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From genes to nanotubes: exploring the UV-resistome in the Andean extremophile Exiguobacterium sp. S17.
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-02-18 DOI: 10.1007/s00792-025-01383-5
Fátima Silvina Galván, Daniel Gonzalo Alonso-Reyes, Virginia Helena Albarracín

Exiguobacterium sp. S17, a polyextremophile isolated from a High-Altitude Andean Lake, exhibits a multi-resistance profile against toxic arsenic concentrations, high UV radiation, and elevated salinity. Here, we characterize the mechanisms underlying the UV resistance of Exiguobacterium sp. S17 (UV-resistome) through comparative genomics within the Exiguobacterium genus and describe morphological and ultrastructural changes using Scanning (SEM) and Transmission (TEM) Electron Microscopy.UV-resistome in Exiguobacterium species ranges from 112 to 132 genes. While we anticipated Exiguobacterium sp. S17 to lead the non-HAAL UV-resistome, it ranked eleventh with 113 genes. This larger UV-resistome in Exiguobacterium spp. aligns with their known adaptation to extreme environments. With SEM/TEM analyses we observed the formation of nanotubes (NTs), a novel finding in Exiguobacterium spp., which increased with higher UV-B doses. These NTs, confirmed to be membranous structures through sensitivity studies and imaging, suggest a role in cellular communication and environmental sensing. Genomic evidence supports the presence of essential NT biogenesis genes in Exiguobacterium sp. S17, further elucidating its adaptive capabilities.Our study highlights the complex interplay of genetic and phenotypic adaptations enabling Exiguobacterium sp. S17 to thrive in extreme UV environments. The novel discovery of NTs under UV stress presents a new avenue for understanding bacterial survival strategies in harsh conditions.

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引用次数: 0
DNA methylation confers epigenetic changes in cold-adapted microorganisms in response to cold stress.
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-02-13 DOI: 10.1007/s00792-025-01381-7
Xuying Bu, Xufeng Dou, Zhe Chen, Lan Liu, Yuxia Mei, Min Ren

DNA methylation modification regulates gene expression during temperature stress. The adaptation mechanisms of cold-adapted microorganisms to low temperatures have been explained at the gene and metabolic levels. However, considering the important epigenetic modification in cells, the role of genomic modification in cold-adapted microorganisms remains underexplored. This study aims to discuss the regulatory role of DNA methylation in the cold response of psychrotroph Exiguobacterium undae TRM 85608. Methylome analysis shows that the methylation level of most genes in the bacterium decreases under cold stress. Combined with transcriptome results, the expression of important cold-response genes such as ABC transporter permease and ATP-binding proteins increases, but their methylation levels decrease, which is associated with a reduction of DNA adenine methyltransferase. We believe that the reduction in genomic methylation modification caused by low temperature is a major factor in stabilizing the normal growth of the cell. The bacterium counteracts cold stress by reducing the expression of methylation modification enzymes and weakening the inhibition of cold-response gene modification. These findings provide new insights into how psychrophilic organisms adapt to low temperatures.

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引用次数: 0
Extremophilic and common fungi in acid brines and their halite.
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-02-11 DOI: 10.1007/s00792-025-01382-6
Kathleen C Benison, John E Hallsworth, Polona Zalar, Miha Glavina, Nina Gunde-Cimerman

Studies of microorganisms in extreme Mars-analog environments have generally overlooked fungi. Here, we document fungi in lake waters, slime, and halite of the acid-saline Lakes Magic and Gneiss in Western Australia with pH 1.4-3.5 and 7-32% total dissolved solids (TDS). Both extremotolerant fungi, including ascomycete Parengyodontium torokii, and relatively common fungi (mesophilic), including Penicillium breviocompactum and Trametes pubescens, were present. Our discovery of P. torokii in halite is among the first known fungal examples of such preservation, and we propose that it has the biological traits of a generalist species. Nine strains of the dominant P. torokii fungi were tested for growth on diverse salts. The presence of mesophilic fungal saprotrophs in these lakes, along with extremophilic fungi, algae, bacteria, and archaea, suggests transport of the former into indigenous lake populations. This reveals a distinction between habitability and preservation potential; not all biosignatures in lake waters or their halite represent organisms that were active in situ. Our results suggest that searches for biosignatures in extreme waters and salt minerals on Earth and Mars should include the possibility of fungi. Additionally, interpretations of microbial communities in both modern brines and the rock record should consider the likelihood of mixed indigenous and transported taxa.

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引用次数: 0
ABC-type salt tolerance transporter genes are abundant and mutually shared among the microorganisms of the hypersaline Sambhar Lake.
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-01-28 DOI: 10.1007/s00792-025-01378-2
Srikanta Pal, Raju Biswas, Abhijit Sar, Arijit Misra, Somasri Dam, Bomba Dam

To fish-out novel salt-tolerance genes, metagenomic DNA of moderately saline sediments of India's largest hypersaline Sambhar Lake was cloned in fosmid. Two functionally-picked clones helped the Escherichia coli host to tolerate 0.6 M NaCl. Deep sequencing of their fosmid DNA insert revealed 32-37% of genes to encode transporters, mostly belonging to ABC (ATP-Binding Cassette)-type, but none specific to channel Na+. The complete metagenome sequence of Sambhar Lake brines, and reanalysed data of twelve other hypersaline metagenome sequences, however, have only around 5% transporter genes, suggesting metagenomic DNA fragments being biasedly-cloned during functional screening. Almost half of the ~ 40 Kb inserts in the two clones was shared, and encode several transporters, and some transposase. This advocates that these transporter-loaded DNA lengths are shuttled among microorganisms of hypersaline environments. Interestingly, one clone showed retarded growth with prominent cell disruptions in scanning electron microscopic images, when fosmid copy number was increased or transporters were NaCl-induced. Its cloned insert exclusively has three genes, encoding a structurally functional ATP-binding protein and its efflux component, whose possible overexpression led to membrane crowding and cell rupture. Thus, microorganisms thriving in hypersaline lakes have plentiful ABC transporters that are mutually shared among themselves. These novel salt tolerance genes have future agricultural biotechnological potential.

为了找出新的耐盐基因,将印度最大的高盐度桑巴尔湖(Sambhar Lake)中等盐度沉积物的元基因组DNA克隆到fosmid中。两个功能性克隆帮助大肠杆菌宿主耐受 0.6 M NaCl。对其fosmid DNA插入物的深度测序显示,32-37%的基因编码转运体,其中大部分属于ABC(ATP结合盒)类型,但没有特异性的Na+通道。然而,桑巴尔湖盐水的完整元基因组序列以及其他 12 个低盐元基因组序列的重新分析数据中只有约 5%的转运体基因,这表明元基因组 DNA 片段在功能筛选过程中被有偏差地克隆。两个克隆中约 40 Kb 的插入片段几乎有一半是共享的,编码多个转运体和一些转座酶。这表明,这些带有转运体的 DNA 长度在低盐环境的微生物中穿梭。有趣的是,在扫描电子显微镜图像中,当 fosmid 复制数量增加或转运体被氯化钠诱导时,一个克隆表现出生长迟缓和明显的细胞破坏。其克隆插入物中仅有三个基因,编码结构上功能性的 ATP 结合蛋白及其外流成分,其过度表达可能导致膜拥挤和细胞破裂。因此,在高盐度湖泊中生长的微生物拥有大量 ABC 转运体,它们之间相互共享。这些新型耐盐基因具有未来农业生物技术的潜力。
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引用次数: 0
Halobellus marinus sp. nov., Halobellus ordinarius sp. nov., Halobaculum rarum sp. nov., and Halorarum halobium sp. nov., halophilic archaea isolated from marine solar salt and a saline lake.
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-01-27 DOI: 10.1007/s00792-025-01379-1
Ling-Rui Zhu, Chidiebele Nwankwo, Jing Hou, Heng-Lin Cui

Four halophilic archaeal strains were isolated from sea salt and a saline lake in China. Based on phylogenetic and phylogenomic analyses, the four strains are related to the genera of Halobellus, Halobaculum, and Halorarum within the family Haloferacaceae. The four strains possess genes responsible for carotenoid synthesis, maintenance of a high internal salt concentration, as well as diverse enzymes with biotechnological potential. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values among these four strains and their related species were lower than the established thresholds proposed for species demarcation. Strains DFY28T, ZY16T, QDC11T, and XH14T were distinguished from related species based on a variety of phenotypic characteristics. The major polar lipids of these four strains were similar to those of respective relatives within the genera Halobellus, Halobaculum, and Halorarum. The phenotypic, phylogenetic, and genome-based analyses suggest that strains DFY28T (= CGMCC 1.17470T = JCM 34310T), ZY16T (= CGMCC 1.17476T = JCM 34311T), QDC11T (= MCCC 4K00127T = KCTC 4308T), and XH14T (= CGMCC 1.17028T = JCM 34145T) represent four novel species of the genera Halobellus, Halobaculum and Halorarum, for which the names Halobellus marinus sp. nov., Halobellus ordinarius sp. nov., Halobaculum rarum sp. nov., and Halorarum halobium sp. nov. are proposed.

{"title":"Halobellus marinus sp. nov., Halobellus ordinarius sp. nov., Halobaculum rarum sp. nov., and Halorarum halobium sp. nov., halophilic archaea isolated from marine solar salt and a saline lake.","authors":"Ling-Rui Zhu, Chidiebele Nwankwo, Jing Hou, Heng-Lin Cui","doi":"10.1007/s00792-025-01379-1","DOIUrl":"https://doi.org/10.1007/s00792-025-01379-1","url":null,"abstract":"<p><p>Four halophilic archaeal strains were isolated from sea salt and a saline lake in China. Based on phylogenetic and phylogenomic analyses, the four strains are related to the genera of Halobellus, Halobaculum, and Halorarum within the family Haloferacaceae. The four strains possess genes responsible for carotenoid synthesis, maintenance of a high internal salt concentration, as well as diverse enzymes with biotechnological potential. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values among these four strains and their related species were lower than the established thresholds proposed for species demarcation. Strains DFY28<sup>T</sup>, ZY16<sup>T</sup>, QDC11<sup>T</sup>, and XH14<sup>T</sup> were distinguished from related species based on a variety of phenotypic characteristics. The major polar lipids of these four strains were similar to those of respective relatives within the genera Halobellus, Halobaculum, and Halorarum. The phenotypic, phylogenetic, and genome-based analyses suggest that strains DFY28<sup>T</sup> (= CGMCC 1.17470<sup>T</sup> = JCM 34310<sup>T</sup>), ZY16<sup>T</sup> (= CGMCC 1.17476<sup>T</sup> = JCM 34311<sup>T</sup>), QDC11<sup>T</sup> (= MCCC 4K00127<sup>T</sup> = KCTC 4308<sup>T</sup>), and XH14<sup>T</sup> (= CGMCC 1.17028<sup>T</sup> = JCM 34145<sup>T</sup>) represent four novel species of the genera Halobellus, Halobaculum and Halorarum, for which the names Halobellus marinus sp. nov., Halobellus ordinarius sp. nov., Halobaculum rarum sp. nov., and Halorarum halobium sp. nov. are proposed.</p>","PeriodicalId":12302,"journal":{"name":"Extremophiles","volume":"29 1","pages":"13"},"PeriodicalIF":2.6,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143046121","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Genomic highlights of the phylogenetically unique halophilic purple nonsulfur bacterium, Rhodothalassium salexigens.
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2025-01-25 DOI: 10.1007/s00792-025-01380-8
Michael T Madigan, Kelly S Bender, Mary N Parenteau, Yukihiro Kimura, Zheng-Yu Wang-Otomo, W Matthew Sattley

Rhodothalassium (Rts.) salexigens is a halophilic purple nonsulfur bacterium and the sole species in the genus Rhodothalassium, which is itself the sole genus in the family Rhodothalassiaceae and sole family in the order Rhodothalassiales (class Alphaproteobacteria). The genome of this phylogenetically unique phototroph comprises 3.35 Mb and is highly chimeric, with nearly half of its genes originating from families other than the Rhodothalassiaceae, many of which lack phototrophic species. Photosynthesis genes in Rts. salexigens are not arranged in a typical photosynthesis gene cluster but are scattered across the genome, suggesting an origin from horizontal transfers. Despite an encoded RuBisCO, autotrophy has not been observed in Rts. salexigens, and enzymes that oxidize common inorganic electron donors are not encoded. Phospholipid biosynthesis in Rts. salexigens is restricted, and phosphoglycerolipids are the only phospholipids present in its intracytoplasmic membranes. Rts. salexigens fixes nitrogen using a Mo-containing nitrogenase and uses ammonia despite previous results that indicated it was a glutamate auxotroph. Glycine betaine is the sole osmolyte in Rts. salexigens, and enzymes are encoded that facilitate both its uptake and its biosynthesis from glycine. The genomic data also support chemotactic swimming motility, growth over a range of salinities, and the production of membrane-strengthening hopanoids.

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引用次数: 0
Mechanism of decolorization and degradation of direct brown D3G by a halo-thermophilic consortium. 直接棕色D3G脱色降解机理研究。
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-12-23 DOI: 10.1007/s00792-024-01376-w
Wenying Wang, Zuotao Zhang, Meichen Sun, Chenlai Li, Mengdi Yan, Chongyang Wang

Azo dye wastewater has garnered significant attention from researchers because of its association with high-temperature, high-salt, and high-alkali conditions. In this study, consortium ZZ efficiently decolorized brown D3G under halophilic and thermophilic conditions. he results indicated that consortium ZZ, which was mainly dominated by Marinobacter, Bacillus, and Halomonas, was achieved decolorization rates ranging from 1 to 10% at temperatures between 40 °C and 50 °C, while maintaining a pH range of 7 to 10 for direct brown D3G degradation. Through the comprehensive utilization of UV-vis spectral analysis, Fourier transform infrared (FTIR), gas chromatography mass spectrometric (GC-MS) techniques, as well as metagenomic analysis, the decolorization and degradation pathway of direct brown by consortium ZZ was proposed. The azo dye reductase, lignin peroxidase, and laccase were also highly expressed in the decolorization process. Additionally, phytotoxicity tests using seeds of Cucumis sativus and Oryza sativa revealed that the intermediates generated showed no significant toxicity compared with distilled water. This investigation elucidated the pivotal contribution of consortium ZZ to azo dye degradation and provided novel theoretical insights along with practical guidance for azo dye treatment at halo-thermophilic conditions.

偶氮染料废水因其与高温、高盐、高碱条件有关而引起了研究人员的极大关注。在本研究中,财团ZZ在亲盐和亲热条件下对褐色D3G进行了高效脱色。结果表明,以Marinobacter、芽孢杆菌和Halomonas为主的菌群ZZ在40 ~ 50℃的温度下脱色率为1% ~ 10%,pH值为7 ~ 10,可直接降解褐色D3G。综合利用紫外-可见光谱分析、傅里叶变换红外(FTIR)、气相色谱-质谱(GC-MS)等技术以及宏基因组分析,提出了财团ZZ对直接棕的脱色降解途径。偶氮染料还原酶、木质素过氧化物酶和漆酶在脱色过程中也有高表达。此外,利用黄瓜和水稻种子进行的植物毒性试验表明,与蒸馏水相比,产生的中间体没有明显的毒性。该研究阐明了络合物ZZ对偶氮染料降解的关键作用,为在晕嗜热条件下处理偶氮染料提供了新的理论见解和实践指导。
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引用次数: 0
Insights into the dynamics and evolution of Rummeliibacillus stabekisii prophages in extreme environments: from Antarctic soil to spacecraft floors. 从南极土壤到航天器地板的极端环境中,对稳定瘤杆菌前体的动力学和进化的见解。
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-12-21 DOI: 10.1007/s00792-024-01377-9
Caroline Martiniuc, Igor Taveira, Fernanda Abreu, Anderson S Cabral, Rodolfo Paranhos, Lucy Seldin, Diogo Jurelevicius

Since prophages can play a multifaceted role in bacterial evolution, this study aims to characterize the virome of Rummeliibacillus stabekisii, a bacterium isolated from different environments, including Antarctic soil and NASA spacecraft floors. From the analyses, it was found that the Antarctic strain, PP9, had the largest number of prophages, including intact ones, indicating potential benefits for survival in adverse conditions. In contrast, other strains harbored predominantly degenerate prophages, suggesting a dynamic process of gene gain and loss during evolution. Furthermore, strain PP9 exhibited polylysogeny, a strategy capable of increasing its competitive advantage by providing a broader spectrum of defensive mechanisms. In addition, evidence demonstrates that prophage regions in PP9 act as hotspots for recombination events, favoring the insertion of different phages and possible antimicrobial resistance genes. Finally, lytic cycle induction experiments revealed at least two intact prophages active in PP9. In this way, understanding the interaction between viruses and bacteria can provide valuable information about microbial evolution and adaptation in extreme environments, such as Antarctica.

由于噬菌体可以在细菌进化中发挥多方面的作用,本研究旨在表征stabekisii rummelibacillus的病毒体,这是一种从不同环境中分离出来的细菌,包括南极土壤和NASA航天器地板。从分析中发现,南极菌株PP9具有最多的噬菌体,包括完整的噬菌体,这表明在不利条件下生存的潜在好处。相比之下,其他菌株主要携带退化的前噬菌体,这表明在进化过程中基因的获得和失去是一个动态的过程。此外,菌株PP9表现出多溶原性,这一策略能够通过提供更广泛的防御机制来增加其竞争优势。此外,有证据表明,PP9中的噬菌体区域是重组事件的热点,有利于不同噬菌体和可能的抗微生物耐药基因的插入。最后,裂解循环诱导实验显示至少有两个完整的原噬菌体在PP9中具有活性。通过这种方式,了解病毒和细菌之间的相互作用可以提供有关微生物在极端环境(如南极洲)中的进化和适应的宝贵信息。
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引用次数: 0
Unraveling the effects of polyhydroxyalkanoates accumulation in Pseudomonas extremaustralis growth and survival under different pH conditions. 不同pH条件下聚羟基烷酸盐积累对极端假单胞菌生长和存活的影响。
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-12-19 DOI: 10.1007/s00792-024-01375-x
María Gabriela Brito, Nancy I López, Laura J Raiger Iustman

Polyhydroxyalkanoates (PHAs) are intracellular polymers that enhance bacterial fitness against various environmental stressors. Pseudomonas extremaustralis 14-3b is an Antarctic bacterium capable of accumulating, short-chain-length PHAs (sclPHAs), composed of C3-C5 monomers, as well as medium-chain-length PHAs (mclPHAs) containing ≥ C6 monomers. Since pH changes are pivotal in bacterial physiology, influencing microbial growth and metabolic processes, we propose that accumulated PHA increases P. extremaustralis fitness to cope with pH changes. To test this, we analyzed the production of sclPHA and mclPHA at different pH levels and its effect on bacterial survival against pH stress. P. extremaustralis was able to grow and accumulate PHA when the culture media pH ranged from 6.0 to 9.5, showing a marked loss of viability outside this range. Additionally, based on the analysis of different PHA-deficient mutants, we found that when exposed to both acidic and alkaline conditions, sclPHA and mclPHA conferred different protection against pH stress, with sclPHA making the main contribution. These results highlight the importance of PHA in supporting survival in pH-stressful environments.

聚羟基烷酸酯(PHAs)是细胞内的聚合物,增强细菌适应各种环境压力。极南假单胞菌14-3b是一种南极细菌,能够积累由C3-C5单体组成的短链长pha (sclpha),以及含有≥C6单体的中链长pha (mclpha)。由于pH值的变化在细菌生理中起着关键作用,影响着微生物的生长和代谢过程,我们认为PHA的积累增加了极南拟南草适应pH值变化的能力。为了验证这一点,我们分析了不同pH水平下sclPHA和mclPHA的产生及其对细菌在pH胁迫下存活的影响。当培养基pH在6.0 ~ 9.5范围内时,极南假单胞菌能够生长和积累PHA,超出此范围后,其生存力明显丧失。此外,基于对不同pha缺陷突变体的分析,我们发现当暴露于酸性和碱性条件下时,sclPHA和mclPHA对pH胁迫具有不同的保护作用,其中sclPHA起主要作用。这些结果强调了PHA在ph应激环境中支持存活的重要性。
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引用次数: 0
Variations on a theme: non-canonical DUF3494 ice-binding proteins. 一个主题的变体:非规范DUF3494冰结合蛋白。
IF 2.6 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-12-13 DOI: 10.1007/s00792-024-01374-y
James A Raymond

Among the many ice-binding proteins (IBPs) found in microorganisms (bacteria, archaea, fungi and algae), the canonical DUF3494 beta-barrel type is the most common. Until now, little variation has been found in this structure: an initial coil leads into an alpha helix that directs the following coils into a reverse stack, with the final coil ending up next to the initial coil. Here, I show that there exist many bacterial proteins whose AlphaFold-predicted structures deviate from the DUF3494 structure so that they are not recognized as belonging to an existing DUF or Pfam family. In these non-canonical DUF3494 (ncDUF3494) proteins, the number of coils in the alpha helix is highly variable, often being as high as 14. The putative ice-binding sides of each of 13 proteins modeled have a well-aligned row of hydrophilic residues, with spacings that are close to the repeat distance on the ice a-axis. A recombinant protein made for one of the proteins showed that it had ice-binding activity, even in the µg/ml range. The ncDUF3494 proteins appear to be found only in bacteria, the great majority of which live in icy habitats. C-terminal PEP-Cterm motifs, which are rare in DUF3494s, are present in most of the ncDUF3494s, possibly indicating a secretory function. The relatively narrow distribution of ncDUF3494 proteins suggests that they are a later development in DUF3494 evolution.

在微生物(细菌、古菌、真菌和藻类)中发现的众多冰结合蛋白(IBPs)中,典型的 DUF3494 β-桶型是最常见的。到目前为止,这种结构几乎没有发现任何变化:初始线圈通向α螺旋,α螺旋引导后面的线圈形成反向堆叠,最后一个线圈在初始线圈旁边结束。在这里,我展示了许多细菌蛋白质,它们的 AlphaFold 预测结构偏离了 DUF3494 结构,因此无法识别它们属于现有的 DUF 或 Pfam 家族。在这些非典型 DUF3494(ncDUF3494)蛋白中,α螺旋中的线圈数量变化很大,通常高达 14 个。建模的 13 个蛋白质中,每个蛋白质的假定冰结合面都有一排排列整齐的亲水残基,其间距接近冰 a 轴上的重复距离。其中一种蛋白质的重组蛋白显示,即使在微克/毫升的范围内,它也具有冰结合活性。ncDUF3494 蛋白似乎只存在于细菌中,而绝大多数细菌都生活在冰雪环境中。大多数 ncDUF3494 蛋白的 C 端 PEP-Cterm 结构在 DUF3494 蛋白中很少见,这可能表明它们具有分泌功能。ncDUF3494蛋白的分布范围相对较窄,这表明它们是DUF3494进化的后期发展。
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引用次数: 0
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Extremophiles
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