A Major Facilitator Superfamily Protein, HepP, Is Involved in Formation of the Heterocyst Envelope Polysaccharide in the Cyanobacterium Anabaena sp. Strain PCC 7120

López‐Igual, Rocío; Lechno‐Yossef, Sigal; Fan, Qing; Herrero, Antonia; Flores, Enrique; Wölk, C. Peter

doi:10.1128/jb.00489-12

Cited by 18 publications

(26 citation statements)

References 61 publications

(70 reference statements)

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“…The Anabaena glucoside transporters could have a role in the recovery of glucosides from extracellular polysaccharides produced under certain physiological conditions, as has been shown to occur in cyanobacterial mats (42). Consistent with this, biomass of the glsC and glsP mutants in old BG11 plates is shiny (not shown), which may be indicative of exopolysaccharide accumulation (33). It is also of interest that although Anabaena has been considered an obligatory photoautotroph (43), recent data suggest that it can grow using fructose, although this sugar has to be provided at a high concentration unless Anabaena is engineered to express a fructose transporter (27,44).…”

Section: Discussionsupporting

confidence: 59%

“…As shown by results with all1711::Tn5-1063 mutant strain FQ163 (33), HepP may be a glucoside transporter that is involved in production of the heterocyst-specific polysaccharide layer and may also mediate sucrose transport. According to BLAST analysis, Alr3705 is the predicted Anabaena genomic product most similar to higher plant sucrose transporters.…”

Section: Figmentioning

confidence: 95%

“…We have previously shown that SepJ-T25 and SepJ-T18 fusions (where the order of protein names denotes N-terminal to C-terminal orientation) are functional in SepJ self-interactions that produce high ␤-galactosidase activity (15,16). As is the case for GlsP, HepP is a predicted integral membrane protein with both the N and C termini in the cytoplasm (33). Because of possible copy number or steric hindrance problems (37, 38), here we tested possible interactions of SepJ-T25 and SepJ-T18 with both N-terminal and C-terminal fusions to T18 and T25, respectively, of each of the glucoside transporter components investigated in this work, GlsC, GlsP, and HepP.…”

Section: Figmentioning

confidence: 99%

“…The Fox Ϫ phenotype denotes inability to grow fixing N 2 under oxic conditions, and it is frequently associated with malformation of the heterocyst envelope, as in the case of the hepP mutant (33). The growth phenotype was investigated here for the glsC and glsP mutants.…”

Section: Figmentioning

confidence: 99%

“…To understand better the role of the transporters identified in this work in the physiology of Anabaena, we investigated their subcellular localization. The localization of HepP in the cytoplasmic membrane of both vegetative cells and heterocysts has been described previously (33). To study the subcellular localization of GlsC and GlsP, strains producing GlsC-GFP and GlsP-GFP fusion proteins were constructed.…”

Section: Figmentioning

confidence: 99%

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

et al. 2017

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When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO 2 through oxygenic photosynthesis and heterocysts that are specialized in N 2 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 ␣-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 CO 2 and N 2 . 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 ␣-glucoside transporters of Anabaena that mediate uptake of esculin and, notably, influence septal structure and the function of septal junctions.

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Section: Discussionsupporting

confidence: 59%

Section: Figmentioning

confidence: 95%

Section: Figmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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

et al. 2017

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Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst‐forming cyanobacterium Anabaena

Burnat

Flores

2014

MicrobiologyOpen

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Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [14C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The Δalr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [14C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the Δalr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium.

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Simultaneous gene inactivation and promoter reporting in cyanobacteria

Chen

et al. 2014

Appl Microbiol Biotechnol

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Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0 % of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72 h, decreased 49.6 % compared to wild-type. Thus, we discovered that All2508 may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms.

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A Major Facilitator Superfamily Protein, HepP, Is Involved in Formation of the Heterocyst Envelope Polysaccharide in the Cyanobacterium Anabaena sp. Strain PCC 7120

Cited by 18 publications

References 61 publications

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

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

Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst‐forming cyanobacterium Anabaena

Simultaneous gene inactivation and promoter reporting in cyanobacteria

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