Differential expression of protein S genes during Myxococcus xanthus development

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.     

Iodination of Myxococcus xanthus during development

Intact cells of Myxococcus xanthus were iodinated with [125I]lactoperoxidase to permit examination of the surface components accessible to labeling during cell development. Vegetative cells, starved on a defined solid medium, aggregated, formed fruiting bodies, and produced myxospores. Cells collected at different stages were iodinated, and their proteins were analyzed by one- and two-dimensional electrophoresis and autoradiography. One-dimensional electrophoresis revealed six iodinated bands in vegetative cell extracts. During development, 10 radioactive bands were detected, 4 of which migrated to the same positions as those of vegetative cells. Only six bands were detected in purified, labeled myxospores. Of these, one band possessed mobility similar to that of labeled vegetative cell proteins, whereas the other bands possessed mobility similar to that detected in developing cells. Analysis of two-dimensional gels indicated that at least 14 proteins were iodinated in vegetative cells, one of which was intensely labeled (protein b). Another of the proteins (protein a) was labeled throughout development. During development, about 30 proteins were iodinated and the prominently labeled ones were designated c, d, e, f, and g. The latter two (proteins f and g) were not detected in purified, iodinated myxospores. The data indicated a pronounced change in surface structure during development; some of the change may be involved in cellular interaction during aggregation.     

Quantifying aggregation dynamics during Myxococcus xanthus development

Under starvation conditions, a swarm of Myxococcus xanthus cells will undergo development, a multicellular process culminating in the formation of many aggregates called fruiting bodies, each of which contains up to 100,000 spores. The mechanics of symmetry breaking and the self-organization of cells into fruiting bodies is an active area of research. Here we use microcinematography and automated image processing to quantify several transient features of developmental dynamics. An analysis of experimental data indicates that aggregation reaches its steady state in a highly nonmonotonic fashion. The number of aggregates rapidly peaks at a value 2- to 3-fold higher than the final value and then decreases before reaching a steady state. The time dependence of aggregate size is also nonmonotonic, but to a lesser extent: average aggregate size increases from the onset of aggregation to between 10 and 15 h and then gradually decreases thereafter. During this process, the distribution of aggregates transitions from a nearly random state early in development to a more ordered state later in development. A comparison of experimental results to a mathematical model based on the traffic jam hypothesis indicates that the model fails to reproduce these dynamic features of aggregation, even though it accurately describes its final outcome. The dynamic features of M. xanthus aggregation uncovered in this study impose severe constraints on its underlying mechanisms.     

Role of autocide AMI in development of Myxococcus xanthus.

A new developmental mutant of Myxococcus xanthus has been isolated by screening TnV insertion mutants for AMI-dependent development in submerged culture. This mutant (ER304) aggregated and sporulated on agar surfaces but required at least 3.8 micrograms of autocide AMI per ml for development in submerged cultures. Spore rescue of ER304 was obtained with the saturated, monounsaturated, and diunsaturated fatty acid fractions of AMI, with specific activities of 68, 115, and 700 U/mg, respectively. In addition, several model fatty acids were capable of rescuing sporulation of ER304; however, there was no correlation between specific lytic activity observed in vegetative cultures and specific rescue activity. Rescue of ER304 was effected during the first ca. 12 h after the initiation of starvation conditions; after this time, addition of AMI or model fatty acids killed the cells. Supernatant fluids of ER304 rescued development in dsg mutants (e.g., DK3260) in submerged cultures, but dsg mutant supernatant fluids were incapable of rescuing ER304 development. The data presented in this article support the idea that the primary mechanism of rescue by AMI is not via lysis, although developmental lysis may be an indirect result of the rescue event. A membrane permeability model is presented to explain the role of autocides in early developmental events in wild-type strains and in the aggregation and sporulation rescue of developmental mutants ER304 and DK3260.     

Methylation of macromolecules during development in Myxococcus xanthus.

Covalent modification of macromolecules can serve to alter their biological activities and is therefore frequently involved in regulation. I examined methylation of proteins and carbohydrates during development and vegetative growth in the procaryote Myxococcus xanthus. Striking differences in the patterns of protein methylation occurred when cell development was induced by nutrient deprivation on solid media and when cells were starved in liquid. In addition, a methylated, protease-resistant macromolecule which contained carbohydrate and which may have been an unusual type of lipopolysaccharide was observed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of methylation patterns in various media and an analysis of the time course of methylation indicated that changes in methylation were part of the developmental pathway which includes aggregation. Induction of development in liquid by glycerol produced no changes in methylation.     

Role of Myxococcus xanthus cell surface antigen 1604 in development.

The inhibition of development of Myxococcus xanthus by monoclonal antibody (MAb) 1604 has been further investigated with two MAbs produced against the affinity-purified cell surface antigen (CSA) 1604. Both of these second-generation MAbs, 4070 and 4054, reacted with the same band at 150 kilodaltons (kDa) on Western immunoblots of lysed and reduced cells. This band was also identified by MAb 1604. However, the affinity-purified CSA was a complex of the two proteins (51 and 23 kDa) and lipopolysaccharide (LPS) that the 150-kDa material comprised. One of the three MAbs, 4070, reacted with LPS on Western immunoblots. Another MAb, 4054, reacted with the 23-kDa protein, and MAb 1604 reacted with the 51-kDa protein found in the CSA complex. Competitive binding studies verified that MAbs 4054 and 1604 identified different epitopes, and MAb 4070 probably reacted with a third epitope of the CSA 1604 complex. MAb 4054 blocked development, although not as thoroughly as MAb 1604 did, when added at 60 micrograms/ml to cells undergoing submerged development. In contrast, MAb 4070 prevented sporulation in submerged development and induced the cells to reaggregate in rings around the initial aggregation centers. A mutant strain of M. xanthus that is deficient in the epitope for MAb 1604 retained the epitope for MAb 4054. The affinity-purified antigen 1604, when added to cells at greater than or equal to 550 ng/ml, altered the appearance of the fruiting bodies and at higher concentrations prevented fruiting body formation. The CSA 1604 moiety responsible for this inhibitory effect is apparently a peptide constituent and not the LPS.     

Two recA genes in Myxococcus xanthus.

Two recA genes, recA1 and recA2, in Myxococcus xanthus were cloned by using the recA gene of Escherichia coli, and their DNA sequences were determined. On the basis of deduced amino acid sequences, RecA1 and RecA2 have 67.0% identity to each other and 60.5 and 60.9% identities to E. coli RecA, respectively. Expression of recA2 was detected in both vegetative and developmental cells by Northern blot (RNA) analysis, and a threefold induction was observed when cells were treated with nalidixic acid. Repeated attempts to isolate a recA2 disruption mutant have failed, while a recA1 disruption mutant was readily isolated. Both the recA1 and recA2 genes expressed in E. coli complement the UV sensitivity of an E. coli recA strain.     

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.     

Changes in cyclic AMP levels during development in Myxococcus xanthus.

Cyclic AMP levels doubled in Myxococcus xanthus under conditions in which cells aggregate and form fruiting bodies. In liquid medium, glycerol- or dimethyl sulfoxide-induced sporulating cultures exhibited a sharp but transient rise in cyclic AMP concentration after 45 min.     

Multicellular development and gliding motility in Myxococcus xanthus

A great deal of progress has been made in the studies of fruiting body development and social gliding in Myxocococcus xanthus in the past few years. This includes identification of the bone fide C-signal and a receptor for type IV pili, and development of a model for the mechanism of adventurous gliding motility. It is anticipated that the next few years will see even more progress as the complete genome sequence is available and genomic and proteomic tools are applied to the study of M. xanthus social behaviors.     

Production of an extracellular milk-clotting activity during development in Myxococcus xanthus.

We describe here an extracellular proteolytic activity secreted during both growth and submerged development by Myxococcus xanthus DK1622. This activity yields the clotting of kappa-casein at pH 6 and is inhibited by specific inhibitors of aspartic proteases. Secretion of this milk-clotting proteolytic activity (of Mcp) is time regulated during the developmental cycle, with a large increase near 9 h poststarvation, but its production does not require cell-cell contact. The lack of secretion of this activity by several developmental mutants in submerged development conditions shows that Mcp production is developmentally regulated.     

黄色粘球菌发育阶段外膜蛋白表达的变化

【目的】黄色粘球菌是研究原核发育的一种模式生物,对其膜蛋白的研究仍然十分缺乏。【方法】利用6种预测软件,在黄色粘球菌的基因组中筛选编码外膜蛋白(OMP)的基因。根据报告基因lacZ,检测这些基因在营养性生长和发育阶段的表达。【结果】基于生物信息学分析,筛选出11个编码外膜蛋白的基因。其中2个基因(MXAN3106和MXAN3883)在发育阶段表达量上升,它们分别编码Secretin家族和Fimbrial usher protein (FUP)家族转运蛋白。其余9个基因在发育起始阶段表达量降低或保持较低水平,它们均编码TonB依赖型受体或外排蛋白。【结论】这些数据提示,黄色粘球菌由生长到发育的转换过程,伴随着膜蛋白表达的显著变化。     

DNA replication during aggregation phase is essential for Myxococcus xanthus development

Previous studies have demonstrated that fruiting body-derived Myxococcus xanthus myxospores contain two fully replicated copies of its genome, implying developmental control of chromosome replication and septation. In this study, we employ DNA replication inhibitors to determine if chromosome replication is essential to development and the exact time frame in which chromosome replication occurs within the developmental cycle. Our results show that DNA replication during the aggregation phase is essential for developmental progression, implying the existence of a checkpoint that monitors chromosome integrity at the end of the aggregation phase.     

Role of two novel two-component regulatory systems in development and phosphatase expression in Myxococcus xanthus

We have cloned a two-component regulatory system (phoR2-phoP2) of Myxococcus xanthus while searching for genes that encode proteins with phosphatase activity, where phoR2 encodes the histidine kinase and phoP2 encodes the response regulator. A second system, phoR3-phoP3, was identified and isolated by using phoP2 as a probe. These two systems are quite similar, sharing identities along the full-length proteins of 52% on the histidine kinases and 64% on the response regulators. The predicted structures of both kinases suggest that they are anchored to the membrane, with the sensor domains being located in the periplasmic space and the kinase domains in the cytoplasm. The response regulators (PhoP2 and PhoP3) exhibit a helix-loop-helix motif typical of DNA-binding proteins in the effector domains located in the C-terminal region. Studies on two single-deletion mutants and one double-deletion mutant have revealed that these systems are involved in development. Mutant fruiting bodies are not well packed, originating loose and flat aggregates where some myxospores do not reshape properly, and they remain as elongated cells. These systems are also involved in the expression of Mg-independent acid and neutral phosphatases, which are expressed during development. The neutral phosphatase gene is especially dependent on PhoP3. Neither PhoP2 nor PhoP3 regulates the expression of alkaline phosphatases and the pph1 gene.