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.

Impact of nano titanium dioxide exposure on cellular structure of Anabaena variabilis and evidence of internalization.

The present study investigated the impact of nano titanium dioxide (nTiO(2) ) exposure on the cellular structures of the nitrogen-fixing cyanobacteria Anabaena variabilis. Results of the present study showed that nTiO(2) exposure led to observable alteration in various intracellular structures and induced a series of recognized stress responses, including production of reactive oxygen species (ROS), appearance and increase in the abundance of membrane crystalline inclusions, membrane mucilage layer formation, opening of intrathylakoidal spaces, and internal plasma membrane disruption. The production of total ROS in A. variabilis cells increased with increasing nTiO(2) doses and exposure time, and the intracellular ROS contributed to only a small fraction (<10%) of the total ROS measured. The percentage of cells with loss of thylakoids and growth of membrane crystalline inclusions increased as the nTiO(2) dose and exposure time increased compared with controls, suggesting their possible roles in stress response to nTiO(2) , as previously shown for metals. Algal cell surface morphology and mechanical properties were modified by nTiO(2) exposure, as indicated by the increase in cell surface roughness and shifts in cell spring constant determined by atomic force microscopy analysis. The change in cell surface structure and increase in the cellular turgor pressure likely resulted from the structural membrane damage mediated by the ROS production. Transmission electron microscopy (TEM) analysis of nTiO(2) aggregates size distribution seems to suggest possible disaggregation of nTiO(2) aggregates when in close contact with microbial cells, potentially as a result of biomolecules such as DNA excreted by organisms that may serve as a biodispersant. The present study also showed, for the first time, with both TEM and Raman imaging that internalization of nTiO(2) particles through multilayered membranes in algal cells is possible. Environ. Toxicol. Chem. 2011; 30:861-869. © 2010 SETAC.

H2O2-induced inhibition of photosynthetic O2 evolution by Anabaena variabilis cells.

Hydrogen peroxide inhibits photosynthetic O2 evolution. It has been shown that H2O2 destroys the function of the oxygen-evolving complex (OEC) in some chloroplast and Photosystem (PS) II preparations causing release of manganese from the OEC. In other preparations, H2O2 did not cause or caused only insignificant release of manganese. In this work, we tested the effect of H2O2 on the photosynthetic electron transfer and the state of OEC manganese in a native system (intact cells of the cyanobacterium Anabaena variabilis). According to EPR spectroscopy data, H2O2 caused an increase in the level of photooxidation of P700, the reaction centers of PS I, and decreased the rate of their subsequent reduction in the dark by a factor larger than four. Combined effect of H2O2, CN-, and EDTA caused more than eight- to ninefold suppression of the dark reduction of P700+. EPR spectroscopy revealed that the content of free (or loosely bound) Mn2+ in washed cyanobacterial cells was ~20% of the total manganese pool. This content remained unchanged upon the addition of CN- and increased to 25-30% after addition of H2O2. The content of the total manganese decreased to 35% after the treatment of the cells with EDTA. The level of the H2O2-induced release of manganese increased after the treatment of the cells with EDTA. Incubation of cells with H2O2 for 2 h had no effect on the absorption spectra of the photosynthetic pigments. More prolonged incubation with H2O2 (20 h) brought about degradation of phycobilins and chlorophyll a and lysis of cells. Thus, H2O2 causes extraction of manganese from cyanobacterial cells, inhibits the OEC activity and photosynthetic electron transfer, and leads to the destruction of the photosynthetic apparatus. H2O2 is unable to serve as a physiological electron donor in photosynthesis.