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Purpose: Allergic diseases have escalated to epidemic levels worldwide, impacting nearly 30% of the global population. Fungi are a significant source of allergens responsible for up to 6% of respiratory diseases in the general population. However, the specific cause of respiratory allergies often remains unidentified. This study aimed to investigate the potential of two common rust fungi, Tranzschelia pruni-spinosae and Phragmidium rubi-idaei , to trigger a proinflammatory response in vitro models representing the upper and lower respiratory tract., Materials and Methods: The BEAS-2B and A549 cell lines simulated upper and lower respiratory endothelial cells. The cytotoxicity of fungal extracts was evaluated using MTT and flow cytometry assays. Cell reactive oxygen species (ROS) production was measured via flow cytometry, while ELISA tests quantified the production of proinflammatory cytokines. Immunofluorescence techniques were employed to assess cell integrity markers., Results: Extracts from T. pruni-spinosae and P. rubi-idaei significantly stimulated the production of proinflammatory cytokines IL-1β and GM-CSF in both cell lines, all of which are associated with the development of allergic responses. The increase in these cytokines and the elevated ROS production were linked to the disruption of epithelial cell junctions., Conclusion: The findings suggest the potential of T. pruni-spinosae and P. rubi-idaei extracts to collectively disrupt the epithelial barrier in the upper and lower respiratory tract by inducing proinflammatory cytokines and the production of reactive oxygen species and metalloproteinases. Although none of the above parameters was spectacularly high, all of them together could cause a decrease in the presence of tight junction proteins, such as E-cadherin and occludin, in epithelial cells., Competing Interests: The authors declare that the research was conducted without any commercial or financial relationship that could be construed as a potential conflict of interest., (© 2025 Sztandera-Tymoczek et al.)

A novel bacterium, designated strain MMK2 T , was isolated from a surface-sterilised root nodule of a Trifolium rubens plant growing in south-eastern Poland. Cells were Gram negative, non-spore forming and rod shaped. The strain had the highest 16S rRNA gene sequence similarity with P. endophytica (99.4%), P. leporis (99.4%) P. rwandensis (98.8%) and P. rodasii (98.45%). Phylogenomic analysis clearly showed that strain MMK2 T and an additional strain, MMK3, should reside in the genus Pantoea and that they were most closely related to P. endophytica and P. leporis. Genome comparisons showed that the novel strain shared 82.96-93.50% average nucleotide identity and 26.2-53. 2% digital DNA:DNA hybridization with closely related species. Both strains produced siderophores and were able to solubilise phosphates. The MMK2 T strain was also able to produce indole-3-acetic acid. The tested strains differed in their antimicrobial activity, but both were able to inhibit the growth of Sclerotinia sclerotiorum 10Ss01. Based on the results of the phenotypic, phylogenomic, genomic and chemotaxonomic analyses, strains MMK2 T and MMK3 belong to a novel species in the genus Pantoea for which the name Pantoea trifolii sp. nov. is proposed with the type strain MMK2 T (= DSM 115063 T  = LMG 33049 T )., (© 2024. The Author(s).)

Polycyclic aromatic hydrocarbons (PAHs) are common xenobiotics that are detrimental to the environment and human health. Bacterial endophytes, having the capacity to degrade PAHs, and plant growth promotion (PGP) may facilitate their biodegradation. In this study, phenanthrene (PHE) utilization of a newly isolated PGP endophytic strain of Pseudomonas chlororaphis 23aP and factors affecting the process were evaluated. The data obtained showed that strain 23aP utilized PHE in a wide range of concentrations (6-100 ppm). Ethyl-acetate-extractable metabolites obtained from the PHE-enriched cultures were analyzed by gas chromatography-mass spectrometry (GC-MS) and thin-layer chromatography (HPTLC). The analysis identified phthalic acid, 3-(1-naphthyl)allyl alcohol, 2-hydroxybenzalpyruvic acid, α -naphthol, and 2-phenylbenzaldehyde, and allowed us to propose that the PHE degradation pathway of strain 23aP is initiated at the 1,2-, 3,4-carbon positions, while the 9,10-C pathway starts with non-enzymatic oxidation and is continued by the downstream phthalic pathway. Moreover, the production of the biosurfactants, mono- (Rha-C 8 -C 8 , Rha-C 10 -C 8:1 , Rha-C 12:2 -C 10 , and Rha-C 12:1 -C 12:1 ) and dirhamnolipids (Rha-Rha-C 8 -C 10 ), was confirmed using direct injection-electrospray ionization-mass spectrometry (DI-ESI-MS) technique. Changes in the bacterial surface cell properties in the presence of PHE of increased hydrophobicity were assessed with the microbial adhesion to hydrocarbons (MATH) assay. Altogether, this suggests the strain 23aP might be used in bioaugmentation-a biological method supporting the removal of pollutants from contaminated environments.

Purpose: Allergic diseases have reached epidemic proportions globally, affecting nearly 30% of the world's population. One of the most prominent sources of allergens is fungi, causing up to 6% of respiratory diseases in the general population. However, the cause of respiratory allergies is not always identifiable. Therefore, we studied the ability of two representatives of common powdery mildew (Erysiphales), Erysiphe palczewskii and Erysiphe convolvuli , to induce a proinflammatory response in in vitro models of the upper and lower respiratory tract., Materials and Methods: Two cell lines, BEAS-2B and A549, were used to mimic upper and lower respiratory epithelial cells. The toxicity of fungal extracts was assessed with MTT and flow cytometry assay. The production of reactive oxygen species in the cells was measured with flow cytometry. ELISA tests were used to determine the production of proinflammatory cytokines. The presence of the cell integrity marker was assessed with the immunofluorescence method., Results: In both cell lines, the extract of E. palczewskii and E. convolvuli microfungi induced marked production of proinflammatory IL-1β, TNF-α, and GM-CSF cytokines involved in developing allergic reactions. The higher levels of these cytokines with higher reactive oxygen species synthesis positively correlated with the disruption of epithelial cell junctions., Conclusion: We conclude that E. palczewskii and E. convolvuli microfungi have strong proinflammatory and proallergenic potential, but this finding needs in vivo confirmation., Competing Interests: The authors declare that the research was conducted without any commercial or financial relationship that could be construed as a potential conflict of interest., (© 2023 Sztandera-Tymoczek et al.)

The effectiveness of phytoremediation is closely related to the various interactions between pollutants, soil particles, rhizosphere microorganisms, and plants. Therefore, the object of current study was a cadmium-tolerant bacterium isolated from the rye rhizosphere, with a high degree of genetic similarity to the genus Chryseobacterium. Chryseobacterium sp. DEMBc1 was able to grow with 36 different BiologGN2 carbon sources and show the adaptation to stress factors such as Cd (100 μg ml -1 ), low temperature (8 °C), and salinity (2% NaCl). Furthermore, it was shown that DEMBc1 had the characteristics of plant growth-promoting microorganisms: it was able to produce ammonia, indole acetic acid, 1-aminocyclopropane-1-carboxylic acid deaminase, and siderophores, as well as solubilize Ca 3 (PO 4 ) 3 . After inoculation with DEMBc1, a significant decrease in the concentration of Cd was observed in the roots of Festuca ovina grown in Cd-polluted soil, compared to the non-inoculated Cd-polluted soil. It was also noticed that DEMBc1 produced a large amount of extracellular polymeric substances that were significantly higher than the cellular biomass. These polymers can form a barrier to reduce the translocation of Cd from the growth medium to the plant roots. According to the current study, DEMBc1 has a stabilizing potential and can decrease the mobility of Cd in the F. ovina rhizosphere, bioaccumulate metals in plant tissues, and effectively improve the bioavailability of nutrients, especially Fe, N, and P in a polluted environments., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Anthropogenic activities generate a high quantity of organic pollutants, which have an impact on human health and cause adverse environmental effects. Monitoring of many hazardous contaminations is subject to legal regulations, but some substances such as therapeutic agents, personal care products, hormones, and derivatives of common organic compounds are currently not included in these regulations. Classical methods of removal of organic pollutants involve economically challenging processes. In this regard, remediation with biological agents can be an alternative. For in situ decontamination, the plant-based approach called phytoremediation can be used. However, the main disadvantages of this method are the limited accumulation capacity of plants, sensitivity to the action of high concentrations of hazardous pollutants, and no possibility of using pollutants for growth. To overcome these drawbacks and additionally increase the efficiency of the process, an integrated technology of bacteria-assisted phytoremediation is being used recently. For the system to work, it is necessary to properly select partners, especially endophytes for specific plants, based on the knowledge of their metabolic abilities and plant colonization capacity. The best approach that allows broad recognition of all relationships occurring in a complex community of endophytic bacteria and its variability under the influence of various factors can be obtained using culture-independent techniques. However, for practical application, culture-based techniques have priority.

Chamaecytisus albus (Spanish broom) is a legume shrub that can be found in only one natural locality in Poland. This specimen is critically endangered; therefore, different actions focusing on protection of this plant in the natural habitat are undertaken, and one of them involves studies of the population of Chamaecytisus albus bacterial endophytes, which in the future could be used as bioprotectants and/or biofertilizers. A collection of 94 isolates was obtained from Spanish broom nodules, and the physiological and genetic diversity of these strains was studied. A few potentially beneficial traits were detected, i.e. secretion of cellulases (66 isolates), production of siderophores (60 isolates), phosphate solubilization (25 isolates), and production of IAA (58 isolates), indole (16 isolates), or HCN (3 isolates). Twenty-nine of the 94 tested isolates were able to induce the development of root nodules in plants grown in vitro and can therefore be assumed as Chamaecytisus albus symbionts. Genome fingerprinting by BOX-PCR, as well as gyrB and nodZ gene sequencing revealed a great genetic diversity of specimens in the collection. The symbiotic isolates were classified in different clades, suggesting they could belong to different species, however, most of them revealed sequence similarity to Bradyrhizobium genus.

Aim: This study investigated the effect of an insect antimicrobial protein, apolipophorin III (apoLp-III), against two newly isolated, identified and characterized clinical strains of Staphylococcus spp. Materials & methods: Both strains were identified by 16S rRNA sequencing and metabolic and phenotypic profiling. The antibacterial activity of apoLp-III was tested using a colony counting assay. ApoLp-III interaction with bacterial cell surface was analyzed by Fourier transform IR spectroscopy. Results: Staphylococcus epidermidis and Staphylococcus capitis were identified. ApoLp-III exerted a dose-dependent bactericidal effect on the tested strains. The differences in the Staphylococcus spp. surface components may contribute to the various sensitivities of these strains to apoLp-III. Conclusion: ApoLp-III may provide a baseline for development of antibacterial preparations against Staphylococcus spp. involved in dermatological problems.

The aim of the study was to assess the incidence, resistance, virulence, and genotypic characteristics of Staphylococcus spp. residing in the gastrointestinal tract of dogs and cats, as a group of animals causing potential contamination of the urban space. A high percentage of strains resistant to penicillin (58%), oxacillin (9%) and tetracycline (60%) were found. All isolates resistant to penicillin, kanamycin, or chloramphenicol carried genes responsible for individual resistance (blaZ, aph(3')-IIIa, and cat (pC194)/cat (pC223), respectively. The mecA gene was detected in 45% of the oxacillin-resistant Staphylococcus pseudintermedius strains. The amplification of DNA fragments surrounding rare restriction sites analysis demonstrated high heterogeneity of genotypic profiles correlating with phenotypic resistance profiles. Multilocus sequence typing analysis classified the methicillin-resistant S. pseudintermedius strains as ST71, ST890, and the totally new ST1047. The presence of a high level of resistance among Staphylococcus strains may suggest a potential risk of transfer of these bacteria between companion animals and humans.

Legionella species synthesize phosphatidylcholine (PC) in two independent pathways: the three-step methylation of phosphatidylethanolamine PMT pathway and the one-step PCS pathway, in which the Pcs enzyme catalyzes the reaction between choline and CDP-diacylglycerol to form PC. Legionella pcs genes encode highly hydrophobic proteins with phosphatidylcholine synthase activity, which contain up to eight transmembrane helices with N- and C-termini located inside the bacterial cell. The comparative analysis of nucleotide sequences of pcs showed that these genes share high sequence identity among members of the Legionellaceae family. Legionella pmtA genes involved in the PMT pathway encoded small cytosolic proteins with putative phosphatidylethanolamine N-methyltransferase activity. The pmtA genes identified in Legionella species had lower sequence identity to each other than the pcs genes. The phylogenetic tree constructed based on the pcs and pmtA gene sequences showed phylogenetic relatedness between Legionella spp. and other bacteria. The utilization of extracellular choline by the four Legionella species leads to changes not only in the lipid components but also in proteins, and the interactions between these components lead to changes in cell surface properties, which result in a decline in induction of proinflammatory cytokines (TNF-α and IL-6)., (© FEMS 2019.)

Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX-PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates.

This is the first report describing isolates from root nodules of Ononis arvensis (field restharrow). The aim of this investigation was to describe the diversity, phylogeny, and plant growth promoting features of microsymbionts of O. arvensis, i.e., a legume plant growing in different places of the southern part of Poland. Twenty-nine bacterial isolates were characterized in terms of their phenotypic properties, genome fingerprinting, and comparative analysis of their 16S rRNA, nodC and acdS gene sequences. Based on the nodC and 16S rRNA gene phylogenies, the O. arvensis symbionts were grouped close to bacteria of the genera Rhizobium and Mesorhizobium, which formed monophyletic clusters. The acdS gene sequences of all the isolates tested exhibited the highest similarities to the corresponding gene sequences of genus Mesorhizobium strains. The presence of the acdS genes in the genomes of rhizobia specific for O. arvensis implies that these bacteria may promote the growth and development of their host plant in stress conditions. The isolated bacteria showed a high genomic diversity and, in the BOX-PCR reaction, all of them (except three) exhibited DNA fingerprints specific only for them. Our studies showed that restharrow isolates formed effective symbiotic interactions with their native host (O. arvensis) and Ononis spinosa but not with Trifolium repens and Medicago sativa belonging to the same tribe Trifolieae as Ononis species and not with Lotus corniculatus, representing the tribe Loteae.

The plant growth-promoting rhizobacteria have developed many different (indirect and direct) mechanisms that have a positive effect on plant growth and development. Strains isolated from Astragalus cicer and Astragalus glycyphyllos root nodules were investigated for their plant growth-promoting properties such as production of indole-3-acetic acid (IAA) and siderophores, phosphate solubilization, ACC deaminase activity, and tolerance to heavy metals. IAA production and P-solubilization were frequent features in the analysed strains, while siderophores were not produced by any of them. In this work, we investigated the presence of the acdS genes and ACC deaminase activities in Astragalaus cicer and A. glycyphyllos microsymbionts, classified within the genus Mesorhizobium. The results demonstrated that the acdS gene is widespread in the genome of Astragalus sp. microsymbionts; however, none of the tested strains showed ACC deaminase activity. The acdS gene sequence similarity of the analysed strains to each other was in the range from 84 to 99 %. On the phylogram of acdS gene sequences of milkvetch, the symbionts clustered tightly with the genus Mesorhizobium bacteria.

In this study, the phylogenetic relationship and taxonomic status of six strains, representing different phenons and genomic groups of Astragalus glycyphyllos symbionts, originating from Poland, were established by comparative analysis of five concatenated housekeeping gene sequences (atpD, dnaK, glnA, recA and rpoB), DNA-DNA hybridization and total DNA G+C content. Maximum-likelihood phylogenetic analysis of combined atpD, dnaK, glnA, recA and rpoB sequence data placed the studied bacteria into the clade comprising the genus Mesorhizobium. In the core gene phylograms, four A. glycyphyllos nodule isolates (AG1, AG7, AG15 and AG27) formed a cluster common with Mesorhizobium ciceri, whereas the two other A. glycyphyllos symbionts (AG17 and AG22) were grouped together with Mesorhizobium amorphae and M. septentrionale. The species position of the studied bacteria was clarified by DNA-DNA hybridization. The DNA-DNA relatedness between isolates AG1, AG7, AG15 and AG27 and reference strain M. ciceri USDA 3383T was 76.4-84.2%, and all these A. glycyphyllos nodulators were defined as members of the genomospecies M. ciceri. DNA-DNA relatedness for isolates AG17 and AG22 and the reference strain M. amorphae ICMP 15022T was 77.5 and 80.1%, respectively. We propose that the nodule isolates AG17 and AG22 belong to the genomic species M. amorphae. Additionally, it was found that the total DNA G+C content of the six test A. glycyphyllos symbionts was 59.4-62.1 mol%, within the range for species of the genus Mesorhizobium.

In this study, the tolerance of Mesorhizobium sp. ACMP18, Mesorhizobium sp. USDA3350, and Mesorhizobium temperatum LMG23931 strains, to cold and freezing were investigated. The ability to withstand freezing at -20 °C and -70 °C for 24 months was different among the studied strains and depended on the cryoprotectant used. The survivability of mesorhizobial strains at -20 °C and -70 °C was significantly improved by some cryoprotectans (glycerol and sucrose/peptone). It is worth noting that the greatest resistance to freezing was detected when stress treatments were performed in glycerol as a cryoprotectant. Using PCR analysis, cspA genes were identified in the studied strains. Their nucleotide sequences were most similar to the sequences of the corresponding genes of the Mesorhizobium species. The expression of the cspA gene in the studied bacteria was analyzed using the RT-PCR technique. The fatty acid composition of the mesorhizobia was determined at 5, 10, 15, and 28 °C. It was noticed that growth temperature significantly affected the fatty acid composition and the amounts of unsaturated fatty acids, especially that of cis-vaccenic acid (18:1ɷ(11)), increased markedly in bacterial cells cultivated at 5, 10, and 15 °C., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We assessed the genomic diversity and genomic relationship of 28 Astragalus glycyphyllos symbionts by three methodologies based on PCR reaction, i.e., RAPD, ERIC-PCR, and AFLP. The AFLP method with one PstI restriction enzyme and selective PstI-GC primer pair had a comparable discriminatory power as ERIC-PCR one and these fingerprinting techniques distinguished among the studied 28 A. glycyphyllos symbionts 18 and 17 genomotypes, respectively. RAPD method was less discriminatory in the genomotyping of rhizobia analyzed and it efficiently resolved nine genomotypes. The cluster analysis of RAPD, ERIC-PCR, and AFLP profiles resulted in a generally similar grouping of the test strains on generated dendrograms supporting a great potential of these DNA fingerprinting techniques for study of genomic polymorphism and evolutionary relationship of A. glycyphyllos nodulators. The RAPD, ERIC-PCR, and AFLP pattern similarity coefficients between A. glycyphyllos symbionts studied was in the ranges 8-100, 18-100, and 23-100%, respectively.

The phylogeny of symbiotic genes of Astragalus glycyphyllos L. (liquorice milkvetch) nodule isolates was studied by comparative sequence analysis of nodA, nodC, nodH and nifH loci. In all these genes phylograms, liquorice milkvetch rhizobia (closely related to bacteria of three species, i.e. Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium ciceri) formed one clearly separate cluster suggesting the horizontal transfer of symbiotic genes from a single ancestor to the bacteria being studied. The high sequence similarity of the symbiotic genes of A. glycyphyllos rhizobia (99-100% in the case of nodAC and nifH genes, and 98-99% in the case of nodH one) points to the relatively recent (in evolutionary scale) lateral transfer of these genes. In the nodACH and nifH phylograms, A. glycyphyllos nodule isolates were grouped together with the genus Mesorhizobium species in one monophyletic clade, close to M. ciceri, Mesorhizobium opportunistum and Mesorhizobium australicum symbiovar biserrulae bacteria, which correlates with the close relationship of these rhizobia host plants. Plant tests revealed the narrow host range of A. glycyphyllos rhizobia. They formed effective symbiotic interactions with their native host (A. glycyphyllos) and Amorpha fruticosa but not with 11 other fabacean species. The nodules induced on A. glycyphyllos roots were indeterminate with apical, persistent meristem, an age gradient of nodule tissues and cortical vascular bundles. To reflect the symbiosis-adaptive phenotype of rhizobia, specific for A. glycyphyllos, we propose for these bacteria the new symbiovar "glycyphyllae", based on nodA and nodC genes sequences.

Binding of human apolipoprotein E (apoE) to Legionella pneumophila lipopolysaccharide was analysed at the molecular level by Fourier-transform infrared spectroscopy, thereby providing biophysical evidence for apoE-L. pneumophila lipopolysaccharide interaction. Atomic force microscopy imaging of apoE-exposed L. pneumophila cells revealed alterations in the bacterial cell surface topography and nanomechanical properties in comparison with control bacteria. The changes induced by apoE binding to lipopolysaccharide on the surface of L. pneumophila cells may participate in: (1) impeding the penetration of host cells by the bacteria; (2) suppression of pathogen intracellular growth and eventually; and (3) inhibition of the development of infection., (© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In this study, the nitrogen fixing Astragalus glycyphyllos symbionts were characterized by phenotypic properties, restriction fragment length polymorphism (RFLP), and sequences of 16S rDNA. The generation time of A. glycyphyllos rhizobia in yeast extract mannitol medium was in the range 4-6 h. The studied isolates exhibited a low resistance to antibiotics, a moderate tolerance to NaCl, assimilated di- and trisaccharides, and produced acid in medium containing mannitol as a sole carbon source. In the cluster analysis, based on 86 phenotypic properties of A. glycyphyllos symbionts and the reference rhizobia, examined isolates and the genus Mesorhizobium strains were placed on a single branch, clearly distinct from other lineages of rhizobial genera. By the comparative analysis of 16S rRNA gene sequences and 16S rDNA-RFLP, A. glycyphyllos nodulators were also identified as the members of the genus Mesorhizobium. On the 16S rDNA sequence phylogram, the representatives of A. glycyphyllos nodule isolates formed a robust, monophyletic cluster together with the Mesorhizobium species at 16S rDNA sequence similarity of these bacteria between 95 and 99 %. Similarly, the cluster analysis of the combined RFLP-16S rDNA patterns, obtained with seven restriction endonucleases, showed that A. glycyphyllos rhizobia are closely related to the genus Mesorhizobium bacteria. The taxonomic approaches used in this paper allowed us to classify the studied bacteria into the genus Mesorhizobium.

The taxonomic status of the Rhizobium sp. K3.22 clover nodule isolate was studied by multilocus sequence analysis (MLSA) of 16S rRNA and six housekeeping chromosomal genes, as well as by a subsequent phylogenic analysis. The results revealed full congruence with the Rhizobium pisi DSM 30132(T) core genes, thus supporting the same taxonomic position for both strains. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence similarity to Rhizobium leguminosarum sv. trifolii, whereas the R. pisi DSM 30132(T)nod genes were most similar to R. leguminosarum sv. viciae. The strains differed in the host range nodulation specificity, since strain K3.22 effectively nodulated red and white clover but not vetch, in contrast to R. pisi DSM 30132(T), which effectively nodulated vetch but was not able to nodulate clover. Both strains had the ability to form nodules on pea and bean but they differed in bean cultivar specificity. The R. pisi K3.22 and DSM 30132(T) strains might provide evidence for the transfer of R. leguminosarum sv. trifolii and sv. viciae symbiotic plasmids occurring in natural soil populations., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

In this study, we have investigated intrinsic salt tolerance of Astragalus cicer microsymbionts (USDA3350, ACMP18) and the role of exogenous glycine betaine in osmoprotection in these bacteria. Salt stress was imposed by NaCl concentrations ranging from 0.5 to 2 %. A. cicer mesorhizobia were capable of tolerating up to 2 % sodium chloride with a population count that was inversely proportional to the salt content. When the extracellular concentration of NaCl was raised to 2 %, the generation time of the UDSA3350 strain in the mid-exponential phase of growth was 3.9-times greater than that in the no-salt control medium, whereas the ACMP18 strain survived under the same conditions but did not multiply. Application of 1 mM glycine betaine into the salt-stressed rhizobium cultures increased the number of culturable bacteria, pointing out that this molecule was involved in restoration of osmotic balance. The decline in A. cicer symbiont viability in the medium with sodium chloride and the osmoprotective role of glycine betaine for these bacteria was confirmed in the experiment using the live/dead Bac Light Bacterial Vibility Kit. Data presented in this study showed the presence of proU-like genes in the genomes of A. cicer rhizobia with high sequence similarity to the genes of the ProU-like system in Sinorhizobium meliloti and the proU operon of Escherichia coli.

The potential role of currency in the spread of pathogenic microflora has been evaluated in many countries. In this study Polish paper notes and the coins in general circulation were assayed for the presence of cultivable bacteria and fungi. Bacterial isolates identification was based on cultural and biochemical characters and by comparison of the 16S rRNA gene sequence. Fungal isolates were recognized with biochemical and morphological criteria. Coagulase-negative staphylococci, (43.6% of the total bacterial count) including Staphylococcus saprophyticus, S. epidermidis, and S. hominis, and Enteroccus spp. (30.8% of the total bacterial count), i.e. E.faecalis, E.faecium and E. durans, were the most numerous bacterial contamination. Penicillium spp., and Aspergillus spp. were the most frequently detected moulds whereas Candida spp. was the most frequent yeast isolated from currency. A visible dependence between the banknote denomination, the physical condition of paper currency, and the number of bacteria and fungi was found. The overall count of bacteria isolated from currency was thousand-fold higher than that of fungal isolates. The total amount of bacteria and fungi recovered from the coins was approximately 2.7-fold lower than that isolated from the notes. In summary, the Polish currency notes were found to be contaminated mainly with commensal bacteria and fungi while the opportunistic pathogenic microorganisms Escherichia coli, Pseudomonas stutzeri and C. albicans were detected at a low frequency.

The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99-100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97-98% for nodC, and 97-100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.

Robinia pseudoacacia microsymbionts from plants growing in Poland and Japan were evaluated for phylogeny and taxonomic position by genomic approach. Based on the comparative analyses of atpD (368 bp) and dnaK (573 bp) gene sequences as well as 16S rDNA restriction analysis (RFLP-16S rDNA), R. pseudoacacia microsymbionts were identified as Mesorhizobium strains. In dnaK and atpD gene phylograms R. pseudoacacia nodulators formed robust, monophyletic clusters with Mesorhizobium species with the nucleotide sequence similarity of 91-98% and 90-98%, respectively. The classification of R. pseudoacacia rhizobia to the genus Mesorhizobium was also supported by amplified 16S rDNA restriction analysis. The studied bacteria formed common clusters with Mesorhizobium species, and their DNA patterns were identical or nearly identical to Mesorhizobium genus strains. When DNA-DNA hybridization was performed, the total DNA of the representative R. pseudoacacia rhizobia exhibited 51-75% relatedness to DNA of Mesorhizobium amorphae ICMP15022 strain and below 41% to DNA of other Mesorhizobium species. These results showed that R. pseudoacacia and M. amorphae belong to the same genomospecies. The G+C content of DNA of R. pseudoacacia two microsymbionts was 59.7 and 60.6 mol% compared to 61-64 mol% across M. amorphae strains.

A multilocus phylogenetic approach was applied to elucidate the phylogeny of Astragalus cicer rhizobia derived from Poland, Ukraine, and Canada. The strains selected for the studies represented three main geographically different phenons of these bacteria. Phylogenetic analyses were performed with three chromosomal housekeeping loci (16S rRNA, atpD, glnII) and three symbiotic genes located on a plasmid (nodA, nodC, nifH). The "core" and "auxiliary" gene trees revealed that A. cicer nodule isolates were intermingled with the strains of Mesorhizobium species, which implies that they are descendents of the same ancestor as mesorhizobia and fall into the Mesorhizobium genus. The noted congruence of the housekeeping and symbiotic gene phylogenies of A. cicer microsymbionts indicates that sym loci are transferred to these bacteria through vertical transmission without a significant participation of intergeneric horizontal gene spread. All the three sym gene sequences of the Polish and Ukrainian A. cicer nodule isolates were more closely related to one another than to the corresponding sequences of the Canadian isolates. The phylogeographic patterns of the sym genes of intercontinental strains point to their relatively long, separate, evolutionary history.

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98-99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771-961 kb megaplasmids.

Three lytic phages (PhiRP1, PhiRP2, and PhiRP3) specific for Robinia pseudoacacia rhizobia were isolated from the soil under black locust. They were characterized by their morphology, host range, and some other properties including DNA molecular weights. Studied phages have been found to belong to Siphoviridae family that comprises viruses with long, and noncontractile tails. They had broad host ranges and effectively lysed not only Robinia pseudoacacia microsymbionts but also different Mesorhizobium species. The phages were homogenous in latent periods (300 min) but heterogeneous in burst sizes (100-200 phage particles per one infected cell) and rise periods (90-120 min). They showed a distinct adsorption rate to Robinia pseudoacacia rhizobia (70.4-93.94%). The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from ca. 82 kb to ca. 105 kb.

DNA polymorphism among 36 Astragalus cicer nodule isolates and 9 reference mesorhizobia was evaluated by a simplified PstI based AFLP procedure with three selective primers: Pst-A, Pst-G, and Pst-GC. The DNA profiles were found to be highly specific for nearly each strain, although DNA bands characteristic for most A. cicer microsymbionts were also noted. The overall topologies of dendrograms, generated by AFLP patterns in PCR reaction with three primers, were very similar to one another and to that constructed by phenotyping. Also the strain compositions in the particular clusters on pheno- and genomograms were in good agreement. The obtained results indicate that AFLP technique can be a useful tool for typing of A. cicer rhizobia as well as for studying their diversity.

Four virulent phages--PhiDl, PhiTl, PhiCYT21, and PhiOS6, infective on Sarothamnus scoparius rhizobia--were isolated from the soil and characterized for morphology, host range, rate of adsorption to bacterial cells, and genome size. New phages were separated into two morphological families: Siphoviridae with long, noncontractile tails (PhiDl, PhiTl) and Myoviridae with long, contractile tails (PhiCYT21, PhiOS6). They were also classified into two groups by a host specificity. One of them included viruses (PhiDl and PhiTl) that lysed S. scoparius bradyrhizobia and Bradyrhizobium sp. (Lupinus) strain Dl, and the second one comprised phages (PhiCYT21 and PhiOS6) that parasitized only Scotch broom native microsymbionts. Phages specific for S. scoparius rhizobia were differentiated not only by morphology and host range but also by a genome size that was in the range from 47,583 to 60,098 b.p.