Martin E. Mulligan's research while affiliated with University of Chicago and other places

Filamentous cyanobacteria such as Anabaena differentiate cells specialized for nitrogen fixation, called heterocysts. A large number of mutants of Anabaena 7120, defective in heterocyst differentiation, were isolated. For some of these, the gene that complements the mutation has been identified by conjugation with a cosmid library of wild-type DNA fragments. One early-acting gene has been sequenced. This gene, het R, is required to initiate differentiation, has no function in vegetative cells, and when present in extra copies in wild-type cells, causes the formation of multiple heterocysts. The organization of the genes for nitrogen fixation in Anabaena was also studied. The nif gene region of vegetative cell DNA of Anabaena 7120 carries two interrupting elements: an 11-kb element inserted near the carboxy terminus of the nifD gene and a 55-kb element inserted near the nifS gene. Both elements are flanked by directly repeated sequences within which site-specific recombination occurs during heterocyst differentiation. The result of these recombinations, in each case, is precise excision of the element and fusion of the heterocyst chromosome. In the case of the 11-kb element, excision leaves behind the intact nifHDK operon. The other rearrangement yields the nifB fdxN nifSU operon, where niifSU have the same significance as their Klebsiella counterparts.

A second nitrogen fixation (nif) operon in the cyanobacterium (blue-green alga) Anabaena (Nostoc) sp. strain PCC 7120 has been identified and sequenced. It is located just upstream of the nifHDK operon and consists of four genes in the order nifB, fdxN, nifS, and nifU. The three nif genes were identified on the basis of their similarity with the corresponding genes from other diazotrophs. The fourth gene, fdxN, codes for a bacterial type ferredoxin (Mulligan, M. E., Buikema, W. J., and Haselkorn, R. (1988) J. Bacteriol. 167, 4406-4410). The four genes are probably transcribed as a single operon, but are expressed at a lower level than the nifHDK operon, and only after a developmentally induced DNA rearrangement occurs that excises a 55-kilobase pair element from within the fdxN gene (Golden, J. W., Mulligan, M. E., and Haselkorn, R. (1987) Nature 327, 526-529; Golden, J. W., Carrasco, C. D., Mulligan, M. E., Schneider, G. J., and Haselkorn, R. (1988) J. Bacteriol. 170, 5034-5041). The promoter for the nifB operon was located by primer extension. Comparison of the nifB 5'-flanking sequence with the nifH 5'-flanking sequence did not reveal any consensus base pairs that would define a nif promoter for Anabaena. The operon contains two instances of 7-base pair directly repeated sequences: seven copies of the repeated sequence are found between the nifB and fdxN genes and six copies are found between the nifS and nifU genes. The function of these repeats is unknown.

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-specific recombination. The first is an 11-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 nifS 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 nifS gene on the chromosome. Although the nifD 11-kb and nifS 55-kb rearrangements both occurred under normal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.

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.

Anabaena is a filamentous cyanobacterium that produces specialized cells, called heterocysts at regular intervals along each filament when deprived of fixed nitrogen under aerobic conditions. heterocysts are anaerobic factories for nitrogen fixation. In Anabaena vegetative-cell DNA, the nifD gene, encoding the alpha subunit of nitrogenase, is interrupted by an 11 000 base pair DNA element. During the differentiation of heterocysts from vegetative cells, this 11 kilobase (kb) element is excised by site-specific recombination between short, directly repeated DNA sequences present at the ends of the element. The excision results in restoration of the nifD coding sequence and of the entire nifHDK transcription unit. A gene has been identified, within the 11 kb element, that is believed to encode the site-specific recombinase responsible for excision of the element during heterocyst differentiation. A second developmentally regulated gene arrangement has also been observed in Anabaena. This event occurs close to the nifS gene and involves a different set of repeated sequences, implying a different site-specific recombination system.

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.

During the differentiation of nitrogen-fixing heterocysts from photosynthetic vegetative cells of the cyanobacterium Anabaena, two DNA rearrangements have been observed. One is the excision, by site-specific recombination between directly repeated sequences, of an 11-kbp element that interrupts the nifD gene. The second occurs next to the nifS gene and involves a different site-specific recombination system.

The nifHDK genes encoding the nitrogenase complex polypeptides of Klebsiella pneumoniae were cloned on a single 6 kbp DNA fragment (Cannon et al 1979 and found to hybridize with DNA from every nitrogen-fixing microorganism tested (Ruvkun, Ausubel 1980; Mazur et al 1980). The cloned Klebsiella nifHDK DNA was subsequently used both to clone and to physically map the homologous genes from other organisms, including various Rhizobia, photosynthetic bacteria, and the cyanobacterium Anabaena. In most cases, the nifHDK gene organization as well as the gene sequences were found to be conserved. In general, the three genes are contiguous and transribed as a single unit. The exceptions are the slow-growing Rhizobia, described elsewhere in this volume, and the heterocyst-forming cyanobacteria, of which Anabaena is the best studied example.