Protease and peptidase activities in growing and sporulating cells and dormant spores of Bacillus megaterium.

Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.     

Biochemical Studies of Bacterial Sporulation and Germination XV. Fatty Acids in Growth, Sporulation, and Germination of Bacillus megaterium

The levels of fatty acids and their distribution were determined in cultures of Bacillus megaterium during growth, sporulation, and germination. Branched-chain pentadecanoates (br-C15) were the principal fatty acids of log-phase cells. Synthesis of branched-chain tetradecanoates (br-C14) during sporulation increased the relative proportion of these branched fatty acids in sporulating cells and in mature spores. The log-phase distribution was reestablished during outgrowth of the spore. The ratio of br-C15 to br-C14 could be radically altered by addition of their respective amino acid precursors, isoleucine and valine, without seriously affecting the sporulation process. The fatty acid composition of each of the purified phospholipids from log-phase cells was the same, indicating that each phospholipid receives a portion of the fatty acid pool present in the cell at the time of its synthesis. Similarly, the fatty acids of each of the spore phospholipids resembled those of the spore extract. Phospholipids accounted for two-thirds of the fatty acids of the log-phase but only one-third of those of the spore.     

Transcription of the genome of adenovirus type 12. III. Maps of stable RNA from productively infected human cells and abortively infected and transformed hamster cells.

The adenovirus type 12-specific mRNA and the stable nuclear RNA from productively infected KB cells, early postinfection, from abortively infected BHK-21 cells, and from the adenovirus type 12-transformed hamster lines T637 and HA12/7 have been mapped on the genome of adenovirus type 12. The intact separated heavy (H) and light (L) strands of adenovirus type 12 DNA have been used to determine the extent of complementarity of the mRNA or nuclear RNA from different cell lines to each of the strands. More precise map positions have been obtained by the use of the H and L complements of the fragments of adenovirus type 12 DNA which were produced with the EcoRI and BamHI restriction endonucleases. The results of the mapping experiments demonstrate that the mRNA's isolated early from productively and abortively infected and from two lines of transformed cells are derived from the same or similar regions of the adenovirus type 12 genome. The map positions on the adenovirus type 12 genome for the mRNA from the cell lines as indicated correspond to regions located approximately between 0 and 0.1 and 0.74 and 0.88 fractional length units on the L strand and to regions between 0.63 and 0.74 and 0.89 and 1.0 fractional length units on the H strand. The HA12/7 line lacks mRNA complementary to the region between 0.74 and 0.88 fractional length units on the L strand. Similar data are found for the nuclear RNA, except that the regions transcribed are more extensive than those observed in mRNA. The polarity of the H strand has its 3'-end on the right terminus in the EcoRI A fragment, and the L strand has its 3'-end on the left terminus in the EcoRI C fragment. Thus, the H strand is transcribed from right to left (1 = leftward strand); and the L strand is transcribed from left to right (r = rightward strand). The designations H and L refer to the relative heavy and light densities of the two strands in polyuridylic-polyguanylic acid-CsCl density gradients. The EcoRI C-H and D-H complements have been shown to be part of the intact L strand; thus, there is a "reversal in heaviness" on the left terminus of the viral DNA.     

The Dictyostelium discoideum spore germination-specific cellulase is organized into functional domains.

During Dictyostelium discoideum spore germination, degradation of the cellulose-containing spore wall is required to allow the amoeba to emerge. The CelA gene, which is transcribed and expressed exclusively during spore germination, codes for a 705-amino-acid protein that has cellulase activity [endo-(1,4)-beta-D-glucanase]. Amoebae transformed by a vector containing the CelA coding sequence or portions of it transcribed from a heterologous promoter expressed and secreted full-length or suitably truncated proteins during vegetative growth when, under normal conditions, these proteins are not made. The gene constructs divided the CelA protein into three domains: a 461-amino-acid N-terminal region that has significant similarity to those of other cellulases and that has been shown to be the catalytic domain; a contiguous 91-residue repeat containing the motif threonine-glutamic acid-threonine-proline, which is glycosylated; and, joined to the repeat, a C-terminal 153-amino-acid sequence that most probably defines a cellulose-binding domain.     

RNA synthesis during spore germination in Bacillus subtilis.

Summary We have analyzed the RNA synthesized during spore germination in Bacillus subtilis. Early in germination there is little incorporation of [³H]uridine into RNA. A large increase in incorporation into RNA was found at 45–60 min into germination which was in part due to increases in the specific activity of the UTP pool. When corrected for specific activity changes, the instantaneous rate of RNA synthesis showed a seven to tenfold increase between 30 and 45 min of germination. Polyacrylamide gel electrophoresis studies showed that the RNA synthesized during germination appeared very similar to the RNA made during vegetative growth. DNA-RNA hybridization studies indicated that mRNA and rRNA were synthesized throughout germination. Their relative proportions remained constant and were very similar to the composition of RNA synthesized during vegetative growth.In partial fulfillment of the requirements for the doctoral degree by A.S. in the Department of Microbiology at the New York University School of Medicine     

Levels of H+ and other monovalent cations in dormant and germinating spores of Bacillus megaterium.

Previous investigators using the extent of uptake of the weak base methylamine to measure internal pH have shown that the pH in the core region of dormant spores of Bacillus megaterium is 6.3 to 6.5. Elevation of the internal pH of spores by 1.6 U had no significant effect on their degree of dormancy or their heat or ultraviolet light resistance. Surprisingly, the rate of methylamine uptake into dormant spores was slow (time for half-maximal uptake, 2.5 h at 24 degrees C). Most of the methylamine taken up by dormant spores was rapidly (time for half-maximal uptake, less than 3 min) released during spore germination as the internal pH of spores rose to approximately 7.5. This rise in internal spore pH took place before dipicolinic acid release, was not abolished by inhibition of energy metabolism, and during germination at pH 8.0 was accompanied by a decrease in the pH of the germination medium. Also accompanying the rise in internal spore pH during germination was the release of greater than 80% of the spores K+ and Na+. The K+ was subsequently reabsorbed in an energy-dependent process. These data indicate (i) that between pH 6.2 and 7.8 internal spore pH has little effect on dormant spore properties, (ii) that there is a strong permeability barrier in dormant spores to movement of charged molecules and small uncharged molecules, and (iii) that extremely early in spore germination this permeability barrier is breached, allowing rapid release of internal monovalent cations (H+, Na+, and K+).     

月腺大戟根总黄酮对尖孢镰刀菌抑制作用的研究

采用生长速率法和孢子萌发法分别测定月腺大戟根总黄酮对尖孢镰刀菌菌丝生长和分生孢子萌发的抑制率,显微观察总黄酮处理尖孢镰刀菌后菌丝体形态结构的变化,并测定菌丝体相对电导率,菌丝体内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性。结果表明:月腺大戟根总黄酮对尖孢镰刀菌菌丝生长和分生孢子萌发均有显著的抑制作用,抑制率随总黄酮浓度增加而增高,20 mg/L时抑制率达100%。总黄酮处理后的尖孢镰刀菌菌丝较细,分支减少,透明度差,液泡数量增多且形成较大的液泡;菌丝体细胞膜透性增加,SOD、CAT活性呈先上升后下降的趋势。以上实验结果可为植物病害生物防治及开发植物源农药方面提供理论依据和实验技术。     

蜜蜂球囊菌菌丝和孢子中全长转录本的差异表达分析

[目的]本研究旨在探究蜜蜂球囊菌Ascosphaera apis(简称“球囊菌”)菌丝(Aam)和孢子(Aas)中的全长转录本与菌丝生长、孢子萌发以及有性生殖的相关性.[方法]将前期获得的Aam和Aas的Nanopore长读段测序数据中的有效读段与球囊菌参考基因组注释的已知转录本进行序列比对,获取全长转录本与已知转录本...     

天柱山黄山松种内与种间竞争的研究

研究无菌条件下不同浓度砷对蜈蚣草的孢子萌发、假根和原叶体生长发育的影响。结果表明,培养基质中As浓度大于30mg/L时,孢子萌发速度、假根和原叶体的生长发育受到抑制,假根的着生点位置发生改变;而与高浓度As影响不同的是,低浓度As(30mg/L)可促进孢子变绿的速率和初期原叶体(播后8d内)的生长。因此,高浓度As能抑制配子体生长发育并改变假根着生点位置,而低浓度As则可促进配子体早期的生长。     

Dictyostelium discoideum gene family contains a long internal amino acid repeat

Two different cDNA clones denoted pTO270-6 and pTO270-11 represent two mRNAs that are developmentally regulated during spore germination in Dictyostelium discoideum. The respective mRNAs are found only during early germination and are not present in other stages of growth or multicellular development. Four different genomic clones that hybridize to sequences that are common to both of the 270 cDNA clones were isolated from Dictyostelium libraries and sequenced. Two are the genes for the two cDNAs, and the other two represent genes that do not seem to be transcribed. All four genomic sequences possess a very unusual internal feature in the deduced protein sequences composed of a monotonous repeat of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The deduced protein corresponding to the 270-6 gene is very similar to avocado (Persea americana) cellulase. Since cellulose in the spore wall has to be digested during spore germination this suggests that this protein may function as an endo-(1,4)-beta-D-glucanase during germination.     

苏云金芽孢杆菌gerA操纵子在芽孢萌发中的作用

芽孢萌发的营养诱导剂通过与特异的萌发受体结合激活下游的萌发过程,从而使芽孢经过一系列的遗传变化及生化反应恢复营养生长.从苏云金芽孢杆菌(Bacillus thuringiensis)中克隆到一个与枯草芽孢杆菌(Bacillus subtilis)gerA操纵子和蜡状芽孢杆菌(Bacillus cereus)gerR操纵子同源的gerA操纵子.苏云金芽孢杆菌gerA操纵子含有3个开放读码框:gerAA、gerAC和gerAB,该操纵子在产孢起始3个小时后开始转录.gerA的破坏阻断了L-丙氨酸诱导的芽孢萌发并且延迟了肌苷诱导的萌发.在L-丙氨酸诱导芽孢萌发的过程中D-环丝氨酸能够提高芽孢的萌发率.