The role of HetN in maintenance of the heterocyst pattern in Anabaena sp. PCC 7120.

The gene hetN encodes a putative oxidoreductase that is known to suppress heterocyst differentiation when present on a multicopy plasmid in Anabaena sp. PCC 7120. To mimic the hetN null phenotype and to examine where HetN acts in the regulatory cascade that controls heterocyst differentiation, we replaced the native chromosomal hetN promoter with the copper-inducible petE promoter. In the presence of copper, heterocyst formation was suppressed in undifferentiated filaments. When hetN expression was turned off by transferring cells to media lacking copper, the filaments initially displayed the wild-type pattern of single heterocysts but, 48 h after the induction of heterocyst formation, a pattern of multiple contiguous heterocysts predominated. Suppression of heterocyst formation by HetN appears to occur both upstream and downstream of the positive regulator HetR: overexpression of hetN in undifferentiated filaments prevents the wild-type pattern of hetR expression as well as the multiheterocyst phenotype normally observed when hetR is expressed from an inducible promoter. Green fluorescent protein fusions show that the expression of hetN in wild-type filaments normally occurs primarily in heterocysts. We propose that HetN is normally involved in the maintenance of heterocyst spacing after the initial heterocyst pattern has been established, but ectopic expression of hetN can also block the initial establishment of the pattern.

Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions.

Heterocyst differentiation in the filamentous cyanobacterium Anabaena PCC 7120 requires a functional hetR gene. Increased expression of the hetR gene is seen in developing and mature heterocysts in response to fixed nitrogen limitation. We mapped four likely transcriptional start sites for hetR and identified a specific transcript that is positively autoregulated. By using the copper-responsive petE promoter from Anabaena PCC 7120 to drive hetR expression, we show that ectopic expression of hetR increases heterocyst frequency and induces heterocyst differentiation under fully repressing conditions. Coexpression of a reporter gene shows that expression from the petE promoter is smoothly induced depending on the amount of copper supplied. In the heterocyst pattern mutant PatA, where terminally positioned heterocysts are formed almost exclusively, expression of the petEhetR fusion does not result in the formation of intercalary heterocysts. These results suggest that although the intracellular concentration of HetR has to be elevated for the differentiation decision, PatA plays a role as well. This role may be in the form of posttranslational modification of HetR, because PatA is a member of the response regulator family of proteins.

The hglK gene is required for localization of heterocyst-specific glycolipids in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant strain 543 of the cyanobacterium Anabaena sp. strain PCC 7120 was originally isolated as a Fox- mutant following chemical mutagenesis. Ultrastructural analysis shows that in nitrogen-replete media the vegetative cells of the mutant are more cylindrical and have thicker septa than those of the wild type, while in nitrogen-free media the mutant heterocysts lack the normal glycolipid layer external to the cell wall. Although this layer is absent, strain 543 heterocysts nevertheless contain heterocyst-specific glycolipids, as determined by thin-layer chromatography. The mutation in strain 543 is in a gene we have named hglK, encoding a protein of 727 amino acids. The wild-type HglK protein appears to contain four membrane-spanning regions followed by 36 repeats of a degenerate pentapeptide sequence, AXLXX. The mutation in strain 543 introduces a termination codon immediately upstream of the pentapeptide repeat region. A mutant constructed by insertion of an antibiotic resistance cassette near the beginning of the hglK gene has the same phenotype as strain 543. We propose that hglK encodes a protein necessary for the localization of heterocyst glycolipids and that this function requires the pentapeptide repeats of the HglK protein.

Characterization of toxin-producing cyanobacteria by using an oligonucleotide probe containing a tandemly repeated heptamer.

Cyanobacteria produce toxins that kill animals. The two main classes of cyanobacterial toxins are cyclic peptides that cause liver damage and alkaloids that block nerve transmission. Many toxin-producing strains from Finnish lakes were brought into axenic culture, and their toxins were characterized. Restriction fragment length polymorphism analysis, probing with a short tandemly repeated DNA sequence found at many locations in the chromosome of Anabaena sp. strain PCC 7120, distinguishes hepatotoxic Anabaena isolates from neurotoxin-producing strains and from Nostoc spp.

A short-filament mutant of Anabaena sp. strain PCC 7120 that fragments in nitrogen-deficient medium.

Strain 129 is a fragmentation mutant of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Growing with fixed nitrogen, this mutant forms filaments that are much shorter than wild-type filaments. Following starvation for fixed nitrogen, strain 129 becomes nearly unicellular and forms few heterocysts, although electron microscopy suggests that proheterocysts form while fragmentation occurs. Starvation for sulfate, phosphate, iron, and calcium does not cause this fragmentation. The affected gene in strain 129, fraC, was cloned by complementation and characterized. It encodes a unique 179-amino-acid protein rich in phenylalanine. Insertional inactivation of the chromosomal copy of fraC results in a phenotype identical to that of strain 129, while complementation using a truncated version of FraC results in only partial complementation of the original mutant. Heterocysts could be induced to form in N-replete cultures of strain 129, as in wild-type cells, by supplying extra copies of the hetR gene on a plasmid. Thus, FraC is required for the integrity of cell junctions in general but is apparently not directly involved in normal differentiation and nitrogen fixation.

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena sp. strain PCC 7120.

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacterium Anabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of the Anabaena ADPGlc PPase gene and its expression in Escherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48,347 Da which is in agreement with the molecular mass determined by SDS-PAGE for the Anabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and the E. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in the Anabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in an E. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the native Anabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be the Anabaena enzyme.

Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120.

Approximately 140 mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on media lacking fixed nitrogen (Fix-) were isolated after mutagenesis with diethyl sulfate and penicillin enrichment. A large cosmid library of wild-type Anabaena sp. strain PCC 7120 DNA was constructed in a mini-RK-2 shuttle vector, and seven mutants representing several morphologically abnormal heterocyst phenotypes were complemented. One of these mutants, 216, failed to differentiate heterocysts. All of these mutants except 216 reduced acetylene under anaerobic conditions, indicating that they are not defective in nitrogen fixation per se. Several cosmids were isolated from each complemented mutant and in most cases showed similar restriction patterns. Comparisons of the complementing cosmids from mutant 216 and two other phenotypically distinct mutants by restriction enzyme analysis identified a common region. This region, when present in either a cosmid or a 9.5-kb NheI subclone, is capable of efficiently complementing all three mutants. A 2.4-kb subclone of this region complements mutant 216 only.

Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120.

Anabaena 7120 mutant 216 fails to differentiate heterocyst. We previously identified a 2.4-kb wild-type DNA fragment able to complement this mutant. We show here that the sequence of this fragment contains a single open reading frame (hetR), encoding a 299-amino-acid protein. Conjugation of deletion subclones of this fragment into strain 216 showed that the hetR-coding region is both necessary and sufficient for complementation of the Het- phenotype. The mutation in 216 is located at nucleotide 535 in the hetR gene, converting a serine at position 179 in the wild-type protein to an asparagine in the mutant. Interruption of the hetR gene in wild-type cells results in a mutant phenotype identical to that of 216. Both 216 and wild-type cells containing wild-type hetR on a plasmid display increased frequency of heterocysts, even on media containing fixed nitrogen. These results suggest that hetR encodes a product that is not only essential for but also controls heterocyst development. This putative regulatory protein lacks known structural motifs characteristic of transcription factors and probably acts at a level one or more steps removed from its target genes.

Expression, nucleotide sequence and mutational analysis of two open reading frames in the nif gene region of Anabaena sp. strain PCC7120.

A 1.8 kb transcript corresponding to a region of the Anabaena 7120 chromosome 4 kb downstream of the nifHDK operon appears 12-18 h after heterocyst induction. The DNA corresponding to this transcript was sequenced and found to contain two open reading frames, designated ORF 1 and ORF 2. Two polypeptides, of 30 kDa and 13 kDa, encoded by these ORFs were expressed in Escherichia coli. An apparent start site for the transcript, detected by S1 nuclease protection, was located 42 bp upstream of the ATG start codon of ORF 1. ORF 2 shows strong sequence similarity to ORF 6 in the nif gene region of Azotobacter vinelandii. ORF 1 was interrupted using a 1.4 kb neomycin resistance cassette and the resulting mutant grew very slowly on medium lacking combined nitrogen. The mutant had 45% of wild-type acetylene reduction activity, which could be complemented by a 2.8 kb EcoRI fragment of wild-type Anabaena DNA containing only ORF 1 and ORF 2. Thus, one or both of these ORFs is required for efficient nitrogen fixation in Anabaena.

Bacterial-type ferredoxin genes in the nitrogen fixation regions of the cyanobacterium Anabaena sp. strain PCC 7120 and Rhizobium meliloti.

The nucleotide sequence of a region located downstream of the nifB gene, both in the cyanobacterium Anabaena sp. strain PCC 7120 and in Rhizobium meliloti, has been determined. This region contains a gene (fdxN) whose predicted polypeptide product strongly resembles typical bacterial ferredoxins. 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.

Conservation of structure and location of Rhizobium meliloti and Klebsiella pneumoniae nifB genes.

Using transposon Tn5-mediated mutagenesis, an essential Rhizobium meliloti nitrogen fixation (nif) gene was identified and located directly downstream of the regulatory gene nifA. Maxicell and DNA sequence analysis demonstrated that the new gene is transcribed in the same direction as nifA and codes for a 54-kilodalton protein. In Klebsiella pneumoniae, the nifBQ operon is located directly downstream of a gene which is structurally and functionally homologous to the R. meliloti nifA gene. The DNA sequences of the K. pneumoniae nifB and nifQ genes (which code for 51- and 20-kilodalton proteins, respectively) were determined. The DNA sequence of the newly identified R. meliloti gene was approximately 50% homologous to the K. pneumoniae nifB gene. R. meliloti does not contain a gene homologous to nifQ directly downstream of nifB. The R. meliloti nifB product shares approximately 40% amino acid homology with the K. pneumoniae nifB product, and 10 of the 12 cysteine residues of the R. meliloti nifB product are conserved with 10 of the 17 cysteine residues of the K. pneumoniae nifB product.

Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae.

We have determined the complete nucleotide sequences of three functionally related nitrogen assimilation regulatory genes from Klebsiella pneumoniae and Rhizobium meliloti. These genes are: 1) The K. pneumoniae general nitrogen assimilation regulatory gene ntrC (formerly called glnG), 2) the K. pneumoniae nif-specific regulatory gene nifA, and 3) an R. meliloti nif-specific regulatory gene that appears to be functionally analogous to the K. pneumoniae nifA gene. In addition to the DNA sequence data, gel-purified K. pneumoniae nifA protein was used to determine the amino acid composition of the nifA protein. The K. pneumoniae ntrC and nifA genes code for proteins of 52,259 and 53,319 d respectively. The R. meliloti nifA gene codes for a 59,968 d protein. A central region within each polypeptide, consisting of approximately 200 amino acids, is between 52% and 58% conserved among the three proteins. Neither the amino termini nor the carboxy termini show any conserved sequences. Together with data that shows that the three regulatory proteins activate promoters that share a common consensus sequence in the -10 (5'-TTGCA-3') and -23 (5'-CTGG-3') regions, the sequence data presented here suggest a common evolutionary origin for the three regulatory genes.

Physical and genetic characterization of Rhizobium meliloti symbiotic mutants.

A set of 19 symbiotic mutants of Rhizobium meliloti obtained by a Tn5 "suicide plasmid" mutagenesis procedure was characterized genetically and physically. As part of this characterization, we showed that R. meliloti strain 1021, like other R. meliloti strains, contains a very large indigenous plasmid (greater than 300 Md) that carries the structural genes for nitrogenase (nifHDK genes). Among the 19 symbiotic mutations studied, at least six were shown to reside on the megaplasmid. By a "walking procedure" we obtained from a cosmid clone bank a set of overlapping cosmids that contained megaplasmid sequences contiguous to nifHDK. A 90 kb region of contiguous DNA from these cosmids was used to probe the mutant strains for rearrangements within this region. The same six mutations that were located on the megaplasmid mapped within the 90 kb region examined, which included the structural genes for nitrogenase (nifHDK). A majority of the mutations characterized in this study could not be correlated with a bona fide Tn5 insertion into a symbiotic gene.

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants.

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.