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.     

Regulation of dev, an operon that includes genes essential for Myxococcus xanthus development and CRISPR-associated genes and repeats

Expression of dev genes is important for triggering spore differentiation inside Myxococcus xanthus fruiting bodies. DNA sequence analysis suggested that dev and cas (CRISPR-associated) genes are cotranscribed at the dev locus, which is adjacent to CRISPR (clustered regularly interspaced short palindromic repeats). Analysis of RNA from developing M. xanthus confirmed that dev and cas genes are cotranscribed with a short upstream gene and at least two repeats of the downstream CRISPR, forming the dev operon. The operon is subject to strong, negative autoregulation during development by DevS. The dev promoter was identified. Its -35 and -10 regions resemble those recognized by M. xanthus sigma(A) RNA polymerase, the homolog of Escherichia coli sigma(70), but the spacer may be too long (20 bp); there is very little expression during growth. Induction during development relies on at least two positive regulatory elements located in the coding region of the next gene upstream. At least two positive regulatory elements and one negative element lie downstream of the dev promoter, such that the region controlling dev expression spans more than 1 kb. The results of testing different fragments for dev promoter activity in wild-type and devS mutant backgrounds strongly suggest that upstream and downstream regulatory elements interact functionally. Strikingly, the 37-bp sequence between the two CRISPR repeats that, minimally, are cotranscribed with dev and cas genes exactly matches a sequence in the bacteriophage Mx8 intP gene, which encodes a form of the integrase needed for lysogenization of M. xanthus.     

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.     

Identification of a vegetative promoter in Myxococcus xanthus. A protein that has homology to histones

A physical map of 330 x 10(3) base-pairs near the replication origin of Myxococcus xanthus chromosome has been established already. Using DNA fragments from this region, Northern blot hybridization analysis was carried out in order to identify the genes expressed during vegetative growth. One of the genes, tentatively designated as vegA, was cloned and its entire DNA sequence was determined. The amino acid sequence of the gene product deduced from the DNA sequence reveals that the VegA protein is a very basic protein with a molecular weight of 18,700. The gene was expressed in Escherichia coli using an expression vector, and its gene product was identified using SDS/polyacrylamide gel electrophoresis. From the results of S1 nuclease mapping, the vegA promoter was found to contain the sequence TAGACA at the -35 region and the sequence AAGGGT at the -10 region. These two regions are separated by 18 nucleotides. Genetic analysis suggests that the vegA gene may be essential for the growth of M. xanthus. From a computer-aided search for homologies to know protein structures, it was found that the VegA protein has homologies to histone H4 of Tetrahymena thermophila and histone H2B of sea urchin.     

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.     

Mutational analysis of the Myxococcus xanthus Omicron4499 promoter region reveals shared and unique properties in comparison with other C-signal-dependent promoters

The bacterium Myxococcus xanthus undergoes multicellular development during times of nutritional stress and uses extracellular signals to coordinate cell behavior. C-signal affects gene expression late in development, including that of Omega4499, an operon identified by insertion of Tn5 lac into the M. xanthus chromosome. The Omega4499 promoter region has several sequences in common with those found previously to be important for expression of other C-signal-dependent promoters. To determine if these sequences are important for Omega4499 promoter activity, the effects of mutations on expression of a downstream reporter gene were tested in M. xanthus. Although the promoter resembles those recognized by Escherichia coli sigma(54), mutational analysis implied that a sigma(70)-type sigma factor likely recognizes the promoter. A 7-bp sequence known as a C box and a 5-bp element located 6 bp upstream of the C box have been shown to be important for expression of other C-signal-dependent promoters. The Omega4499 promoter region has C boxes centered at -33 and -55 bp, with 5-bp elements located 7 and 8 bp upstream, respectively. A multiple-base-pair mutation in any of these sequences reduced Omega4499 promoter activity more than twofold. Single base-pair mutations in the C box centered at -33 bp yielded a different pattern of effects on expression than similar mutations in other C boxes, indicating that each functions somewhat differently. An element from about -81 to -77 bp exerted a twofold positive effect on expression but did not appear to be responsible for the C-signal dependence of the Omega4499 promoter. Mutations in sigD and sigE, which are genes that encode sigma factors, reduced expression from the Omega4499 promoter. The results provide further insight into the regulation of C-signal-dependent genes, demonstrating both shared and unique properties among the promoter regions so far examined.     

Cloning, expression, and characterization of the lon gene of Erwinia amylovora: evidence for a heat shock response.

The gene encoding the Lon protease of Erwinia amylovora has been cloned by complementation of an Escherichia coli lon mutant. Analysis of the determined nucleotide sequence of the lon gene revealed extensive homology to the nucleotide sequences of cloned lon genes from E. coli, Myxococcus xanthus, and Bacillus brevis. The predicted amino acid sequence of the E. amylovora Lon protease was 94, 59, and 54% identical to the predicted amino acid sequences of the Lon proteases of E. coli, M. xanthus, and B. brevis, respectively. The -10 and -35 promoter regions of the cloned lon gene had extensive homology to the respective consensus sequences of E. coli heat shock promoters. Promoter mapping of the lon gene located the start site 7 bases downstream of the -10 region. Cloning of the lon promoter upstream of a cat reporter gene demonstrated that expression of the E. amylovora lon gene was inducible by a heat shock. This is the first demonstration of a heat shock-regulated gene in E. amylovora. Site-directed mutagenesis of the -10 region of the lon promoter confirmed that the heat shock expression of the E. amylovora lon gene may be mediated by a sigma 32-like factor. Insertional inactivation of the E. amylovora chromosomal lon gene confirmed that the lon gene was not essential for either vegetative growth or infection of apple seedlings. E. amylovora lon mutants had increased sensitivity to UV irradiation and elevated levels of extracellular polysaccharide, suggesting comparable roles for the Lon proteases in both E. amylovora and E. coli.