福建红曲醋中产酸菌株的分离及鉴定

添加含有高浓度乙醇的红曲酒至福建传统红曲醋醋母中富集产酸菌株,采用高浓度乙醇平板,依据溶解圈指标从福建传统红曲醋液体循环工艺样品中分离出7株产酸菌株。综合菌株形态、生理生化实验以及16S r DNA序列测定等信息,确定这7株菌株分类地位为变形菌门(Proteobacteria)α-变形菌纲(Alphaproteobacteria)红螺菌目(Rhodospirillales)醋酸菌科(Acetobacteraceae)葡糖酸醋杆菌属(Gluconacetobacter),其中菌株Y5052、Y5054、Y5072、Y5092鉴定为斯氏葡糖酸醋杆菌(Gluconacetobacter swingsii);菌株Y5032、Y5033、Y5071鉴定为欧洲葡糖酸醋杆菌(Gluconacetobacter europaeus)。测定这些菌株的产酸能力,菌株Y5052产酸量最低,7 d达15 g/L;菌株Y5054产酸能力最强,7 d产酸量达57.0 g/L。     

醋酸菌中CRISPR位点的比较基因组学与进化分析

CRISPR (Clustered regularly interspaced short palindromic repeats)是近几年发现的一种广泛存在于细菌和古菌中,能够应对外源DNA干扰(噬菌体、病毒、质粒等),并提供免疫机制的重复序列结构。CRISPR系统通常由同向重复序列、前导序列、间隔序列和CRISPR相关蛋白组成。本研究以醋酸发酵中常见3个属醋杆菌属(Acetobacter)、葡糖醋杆菌属(Gluconacetobacter)和葡糖杆菌属(Gluconobacter)的48个菌株为研究对象,通过其基因组上CRISPR相关基因序列的生物信息学分析,探索CRISPR位点在醋酸菌中的多态性及其进化模式。结果表明48株醋酸菌中有32株存在CRISPR结构,大部分CRISPR-Cas结构属于type I-E和type I-C类型。除了葡糖杆菌属外,葡糖醋杆菌属和醋杆菌属中的部分菌株含有II类的CRISPR-Cas系统结构(CRISPR-Cas9)。来自不同属菌株的CRISPR结构中重复序列具有较强的保守性,而且部分菌株CRISPR结构中的前导序列具有保守的motif (与基因的转录调控有关)及启动子序列。进化树分析表明cas1适合用于醋酸菌株的分类,而不同菌株间cas1基因的进化与重复序列的保守性相关,预示它们可能受相似的功能选择压力。此外,间隔序列的数量与噬菌体数量及插入序列(Insertion sequence, IS)数量有正相关的趋势,说明醋酸菌在进化过程中可能正不断受新的外源DNA入侵。醋酸菌中CRISPR结构位点的分析,为进一步研究不同醋酸菌株对醋酸胁迫耐受性差异及其基因组稳定性的分子机制奠定了基础。     

山西醋醅中醋酸菌的分离及初步鉴定

目的从山西省某醋厂能正常发酵的醋醅中分离出优势醋酸菌株并加以鉴定。方法经过菌种的增殖培养,采用稀释涂布法分离菌株,得到127株醋酸菌,再经过初筛和复筛,筛选出9株产醋酸优势菌株,对9株优势菌株进行传代培养。结果筛选出在传代培养过程中,产醋酸酸度高且产量稳定的菌株为L4,其产酸量为66.92 g/L,酒精转化率为72.42%。结论根据菌株L4形态观察及生理生化特征初步判定为醋酸菌属醋化醋杆菌奥尔兰亚种。     

CRISPR/dCas9干扰bcsD基因表达调控细菌纤维素结构

木葡糖酸醋杆菌(Gluconacetobacter xylinus)是细菌纤维素的主要生产菌株。在该菌中,BcsD是纤维素合酶的亚基之一,参与细菌纤维素的组装过程。利用CRISPR/dCas9系统调控bcsD基因的表达量,获得了一系列bcsD基因表达量不同的木葡糖酸醋杆菌。通过分析细菌纤维素的结构特征发现,细菌纤维素的结晶度和孔隙率随着木葡糖酸醋杆菌中bcsD表达量的变化而发生改变。其中孔隙率的变化范围在59.95%–84.05%之间,结晶度的变化范围在74.26%–93.75%之间,而细菌纤维素的产量并未因bcsD的表达量变化而发生显著下降。结果表明,bcsD的表达量低于55.34%后,细菌纤维素的孔隙率显著上升,并且细菌纤维素的结晶度与bcsD的表达量呈正相关。最终,通过干扰bcsD基因的表达,实现了一步发酵木葡糖酸醋杆菌获得了产量稳定且结构不同的细菌纤维素。     

氧化葡糖杆菌1.637山梨糖还原酶基因sr功能初步研究

目的:研究山梨糖还原酶基因sr(B932_3022)在氧化葡糖杆菌1.637生长代谢中的作用。方法:构建sr基因同源敲除质粒p GX-srup-Gm-down,PCR扩增得到打靶片段,电击转化导入氧化葡糖杆菌1.637,通过庆大霉素抗性和PCR筛选sr基因重组敲除菌,分别以山梨醇和山梨糖为碳源考察野生菌与敲除菌生长和山梨糖代谢的差异。结果:抗性和PCR验证结果显示敲除了氧化葡糖杆菌1.637的sr基因;与野生菌相比,敲除菌生长出现滞后,以山梨醇为碳源时敲除菌山梨糖积累增多,而以山梨糖为碳源时山梨糖消耗减少。结论:氧化葡糖杆菌1.637的sr基因敲除影响菌株前期生长与山梨糖代谢。     

广东南岭森林土壤中可培养细菌多样性

广东南岭森林土壤中蕴藏着丰富的生物资源,但对其中的可培养细菌种类仍缺乏系统了解。本研究采用贫营养型的R2A培养基和富营养型的TSA培养基对南岭森林土壤中细菌进行了分离,获得细菌408株,分别从属于厚壁菌门、变形菌门、放线菌门和拟杆菌门的35属。其中的优势类群为厚壁菌门,占分离总数量的71%。在属水平,芽胞杆菌及其近缘属为优势类群。除芽胞杆菌外,假单胞菌、伯克霍尔德氏菌、草酸杆菌科Collimonas属和罗丹诺杆菌科Dyella属是分离获得的主要类群。R2A培养基在分离革兰氏阴性的变形菌门菌株方面表现出一定的偏好性,而TSA培养基分离得到的更多为快速生长的芽胞杆菌及其近缘的革兰氏阳性细菌。发现了15属的菌株具有一定的水解酶活性,大多表现出对淀粉和牛奶的水解活性,对有机磷的水解性能优于对无机磷的水解。降解纤维素的菌株则主要集中于芽胞杆菌及其近缘属中。发现了潜在新物种26株,分布于芽胞杆菌、Dyella、类芽孢杆菌等9属中。本研究仅使用了两种营养类型的培养基,进一步借助培养组学技术有望能更加全面反映南岭森林土壤中的可培养微生物多样性。     

大肠杆菌ptsG基因的敲除及其对L-phe生产的影响

L-phe是重要的食品和医药中间体,用大肠杆菌发酵葡萄糖生成phe时,对葡糖糖转运起重要作用的磷酸烯醇丙酮酸糖磷酸转移酶系统(PTS)对phe产量合成有很大影响,在大肠杆菌PTS系统中,葡糖糖主要由ptsG基因编码的葡萄糖特异性转运蛋白酶ⅡCBGlc转运入细胞,通过基因敲除技术获取ptsG缺陷菌株,可以减少菌株对葡糖糖的摄取,减少乙酸的生成,利于菌株的高密度发酵和相关代谢中间物获得。利用Red同源重组技术将大肠杆菌染色体上的ptsG基因进行敲除,得到PTS缺陷菌株MD-ptsG-。该菌株在以葡萄糖为惟一碳源的培养基中摇瓶培养,菌密度为对照菌株的3.5倍,L-phe产量提高12%。     

Knockout of ptsG Gene in Escherichia coli and Its Influence on Production of L-phenylalanine

L-phe 是重要的食品和医药中间体,用大肠杆菌发酵葡萄糖生成 phe 时,对葡糖糖转运起重要作用的磷酸烯醇丙酮酸糖磷酸转移酶系统(PTS)对 phe 产量合成有很大影响,在大肠杆菌 PTS 系统中,葡糖糖主要由 ptsG 基因编码的葡萄糖特异性转运蛋白酶ⅡCBGlc转运入细胞,通过基因敲除技术获取ptsG缺陷菌株,可以减少菌株对葡糖糖的摄取,减少乙酸的生成,利于菌株的高密度发酵和相关代谢中间物获得.利用 Red 同源重组技术将大肠杆菌染色体上的 ptsG 基因进行敲除,得到 PTS 缺陷菌株 MD-ptsG-.该菌株在以葡萄糖为惟一碳源的培养基中摇瓶培养,菌密度为对照菌株的3.5倍,L-phe 产量提高12％.     

利用转座系统在葡糖杆菌中表达山梨糖脱氢酶

目的：利用Mini—Tn5转座系统在葡糖杆菌中表达山梨糖脱氢酶（SDH）。方法：分离得到从山梨醇产糖的快生型小菌Y25K2,利用PCR方法扩增并分析快生型小菌的16SrDNA;构建pUT-mini—Tn5-Tet转座载体,将SDH基因（sdh）插入该载体,利用接合转移,将sdh整合至快生型小菌Y25K2的染色体,通过Western印迹检测SDH的表达。结果：16SrDNA鉴定结果初步表明快生型小菌为葡糖杆菌;构建得到pUT-mini—Tn5-Tet-sdh,将sdh整合至快生型菌Y25K2基因组,并检测到其在快生型小菌Y25K2中的表达。结论：利用Mini—Tn5转座系统在葡糖杆菌中表达了山梨糖脱氢酶。     

一株东方醋酸杆菌分离与其促进果蝇生长发育

【目的】分离与鉴定黑腹果蝇体内醋酸杆菌,并研究其对宿主生长发育的促进作用。【方法】利用醋酸杆菌选择性培养基分离果蝇肠道醋酸杆菌;通过革兰氏染色和16S rRNA基因比对鉴定菌种;肠道定植实验验证共生关系;发育历期和生长速率实验检测其促进果蝇生长作用;免疫荧光染色技术检测肠道细胞增殖;RT-PCR法检测促生长的分子标志物和相关的信号通路。【结果】菌株为东方醋酸杆菌(Acetobacter orientalis),可以持续地定植在果蝇肠道及其培养基中,并且明显促进果蝇的生长。东方醋酸杆菌通过胰岛素信号通路增加肠分裂细胞的数量和促进蜕皮激素的分泌。【结论】东方醋酸杆菌是果蝇的一种共生菌,对果蝇肠道结构和机体发育具有重要的作用。     

Gluconobacter oxydans: its biotechnological applications

Gluconobacter oxydans is a gram-negative bacterium belonging to the family Acetobacteraceae. G. oxydans is an obligate aerobe, having a respiratory type of metabolism using oxygen as the terminal electron acceptor. Gluconobacter strains flourish in sugary niches e.g. ripe grapes, apples, dates, garden soil, baker's soil, honeybees, fruit, cider, beer, wine. Gluconobacter strains are non-pathogenic towards man and other animals but are capable of causing bacterial rot of apples and pears accompanied by various shades of browning. Several soluble and particulate polyol dehydrogenases have been described. The organism brings about the incomplete oxidation of sugars, alcohols and acids. Incomplete oxidation leads to nearly quantitative yields of the oxidation products making G. oxydans important for industrial use. Gluconobacter strains can be used industrially to produce L-sorbose from D-sorbitol; D-gluconic acid, 5-keto- and 2-ketogluconic acids from D-glucose; and dihydroxyacetone from glycerol. It is primarily known as a ketogenic bacterium due to 2,5-diketogluconic acid formation from D-glucose. Extensive fermentation studies have been performed to characterize its direct glucose oxidation, sorbitol oxidation, and glycerol oxidation. The enzymes involved have been purified and characterized, and molecular studies have been performed to understand these processes at the molecular level. Its possible application in biosensor technology has also been worked out. Several workers have explained its basic and applied aspects. In the present paper, its different biotechnological applications, basic biochemistry and molecular biology studies are reviewed.     

Biochemistry and biotechnological applications of <Emphasis Type="Italic">Gluconobacter</Emphasis> strains

The genus Gluconobacter belongs to the group of acetic acid bacteria, which are characterized by their ability to incompletely oxidize a wide range of carbohydrates and alcohols. The corresponding products (aldehydes, ketones and organic acids) are excreted almost completely into the medium. In most cases, the reactions are catalyzed by dehydrogenases connected to the respiratory chain. Since the reactive centers of the enzymes are oriented towards the periplasmic space, transport of substrates and products into, and out of, the cell is not necessary. Thus, rapid accumulation of incompletely oxidized products in the medium is facilitated. These organisms are able to grow in highly concentrated sugar solutions and at low pH-values. High oxidation rates correlate with low biomass production, which makes Gluconobacter strains interesting organisms for industrial applications. Modern fermentation processes, such as the production of L-sorbose (vitamin C synthesis) and 6-amino- L-sorbose (synthesis of the antidiabetic drug miglitol) are carried out with members of this genus. Other important products are dihydroxyacetone, gluconate and ketogluconates. The bacteria belonging to the genus Gluconobacter exhibit extraordinary uniqueness not only in their biochemistry but also in their growth behavior and response to extreme culture conditions. This uniqueness makes them ideal organisms for microbial process development.     

Asaia lannaensis sp. nov., a new acetic acid bacterium in the Alphaproteobacteria

Asaia lannaensis sp. nov. was described for two strains isolated from flowers of the spider lily collected in Chiang Mai, Thailand. The isolates produced acetic acid from ethanol on ethanol/calcium carbonate agar, differing from the type strains of Asaia bogorensis, Asaia siamensis, and Asaia krungthepensis, but did not grow in the presence of 0.35% acetic acid (v/v). The new species is the fourth of the genus Asaia, the family Acetobacteraceae.     

Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences

Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.     

The genus Gluconobacter oxydans: comprehensive overview of biochemistry and biotechnological applications

The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in "historical" production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.     

Identification of acetic acid bacteria isolated from Indonesian sources,especially of isolates classified in the genus Gluconobacter

Sixty-four strains of acetic acid bacteria were isolated from Indonesian sources such as fruits, flowers, and fermented foods by the enrichment culture at pH 3.5. Forty-five strains were routinely identified as Acetobacter strains because of their oxidation of acetate and lactate to carbon dioxide and water and their Q-9 isoprenolog, corresponding to 70% of all the 64 acetic acid bacteria isolated. Eight isolates were identified as Gluconacetobacter strains because of their oxidation of acetate and lactate and their Q-10 isoprenolog, occupying 13% of all the isolates. The remaining 11 isolates, accommodated in the genus Gluconobacter because of no oxidation of acetate and lactate and because of their Q-10 isoprenolog, accounted for 17% of all the isolates. They were divided into two groups based on DNA base compositions. One comprised the seven isolates, which had high G1C contents of DNA ranging from 60.3 to 63.5 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter oxydans at values of 64-94% of DNA relatedness. The other comprised the remaining four isolates, which had low G+C contents of DNA ranging from 57.5 to 57.7 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter frateurii at values of 63-77% of DNA relatedness. The high values of DNA relatedness, 84 to 96%, were obtained between the type strains of Gluconobacter cerinus and Gluconobacter asaii.     

Identification of strains assigned to the genus Gluconobacter Asai 1935 based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer regions

Thirteen reference strains, including the type strains of the type species of the genus Gluconobacter, Gluconobacter oxydans (NBRC 14819T), Gluconobacter cerinus (NBRC 3267T), and Gluconobacter frateurii (IFO 3264T) were examined for their species identification based on the sequence and the restriction analyses of the 16S-23S rDNA internal transcribed spacer (ITS) regions. A phylogenetic tree constructed by the neighbor-joining method represented three clusters corresponding respectively to the three species, G. oxydans, G. cerinus, and G. frateurii. The type strain of Gluconobacter asaii (NBRC 3276T), which is a junior subjective synonym of G. cerinus, was included completely in the G. cerinus cluster. Several restriction endonucleases discriminating the three species from one another were selected by computer analyses: Bsp1286I, MboII, SapI, Bpu10I, EarI, BsiHKAI, and FatI. On digestion of the PCR products with restriction endonucleases Bsp1286I and MboII, all the restriction patterns coincided with those of the type strains of the three species except for strain NBRC 3251. This strain gave a different pattern from the type strain of G. frateurii, when digested with MboII. However, strain 3251 was included phylogenetically in the G. frateurii cluster. All the reference strains were thus identified at the species level by the sequence and the restriction analyses of the 16S-23S rDNA ITS regions.     

Nitrogen Fixation in <Emphasis Type="Italic">Asaia</Emphasis> sp. (Family Acetobacteraceae)

The genus Asaia (family Acetobacteraceae) was first introduced with a single species—Asaia bogorensis and later six more species were described namely A. siamensis, A. krungthepensis, A. lannaensis, A. platycodi, A. prunellae, and A. astilbes. Acetobacteraceae family has been divided into ten genera but, only three of them include nitrogen fixing species: Gluconacetobacter, Acetobacter, and Swaminathania. This article originated from our study primarily aimed to isolate new endosymbiotic nitrogen fixer among Acetobacteraceae during which we have isolated, for the first time in India, four different strains of Asaia sp. from three different sources: Michalia champaca flower, Anopheles mosquito, and ant Tetraponera rufonigra. All the endosymbiotic strains isolated possess the ability to fix nitrogen. Evidence for both nitrogenase activity and the presence of nifH gene in isolated Asaia sp. is presented. Asaia bogorensis (MTCC 4041^T) and A. siamensis (MTCC 4042^T), two of the validated type strains available from the repository, were tested positive for the presence of functional nitrogenase. The nifH gene sequences from these type strains were also confirmed and compared with other nitrogen fixing members of the family Acetobacteraceae. Our result corroborate with the previous reports that Asaia sp. are indeed widely distributed in nature but this is the first time demonstration of their functional nitrogenase activity. This study shows Asaia sp. as fourth genera of nitrogen fixing bacteria in the family Acetobacteraceae.     

Distribution and Solubilization of Particulate Gluconate Dehydrogenase and Particulate 2-Ketogluconate Dehydrogenase in Acetic Acid Bacteria

The distribution of two particulate enzymes, gluconate dehydrogenase (GDH) and 2-ketogluconate dehydrogenase (2KGDH), was investigated with cell free extract through 26 strains of genus Acetobacter and genus Gluconobacter. GDH activity was found in the cell free extracts from all strains of genus Gluconobacter and two species of genus Acetobacter, A. aceti and A. aurantium. High activity of 2KGDH was also found in the pigment-producing strains of genus Gluconobacter.

Best solubilization of particulate enzymes was attained with the highest recovery when 10 mg of Triton X–100 and 30 mg of protein of particulate fractions in 1 ml of 0.01 m phosphate buffer, pH 6.0, are incubated for 9 hr at 5°C with continuous stirring.

By comparison of the total enzyme activity of particulate enzymes with that of NAD(P)-linked enzymes in the cell free extract, it was obvious that the formation of ketogluconates by particulate enzymes was much more predominant, roughly over 100 times higher, as that of NAD(P)-linked enzymes.