Characterization of IsaA and SceD, two putative lytic transglycosylases of Staphylococcus aureus

Bacterial cell wall peptidoglycan is a dynamic structure requiring hydrolysis to allow cell wall growth and division. Staphylococcus aureus has many known and putative peptidoglycan hydrolases, including two likely lytic transglycosylases. These two proteins, IsaA and SceD, were both found to have autolytic activity. Regulatory studies showed that the isaA and sceD genes are partially mutually compensatory and that the production of SceD is upregulated in an isaA mutant. The expression of sceD is also greatly upregulated by the presence of NaCl. Several regulators of isaA and sceD expression were identified. Inactivation of sceD resulted in impaired cell separation, as shown by light microscopy, and "clumping" of bacterial cultures. An isaA sceD mutant is attenuated for virulence, while SceD is essential for nasal colonization in cotton rats, thus demonstrating the importance of cell wall dynamics in host-pathogen interactions.     

Foreign gene expression (beta-galactosidase) during the cell cycle phases in recombinant CHO cells

Recombinant mammalian cultures for heterologous gene expression typically involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation of intracellular foreign protein in single cells during the cell cycle of Chinese hamster ovary (CHO) cells transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the lacZ gene for bacterial beta-galactosidase (a nonsecreated protein). The lacZ gene was under the control of the constitutive cytomegalovirus promoter. These normally attachment-grown cells were adapted to suspension culture in 10(-7) M methotrexate, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in the exponential growth phase, early plateau phase, and inhibited traverse of the cell cycle during exponential growth. The results showed that the beta-galactosidase production rate is higher in the S phase than that in the G1 or G2/M phases. Also, when cell cycle progression was stopped at the S phase by addition of aphidicolin, beta-galactosidase content in single cells was higher than that in exponential phase or plateau phase cells and increased with increasing culture time. Although the cells did not continue to divide after aphidicolin addition, the production of beta-galactosidase per unit volume of culture was very similar to that in normal exponential growth. (c) 1993 John Wiley & Sons, Inc.     

铜绿假单胞菌噬菌体K5中DNA聚合酶等基因在宿主中的表达

本研究分析了铜绿假单胞菌噬菌体K5基因在宿主中的表达及其影响因素. 通过测定融合报告基因dnaP-lacZ、capP-lacZ、bapP-lacZ和rdr-lacZ编码的β 半乳糖苷酶活力,分析了噬菌体K5相关基因的表达水平,发现噬菌体K5的不同基因在宿主细胞内表达水平存在较大差异,其中噬菌体K5的DNA聚合酶基因dnaP的表达水平最高,而主要衣壳蛋白基因capP的表达水平最低. 加入噬菌体后,除二磷酸核糖核苷酸还原酶基因rnr外,其它基因的表达水平均有明显提高,说明噬菌体自身因子能够调控噬菌体部分基因在宿主细胞中的表达. 进一步分析显示,噬菌体基因在对数生长前期细胞中的表达水平显著高于平衡期. 同时,噬菌体感染对数生长前期的宿主菌,其释放量为12.8 PFU/感染中心,是平衡期释放量的9.2倍. 噬菌体以对数生长期宿主为指示菌时噬菌体的滴度为4.7×108 PFU/mL,而以平衡期宿主菌为指示菌噬菌体K5滴度仅能达到2.5×104 PFU/mL,噬菌体K5的裂解能力显著降低. 这些结果对研究噬菌体与宿主细胞的相互作用机制具有重要作用.     

Chromatin‐level regulation of the fragmented dothistromin gene cluster in the forest pathogen Dothistroma septosporum

Genes required for fungal secondary metabolite production are usually clustered, co‐regulated and expressed in stationary growth phase. Chromatin modification has an important role in co‐regulation of secondary metabolite genes. The virulence factor dothistromin, a relative of aflatoxin, provided a unique opportunity to study chromatin level regulation in a highly fragmented gene cluster that is switched on during early exponential growth phase. We analysed three histone modification marks by ChIP‐qPCR and gene deletion in the pine pathogen Dothistroma septosporum to determine their effects on dothistromin gene expression across a time course and at different loci of the dispersed gene cluster. Changes in gene expression and dothistromin production were associated with changes in histone marks, with higher acetylation (H3K9ac) and lower methylation (H3K9me3, H3K27me3) during early exponential phase at the onset of dothistromin production. But while H3K27me3 directly influenced dothistromin genes dispersed across chromosome 12, effects of H3K9 acetylation and methylation were orchestrated mainly through a centrally located pathway regulator gene DsAflR. These results revealed that secondary metabolite production can be controlled at the chromatin‐level despite the genes being dispersed. They also suggest that patterns of chromatin modification are important in adaptation of a virulence factor for a specific role in planta.     

Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases

Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance.     

Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus

The YycG/YycF essential two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low-G+C gram-positive bacteria, including several pathogens such as Staphylococcus aureus. By studying growth of S. aureus cells where the yyc operon is controlled by an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter, we have shown that this system is essential in S. aureus during growth at 37 degrees C and that starvation for the YycG/YycF regulatory system leads to cell death. During a previous study of the YycG/YycF TCS of B. subtilis, we defined a potential YycF consensus recognition sequence, consisting of two hexanucleotide direct repeats, separated by five nucleotides [5'-TGT(A/T)A(A/T/C)-N(5)-TGT(A/T)A(A/T/C)-3']. A detailed DNA motif analysis of the S. aureus genome indicates that there are potentially 12 genes preceded by this sequence, 5 of which are involved in virulence. An in vitro approach was undertaken to determine which of these genes are controlled by YycF. The YycG and YycF proteins of S. aureus were overproduced in Escherichia coli and purified. Autophosphorylation of the YycG kinase and phosphotransfer to YycF were shown in vitro. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the promoter region of the ssaA gene, encoding a major antigen and previously suggested to be controlled by YycF. YycF was also shown to bind specifically to the promoter regions of two genes, encoding the IsaA antigen and the LytM peptidoglycan hydrolase, in agreement with the proposed role of this system in controlling virulence and cell wall metabolism.     

Analysis of growth phase regulated KatA and CatE and their physiological roles in determining hydrogen peroxide resistance in Agrobacterium tumefaciens

Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE. In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined. katA and catE were found to be independently regulated in a growth phase dependent manner. KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase. Only small increases in H2O2 resistance levels were detected as cells entering stationary phase. The katA mutant was more sensitive to H2O2 than the parental strain during both exponential and stationary phase. Inactivation of catE alone did not significantly change the level of H2O2 resistance. However, the katA catE double mutant was more sensitive to H2O2 during both exponential and stationary phase than either of the single catalase mutants. The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A. tumefaciens from H2O2 toxicity during all phases of growth. Catalase-peroxidase activity (KatA) was required for full H2O2 resistance. The expression patterns of the two catalases in A. tumefaciens reflect their physiological roles in the protection against H2O2 toxicity, which are different from other bacteria.     

Overproduction, purification and characterization of the HPB12-L24 ribosomal protein of Bacillus subtilis

Abstract Polyhydroxyalkanoate (PHA) accumulation and the morphology of PHA inclusion bodies were examined in Bacillus megaterium , strain 11561. Our results show a pattern of PHA degradation and synthesis, and of inclusion body growth and proliferation not previously reported. Degradation of PHA in the lag phase was followed by synthesis of PHA at an accelerating rate during exponential growth. PHA accumulation reached a maximum rate at late exponential/early stationary phase and the rate declined to a lower steady state in the stationary phase. During exponential and early stationary phase growth, PHA had a faster doubling rate than that of total cell biomass (w/w). Results of the morphology studies suggest that PHA inclusion bodies proliferated by budding and reached maximum size by early stationary phase growth. This pattern was observed in minimal and in rich media.     

Expression and characterization of alkaline phosphatases during differentiation of human pancreatic cancer (Capan-1) cells in culture.

Human pancreatic cells of the Capan-1 cell line differentiate in culture. During the exponential growth phase, the cells are undifferentiated, only becoming differentiated during the stationary phase. The formation of domes in this phase is related to the exchange of water and electrolytes. The present study was designed to characterize the localization and expression of alkaline phosphatases (AP) in Capan-1 cells during growth in culture. Biochemical, cytoenzymatic and immunocytochemical methods were employed combined with light and electron microscopic examination. AP essentially of the placental type were expressed progressively during the exponential growth phase, and were seen to be distributed over the surface of the Capan-1 cells. In the stationary phase, the AP became localized on the surface of microvilli. The precipitates of the enzyme reaction highlighted regular four-bodied structures. Biochemical assays showed a progressive increase in activity of this enzyme in cells during both the exponential and stationary growth phases. However, in the stationary phase between days 7 and 8, there was a fall in enzyme activity, with a corresponding increase in this activity in the culture medium. Cytological examination indicated that this fall could be accounted for by loss of AP-positive membranes by vesiculization of apical microvilli and release of microvesicles into the culture medium. Immunoblots showed that Capan-1 cells expressed two types of AP, a placental type (70 kDa) and to a lesser extent a liver type (80 kDa). Expression of the placental type was attributed to a neoplastic derepression of the coding gene, while the liver type was assumed to be a normal gene expression of human duct cells. The placental type AP might thus serve as a marker of transformation, and the liver type as a marker of differentiation.     

Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes

Abstract The regulation of the spvR promoter from the Salmonella dublin virulence plasmid was monitored using proter-reporter gene fusion constructs. Activity was dependent upon the presence of the spv region and was affected by the number of copies of the spv region present with the cell. Activity remained constant throughout exponential growth, and increased rapidly with the onset of stationary phase, under both aerobic and anaerobic conditions. Additionally, the level of spvR expression was controlled by the availability of iron, activity being greatest under low iron conditions in stationary phase. The spvA gene product negatively regulated spvR expression in a dose-dependent manner, indicating that SpvA provides a negative feedback mechanism for this operon.     

Fungal melanin inhibitor and related compounds from Penicillium decumbens

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.