Michał Złoch

Metabolic potential and community structure of endophytic and rhizosphere bacteria associated with the roots of the halophyte Aster tripolium L.

The submitted work assumes that the abundance and diversity of endophytic and rhizosphere microorganisms co-existing with the halophytic plant Aster tripolium L. growing in a salty meadow in the vicinity of a soda factory (central Poland) represent unique populations of cultivable bacterial strains. Endophytic and rhizosphere bacteria were (i) isolated and identified based on 16S rDNA sequences; (ii) screened for nifH and acdS genes; and (iii) analyzed based on selected metabolic properties. Moreover, total microbial biomass and community structures of the roots (endophytes), rhizosphere and soil were evaluated using a cultivation-independent technique (PLFA) to characterize plant-microbial interactions under natural salt conditions. The identification of the isolated strains showed domination by Gram-positive bacteria (mostly Bacillus spp.) both in the rhizosphere (90.9%) and roots (72.7%) of A. tripolium. Rhizosphere bacterial strains exhibited broader metabolic capacities, while endophytes exhibited higher specificities for metabolic activity. The PLFA analysis showed that the total bacterial biomass decreased in the following order (rhizosphere<soil<endophytes) and confirmed the dominance of Gram-positive endophytic bacteria in the roots of the halophyte. The described strain collection provides a valuable basis for a subsequent applications of bacteria in improvement of site adaptation of plants in saline soils.

Transcriptomic profiling of the salt stress response in excised leaves of the halophyte Beta vulgaris ssp. maritima.

Beta vulgaris ssp. maritima is a halophytic relative of cultivated beets. In the present work a transcriptome response to acute salt stress imposed to excised leaves of sea beet was investigated. Salt treatments consisted of adding NaCl directly to the transpiration stream by immersing the petioles of excised leaves into the salt solutions. Sequencing libraries were generated from leaves subjected to either moderate or strong salt stress. Control libraries were constructed from untreated leaves. Sequencing was performed using the Illumina MiSeq platform. We obtained 32970 unigenes by assembling the pooled reads from all the libraries with Trinity software. Screening the nr database returned 18,362 sequences with functional annotation. Using the reference transcriptome we identified 1,246 genes that were differentially expressed after 48 h of NaCl stress. Genes related to several cellular functions such as membrane transport, osmoprotection, molecular chaperoning, redox metabolism or protein synthesis were differentially expressed in response to salt stress. The response of sea beet leaves to salt treatments was marked out by transcriptomic up-regulation of genes related to photosynthetic carbon fixation, ribosome biogenesis, cell wall-building and cell wall expansion. Furthermore, several novel and undescribed transcripts were responsive to salinity in leaves of sea beet.

Metabolic profiles of microorganisms associated with the halophyte Salicornia europaea in soils with different levels of salinity

Abstract: Plants have evolved a wide range of adaptation mechanisms that enable them to grow and develop in environments of high soil salinity. We examine the plant—microbial interactions of Salicornia europaea (Herbaceae) as potential mechanisms responsible for the high tolerance of halophytes to saline conditions. We evaluated the impact of different NaCl concentrations on microorganisms associated with S. europaea growing at 2 saline sites in central Poland: a salty meadow in the vicinity of a soda factory (anthropogenic salinity) and an area affected by natural brine (landscape park). The community-level physiological profiles of microorganisms associated with the plant were determined for endophytes from the roots, rhizosphere, and salt-affected soil. The highest total microbial metabolic activity, irrespective of the test site, was always in the rhizosphere, while the activity of endophytic and soil populations depended on salinity. In general, a higher total metabolic activity was observed at the test site exhibiting higher salt concentration. We propose that increased metabolic activity of microbial populations under increased salt stress can significantly contribute to the site adaptation of halophytes to saline conditions.

Modeling of phytoextraction efficiency of microbially stimulated Salix dasyclados L. in the soils with different speciation of heavy metals

ABSTRACT Bioaugmentation of soils with selected microorganisms during phytoextraction can be the key solution for successful bioremediation and should be accurately calculated for different physicochemical soil properties and heavy metal availability to guarantee the universality of this method. Equally important is the development of an accurate prediction tool to manage phytoremediation process. The main objective of this study was to evaluate the role of three metallotolerant siderophore-producing Streptomyces sp. B1–B3 strains in the phytoremediation of heavy metals with the use of S. dasyclados L. growing in four metalliferrous soils as well as modeling the efficiency of this process based on physicochemical and microbiological properties of the soils using artificial neural network (ANN) analysis. The bacterial inoculation of plants significantly stimulated plant biomass and reduced oxidative stress. Moreover, the bacteria affected the speciation of heavy metals and finally their mobility, thereby enhancing the uptake and bioaccumulation of Zn, Cd, and Pb in the biomass. The best capacity for phytoextraction was noted for strain B1, which had the highest siderophore secretion ability. Finally, ANN model permitted to predict efficiency of phytoextraction based on both the physicochemical properties of the soils and the activity of the soil microbiota with high precision.

Identification of Microorganisms by Modern Analytical Techniques

Rapid detection and identification of microorganisms is a challenging and important aspect in a wide range of fields, from medical to industrial, affecting human lives. Unfortunately, classical methods of microorganism identification are based on time-consuming and labor-intensive approaches. Screening techniques require the rapid and cheap grouping of bacterial isolates; however, modern bioanalytics demand comprehensive bacterial studies at a molecular level. Modern approaches for the rapid identification of bacteria use molecular techniques, such as 16S ribosomal RNA gene sequencing based on polymerase chain reaction or electromigration, especially capillary zone electrophoresis and capillary isoelectric focusing. However, there are still several challenges with the analysis of microbial complexes using electromigration technology, such as uncontrolled aggregation and/or adhesion to the capillary surface. Thus, an approach using capillary electrophoresis of microbial aggregates with UV and matrix-assisted laser desorption ionization time-of-flight MS detection is presented.

The influence of different pH on the electrophoretic behaviour of Saccharomyces cerevisiae modified by calcium ions

The effect of a different pH on Saccharomyces cerevisiae cells modified with calcium ions was investigated by the capillary zone electrophoresis technique. For the identification of the wild strain of S. cerevisiae, the ribosomal nucleic acid sequencing and internal transcribed spacer sequencing as well as spectrometric approach were applied. The potentiometric titration and Fourier transform infrared spectroscopy have shown the occurrence of active functional groups such as carboxyl, amine/hydroxyl, phosphate/hydrogen phosphate groups on the surface of native yeast cells. Moreover, the spectroscopy study in a medium infrared range was carried out to identify the functional groups of yeast cells that participate in calcium ions binding interaction. Furthermore, the microscopic and spectrometric analysis shows that the pH value of the calcium ions solution has a significant effect on the intensity yeast cells clumping. Additionally, the impact of yeast cell clumping on the electrophoretic behaviours was examined. The modification of surface functional groups by calcium ions significantly affected the efficiency of electrophoretic separation. However, these changes did not affect the accuracy of S. cerevisiae identification by MALDI equipment with BioTyper platform. These results form the analytical solution for coupling of electrophoresis and MALDI-TOF MS technique.

Cadmium-induced changes in the production of siderophores by a plant growth promoting strain of Pseudomonas fulva

The impact of increasing Cd2+ exposure on the quality and quantity of siderophores produced by a plant growth promoting Pseudomonas fulva strain was tested to gain insight into the degree of change. P. fulva was cultured in the increasing concentrations of Cd2+ (0, 0.5, 1.0, 2.0 mM). The secreted siderophores were separated by HPLC and characterized by UHPLC‐QTOF/MS. In the presence of 2 mM Cd2+ synthesis of siderophores (hydroxamates, catecholates, phenolates) was mitigated compared to the treatments with lower concentrations of Cd2+ (0.5 and 1 mM). Increased synthesis of catecholates in 0.5 and 1 mM Cd2+ and of phenolates in 0.5–2 mM Cd2+ was revealed compared to the variant without Cd2+. Out of seven different hydroxamates, the secretion of ferrioxamine E was significantly decreased in the highest Cd2+ concentration. Two additional ferrioxamines, X2 and D2, were secreted independent of the presence or absence of Cd2+. Exposure to Cd2+ change the composition of siderophores secreted by P. fulva with selective promotion of catecholates and phenolates at the expense of hydroxamates. Successful adaptation in a Cd‐contaminated soil in the frame of practical applications to promote phytoremediation can be assumed.