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Turbidimetric bioassays: A solution to antimicrobial activity detection in asymptomatic bacteriuria isolates against uropathogenic Escherichia coli 比浊生物测定法:无症状菌尿分离物对尿路致病性大肠杆菌抗菌活性检测的解决方案
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-05-06 DOI: 10.1002/mbo3.1411
Ciara Kenneally, Craig P. Murphy, Roy D. Sleator, Eamonn P. Culligan

Traditional bacteriocin screening methods often face limitations due to diffusion-related challenges in agar matrices, which can prevent the peptides from reaching their target organism. Turbidimetric techniques offer a solution to these issues, eliminating diffusion-related problems and providing an initial quantification of bacteriocin efficacy in producer organisms. This study involved screening the cell-free supernatant (CFS) from eight uncharacterized asymptomatic bacteriuria (ABU) isolates and Escherichia coli 83972 for antimicrobial activity against clinical uropathogenic E. coli (UPEC) strains using turbidimetric growth methods. ABU isolates exhibiting activity against five or more UPEC strains were further characterized (PUTS 37, PUTS 58, PUTS 59, S-07-4, and SK-106-1). The inhibition of the CFS by proteinase K suggested that the antimicrobial activity was proteinaceous in nature, potentially bacteriocins. The activity of E. coli PUTS 58 and SK-106-1 was enhanced in an artificial urine medium, with both inhibiting all eight UPECs. A putative microcin H47 operon was identified in E. coli SK-106-1, along with a previously identified microcin V and colicin E7 in E. coli PUTS 37 and PUTS 58, respectively. These findings indicate that ABU bacteriocin-producers could serve as viable prophylactics and therapeutics in the face of increasing antibiotic resistance among uropathogens.

传统的细菌素筛选方法往往受到琼脂基质中与扩散有关的难题的限制,这些难题会阻碍肽到达目标生物体。比浊技术为这些问题提供了一种解决方案,它消除了与扩散相关的问题,并能初步量化细菌素在生产者生物体中的效力。本研究采用浊度生长法对来自 8 个未定性无症状菌尿症(ABU)分离物和大肠杆菌 83972 的无细胞上清液(CFS)进行筛选,以确定其对临床尿路致病性大肠杆菌(UPEC)菌株的抗菌活性。对五株或五株以上 UPEC 菌株具有活性的 ABU 分离物(PUTS 37、PUTS 58、PUTS 59、S-07-4 和 SK-106-1)被进一步鉴定。蛋白酶 K 对 CFS 的抑制作用表明,其抗菌活性本质上是蛋白性的,可能是细菌素。在人工尿培养基中,大肠杆菌 PUTS 58 和 SK-106-1 的活性增强,两者都能抑制所有八种 UPECs。在大肠杆菌 SK-106-1 中发现了一个推定的微素 H47 操作子,以及之前在大肠杆菌 PUTS 37 和 PUTS 58 中分别发现的微素 V 和大肠杆菌素 E7。这些研究结果表明,面对泌尿道病原体对抗生素的耐药性不断增加,ABU 细菌素生产者可作为可行的预防和治疗药物。
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引用次数: 0
Utilizing novel Escherichia coli-specific conserved signature proteins for enhanced monitoring of recreational water quality 利用新型大肠埃希氏菌特异性保守特征蛋白加强对休闲水质的监测
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-04-29 DOI: 10.1002/mbo3.1410
Faizan Saleem, Enze Li, Kevin L. Tran, Bashudev Rudra, Thomas A. Edge, Herb E. Schellhorn, Radhey S. Gupta

Escherichia coli serves as a proxy indicator of fecal contamination in aquatic ecosystems. However, its identification using traditional culturing methods can take up to 24 h. The application of DNA markers, such as conserved signature proteins (CSPs) genes (unique to all species/strains of a specific taxon), can form the foundation for novel polymerase chain reaction (PCR) tests that unambiguously identify and detect targeted bacterial taxa of interest. This paper reports the identification of three new highly-conserved CSPs (genes), namely YahL, YdjO, and YjfZ, which are exclusive to E. coli/Shigella. Using PCR primers based on highly conserved regions within these CSPs, we have developed quantitative PCR (qPCR) assays for the evaluation of E. coli/Shigella species in water ecosystems. Both in-silico and experimental PCR testing confirmed the absence of sequence match when tested against other bacteria, thereby confirming 100% specificity of the tested CSPs for E. coli/Shigella. The qPCR assays for each of the three CSPs provided reliable quantification for all tested enterohaemorrhagic and environmental E. coli strains, a requirement for water testing. For recreational water samples, CSP-based quantification showed a high correlation (r > 7, p < 0.01) with conventional viable E. coli enumeration. This indicates that novel CSP-based qPCR assays for E. coli can serve as robust tools for monitoring water ecosystems and other critical areas, including food monitoring.

大肠杆菌是水生生态系统中粪便污染的替代指标。应用 DNA 标记,如保守特征蛋白(CSPs)基因(特定类群的所有物种/菌株所独有),可为新型聚合酶链式反应(PCR)测试奠定基础,从而明确识别和检测感兴趣的目标细菌类群。本文报告了三个新的高保守 CSP(基因)的鉴定结果,即 YahL、YdjO 和 YjfZ,它们是大肠杆菌/志贺氏菌独有的。利用基于这些 CSP 高度保守区域的 PCR 引物,我们开发出了定量 PCR(qPCR)检测方法,用于评估水生态系统中的大肠杆菌/志贺菌物种。在对其他细菌进行测试时,我们都通过室内和实验 PCR 测试确认了序列匹配的缺失,从而证实了所测试的 CSP 对大肠杆菌/志贺氏菌具有 100% 的特异性。三种 CSP 中每一种的 qPCR 检测都能对所有检测的肠出血性大肠杆菌和环境大肠杆菌菌株进行可靠的定量,这也是水质检测的一项要求。对于娱乐性水样,基于 CSP 的定量与传统的大肠杆菌计数显示出很高的相关性(r > 7, p < 0.01)。这表明,基于 CSP 的新型大肠杆菌 qPCR 检测方法可作为监测水生态系统和其他关键领域(包括食品监测)的有力工具。
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引用次数: 0
Multidrug-resistant Stenotrophomonas maltophilia in residential aged care facilities: An emerging threat 养老院中的嗜麦芽霉单胞菌对多种药物产生耐药性:新出现的威胁
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-04-29 DOI: 10.1002/mbo3.1409
Sylvia A. Sapula, Bradley J. Hart, Naomi L. Siderius, Anteneh Amsalu, Jack M. Blaikie, Henrietta Venter

Stenotrophomonas maltophilia is a multidrug-resistant (MDR), Gram-negative bacterium intrinsically resistant to beta-lactams, including last-resort carbapenems. As an opportunistic pathogen, it can cause serious healthcare-related infections. This study assesses the prevalence, resistance profiles, and genetic diversity of S. maltophilia isolated from residential aged care facilities (RACFs). RACFs are known for their overuse and often inappropriate use of antibiotics, creating a strong selective environment that favors the development of bacterial resistance. The study was conducted on 73 S. maltophilia isolates recovered from wastewater and facility swab samples obtained from three RACFs and a retirement village. Phenotypic and genotypic assessments of the isolates revealed high carbapenem resistance, exemplifying their intrinsic beta-lactam resistance. Alarmingly, 49.3% (36/73) of the isolates were non-wild type for colistin, with minimum inhibitory concentration values of > 4 mg/L, and 11.0% (8/73) were resistant to trimethoprim-sulfamethoxazole. No resistance mechanisms were detected for either antimicrobial. Genotypic assessment of known lineages revealed isolates clustering with Sm17 and Sm18, lineages not previously reported in Australia, suggesting the potential ongoing spread of MDR S. maltophilia. Lastly, although only a few isolates were biocide tolerant (2.7%, 2/73), their ability to grow in high concentrations (64 mg/L) of triclosan is concerning, as it may be selecting for their survival and continued dissemination.

嗜麦芽血单胞菌是一种耐多药(MDR)革兰氏阴性细菌,对β-内酰胺类药物(包括最后的碳青霉烯类)具有内在耐药性。作为一种机会性病原体,它可引起严重的医疗相关感染。本研究评估了从养老院(RACF)中分离出的嗜麦芽糖酵母菌的流行率、耐药性特征和遗传多样性。众所周知,养老院过度使用抗生素,而且往往使用不当,这就形成了一个有利于细菌耐药性发展的强大选择性环境。这项研究针对从三家 RACF 和一家退休村的废水和设施拭子样本中分离出的 73 个嗜麦芽汁酵母菌进行了研究。对分离菌株的表型和基因型评估显示,这些菌株对碳青霉烯类耐药性较高,体现了其固有的β-内酰胺耐药性。令人担忧的是,49.3%(36/73)的分离株对可乐定具有非野生型耐药性,最低抑菌浓度值为 4 毫克/升,11.0%(8/73)的分离株对三甲双胍-磺胺甲噁唑具有耐药性。对这两种抗菌素均未发现耐药机制。对已知菌系的基因型评估显示,分离物与 Sm17 和 Sm18 菌系聚集在一起,而这两个菌系以前在澳大利亚未曾报道过,这表明嗜麦芽糖酵母菌的 MDR 有可能正在蔓延。最后,虽然只有少数分离物对杀菌剂有耐受性(2.7%,2/73),但它们在高浓度(64 毫克/升)三氯生中的生长能力令人担忧,因为这可能会促进它们的生存和继续传播。
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引用次数: 0
Biosynthetic potential of the sediment microbial subcommunities of an unexplored karst ecosystem and its ecological implications 未开发岩溶生态系统沉积物微生物亚群落的生物合成潜力及其生态影响
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-04-09 DOI: 10.1002/mbo3.1407
Pablo Suárez-Moo, Alejandra Prieto-Davó

Microbial communities from various environments have been studied in the quest for new natural products with a broad range of applications in medicine and biotechnology. We employed an enrichment method and genome mining tools to examine the biosynthetic potential of microbial communities in the sediments of a coastal sinkhole within the karst ecosystem of the Yucatán Peninsula, Mexico. Our investigation led to the detection of 203 biosynthetic gene clusters (BGCs) and 55 secondary metabolites (SMs) within 35 high-quality metagenome-assembled genomes (MAGs) derived from these subcommunities. The most abundant types of BGCs were Terpene, Nonribosomal peptide-synthetase, and Type III polyketide synthase. Some of the in silico identified BGCs and SMs have been previously reported to exhibit biological activities against pathogenic bacteria and fungi. Others could play significant roles in the sinkhole ecosystem, such as iron solubilization and osmotic stress protection. Interestingly, 75% of the BGCs showed no sequence homology with bacterial BGCs previously reported in the MiBIG database. This suggests that the microbial communities in this environment could be an untapped source of genes encoding novel specialized compounds. The majority of the BGCs were identified in pathways found in the genus Virgibacillus, followed by Sporosarcina, Siminovitchia, Rhodococcus, and Halomonas. The latter, along with Paraclostridium and Lysinibacillus, had the highest number of identified BGC types. This study offers fresh insights into the potential ecological role of SMs from sediment microbial communities in an unexplored environment, underscoring their value as a source of novel natural products.

人们研究了各种环境中的微生物群落,以寻找新的天然产品,这些产品在医学和生物技术领域有着广泛的应用。我们采用富集方法和基因组挖掘工具,研究了墨西哥尤卡坦半岛喀斯特生态系统中沿海天坑沉积物中微生物群落的生物合成潜力。通过研究,我们在这些亚群落的 35 个高质量元基因组组装基因组(MAGs)中发现了 203 个生物合成基因簇(BGCs)和 55 个次级代谢产物(SMs)。最丰富的 BGC 类型是萜烯、非核糖体肽合成酶和 III 型多酮合成酶。以前曾有报道称,一些在硅学中鉴定出的 BGCs 和 SMs 具有抗病原菌和真菌的生物活性。其他一些则可能在水坑生态系统中发挥重要作用,如铁增溶和渗透压保护。有趣的是,75%的BGCs与MiBIG数据库中之前报道的细菌BGCs没有序列同源性。这表明,这种环境中的微生物群落可能是编码新型特化化合物基因的一个尚未开发的来源。大多数 BGCs 都是在 Virgibacillus 属的途径中发现的,其次是 Sporosarcina、Siminovitchia、Rhodococcus 和 Halomonas。后者与副梭菌和溶血芽孢杆菌一起,拥有数量最多的已鉴定 BGC 类型。这项研究对未开发环境中沉积物微生物群落中的 SMs 的潜在生态作用提供了新的见解,强调了它们作为新型天然产品来源的价值。
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引用次数: 0
Characterizing arginine, ornithine, and putrescine pathways in enteric pathobionts 鉴定肠道致病菌中的精氨酸、鸟氨酸和腐胺途径
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-04-01 DOI: 10.1002/mbo3.1408
Ian M. Lillie, Charles E. Booth, Adelaide E. Horvath, Matthew Mondragon, Melinda A. Engevik, Thomas D. Horvath

Arginine-ornithine metabolism plays a crucial role in bacterial homeostasis, as evidenced by numerous studies. However, the utilization of arginine and the downstream products of its metabolism remain undefined in various gut bacteria. To bridge this knowledge gap, we employed genomic screening to pinpoint relevant metabolic targets. We also devised a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method to measure the levels of arginine, its upstream precursors, and downstream products in cell-free conditioned media from enteric pathobionts, including Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Pseudomonas fluorescens, Acinetobacter baumannii, Streptococcus agalactiae, Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis. Our findings revealed that all selected bacterial strains consumed glutamine, glutamate, and arginine, and produced citrulline, ornithine, and GABA in our chemically defined medium. Additionally, E. coli, K. pneumoniae, K. aerogenes, and P. fluorescens were found to convert arginine to agmatine and produce putrescine. Interestingly, arginine supplementation promoted biofilm formation in K. pneumoniae, while ornithine supplementation enhanced biofilm formation in S. epidermidis. These findings offer a comprehensive insight into arginine-ornithine metabolism in enteric pathobionts.

大量研究证明,精氨酸-鸟氨酸代谢在细菌平衡中发挥着至关重要的作用。然而,精氨酸的利用及其代谢的下游产物在各种肠道细菌中仍未确定。为了弥补这一知识空白,我们采用了基因组筛选技术来确定相关的代谢靶标。我们还设计了一种有针对性的液相色谱-串联质谱(LC-MS/MS)代谢组学方法,以测量肠道致病菌(包括大肠埃希菌、产气克雷伯氏菌、肺炎克雷伯氏菌、流感假单胞菌等)无细胞条件培养基中精氨酸、其上游前体和下游产物的水平。肺炎双球菌、荧光假单胞菌、鲍曼不动杆菌、无乳链球菌、表皮葡萄球菌、金黄色葡萄球菌和粪肠球菌。我们的研究结果表明,在化学定义的培养基中,所有选定的细菌菌株都消耗谷氨酰胺、谷氨酸和精氨酸,并产生瓜氨酸、鸟氨酸和 GABA。此外,还发现大肠杆菌、肺炎双球菌、产气荚膜杆菌和荧光团菌能将精氨酸转化为矢车菊碱并产生腐胺。有趣的是,补充精氨酸可促进肺炎双球菌生物膜的形成,而补充鸟氨酸可促进表皮葡萄球菌生物膜的形成。这些发现为精氨酸-鸟氨酸在肠道致病菌中的代谢提供了一个全面的视角。
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引用次数: 0
What is the role of microbial biotechnology and genetic engineering in medicine? 微生物生物技术和基因工程在医学中的作用是什么?
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-03-31 DOI: 10.1002/mbo3.1406
Fernando Santos-Beneit

Microbial products are essential for developing various therapeutic agents, including antibiotics, anticancer drugs, vaccines, and therapeutic enzymes. Genetic engineering techniques, functional genomics, and synthetic biology unlock previously uncharacterized natural products. This review highlights major advances in microbial biotechnology, focusing on gene-based technologies for medical applications.

微生物产品对于开发各种治疗药物(包括抗生素、抗癌药物、疫苗和治疗酶)至关重要。基因工程技术、功能基因组学和合成生物学解锁了以前未表征的天然产品。本综述重点介绍了微生物生物技术的主要进展,重点是基于基因的医疗应用技术。
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引用次数: 0
Using meta-analysis to understand the impacts of dietary protein and fat content on the composition of fecal microbiota of domestic dogs (Canis lupus familiaris): A pilot study 利用荟萃分析了解膳食蛋白质和脂肪含量对家犬粪便微生物群组成的影响:一项试点研究。
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-03-21 DOI: 10.1002/mbo3.1404
Francis D. Phimister, Rachel C. Anderson, David G. Thomas, Michelle J. Farquhar, Paul Maclean, Ruy Jauregui, Wayne Young, Christina F. Butowski, Emma N. Bermingham

The interplay between diet and fecal microbiota composition is garnering increased interest across various host species, including domestic dogs. While the influence of dietary macronutrients and their associated microbial communities have been extensively reviewed, these reviews are descriptive and do not account for differences in microbial community analysis, nor do they standardize macronutrient content across studies. To address this, a meta-analysis was performed to assess the impact of dietary crude protein (“protein”) and dietary crude fat (“fat”) on the fecal microbiota composition in healthy dogs. Sixteen publications met the eligibility criteria for the meta-analysis, yielding a final data set of 314 dogs. Diets were classed as low, moderate, high, or supra in terms of protein or fat content. Sequence data from each publication were retrieved from public databases and reanalyzed using consistent bioinformatic pipelines. Analysis of community diversity indices and unsupervised clustering of the data with principal coordinate analysis revealed a small effect size and complete overlap between protein and fat levels at the overall community level. Supervised clustering through random forest analysis and partial least squares-discriminant analysis indicated alterations in the fecal microbiota composition at a more individual taxonomic level, corresponding to the levels of protein or fat. The Prevotellaceae Ga6A1 group and Enterococcus were associated with increasing levels of protein, while Allobaculum and Clostridium sensu stricto 13 were associated with increasing levels of fat. Interestingly, the random forest analyses revealed that Sharpea, despite its low relative abundance in the dog's fecal microbiome, was primarily responsible for the separation of the microbiome for both protein and fat. Future research should focus on validating and understanding the functional roles of these relatively low-abundant genera.

在包括家犬在内的各种宿主物种中,饮食与粪便微生物群组成之间的相互作用正引起越来越多的关注。虽然膳食宏量营养素及其相关微生物群落的影响已被广泛综述,但这些综述都是描述性的,没有考虑到微生物群落分析的差异,也没有对不同研究中的宏量营养素含量进行标准化。为此,我们进行了一项荟萃分析,以评估膳食粗蛋白("蛋白质")和膳食粗脂肪("脂肪")对健康狗粪便微生物群组成的影响。有 16 篇文献符合荟萃分析的资格标准,最终获得了 314 只狗的数据集。日粮的蛋白质或脂肪含量被分为低、中、高或超高。从公共数据库中检索了每篇论文的序列数据,并使用一致的生物信息学管道进行了重新分析。群落多样性指数分析和主坐标分析法对数据进行的无监督聚类显示,在整个群落水平上,蛋白质和脂肪含量之间的影响很小,而且完全重叠。通过随机森林分析和偏最小二乘判别分析进行的有监督聚类表明,粪便微生物群组成在更个体化的分类水平上发生了变化,与蛋白质或脂肪水平相对应。前孢子菌科 Ga6A1 组和肠球菌与蛋白质含量的增加有关,而 Allobaculum 和严格意义上的梭状芽孢杆菌 13 则与脂肪含量的增加有关。有趣的是,随机森林分析表明,尽管沙佩氏菌在狗粪便微生物组中的相对丰度较低,但它却是蛋白质和脂肪微生物组分离的主要原因。未来的研究应侧重于验证和了解这些相对低丰度菌属的功能作用。
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引用次数: 0
Genomic characterization of extended-spectrum beta-lactamase-producing and carbapenem-resistant Escherichia coli from urban wastewater in Australia 澳大利亚城市污水中产广谱β-内酰胺酶和耐碳青霉烯类大肠埃希菌的基因组特征。
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-03-15 DOI: 10.1002/mbo3.1403
Zillur Rahman, Mary-Louise McLaws, Torsten Thomas

This study investigates extended-spectrum beta-lactamase-producing and carbapenem-resistant Escherichia coli isolates from Sydney's wastewater. These isolates exhibit resistance to critical antibiotics and harbor novel resistance mechanisms. The findings highlight the importance of wastewater-based surveillance in monitoring resistance beyond the clinical setting.

本研究调查了悉尼废水中分离出的产生广谱β-内酰胺酶和耐碳青霉烯类的大肠埃希菌。这些分离菌株表现出对关键抗生素的耐药性,并隐藏着新型耐药机制。研究结果凸显了基于废水的监测对临床环境以外的耐药性监测的重要性。
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引用次数: 0
Effects of color variation and physiological state on ascidian microbiomes 颜色变化和生理状态对腹足类微生物群的影响
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-03-13 DOI: 10.1002/mbo3.1405
Samantha K. Morrison, Patrick M. Erwin, Susanna López-Legentil

Ascidians, known for their color variation, host species-specific microbial symbiont communities. Some ascidians can also transition into a nonfiltering (resting) physiological state. Recent studies suggest that the microbial symbiont communities may vary across different physiological states and color morphs of the host. The colonial ascidian, Polyclinum constellatum, which exhibits several color morphs in the Caribbean Sea, periodically ceases its filtering activity. To investigate if color variation in P. constellatum is indicative of sibling speciation, we sequenced fragments of the ribosomal 18S rRNA and the mitochondrial cytochrome oxidase subunit I genes. Additionally, we sequenced a fragment of the 16S rRNA gene to characterize the microbial communities of two common color morphs (red and green) in colonies that were either actively filtering (active) or nonfiltering (resting). Phylogenetic analyses of both ascidian genes resulted in well-supported monophyletic clades encompassing all color variants of P. constellatum. Interestingly, no significant differences were observed among the microbial communities of the green and red morphs, suggesting that color variation in this species is a result of intraspecific variation. However, the host's physiological state significantly influenced the microbial community structure. Nonfiltering (resting) colonies hosted higher relative abundances of Kiloniella (Alphaproteobacteria) and Fangia (Gammaproteobacteria), while filtering colonies hosted more Reugeria (Alphaproteobacteria) and Endozoicomonas (Gammaproteobacteria). This study demonstrates that microbial symbiont communities serve as reliable indicators of the taxonomic state of their host and are strongly influenced by the host's feeding condition.

Ascidians 以其颜色变化而闻名,它们寄生于特定物种的微生物共生群落。有些腹足类还能过渡到非过滤(静止)生理状态。最近的研究表明,微生物共生群落可能因宿主的不同生理状态和颜色形态而异。在加勒比海有几种颜色形态的聚合腹足类(Polyclinum constellatum)会周期性地停止过滤活动。为了研究星座聚伞花序的颜色变异是否表明同胞物种的分化,我们对核糖体 18S rRNA 和线粒体细胞色素氧化酶亚单位 I 基因片段进行了测序。此外,我们还对 16S rRNA 基因片段进行了测序,以确定两种常见颜色形态(红色和绿色)的微生物群落的特征。对这两种腹足类基因的系统发育分析结果表明,单系支系得到了很好的支持,涵盖了所有颜色变体的P. constellatum。有趣的是,在绿色和红色形态的微生物群落中没有观察到明显的差异,这表明该物种的颜色变异是种内变异的结果。不过,宿主的生理状态对微生物群落结构有很大影响。非过滤(静止)菌落寄生了相对丰度较高的Kiloniella(低等蛋白菌)和Fangia(高等蛋白菌),而过滤菌落则寄生了较多的Reugeria(低等蛋白菌)和Endozoicomonas(高等蛋白菌)。这项研究表明,微生物共生群落是宿主分类状态的可靠指标,并受到宿主摄食状况的强烈影响。
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引用次数: 0
Complete nucleotide sequence and comparative genomic analysis of microcin B17 plasmid pMccB17 微量元素 B17 质粒 pMccB17 的完整核苷酸序列和比较基因组分析。
IF 3.4 3区 生物学 Q1 Immunology and Microbiology Pub Date : 2024-03-05 DOI: 10.1002/mbo3.1402
Mayokun Ajeigbe, Stephen Childs, Timothy A. Paget, Lewis E. H. Bingle

We present a comprehensive sequence and bioinformatic analysis of the prototypical microcin plasmid, pMccb17, which includes a definitive sequence for the microcin operon, mcb. Microcin B17 (MccB17) is a ribosomally synthesized and posttranslationally modified peptide produced by Escherichia coli. It inhibits bacterial DNA gyrase similarly to quinolone antibiotics. The mcb operon, which consists of seven genes encoding biosynthetic and immunity/export functions, was originally located on the low copy number IncFII plasmid pMccB17 in the Escherichia coli strain LP17. It was later transferred to E. coli K-12 through conjugation. In this study, the plasmid was extracted from the E. coli K-12 strain RYC1000 [pMccB17] and sequenced twice using an Illumina short-read method. The first sequencing was conducted with the host bacterial chromosome, and the plasmid DNA was then purified and sequenced separately. After assembly into a single contig, polymerase chain reaction primers were designed to close the single remaining gap via Sanger sequencing. The resulting complete circular DNA sequence is 69,190 bp long and includes 81 predicted genes. These genes were initially identified by Prokka and subsequently manually reannotated using BLAST. The plasmid was assigned to the F2:A-:B- replicon type with a MOBF12 group conjugation system. A comparison with other IncFII plasmids revealed a large proportion of shared genes, particularly in the conjugative plasmid backbone. However, unlike many contemporary IncFII plasmids, pMccB17 lacks transposable elements and antibiotic resistance genes. In addition to the mcb operon, this plasmid carries 25 genes of unknown function.

我们对原型 microcin 质粒 pMccb17 进行了全面的序列和生物信息学分析,其中包括 microcin 操作子 mcb 的确定序列。microcin B17(MccB17)是由大肠杆菌产生的一种经核糖体合成和翻译后修饰的多肽。它对细菌 DNA 回旋酶的抑制作用与喹诺酮类抗生素类似。mcb 操作子由 7 个编码生物合成和免疫/输出功能的基因组成,最初位于大肠杆菌菌株 LP17 中的低拷贝数 IncFII 质粒 pMccB17 上。它后来通过共轭转入大肠杆菌 K-12。本研究从大肠杆菌 K-12 菌株 RYC1000 [pMccB17] 中提取了该质粒,并使用 Illumina 短读方法进行了两次测序。第一次测序与宿主细菌染色体一起进行,然后对质粒 DNA 进行纯化和单独测序。组装成单个序列后,设计聚合酶链式反应引物,通过桑格测序填补剩余的单个缺口。最后得到的完整环状 DNA 序列长 69,190 bp,包括 81 个预测基因。这些基因最初由 Prokka 鉴定,随后使用 BLAST 进行了人工重新标注。该质粒属于 F2:A-:B- 复制子类型,具有 MOBF12 组连接系统。与其他 IncFII 质粒的比较显示,该质粒有很大一部分共享基因,特别是在共轭质粒骨架中。然而,与许多当代的 IncFII 质粒不同,pMccB17 缺乏转座元件和抗生素抗性基因。除了 mcb 操作子外,该质粒还携带了 25 个功能未知的基因。
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