Two marine, unicellular aerobic nitrogen-fixing cyanobacteria, Cyanothece strain BH63 and Cyanothece strain BH68, were isolated from the intertidal sands of the Texas Gulf coast in enrichment conditions designed to favor rapid growth. By cell morphology, ultrastructure, a GC content of 40%c, and aerobic nitrogen fitation ability, these strains were assigned to the genus Cyanothece. These strains can use molecular nitrogen as the sole nitrogen source and are capable of photoheterotrophic growth in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and glycerol. The strains demonstrated a doubling time of 10 to 14 h in the presence of nitrate and 16 to 20 h under nitrogen-fixing conditions. Rapid growth of nitrogen-fixing cultures can be obtained in continuous light even when the cultures are continuously shaken or bubbled with air. Under 12-h alternating light and dark cycles, the aerobic nitrogenase activity was confined to the dark phase. The typical rates of aerobic nitrogenase activity in Cyanothece strains BH63 and BH68 were 1,140 and 1,097 nmol of C2H2 reduced per mg (dry weight) per h, respectively, and nitrogenase activity was stimulated twofold by light. Ultrastructural observations revealed that numerous inclusion granules formed between the photosynthetic membranes in cells grown under nitrogen-fixing conditions. These Cyanothece strains possess many characteristics that make them particularly attractive for a detailed analysis of the interaction of nitrogen fixation and photosynthesis in an aerobic diazotroph.In all nitrogen-fixing organisms, the nitrogen fixation process is carried out by nitrogenase, an extremely oxygensensitive enzyme. Microorganisms have developed various strategies to protect their nitrogenase from oxygen inhibition (2, 5, 6). Among the nitrogen-fixing microorganisms, the cyanobacteria occupy a unique position because these are the only oxygenic photosynthetic organisms capable of nitrogen fixation under aerobic conditions (13). Nitrogen fixation has been reported for all three major morphological groups of cyanobacteria: heterocystous filamentous, nonheterocystous filamentous, and unicellular forms (5, 13). The oxygen protection mechanisms employed by these organisms vary considerably. In the heterocystous filamentous strains, about 5 to 10% of the cells undergo morphological differentiation into specialized cells called heterocysts under nitrogen-fixing conditions (13,49). In this arrangement, nitrogen fixation and photosynthetic oxygen evolution are spatially separated, so that oxygenic photosynthesis takes place in vegetative cells and nitrogen fixation occurs in heterocysts. The fixed nitrogen from these cells is exported to the neighboring vegetative cells, and the reductant for nitrogen fixation is imported from the vegetative cells (5, 49).There are many genera of nonheterocystous cyanobacteria, both filamentous and unicellular, that are capable of nitrogen fixation. The nonheterocystous filamentous forms have been placed in the genera Trichodesmium, Oscillatoria, an...

The molecular chaperonins such as GroEL are now widely regarded as essential components for the stabilization of integral membrane or secretory proteins before membrane insertion or translocation, as well as for the assembly of macromolecular complexes such as ribulose bisphosphate carboxylase-oxygenase. The groESL operon of Synechococcus sp. strain PCC 7942 was cloned as two independent lacZ-groEL translational fusions by immunoscreening a lambda ZAP genomic expression library and then sequenced. The derived amino acid sequences of the GroES and GroEL proteins demonstrated very high levels of amino acid identity with cognate chaperonins from bacteria and chloroplasts. The bicistronic 2.4-kilobase transcript from this operon, barely detectable in RNA preparations from cells grown at 30 degrees C, accumulated approximately 120-fold in preparations from cells grown for 20 min at 45 degrees C. Under these conditions, GroEL protein accumulated to 10-fold-higher levels. Primer extension analysis was used to identify a cyanobacterial heat shock promoter located at -81 base pairs from the groES initiation codon. The transcriptional -10 and -35 sequences differ slightly from Escherichia coli consensus heat shock promoter sequences.

The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is involved in photosystem II water oxidation was cloned and sequenced from the genome of the cyanobacterium Anacystis nidulans R2. The gene (here designated woxA) was shown to be present in a single copy. The deduced amino acid sequence indicated that the translation product consisted of 277 amino acid residues with a Mr of 29,306. The comparison of the sequence with that of mature MSP from spinach chloroplasts suggested that the translation product is a precursor whose amino-terminal 28 amino acid residues represent the signal peptide for the protein to cross the thylakoid membrane into the lumen. The length of the putative signal peptide was less than half that of the transit peptide for thylakoid-lumenal proteins of higher plants, whereas the structural prorfle of the putative signal peptide was similar to that of the carboxyl-terminal portion of the higher plant transit peptides. The amino acid sequence of the mature A. nidulans R2 MSP showed rather low homology (48-49%) to higher plant MSPs, but the conserved amino acid residues appeared to be clustered. Five clusters were tentatively assigned, in which the homology values were in a range of 66-70%. Domains essential for the functioning of MSP are expected to be situated in these clusters. It is of note that the two cysteine residues in MSP were conserved, and the disulfide linkage between them may play an important role in maintaining the tertiary structure of MSP.Photosynthetic water oxidation is closely associated with photosystem II (PSII) activity, and many of the structural components responsible for this mechanism are located at the lumenal surface of the thylakoid membrane. The minimum unit capable of photooxidizing water seems to contain five intrinsic proteins and one extrinsic protein (1-3)-namely, the 47-kDa and 43-kDa chlorophyll-a-binding proteins, the D1 and D2 proteins, cytochrome b559, and the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein). The MSP is involved in water oxidation but not in the photochemical charge separation or in electron transport on the reducing side of PSII (4). Biochemical studies on 02-evolving PSII preparations suggest that MSP binds to the putative Mn-binding protein and keeps two of the four Mn atoms associated with PSII (5). The intrinsic Mn-binding protein has not yet been identified but may be the D1 or D2 protein (6). The MSP seems to stabilize the Mn cluster and allows the water-oxidizing center to operate with the best efficiency (5).The spinach MSP is physicochemically and chemically well characterized (7), and its amino acid sequence is reported to have partial homology to the putative Mn-binding site of bacterial Mn superoxide dismutases (8). In higher plants, it is suggested that the MSP is encoded by nuclear DNA and that the precursor synthesized in the cytoplasm is posttransiationally transported into the chloroplast (9, 10). The precursor must undergo at least two processing steps t...

The aerobic nitrogen-fixing cyanobacterium, Cyanothece sp. BH68K produces non-mucoid variants defective in exopolysaccharide (EPS) production at a high frequency. The EPS-producing wild-type colonies (EPS(+)) have a characteristic smooth and shiny appearance which allows them to be easily distinguished from the EPS(-) variants. When grown on agar plates lacking a source of combined nitrogen, the EPS(-) variants exhibited a yellow phenotype typical of nitrogen starvation. These EPS(-) variants showed varying degrees of reversion back to the EPS(+) phenotype. After reversion, they exhibited normal diazotrophic growth on agar plates. Alcian blue and ruthenium red staining indicated that the EPS is an acidic polysaccharide, which is present as a loose network around the cell, and which can be completely removed by low speed centrifugation. The accumulation of EPS takes place mainly during the stationary phase. All EPS(-) variants failed to produce any EPS. Analysis of growth of wild-type and EPS(-) variants revealed that EPS production is beneficial for diazotrophic growth on solid medium, but not in liquid medium. In addition, EPS phenotypic alteration may have some advantage in the dispersal of cells from one place to another in the natural environment.

We describe the cloning and sequencing of a gene from the cyanobacterium Synechococcus sp. strain PCC7942, designated irpA (iron-regulated protein A), that encodes for a protein involved in iron acquisition or storage. Polyclonal antibodies raised against proteins which accumulate during iron-deficient growth were used as probes to isolate immunopositive clones from a lambda gt11 genomic expression library. The clone, designated lambda gtAN26, carried a 1.7-kilobase (kb) chromosomal DNA insert and was detected by cross-reactivity with antibody against a 36-kilodalton protein. It was possible to map a 20-kb portion of the chromosome with various DNA probes from lambda gt11 and lambda EMBL-3 clones, and Southern blot analysis revealed that the irpA gene was present in a single copy and localized within a 1.7-kb PstI fragment. DNA sequencing revealed an open reading frame of 1,068 nucleotides capable of encoding 356 amino acids which yields a protein with a molecular weight of 38,584. The hydropathy profile of the polypeptide indicated a putative N-terminal signal sequence of 44 amino acid residues. IrpA is a cytoplasmic membrane protein as determined by biochemistry and electron microscopy immunocytochemistry. The upstream region of the irpA gene contained a consensus sequence similar to the aerobactin operator in Escherichia coli. This fact, plus a mutant with a mutation in irpA that is unable to grow under iron-deficient conditions, led us to suggest that irpA is regulated by iron and that the gene product is involved in iron acquisition or storage.

The gene (cbpA) press). This research has involved primarily the regulation of phycobilisome biosynthesis (15,16,44; Bryant, in press), but some very interesting work has been performed on gas vesicle formation (transcription of gvp genes) during hormogonia differentiation in Calothrix strain PCC 7601 (44). Taken together, these studies have shown that both transcriptional and posttranscriptional control are involved in photoregulation of these processes. Although no photoreceptor has yet been isolated, the physiological evidence strongly suggests that such molecules exist and mediate responses to light wavelength (16). We would like to extend such studies to other genes, particularly those that code for membrane proteins.We have been involved with an analysis of membrane components in the unicellular cyanobacteria, with particular emphasis on the photosynthetic membrane (reviewed in references 4 and 41). It has become increasingly clear that there are three distinct membrane systems in the cyanobacteria (photosynthetic thylakoids and the inner and outer envelope membranes), each with its own structural and

Procedures for isolating RNA from bacteria involve disruption of the cells, followed by steps to separate the RNA from contaminating DNA and protein. Lysis strategies differ in the protocols presented in this unit, including chemical degradation of gram-negative cell walls using sucrose/detergent or lysozyme, and sonication to break open gram-positive cell walls. Combinations of enzymatic degradation, organic extraction, and alcohol or salt precipitation are employed in the procedures to isolate the RNA from other cellular components, and various inhibitors of ribonuclease activity (diethylpyrocarbonate, vanadyl-ribonucleoside complex, and aurintricarboxylic acid) are described. If extremely high-quality RNA is required (e.g., for gene expression studies), instructions are provided for CsCl step-gradient centrifugation to remove all traces of contaminating DNA.

In cyanobacteria, ammonium represses expression of proteins involved in nitrogen fixation and assimilation. The global nitrogen regulator gene ntcA encodes a DNA-binding protein, NtcA, that is a transcriptional activator of genes subject to nitrogen control. We report the cloning and sequencing of the ntcA gene from a nitrogen-fixing unicellular cyanobacterium, Cyanothece sp. strain BH68K. The gene comprises 678 nucleotides, and the deduced NtcA protein contains 226 amino acids with a predicted molecular weight of 25,026. In addition, ntcA mRNA levels were measured in cells grown under different nitrogen regimes. Under nitrogenfixing conditions, ntcA transcripts were weakly expressed. Furthermore, ntcA expression was diminished or inversely proportional to nifHDK expression. Conversely, ntcA expression increased in nitrate-grown cells, and a concentration-dependent increase was seen in ammonium-grown cells up to 1 mM NH 4 Cl. These results indicate that ntcA is involved more in nitrogen assimilation than in nitrogen fixation and also imply that the rhythmic expression of ntcA and nifHDK transcription may be under the control of a circadian clock.