M. M. Babykin

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.

Cloning and Molecular Analysis of the Gene PRQR Controlling Resistance to Paraquat in Synechocystis sp. PCC 6803

Oxygen-evolving photosynthetic cells must possess efficient systems for protection against active oxygen species generated by endogenous mechanisms and various environmental factors. Redox-cycling agents, such as paraquat and menadione, can induce the formation of active radicals causing an oxidative stress [1]. Paraquat accepts electrons from Photosystem I and its cation radical reduces oxygen to superoxide which is converted to hydrogen peroxide. The exact site of paraquat action is uncertain. It was suggested that Fe-S center B of Photosystem I is the main site of electron donation to paraquat [2]. The enhancement of resistance to paraquat in plants and bacteria may be associated with alterations in transport of the drug, failure to interact with cell targets, elevation of activities of enzymes involved in detoxification of paraquat, scavenging of active oxygen species or repair of oxidative damage [3,4]. An adaptive response to oxidative stress in some bacteria is mediated by regulatorysoxRS and oxyR gene systems which control at the transcriptional level the induction of antioxidant defensive proteins [1,4]. Bacterial soxR-constitutive mutants are characterised by an increased resistance to redox-cycling agents [4,5]. A number of cyanobacterial mutants resistant to paraquat treatment have been isolated [6-8], but the genetic nature of this resistance has not been identified. Cloning and molecular analysis of genes controlling resistance to redox-cycling agents can help to understand the nature of cell targets for these agents and the defence mechanisms against oxidative stress and photoinactivation in cyanobacteria. Here, we report the cloning and sequencing of a novel gene of Synechocystis sp. PCC 6803, mutation in which results in elevated cross-resistance to paraquat and menadione.

TonB-Dependent Utilization of Dihydroxamate Xenosiderophores in Synechocystis sp. PCC 6803

In Gram-negative bacteria, transport of ferric siderophores through outer membrane is a complex process that requires specific outer membrane transporters and energy-transducing TonB–ExbB–ExbD system in the cytoplasmic membrane. The genome of the non-siderophore-producing cyanobacterium Synechocystis sp. PCC 6803 encodes all putative components of the siderophore-mediated iron uptake system. So far, there has been no experimental evidence for the existence of such a pathway in this organism. On the contrary, its reductive iron uptake pathway has been studied in detail. We demonstrate that Synechocystis sp. PCC 6803 is capable of using dihydroxamate xenosiderophores, either ferric schizokinen (FeSK) or a siderophore of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 (SAV), as the sole source of iron. Inactivation of the tonB gene or the exbB1–exbD1 gene cluster resulted in an inability to utilize these siderophores. At the same time, the inactivation of the feoB gene encoding FeoB plasma membrane ferrous iron transporter, or one of the futB or futC genes encoding permease and ATPase subunit of FutABC ferric iron transporter, did not impair the ability of cells to utilize FeSK or SAV as the sole source of iron for growth. Our data suggest that cyanobacterium Synechocystis sp. PCC 6803 is capable of acquiring iron–siderophore complexes in a TonB-dependent manner without iron reduction in the periplasm.

In Cis and In Trans Autorepression of the prqR Gene in Cyanobacterium Synechocystis sp. PCC 6803

Genetic analysis of the allele interactions was carried out with the use of recombinant plasmids and reporter genes to study the autorepressor function of prqR, which negatively regulates the prqR–prqA operon and the response to oxidative stress inducer methyl viologen (MV) in cyanobacterium Synechocystis sp. PCC 6803. The wild-type prqR cloned in Escherichia colishowed negative autoregulation and suppressed in trans the derepressed mutant alleles. Frameshift mutation C134fs, which was introduced in prqR by site-directed mutagenesis, impaired the autoregulation, implicating the PrqR C-terminal domain in transcriptional repression. Missense mutation C134S, changing the only redox-sensitive Cys of PrqR, had no effect on prqR expression, indicating that oxidation and consequent disulfide bridging of two PrqR molecules was not responsible for MV-induced autorepression of prqR. Analysis of the prqR–prqA deletion derivatives lacking the promoter and most of prqR revealed weak uncontrollable expression of reporter cat, testifying to the existence of a constitutive promoter in prqA responsible for MV resistance. The interaction of the wild-type and mutant prqR alleles in Synechocystis cells revealed a cis-dominant character of the impairment of prqR autoregulation. Stimulation of in cis autorepression of prqR was assumed to contribute to the induction of systems protecting cyanobacteria against oxidative stress.

Inversion of phototaxis in cells of Synechocystis sp. PCC 6803 determined by a mutation in the regulatory gene prqR

Phototaxis, positive (movement toward the light source) or negative (from the light source) mediates the adaptation of cyanobacteria to varying wave lengths and illumination intensity. The transcription regulator PrqR of the family TetR is known as a repressor of the prqRA operon controlling resistance to the oxidative stress inducer methyl viologen in the cyanobacterium Synechocystis sp. PCC 6803. However, it was shown in this work that mutation prqRL17Q affecting the DNA-binding domain of the PrqR protein, which causes derepression of the prqRA operon and enhances cell resistance to methyl viologen, additionally determines negative phototaxis induced with white light and red light of low intensity. The inactivation of gene prqA did not affect cell motility in mutant PqR carrying mutation prqRL17Q and in the wild-type strain characterized by positive phototaxis appearing in response to the light of low intensity. Moreover, a mutant with deletion prqR did not differ from the wild-type strain with respect to phototaxis type suggesting that the specificity of the regulator protein was changed in cells carrying prqRL17Q mutation. Note that changes in transcription of pilA genes that control biogenesis of pili providing for cell motility were not detected in mutant PqR, and, in agreement with data of atomic force microscopy, the type of pili formation is identical in prqR mutants and the wild-type strain. Meanwhile, mutant PqR manifested a decrease in transcription of gene taxD1 encoding the photoreceptor of red light that is required for the positive phototaxis of cyanobacteria. These data imply that mutation prqRL17Q changes the specificity of the PrqR repressor protein and thereby affects the regulation of phototaxis at the level of photoperception and signal transduction in cells.

Atomic Force Microscopy Study of Pili in the Cyanobacterium Synechocystis SP. PCC 6803

Atomic force microscopy (AFM) is now widely used method in life sciences, specifically for investigation of the microbial surfaces. Continuing the AFM research of various bacteria we studied pilus-like appendages of the unicellular cyanobacterium Synechocystis sp. PCC 6803. As known the Synechocystis 6803 wild type cells produce pili of two morphotypes distinguished by diameter, length, morphology and relative abundance. Thick pili (one morphotype) are identified with well-known bacterial type IV pili responsible for cell gliding motility; the function of thin pili (another morphotype) is still unknown. AFM has revealed differences in piliation of the motile (wild type) and non-motile (spontaneous mutant) strains of the cyanobacterium and allowed estimating real dimensions and quantity of pili. According to the data obtained non-motile mutant cells possess thick pili increased in number (to 3—5 times) and length (more than to 10 times) relative to the wild type cells, however, both strains do not differ in thin pili. The advantages of AFM over conventional electron microscopic techniques in comparative morphology of the bacterial cells are discussed.