Cyanobacterial Catalase Activity Prevents Oxidative Stress Induced by Pseudomonas fluorescens DUS1-27 from Inhibiting Brassica napus L. (canola) Growth.

Plant growth-promoting bacteria (PGPB) inhabit the rhizosphere of plants and are capable of enhancing plant growth through a number of mechanisms. A strain of Pseudomonas fluorescens DUS1-27 was identified as a potential PGPB candidate based on its ability to increase the growth of Brassica napus L. (canola) over that of uninoculated control plants in a soil-based system. The same P. fluorescens isolate was found to reduce plant growth in a hydroponic growth system, with plants showing the symptoms of a microbe-associated molecular pattern (MAMP) response to the bacteria. The amperometric quantification of H2O2, fluorescence-based total peroxidase assays, and quantification of catalase gene expression levels using qRT-PCR revealed that oxidative stress reduced plant growth in the hydroponic system. The addition of the cyanobacterium Nostoc punctiforme (known to have high catalase activity levels) in the hydroponic system as a co-inoculant reduced oxidative stress (49.7% decrease in H2O2 concentrations) triggered by the addition of P. fluorescens DUS1-27, thereby enabling plants to grow larger than uninoculated control plants. These results show the advantage of inoculating with multiple bacteria to promote plant growth and, for the first time, demonstrate that N. punctiforme beneficially assists plants under oxidative stress through its catalase activity in planta.

Altered expression of two zinc transporters, SLC30A5 and SLC30A6, underlies a mammary gland disorder of reduced zinc secretion into milk.

Two cases of zinc deficiency in breastfed neonates were investigated where zinc levels in the mothers' milk were reduced by more than 75 % compared to normal. The objective of this study was to find the molecular basis of the maternal zinc deficiency condition. Significant reductions in mRNA expression and protein levels of the zinc transporters SLC30A5 and SLC30A6 were found in maternal tissue, suggesting a causal link to the zinc-deficient milk. Novel splice variants of the SLC30A6 transcript were detected. No modifications were found in coding regions, or in transcription binding sites of promoter regions or in 5' and 3' untranslated regions of both transporters in lymphoblasts and fibroblasts isolated from both mothers. Altered DNA methylation in SLC30A5 at two CpG sites was detected and may account for the reduced levels of SLC30A5 mRNA and protein in lymphoblasts. Reduced SLC30A6 mRNA and protein levels in lymphoblasts may be secondary to reduced SLC30A5 expression, as they function as a heterodimer in zinc transport. In conclusion, two cases of zinc deficiency are linked to low levels of the SLC30A5 and SLC30A6 zinc transporters. These two zinc transporters have not been previously associated with zinc deficiency in milk.

Molecular and cellular characterisation of the zinc uptake (Znu) system of Nostoc punctiforme.

Metal homoeostasis in cyanobacteria is based on uptake and export systems that are controlled by their own regulators. This study characterises the zinc uptake (Znu) system in Nostoc punctiforme. The system was found to comprise of three subunits in an ACB operon: a Zn(2+)-binding protein (ZnuA18), a transmembrane domain (ZnuB) and an ATPase (ZnuC). These proteins are encoded within the znu operon regulated by a zinc uptake transcription repressor (Zur). Interestingly, a second Zn(2+)-binding protein (ZnuA08) was also identified at a distal genomic location. Interactions between components of the ZnuACB system were investigated using knockouts of the individual genes. The znuA08(-), znuA18(-), znuB(-) and znuC(-) mutants displayed overall reduced znuACB transcript levels, suggesting that all system components are required for normal expression of znu genes. Zinc uptake assays in the Zn(2+)-binding protein mutant strains showed that the disruption of znuA18 had a greater negative effect on zinc uptake than disruption of znuA08. Complementation studies in Escherichia coli indicated that both znuA08 and znuA18 were able to restore zinc uptake in a znuA(-) mutant, with znuA18 permitting the highest zinc uptake rate. The N. punctiforme zur was also able to complement the E. coli zur(-) mutant.

Bioinformatic and expression analyses of genes mediating zinc homeostasis in Nostoc punctiforme.

Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn(2+) over 14 days showed that ZnCl(2) levels above 22 microM significantly reduced cell viability. After 3 days in 22 microM ZnCl(2), ca. 12% of the Zn(2+) was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 microM relative to 0 microM treatment. Radiolabeled (65)Zn showed Zn(2+) uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn(2+) uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 microM ZnCl(2) within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.