Four New Genes of Cyanobacterium Synechococcus elongatus PCC 7942 Are Responsible for Sensitivity to 2-Nonanone.

Microbial volatile organic compounds (VOCs) are cell metabolites that affect many physiological functions of prokaryotic and eukaryotic organisms. Earlier we have demonstrated the inhibitory effects of soil bacteria volatiles, including ketones, on cyanobacteria. Cyanobacteria are very sensitive to ketone action. To investigate the possible molecular mechanisms of the ketone 2-nonanone influence on cyanobacterium Synechococcus elongatus PCC 7942, we applied a genetic approach. After Tn5-692 transposon mutagenesis, several 2-nonanone resistant mutants have been selected. Four different mutant strains were used for identification of the impaired genes (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732, Synpcc7942_0726) that encode correspondingly: 1) a murein-peptide ligase Mpl that is involved in the biogenesis of cyanobacteria cell wall; 2) a putative ABC transport system substrate-binding proteins MlaD, which participates in ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane by aberrantly localized phospholipids transport from outer to inner membranes of bacterial cells; 3) a conserved hypothetical protein that is encoding by gene belonging to phage gene cluster in Synechococcus elongatus PCC 7942 genome; 4) a protein containing the VRR-NUC (virus-type replication-repair nuclease) domain present in restriction-modification enzymes involved in replication and DNA repair. The obtained results demonstrated that 2-nonanone may have different targets in Synechococcus elongatus PCC 7942 cells. Among them are proteins involved in the biogenesis and functioning of the cyanobacteria cell wall (Synpcc7942_1362, Synpcc7942_0351, Synpcc7942_0732) and protein participating in stress response at DNA restriction-modification level (Synpcc7942_0726). This paper is the first report about the genes that encode protein products, which can be affected by 2-nonanone.

Microbial Communities of Polymetallic Deposits' Acidic Ecosystems of Continental Climatic Zone With High Temperature Contrasts.

Acid mine drainage (AMD) systems are globally widespread and are an important source of metal pollution in riverine and coastal systems. Microbial AMD communities have been extensively studied for their ability to thrive under extremely acidic conditions and for their immense contribution to the dissolution of metal ores. However, little is known on microbial inhabitants of AMD systems subjected to extremely contrasting continental seasonal temperature patterns as opposed to maritime climate zones, experiencing much weaker annual temperature variations. Here, we investigated three types of AMD sites in Eastern Transbaikalia (Russia). In this region, all surface water bodies undergo a deep and long (up to 6 months) freezing, with seasonal temperatures varying between -33 and +24°C, which starkly contrasts the common well-studied AMD environments. We sampled acidic pit lake (Sherlovaya Gora site) located in the area of a polymetallic deposit, acidic drainage water from Bugdaya gold-molybdenum-tungsten deposit and Ulan-Bulak natural acidic spring. These systems showed the abundance of bacteria-derived reads mostly affiliated with Actinobacteria, Acidobacteria, Alpha- and Gammaproteobacteria, chloroplasts, Chloroflexi, Bacteroidetes, and Firmicutes. Furthermore, candidate taxa "Ca. Saccharibacteria" (previously known as TM7), "Ca. Parcubacteria" (OD1) and WPS-2 were represented in substantial quantities (10-20%). Heterotrophy and iron redox cycling can be considered as central processes of carbon and energy flow for majority of detected bacterial taxa. Archaea were detected in low numbers, with Terrestrial Miscellaneous Euryarchaeal Group (TMEG), to be most abundant (3%) in acidic spring Ulan-Bulak. Composition of these communities was found to be typical in comparison to other AMD sites; however, certain groups (as Ignavibacteriae) could be specifically associated with this area. This study provides insight into the microbial diversity patterns in acidic ecosystems formed in areas of polymetallic deposits in extreme continental climate zone with contrasting temperature parameters.

Inhibition of cyanobacterial photosynthetic activity by natural ketones.

Microbial volatiles have a significant impact on the physiological functions of prokaryotic and eukaryotic organisms. Various ketones are present in volatile mixtures produced by plants, bacteria, and fungi. Our earlier results demonstrated the inhibitory effects of soil bacteria volatiles, including ketones, on cyanobacteria. In this work, we thoroughly examined the natural ketones, 2-nonanone and 2-undecanone to determine their influence on the photosynthetic activity in Synechococcus sp. PCC 7942. We observed for the first time that the ketones strongly inhibit electron transport through PSII in cyanobacteria cells in vivo. The addition of ketones decreases the quantum yield of primary PSII photoreactions and changes the PSII chlorophyll fluorescence induction curves. There are clear indications that the ketones inhibit electron transfer from QA to QB , electron transport at the donor side of PSII. The ketones can also modify the process of energy transfer from the antenna complex to the PSII reaction center and, by this means, increase both chlorophyll fluorescence quantum yield and the chlorophyll excited state lifetime. At the highest tested concentration (5 mM) 2-nonanone also induced chlorophyll release from Synechococcus cells that strongly indicates the possible role of the ketones as detergents.

The Cyanotoxin BMAA Induces Heterocyst Specific Gene Expression in Anabaena sp. PCC 7120 under Repressive Conditions.

Cyanobacteria synthesize neurotoxic ß-N-methylamino-l-alanine (BMAA). The roles of this non-protein amino acid in cyanobacterial cells are insufficiently studied. During diazotrophic growth, filamentous cyanobacteria form single differentiated cells, called heterocysts, which are separated by approximately 12⁻15 vegetative cells. When combined nitrogen is available, heterocyst formation is blocked and cyanobacterial filaments contain only vegetative cells. In the present study, we discovered that exogenous BMAA induces the process of heterocyst formation in filamentous cyanobacteria under nitrogen-replete conditions that normally repress cell differentiation. BMAA treated cyanobacteria form heterocyst-like dark non-fluorescent non-functional cells. It was found that glutamate eliminates the BMAA mediated derepression. Quantitative polymerase chain reaction (qPCR) permitted to detect the BMAA impact on the transcriptional activity of several genes that are implicated in nitrogen assimilation and heterocyst formation in Anabaena sp. PCC 7120. We demonstrated that the expression of several essential genes increases in the BMAA presence under repressive conditions.

Stress effects of cyanotoxin ß-methylamino-L-alanine (BMAA) on cyanobacterial heterocyst formation and functionality.

Various species of cyanobacteria, diatoms and dinoflagellates are capable of synthesizing the non-proteinogenic neurotoxic amino acid ß-N-methylamino-L-alanine (BMAA), which is known to be a causative agent of human neurodegeneration. Similar to most cyanotoxins, the biological and ecological functions of BMAA in cyanobacteria are unknown. In this study, we show for the first time that BMAA, in micromolar amounts, inhibits the formation of heterocysts (specialized nitrogen-fixing cells) in heterocystous, diazotrophic cyanobacteria [Anabaena sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), Nostoc sp. strain 8963] under conditions of nitrogen starvation. The inhibitory effect of BMAA is abolished by the addition of glutamate. To understand the genetic reason for the observed phenomenon, we used qPCR to study the expression of key genes involved in cell differentiation and nitrogen metabolism in the model cyanobacterium Anabaena sp. PCC 7120. We observed that in the presence of BMAA, Anabaena sp. PCC 7120 does not express two essential genes associated with heterocyst differentiation, namely, hetR and hepA. We also found that addition of BMAA to cyanobacterial cultures with mature heterocysts inhibits nifH gene expression and nitrogenase activity.

Inhibitory and toxic effects of volatiles emitted by strains of Pseudomonas and Serratia on growth and survival of selected microorganisms, Caenorhabditis elegans, and Drosophila melanogaster.

In previous research, volatile organic compounds (VOCs) emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans). VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds--ketones (2-nonanone, 2-heptanone, 2-undecanone) and dimethyl disulfide--were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions.