Lee Kroos

Developmental cheating in the social bacterium Myxococcus xanthus

Cheating is a potential problem in any social system that depends on cooperation and in which actions that benefit a group are costly to individuals that perform them. Genetic mutants that fail to perform a group-beneficial function but that reap the benefits of belonging to the group should have a within-group selective advantage, provided that the mutants are not too common. Here we show that social cheating exists even among prokaryotes. The bacterium Myxococcus xanthus exhibits several social behaviours, including aggregation of cells into spore-producing fruiting bodies during starvation. We examined a number of M. xanthus genotypes that were defective for fruiting-body development, including several lines that evolved for 1,000 generations under asocial conditions and others carrying defined mutations in developmental pathways, to determine whether they behaved as cheaters when mixed with their developmentally proficient progenitor. Clones from several evolved lines and two defined mutants exhibited cheating during development, being over-represented among resulting spores relative to their initial frequency in the mixture. The ease of finding anti-social behaviours suggests that cheaters may be common in natural populations of M. xanthus.

A forespore checkpoint for mother cell gene expression during development in B. subtilis

Gene expression in the mother cell compartment of sporulating cells of B. subtilis is partly governed by the mother cell RNA polymerase sigma factor sigma K. Paradoxically, sigma K-directed gene expression also depends on sigma G, the product of the forespore compartment regulatory gene spoIIIG, and on other forespore regulatory proteins. We now identify mutations in the genes bofA and bofB that relieve the dependence of mother cell gene expression on forespore regulatory proteins but not on sigma K. We establish that the dependence of mother cell gene expression on the forespore regulatory proteins is mediated at the level of the conversion of pro-sigma K to its mature, active form. We propose that the bofA and/or bofB proteins govern this conversion in response to a signal generated by the forespore. Activation of pro-sigma K could be a checkpoint for coordinating gene expression between the mother cell and forespore compartments of the developing sporangium.

CONTROL OF SIGMA FACTOR ACTIVITY DURING BACILLUS SUBTILIS SPORULATION

When starved, Bacillus subtilis undergoes asymmetric division to produce two cell types with different fates. The larger mother cell engulfs the smaller forespore, then nurtures it and, eventually, lyses to release a dormant, environmentally resistant spore. Driving these changes is a programme of transcriptional gene regulation. At the heart of the programme are σ factors, which become active at different times, some only in one cell type or the other, and each directing RNA polymerase to transcribe a different set of genes. The activity of each σ factor in the cascade is carefully regulated by multiple mechanisms. In some cases, novel proteins control both σ factor activity and morphogenesis, co‐ordinating the programme of gene expression with morphological change. These bifunctional proteins, as well as other proteins involved in σ factor activation, and even precursors of σ factors themselves, are targeted to critical locations, allowing the mother cell and forespore to communicate with each other and to co‐ordinate their programmes of gene expression. This signalling can result in proteolytic σ factor activation. Other mechanisms, such as an anti‐σ factor and, perhaps, proteolytic degradation, prevent σ factors from becoming active in the wrong cell type. Accessory transcription factors modulate RNA polymerase activity at specific promoters. Negative feedback loops limit σ factor production and facilitate the transition from one σ factor to the next. Together, the mechanisms controlling σ factor activity ensure that genes are expressed at the proper time and level in each cell type.

Yapsins Are a Family of Aspartyl Proteases Required for Cell Wall Integrity in Saccharomyces cerevisiae

ABSTRACT The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Δ2Δ mutant and the quintuple yapsin mutant (5ypsΔ) undergo osmoremedial cell lysis at 37°C. The cell walls of both 5ypsΔ and yps1Δ2Δ mutants have decreased amounts of 1,3- and 1,6-β-glucan. Although there is decreased incorporation of both 1,3- and 1,6-β-glucan in the 5ypsΔ mutant in vivo, in vitro specific activity of both 1,3- and 1,6-β-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsΔ. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Δ mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.

Sporulation regulatory protein gerE from Bacillus subtilis binds to and can activate or repress transcription from promoters for mother-cell-specific genes

The mother-cell line of gene expression during sporulation in Bacillus subtilis is a hierarchical cascade consisting of at least four temporally controlled gene sets, the first three of which each contain a regulatory gene for the next gene set in the pathway. gerE, a member of the penultimate gene set, is a regulatory gene whose products is required for the transcriptional activation of genes (coat protein genes cotB and cotC) in the last gene set. The gerE product also influences the expression of other members of the penultimate gene set (coat protein genes cotA and cotD appear to be repressed and activated, respectively). We now report that the purified product of gerE (GerE) is a DNA-binding protein that adheres to the promoters for cotB and cotC. We also show that GerE stimulates cotB and cotC transcription in vitro by RNA polymerase containing the mother-cell sigma factor sigma K. These findings support the view that GerE is a positively acting, regulatory protein whose appearance at a late stage of development directly activates the transcription of genes in the last known temporal class of mother-cell-expressed genes. In addition, GerE stimulates cotD transcription and inhibits cotA transcription in vitro by sigma K RNA polymerase, as expected from in vivo studies, and, unexpectedly, profoundly inhibits in vitro transcription of the gene (sigK) that encodes sigma K. The effects of GerE on cotD and sigK transcription are just the opposite of the effects exerted by the earlier-appearing, mother-cell regulatory protein spoIIID, suggesting that the ordered appearance of first SpoIIID, then GerE, ensures proper flow of the regulatory cascade controlling gene expression in the mother cell.

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.

Sporulation and enterotoxin (CPE) synthesis are controlled by the sporulation-specific sigma factors SigE and SigK in Clostridium perfringens.

Clostridium perfringens is the third most frequent cause of bacterial food poisoning annually in the United States. Ingested C. perfringens vegetative cells sporulate in the intestinal tract and produce an enterotoxin (CPE) that is responsible for the symptoms of acute food poisoning. Studies of Bacillus subtilis have shown that gene expression during sporulation is compartmentalized, with different genes expressed in the mother cell and the forespore. The cell-specific RNA polymerase sigma factors sigma(F), sigma(E), sigma(G), and sigma(K) coordinate much of the developmental process. The C. perfringens cpe gene, encoding CPE, is transcribed from three promoters, where P1 was proposed to be sigma(K) dependent, while P2 and P3 were proposed to be sigma(E) dependent based on consensus promoter recognition sequences. In this study, mutations were introduced into the sigE and sigK genes of C. perfringens. With the sigE and sigK mutants, gusA fusion assays indicated that there was no expression of cpe in either mutant. Results from gusA fusion assays and immunoblotting experiments indicate that sigma(E)-associated RNA polymerase and sigma(K)-associated RNA polymerase coregulate each others expression. Transcription and translation of the spoIIID gene in C. perfringens were not affected by mutations in sigE and sigK, which differs from B. subtilis, in which spoIIID transcription requires sigma(E)-associated RNA polymerase. The results presented here show that the regulation of developmental events in the mother cell compartment of C. perfringens is not the same as that in B. subtilis and Clostridium acetobutylicum.

Regulation of σ factor activity during Bacillus subtilis development

Progression of Bacillus subtilis through a series of morphological changes is driven by a cascade of sigma (sigma) factors and results in formation of a spore. Recent work has provided new insights into the location and function of proteins that control sigma factor activity, and has suggested that multiple mechanisms allow one sigma factor to replace another in the cascade.

Myxobacteria, Polarity, and Multicellular Morphogenesis

Myxobacteria are renowned for the ability to sporulate within fruiting bodies whose shapes are species-specific. The capacity to build those multicellular structures arises from the ability of M. xanthus to organize high cell-density swarms, in which the cells tend to be aligned with each other while constantly in motion. The intrinsic polarity of rod-shaped cells lays the foundation, and each cell uses two polar engines for gliding on surfaces. It sprouts retractile type IV pili from the leading cell pole and secretes capsular polysaccharide through nozzles from the trailing pole. Regularly periodic reversal of the gliding direction was found to be required for swarming. Those reversals are generated by a G-protein switch which is driven by a sharply tuned oscillator. Starvation induces fruiting body development, and systematic reductions in the reversal frequency are necessary for the cells to aggregate rather than continue to swarm. Developmental gene expression is regulated by a network that is connected to the suppression of reversals.

Evidence that SpoIVFB Is a Novel Type of Membrane Metalloprotease Governing Intercompartmental Communication during Bacillus subtilis Sporulation

Processing of pro-sigma(K) in the mother cell compartment of sporulating Bacillus subtilis involves SpoIVFB and is governed by a signal from the forespore. SpoIVFB has an HEXXH motif characteristic of metalloproteases embedded in one of its transmembrane segments. Several conservative single amino acid changes in the HEXXH motif abolished function. However, changing the glutamic acid residue to aspartic acid, or changing the isoleucine residue that precedes the motif to proline, permitted SpoIVFB function. Only one other putative metalloprotease, site 2 protease has been shown to tolerate aspartic acid rather than glutamic acid in its HEXXH sequence. Site 2 protease and SpoIVFB share a second region of similarity with a family of putative membrane metalloproteases. A conservative change in this region of SpoIVFB abolished function. Interestingly, SpoIVFA increased the accumulation of certain mutant SpoIVFB proteins but was unnecessary for accumulation of wild-type SpoIVFB.