Microbiology-Sgm最新文献

The filamentous actinomycete Actinoplanes missouriensis develops terminal sporangia on substrate mycelia via short sporangiophores. Each sporangium, surrounded by an outer envelope, contains a few hundred spores encapsulated by the sporangium matrix. In this study, we identified a spore surface-displayed protein, SspA, that is required for the structural strength of sporangia in A. missouriensis through suppressor screening using a spore release-deficient mutant. SspA has a sortase-dependent cell wall-localizing signal, and its mature part is predicted to be a putative intrinsically disordered protein. An sspA null mutant (ΔsspA) strain formed sporangia, but the mutant sporangia were highly fragile and collapsed immediately to release spores when suspended in aquatic solutions. Transmission electron microscopy revealed that the ΔsspA sporangia did not mature normally; the electron-dense sporangium matrix was not observed in the peripheral region of each spore, and the outer envelope of some sporangia was damaged. Peptide-tagged SspA proteins produced in the ΔsspA strain were detected on the surface of the zoospores using the HiBiT system. The heat tolerance of ΔsspA zoospores was higher than that of wild-type zoospores, suggesting that SspA influences the frequency of cross-bridges in the cell wall peptidoglycan. Phenotypic changes in the ΔsspA strain were restored by introducing sspA with its own promoter into the ΔsspA strain. These results demonstrate that SspA is a sporangiospore cell wall-anchored protein required for the formation of rigid sporangium structures in A. missouriensis. It is speculated that SspA is involved in the production of the sporangium matrix polysaccharides.

Could codon composition condition the immediate success and the orientation of horizontal gene transfer? Horizontal gene transfer represents a change in the genome of expression of the transferred gene, and experimental evidence has accumulated indicating that the codon composition of a sequence is an important determinant of its compatibility with the translation machinery of the genome in which it is expressed. This suggests that codon composition influences the phenotype and the fitness conferred by a transferred gene and thus the immediate success of the transfer. To directly test this hypothesis, we characterized the resistance conferred by synonymous variants of a gentamicin resistance gene in three bacterial species: Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. The strongest determinant of the resistance level conferred was the species in which the resistance gene was transferred, very likely because of important differences in the copy number of the plasmid carrying the gene. Significant differences in resistance were also found between synonymous variants within each of the three species, but more importantly, there was a strong interaction between species and variant: variants conferring high resistance in one species confer low resistance in another. However, the similarity in codon usage between the synonymous variants and the host genome only explained part of the phenotypic differences between variants in one species, P. aeruginosa. Further investigation of alternative explanations did not reveal common universal mechanisms across our three bacterial species. We conclude that codon composition can be a determinant of post-horizontal gene transfer success. However, there are multiple paths leading from synonymous sequence to phenotype, and sensitivity to these different paths is species-specific.

Bacterial pathogens sense and respond to host-derived metabolites to regulate virulence and establish successful infections. d-Serine, an amino acid abundant in some extraintestinal environments but scarce in the intestine, functions as a key niche-specific signal that influences the tissue tropism of different Escherichia coli pathotypes. In enterohaemorrhagic E. coli, a major foodborne pathogen causing severe human disease, d-serine exposure triggers significant transcriptional changes distinct from those seen in extraintestinal pathotypes. Recent publication of our extensive d-serine pathotype transcriptome data on the open database MORF included genome reannotation. This revealed the previously unannotated small regulatory RNA SgrS as being the most significantly upregulated transcript in response to d-serine exposure. Despite its canonical role in managing glucose-phosphate stress by inhibiting glucose uptake, here, we show that d-serine-mediated growth inhibition occurs independently of SgrS and extends beyond glucose metabolism, affecting growth across diverse carbon sources with varying transport mechanisms. Growth inhibition persisted following the deletion of sgrS and could not be alleviated by pyruvate or pantothenate supplementation, while l-serine provided partial rescue, suggesting disruption of fundamental metabolic processes. The novel observation that d-serine induces SgrS suggests a wider regulatory role beyond managing glucose phosphate stress during impeded glycolysis. Moreover, we describe key distinctions between d-serine toxicity in EHEC and previous reports with laboratory E. coli strains, highlighting the importance of strain-specific metabolic and regulatory architecture in response to host-associated cues.

Chitin, an essential structural component of most fungal cell walls, is produced by transmembrane proteins called chitin synthases (CHSs). Our previous study identified novel basidiomycete-specific chss (chsb) clades (BI-BIII) and suggested functional differences between the chsb genes of clade BII and BIII. This study, together with our previous work, presents the first comprehensive functional analysis and identification of clade-specific roles of chsb genes in vegetative hyphae of the white-rot fungus Pleurotus ostreatus. Using homologous recombination, we disrupted chsb1 (clade BI) and simultaneously disrupted chsb2 and chsb3 (clade BII) to investigate their roles in vegetative growth, cell wall biosynthesis and stress response. Deletion of chsb1 led to reduced colony growth, impaired aerial hyphae formation, thinner cell walls and significant cell wall remodelling. In contrast, single disruptions of chsb2 or chsb3 caused mild phenotypes, while double disruption of these genes resulted in severe growth defects, complete loss of aerial hyphae, and abnormal septation. Relative amounts of chitin were increased in the Δchsb1 and Δchsb2Δchsb3 strains, whereas β-glucan was decreased, which is likely related to cell wall thinning. Together with our previous study, these results reveal clear functional differentiation between chsb clades: clade BI influences the relative percentage of cell wall components, as well as radial and aerial hyphal growth; clade BII affects overall growth, cell wall component and septum formation; and clade BIII appears to have a more specific role in aerial hyphae development. These findings advance our understanding of cell wall evolution in filamentous basidiomycetes.

Candida albicans uniquely possesses an expanded family of genes (the TLO gene family) that encodes 10-15 paralogues of the Med2 component of the transcriptional regulator Mediator. Previous studies have shown that TLO null mutants are unable to form hyphae and are hypersensitive to environmental stress. However, the reason for the TLO gene expansion remains unclear, and the current study aimed to determine if reduction in the TLO family copy number affected virulence. In order to investigate this, we used CRISPR-Cas9 mutagenesis to generate two TLO-depleted mutants: one mutant retaining only TLOβ2 (CaTLO2) and the second mutant containing only TLOγ5 (CaTLO5). Both TLO-depleted mutants exhibited increased filamentous growth, increased susceptibility to specific stresses and reduced virulence in a murine model of oropharyngeal candidiasis (OPC). In vitro, the CaTLO5 mutant also exhibited impaired hyphal escape from macrophages and reduced hyphal invasion of oral keratinocytes. We then investigated if complementation with TLOα1, a gene previously shown to restore wild-type growth in a Δtlo null mutant, could restore virulence. In vitro infection models showed that TLOα1 could restore true hypha formation, epithelial invasion and hyphal escape from macrophages in the CaTLO5 background. The murine OPC model showed that TLOα1 could restore wild-type virulence in both CaTLO2 and CaTLO5 strains, suggesting an essential role for α-TLO in oral mucosal infection. Together, these findings highlight the functional specialization between the α, β and γ TLO gene groups and establish α-TLO as a major regulator of virulence in C. albicans.

Quantitative reverse transcription PCR (RT-qPCR) is a popular and reliable tool for monitoring fluctuations in functional bacterial gene expression. A necessary step of the qRT-qPCR process is the use of a reference gene, which acts to distinguish between technical bias and true biological variation. Many reference genes have been defined for bacterial species; however, few studies have validated their stability across strain types and environmental test conditions. In this study of Pseudomonas aeruginosa, the expression consistency of seven commonly used reference genes (rpoD, proC, rpoS, 16S, algD, gyrA and ampC) was assessed in P. aeruginosa laboratory (PAO1) and clinical (LESB65) isolates grown in Lysogeny broth, synthetic cystic fibrosis (CF) media 2 (SCFM2) and CF lung media (CFLM) at various growth time points (2, 6, 24 and 72 h). The stability of the reference genes was then ranked using the RefFinder programme, and three differentially ranked (rpoS, 16S and ampC) were used to interpret the expression of a Pseudomonas virulence-related gene (exoS). The results showed that 16S was the only reference gene that was quantifiably expressed by both P. aeruginosa strains grown in all media types at all growth times. Furthermore, analysing the expression of exoS with different reference genes significantly influenced the calculated expression of exoS in SCFM2 and CFLM. This study has identified a suitable reference gene for RT-qPCR with P. aeruginosa grown in complex respiratory-mimicking media. The results presented here also highlight the importance of validating reference gene expression under the chosen experimental conditions and increase our understanding of how pathogen biology can fluctuate across diverse conditions. Such knowledge is paramount for the development of novel therapeutics, including antimicrobials and anti-virulence agents.

Nitrogen-fixing microbes are a primary contributor of this important nutrient to the global nitrogen cycle. Biological nitrogen fixation (BNF) through the enzyme nitrogenase requires extensive energy that in whole cells is generally studied during the oxidation of carbohydrates such as sugars. The nitrogen-fixing bacterium Azotobacter vinelandii is a model diazotroph for the study of aerobic BNF. Much is known about metabolism in A. vinelandii when cultured on a simple medium where energy is provided primarily in the form of sucrose or glucose. Outside of the laboratory, this soil bacterium grows on metabolites primarily derived from plant root exudates or from the degradation of dead plant matter. In this work, we expand on previous studies looking at genes that are essential to BNF in A. vinelandii when grown on sucrose medium using transposon sequencing (Tn-seq). We applied Tn-seq to determine the genes essential to growth when the medium was shifted to acetate, succinate or glycerol as the primary carbon and energy source to fuel both growth and BNF. A global overview of the genes of central metabolism and those directing substrates toward central metabolism, along with a selection of unexpected genes that were essential for specific growth substrates, is provided.

Single-copy chromosomal integration systems are essential tools for stable gene expression in bacteria, minimizing variability associated with plasmid-based systems. The Tn7 transposon-based system is widely used for this purpose, and one important application is the generation of reporter systems, such as the bioluminescent luxCDABE operon (lux). However, current Tn7-lux vectors exhibit undesirable background expression due to cryptic promoter activity near the antibiotic resistance cassette. Here, we report the construction of an improved vector, pTn7-lux-B0015, incorporating a strong synthetic terminator upstream of the lux operon. This modification effectively eliminated basal luminescence in the absence of a promoter and enhanced the dynamic range and responsiveness of the reporter. Using a Xanthomonas citri type III secretion system promoter as a model, we demonstrate that pTn7-lux-B0015 enables more accurate detection of gene expression under relevant growth conditions. This vector provides a valuable tool for the development of precise and tunable bioluminescent reporters in bacterial systems.

Desiccation tolerance is central to the pathogenic success of the opportunistic pathogen Acinetobacter baumannii, allowing its survival on hospital surfaces in the absence of water and nutrients for months at a time, compromising surface decontamination and aiding cross-contamination between staff and patients. Despite the importance of desiccation tolerance, the regulation underpinning this behaviour remains largely elusive. In this work, transcriptomic analyses of desiccated cells revealed phenylacetic acid (PAA) catabolism as an essential mediator of desiccation tolerance. We subsequently demonstrate that deletion of the paa operon abolished the clonogenicity of desiccated cells. Strikingly, these A. baumannii cells remained viable by entering the viable but non-culturable (VBNC) state, a means to survive extreme stressors like antibiotic exposure. Furthermore, we uncover that PAA catabolism is necessary to mediate PAA-driven biofilm regulation. These findings highlight PAA catabolism as a modulator of biofilm formation and a key pathway for entry into the VBNC state in response to desiccation. This reveals PAA catabolism as a target for novel infection prevention strategies.

There is an urgent need to identify new chemical compounds with novel modes of action to help manage the antimicrobial resistance crisis. Fungi are prolific producers of secondary metabolites, including those with antimicrobial properties, and contain biosynthetic gene clusters that awaken only under certain growth conditions. In recent years, a wealth of novel fungal biosynthetic pathways and compounds have been identified, suggesting fungi remain a viable source for developing new antimicrobials. The International Collection of Microorganisms from Plants (ICMP) contains thousands of fungi and bacteria primarily sourced from Aotearoa New Zealand. Here, we report the results of our efforts to screen 32 fungal ICMP isolates for activity against Escherichia coli, a leading cause of deaths attributable to antimicrobial resistance. We used a 'one strain-many compounds' approach, growing the ICMP isolates on seven different media with different pH and various carbon and nitrogen sources. We also tested the isolates for activity at various ages. Our results indicate that several of the tested fungi possess anti-E. coli activity and are suitable for further study. Our results also provide further strong evidence for the impact of media on both fungal growth and bioactivity.