Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120

In response to combined nitrogen starvation in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 is able to develop a particular cell type, called a heterocyst, specialized in molecular nitrogen fixation. Heterocysts are regularly intercalated among vegetative cells and represent 5-10% of all cells along each filament. In unicellular cyanobacteria, the key Krebs cycle intermediate, 2-oxoglutarate (2-OG), has been suggested as a nitrogen status signal, but in vivo evidence is still lacking. In this study we show that nitrogen starvation causes 2-OG to accumulate transiently within cells of Anabaena PCC 7120, reaching a maximal intracellular concentration of approximately 0.1 mM 1 h after combined nitrogen starvation. A nonmetabolizable fluorinated 2-OG derivative, 2,2-difluoropentanedioic acid (DFPA), was synthesized and used to demonstrate the signaling function of 2-OG in vivo. DFPA is shown to be a structural analogue of 2-OG and the process of its uptake and accumulation in vivo can be followed by (19)F magic angle spinning NMR because of the presence of the fluorine atom and its chemical stability. DFPA at a threshold concentration of 0.3 mM triggers heterocyst differentiation under repressing conditions. The multidisciplinary approaches using synthetic fluorinated analogues, magic angle spinning NMR for their analysis in vivo, and techniques of molecular biology provide a powerful means to identify the nature of the signals that remain unknown or poorly defined in many signaling pathways.     

Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways

When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-ras(G12D)) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-ras(G12D) leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-ras(G12D) fail to constitutively activate but rather hyperactivate cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-ras(G12D) display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies.     

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca(2+)-binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. The Ca(2+) reporter protein obelin was used to show that mature heterocysts had a high intracellular free Ca(2+)concentration {[Ca(2+)](i)}, and immunoblotting showed that CcbP was absent from heterocysts. A regular pattern of cells with higher [Ca(2+)](i) was established during heterocyst differentiation before the appearance of proheterocysts. A rapid increase of [Ca(2+)](i) could be detected 4 h after the removal of combined nitrogen, and this increase was suppressed by excessive CcbP. These results suggest that Ca(2+) ions play very important roles in hetR induction and heterocyst differentiation.     

Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp. PCC 7120, can be separated by mutation

The HetR protein has long been recognized as a key player in the regulation of heterocyst development. HetR is known to possess autoproteolytic and DNA-binding activities. During a search for mutants of Anabaena sp. PCC 7120 that can overcome heterocyst suppression caused by overexpression of the patS gene, which encodes a negative regulator of differentiation, a bypass mutant strain, S2-45, was isolated that produced a defective pattern (Pat phenotype) of irregularly spaced single and multiple contiguous heterocysts (Mch phenotype) in combined nitrogen-free medium. Analysis of the S2-45 mutant revealed a R223W mutation in HetR, and reconstruction in the wild-type background showed that this mutation was responsible for the Mch phenotype and resistance not only to overexpressed patS, but also to overexpressed hetN, another negative regulator of differentiation. Ectopic overexpression of the hetRR223W allele in the hetRR223W background resulted in a conditionally lethal (complete differentiation) phenotype. Analysis of the heterocyst pattern in the hetRR223W mutant revealed that heterocysts differentiate essentially randomly along filaments, indicating that this mutation results in an active protein that is insensitive to the major signals governing heterocyst pattern formation. These data provide genetic evidence that, apart from being an essential activator of differentiation, HetR plays a central role in the signaling pathway that controls the heterocyst pattern.     

The patA gene product, which contains a region similar to CheY of Escherichia coli, controls heterocyst pattern formation in the cyanobacterium Anabaena 7120.

In Anabaena 7120, heterocysts (cells specialized for nitrogen fixation) develop at the ends of filaments and at intervals within each filament. We have isolated a mutant Anabaena strain that develops heterocysts mostly at the ends of filaments. This mutant, PAT-1, grows poorly under nitrogen-fixing conditions. The wild-type gene that complements the mutation in PAT-1, called patA, was cloned and sequenced. The predicted PatA protein contains 379 amino acids distributed among three "domains" based on predictions of hydropathy and flexibility. The carboxyl-terminal domain is very similar to that of CheY and other response regulators in two-component regulatory systems in eubacteria. The patA mutation suppresses the multiheterocyst phenotype produced by extra copies of the wild-type hetR gene described previously, suggesting that PatA and HetR are components of the same environment-sensing regulatory circuit in Anabaena.     

Expression of a bacterial mtlD gene in transgenic tobacco leads to production and accumulation of mannitol.

A bacterial gene encoding mannitol-1-phosphate dehydrogenase, mtlD, was engineered for expression in higher plants. Gene constructions were stably incorporated into tobacco plants. The mtlD gene was expressed and translated into a functional enzyme in tobacco, resulting in the synthesis and accumulation of mannitol, which was identified by NMR and mass spectroscopy. Mannitol concentrations exceeded 6 mumol/g (fresh weight) in the leaves and in the roots of some transformants, whereas this sugar alcohol was not detected in these organs of wild-type tobacco plants or of untransformed tobacco plants that underwent the same regeneration scheme. These experiments demonstrate that branch-points in plant carbohydrate metabolism can be generated by which novel gene products can utilize endogenous substrates to divert metabolic energy into novel compounds. Additionally, the system described here allows for physiological studies in which the responses of wild-type and transgenic tobacco to various environmental stimuli can be compared directly. Such studies will facilitate our understanding of the roles of sugar alcohols (e.g., in stress tolerance) in higher plants.     

A gene encoding a protein related to eukaryotic protein kinases from the filamentous heterocystous cyanobacterium Anabaena PCC 7120.

Protein kinases play essential roles in the development of eukaryotic cells. These enzymes display various degrees of sequence similarity in their catalytic domains. This conservation has allowed the identification of protein kinases in a variety of organisms, including the Gram-negative bacterium Myxococcus xanthus. In this study, sequences related to those encoding eukaryotic protein kinases were amplified by PCR from DNA of Anabaena PCC 7120, a filamentous cyanobacterium that differentiates cells specifically for nitrogen fixation, called heterocysts, under conditions of combined nitrogen limitation. Results from Southern hybridization and sequencing of PCR products suggest the presence of a family of similar protein kinases in this strain. One of the corresponding genes (pknA) was isolated from a gene library. The N-terminal region of its amino acid sequence shows significant similarity to the catalytic domains of eukaryotic-type protein kinases. Expression of this gene was found to be developmentally regulated. Inactivation of pknA led to colonies that appeared light green and rough in the absence of combined nitrogen. Mutant filaments produce fewer heterocysts than wild-type ones. These results suggest that pknA is required for both normal cellular growth and differentiation of Anabaena PCC 7120.     

Regulation of differentiation of murine progenitor cells derived from blast cell colonies under serum-deprived conditions

We have examined the effect of interleukin 3 (IL-3), granulocyte-macrophage (GM)-, granulocyte (G)-, and macrophage (M)-colony-stimulating factors (CSFs) on the induction of GM colonies from highly enriched murine hematopoietic progenitor cells under serum-deprived conditions. Each growth factor was tested alone or in combination with suboptimal concentrations of the others. The effect of each CSF on GM colony growth in fetal bovine serum (FBS)-supplemented cultures of unfractionated marrow cells is reported for comparison. GM-CSF induced GM colony growth in serum-deprived cultures of purified progenitor cells to the same extent as in FBS-supplemented cultures of unfractionated marrow cells. In contrast, IL-3 was only one-tenth as active in promoting the growth of enriched progenitor cells under serum-deprived conditions when compared with its effect on colony growth from unfractionated marrow. M-CSF and G-CSF were almost completely ineffective in both cases. G-CSF induction of GM colony growth from purified progenitor cells was restored by addition of suboptimal concentrations of IL-3 or GM-CSF, suggesting that either IL-3 or GM-CSF is required to observe the effect of G-CSF. Addition of G-CSF to GM-CSF-stimulated cultures did not increase the maximal number of colonies detected, indicating that these two growth factors may act on the same subset of progenitor cells. Addition of GM-CSF or IL-3 to IL-3- or GM-CSF-stimulated cultures, respectively, increased by 40% the maximal number of colonies detected, suggesting that these two factors act on at least partially separate subsets of GM progenitors. These data parallel the recent observations on the control of human GM colony formation under FBS-deprived conditions and support a model for the control of myeloid differentiation that requires the interplay of different growth factors.     

A retroviral promoter is sufficient to convert proto-src to a transforming gene that is distinct from the src gene of Rous sarcoma virus.

The src genes of four natural isolates of avian sarcoma viruses differ from cellular proto-src in two genetic substitutions: the promoter of the cellular gene is replaced by a retroviral counterpart, and at least six codons from the 3' terminus are replaced by retroviral or heterologous cell-derived elements. Since virus constructs with a complete proto-src coding region failed to transform avian cells but acquired transforming function by point mutations of various codons, it has been proposed that point mutation is sufficient to convert proto-src to a transforming gene. However, promoter substitution is sufficient to convert two other proto-onc genes, proto-ras and proto-myc, to retroviral transforming genes. In view of this, we have reexamined whether promoter substitution, point mutation, or both are necessary to convert proto-src into a retroviral transforming gene. It was found that a recombinant virus (RpSV), in which the src gene of Rous sarcoma virus (RSV) was replaced by the complete coding region of proto-src, transformed quail and chicken embryo cells. The oncogene of RpSV differs from the src gene of RSV in three genetic properties: (i) it is weaker--e.g., transformed cells are flatter; (ii) it is slower--e.g., focus formation takes 9 to 12 days compared to 4 days for RSV; and (iii) its host range is narrower than that of RSV--e.g., only subsets of heterogeneous embryo cells are transformed by RpSV even after weeks or months. Replacement of the proto-src 3' terminus of RpSV by that of src from RSV generates a recombinant virus (RpvSV) that equals RSV in transforming function. It is concluded that a retroviral promoter, naturally substituted via illegitimate recombination with retroviruses, is sufficient to convert at least three proto-onc genes, src, myc, and ras, to retroviral transforming genes.     

Sequence of the nifD gene coding for the alpha subunit of dinitrogenase from the cyanobacterium Anabaena

The nucleotide sequence of nifD, the structural gene for the α subunit of dinitrogenase from Anabaena 7120, has been determined. The coding sequence contains 1,440 nucleotides, which predict an amino acid sequence of 480 residues and M_r of 54,283. The predicted sequence contains eight cysteines, of which five are conserved with respect to adjoining sequences and position relative to the α subunits of dinitrogenase from Azotobacter, Clostridium, and Klebsiella. Because there are also five conserved cysteines in the β subunit of Anabaena dinitrogenase [Mazur, B. J. & Chiu, C.-F. (1982) Proc. Natl. Acad. Sci. USA 79, 6782-6786], the number of cysteine residues participating as ligands to FeS clusters is likely to be 20 per α₂β₂ tetramer. This number is sufficient to accommodate the known four Fe₄S₄ clusters, leaving at least four cysteines to be shared among the two FeMo cofactors and the more poorly characterized two-iron center. Although the α- and β-subunit gene sequences are not recognizably homologous, their secondary structures, predicted from the sequences, indicate similar domains around three of the conserved cysteine residues.     

Is the product of the src gene a promoter?

Addition of a potent promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), to primary avian tendon or chicken embryo fibroblast cells infected with a temperature-sensitive mutant of Rous sarcoma virus produced a complete transformed phenocopy at the nonpermissive temperature by the criteria tested. While normal, uninfected cultures also shifted towards a transformed phenotype after TPA addition, they did not achieve the same degree of morphological and biochemical alteration seen in virus-infected, TPA-treated cells. It is proposed that viral carcinogenesis, despite its rapidity, may occur in two stages: an "initiation" step caused by expression of a part of viral genome other than src (or by integration) and a promotion step (itself a multistep process) caused by the activation of the src gene. The src gene product could be enhanced or replaced by other promoting agents.