CodY, a highly conserved protein in the low G + C, gram-positive bacteria, regulates the expression of many Bacillus subtilis genes that are induced as cells make the transition from rapid exponential growth to stationary phase and sporulation. This transition has been associated with a transient drop in the intracellular pool of GTP. Many stationary-phase genes are also induced during exponential-growth phase by treatment of cells with decoyinine, a GMP synthetase inhibitor. The effect of decoyinine on an early-stationary-phase gene is shown here to be mediated through CodY and to reflect a reduction in guanine nucleotide accumulation. CodY proved to bind GTP in vitro. Moreover, CodY-mediated repression of target promoters was dependent on a high concentration of GTP, comparable to that found in rapidly growing exponential-phase cells. Because a codY-null mutant was able to sporulate under conditions of nutrient excess, CodY also appears to be a critical factor that normally prevents sporulation under such conditions. Thus, B. subtilis CodY is a novel GTP-binding protein that senses the intracellular GTP concentration as an indicator of nutritional conditions and regulates the transcription of early-stationary-phase and sporulation genes, allowing the cell to adapt to nutrient limitation. Our understanding of the relationship between environmental signals and global changes in gene expression is limited by the difficulty in identifying intracellular signaling molecules that interact with key regulatory proteins. This gap is particularly apparent for cases of general nutrient limitation. When Bacillus subtilis cells encounter nutrient limitation and enter stationary phase, a variety of adaptive processes-such as genetic competence, secretion of macromolecule-degrading enzymes, import of secondary nutrients, activation of metabolic pathways, chemotaxis and motility, production of antibiotics, and sporulation-are initiated (Sonenshein 1989). A network of global regulatory proteins modulates the cell's response and regulates the choice between adaptation to poor growth conditions and sporulation (Sonenshein 1989(Sonenshein , 2000Burkholder and Grossman 2000), but the specific signals to which these regulators respond have remained a mystery.Many B. subtilis genes that are expressed early in stationary phase are repressed by CodY (Table 1). Preliminary results indicate that CodY also contributes to regulation of at least two genes (citB, spo0A) whose products are necessary for sporulation (M. Ratnayake- Lecamwasam and A.L. Sonenshein, unpubl. Bolotin et al. 1999).CodY was first identified as a repressor of the B. subtilis dipeptide transport (dpp) operon and was found to be active when cells are grown with an excess of glucose or Casamino acids (CAA) as reported by Slack et al. (1995). During vegetative growth, the dpp operon is also directly repressed by AbrB, a second global regulator of earlystationary-phase genes Strauch 1993;Serror and Sonenshein 1996b), but the repressive effects of nutrient excess are mediat...

Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using a-and f-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globinrelated DNA sequences. We detect specific, high-stringency hybridization of the a-globin probe to genomic DNAs of both white-and red-blooded species, whereas the 8-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of a-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes p-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult ,3-globin locus prior to the diversification of the clade.In 1954 Ruud (1) published the first systematic analysis of the "white" blood of an antarctic icefish, Chaenocephalus aceratus. He reported that fresh blood was nearly transparent, contained leukocytes at 1% by volume, but lacked erythrocytes and the respiratory transport pigment hemoglobin. Furthermore, the oxygen-carrying capacity of C. aceratus blood was approximately 10% that of two red-blooded notothenioids. Subsequent investigations extended these observations to other icefish species and revealed that icefish blood contains small numbers of "erythrocyte-like" cells that, nevertheless, are devoid of hemoglobin (2, 3). Thus, limited to oxygen physically dissolved in their blood, the icefishes have evolved compensatory physiological and circulatory adaptations-e.g., modest suppression of metabolic rates, enhanced gas exchange by large, well-perfused gills and cutaneous respiration, and large increases in cardiac output and blood volume-that ensure adequate oxygenation of their tissues (4, 5).We have initiated studies to determine the status of globin genes in channichthyid genomes and to evaluate potential evolutionary mechanisms leading to the hemoglobinless phenotype. Icefishes evolved from the red-blooded Notothenioidei (6, 7), which, in contrast to temperate fishes, are characterized by a paucity of hemoglobin forms (7-10). Adults of the family Nototheniidae (antarctic rockcods) generally possess a major hemoglobin, Hb 1 (-95% of the total), and a second, minor hemoglobin, Hb 2, that differ in their a chains (al and a2, respectively) (11-13). The more phyletically derived harpagiferids and bathydraconids have a single hemoglobin. The trend toward reduced hemoglobin multiplicity in t...

The roles of the CcpC, CodY, and AbrB proteins in regulation of the Bacillus subtilis aconitase (citB) gene were found to be distinct and to vary with the conditions and phase of growth. CcpC, a citrate-inhibited repressor that is the primary factor regulating citB expression in minimal-glucose-glutamine medium, also contributed to repression of citB during exponential-phase growth in broth medium. A null mutation in codY had no effect on citB expression during growth in minimal medium even when combined with ccpC and abrB mutations. However, a codY mutation slightly relieved repression during exponential growth in broth medium and completely derepressed citB expression when combined with a ccpC mutation. An abrB mutation led to decreased expression of citB during stationary phase in both broth and minimal medium. All three proteins bound in vitro to specific and partially overlapping sites within the citB regulatory region. Interaction of CcpC and CodY with the citB promoter region was partially competitive.

Bacillus subtilis CodY protein is the best-studied member of a novel family of global transcriptional regulators found ubiquitously in low-G؉C gram-positive bacteria. As for many DNA-binding proteins, CodY appears to have a helix-turn-helix (HTH) motif thought to be critical for interaction with DNA. This putative HTH motif was found to be highly conserved in the CodY homologs. Site-directed mutagenesis was used to identify amino acids within this motif that are important for DNA recognition and binding. The effects of each mutation on DNA binding in vitro and on the regulation of transcription in vivo from two target promoters were tested. Each of the mutations had similar effects on binding to the two promoters in vitro, but some mutations had differential effects in vivo.

The icefishes of the Southern Ocean (family Channichthyidae, suborder Notothenioidei) are unique among vertebrates in their inability to synthesize hemoglobin. We have shown previously (Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella, L., Ciaramella, M., di Prisco, G., and Detrich, H. W., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1817-1821) that icefishes retain inactive genomic remnants of adult notothenioid ␣-globin genes but have lost the gene that encodes adult ␤-globin. Here we demonstrate that loss of expression of the major adult ␣-globin, ␣1, in two species of icefish (Chaenocephalus aceratus and Chionodraco rastrospinosus) results from truncation of the 5 end of the notothenioid ␣1-globin gene. The wild-type, functional ␣1-globin gene of the Antarctic yellowbelly rockcod, Notothenia coriiceps, contains three exons and two A ؉ T-rich introns, and its expression may be controlled by two or three distinct promoters. Retained in both icefish genomes are a portion of intron 2, exon 3, and the 3-untranslated region of the notothenioid ␣1-globin gene. The residual, nonfunctional ␣-globin gene, no longer under positive selection pressure for expression, has apparently undergone random mutational drift at an estimated rate of 0.12-0.33%/million years. We propose that abrogation of hemoglobin synthesis in icefishes most likely resulted from a single mutational event in the ancestral channichthyid that deleted the entire ␤-globin gene and the 5 end of the linked ␣1-globin gene.