We describe a simple algorithm for computing a homology score for Escherichia coli promoters based on DNA sequence alone. The homology score was related to 31 values, measured in vitro, of RNA polymerase selectivity, which we define as the product KBk2, the apparent second order rate constant for open complex formation. We found that promoter strength could be predicted to within a factor of +/-4.1 in KBk2 over a range of 10(4) in the same parameter. The quantitative evaluation was linked to an automated (Apple II) procedure for searching and evaluating possible promoters in DNA sequence files.

A gene (designated ecaA) encoding a vertebrate-like (␣-type) carbonic anhydrase (CA) has been isolated from two disparate cyanobacteria, Anabaena sp. strain PCC 7120 and Synechococcus sp. strain PCC 7942. The deduced amino acid sequences correspond to proteins of 29 and 26 kDa, respectively, and revealed significant sequence similarity to human CAI and CAII, as well as Chlamydomonas CAHI, including conservation of most active-site residues identified in the animal enzymes. Structural similarities between the animal and cyanobacterial enzymes extend to the levels of antigenicity, as the Anabaena protein cross-reacts with antisera derived against chicken CAII. Expression of the cyanobacterial ecaA is regulated by CO 2 concentration and is highest in cells grown at elevated levels of CO 2 . Immunogold localization using an antibody derived against the ecaA protein indicated an extracellular location. Preliminary analysis of Synechococcus mutants in which ecaA has been inactivated by insertion of a drug resistance cassette suggests that extracellular carbonic anhydrase plays a role in inorganic-carbon accumulation by maintaining equilibrium levels of CO 2 and HCO 3 ؊ in the periplasm.Carbonic anhydrase (CA), a zinc metalloenzyme, catalyzes the reversible hydration of CO 2 and plays a significant role in such processes as pH homeostasis, respiratory gas exchange, photosynthesis, and ion transport (3,6,24). On the basis of distinct amino acid sequences (including active-site regions), three major CA groups have been described: (i) the ␣, or "eukaryotic," group, which includes CA isoforms found in vertebrates; (ii) the ␤, or "bacterial," group, which includes CA enzymes in eubacteria and structurally similar CA isoforms localized in the higher-plant chloroplast and cytosol; and (iii) a recently identified ␥, or "archaebacterial," group of CAs thought to play a role in acetate metabolism (1, 12). Each of these CA types is presumed to have evolved independently (12). In the cyanobacteria, a ␤-type CA has been identified previously and was shown to be required for photosynthesis at air levels of CO 2 (8). Efficient photosynthetic inorganic carbon (C i ) assimilation by cyanobacteria at these limiting levels of C i requires the operation of CO 2 -concentrating mechanisms (CCMs) (3,4,6,13). The activity of CCMs results in the energy-dependent transport of both HCO 3 Ϫ and CO 2 across the cytoplasmic membrane and the formation of a large intracellular pool of C i which is used during photosynthesis. Along with as yet unidentified CO 2 /HCO 3 Ϫ transporter proteins and energization components involving NADP(H) dehydrogenasedependent photosystem I electron flow (14, 19), a proteinencapsulated aggregate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) called a "carboxysome" is an integral part of a cyanobacterial CCM. It is hypothesized that associated with the cyanobacterial carboxysome is the ␤-type CA, a product of the icfA (also designated ccaA) gene (8,20,27). This CA exhibits significant amino acid sequence si...

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated heterocysts in response to a lack of combined nitrogen. Heterocysts are found approximately every 10th cell along the filament and are morphologically and biochemically specialized for nitrogen fixation. At least two DNA rearrangements occur during heterocyst differentiation in Anabaena sp. strain PCC 7120, both the result of developmentally regulated site-specffic recombination. The first is an l1-kilobase-pair (kb) deletion from within the 3' end of the nifD gene. The second rearrangement occurs near the nifS gene but has not been completely characterized. The DNA sequences found at the recombination sites for each of the two rearrangements show no similarity to each other. To determine the topology-of the rearrangement near the nitS gene, cosmid libraries of vegetative-cell genomic DNA were constructed and used to clone the region of the chromosome involved in the rearrangement. Cosmid clones which spanned the DNA separating the two recombination sites that define the ends of the element were obtained. The restriction map of this region of the chromosome showed that the rearrangement was the deletion of a 55-kb DNA element from the heterocyst chromosome. The excised DNA was neither degraded nor amplified, and its function, if any, is unknown. The 55-kb element was not detectably transcribed in either vegetative cells or heterocysts. The deletion resulted in placement of the rbcLS operon about 10 kb from the nijS gene on the chromosome. Although the nipD 11-kb and nifS 55-kb rearrangements both occurred under normnal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 is accompanied by two developmentally regulated genome rearrangements (8,9,14). Unlike most DNA rearrangements in procaryotic organisms, such as the movement of transposable elements and phase variation inversions which occur at very low frequencies (33), rearrangement of the heterocyst genome is tightly regulated by an environmentally induced developmental sequence.Heterocysts are highly specialized, terminally differentiated cells which reduce atmospheric dinitrogen to ammonia and then export the fixed nitrogen to neighboring vegetative cells in the form of glutamine (reviewed in references 12 and 37). Differentiation of a vegetative cell into a heterocyst is repressed either by the presence of an external source of combined nitrogen such as ammonia or by the close proximity of neighboring heterocysts in the filament. In the absence of a combined nitrogen source, approximately 10% of the cells along a filament form heterocysts at a relatively constant interval, producing a simple one-dimensional pattern of development.Global changes in gene expression occur during heterocyst differentiation (5, 22). Most notably, nitrogen fixation (nif) genes are induced an...

In the absence of a combined nitrogen source, such as ammonia, approximately every tenth vegetative cell along filaments of the cyanobacterium Anabaena develops into a heterocyst, a terminally differentiated cell that is morphologically and biochemically specialized for nitrogen fixation. At least two specific DNA rearrangements involving the nitrogen-fixation (nif) genes occur during heterocyst differentiation, one within the nifD gene and the other near the nifS gene. The two rearrangements have several properties in common. Both occur quantitatively in all heterocyst genomes, both occur at approximately the same developmental time, late in the process of heterocyst differentiation, and both result from site-specific recombination between short repeated DNA sequences. We report here the nucleotide sequences found at the site of recombination near the nifS gene. These sequences differ from those found previously for the nifD rearrangement, suggesting that the two rearrangements are catalysed by different enzymes and may be regulated independently. We also show that the nifS gene is transcribed only from rearranged genomes.

The glbN gene of Nostoc commune UTEX 584 is juxtaposed to nifU and nifH, and it encodes a 12-kDa monomeric hemoglobin that binds oxygen with high affinity. In N. commune UTEX 584, maximum accumulation of GlbN occurred in both the heterocysts and vegetative cells of nitrogen-fixing cultures when the rate of oxygen evolution was repressed to less than 25 mol of O 2 mg of chlorophyll a ؊1 h ؊1 . Accumulation of GlbN coincided with maximum synthesis of NifH and ferredoxin NADP ؉ oxidoreductase (PetH or FNR). A total of 41 strains of cyanobacteria, including 40 nitrogen fixers and representing 16 genera within all five sections of the cyanobacteria were screened for the presence of glbN or GlbN. glbN was present in five Nostoc strains in a single copy. Genomic DNAs from 11 other Nostoc and Anabaena strains, including Anabaena sp. strain PCC 7120, provided no hybridization signals with a glbN probe. A constitutively expressed, 18-kDa protein which cross-reacted strongly with GlbN antibodies was detected in four Anabaena and Nostoc strains and in Trichodesmium thiebautii. The nifU-nifH intergenic region of Nostoc sp. strain MUN 8820 was sequenced (1,229 bp) and was approximately 95% identical to the equivalent region in N. commune UTEX 584. Each strand of the DNA from the nifU-nifH intergenic regions of both strains has the potential to fold into secondary structures in which more than 50% of the bases are internally paired. Mobility shift assays confirmed that NtcA (BifA) bound a site in the nifU-glbN intergenic region of N. commune UTEX 584 approximately 100 bases upstream from the translation initiation site of glbN. This site showed extensive sequence similarity with the promoter region of glnA from Synechococcus sp. strain PCC 7942. In vivo, GlbN had a specific and prominent subcellular location around the periphery of the cytosolic face of the cell membrane, and the protein was found solely in the soluble fraction of cell extracts. Our hypothesis is that GlbN scavenges oxygen for and is a component of a membrane-associated microaerobically induced terminal cytochrome oxidase.

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We show that the occurrence and homology score (1) of promoter-sites in DNA depends upon the base composition of the DNA. We used simple probability theory to calculate the mean homology score expected for all promoter-sites that had a specific match in the canonical hexamers. By using the square root of this mean score as a measure of significance, we objectively classify all promoter-sites which are reported. We tested the theoretical approach in two ways. First, we used the program (PROMSEARCH)' to analyzes 150,000 base-pairs of random sequence DNA with different base compositions and we found excellent agreement with the theoretical predictions. Our second test was the analysis of a number of sequences drawn from the GENBANK DNA sequence database. We have analyzed 20 bacterial and bacteriophage sequences, which consisted of at least one operon, for promoter-sites. We found no absolute preference for promoter-sites within noncoding regions. We show the results of analyzing the phages X, T7 and fd, and the E. coli lac operon. The major known promoters in these sequences were all found correctly. We discuss the question of the location of a number of minor promoter-sites and show how PROMSEARCH can be used to help identify the correct location of the promoter. This approach can be applied to the search for any DNA site and should allow greater objectivity when comparing DNA sequences for meaningful subsequences.

The nucleotide sequence of a region located downstream of the nifB gene, both in the cyanobacterium Anabaena sp. strain PCC 7120 and in Rhiizobium melloti, has been determined. This region contains a gene (fdxN) whose predicted polypeptide product strongly resembles typical bacterial ferredoins. Cyanobacteria have not previously been shown to contain bacterial-type ferredoxins. The presence of this gene suggests that nitrogen-fixing cyanobacteria have at least four distinct ferredoxins.The filamentous cyanobacterium Anabaena sp. strain PCC 7120 will, if subjected to starvation for a source of fixed nitrogen, undergo a developmental change in which approximately every 10th cell differentiates to form a heterocyst (12). The heterocyst provides an anaerobic environment in which nitrogenase can function. Prior to expression of the nitrogen fixation (nif) genes, at least two DNA rearrangements must occur (10, 11). In one rearrangement, 11 kilobase pairs of DNA is excised from within the 3' end of the nifD gene, thereby restoring the nipD reading frame and allowing expression of an intact nifHDK operon. The second rearrangement is now known to be the excision of approximately 55 kilobase pairs (J.