José E. Frías

Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC7120

The cyanobacterial ntcA gene encodes a DNA‐binding protein that belongs to the Crp family of bacterial transcriptional regulators. In this work, we describe the isolation of an ntcA insertional mutant of the dinitrogen‐fixing, heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120. The Anabaena ntcA mutant was able to use ammonium as a source of nitrogen for growth, but was unable to assimilate atmospheric nitrogen (dinitrogen) or nitrate. Nitrogenase and enzymes of the nitrate reduction system were not synthesized in the ntcA mutant under derepressing conditions, and glutamine synthetase levels were lower in the mutant than in the wild‐type strain. In the ntcA mutant, in response to removal of ammonium, accumulation of mRNA of the genes encoding nitrogenase (nifHDK), nitrite reductase (nir, the first gene of the nitrate assimilation operon), and glutamine synthetase (glnA) was not observed. A transcription start point of the Anabaena glnA gene (corresponding to RNA1), that has been shown to be used preferentially after nitrogen step‐down, was not used in the ntcA insertional mutant. Heterocyst development (which is necessary for the aerobic fixation of dinitrogen) and induction of hetR (a regulatory gene that is required for heterocyst development) were also impaired in the ntcA mutant. These results showed that the ntcA gene product, NtcA, is required in Anabaena sp. PCC 7120 for the expression of genes encoding proteins involved in the assimilation of nitrogen sources alternative to ammonium including dinitrogen and nitrate, and that the process of heterocyst development is also controlled by NtcA.

Photosynthetic nitrate assimilation in cyanobacteria.

Nitrate uptake and reduction to nitrite and ammonium are driven in cyanobacteria by photosynthetically generated assimilatory power, i.e., ATP and reduced ferredoxin. High-affinity nitrate and nitrite uptake takes place in different cyanobacteria through either an ABC-type transporter or a permease from the major facilitator superfamily (MFS). Nitrate reductase and nitrite reductase are ferredoxin-dependent metalloenzymes that carry as prosthetic groups a [4Fe–4S] center and Mo-bis-molybdopterin guanine dinucleotide (nitrate reductase) and [4Fe–4S] and siroheme centers (nitrite reductase). Nitrate assimilation genes are commonly found forming an operon with the structure: nir (nitrite reductase)-permease gene(s)-narB (nitrate reductase). When the cells perceive a high C to N ratio, this operon is transcribed from a complex promoter that includes binding sites for NtcA, a global nitrogen-control regulator that belongs to the CAP family of bacterial transcription factors, and NtcB, a pathway-specific regulator that belongs to the LysR family of bacterial transcription factors. Transcription is also affected by other factors such as CnaT, a putative glycosyl transferase, and the signal transduction protein PII. The latter is also a key factor for regulation of the activity of the ABC-type nitrate/nitrite transporter, which is inhibited when the cells are incubated in the presence of ammonium or in the absence of CO2. Notwithstanding significant advance in understanding the regulation of nitrate assimilation in cyanobacteria, further post-transcriptional regulatory mechanisms are likely to be discovered.

Activation of the Anabaena nir operon promoter requires both NtcA (CAP family) and NtcB (LysR family) transcription factors

A region of the genome of the heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120 containing the ntcB gene was identified. This region is located upstream from the nir operon involved in nitrate assimilation in this cyanobacterium. An Anabaena ntcB mutant was able to use ammonium and dinitrogen as sources of nitrogen for growth but was unable to assimilate nitrate. Enzymes of the nitrate reduction system were not synthesized in the ntcB mutant under derepression conditions. The transcription start‐point of the Anabaena nir operon, which has been shown to be subjected to ammonium‐stimulated repression and whose expression requires the global nitrogen regulator NtcA, was only weakly used in the ntcB mutant. The expression of the ntcB gene in strain PCC 7120 was also subjected to repression by ammonium and was found to take place from an NtcA‐activated promoter located 31 bp upstream from the start of the ntcB gene. NtcB binds to the nir promoter region in vitro and protects a region localized just upstream from the NtcA‐binding site in footprinting assays. These results showed that NtcB, a LysR‐family protein, is required in addition to NtcA, a CAP‐family protein, for the expression of genes encoding proteins specifically involved in nitrate assimilation in Anabaena sp. PCC 7120.

Open Reading Frame all0601 from Anabaena sp. Strain PCC 7120 Represents a Novel Gene, cnaT, Required for Expression of the Nitrate Assimilation nir Operon

Expression of the nitrate assimilation nir operon in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 requires the action of both the global nitrogen control transcription factor NtcA and the pathway-specific transcriptional regulator NtcB. In the genome of this cyanobacterium, the ntcB gene is found in a cluster of genes located in the complementary strand, upstream from the nir operon. Just downstream of ntcB, there is an open reading frame, all0601 (previously designated orf356 and now designated the cnaT gene), that putatively encodes a protein similar to proteins with glycosyl transferase activity and that is also present clustered together with ntcB homologues or nitrate assimilation structural genes in other cyanobacterial genomes. An insertional mutant of cnaT was generated and found to be unable to assimilate nitrate, although it could use ammonium or dinitrogen as a source of nitrogen for growth. In the mutant, under derepression conditions, nir operon mRNA (as determined by RNA-DNA hybridization and primer extension analysis) and enzymes of the nitrate reduction system (i.e., nitrate reductase and nitrite reductase) were expressed at low or undetectable levels. Inactivation of cnaT did not impair expression of ntcB, and expression of cnaT itself was constitutive and regulated by neither NtcA nor NtcB. Regulation of expression of the nir operon in Anabaena sp. strain PCC 7120 by CnaT and the previously described regulatory elements, NtcA and NtcB, is discussed.

Negative Regulation of Expression of the Nitrate Assimilation nirA Operon in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, expression of the nitrate assimilation nirA operon takes place in the absence of ammonium and the presence of nitrate or nitrite. Several positive-action proteins that are required for expression of the nirA operon have been identified. Whereas NtcA and NtcB exert their action by direct binding to the nirA operon promoter, CnaT acts by an as yet unknown mechanism. In the genome of this cyanobacterium, open reading frame (ORF) all0605 (the nirB gene) is found between the nirA (encoding nitrite reductase) and ntcB genes. A nirB mutant was able to grow at the expense of nitrate as a nitrogen source and showed abnormally high levels of nirA operon mRNA both in the presence and in the absence of nitrate. This mutant showed increased nitrate reductase activity but decreased nitrite reductase activity, an imbalance that resulted in excretion of nitrite, which accumulated in the extracellular medium, when the nirB mutant was grown in the presence of nitrate. A nirA in-frame deletion mutant also showed a phenotype of increased expression of the nirA operon in the absence of ammonium, independent of the presence of nitrate in the medium. Both NirB and NirA are therefore needed to keep low levels of expression of the nirA operon in the absence of an inducer. Because NirB is also needed to attain high levels of nitrite reductase activity, NirA appears to be a negative element in the nitrate regulation of expression of the nirA operon in Anabaena sp. strain PCC 7120.

Divisome-dependent subcellular localization of cell–cell joining protein SepJ in the filamentous cyanobacterium Anabaena

Heterocyst‐forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA‐dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two‐hybrid system. We found SepJ self‐interaction and a specific interaction with FtsQ, confirmed by co‐purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity.

Ar + NO microwave plasmas for Escherichia coli sterilization

Ar + NO microwave discharges are used for sterilization and the results are compared with additional experiments with Ar, O2 and N2–O2 plasma mixtures. The NO* species produced in the Ar–NO mixtures remain up to long distances from the source, thus improving the sterilization efficiency of the process. E. coli individuals exposed to the Ar + NO plasma undergo morphological damage and cell lysis. Combined effects of etching (by O* and Ar* species) and UV radiation (from deactivation of NO* species) are responsible for the higher activity found for this plasma mixture.

Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120

UNLABELLED Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. IMPORTANCE Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria assimilate nitrate, but regulation of the nitrate assimilation system varies in different cyanobacterial groups. In the N2-fixing, heterocyst-forming cyanobacteria, the nirA operon, which includes the structural genes for the nitrate assimilation system, is expressed in the presence of nitrate or nitrite if ammonium is not available to the cells. Here we studied the genes required for production of an active nitrate reductase, providing information on the nitrate-dependent induction of the operon, and found evidence for possible protein-protein interactions in the maturation of nitrate reductase and nitrite reductase.Plan Nacional de Investigacion y European Regional Development Fund BFU2008-03811 BFU2011-22762

Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO2 through oxygenic photosynthesis and heterocysts that are specialized in N2 fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was inhibited by the α-glucosides sucrose and maltose. Analysis of Anabaena mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of Anabaena was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in Anabaena, glucoside transporters influence the structure and function of septal junctions.IMPORTANCE Heterocyst-forming cyanobacteria have the ability to perform oxygenic photosynthesis and to assimilate atmospheric CO2 and N2 These organisms grow as filaments that fix these gases specifically in vegetative cells and heterocysts, respectively. For the filaments to grow, these types of cells exchange nutrients, including sucrose, which serves as a source of reducing power and of carbon skeletons for the heterocysts. Movement of sucrose between cells in the filament takes place through septal junctions and has been traced with a fluorescent sucrose analog, esculin, that can be taken up by the cells. Here, we identified α-glucoside transporters of Anabaena that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions.