An effective sporulation of Myxococcus xanthus requires glycogen consumption via Pkn4-activated 6-phosphofructokinase

6-Phosphofructokinase (PFK) is a key enzyme for glycolysis in both prokaryotes and eukaryotes. Previously, it was found that the activity of Myxococcus xanthus PFK increased 2.7-fold upon phosphorylation at Thr-226 by the Ser/Thr kinase Pkn4. The pkn4 gene is located 18 bp downstream of the pfk gene forming an operon, and both genes are expressed during vegetative growth and development. Here, we show that glycogen, which accumulates during stationary phase and early in development, is consumed during sporulation. A pfk-pkn4 deletion strain accumulated glycogen at a higher level than the wild-type strain, was unable to consume glycogen during developmental progression and exhibited a poor spore yield. From genetic complementation analysis of the pfk-pkn4 deletion strain with the pfk and pkn4 genes, it was found that glycogen consumption and a high spore yield require not only the pfk gene but also the pkn4 gene. Furthermore, phosphorylation is critical for glycogen consumption because the pfk gene engineered to express the mutant PFK (Thr-226-Ala) did not complement a pfk mutant. We propose that glycogen metabolism in M. xanthus is regulated in a similar manner to that in eukaryotes requiring a protein Ser/Thr kinase.     

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.     

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

The Myxococcus xanthus gene, pkn9 , encodes a protein that contains significant homology with eukaryotic Ser/Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus . This region also shows 29, 25 and 29% identity with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I–III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Two-dimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.     

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.     

Effects of overexpression of Pkn2, a transmembrane protein serine/threonine kinase, on development of Myxococcus xanthus.

Pkn2 is a putative transmembrane protein serine/threonine kinase required for normal development of Myxococcus xanthus. The effect of Pkn2 overexpression on development of M. xanthus was examined by expressing pkn2 under the control of a kanamycin promoter. Pkn2 was clearly detected by Western blot (immunoblot) analysis in the overexpression strain (the PKm/pkn2 strain) but could not be detected in the wild-type strain. Overexpressed Pkn2 was located almost exclusively in the membrane fraction, suggesting that Pkn2 is a transmembrane receptor-type protein Ser/Thr kinase. The PKm/pkn2 strain formed fruiting bodies more slowly than the wild-type strain, in contrast to a Pkn2 deletion strain, the delta pkn2 strain, which developed faster than the wild-type strain. However, spore production was reduced in both the PKm/pkn2 and delta pkn2 strains. These data suggest that Pkn2 functions as a negative regulator for fruiting-body formation and that the proper level of Pkn2 is necessary for maximum myxospore yield.     

Factors that modulate the Pkn4 kinase cascade in Myxococcus xanthus

Myxococcus xanthus, a gram-negative developmental bacterium, contains a large number of protein Ser/Thr kinases (PSTKs). Among these PSTKs, Pkn4 is shown to be 6-phosphofructokinase (PFK) kinase. PFK associates with the regulatory domain of Pkn4 (Pkn4RD) and is activated 2.7-fold upon phosphorylation at Thr-226 by Pkn4. The activation of PFK is required to consume glycogen accumulated during early development and is essential for efficient sporulation. Three new factors, MkapA, MkapB and MkapC have been identified that associate with Pkn4 by the yeast two-hybrid screen and each contains well-known protein-protein interaction domains. MkapB interacts with Pkn4 in a phosphorylation-dependent manner and remains associated with Pkn4 after its phosphorylation. Binding of MkapB to Pkn4 prevents the interaction of Pkn4 with PFK and consequently PFK phosphorylation and activation. A pfk-pkn4 deletion mutant accumulates glycogen at a rate two folds higher than the parent strain, DZF1, at the stationary phase and early development stage, it is unable to consume glycogen during development and produces only 3.4% of the DZF1 spore yield. In contrast, an mkapB deletion mutant exhibits a 24 h delay in fruiting body formation, accumulates less glycogen in the stationary phase and gives rise to 6.4% of the DZF1 spore yield. In addition to Pkn4, MkapA associates with other membrane-associated PSTKs, Pkn1, Pkn2, Pkn8 and Pkn9, while MkapB associates with Pkn8 and Pkn9, and MkapC with Pkn8. These results indicate that there are complex PSTK networks in M. xanthus sharing common modulating factors.     

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。