Microbiology-Sgm最新文献

Flagella are widely distributed appendages in bacteria with well-characterized functions in motility and chemotaxis. They also interact directly with hosts and, due to their ubiquity, are potent immune elicitors for hosts from both the plant and animal kingdoms. Furthermore, flagella have been shown to facilitate attachment for several different bacterial species, including several plant-associated bacteria to plant hosts. We previously demonstrated binding of flagella from Escherichia coli to ionic lipids in plant plasma membranes for horticultural species and Arabidopsis thaliana. As such, flagella could be considered as a generic colonization factor, especially in the early stages of the interactions. Therefore, we tested whether flagella from a genetically related species of plant pathogen, Pectobacterium atrosepticum, mediated binding to its susceptible plant host, potato, in a similar manner to E. coli. Surprisingly, flagella containing the filament flagellin from P. atrosepticum did not confer any binding advantage to potato roots. Furthermore, there was no direct interaction between purified flagella and potato membrane lipids (charged or uncharged). The binding capacity of Pectobacterium to potato is dependent upon the motility function of flagella, as both flagella-deficient and motor-deficient mutants were reduced in their binding to potato roots.

Bacillus velezensis HG88 was isolated from ileal mucosa samples of egg layer hens that were raised without the use of antibiotics. Its cell-free supernatant (CFS) was found to inhibit the growth of Clostridium perfringens, the causative agent of necrotic enteritis in chickens. The inhibitory compound was determined to be proteinaceous due to its susceptibility to protease digestion. The antimicrobial activity was specific towards C. perfringens, as the CFS did not inhibit the growth of Gram-positive or Gram-negative bacteria across nine different species and two yeast fungi. Separation of proteins from the CFS followed by peptide mass fingerprinting and genomic analyses of the strain enabled the identification of a putative antibacterial peptide with an export signal for secretion from the cell. The peptide from B. velezensis HG88, named IP_HG88, has sequence similarity to bacterial SH3 domains that are known to bind to the peptide portion of peptidoglycan. The gene encoding this peptide was cloned, and the peptide was purified from recombinant Escherichia coli as an N-terminal His-tagged peptide. The IP_HG88 with or without the His-tag inhibited the growth of C. perfringens with a minimum bactericidal concentration of ~57.0 or 39.1 µg ml^-1, respectively. The 3D structure of IP_HG88 was also predicted using AlphaFold 2.0.

This study investigates the role of polyamine biosynthesis in the pathogenesis of the bacterial phytopathogen Pseudomonas syringae pv. tomato. Through a comprehensive phenotypic analysis of mutant strains affected in the synthesis of putrescine and spermidine, we reveal a complex interplay between this metabolic pathway and bacterial virulence. Disruption of putrescine synthesis impairs a variety of virulence traits such as motility, biofilm formation, siderophore production, prevention of plant stomatal closure and the functionality of the type III secretion system. This is reversed by reintroducing the deleted genes, but not by the supplementation of culture media with putrescine or apoplastic washing fluids (AWF). Similarly, suppression of spermidine biosynthesis results in a comparable phenotype. However, in this case, the wild-type phenotype is restored by adding spermidine, AWF or expressing the spermidine synthase gene. We conclude that both putrescine and spermidine are important for bacterial virulence and that plant-derived spermidine can partially compensate for bacterial needs. Accordingly, whereas putrescine deficiency leads to a hypovirulent phenotype, spermidine synthesis perturbation does not affect plant colonization. These findings emphasize the critical role of polyamine metabolism in the plant invasion process by bacterial pathogens.

In the absence of the broadly conserved deaminase RidA (Reactive intermediate deaminase A), Salmonella enterica and other organisms accumulate the reactive enamine species 2-aminoacrylate (2AA). Free 2AA, generated from serine by the serine/threonine dehydratase IlvA, reacts with and covalently inactivates a subset of pyridoxal 5'-phosphate-dependent enzymes. The metabolic stress caused by 2AA generates growth defects in S. enterica, including (i) when l-alanine is used as a nitrogen source, (ii) when pyruvate is used as a carbon source or (iii) in the presence of exogenous serine. Although the enzymatic targets of 2AA are consistent between growth conditions, the consequences of 2AA-dependent damage differ depending on the distribution of metabolic flux required in different conditions. Analysing the suppressors of a ridA mutant has furthered our understanding of the RidA stress paradigm and, more generally, how a metabolic network responds to perturbation. Many such suppressors modulate the metabolic network to eliminate 2AA production by IlvA. Here, we describe that eliminating the MetJ transcriptional repressor allows a ridA mutant to grow in the presence of 2AA stress in each of the three conditions. The mechanisms by which a ΔmetJ suppresses a ridA mutant are nuanced and medium-dependent, emphasizing that consequences of 2AA stress differ based on environmental and metabolic context.

Culturomics approaches have advanced microbial research by enabling the high-throughput isolation and characterization of a broader range of bacterial taxa, including some previously considered unculturable. Here, we present the testing and optimization of a protocol for isolating and identifying hundreds of cultivable microbes from field-grown plants. This protocol was tested and optimized using the root microbiomes of field-grown corn and pea plants under varying environmental conditions in ND, USA. By employing dilution-to-extinction culturing and a two-step barcoding PCR strategy targeting the V4 region of the 16S rRNA gene, we identified over 200 unique bacterial isolates. The optimized bioinformatic pipeline, built around the DADA2 package, ensured accurate amplicon sequence variant detection and taxonomy assignment. The resulting bacterial isolates span diverse phylogenetic groups, including plant-associated taxa known for promoting plant growth and mitigating stress. Our findings highlight the value of culturomics in generating microbial collections for synthetic community design and advancing plant-microbe interaction research. The protocol's scalability, cost-effectiveness and robust performance demonstrate its potential for widespread application in agricultural microbiome studies.

Streptococcus mutans is commonly associated with the development of dental caries worldwide. Due to their specificity for S. mutans, phage represents a promising avenue for future targeted therapeutic strategies. In this study, we investigated how phage resistance develops in S. mutans. As a model phage, we used ɸAPCM01, which is known to infect a serotype e strain. We isolated and sequenced the genomes of 15 spontaneous resistant mutants and found that 10 had acquired novel clustered regularly interspaced short palindromic repeats (CRIPSR) spacers targeting the phage, with a total of 18 new spacers identified. Additionally, eight strains contained mutations in rhamnose-glucose polysaccharide biosynthetic genes, three of which also acquired spacers. Only the rgp mutants exhibited defects in phage adsorption, supporting the role of these cell surface glycans as the phage receptor. Mutations in rgpF and the newly identified gene rgpX led to severe cell division defects and impaired biofilm formation, the latter of which was also shared by an rgpD mutant. Thus, rgp mutations confer phage resistance but impose severe fitness costs, limiting pathogenic potential. Surprisingly, we found that ɸAPCM01 was capable of binding to and injecting its genome into UA159, a model serotype c strain. However, UA159 was resistant to infection due to an unknown post-entry defence mechanism. Consequently, ɸAPCM01 has the potential to infect both major serotypes associated with dental caries.

Evolutionary history encompasses both genetic and phenotypic bacterial differences; however, the extent to which this history influences drug response and antimicrobial resistance (AMR) adaptation remains unclear. Historical contingencies arise when elements from an organism's past leave lasting effects on the genome, altering the paths available for adaptation. Here, we compare two diverging reference strains of Acinetobacter baumannii, representative of archaic and contemporary infections, to study the impact of deep historical differences shaped by decades of adaptation in varying antibiotic and host pressures. We evaluated these effects by comparing immediate and adaptive responses to the last-resort antibiotic, tigecycline (TGC). The strains demonstrated divergent transcriptional responses, suggesting that baseline transcript levels may dictate global responses to antibiotics. Experimental evolution in TGC revealed clear differences in population dynamics, with hard sweeps in populations founded by one strain and no mutations reaching fixation in the other strain. AMR was acquired through predictable mechanisms of increased efflux and drug target modification; however, efflux targets were dictated by strain background. Genetic adaptation may outweigh historic differences in transcriptional networks, as evolved populations no longer show transcriptomic signatures of drug response. Importantly, fitness-resistance trade-offs were only observed in lineages evolved from the archaic strain, while the contemporary reference isolate suffered no fitness defects. This suggests that decades of adaptation to antibiotics resulted in pre-existing compensatory mechanisms in the more contemporary isolate, an important example of a beneficial effect of historical contingencies.

Kinamycin biosynthesis is a complex process that has been extensively studied over the years, yet specific enzymatic steps continue to be unveiled. A diazo group present in the molecule is responsible for the promising antitumour activity of kinamycins, but its installation in the specific strain Streptomyces ambofaciens has yet to be characterized. In this study, we explore the diazo functionalization of kinamycin in this strain. A FAD-dependent monooxygenase is identified, which is essential for kinamycin biosynthesis. In its absence, stealthin C accumulates instead, likely as a pathway shunt product. Furthermore, as a result of the position of the gene encoding this monooxygenase, named alp2F, we also propose new boundaries of the kinamycin biosynthetic gene cluster, resulting in a large cluster spanning over 72 kb. This work paves the way for the continued understanding of the biosynthetic steps that are characteristic of diazo-containing natural products and provides new biocatalysts for molecular engineering and accelerates bioactive compounds production.

The prevalence of diseases caused by pathogenic fungi of the Candida genus is currently a significant problem, particularly due to the emerging antifungal drug resistance and increasing number of immunocompromised individuals susceptible to opportunistic infections. Recently, it has been shown that fungal extracellular vesicles (EVs) - nanometre-sized structures of cellular origin, equipped with varied cargo enclosed by lipid bilayer - may play a vital role in the response of pathogen cells to antifungal treatment. In this work, we demonstrated that Candida albicans yeast cells grown at the subinhibitory concentrations of three commonly used antifungal drugs - amphotericin B, fluconazole and caspofungin - released a greater number of EVs than fungal cells grown under drug-free conditions. Moreover, these EVs exhibited some variability in size and protein composition, yet they consistently induced the production of the pro-inflammatory cytokine IL-8 by THP-1 macrophage-like cells at levels comparable to control EVs. In studies using the invertebrate model organism Galleria mellonella, EVs released by cells exposed to antifungals did not cause a significant increase in larval mortality, similar to control EVs, although they triggered a remarkably lower activation of phenol oxidase in larval haemolymph. In addition, EVs produced in the presence of caspofungin interacted more noticeably with the membrane of U-937 pro-monocytic cells as indicated by measurements of zeta potential changes. Furthermore, tested EVs contributed to increased tolerance of C. albicans cells to the antifungal drugs. These observations underscore the unmissable role of EVs in the response of pathogen cells to antifungal treatment and highlight the importance of understanding EV functionalities in antifungal resistance.