The effects of changes in mitochondrial DNA in cucumber (Cucumis sativus L.) mosaic mutant (MSC16) on respiration, photosynthesis and photorespiration were analyzed under non-stressed conditions. Decreased respiratory capacity of complex I in MSC16 mitochondria was indicated by lower respiration rates of intact mitochondria with malate and by rotenone-inhibited NADH or malate oxidation in the presence of alamethicin. Moreover, blue native PAGE indicated decreased intensity of protein bands of respiratory chain complex I in MSC16 leaves. Concerning the redox state, complex I impairment could be compensated to some extent by increased external NADH dehydrogenases (ND(ex)NADH) and alternative oxidase (AOX) capacity, the latter presenting differential expression in the light and in the dark. Although MSC16 mitochondria have a higher AOX protein level and an increased capacity, the AOX activity measured in the dark conditions by oxygen discrimination technique is similar to that in wild-type (WT) plants. Photosynthesis induction by light followed different patterns in WT and MSC16, suggesting changes in feedback chloroplast DeltapH caused by different adenylate levels. At steady-state, net photosynthesis was only slightly impaired in MSC16 mutants, while photorespiration rate (PR) was significantly increased. This was the result of large decreases in both stomatal and mesophyll conductance to CO2, which resulted in a lower CO2 concentration in the chloroplasts. The observed changes on CO2 diffusion caused by mitochondrial mutations open a whole new view of interaction between organelle metabolism and whole tissue physiology. The sum of all the described changes in photosynthetic and respiratory metabolism resulted in a lower ATP availability and a slower plant growth.

The MSC16 cucumber (Cucumis sativus L.) mutant with lower activity of mitochondrial Complex I was used to study the influence of mitochondrial metabolism on whole cell energy and redox state. Mutant plants had lower content of adenylates and NADP(H) whereas the NAD(H) pool was similar as in wild type. Subcellular compartmentation of adenylates and pyridine nucleotides were studied using the method of rapid fractionation of protoplasts. The data obtained demonstrate that dysfunction of mitochondrial respiratory chain decreased the chloroplastic ATP pool. No differences in NAD(H) pools in subcellular fractions of mutated plants were observed; however, the cytosolic fraction was highly reduced whereas the mitochondrial fraction was more oxidized in MSC16, as compared to WTc. The NADP(H) pool in MSC16 protoplasts was greatly decreased and the chloroplastic NADP(H) pool was more reduced, whereas the extrachloroplastic pool was much more oxidized, than in WTc protoplast. Changes in nucleotides distribution in cucumber MSC16 mutant were compared to changes found in tobacco (Nicotiana sylvestris) CMS II mitochondrial mutant. In contrast to MSC16 cucumber, the content of adenylates in tobacco mutant was much higher than in tobacco wild type. The differences were more pronounced in leaf tissue collected after darkness than in the middle of the photoperiod. Results obtained after tobacco protoplast fractionating showed that the increase in CMS II adenylate content was mainly due to a higher level in extrachloroplast fraction. Both mutations have a negative effect on plant growth through perturbation of chloroplast/mitochondrial interactions.

The widespread emergence of bacterial resistance to existing antibiotics forces the development of new therapeutic agents. The use of short modified oligonucleotides, such as peptide nucleic acids (PNAs), seems a promising strategy. However, the uptake of such oligonucleotides is limited by the bacterial cell wall and is species-dependent. Therefore, new carriers for PNAs should be extensively explored. In this study, we examined the antibacterial activity of vitamin B 12 −PNA conjugates. Vitamin B 12 was covalently linked to a PNA oligomer targeted at the mRNA of an essential acpP gene encoding acyl carrier protein in Escherichia coli. PNA−vitamin B 12 conjugates were synthesized using the Cu(I)-catalyzed 1,3-dipolar cycloaddition. We examined two types of linkers between vitamin B 12 and PNA, including a cleavable disulfide bond. As a positive control for PNA uptake, we used PNA conjugated to the most widely used cell-penetrating peptide (KFF) 3 K. We found that vitamin B 12 −PNA conjugates inhibit E. coli growth at a concentration of 5 μM, similar as (KFF) 3 K−PNA. We also showed that vitamin B 12 −PNA conjugates are stable in the presence of biological media. This study provides the foundation for improving and developing PNAs conjugated to vitamin B 12 as antibacterials.

The majority of antibiotics used in the clinic target bacterial protein synthesis. However, the widespread emergence of bacterial resistance to existing drugs creates a need to discover or develop new therapeutic agents. Ribosomal RNA (rRNA) has been a target for numerous antibiotics that bind to functional rRNA regions such as the peptidyl transferase center, polypeptide exit tunnel, and tRNA binding sites. Even though the atomic resolution structures of many ribosome-antibiotic complexes have been solved, improving the ribosome-acting drugs is difficult because the large rRNA has a complicated 3D architecture and is surrounded by numerous proteins. Computational approaches, such as structure-based design, often fail when applied to rRNA binders because electrostatics dominate the interactions and the effect of ions and bridging waters is difficult to account for in the scoring functions. Improving the classical anti-ribosomal agents has not proven particularly successful and has not kept pace with acquired resistance. So one needs to look for other ways to combat the ribosomes, finding either new rRNA targets or totally different compounds. There have been some efforts to design translation inhibitors that act on the basis of the sequence-specific hybridization properties of nucleic acid bases. Indeed oligonucleotides hybridizing with functional regions of rRNA have been shown to inhibit translation. Also, some peptides have been shown to be reasonable inhibitors. In this review we describe these nonconventional approaches to screening for ribosome inhibition and function of particular rRNA regions. We discuss inhibitors against rRNA that may be designed according to nucleotide sequence and higher order structure.

The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2'-O-methyl oligoribonucleotides (2'-OMe RNAs). Further, the inhibition efficiencies of the designed ribosome-interfering 2'-OMe RNAs were tested using a β-galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC values below 1 μM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2'-OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double-filter retention assay. Further, we applied heat-shock chemical transformation to introduce 2'-OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC in the cell-free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter-subunit bridges can help design efficient antisense oligomers to probe the ribosome function.