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Prevalence of mutations associated with tolerance to chlorhexidine and other cationic biocides among Proteus mirabilis clinical isolates. 在奇异变形杆菌临床分离株中与氯己定和其他阳离子杀菌剂耐受性相关的突变发生率。
IF 3.5 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-07-01 DOI: 10.1099/mic.0.001580
Vicky Bennett, Ocean E Clarke, Maryam Y Ravari, James D Winslow, Matthew E Wand, Andrew Preston, Emma L Denham, J Mark Sutton, Brian V Jones

Proteus mirabilis is a frequent cause of catheter-associated urinary tract infection and often exhibits high tolerance to chlorhexidine (CHD), a biocide used widely in healthcare settings. We previously demonstrated that inactivation of the smvR repressor (leading to overexpression of the smvA efflux system), truncation of the MltA-interacting protein MipA and aspects of lipopolysaccharide (LPS) structure modulate CHD susceptibility in this organism. However, the prevalence of these mechanisms among P. mirabilis clinical isolates, the conditions under which they can be acquired and their impact on susceptibility to other cationic biocides require further study. Through phenotypic and genomic analysis of a panel of 78 P. mirabilis clinical isolates, we have confirmed that deleterious mutations in smvR commonly arise in P. mirabilis and are significantly associated with reduced susceptibility to CHD and other cationic biocides. Mutations in mipA were also associated with CHD tolerance. Conversely, mutations in smvA and the rppA response regulator (which governs lipid A modifications that alter LPS surface charge) were associated with increased susceptibility to several biocides. Several isolates harbouring smvR mutations displayed incongruous phenotypes, exhibiting relatively modest CHD tolerance, which could not be accounted for by co-occurring mutations in smvA and rppA or defects in LPS (as assessed by polymyxin B susceptibility). Further analysis of these isolates revealed mutations in the LPS core biosynthesis gene waaG, leading to LPS truncation from the inner core region. Directed evolution experiments further reinforced the importance of smvR inactivation in biocide adaptation in P. mirabilis and demonstrated that relevant mutations can be selected for by exposure to CHD concentrations up to four times lower than the minimum inhibitory concentration. Taken together, these results expand our understanding of mechanisms underlying tolerance to cationic biocides in this species and provide evidence for common mechanisms of cationic biocide tolerance.

奇异变形杆菌是导尿管相关性尿路感染的常见原因,通常对氯己定(CHD)具有高耐受性,氯己定是一种广泛用于医疗保健机构的杀菌剂。我们之前证明smvR抑制因子失活(导致smvA外排系统过表达),mlta相互作用蛋白MipA的截断以及脂多糖(LPS)结构方面调节了这种生物的冠心病易感性。然而,这些机制在P. mirabilis临床分离株中的流行程度,它们可以获得的条件以及它们对其他阳离子杀菌剂敏感性的影响需要进一步研究。通过对78个奇异假单胞菌临床分离株的表型和基因组分析,我们证实了smvR的有害突变通常出现在奇异假单胞菌中,并且与降低冠心病和其他阳离子杀菌剂的易感性显著相关。mipA突变也与冠心病耐受性有关。相反,smvA和rppA反应调节因子(控制脂质A修饰,改变LPS表面电荷)的突变与几种杀菌剂的易感性增加有关。一些携带smvR突变的分离株表现出不一致的表型,表现出相对适度的冠心病耐受性,这不能通过smvA和rppA共同发生的突变或LPS缺陷来解释(通过多粘菌素B敏感性评估)。对这些分离物的进一步分析显示,LPS核心生物合成基因waaG发生突变,导致LPS从核心区域截断。定向进化实验进一步强化了smvR失活在P. mirabilis杀菌剂适应中的重要性,并证明暴露于比最低抑制浓度低4倍的CHD浓度下可以选择相关突变。综上所述,这些结果扩大了我们对该物种对阳离子杀菌剂耐受机制的理解,并为阳离子杀菌剂耐受的共同机制提供了证据。
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引用次数: 0
KpSC-ID: a multiplex real-time PCR assay for the simultaneous detection of the Klebsiella pneumoniae species complex and specific identification of Klebsiella pneumoniae, Klebsiella quasipneumoniae and Klebsiella variicola. KpSC-ID:多重实时荧光定量PCR同时检测肺炎克雷伯菌菌种复合体,特异鉴定肺炎克雷伯菌、准肺炎克雷伯菌和痘克雷伯菌。
IF 3.5 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-07-01 DOI: 10.1099/mic.0.001587
Grainne McAndrew, Elodie Barbier, Carla Rodrigues, Pascal Piveteau, Sylvain Brisse, Kate Reddington

The Klebsiella pneumoniae species complex (KpSC) comprises five closely related bacterial species, namely Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola, Klebsiella quasivariicola and Klebsiella africana. The KpSC is ubiquitous in the environment and is also an important human pathogen, particularly associated with healthcare-associated infections. The accurate detection and differentiation of the KpSC is challenging owing to the close phenotypic and genotypic identity (93-95% average nucleotide identity) shared between these members. Current diagnostic assays either fail to detect and identify all KpSC members or misidentify some KpSC members as K. pneumoniae sensu stricto. It is currently estimated that ~20% of human infections are caused by members of the KpSC other than K. pneumoniae. This leads to underreporting of some KpSC members in both clinical and environmental settings, which impacts our understanding of the importance of each species. Furthermore, it limits our understanding of the global and local epidemiological impact of some members of the KpSC. In this study, a rapid multiplex real-time PCR assay (KpSC-ID) was designed and developed to detect all KpSC members while simultaneously identifying the predominant human pathogens K. pneumoniae, K. quasipneumoniae and K. variicola. Assay performance was verified in silico using a panel of over 1,000 publicly available genome sequences and experimentally validated using a panel of genomic DNA extracted from 54 Enterobacteriaceae. The assay displayed excellent specificity against over 1,000 genome sequences tested in silico. During in vitro validation, the pan-KpSC assay detected each (29/29) KpSC species and strains tested. For the species-specific assays, 100% specificity was demonstrated in the K. pneumoniae, K. quasipneumoniae and K. variicola assays, respectively. Sensitivity of 10 genomic equivalents was demonstrated for each assay. Ultimately, the diagnostic assay developed in this study can improve our understanding of the significance of KpSC members, which is important when investigating their routes of transmission and epidemiology.

肺炎克雷伯菌物种复合体(KpSC)由五个密切相关的细菌物种组成,即肺炎克雷伯菌、准肺炎克雷伯菌、水痘克雷伯菌、准水痘克雷伯菌和非洲克雷伯菌。KpSC在环境中无处不在,也是一种重要的人类病原体,特别是与医疗保健相关的感染有关。由于这些成员之间具有密切的表型和基因型同一性(平均核苷酸同一性为93-95%),因此准确检测和分化KpSC具有挑战性。目前的诊断方法要么不能检测和识别所有KpSC成员,要么将某些KpSC成员错误地识别为严格感肺炎克雷伯菌。目前估计,约20%的人类感染是由肺炎克雷伯菌以外的KpSC成员引起的。这导致在临床和环境设置中一些KpSC成员的少报,这影响了我们对每个物种重要性的理解。此外,它限制了我们对KpSC某些成员的全球和当地流行病学影响的理解。本研究设计并开发了一种快速多重实时PCR检测方法(KpSC- id),用于检测所有KpSC成员,同时鉴定主要的人类病原体肺炎克雷伯菌、准肺炎克雷伯菌和天花克雷伯菌。使用超过1000个公开可用的基因组序列在计算机上验证了分析性能,并使用从54个肠杆菌科提取的基因组DNA进行了实验验证。该分析对超过1000个基因组序列的硅测试显示出极好的特异性。在体外验证过程中,pan-KpSC法检测到每个(29/29)KpSC种和菌株。对于种特异性检测,肺炎克雷伯菌、准肺炎克雷伯菌和天花克雷伯菌检测分别具有100%的特异性。每项检测均显示了10个基因组等效物的敏感性。最终,本研究开发的诊断方法可以提高我们对KpSC成员意义的理解,这在调查其传播途径和流行病学时非常重要。
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引用次数: 0
Improved efficiency of two-step amplicon PCR using an acoustic liquid handler. 利用声波液体处理器提高了两步扩增PCR的效率。
IF 3.5 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-07-01 DOI: 10.1099/mic.0.001579
Brooke R Benz, Eglantina Lopez-Echartea, Briana K Whitaker, Thomas Baldwin, Barney A Geddes

The improvement in next-generation sequencing technologies has reduced the costs of sequencing significantly. However, library preparation costs for amplicon sequencing have remained largely unchanged - which is ultimately the cost-limiting step in processing large numbers of microbiome samples. Acoustic liquid handlers can transfer volumes as low as 2.5 nl and have been used to miniaturize several different molecular and cellular assays, including single-step PCR amplicon library preparations. However, there are no current methods available for a two-step library preparation process using an acoustic liquid handler. In this study, we tested the efficiency of an acoustic liquid handler to automate the PCRs and library quantification while also incorporating automated library bead cleanup. We compared the material usage and costs for library preparation and sequencing results of this automated method to the standard, manual method. The automated protocol was able to reduce both PCR reaction volumes fivefold and increased efficiency for library preparation by ~32% without affecting bacterial community compositions. The associated increase in the efficiency of our automated method will allow for greater throughput in sequencing hundreds of microbiome samples without affecting the quality of those sequences.

新一代测序技术的进步大大降低了测序成本。然而,扩增子测序的文库制备成本基本保持不变,这是处理大量微生物组样品的最终成本限制步骤。声波液体处理机可以传输低至2.5 nl的体积,并已用于小型化几种不同的分子和细胞分析,包括单步PCR扩增文库制备。然而,目前还没有使用声波液体处理器的两步文库制备方法。在这项研究中,我们测试了声波液体处理器的效率,以自动化pcr和文库定量,同时还结合了自动化文库头清理。我们将这种自动化方法的文库制备和测序结果的材料使用量和成本与标准的人工方法进行了比较。自动化方案能够在不影响细菌群落组成的情况下将PCR反应体积减少5倍,并将文库制备效率提高约32%。我们的自动化方法效率的相关提高将允许在不影响这些序列的质量的情况下对数百个微生物组样品进行测序的更大吞吐量。
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引用次数: 0
Comparative genomic analysis of Latilactobacillus sakei strains provides new insights into their association with different niche adaptations. 酒井乳酸杆菌菌株的比较基因组分析为它们与不同生态位适应的关系提供了新的见解。
IF 2.6 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-07-01 DOI: 10.1099/mic.0.001578
Kohei Ito, Yutaro Ito

Latilactobacillus sakei, a lactic acid bacterium in diverse environments such as fermented foods, meat and the human gastrointestinal tract, exhibits significant genetic diversity and niche-specific adaptations. This study conducts a comprehensive comparative genomic analysis of 29 complete L. sakei genomes to uncover the genetic mechanisms underlying these adaptations. Phylogenetic analysis divided the species into three distinct clades that did not correlate with the source of isolation and did not suggest any niche-specific evolutionary direction. The pan-genome analysis revealed a substantial core genome alongside a diverse genetic repertoire, indicating both high genetic conservation and adaptability. Predicted growth rates based on codon use bias analysis suggest that L. sakei strains have an overall faster growth rate and may be able to efficiently dominate in competitive environments. Plasmid analysis revealed a variety of plasmids carrying genes essential for carbohydrate metabolism, enhancing L. sakei's ability to thrive in various fermentation substrates. It was also found that the number of genes belonging to the GH1 family amongst sugar metabolism-related genes present on chromosomes and plasmids varies between strains and that AA1, which is involved in alcohol oxidation, has been acquired from plasmids. blast analysis revealed that some strains have environmental adaptation gene clusters of cell surface polysaccharides that may mediate attachment to food and mucosa. The knowledge gleaned from this study lays a solid foundation for future research aimed at harnessing the genetic traits of L. sakei strains for industrial and health-related applications.

酒井乳杆菌是一种存在于多种环境中的乳酸菌,如发酵食品、肉类和人类胃肠道,它表现出显著的遗传多样性和生态位特异性适应。本研究对29个完整的白井l.s akei基因组进行了全面的比较基因组分析,以揭示这些适应的遗传机制。系统发育分析将该物种划分为三个不同的分支,这与隔离来源无关,也没有提出任何特定于生态位的进化方向。泛基因组分析揭示了一个实质性的核心基因组以及多样化的遗传库,表明高度的遗传保守性和适应性。基于密码子使用偏差分析的预测增长率表明,L. sakei菌株总体上具有更快的增长率,并且可能能够在竞争环境中有效地占主导地位。质粒分析揭示了多种携带碳水化合物代谢必需基因的质粒,增强了L. saki在各种发酵底物中茁壮成长的能力。研究还发现,在染色体和质粒上存在的糖代谢相关基因中,属于GH1家族的基因数量因菌株而异,并且从质粒中获得了参与酒精氧化的AA1。Blast分析显示,一些菌株具有细胞表面多糖的环境适应基因簇,可能介导对食物和粘膜的附着。从这项研究中收集到的知识为未来的研究奠定了坚实的基础,旨在利用L. sakei菌株的遗传性状用于工业和健康相关的应用。
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引用次数: 0
Antibiotic use in rural Kenyan livestock: navigating misuse, experience gaps and AMR risks. 肯尼亚农村牲畜抗生素使用:应对滥用、经验差距和抗生素耐药性风险。
IF 2.6 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-07-01 DOI: 10.1099/mic.0.001582
Lucy Obolensky, Esbon Wambugu, Edna K Kubai, Iain Doig, Miriam Beattie, Michael J Dillon

Antimicrobial resistance (AMR) is an escalating global health threat, with the greatest risk observed in low- to middle-income countries, particularly in the global south. The World Health Organization advocates for a One Health approach to address AMR, promoting collaboration across sectors, including in agriculture. This study aims to enhance understanding of antimicrobial use and stewardship in livestock within pastoralist communities in northern Kenya, where there is limited information. The study employed a qualitative approach, using semi-structured interviews to gather data on farming practices and antibiotic use. Interviews were conducted by trained volunteers proficient in Swahili and Ma (a Maasai language), across four pastoralist communities in northern Kenya in December 2023. The data were then thematically analysed by four researchers. Fifty-one individuals participated in the study. Thematic analysis revealed several key insights, including the widespread misuse of antibiotics, often used on intuition and without professional support. A notable barrier to appropriate use was the lack of veterinary advice, with many participants relying on agrovets or past experience for guidance. Cross-use of antibiotics, such as administering animal antibiotics to humans, was also observed. Awareness of AMR was limited, and leftover antibiotics were often saved or shared across communities. The findings from this study underscore the critical need for targeted education and training within these communities.

抗微生物药物耐药性(AMR)是一个不断升级的全球健康威胁,在中低收入国家,特别是在全球南方国家,风险最大。世界卫生组织倡导以“一个健康”方针解决抗生素耐药性问题,促进包括农业在内的各部门合作。本研究旨在加强对肯尼亚北部牧民社区牲畜抗微生物药物使用和管理的了解,那里的信息有限。该研究采用了定性方法,使用半结构化访谈来收集有关农业实践和抗生素使用的数据。2023年12月,由精通斯瓦希里语和马语(一种马赛语)的训练有素的志愿者在肯尼亚北部的四个牧民社区进行了采访。然后,四位研究人员对这些数据进行了主题分析。51个人参与了这项研究。专题分析揭示了几个关键的见解,包括抗生素的广泛滥用,经常凭直觉使用,没有专业支持。适当使用的一个显著障碍是缺乏兽医咨询意见,许多参与者依靠agrovets或过去的经验作为指导。还观察到交叉使用抗生素,例如给人类使用动物抗生素。对抗菌素耐药性的认识有限,剩余的抗生素往往被保存起来或在社区间共享。这项研究的结果强调了在这些社区进行有针对性的教育和培训的迫切需要。
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引用次数: 0
Pectobacterium atrosepticum SCRI1043 flagella mediate adherence to potato plants indirectly through motility. 败腐乳杆菌SCRI1043鞭毛通过运动性间接介导对马铃薯植株的粘附。
IF 3.5 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-07-01 DOI: 10.1099/mic.0.001588
Ashleigh Holmes, Sonia Humphris, Jacqueline Marshall, Yannick Rossez, Ian Toth, Nicola J Holden

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.

鞭毛是细菌中广泛分布的附属物,具有良好的运动和趋化功能。它们也直接与宿主相互作用,由于它们无处不在,它们是植物和动物王国宿主的有效免疫激发子。此外,鞭毛已被证明可以促进几种不同细菌的附着,包括几种与植物相关的细菌与植物宿主的附着。我们之前在园艺物种和拟南芥中证实了大肠杆菌鞭毛与植物质膜离子脂质的结合。因此,鞭毛可以被认为是一种通用的定植因子,特别是在相互作用的早期阶段。因此,我们测试了一种遗传相关的植物病原体——萎败Pectobacterium atrosepticum的鞭毛是否以类似于大肠杆菌的方式介导其与易感植物宿主马铃薯的结合。令人惊讶的是,含有atrosepticum长丝鞭毛蛋白的鞭毛并没有给马铃薯根系带来任何结合优势。此外,纯化鞭毛与马铃薯膜脂(带电或不带电)之间没有直接相互作用。Pectobacterium与马铃薯的结合能力取决于鞭毛的运动功能,因为鞭毛缺陷和运动缺陷突变体与马铃薯根部的结合能力都降低了。
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引用次数: 0
Purification and characterization of a novel antibacterial peptide against Clostridium perfringens. 一种新型产气荚膜梭菌抗菌肽的纯化及特性研究。
IF 2.6 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-07-01 DOI: 10.1099/mic.0.001573
Alex Novodvorski, Avalene Kong, Hai Yu, Dion Lepp, Ashley Brott, Jason Carere, Stephen Seah, Joshua Gong

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 IPHG88, 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 IPHG88 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 IPHG88 was also predicted using AlphaFold 2.0.

从不使用抗生素饲养的蛋鸡回肠黏膜样品中分离得到一株韦氏芽孢杆菌HG88。其无细胞上清液(CFS)对鸡坏死性肠炎病原菌产气荚膜梭菌(Clostridium perfringens)有抑制作用。由于对蛋白酶消化的敏感性,该抑制化合物被确定为蛋白质。CFS对9种革兰氏阳性或革兰氏阴性菌和2种酵母菌的生长均无抑制作用,对产气荚膜梭菌具有特异性抑菌活性。从CFS中分离蛋白质,然后对菌株进行肽质量指纹图谱和基因组分析,鉴定出一种假定的抗菌肽,该肽具有输出信号,可从细胞分泌。来自B. velezensis HG88的肽被命名为IPHG88,其序列与已知与肽聚糖肽部分结合的细菌SH3结构域相似。克隆了该肽的编码基因,并从重组大肠杆菌中纯化得到n端his标记肽。带His-tag和不带His-tag的IPHG88对产气荚膜荚膜梭菌的抑制作用最低浓度分别为~57.0和39.1µg ml-1。利用AlphaFold 2.0对IPHG88的三维结构进行预测。
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引用次数: 0
The influence of the polyamine synthesis pathways on Pseudomonas syringae virulence and plant interaction. 多胺合成途径对丁香假单胞菌毒力及植物互作的影响。
IF 2.6 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-06-01 DOI: 10.1099/mic.0.001569
Leandro Solmi, Franco Rubén Rossi, Fernando Matías Romero, Marcel Bach-Pages, Gail M Preston, Andrés Gárriz

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.

本研究探讨了多胺生物合成在丁香假单胞菌发病机制中的作用。番茄。通过对受腐胺和亚精胺合成影响的突变菌株的综合表型分析,我们揭示了这种代谢途径与细菌毒力之间的复杂相互作用。腐胺合成的破坏会损害多种毒力性状,如运动、生物膜的形成、铁载体的产生、植物气孔关闭的预防和III型分泌系统的功能。这可以通过重新引入缺失的基因来逆转,但不能通过补充腐胺或外胞质洗涤液(AWF)的培养基来逆转。同样,抑制亚精胺生物合成也会导致类似的表型。然而,在这种情况下,通过添加亚精胺、AWF或表达亚精胺合成酶基因,恢复了野生型表型。我们得出结论,腐胺和亚精胺对细菌的毒力都很重要,植物衍生的亚精胺可以部分补偿细菌的需要。因此,尽管腐胺缺乏导致低毒性表型,亚精胺合成扰动不影响植物定植。这些发现强调了多胺代谢在细菌病原体入侵植物过程中的关键作用。
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引用次数: 0
Perturbation of the MetJ regulon impacts the consequences of 2-aminoacrylate stress in Salmonella enterica. MetJ调控的扰动影响肠沙门氏菌中2-氨基丙烯酸酯应激的后果。
IF 2.6 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-06-01 DOI: 10.1099/mic.0.001572
Bryce R Sawyer, Wangchen Shen, Diana M Downs

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.

在缺乏广泛保守的脱氨酶RidA(反应性中间脱氨酶A)的情况下,肠沙门氏菌和其他生物会积累反应性脱胺物质2-氨基丙烯酸酯(2AA)。游离2AA由丝氨酸/苏氨酸脱水酶IlvA产生,与吡哆醛5'-磷酸依赖酶发生反应并共价失活。2AA引起的代谢应激在肠链球菌中产生生长缺陷,包括(i)当l-丙氨酸被用作氮源时,(ii)当丙酮酸被用作碳源时,或(iii)当外源丝氨酸存在时。尽管在不同的生长条件下,2AA的酶促靶点是一致的,但2AA依赖性损伤的后果取决于不同条件下所需代谢通量的分布。分析ridA突变体的抑制因子进一步加深了我们对ridA应激模式的理解,更一般地说,是对代谢网络如何响应扰动的理解。许多此类抑制因子调节代谢网络以消除IlvA产生的2AA。在这里,我们描述了去除MetJ转录抑制因子允许ridA突变体在存在2AA胁迫的三种情况下生长。ΔmetJ抑制ridA突变的机制是微妙的和中等依赖的,强调2AA应激的后果是基于环境和代谢背景而不同的。
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引用次数: 0
Culturomics from field-grown crop plants using dilution to extinction, two-step library preparation and amplicon sequencing. 利用稀释至灭绝、两步文库制备和扩增子测序对大田作物进行培养组学分析。
IF 2.6 4区 生物学 Q3 MICROBIOLOGY Pub Date : 2025-06-01 DOI: 10.1099/mic.0.001571
Eglantina Lopez-Echartea, Nicholas Dusek, Mallory Misialek, Mohammad Al Mahmud-Un-Nabi, Riley Williamson, Komal Marathe, Barney A Geddes

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.

培养组学方法通过高通量分离和表征更广泛的细菌分类群,包括一些以前认为不可培养的细菌,从而促进了微生物研究。在这里,我们提出了一个方案的测试和优化,从田间种植的植物中分离和鉴定数百种可培养微生物。在美国ND的不同环境条件下,利用大田种植的玉米和豌豆植株的根微生物组对该方案进行了测试和优化。通过稀释至灭活培养和针对16S rRNA基因V4区的两步条形码PCR策略,我们鉴定出200多个独特的细菌分离株。围绕DADA2包构建的优化生物信息学管道确保了准确的扩增子序列变异检测和分类分配。由此产生的细菌分离株跨越不同的系统发育类群,包括促进植物生长和减轻压力的植物相关类群。我们的研究结果突出了培养组学在合成群落设计和推进植物-微生物相互作用研究中产生微生物收集的价值。该协议的可扩展性、成本效益和强大的性能表明其在农业微生物组研究中的广泛应用潜力。
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