Myxococcus xanthus develops species-specific multicellular fruiting bodies. Starting from a uniform mat of cells, some cells enter into nascent fruiting body aggregates, whereas other cells remain outside. The cells within the fruiting body differentiate from rods into spherical, heat-resistant spores, whereas the cells outside the aggregates, called peripheral cells, remain rod-shaped. Early developmentally regulated genes are expressed in peripheral cells as well as by cells in the fruiting bodies. By contrast, late developmental genes are only expressed by cells within the nascent fruiting bodies. The data show that peripheral cells begin to develop, but are unable to express genes that are switched on later than about 6 h after the start of development. All of the genes whose expression is limited to the fruiting body are dependent on C-signaling either directly or indirectly, whereas the genes that are equally expressed in peripheral rods and in fruiting body cells are not. One of the C-signal-dependent and spatially patterned operons is called dev, and the dev operon has been implicated in the process of sporulation. It is proposed that expression of certain genes, including those of the dev operon, is limited to the nascent fruiting body because fruiting body cells engage in a high level of C-signaling. Peripheral cells do less C-signaling than fruiting body cells, because they have a different spatial arrangement and are at lower density. As a consequence, peripheral cells fail to express the late genes necessary for spore differentiation.spatial pattern ͉ positive feedback ͉ Myxobacteria ͉ cell-cell interaction H ow spatial patterns of differentiated cells arise is a central issue for animal and plant development. Myxococcus xanthus and other myxobacteria differentiate spores in response to nutrient deprivation. Although most bacteria sporulate individually, myxobacteria build large structured masses of spores, called fruiting bodies. Under nutrient-rich conditions, M. xanthus grows and divides as rod-shaped cells. When its development is induced by starvation, a hundred thousand cells contribute to building a fruiting body, whose shape is speciesspecific. Cells that have entered into the fruiting body finally differentiate into environmentally resistant myxospores, which can survive years without nutrients. However, not all of the starvation-induced cells become spores. Cells within fruiting bodies become spores, cells outside and between these multicellular structures remain rod-shaped and nonresistant (1). These cells, called peripheral rod cells, never become spores, despite their synthesis of two sporulation proteins, Tps and C (1). Dworkin and Gibson (2) showed that every cell innately has the capacity to become a spore. A difference in the developmental fate of peripheral rods and of fruiting body cells constitutes a spatial pattern that needs to be explained.Most patterns involve cell-to-cell signaling, and sporulation depends on C-signaling. Ordinarily, each cell is simultaneously a transmitter...

The multicellular developmental cycle of Myxococcus xanthus requires large-scale changes in gene transcription, and recent findings indicate that NtrC-like activators play a prominent role in regulating these changes. In this study, we made insertions in 28 uncharacterized ntrC-like activator (nla) genes and found that eight of these insertions cause developmental defects. Hence, these results are consistent with the idea that M. xanthus uses a series of different NtrC-like activators during fruiting body development. Four of the eight developmental mutants we identified have motility defects. The nla1, nla19, and nla23 mutants show S-motility defects, while the nla24 mutant shows defects in both S-motility and A-motility. During development, aggregation of the nla1, nla19, and nla23 mutants is delayed slightly and the nla24 mutant shows no signs of aggregation or sporulation. The nla4, nla6, nla18, and nla28 mutants have no appreciable loss in motility, but they fail to aggregate and to sporulate normally. The nla18 mutant belongs to a special class of developmental mutants whose defects can be rescued when they are codeveloped with wild-type cells, suggesting that nla18 fails to produce a cell-cell signal required for development. The three remaining activator mutants, nla4, nla6, and nla28, appear to have complex developmental phenotypes that include deficiencies in cell-cell developmental signals. The social lifestyle of the gram-negative soil bacteriumMyxococcus xanthus is rather unusual in the prokaryotic world. In nature, large groups of M. xanthus cells feed on prey bacteria to obtain their nutrients. When this nutrient supply is depleted, M. xanthus cells initiate a complex developmental program that culminates in the formation of a multicellular fruiting body. Once the fruiting body is molded into its final form, individual rod-shaped cells within the fruiting body differentiate into a specialized and dormant cell (a spherical spore) that is resistant to many forms of environmental stress (for reviews, see references 8 and 53).All of the morphological events that occur during M. xanthus development are accompanied by large-scale changes in gene expression, and the products of many of these genes are absolutely required for normal development (14,35,36,43,47,57,60). The temporal and spatial expression of developmental genes is coordinated by a series of cell-cell signals; each signal is required for the expression of different sets of genes (7,21,26,32,34,39,41,42). For example, A-signal is required for gene expression during all stages of M. xanthus development, while C-signal is only required for gene expression during the later stages of development.Recent findings indicate that M. xanthus uses 54 -like promoters to drive expression of many developmentally regulated genes; the hallmarks of 54 promoters are recognition sequences located around Ϫ12 and Ϫ24 bp upstream of the transcriptional start site (13,15,20,30,50,64,66; J. S. Jakobsen, E. Licking, and D. Kaiser, personal communication). Moreover, e...

The signal transduction networks that initiate multicellular development in bacteria remain largely undefined. Here, we report that Myxococcus xanthus regulates entry into its multicellular developmental program using a novel strategy: a cascade of transcriptional activators known as enhancer binding proteins (EBPs). The EBPs in the cascade function in sequential stages of early development, and several lines of evidence indicate that the cascade is propagated when EBPs that function at one stage of development directly regulate transcription of an EBP gene important for the next developmental stage. We also show that the regulatory cascade is designed in a novel way that extensively expands on the typical use of EBPs: Instead of using only one EBP to regulate a particular gene or group of genes, which is the norm in other bacterial systems, the cascade uses multiple EBPs to regulate EBP genes that are positioned at key transition points in early development. Based on the locations of the putative EBP promoter binding sites, several different mechanisms of EBP coregulation are possible, including the formation of coregulating EBP transcriptional complexes. We propose that M. xanthus uses an EBP coregulation strategy to make expression of EBP genes that modulate stage-stage transitions responsive to multiple signal transduction pathways, which provide information that is important for a coordinated decision to advance the developmental process.

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 A RNA polymerase, the homolog of Escherichia coli 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.

In the bacterium Myxococcus xanthus, starvation triggers the formation of multicellular fruiting bodies containing thousands of stress-resistant spores. Recent work showed that fruiting body development is regulated by a cascade of transcriptional activators called enhancer binding proteins (EBPs). The EBP Nla6 is a key component of this cascade; it regulates the promoters of other EBP genes, including a downstream-functioning EBP gene that is crucial for sporulation. In recent expression studies, hundreds of Nla6-dependent genes were identified, suggesting that the EBP gene targets of Nla6 may be part of a much larger regulon. The goal of this study was to identify and characterize genes that belong to the Nla6 regulon. Accordingly, a direct repeat [consensus, C(C/A)ACGNNGNC] binding site for Nla6 was identified using in vitro and in vivo mutational analyses, and the sequence was subsequently used to find 40 potential developmental promoter (88 gene) targets. We showed that Nla6 binds to the promoter region of four new targets (asgE, exo, MXAN2688, and MXAN3259) in vitro and that Nla6 is important for their normal expression in vivo. Phenotypic studies indicate that all of the experimentally confirmed targets of Nla6 are primarily involved in sporulation. These targets include genes involved in transcriptional regulation, cell-cell signal production, and spore differentiation and maturation. Although sporulation occurs late in development, all of the developmental loci analyzed here show an Nla6-dependent burst in expression soon after starvation is induced. This finding suggests that Nla6 starts preparing cells for sporulation very early in the developmental process. IMPORTANCEBacterial development yields a remarkable array of complex multicellular forms. One such form, which is commonly found in nature, is a surface-associated aggregate of cells known as a biofilm. Mature biofilms are structurally complex and contain cells that are highly resistant to antibacterial agents. When starving, the model bacterium Myxococcus xanthus forms a biofilm containing a thin mat of cells and multicellular structures that house a highly resistant cell type called a myxospore. Here, we identify the promoter binding site of the transcriptional activator Nla6, identify genes in the Nla6 regulon, and show that several of the genes in the Nla6 regulon are important for production of stress-resistant spores in starvation-induced M. xanthus biofilms. Bacterial development yields a remarkable array of complex multicellular forms. Arguably, one of the most interesting forms of bacterial multicellularity is a surface-associated aggregate of cells known as a biofilm (1). Mature biofilms are complex and often contain highly ordered structural features, such as towers of cells. Particular sets of genes must be expressed at each stage in the development of biofilms; however, the regulation and function of such genes are not well understood in the vast majority of bacteria. In this study, we identified a set of developmental promote...