Analysis of dofA,a fruA-dependent developmental gene,and its homologue,dofB, in Myxococcus xanthus

The developmentally regulated gene dofA, identified from pulse-labeling experiments by two-dimensional gel electrophoresis, and its homologue, dofB, were cloned and characterized in Myxococcus xanthus. Deletion of dofA and dofB did not affect the vegetative growth and development of M. xanthus. dofA was specifically expressed during development, while dofB expression was observed during vegetative growth and development. The dofA-lacZ fusion was introduced into a fruA mutant and A, B, C, D, and E extracellular signal mutants. The pattern of dofA expression in the C signal mutant was similar to that of the wild-type strain, while dofA expression was not detected in the fruA mutant. These results are consistent with those of the pulse-labeling experiments. dofA expression was reduced in A and E signal mutants, whereas dofA expression was delayed in B and D signal mutants. The patterns of expression of the dofA gene in the fruA mutant and the five signal mutants are strikingly similar to that of the tps gene, which encodes protein S, a major component of the outer surface of the myxospore; this result suggests that the dofA and tps genes are similarly regulated. The involvement of a highly GC-rich inverted repeat sequence (underlined), CGGCCCCCGATTCGTCGGGGGCCG, in developmentally regulated dofA expression is suggested.     

粘细菌转录因子FruA的表达、纯化及调控作用

ＦｒｕＡ是粘细菌（Ｍｙｘｏｃｏｃｃｕｓ ｘａｎｔｈｕｓ）发育所必需的转录因子,影响着一系列发育特异性基因的表达,在大肠杆菌中表达了带组氨酸标记的ＦｒｕＡ,并用镍离子层析进行快速分离纯化。凝胶阻滞试验提示ＦｒｕＡ对靶基因的调控可能需要其它因子的参与。     

黄色黏细菌转录因子FruA对自身基因的表达不具自调节作用

 粘细菌是研究多细胞结构形态发生机制的良好模型.FruA是粘细菌发育所必需的一种 关键性转录因子, 调节一系列发育相关基因的表达,本文研究FruA对自身基因是否存在反馈调节从而导致发育后期fruA表达水平的下调.以野生型粘细菌模式菌株DK1622为基础构建fruA基因敲除突变株DK1622ΔfruA,再将fruA-lacZ转录融合载体pMF1A整合入fruA突变株染色体attB, 获得重组菌株DK1622ΔfruA/pMF1A,通过检测β-半乳糖苷酶活性来确认FruA对自身基因的表达水平是否有影响. 结果表明fruA调控序列完整的fruA-lacZ转录融合体β-半乳糖苷酶活性在DK1622/pMF1A和DK1622ΔfruA/pMF1A之间无明显差异, 即fruA表达产物作为一种转录因子对自身基因的转录没有调节作用,黏细菌发育后期fruA表达水平的下降存在其它调节机制.     

粘细菌转录因子FruA与结合蛋白FruB协同参与发育基因的调控

粘细菌中一系列发育相关基因受到转录因子ＦｒｕＡ的调节。用亲和层析法从粘细菌分离出另一种与ＦｒｕＡ结合的蛋白因子ＦｒｕＢ。实验表明,ＦｒｕＢ可以被粘细菌细胞膜上的蛋白激酶磷酸化,磷酸化后的ＦｒｕＢ与ＦｒｕＡ形成复合物,此复合物通过与靶基因顺式元件的结合来调节基因的表达。有助于深入理解ＦｒｕＡ对发育相关基因的调节作用机制。     

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.     

Mutational analysis of the fruA promoter region demonstrates that C-Box and 5-base-pair elements are important for expression of an essential developmental gene of Myxococcus xanthus

Myxococcus xanthus uses extracellular signals during development to regulate gene expression. C-signaling regulates the expression of many genes induced after 6 h into development. FruA is a protein that is necessary for cells to respond to C-signaling, but expression of the fruA gene does not depend on C-signaling. Yet the fruA promoter region has a C box and a 5-bp element, similar to the promoter regions of several C-signal-dependent genes, where these sequences are crucial. Here, we show that the C box and 5-bp elements are important for expression of fruA, demonstrating for the first time that these sequences play a role in the expression of a gene that does not depend on C-signaling and is required for M. xanthus development.     

Differential localization of the Streptococcus mutans GS-5 fructan hydrolase enzyme, FruA

Abstract Streptococcus mutans GS-5 synthesizes an exo-β-d-fructosidase, FruA, capable of degrading levans, inulins, sucrose and raffinose, with the greatest activity on levans. A previous analysis of the deduced amino acid sequence of the FruA protein revealed the presence of a C-terminus with an LPXTGX membrane sorting sequence and membrane spanning domain, characteristic of many Gram-positive cocci surface proteins. Here it is demonstrated that FruA, which had been previously shown to exist almost exclusively as an extracellular enzyme, can be detected in significant proportions at the surface of S. mutans cells. Moreover, growth of S. mutans GS-5 at steady state in continuous culture at pH values of 7.0, 6.0, or 5.0 revealed that the amount of cell-associated enzyme increased with decreasing pH values, such that roughly 50% of the total fructanase activity of pH 5.0-grown organisms was cell-associated. This result was confirmed using anti-recombinant-FruA antisera in Western blotting of culture supernate and cell-associated enzyme preparations from chemostat-grown cells. Incubation of S. mutans at pH values of 5.0, 6.0 or 7.0 in buffered media yielded results similar to those observed in the chemostat experiments. The release of FruA from S. mutans was also shown to be inhibitable by copper, which is known to interfere with the release of the surface adhesin, P1, from intact cells and protoplasts of S. mutans . These data provide evidence for a unique post-translational mechanism for the regulation of the catabolism of polysaccharides by bacteria. The control of degradation of plaque fructans by modulation of the release of the fructanase enzyme from S. mutans may play a critical role in the temporal and spatial separation of the synthesis and degradation of dental plaque fructans.