Microbiology-Sgm最新文献

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger regulating many physiological processes in bacteria. In the oral commensal species Streptococcus mitis, c-di-AMP is involved in regulating metabolism, growth, colony morphology, chain length, biofilm formation and DNA stress tolerance. However, no c-di-AMP-regulated effector proteins have yet been characterized in S. mitis. In this study, we first show that a ΔcdaA mutant, unable to produce c-di-AMP, grows slowly under low environmental potassium conditions. Growth of the cdaA mutant was not restored by reintroducing cdaA in the original locus (KBcdaA). Whole-genome sequencing of multiple KBcdaA isolates revealed secondary mutations in a putative potassium transporter. The mutations were predicted to result in the truncation of the protein or the alteration of a conserved glycine residue essential for selective potassium uptake, disrupting protein function. A Δpde2 mutant overproducing c-di-AMP survived poorly under high environmental sodium concentrations. We then characterized the potassium transporter regulator protein TrkA. Biochemical analyses of the purified recombinant TrkA protein revealed that it specifically binds c-di-AMP with high affinity in vitro. Using deletion mutants of trkA, we demonstrate that TrkA is essential for growth under low environmental potassium conditions. Ultra-high-performance liquid chromatography coupled to tandem mass spectrometry revealed lower c-di-AMP concentration in the ΔtrkA mutant compared to the WT. This was not due to transcriptional regulation of the expression of the c-di-AMP turnover proteins CdaA, Pde1 or Pde2. C-di-AMP production is not affected by the extracellular potassium concentrations under the conditions tested. We also demonstrate a potential role of TrkA in UV stress tolerance but do not characterize the mechanism in this study.

Non-encapsulated Streptococcus pneumoniae (NESp) represent up to 19% of circulating pneumococci and exhibit high rates of genetic exchange and antimicrobial resistance. Saliva is increasingly used as a pneumococcal carriage study specimen, and we recently developed a qPCR assay to enhance carriage surveillance and characterization of NESp in saliva. Previous work has established that pneumococci remain viable in unsupplemented saliva for extended periods under various conditions. However, these findings may not be applicable to NESp. Therefore, to ensure the robustness of NESp detection in saliva-based carriage studies, we evaluated the impact of transport and storage conditions of saliva samples on NESp detection. Six NESp strains from two clinically relevant NESp null-capsule clades (NCCs), NCC1 (carrying pspK) and NCC2 (carrying aliC and aliD), were spiked into pneumococcus (lytA)-negative saliva and incubated through various temperatures and freeze-thaw conditions. Endpoints were processed using either culture enrichment (CE) and DNA extraction (CE-DNA), or an extraction-free method without CE, before testing for lytA using qPCR. Detection stability was assessed using linear regression modelling over temperature, time and freeze-thaws. Following CE-DNA, detection of NESp remained stable for ≤24 or ≤72 h when stored at room temperature or 4 °C, respectively, and over two freeze-thaw cycles (-80 °C), with glycerol supplementation providing slight benefits. Stability of detection when using CE-DNA depended on NCC; detection of NCC2 strains was lower and less stable than NCC1. Compared to CE-DNA, extraction-free detection was more stable, with no significant loss over 72 h at room temperature and over three freeze-thaw cycles, and negligible differences in detection between NCC1 and NCC2 strains. Additionally, extraction-free detection of NCC1, and less so NCC2, increased over the first 24 h when stored at 20-30 °C, suggesting growth of the NESp strains in saliva. Testing of ΔaliCaliD and ΔpspK mutants revealed that these genes increased in vitro viability when cultured in broth but did not significantly alter competitive fitness during saliva CE. The NCC1 NESp strains tested exhibited similar stability patterns in unsupplemented saliva as encapsulated pneumococci. However, the NCC2 strains tested here were less resilient during CE, likely due to competition with other oral microbes. Therefore, recovery of NCC2 NESp may be impacted by transport and storage conditions, leading to an underestimation of carriage prevalence when tested using CE-based methods. For the reliable carriage surveillance of NESp, samples should be stored at 4 °C soon after collection and at -80 °C within 72 h. Methods which directly detect DNA without CE may provide a less biassed accounting of NCC2 strains.

Honey bee (Apis mellifera) larvae are susceptible to the bacterial pathogen Paenibacillus larvae, which causes severe damage to bee colonies. Antibiotic treatment requires veterinary supervision in the USA, is not used in many parts of the world, perpetuates problems associated with antibiotic resistance and may necessitate residual testing in bee products. There is interest in using bacteriophages to treat infected colonies (bacteriophage therapy), and several trials are promising. Nevertheless, the safety of using biological agents in the environment must be scrutinized. In this study, we analysed the ability of P. larvae to evolve resistance to several different bacteriophages. We found that bacteriophage resistance rapidly developed in culture but often results in growth defects. Mutations in the bacteriophage-resistant isolates are concentrated in genes encoding potential surface receptors but are also observed in genes controlling general cellular functions and in two cases - lysogeny. Testing one of these isolates in bee larvae, we found it to have reduced virulence compared to the parental P. larvae strain. We also found that bacteriophages are likely able to counteract resistance evolution. This work suggests that while bacteriophage resistance may arise, its impact will likely be mitigated by reduced pathogenicity and secondary bacteriophage mutations that overcome resistance.

Bacterial pathogens employ a diverse array of virulence factors to colonize and subsequently elicit disease in their host. These factors are often subject to extensive regulation at the transcriptional level to ensure that their expression is timely. Although many pathogens use bespoke transcription factors that primarily target virulence genes, global transcription factors also sometimes play a role in controlling these genes. Enteroaggregative Escherichia coli (EAEC) is a significant cause of watery and mucoid diarrhoea globally. The organism colonizes the small intestine before producing toxins that elicit disease, using a multitude of virulence factors that are encoded both chromosomally and on virulence plasmids. In this work, we have studied the cAMP receptor protein (CRP), a well-characterized bacterial global transcription factor, focusing on its role in the pathogenicity of the prototype EAEC strain 042. We show that, although most functional CRP binding sites on the chromosome are conserved between E. coli K-12 and 042, CRP has been co-opted to couple the expression of some virulence genes to the nutritional state of the cell. We report novel mechanisms for CRP-dependent regulation of genes whose products contribute to the maturation of a bacterial antibiotic, export of a polysaccharide capsule and production of a putative adhesin.

Rare actinomycetes are increasingly recognised as a valuable yet underexplored source of bioactive compounds with significant biomedical potential. While it is well established that bacteria have evolved adaptive mechanisms to withstand environmental stressors, such as variations in temperature, salinity or pH, our understanding of how these abiotic parameters influence bacterial metabolism remains limited. This has important implications not only for laboratory cultivation but also for predicting microbial behaviour in natural ecosystems. In this study, we investigated the effect of temperature on specialized metabolite production by the genus Rhodococcus. Seven strains isolated from marine sediments in three regions - Scotland, the sub-Arctic and Antarctica - were cultured at 20, 25 and 30 °C. Strain-specific growth curves were generated to normalize metabolite extraction at equivalent growth stages, resulting in a total of 54 Rhodococcus metabolite extracts. Liquid chromatography-high-resolution mass spectrometry analysis combined with molecular networking revealed that lower cultivation temperatures reduced bacterial biomass and delayed the onset of the stationary phase, and strain Rhodococcus KRD197 exhibited temperature shifts in metabolism that were associated with alterations in carbohydrate and fatty acid metabolism, potentially linked to osmotic regulation and cell membrane adaptation. These findings highlight the impact of temperature on Rhodococcus-specialized metabolism and support the potential of rare actinomycetes from extreme environments for expanding chemistry from these understudied genera.

Resumption of the planktonic phase of the Bacillus cereus cell cycle necessitates degradation of certain morphological structures and physiological features that confer metabolic dormancy and multi-factorial resistance properties to the spore form of the bacterium. Depolymerization of the peptidoglycan cortex, which is crucial to maintenance of spore dormancy, constitutes a major germination event and is conducted by a complement of spore cortex lytic enzymes that are active only during spore germination. This work reports on the structure and function of the major cortex lytic enzymes in B. cereus spores, revealing insight to their location, individual contributions to germination when triggered by different routes and regions of the SleB protein that are important for mediating interactions with its peptidoglycan substrate. The effect of null mutations to lipoproteins of the YlaJ/YhcN family on spore properties is also characterized, revealing parallels with prior observations concerning YlaJ's influence on SleB activity during germination. Finally, a structural model of a putative SleB-YpeB-YlaJ complex is presented. The model, which was subject to an initial validation by evolutionary covariance analysis and site-directed mutagenesis, reveals how the SleB protein might be held in an inactive state courtesy of its interactions with YpeB and YlaJ during spore dormancy, potentially shedding light on a long-standing puzzle in spore germination.

Actinobacteria are important for industrial production of antibiotics, fine chemicals and food and a source of new compounds for drug discovery. Their central metabolism is regulated by a conserved protein GarA that is unique to the Actinobacteria and has been studied in Mycobacterium tuberculosis and Corynebacterium glutamicum. GarA regulates the TCA cycle and glutamate metabolism by direct binding to enzymes to modulate their activity on glutamate and alpha-ketoglutarate. Given the importance of the TCA cycle in the synthesis of acyl-CoA precursors for antibiotic biosynthesis, and increasing evidence for the role of nitrogen regulators in control of secondary metabolism, we hypothesized that engineering GarA could be used to enhance production of valuable metabolites. His₆-tagged GarA was introduced into Saccharopolyspora erythraea, an overproducer of the polyketide antibiotic erythromycin. Phosphorylation of GarA was detected at the N-terminal ETTS motif, suggesting that it is regulated by protein kinases like in M. tuberculosis. GarA expression was observed at all growth stages, and a truncated form lacking the phosphorylation site accumulated during late fermentation. Engineered S. erythraea expressing phosphoablative GarA produced twofold more erythromycin, both in standard fermentation broth and in minimal medium. To investigate the mechanism for the increased titre, the engineered strain was characterized for transcription of erythromycin biosynthetic genes, as well as its ability to metabolize glutamate and its intracellular and extracellular aa content. The observed alterations in aa metabolism are consistent with the role of GarA as a TCA cycle regulator that may influence precursor supply for polyketide biosynthesis.

Persulcatusin (IP) is a tick defensin isolated from Ixodes persulcatus and is composed of 38 aa (molecular weight of 4,200). IP exhibits potent antimicrobial activity against Staphylococcus aureus, including drug-resistant strains, such as methicillin- and vancomycin-resistant S. aureus. Despite its potential use as an anti-S. aureus drug, its application remains underdeveloped because of several limitations, such as manufacturing costs and in vivo safety. Here, the combined effect of IP and other conventional antibiotics and antimicrobial proteins/peptides against S. aureus bacterial infections was investigated. Combinations of several antimicrobial compounds, including β-lactams, peptide antibiotics and lytic enzymes, showed a synergistic effect against S. aureus with a fractional inhibitory concentration index (FICI) of ≤0.75. In contrast, IP had an additive or irrelevant effect with a FICI of 1.0-2.0 when combined with several antibiotics such as chloramphenicol, gentamycin, kanamycin, erythromycin or vancomycin. Interestingly, S. aureus cells pretreated with IP for a short time demonstrated reduced susceptibility to daptomycin. Furthermore, it was determined that the mode of bactericidal activity of IP was substantially different in growth and non-growth states, suggesting that the mechanism of action of IP was associated with the inhibition of bacterial biosynthesis. These findings indicated that the combined effect of IP and conventional antibiotics has the potential to be used as an effective antimicrobial drug against S. aureus. Furthermore, it also suggested that an unknown mechanism of action of IP was associated with the inhibition of bacterial cell biosynthesis.

Gram-positive bacteria commonly employ autoinducing peptide (AIP) signal molecules to co-ordinate gene expression at the population level. This primer provides a basic overview of agr-dependent quorum sensing systems and outlines how AIPs are produced and sensed, what they control and the importance of agr for both inter-bacterial and host-pathogen interactions.

In this study, the liquid-liquid co-culture method was applied using faecal samples and specific bacterial species as growth-supporting bacteria. We aimed to isolate new, difficult-to-culture bacterial species using metabolites produced by supportive bacteria to promote the growth of small bacteria selected using filter treatment. This study aimed to identify the supporting bacteria and their metabolites that promote the growth of these isolates. Analysis of the 16S rRNA gene sequences of the isolates obtained by co-culture revealed that they were Waltera spp., Roseburia spp. and Phascolarctobacterium faecium. Roseburia spp. and Waltera spp. were isolated from several faecal samples, suggesting that they were specifically isolated using this culture method. We focused on Waltera spp. isolated from several faecal samples with unique shapes, from long to short or thin cells. The growth of Waltera spp. was not promoted by co-culture on the agar medium, suggesting that growth was only promoted by liquid-liquid co-culture. The growth of the selected small-sized Waltera spp. was promoted by co-culture, whereas the growth of the unfiltered long-cell Waltera sp. strain was suppressed by co-culture. The selected small Waltera spp. did not grow when the supporting bacterial supernatant was added, suggesting that the supporting bacteria and Waltera spp. had a symbiotic relationship through the continuous exchange of metabolites. Co-cultured supporting bacteria (diluted faecal samples) with selected small-sized Waltera spp. were predominantly Bacteroides thetaiotaomicron and Escherichia coli, compared with monoculture diluted faecal samples. We further confirmed the growth of filtered Waltera spp. by co-culturing them with B. thetaiotaomicron and E. coli. Additionally, when B. thetaiotaomicron and E. coli were co-cultured with the selected small Waltera spp., some nutrients and metabolites were reduced. Decreased metabolites were added to the medium, and selected small-sized Waltera spp. were cultured, but Waltera spp. did not grow. Therefore, it was again strongly suggested that continuous co-culturing with the supporting bacteria was important for the growth of Waltera spp. The liquid-liquid co-culture method used in this study can be used to isolate new and unique bacterial species from any environment, not just the gut microbiome. Furthermore, this co-culture method helped identify supporting bacteria and understand metabolite variations.