Ana Valladares

Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development

Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR, and induction of hetR upon nitrogen step‐down depends on NtcA. The use of two out of the four transcription start points (tsps) described for the hetR gene (those located at positions –728 and –271) was found to be dependent on NtcA, and the use of the tsp located at position –271 was also dependent on HetR. Thus, autoregulation of hetR could take place via the activation of transcription from this tsp. Expression of ntcA in nitrogen‐fixing cultures was higher than in cells growing in the presence of ammonium or nitrate, and high expression of ntcA under nitrogen deficiency resulted from an increased use of tsps located at positions –180 and –49. The induction of the use of these tsps did not take place in ntcA or hetR mutant strains. These results indicate a mutual dependency in the induction of the regulatory genes hetR and ntcA that takes place in response to nitrogen step‐down in Anabaena cells. Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA‐dependent tsp was, however, not dependent on HetR.

An ABC‐type, high‐affinity urea permease identified in cyanobacteria

Urea is an important nitrogen source for many microorganisms, but urea active transporters have not been characterized at a molecular level in any bacterium. Cells of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 exhibited the capacity to take up [14C]‐urea from low‐concentration (<1 μM) urea solutions. The Ks of Anabaena cells for urea was about 0.11 μM, and the observed uptake activity involved the transport and metabolism of urea. In contrast to urease, which was constitutively ex‐pressed, expression of the high‐affinity urea uptake activity was subjected to nitrogen control. In an Anabaena ureG (urease–) mutant, a concentrative, active transport of urea could be demonstrated. We found that a mutant of open reading frame (ORF) sll0374 from the Synechocystis genomic sequence lacked urea transport activity. This ORF encoded a conserved component of an ABC‐type transporter, but it is not clustered together with any other possible transporter‐encoding gene. An Anabaena homologue of sll0374, urtE, was isolated and found to be part of a cluster of genes, urtABCDE, putatively encoding all the elements of an ABC‐type permease. Although the longest transcript that we could detect only covered urtABC, the impairment of urea transport by inactivation of urtA, urtB or urtE suggested that the whole gene cluster is expressed producing the urea permease. Expression was induced under nitrogen‐limiting conditions, and a complex promoter regulated by the cyanobacterial global nitrogen control transcription factor NtcA was found upstream from urtA. Our work adds urea to the known substrates of the versatile class of ABC‐type transporters and suggests the involvement of a transporter of this superfamily in urea scavenging by some bacteria in natural environments.

Cytochrome c oxidase genes required for nitrogenase activity and diazotrophic growth in Anabaena sp. PCC 7120

N2 fixation is an O2‐sensitive process and some filamentous diazotrophic cyanobacteria that grow performing oxygenic photosynthesis confine their N2 fixation machinery to heterocysts, specialized cells that maintain a reducing environment adequate for N2 fixation. Respiration is thought to contribute to the diazotrophic metabolism of heterocysts and the genome of the heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120 bears three gene clusters putatively encoding cytochrome c oxidases. Transcript analysis of these cox gene clusters through RNA/DNA hybridization identified two cox operons, cox2 and cox3, that are induced after nitrogen step‐down in an NtcA‐ and HetR‐dependent manner and appear to be expressed specifically in heterocysts. In contrast, cox1 was expressed only in vegetative cells. Expression of cox2 and cox3 occurred at an intermediate stage (about 9 h) during the process of heterocyst development following nitrogen step‐down. Inactivation of genes in the two inducible cox operons, but not separately in either of them, strongly reduced nitrogenase activity and prevented diazotrophic growth in aerobic conditions. These results show that the nitrogen‐regulated cytochrome c oxidase‐type respiratory terminal oxidases Cox2 and Cox3 are essential for heterocyst function in Anabaena sp. PCC 7120.

Heterocyst Development and Diazotrophic Metabolism in Terminal Respiratory Oxidase Mutants of the Cyanobacterium Anabaena sp. Strain PCC 7120

Heterocyst development was analyzed in mutants of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bearing inactivated cox2 and/or cox3 genes, encoding heterocyst-specific terminal respiratory oxidases. At the morphological level, the cox2 cox3 double mutant (strain CSAV141) was impaired in membrane reorganization involving the so-called honeycomb system that in the wild-type strain is largely or exclusively devoted to respiration, accumulated glycogen granules at conspicuously higher levels than the wild type (in both vegetative cells and heterocysts), and showed a delay in carboxysome degradation upon combined nitrogen deprivation. Consistently, chemical analysis confirmed higher accumulation of glycogen in strain CSAV141 than in the wild type. No impairment was observed in the formation of the glycolipid or polysaccharide layers of the heterocyst envelope, consistent with the chemical detection of heterocyst-specific glycolipids, or in the expression of the heterocyst-specific genes nifHDK and fdxH. However, nitrogenase activity under oxic conditions was impaired in strain CSAV135 (cox3) and undetectable in strain CSAV141 (cox2 cox3). These results show that these dedicated oxidases are required for normal development and performance of the heterocysts and indicate a central role of Cox2 and, especially, of Cox3 in the respiratory activity of the heterocysts, decisively contributing to protection of the N(2) fixation machinery against oxygen. However, in contrast to the case for other diazotrophic bacteria, expression of nif genes in Anabaena seems not to be affected by oxygen.

Constitutive and nitrogen-regulated promoters of the petH gene encoding ferredoxin:NADP+ reductase in the heterocyst-forming cyanobacterium Anabaena sp.

Determination of the putative transcription start points of the petH gene encoding ferredoxin:NADP+ reductase in the heterocyst‐forming cyanobacteria Anabaena sp. PCC 7119 and PCC 7120 showed that this gene is transcribed from two promoters, one constitutively used under different conditions of nitrogen nutrition and the other one used in cells subjected to nitrogen stepdown and in nitrogen‐fixing filaments. The latter promoter, whose use was NtcA‐dependent but HetR‐independent, was functional in heterocysts. The N‐control transcriptional regulator NtcA was observed to bind in vitro to this promoter. For the sake of comparison, the transcription start points of the nifHDK operon in strain PCC 7120 and binding of NtcA to the nifHDK promoter were also examined.

Transcription Activation by NtcA and 2-Oxoglutarate of Three Genes Involved in Heterocyst Differentiation in the Cyanobacterium Anabaena sp. Strain PCC 7120

In Anabaena sp. strain PCC 7120, differentiation of heterocysts takes place in response to the external cue of combined nitrogen deprivation, allowing the organism to fix atmospheric nitrogen in oxic environments. NtcA, a global transcriptional regulator of cyanobacteria, is required for activation of the expression of multiple genes involved in heterocyst differentiation, including key regulators that are specific to the process. We have set up a fully defined in vitro system, which includes the purified Anabaena RNA polymerase, and have studied the effects of NtcA and its signaling effector 2-oxoglutarate on RNA polymerase binding, open complex formation, and transcript production from promoters of the hetC, nrrA, and devB genes that are activated by NtcA at different stages of heterocyst differentiation. Both RNA polymerase and NtcA could specifically bind to the target DNA in the absence of any effector. 2-Oxoglutarate had a moderate positive effect on NtcA binding, and NtcA had a limited positive effect on RNA polymerase recruitment at the promoters. However, a stringent requirement of both NtcA and 2-oxoglutarate was observed for the detection of open complexes and transcript production at the three investigated promoters. These results support a key role for 2-oxoglutarate in transcription activation in the developing heterocyst.

The NtcA-Dependent P1 Promoter Is Utilized for glnA Expression in N2-Fixing Heterocysts of Anabaena sp. Strain PCC 7120

Expression of the glnA gene encoding glutamine synthetase, a key enzyme in nitrogen metabolism, is subject to a variety of regulatory mechanisms in different organisms. In the filamentous, N(2)-fixing cyanobacterium Anabaena sp. strain PCC 7120, glnA is expressed from multiple promoters that generate several transcripts whose abundance is influenced by NtcA, the transcription factor exerting global nitrogen control in cyanobacteria. Whereas RNA(I) originates from a canonical NtcA-dependent promoter (P(1)) and RNA(II) originates from a sigma(70)-type promoter (P(2)), RNA(IV) is influenced by NtcA but the corresponding promoter (P(3)) does not have the structure of NtcA-activated promoters. Using RNA isolated from Anabaena filaments grown under different nitrogen regimens, we observed, in addition to these transcripts, RNA(V), which has previously been detected only in in vitro transcription assays and should originate from P(4). However, in heterocysts, which are differentiated cells specialized in N(2) fixation, RNA(I) was the almost exclusive glnA transcript. Analysis of P(glnA)::lacZ fusions containing different fragments of the glnA upstream region confirmed that fragments carrying P(1), P(2), or P(3) and P(4) have the ability to promote transcription. Mutation of the NtcA-binding site in P(1) eliminated P(1)-directed transcription and allowed increased use of P(2). The NtcA-binding site in the P(1) promoter and binding of NtcA to this site appear to be key factors in determining glnA gene expression in vegetative cells and heterocysts.

Interaction of FurA from Anabaena sp. PCC 7120 with DNA: A Reducing Environment and the Presence of Mn2+ are Positive Effectors in the Binding to isiB and furA Promoters

The Fur (ferric uptake regulator) protein is a global regulator in most prokaryotes that controls a large number of genes. Fur is a classical repressor that uses ferrous iron as co-repressor and binds to specific DNA sequences (iron boxes) as a dimer. Three different genes coding for Fur homologues have been identified in Anabaena sp. PCC 7120. FurA controls the transcription of flavodoxin, the product of the isiB gene, and is moderately autoregulated. In this work, the promoter of the furA gene was defined and the FurA protected regions in the furA and isiB promoters were identified, showing that the binding sites for Anabaena FurA contain A/T-rich sequences with a variable arrangement compared to the conventional 19-base pair Fur consensus. The influence of different factors on the interaction between FurA and the promoters was evaluated in vitro. The affinity of FurA for the DNA targets was significantly affected by the redox status of this regulator and the presence of Mn2+. The optimal binding conditions were observed in the presence of both Mn2+ and DTT. Those results suggest that, in addition to iron availability, FurA–DNA interaction is modulated by redox conditions.

The interplay between siderophore secretion and coupled iron and copper transport in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120.

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P(trc)) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P(trc)-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120.

FurA is the master regulator of iron homeostasis and modulates the expression of tetrapyrrole biosynthesis genes in Anabaena sp. PCC 7120

Knowledge on the regulatory mechanisms controlling iron homeostasis in cyanobacteria is limited. In Anabaena sp. PCC 7120, the ferric uptake regulator FurA is a constitutive and essential protein whose expression is induced under iron deprivation. Our previous analyses have shown that this protein acts as a global transcriptional regulator, controlling the expression of several genes belonging to different functional categories, including schT, a gene coding for a TonB-dependent schizokinen transporter. In the present study we analysed the impact of FurA overexpression and iron availability on the transcriptional modulation of a broad range of Anabaena iron uptake, transport, storage and cellular iron utilization mechanisms, including enzymes involved in siderophore biosynthesis, TonB-dependent siderophore outer membrane transporters, siderophore periplasmic binding proteins, ABC inner membrane permeases, ferritin Dps family proteins, and enzymes involved in tetrapyrrole biosynthesis. By combining reverse transcription-PCR analyses, electrophoretic mobility shift assays and DNase I footprinting experiments, we defined a variety of novel direct iron-dependent transcriptional targets of this metalloregulator, including genes encoding at least five enzymes involved in the tetrapyrrole biosynthesis pathway. The results unravel the role of FurA as the master regulator of iron homeostasis in Anabaena sp. PCC 7120, providing new insights into the Fur regulons in cyanobacteria.