNA fragments induced by ampicillin. Sixty clinical isolates, with different combinations of susceptibility-resistance to each antibiotic, were co-incubated with the EUCAST breakpoints of susceptibility of ciprofloxacin and ampicillin. The morphological assay correctly categorized all the strains for each antibiotic in 60 min, demonstrating the feasible independent evaluation of a mixture of quinolone and beta-lactam. The rapid phenotypic assay may shorten the incubation times and necessary microbial mass currently required for evaluation. Full article

The spatial organization of bacterial chromosomes is crucial for cellular functions. It remains unclear how bacterial chromosomes adapt to high-temperature stress. This study delves into the 3D genome architecture and transcriptomic responses of Escherichia coli under heat-stress conditions to unravel the intricate interplay between the chromosome structure and environmental cues. By examining the role of macrodomains, chromosome interaction domains (CIDs), and nucleoid-associated proteins (NAPs), this work unveils the dynamic changes in chromosome conformation and gene expression patterns induced by high-temperature stress. It was observed that, under heat stress, the short-range interaction frequency of the chromosomes decreased, while the long-range interaction frequency of the Ter macrodomain increased. Furthermore, two metrics, namely, Global Compactness (GC) and Local Compactness (LC), were devised to measure and compare the compactness of the chromosomes based on their 3D structure models. The findings in this work shed light on the molecular mechanisms underlying thermal adaptation and chromosomal organization in bacterial cells, offering valuable insights into the complex inter-relationships between environmental stimuli and genomic responses. Full article

Theoretical and experimental approaches have been applied to study the polymer physics underlying the compaction of DNA in the bacterial nucleoid. Knowledge of the compaction mechanism is necessary to obtain a mechanistic understanding of the segregation process of replicating chromosome arms (replichores) during the cell cycle. The first part of this review discusses light microscope observations demonstrating that the nucleoid has a lower refractive index and thus, a lower density than the cytoplasm. A polymer physics explanation for this phenomenon was given by a theory discussed at length in this review. By assuming a phase separation between the nucleoid and the cytoplasm and by imposing equal osmotic pressure and chemical potential between the two phases, a minimal energy situation is obtained, in which soluble proteins are depleted from the nucleoid, thus explaining its lower density. This theory is compared to recent views on DNA compaction that are based on the exclusion of polyribosomes from the nucleoid or on the transcriptional activity of the cell. These new views prompt the question of whether they can still explain the lower refractive index or density of the nucleoid. In the second part of this review, we discuss the question of how DNA segregation occurs in Escherichia coli in the absence of the so-called active ParABS system, which is present in the majority of bacteria. How is the entanglement of nascent chromosome arms generated at the origin in the parental DNA network of the E. coli nucleoid prevented? Microscopic observations of the position of fluorescently-labeled genetic loci have indicated that the four nascent chromosome arms synthesized in the initial replication bubble segregate to opposite halves of the sister nucleoids. This implies that extensive intermingling of daughter strands does not occur. Based on the hypothesis that leading and lagging replichores synthesized in the replication bubble fold into microdomains that do not intermingle, a passive four-excluding-arms model for segregation is proposed. This model suggests that the key for segregation already exists in the structure of the replication bubble at the very start of DNA replication; it explains the different patterns of chromosome arms as well as the segregation distances between replicated loci, as experimentally observed. Full article

Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from Sinorhizobium meliloti plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from Sinorhizobium meliloti by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS. We show that MucR purified from Sinorhizobium meliloti can bind DNA and recognize the region upstream of the ndvA gene in EMSA, revealing that this gene is a direct target of MucR. Although MucR DNA binding activity was already described, a detailed characterization of Sinorhizobium meliloti DNA targets has never been reported. We, thus, analyze sequences recognized by MucR in the rem gene promoter, showing that this protein recognizes AT-rich sequences and does not require a consensus sequence to bind DNA. Furthermore, we investigate the dependence of MucR DNA binding on the length of DNA targets. Taken together, our studies establish MucR from Sinorhizobium meliloti as a member of a new family of Histone-like Nucleoid Structuring (H-NS) proteins, thus explaining the multifaceted role of this protein in many species of alpha-proteobacteria. Full article

Chromosome segregation in Pseudomonas aeruginosa is assisted by the tripartite ParAB–parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB) and its target parS sequence(s). ParB forms a nucleoprotein complex around four parSs (parS1–parS4) that overlaps oriC and facilitates relocation of newly synthesized ori domains inside the cells by ParA. Remarkably, ParB of P. aeruginosa also binds to numerous heptanucleotides (half-parSs) scattered in the genome. Here, using chromatin immunoprecipitation-sequencing (ChIP-seq), we analyzed patterns of ParB genome occupancy in cells growing under conditions of coupling or uncoupling between replication and cell division processes. Interestingly, a dissipation of ParB–parS complexes and a shift of ParB to half-parSs were observed during the transition from the exponential to stationary phase of growth on rich medium, suggesting the role of half-parSs in retaining ParB on the nucleoid within non-dividing P. aeruginosa cells. The ChIP-seq analysis of strains expressing ParB variants unable to dislocate from parSs showed that the ParB spreading ability is not required for ParB binding to half-parSs. Finally, a P. aeruginosa strain with mutated 25 half-parSs of the highest affinity towards ParB was constructed and analyzed. It showed altered ParB coverage of the oriC region and moderate changes in gene expression. Overall, this study characterizes a novel aspect of conserved bacterial chromosome segregation machinery. Full article

We have developed a de novo method for the identification of dispersed repeats based on the use of random position-weight matrices (PWMs) and an iterative procedure (IP). The created algorithm (IP method) allows detection of dispersed repeats for which the average number of substitutions between any two repeats per nucleotide (x) is less than or equal to 1.5. We have shown that all previously developed methods and algorithms (RED, RECON, and some others) can only find dispersed repeats for x ≤ 1.0. We applied the IP method to find dispersed repeats in the genomes of E. coli and nine other bacterial species. We identify three families of approximately 1.09 × 10⁶, 0.64 × 10⁶, and 0.58 × 10⁶ DNA bases, respectively, constituting almost 50% of the complete E. coli genome. The length of the repeats is in the range of 400 to 600 bp. Other analyzed bacterial genomes contain one to three families of dispersed repeats with a total number of 10³ to 6 × 10³ copies. The existence of such highly divergent repeats could be associated with the presence of a single-type triplet periodicity in various genes or with the packing of bacterial DNA into a nucleoid. Full article