James W. Golden

Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena

Nitrogen fixation by the cyanobacterium Anabaena is carried out in heterocysts, specialized, non-dividing cells which differentiate under conditions of ammonia or nitrate deprivation. In Anabaena, heterocyst differentiation is accompanied by rearrangement of some nitrogen fixation genes. A site-specific recombination between an 11 base-pair direct repeat sequence flanking the nif K and nif D genes removes 11 kilobases of intervening DNA, resulting in juxtaposition of the two genes and an alteration of the nif D protein-coding sequence.

Heterocyst formation in Anabaena

Heterocystous cyanobacteria grow as multicellular organisms with a distinct one-dimensional developmental pattern of single nitrogen-fixing heterocysts separated by approximately ten vegetative cells. Several genes have been identified that are required for heterocyst development and pattern formation. A key regulator, HetR, has been recently shown to be aserine-type protease.

Suppression of heterocyst differentiation in Anabaena PCC 7120 by a cosmid carrying wild-type genes encoding enzymes for fatty acid synthesis

A cosmid containing a wild-type Anabaena PCC 7120 DNA fragment was found to suppress heterocyst differentiation, creating a Het phenotype in an otherwise wild-type strain. Curing of the cosmid restored the full wild-type Het+ Nif+ phenotype. The cosmid contains at least four genes encoding proteins with significant sequence similarity to enzymes involved in the synthesis of fatty acids. Selection for Nif+ revertants of the suppressed strain yielded modified cosmids, one of which contained a 10.2-kb transposon, Tas1, inserted into the promoter region of a gene encoding a protein with acyl carrier and beta-keto reductase domains. This gene, called hetN, was shown previously by Black and Wolk (J. Bacteriol. (1994) 176, 2282-2292) to inhibit heterocyst differentiation when present alone on a plasmid. Oddly, hetN gene transcription is detected later than 6 h into heterocyst differentiation.

PlmA, a New Member of the GntR Family, Has Plasmid Maintenance Functions in Anabaena sp. Strain PCC 7120

The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 maintains a genome that is divided into a 6.4-Mb chromosome, three large plasmids of more that 100 kb, two medium-sized plasmids of 55 and 40 kb, and a 5.5-kb plasmid. Plasmid copy number can be dynamic in some cyanobacterial species, and the genes that regulate this process have not been characterized. Here we show that mutations in an open reading frame, all1076, reduce the numbers of copies per chromosome of several plasmids. In a mutant strain, plasmids pCC7120delta and pCC7120zeta are both reduced to less than 50% of their wild-type levels. The exogenous pDU1-based plasmid pAM1691 is reduced to less than 25% of its wild-type level, and the plasmid is rapidly lost. The peptide encoded by all1076 shows similarity to members of the GntR family of transcriptional regulators. Phylogenetic analysis reveals a new domain topology within the GntR family. PlmA homologs, all coming from cyanobacterial species, form a new subfamily that is distinct from the previously identified subfamilies. The all1076 locus, named plmA, regulates plasmid maintenance functions in Anabaena sp. strain PCC 7120.

Identification and Inactivation of Three Group 2 Sigma Factor Genes in Anabaena sp. Strain PCC 7120

Three new Anabaena sp. strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized. Insertional inactivation of sigD, sigE, and sigF genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of sigD and sigE mutant strains to establish diazotrophic growth. A sigD sigE double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation. This double mutant could be complemented by wild-type copies of sigD or sigE, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants. However, the sigE gene was required for lysogenic development of the temperate cyanophage A-4L. Several other combinations of double mutations, especially sigE sigF, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation. We found no evidence for developmental regulation of the sigma factor genes. luxAB reporter fusions with sigD, sigE, and sigF all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown. Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups. Three morphologically and physiologically distant strains, Anabaena sp. strain PCC 7120, Synechococcus sp. strain PCC 7002, and Synechocystis sp. strain PCC 6803 each contain representatives of four subgroups. Unlike unicellular strains, Anabaena sp. strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria.

Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

patS Minigenes Inhibit Heterocyst Development of Anabaena sp. Strain PCC 7120

The patS gene encodes a small peptide that is required for normal heterocyst pattern formation in the cyanobacterium Anabaena sp. strain PCC 7120. PatS is proposed to control the heterocyst pattern by lateral inhibition. patS minigenes were constructed and expressed by different developmentally regulated promoters to gain further insight into PatS signaling. patS minigenes patS4 to patS8 encode PatS C-terminal 4 (GSGR) to 8 (CDERGSGR) oligopeptides. When expressed by P(petE), P(patS), or P(rbcL) promoters, patS5 to patS8 inhibited heterocyst formation but patS4 did not. In contrast to the full-length patS gene, P(hepA)-patS5 failed to restore a wild-type pattern in a patS null mutant, indicating that PatS-5 cannot function in cell-to-cell signaling if it is expressed in proheterocysts. To establish the location of the PatS receptor, PatS-5 was confined within the cytoplasm as a gfp-patS5 fusion. The green fluorescent protein GFP-PatS-5 fusion protein inhibited heterocyst formation. Similarly, full-length PatS with a C-terminal hexahistidine tag inhibited heterocyst formation. These data indicate that the PatS receptor is located in the cytoplasm, which is consistent with recently published data indicating that HetR is a PatS target. We speculated that overexpression of other Anabaena strain PCC 7120 RGSGR-encoding genes might show heterocyst inhibition activity. In addition to patS and hetN, open reading frame (ORF) all3290 and an unannotated ORF, orf77, encode an RGSGR motif. Overexpression of all3290 and orf77 under the control of the petE promoter inhibited heterocyst formation, indicating that the RGSGR motif can inhibit heterocyst development in a variety of contexts.

Cell-type specificity of the Anabaena fdxN-element rearrangement requires xisH and xisI.

The fdxN element, along with two other DNA elements, is excised from the chromosome during heterocyst differentiation in Anabaena sp. strain PCC 7120. Previous work showed that rearrangement of the fdxN element requires the xisF gene, which encodes a site‐specific recombinase, and suggested that at least one other heterocyst‐specific factor is involved. Here we report that the xisH and xisl genes are necessary for the heterocyst‐specific excision of the fdxN element. Deletion of a 3.2 kb region downstream of the xisF gene blocked the fdxN‐element rearrangement in hetero‐cysts. The 3.2 kb deletion was complemented by the two overlapping genes xisH and xisl. Interestingly, extra copies of xlsHI on a replicating plasmid resulted in the xisF‐dependent excision of the fdxN element in vegetative cells. Therefore, xisHI are involved in the control of cell‐type specificity of the fdxN rearrangement. The xisHI genes had no effect on the two other DNA rearrangements. The xisHl‐induced excision of the fdxN element produced strains lacking the element and demonstrates that the 55 kb element contains no essential genes. xisH and xisl do not show similarity to any known genes.

Heterocyst-Specific Excision of the Anabaena sp. Strain PCC 7120 hupL Element Requires xisC

In nitrogen-limiting conditions, approximately 10% of the vegetative cells in filaments of the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 differentiate into nitrogen-fixing heterocysts. During the late stages of heterocyst differentiation, three DNA elements, each embedded within an open reading frame, are programmed to excise from the chromosome by site-specific recombination. The DNA elements are named after the genes that they interrupt: nifD, fdxN, and hupL. The nifD and fdxN elements each contain a gene, xisA or xisF, respectively, that encodes the site-specific recombinase required for programmed excision of the element. Here, we show that the xisC gene (alr0677), which is present at one end of the 9,435-bp hupL element, is required for excision of the hupL element. A strain in which the xisC gene was inactivated showed no detectable excision of the hupL element. hupL encodes the large subunit of uptake hydrogenase. The xisC mutant forms heterocysts and grows diazotrophically, but unlike the wild type, it evolved hydrogen gas under nitrogen-fixing conditions. Overexpression of xisC from a plasmid in a wild-type background caused a low level of hupL rearrangement even in nitrogen-replete conditions. Expression of xisC in Escherichia coli was sufficient to produce rearrangement of an artificial substrate plasmid bearing the hupL element recombination sites. Sequence analysis indicated that XisC is a divergent member of the phage integrase family of recombinases. Site-directed mutagenesis of xisC showed that the XisC recombinase has functional similarity to the phage integrase family.

Developmental rearrangement of cyanobacterial nitrogen-fixation genes

Abstract 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.