Abstracts of research studies conducted in the Biochemistry and Microbiology Department in 2020

Project leader: Krzysztof Pawłowski. collaborators: Małgorzata Dudkiewicz, Marcin Gradowski, Bartłomiej Deszcz (Ph.D. student), Marianna Krysińska (Ph.D. student), Bartosz Baranowski (Ph.D. student, IBB PAN).

We performed a bioinformatics survey of bacterial kinase families (article in Bioch. Soc. Trans.). We built a catalogue of kinases from bacteria of the genus Legionella, including almost 20 novel families (article in preparation).

We characterised bioinformatically novel effector ADP-ribosyltransferase (ART) domains from the pathogenic fungi Magnaporthe oryzae (experimental work ongoing, collaboration with University of Texas Souhwestern, Dallas - UTSW). We described two novel ART families in the bacterium Legionella pneumophila (paper in press in J. Biol. Chem., more experimental work ongoing - UTSW). We also characterised bioinformatically the evolutionarily conserved eukaryotic ADP-ribosyltransferase LRRC9 (preprint on bioRxiv, article in revision in PeerJ).

We analised bioinformatically kinase and substrate networks related to survival in melanoma (article in preparation, collaboration with Lund University).

We built a bioinformatics server for functional analysis of genomic neighborhoods and gene co-occurrence.

Project leader: Tomasz Stępkowski, collaborators: Joanna Banasiewicz, Barbara Łotocka, Małgorzata Dudkiewicz, Krzysztof Pawłowski.The importance of geographical isolation of South America in evolution of nodule bacteria of the genus Bradyrhizobium in the Neotropical Floristic Kingdom. NCN Opus, 2015-2020,

The project covered three main themes. The first concerned the phylogenetic analyses of rhizobial isolates originating from root nodules formed on legume-trap plants grown in pots containing soil samples collected in the Rio Grande do Sul state area. Soil samples came from two eco-climatic zones - tropical lowland meadows and areas previously included in the subtropical araucaria forest that once covered a volcanic plateau known as the Campos de Cima da Serra. Mainly Macroptilium atropurpureum - a species commonly used in the isolation of rhizobium strains from tropical soil samples and several local species - was used as trap plants. Phylogenetic studies of the isolated Bradyrhizobium strains revealed the presence of many, potentially new species and the quantitative advantage of some phylogenetic groups. Moreover, isolates representing new genera within the order of Rhizobiales were obtained. However, authentication tests will be required to determine their symbiotic properties.

The second issue concerned the development and application of metagenomic methods based on the amplification of the nifD gene encoding the alpha nitrogenase subunit. The aim of this part of the project was to amplify this gene within Bradyrhizobium, which was supposed to enable the identification of both dominant and rare taxa within this genus. Phylogenetic analysis of the obtained sequences revealed the presence of 19 new groups (apart from the 19 already described), confirming the dominance of the groups described in the "classical" part of the project. This method showed a very large scale of nifD gene sequence diversity within this bacterial genus.

The third theme was related to the identification of Bradyrhizobium strains that emerged in Brazil's soils as a result of the uncontrolled spread of invasive Fabaceae and the associated migration of their natural bacterial symbionts. Our research carried out mainly with metagenomic methods showed the presence of strains representing Clade I - the dominant group in Australia in the places of Australian acacia species occurrence and strains characteristic for Europe in the places of the occurrence of European gorse. This indicates the significant changes in the composition of soil microbiomes in Brazil as a result of the introduction of alien species of the Fabaceae.

Project leader: Tomasz Stępkowski, collaborator: Joanna Banasiewicz The application of phylogeographic and metagenomic approaches for assessing the impact of invasive Australian Acacia species on rhizobium communities in the Mpumalanga and KwaZULU Natal Provinces of south Africa – BRADYAF. NCBiR współpraca dwustronna polsko-południowoafrykańska, 2019-2022,

The project assumes the application of both traditional and metagenomic approaches. Due to the complications resulting from the global COVID-19 pandemic, we were forced to focus on the metagenomic part of the project and delegate the "classical" part to our partners from the University of Pretoria. In the ongoing research, we are working on the development of research methods enabling deep analysis of the structure of the microbial community within the selected taxon, in this case, within the order Rhizobiales. At present, we are at the stage of developing methods for the amplification of selected marker genes, using the experience gained during the implementation of our project financed by the National Science Center.

Project leader: Urszula Jankiewicz, collaborator: Arletta Kochańska – Jezierska

Limiting the susceptibility of plants to abiotic stresses in the presence of bacteria synthesizing 1-aminocyclopropane 1-carboxylic acid deaminase (ACC).

Bacteria with the acdS gene encoding ACC deaminase, which is responsible for the breakdown of ethylene into ammonia and α-ketobutyrate, are of interest due to their plant growth promoting properties, especially in the presence of biotic and abiotic stressors. Ethylene is a gaseous plant hormone synthesized in plant tissues from its precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Ethylene is involved in many plant development processes, e.g. tissue differentiation or aging processes, as well as regulates the response of plants to both abiotic and abiotic stresses. salinity, drought or the presence of heavy metal ions.

As a result of the conducted experiments, bacteria with the ability to synthesize active ACC deaminase were isolated and identified. The enzymatic activity was determined by the spectrophotometric method. Sequence analysis of the gene encoding the 16S rRNA showed that all active bacterial isolates belonged to the genera Pseudomonas and Bacillus. Tomato plants, subjected to salt stress, performed much better in the presence of deaminase synthesizing bacteria than those grown without inoculation with such bacteria.

Project leader: Arletta Kochańska-Jeziorska, Urszula Jankiewicz

The molecular language of bacteria and its application in the induction of plant resistance.

Until recently, bacteria have been regarded as organisms, unable to communicate. The discovery of quorum sensing (QS) has become a phenomenon and opened up many new possibilities. QS is a bacterial communication system that depends on the population density of these bacteria. It plays an important role in the regulation of many genes, e.g. bioluminescence, virulence, biofilm formation, and also coordinates and controls the behaviour of individual cells in a population. The mediator in QS communication is small signal molecules - autoinductors. These molecules are of great interest due to their action not only on bacteria but also on eukaryotic organisms, as some of them can positively affect plant growth and plants health. Cell signalling is especially important in extreme environments and can play a key role in enhancing microbial biodiversity. Deliberate blocking of autoinductors produced by pathogens may become a breakthrough in times of increasing antibiotic resistance among bacteria. On the other hand, the use of specific signalling molecules or the bacteria that produce them on plants can affect the positive response of the plant and manifest itself in resistance to pathogen attacks or salt stress.