Vladislav V. Zinchenko

Stress Sensors and Signal Transducers in Cyanobacteria

In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress.

Eukaryotic-like Ser/Thr protein kinases SpkC/F/K are involved in phosphorylation of GroES in the cyanobacterium Synechocystis

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.

Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus.

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.

Regulation systems for stress responses in cyanobacteria

The article reviews the main systems that regulate gene expression in cyanobacteria in response to various treatments: low and high temperatures, salt, hyperosmotic and oxidative stresses. The systems for perception of light are also reviewed. Functional characteristics are presented for known two-component regulatory systems, eukaryotic-type serine-threonine protein kinases, σ-subunits of RNA-polymerase, DNA-binding transcription factors. Different mechanisms of perception of stress signals are analyzed, including changes in DNA supercoiling under different stress conditions.

Regulatory Role of Membrane Fluidity in Gene Expression

Plants and other photosynthetic organisms experience frequent changes in environment. Their ability to survive depends on their capacity to acclimate to such changes. In particular, fluctuations in temperature and/or osmolarity affect the fluidity of cytoplasmic and thylakoid membranes. The molecular mechanisms responsible for the perception of changes in membrane fluidity have not been fully characterized. However, the analysis of genome-wide gene expression with DNA microarrays has provided a powerful new approach to studies of the contribution of membrane fluidity to gene expression and to the identification of environmental sensors. In this chapter, we summarize the knowledge on the mechanisms that regulate membrane fluidity, on putative sensors that perceive changes in membrane fluidity, and on the subsequent expression of genes that ensures acclimation to a new set of environmental conditions.

A novel ATP-binding cassette transporter is responsible for resistance to viologen herbicides in the cyanobacterium Synechocystis sp. PCC 6803

The charged quaternary ammonium compounds – methyl, ethyl and benzyl viologens – generate reactive oxygen species in photosynthetic cells. Three independent methyl viologen‐resistant spontaneous mutants of Synechocystis sp. PCC 6803 were identified, in which the conserved R115 residue of the Slr1174 protein was replaced with G115, L115 and C115. The Slr1174 protein of the DUF990 family is related to the permease subunit of an ABC‐2‐type transporter and its R115 mutation was found to be solely responsible for the observed methyl viologen resistance. Bioinformatic analysis showed that in various bacterial genomes, two genes encoding another permease subunit and the ATPase component of an ATP‐binding cassette transporter form putative operons with slr1174 orthologs, suggesting that the protein products of these genes may form functional transporters. The corresponding genes in Synechocystis sp. PCC 6803, i.e. slr0610 for the permease and slr1901 for the ATPase, did not form such an operon. However, insertional inactivation of any slr1174, slr0610 or slr1901 genes in both the wild‐type and the R115‐resistant mutant resulted in increased sensitivity to methyl, ethyl and benzyl viologens; moreover, single‐ and double‐insertion mutants did not differ in their viologen sensitivity. Our data suggest that Slr1901, Slr1174 and Slr0610 form a heteromeric ATP‐binding cassette‐type viologen exporter, in which each component is critical for viologen extrusion. Because the greatest increase in mutant sensitivity was observed in the case of ethyl viologen, the three proteins have been named EvrA (Slr1901), EvrB (Slr1174) and EvrC (Slr0610). This is the first report of a function for a DUF990 family protein.

On the involvement of the regulatory gene prqR in the development of resistance to methyl viologen in cyanobacterium Synechocystis sp. PCC6803

The role of the prqR gene in the regulation of the adaptive response of the cyanobacteriumSynechocystis sp. PCC6803 to the oxidative stress induced with methyl viologen (MV) was studied. For this, mRNA levels of prqR and the genes, which may be involved in the control of resistance to MV, was determined by means of Northern blot hybridization in wild-type cells and in the MV-resistant Prq20 mutant with a mutation located in the DNA-binding domain of the PrqR protein. It was revealed that the prqR gene belongs to the prqR–prqA operon and downregulates its transcription. In cells of the wild-type strain treated with MV, the autorepressor activity of the PrqR protein enhances and transcript levels of mvrA and sodB genes encoding respectively an assumed transporter protein and iron-containing superoxide dismutase increases significantly. The prqR gene may be involved in the negative, indirect control of transcription of these genes. In this connection, the Prq20 mutant is characterized by an MV-independent derepression of the prqR–prqA operon and by a slightly increased transcript levels of mvrA and sodBgenes not stimulated by MV. Moreover, the expression of mvrA and sodB genes was significantly lower than in wild-type cells after the MV treatment. On the strength of this evidence, it is assumed that the main mechanism underlying for the resistance to MV in the Prq20 mutant is derepression of the prqAgene, the product of which is homologous to multidrug transporters, the drug efflux proteins.

Antibiotic resistance of potential probiotic bacteria of the genus Lactobacillus from human gastrointestinal microbiome

Thirteen Lactobacillus strains isolated from the gastrointestinal microbiome of people from the territory of the former Soviet Union have been studied for resistance to 15 antibiotics of different nature, namely, penicillins, aminoglycosides, macrolides, lincosamides, tetracyclines, chloramphenicol, and rifampicin. The strains included four strains of L. plantarum, four of L. helveticus, three of L. casei/paracasei, one of L. rhamnosus, and one of L. fermentum. All strains showed relative sensitivity to ampicillin, chloramphenicol, rifampicin, roxithromycin, erythromycin, and azithromycin, while none of them were sensitive to all tested antibiotics. L. plantarum strains had the broadest resistance spectra: one strain was resistant to tetracycline and three aminoglycosides and three strains were resistant to tetracycline and five aminoglycosides; one strain demonstrated high resistance to clindamycin and two strains to lincomycin. At the same time, two L. plantarum strains demonstrated resistance to benzylpenicillin coupled with sensitivity to ampicillin, another β-lactam antibiotic. Such resistance was clearly not related to the β-lactamase activity and could be explained by a specific mutation in one of the penicillin-binding proteins of the cell wall. Strains of L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum exhibited cross resistance to two to five different aminoglycosides. A PCR test of the resistance determinants for the widely clinically used antibiotics, tetracycline, chloramphenicol, and erythromycin revealed the presence of the tetM gene of conjugative transposon in L. casei/paracasei and two L. helveticus strains. Nucleotide sequence analysis of the amplified tetM fragments demonstrated their high homology with the tetM genes of Enterococcus faecalis and Streptococcus pneumoniae. The strains carrying tetM were tested for the genes of replication and conjugative transfer of plasmids in lactic acid bacteria. The results indicated that these strains contain genes identical or highly homologous to the rep and trsK genes of the plca36 plasmid and rep gene of the pLH1 and pLJ1 plasmids of lactic acid bacteria. The tetM gene is probably not expressed in strains sensitive to the corresponding antibiotic. However, the investigated lactobacilli cannot be directly used as probiotics, as they may serve as a source of genes for antibiotic resistance in the human microbiome.

The prqA and mvrA Genes Encoding Drug Efflux Proteins Control Resistance to Methyl Viologen in the Cyanobacterium Synechocystis sp. PCC 6803

Derivatives with insertional inactivation of prqA and mvrAgenes were obtained and studied in the Synechocystis sp. PCC 6803 wild-type strain and in the mutant Prq20 resistant to methyl viologen (MV). It was shown that the formation of resistance to MV is associated with the operation of two systems: constitutive and inducible. TheprqAgene encoding drug efflux protein controls the constitutive system of cell resistance to MV. Derepression of the prqA gene is the main reason for an enhanced MV resistance in the Prq20 mutant with impaired repressor function of the PrqR protein. The mvrA gene encoding the transmembrane protein from the family of transporters of sugar and other compounds controls the inducible MV resistance. It is assumed that the MvrA protein is required for efficient elimination from cells of toxic substances formed upon oxidative stress or participates in the repair of membranes destroyed by oxidants. The data obtained demonstrated for the first time that transport systems are involved in the development of MV resistance in photosynthetic organisms.

Rapid separation of DNAs by buoyant density in three-layer CsCl gradients

The formation of three-layer CsCl gradients with a narrow middle layer containing the analyzed material allows the separation of macrospecies according to their buoyant densities during the first hours of centrifugation before equilibrium is attained. The method can be used for the isolation and purification of various DNA molecules, such as mitochondrial and plasmid DNAs.