ThetrpEGene Negatively Regulates Differentiation of Heterocysts at the Level of Induction in Anabaena sp. Strain PCC 7120

Abstract

Levels of 2-oxoglutarate (2-OG) reflect nitrogen status in many bacteria. In heterocystous cyanobacteria, a spike in the 2-OG level occurs shortly after the removal of combined nitrogen from cultures and is an integral part of the induction of heterocyst differentiation. In this work, deletion of one of the two annotated trpE genes in Anabaena sp. strain PCC 7120 resulted in a spike in the 2-OG level and subsequent differentiation of a wild-type pattern of heterocysts when filaments of the mutant were transferred from growth on ammonia to growth on nitrate. In contrast, 2-OG levels were unaffected in the wild type, which did not differentiate under the same conditions. An inverted-repeat sequence located upstream of trpE bound a central regulator of differentiation, HetR, in vitro and was necessary for HetR-dependent transcription of a reporter fusion and complementation of the mutant phenotype in vivo. Functional complementation of the mutant phenotype with the addition of tryptophan suggested that levels of tryptophan, rather than the demonstrated anthranilate synthase activity of TrpE, mediated the developmental response of the wild type to nitrate. A model is presented for the observed increase in 2-OG in the trpE mutant. The Krebs cycle intermediate 2-oxoglutarate (2-OG) is an intracellular indicator of nitrogen limitation in many bacteria (reviewed in reference 1). It is the carbon skeleton for assimilation of ammonia in bacteria that primarily use the GS-GOGAT system, and therefore, the concentration of 2-OG rises when ammonia is not available. Sensors of 2-OG include the ubiquitous PII superfamily of proteins and the CRP family protein NtcA, which in cyanobacteria functionally replaces the NtrB-NtrC system of enteric bacteria (reviewed in reference 2). Direct interaction with 2-OG increases the affinity of the NtcA transcriptional regulator for DNA targets and is necessary for initiation of transcription (3) and its interaction with PII frees PipX (4), which is thought to stabilize the active form of NtcA (5). NtcA regulates the expression of genes required for transformation of inorganic forms of nitrogen such as nitrate, nitrite, and dinitrogen into ammonia, as well as the developmental program of heterocystous cyanobacteria.Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates nitrogen-fixing heterocysts when a source of combined nitrogen is scarce (reviewed in references 6 and 7). During growth with a combined nitrogen source, usually ammonia or nitrate under laboratory conditions, Anabaena filaments are comprised exclusively of photosynthetic vegetative cells. When combined nitrogen is absent, however, a periodic pattern of morphologically distinct, nitrogen-fixing heterocyst cells is formed at approximately every 10th cell in the filament. The cascade of events involved in heterocyst differentiation can be divided into four phases: induction of differentiation, pattern formation, commitment to differentiation, and heterocyst maturation (8).The induction phase begins with ...

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120

“…The coding regions of murB and murC were amplified by PCR from Anabaena chromosomal DNA with the primer pairs murB-BamHI-F and murBSacI-R and murC-BamHI-F and murC-SacI-R, respectively. The products were cloned as BamHI-SacI fragments into the same sites of pPJAV360 (41) to create pPJAV371 (P BAD -murB) and pPJAV372 (P BADmurC).…”

Preventing Accidental Heterocyst Development in Cyanobacteria

“…CalA is not the first player reported to suppress heterocyst differentiation in Anabaena. A trpE mutant displayed a similar, and even much stronger, phenotype compared to that resulting from a knockdown of calA (29) (Fig. 1).…”

“…Anabaena has two genes encoding anthranilate synthase in the tryptophan biosynthesis pathway. One of the two, trpE, is a member of the hetR regulon (29). The trpE mutant forms about 8% heterocysts when cultured in the presence of nitrate, similar to the wild type under combined nitrogen deprivation; ammonium, on the other hand, can still repress heterocyst differentiation.…”

“…Tryptophan synthesis requires glutamine as a precursor. According the proposed model (29), tryptophan can be transformed by tryptophan transaminase, in a 2-OG-dependent manner, to glutamate and 3-indole pyruvate. In the absence of TrpE, less 2-OG would be consumed, resulting in a transient accumulation of 2-OG.…”

Mathematical models of nitrogen-fixing cell patterns in filamentous cyanobacteria

“…hetR transcription has the same regulation as ntcA plus the inhibition from patS. In (2015). When combined nitrogen (cN) is provided to the cell, there is only one stable fixed point (A), which corresponds to a state in which the production of both HetR and PatS is minimum (vegetative state).…”