等离子体射流灭活液体中铜绿假单胞菌的研究

【目的】测定等离子射流对铜绿假单胞菌(Pseudomonas aeruginosa)的灭活效果,探究低温等离子体射流的杀菌机理。【方法】采用平板计数法测定等离子体射流的杀菌效果,荧光显微镜、透射电镜观察等离子体作用后菌体结构的变化,蛋白浓度测定和SDS-PAGE电泳检测菌液上清液中可溶性蛋白的泄漏量。【结果】等离子体射流处理铜绿假单胞菌菌液5 min,杀灭率可达到99.9%以上。透射电镜观察可见细菌菌体结构发生改变,细胞壁、细胞膜损伤破裂,细胞内容物泄露。进一步对处理铜绿假单胞菌上清液中的蛋白质含量变化进行检测,结果显示随着处理时间的增加,上清液中蛋白质含量持续增加,在2 min时达到最大值。【结论】等离子体射流可以通过破坏细胞结构造成细胞质泄露,使其丧失正常的细胞功能,从而达到快速有效地杀灭铜绿假单胞菌的效果。     

枯草芽胞杆菌活菌制剂促进烧伤创面愈合的实验研究

目的　探讨枯草芽胞杆菌活菌制剂对烧伤创面愈合的影响。方法　通过大鼠深Ⅱ°烧伤模型,使用枯草芽胞杆菌活菌制剂,观察烧伤创面愈合过程中,成纤维细胞增殖周期的变化,以及测定羟脯氨酸（OHP）的含量,同时记录烧伤创面愈合时间,从而评价该制剂对烧伤创面愈合的影响。结果　应用枯草芽胞杆菌活菌制剂可促进成纤维细胞分裂、增殖,胶原含量提高,创面愈合时间明显缩短。结论　枯草芽胞杆菌活菌制剂具有促进烧伤创面愈合的作用。     

苏云金芽胞杆菌cry26Aa和cry28Aa基因共表达可在芽胞外壁内侧形成伴胞晶体

苏云金芽胞杆菌幕虫亚种的伴胞晶体在预芽胞外壁内侧形成,呈现晶体芽胞粘连的现象。根据已发表的cry26Aa1和cry28Aa1基因序列设计引物,从苏云金芽胞杆菌幕虫亚种T02中扩增得到cry26Aa和cry28Aa基因,通过穿梭载体将这两个基因分别和同时转化到苏云金芽胞杆菌无晶体突变株BMB171后,透射电镜下可在芽胞外壁内侧和外侧同时观察到伴胞晶体,而单独表达时可在芽胞外壁外侧观察到伴胞晶体。结果表明,伴胞晶体在芽胞外壁内侧表达不单独依赖于启动子的时空调控,可能还受到晶体蛋白相互作用的影响。     

需氧芽胞杆菌芽胞核心组分的研究现状

芽胞核心作为芽胞的原生质体,实际上是处于休眠状态的细胞,其化学组化比较复杂,核酸,蛋白质,水,无机离子以及有机小分子共同构成芽胞核心特有的“内环境”各种化学组分的含量及存在的形式均与芽胞工能特性尤其抗性密切相关,对它们的深入研究有助于进一步揭示芽胞抗生的有关机制,本文综述对需氧芽胞杆菌核心组分研究。     

苏云金芽胞杆菌cry26Aa和cry28Aa基因共表达可在芽胞外壁内侧形成伴胞晶体

苏云金芽胞杆菌幕虫亚种的伴胞晶体在预芽胞外壁内侧形成,呈现晶体芽胞粘连的现象。根据已发表的cry26Aa1和cry28Aa1基因序列设计引物,从苏云金芽胞杆菌幕虫亚种T02中扩增得到cry26Aa和cry28Aa基因,通过穿梭载体将这两个基因分别和同时转化到苏云金芽胞杆菌无晶体突变株BMB171后,透射电镜下可在芽胞外壁内侧和外侧同时观察到伴胞晶体,而单独表达时可在芽胞外壁外侧观察到伴胞晶体。结果表明,伴胞晶体在芽胞外壁内侧表达不单独依赖于启动子的时空调控,可能还受到晶体蛋白相互作用的影响。     

耐碱芽胞杆菌木聚糖酶的形成条件及特性

通过碱性选择平板分离到耐碱的芽胞杆菌B－141菌株。该菌在碱性（pH10）条件及木聚糖存在下能产生胞外木聚精酶。该酶最适反应温度为60℃,在60℃以下基本稳定;酶反应的最适pH为3～7,但在碱性条件下稳定,在pH10环境处理60min,仍保持约70％的活性。从TLC分析可知,该酶作用于燕麦木聚糖时,主要产物为大于三体的寡糖。     

拮抗Bacillus subtilis的筛选及发酵条件对拮抗能力的影响

筛选了3株对大肠埃希菌具有拮抗活性的枯草芽胞杆菌,并对这3个菌株的拮抗能力最强的Bf菌株的发酵条件进行了初步研究。先将不同枯草芽胞杆菌菌株与株大肠埃希菌进行拮抗试验,根据抑菌率,筛选出对大肠埃希菌拮抗效果较好的枯草芽胞杆菌Bf。然后,在不同pH值、温度以及不同的培养时间下观察记录Bf对大肠埃希菌的拮抗作用。根据实验结果分析得到:筛选到的枯草芽胞杆菌在pH值为7.0,37℃下培养40 h后抑菌效果最好。     

棉苗微生态学研究Ⅰ.正常棉苗内生菌分离和某些生化特性测定

从4个不同棉花品种体内分离到内生菌503株,鉴定了102株,分别属于假单胞杆菌属、黄单胞杆菌属、芽胞杆菌属和欧文氏菌属。分3个不同生长期测定了4个品种的可溶性蛋白含量和超氧化物歧化酶(SOD)、过氧化物酶(POD)活性变化。结果表明,4个品种棉花SOD活性在种期(S₁)、芽期(S₂)和苗期(S₃)呈下降趋势;不同品种、不同生长期的可溶性蛋白含量、POD活性存在极显着差异,品种与生长期还存在显着互作效应。     

鸡肠源芽胞杆菌的分离、鉴定和抗菌活性

从人工饲养的健康三黄肉鸡的盲肠和大肠中分离出10株芽胞杆菌,对其进行菌落形态观察、革兰染色、芽胞染色和生理生化特性鉴定,确定为枯草芽胞杆菌、地衣芽胞杆菌、蜡状芽胞杆菌、巨大芽胞杆菌、球形芽胞杆菌、短小芽胞杆菌、凝结芽胞杆菌。同时测定了它们对动物病原性大肠埃希菌和链球菌的抑菌活性。其中有9株芽胞杆菌对大肠埃希菌有抑制作用,8株芽胞杆菌对链球菌有抑制作用。结果表明芽胞杆菌分离株Y2、Y3、Y4、Y5、Y6、Y7、Y8具有作为益生菌开发的潜力。     

小菌虫肠道可培养细菌的分离鉴定 及产消化酶活性分析

摘要：目的 分离小菌虫肠道可培养细菌,并研究其产消化酶活性,探讨肠道细菌对小菌虫消化食物的影响。方法 采用传统细菌分离培养方法分离小菌虫肠道细菌,利用16S rDNA序列进行细菌分子鉴定;利用筛选培养基鉴别各细菌的产蛋白酶、脂肪酶、淀粉酶和纤维素酶活性。结果 在小菌虫肠道中分离到4种可培养细菌,分别是枯草芽胞杆菌（Bacillus subtilis）、肺炎克雷伯菌（Klebsiella pneumoniae）、Pseudocitrobacter faecalis和芽胞杆菌（Bacillus sp.）。其中,2种芽胞杆菌属细菌有产消化酶活性。枯草芽胞杆菌有产纤维素酶、淀粉酶和蛋白酶活性;芽胞杆菌仅有产蛋白酶活性,但产酶能力低于枯草芽胞杆菌。结论 小菌虫肠道细菌中可培养细菌结构简单,但其中的芽胞杆菌属细菌有产纤维素酶、淀粉酶和蛋白酶能力,说明小菌虫肠道中的2种芽胞杆菌属细菌可能有协助小菌虫进行食物消化的功能。     

Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination

Aims:  To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat.
Methods:  The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined.
Results:  Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50°C.
Conclusions:  Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed.
Significance and Impact of the Study:  The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus .     

Mechanisms of induction of germination of Bacillus subtilis spores by high pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

Structure of Methylosinus trichosporium exospores

Methylosinus trichosporium exospores did not display a well-defined cortex or an exosporium. A thick, electron-dense exospore wall was characteristic of the exospores. Located on the exterior of the exospore wall was a cell wall to which a well-defined capsule was attached. An extensive lamellar intracytoplasmic membrane system characteristic of the kind in vegetative cells of this bacterium was present along the interior periphery of the exospore wall. Upon germination of M. trichosporium exospores, the thick exospore wall gradually disappeared and a germ tube formed. The intracytoplasmic membranes of the exospores extended into the germ tube which did not possess the extensive fibrillar capsule observed on the dormant exospore. Cup-shaped exospores which have an ultrastructure similar to that of mature exospores except that they are invaginated also germinated upon exposure to methane.     

Mechanisms of Induction of Germination of Bacillus subtilis Spores by High Pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

GerN, an antiporter homologue important in germination of Bacillus cereus endospores

A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this B. cereus gene, named gerN, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to L-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 microM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 microM L-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of L-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.     

Summer meeting 2013 – when the sleepers wake: the germination of spores of Bacillus species

Spores of Bacillus species can remain dormant and resistant for years, but can rapidly ‘come back to life’ in germination triggered by agents, such as specific nutrients, and non‐nutrients, such as CaDPA, dodecylamine and hydrostatic pressure. Major events in germination include release of spore core monovalent cations and CaDPA, hydrolysis of the spore cortex peptidoglycan (PG) and expansion of the spore core. This leads to a well‐hydrated spore protoplast in which metabolism and macromolecular synthesis begin. Proteins essential for germination include the GerP proteins that facilitate germinant access to spores' inner layers, germinant receptors (GRs) that recognize and respond to nutrient germinants, GerD important in rapid GR‐dependent germination, SpoVA proteins important in CaDPA release and cortex‐lytic enzymes that degrade cortex PG. Rates of germination of individuals in spore populations are heterogeneous, and methods have been developed recently to simultaneously analyse the germination of multiple individual spores. Spore germination heterogeneity is due primarily to large variations in GR levels among individual spores, with spores that germinate extremely slowly and termed superdormant having very low GR levels. These and other aspects of spore germination will be discussed in this review, and major unanswered questions will also be discussed.     

Effects of cortex peptidoglycan structure and cortex hydrolysis on the kinetics of Ca(2+)-dipicolinic acid release during Bacillus subtilis spore germination

The kinetic parameters of the release of Ca(2+)-dipicolinic acid (CaDPA) during germination of spore populations and multiple individual spores of Bacillus subtilis strains with major alterations in the structure of the spore peptidoglycan (PG) cortex or lacking one or both of the two redundant enzymes involved in cortex hydrolysis (cortex-lytic enzymes [CLEs]) were determined. The lack of the CLE CwlJ greatly slowed CaDPA release with a germinant receptor (GR)-dependent germinant, l-valine, or a non-GR-dependent germinant, dodecylamine. The absence of the cortex-specific PG modification muramic acid-δ-lactam also increased the time needed for full CaDPA release during germination with both types of germinants. In contrast, increased cortex PG cross-linking was associated with faster times for initiation of CaDPA release with both l-valine and dodecylamine but not with faster CaDPA release once this release had been initiated. These data suggest that the precise structure of the spore cortex plays a significant role in determining the timing and the rate of CaDPA release during B. subtilis spore germination and, further, that this effect is independent of effects of GRs.     

Monitoring of Commitment,Blocking, and Continuation of Nutrient Germination of Individual Bacillus subtilis Spores

Short exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores'' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ∼7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (T_release) greater than the time of germinant removal (T_b) increased, while the time T_lag − T_b, where T_lag represents the time when rapid DPA release began, was decreased but rapid DPA release times (ΔT_release = T_release − T_lag) were increased; Factors affecting average T_release values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants.     

Characterization of bacterial spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers

This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single bacterial spores. The characterization consists of the following steps: (i) loading heat-activated dormant spores into a temperature-controlled microscope sample holder containing a germinant solution plus a nucleic acid stain; (ii) capturing a single spore with optical tweezers; (iii) simultaneously measuring phase-contrast images, Raman spectra and fluorescence images of the optically captured spore at 2- to 10-s intervals; and (iv) analyzing the acquired data for the loss of spore refractility, changes in spore-specific molecules (in particular, dipicolinic acid) and uptake of the nucleic acid stain. This information leads to precise correlations between various germination events, and takes 1-2 h to complete. The method can also be adapted to use multi-trap Raman spectroscopy or phase-contrast microscopy of spores adhered on a cover slip to simultaneously obtain germination parameters for multiple individual spores.     

Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.