Nutritional status regulates algicidal activity of Aeromonas sp. L23 against cyanobacteria and green algae.

Algicidal bacteria have received broad acceptance as an ecofriendly tool for controlling harmful algal blooms. However, their practical application is still limited to the lab-scale tests due to the complex alga-bacterium interactions in different nutrient statuses. In this study, the Aeromonas sp. L23 that exhibit relatively wide-spectrum in algicidal activity was isolated from a eutrophic agricultural lake. The physiological response of cyanobacteria and green to the algicidal activity under varied nutritional status were studied in an alga-bacterial co-culture. The algicidal activities of L23 against Microcystis aeruginosa UTEX LB 2385, Microcystis aeruginosa NHSB, Anabaena variabilis AG10064, Scenedesmus quadricauda AG10003, and Chlorella vulgaris AG10034 were 88 ± 1.2%, 94 ± 2.6%, 93 ± 0.5%, 82 ± 1.1%, and 47 ± 0.9%, respectively. The L23 cells had low algicidal activity in cell pellet (3%-9%) compared with the cell-free supernatant (78%-93%), indicating that the activity is induced by extracellular substances. Adding glucose, NaNO3, NH4Cl, and KH2PO4 to the co-culture raised the algicidal activity of the L23 against green algae by 5%-50%. Conversely, a 10%-20% decrease in activity occurred against the target cyanobacteria except M. aeruginosa UTEX LB 2385. These results indicated that the interspecific algicidal activity changes according to the nutritional status, which means that the alga-bacterium interaction will be more complex in the field where the nutritional status changes from time to time.

Light-controlled carotenoid transfer between water-soluble proteins related to cyanobacterial photoprotection.

Carotenoids are lipophilic pigments with multiple biological functions from coloration to vision and photoprotection. Still, the number of water-soluble carotenoid-binding proteins described to date is limited, and carotenoid transport and carotenoprotein maturation processes are largely underexplored. Recent studies revealed that CTDHs, which are natural homologs of the C-terminal domain (CTD) of the orange carotenoid protein (OCP), a photoswitch involved in cyanobacterial photoprotection, are able to bind carotenoids, with absorption shifted far into the red region of the spectrum. Despite the recent discovery of their participation in carotenoid transfer processes, the functional roles of the diverse family of CTDHs are not well understood. Here, we characterized CTDH carotenoproteins from Anabaena variabilis (AnaCTDH) and Thermosynechococcus elongatus and examined their ability to participate in carotenoid transfer processes with a set of OCP-derived proteins. This revealed that carotenoid transfer occurs in several directions guided by different affinities for carotenoid and specific protein-protein interactions. We show that CTDHs have higher carotenoid affinity compared to the CTD of OCP from Synechocystis, which results in carotenoid translocation from the CTD into CTDH via a metastable heterodimer intermediate. Activation of OCP by light, or mutagenesis compromising the OCP structure, provides AnaCTDH with an opportunity to extract carotenoid from the full-length OCP, either from Synechocystis or Anabaena. These previously unknown reactions between water-soluble carotenoproteins demonstrate multidirectionality of carotenoid transfer, allowing for efficient and reversible control over the carotenoid-mediated protein oligomerization by light, which gives insights into the physiological regulation of OCP activity by CTDH and suggests multiple applications.

Energy transfer in Anabaena variabilis filaments adapted to nitrogen-depleted and nitrogen-enriched conditions studied by time-resolved fluorescence.

Nitrogen is among the most important nutritious elements for photosynthetic organisms such as plants, algae, and cyanobacteria. Therefore, nitrogen depletion severely compromises the growth, development, and photosynthesis of these organisms. To preserve their integrity under nitrogen-depleted conditions, filamentous nitrogen-fixing cyanobacteria reduce atmospheric nitrogen to ammonia, and self-adapt by regulating their light-harvesting and excitation energy-transfer processes. To investigate the changes in the primary processes of photosynthesis, we measured the steady-state absorption and fluorescence spectra and time-resolved fluorescence spectra (TRFS) of whole filaments of the nitrogen-fixing cyanobacterium Anabaena variabilis at 77 K. The filaments were grown in standard and nitrogen-free media for 6 months. The TRFS were measured with a picosecond time-correlated single photon counting system. Despite the phycobilisome degradation, the energy-transfer paths within phycobilisome and from phycobilisome to both photosystems were maintained. However, the energy transfer from photosystem II to photosystem I was suppressed and a specific red chlorophyll band appeared under the nitrogen-depleted condition.

Simultaneous gene inactivation and promoter reporting in cyanobacteria.

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.

Exogenous osmolytes suppresses the toxic effects of malathion on Anabaena variabilis.

Role of osmolytes is though well established for salt, drought and chilling stress, but their role in pesticide stress is yet to be explored thoroughly. The sporadic information covers our previous studies on proline with respect to endosulfan and carbaryl pesticides in cyanobacteria. Therefore, during the present investigation importance of osmolytes (exogenous and endogenous) is studied in cyanobacterial biofertilizer Anabaena variabilis in the presence of 25, 50, 75 and 100 µg mL(-1) malathion pesticide. Present investigation has two parts. In the first part we showed that malathion exert its toxic effect on growth (biomass) via. malondialdehyde (MDA) and hydrogen peroxide (H2O2). This was associated with quantitative enhancement of endogenous osmolytes (proline, sucrose, mannitol, trehalose and glycogen). In the second part effort was made to corelate effect of exogenous addition of osmolytes (which were detected in the first part of this study) on growth and antioxidant enzymes [like superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX)] of A. variabilis in the presence of 100 µg mL(-1) malathion. Surprisingly it was observed that exogenous osmolytes gave additional protection to the organism. The order of protection provided by osmolytes was as trehalose>glycogen>sucrose>mannitol>proline in A. variabilis.

The all0458/lti46.2 gene encodes a low temperature-induced Dps protein homologue in the cyanobacteria Anabaena sp. PCC 7120 and Anabaena variabilis M3.

DNA-binding proteins from starved cells (Dps), which are encoded by many bacterial genomes, protect genomic DNA via non-specific DNA binding, as well as inhibition of free radical formation by chelating Fe(II). In the filamentous cyanobacterium Anabaena, the second gene (lti46.2) in the low temperature-induced gene operon lti46 in strain M3 was found to encode a homologue of Dps, but for a long time this gene remained poorly characterized. A gene cluster, all0459-all0458-all0457, was found later to be 100% identical to the lti46 gene cluster in a closely related strain, PCC 7120. In the present study, we detected ferroxidase activity of the Lti46.2/All0458 protein, which formed a dodecamer, as found in other Dps proteins. In addition, three homologues of all0458 were found in strain PCC 7120, namely, all1173, alr3808 and all4145. We analysed expression of the lti46 or all0459-8-7 gene cluster in both strains, M3 and PCC 7120, and confirmed its induction by low temperature. We found that the All0458-GFP fusion protein and the All1173-GFP fusion protein were localized to the nucleoids. In the all0458 null mutant, the transcript of the alr3808 gene accumulated. These results suggest that there might be complex cooperation of various members of the dps family in protecting the genome from environmental stresses such as changing temperature.

Role of the 5'-UTR in accumulation of the rbpA1 transcript at low temperature in the cyanobacterium Anabaena variabilis M3.

The expression of the rbp genes, which encode RNA-binding proteins with a single RNA-recognition motif and a glycine-rich sequence, is known to increase at low temperature in cyanobacteria. We previously showed that their regulation involved both transcription and mRNA stability. In the present study, various reporter constructs with deletions and mutations were used to analyze this regulation, revealing that at least the following three elements are involved. First, a putative enhancer element is located within the upstream gene. Second, the rbpA1 transcript is dramatically stabilized by a large stem-loop structure located at the 5' terminus. Third, the transcript is also destabilized by a downstream box located within the coding region.