Márton Palatinszky

Bacillus aurantiacus sp. nov., an alkaliphilic and moderately halophilic bacterium isolated from Hungarian soda lakes

Three alkaliphilic and moderately halophilic strains designated K1-5T, K1-10 and B1-1, characterized by optimal growth at pH 9.0-10.0 and at 3-7 % (w/v) NaCl, were isolated from extremely shallow, alkaline soda lakes located in Hungary. Cells of the strains are Gram-positive, straight rods and form a central to subterminal, ellipsoidal endospore. The isolates are strictly aerobic, catalase-positive, oxidase-negative and contain a peptidoglycan of type A1 gamma based on meso-diaminopimelic acid. In strain K1-5T, menaquinone-7 (MK-7) is the predominant isoprenoid quinone and anteiso-C15 : 0 is the major cellular fatty acid. The DNA G+C content of strain K1-5T is 42.9 mol%. 16S rRNA gene-based phylogenetic analysis revealed that the strains exhibit levels of sequence similarity of less than 95.8 % to known Bacillus species. According to the polyphasic characterization, the strains represent a novel species, for which the name Bacillus aurantiacus sp. nov. is proposed. The type strain is K1-5T (=DSM 18675T =CCM 7447T =NCAIM B002265T).

Preferential ligation during TA-cloning of multitemplate PCR products--a factor causing bias in microbial community structure analysis.

The description of microbial community structure is always biased by the selectivity of the methods applied. Although TA cloning of PCR amplified community DNA is one of the most widely used techniques in bacterial community analysis, no thorough comparative testing has been carried out on different TA cloning systems. In this study, we measured and compared the selectivity of two widely used TA-cloning kits in experimental setups where the length heterogeneity of the inserts modeled the natural length variation of the 16S rRNA gene and the 16S-23S intergenic spacer region. Both TOPO TA (Invitrogen, CA USA) and pGem-T vector system (Promega, WI USA) cloning kits showed significant and reproducible insert size related selectivity. The effect of ligation time and temperature was also studied in case of the pGem-T vector system. We compared the performance of the two cloning kits on an environmental sample, along with a semiquantitative community fingerprinting method to gain reference data free of cloning bias. The two clone libraries showed significantly different compositions, and were also differing from the community structure revealed by length heterogeneity PCR.

Single-nucleotide primer extension assay for detection and sequence typing of "Dehalococcoides" spp.

ABSTRACT A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus “Dehalococcoides”. The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.

Phylogenetic Diversity of Bacterial Communities Associated with Sulfurous Karstic Well Waters of a Hungarian Spa

The composition of bacterial communities associated with waters of one thermal and three sulfurous lukewarm wells of cavernous limestone aquifers in the Southwestern part of Hungary was studied in 2007 and 2008 by microscopy and culture-independent clone library analysis targeting the 16S rRNA gene. The presence of Thiothrix-like filamentous bacteria was observed by microscopy in the lukewarm samples. From the clone libraries, a high proportion of phylotypes belonging to Proteobacteria (Rhodobacter, Sphingopyxis, Phenylobacterium, Ochrobactrum, Methylobacterium, Sulfuritalea, Thiobacillus, Limnobacter, Acidovorax, Xanthomonadaceae, Thiothrix, Lysobacter, Pseudoxanthomonas, Desulfopila, Desulfocapsa, Desulforhopalus, Sulfuricurvum, Sulfurimonas, Sulfurospirillum) was revealed. In addition, phylotypes of Acidobacteria, Chloroflexi, Sphingobacteria, Clostridia, Aquificae, Deferribacteres and Chlorobi were detected. On the basis of their habitat preference 21 out of a total of 39 different phylotypes represented mesophilic and thermophilic sulfur bacteria. From the lukewarm samples large numbers of clones were affiliated with uncultured Epsilonproteobacteria clones common in sulfurous hydrothermal vents. From the thermal sample, several clones were in the closest relationship to the Sulfuritalea hydrogenivorans, a novel sulfur-oxidizing Betaproteobacterium.

Comparison of soil microbial communities from two distinct karst areas in Hungary

Karst areas belong to the most exposed terrestrial ecosystems, therefore their study have a priority task in Hungary, as well. The aim of this study was to compare the structure, activity and diversity of soil microbial communities from two distinct Hungarian karst areas (Aggtelek NP and Tapolca-basin). Soil samples were taken three times from 6 distinct sites, from different depths. Soil microbial biomass C (MBC), microbial biomass N (MBN), basal respiration (BRESP) and substrate induced respiration (SIR) were measured. The phylogenetic diversity of bacterial communities was compared by Denaturing Gradient Gel Electrophoresis (DGGE). The highest MBC, MBN, BRESP and SIR values were measured in the rendzina soil from Aggtelek. On the basis of biomass and respiration measurements, microbial communities differentiated mainly according to soil depths whereas DGGE profiles of bacterial communities resulted in groups mainly according to sampling sites.

A POLYPHASIC STUDY ON THE SPECIES DIVERSITY OF THE SEDIMENT MICROBIOTA OF LAKE HÉVÍZ

Lake Hévíz is the largest natural thermal lake of Europe, harboring special bacterial communities. The aim of the present study was to gain information about the distribution and species diversity of the sediment microbiota, with special focus on Actinobacteria, by using cultivation-based and -independent molecular methods. Samples from two depths were taken in two different locations in October 2007. 245 strains were isolated, grouped to 85 OTUs by ARDRA, and identified by 16S rDNA sequencing. Most of the strains showed highest sequence similarity with Bacillus and related genera. Strains belonging to the phylum Actinobacteria were identified as members of Arthrobacter, Brachybacterium, Brevibacterium, Curtobacterium, Friedmanniella, Gordonia, Kocuria, Microbacterium, Micrococcus, Micromonospora, Mycobacterium, Rhodococcus, Streptomyces and Williamsia . Two clone libraries were constructed from H3M and H4M samples, providing 288 and 192 clones which were grouped to 150 and 125 OTUs, respectively, by ARDRA. The two most abundant group of the H4M library were OP8-related. The phylum Proteobacteria was represented mostly by delta-Proteobacteria, other relevant groups were Cyanobacteria, Bacteroidetes, Acidobacteria and beta-Proteobacteria. The H3M library was dominated by Cyanobacteria, Verrucomicrobia, beta-Proteobacteria, gamma-Proteobacteria and delta-Proteobacteria. Chloroflexi, Bacteroidetes, Spirochetes and Firmicutes were scarce. Results from the clone libraries were compared to the length-heterogeneity-PCR fingerprints of the communities.

Spatial and temporal changes of bacterial communities inhabiting the well waters of Harkány spa

In this study, changes in the bacterial community composition of the well waters of Harkány Spa were examined. Physical and chemical properties of mixing subsurface cold and thermal karst waters were correlated to shifts in bacterial community structures analyzed by denaturing gradient gel electrophoresis (DGGE) and principal component analysis (PCA). In addition, mineral components of the pellets were investigated by scanning electron microscopy. Samples from the effluent waters of Büdöstapolca I and II, Matty and Thermal VI wells were taken seasonally in 2007 and 2008. The comparison of the results of DGGE and PCA analyses showed that bacterial communities from the Büdöstapolca wells were distinct from those of Matty and Thermal VI, but seasonal changes were not detected. According to the phylogenetic analysis of the excised DGGE bands, presence of chemolithotrophic Proteobacteria (Thiobacillus, Thiothrix, and distant relatives of Sulfurospirillum) were typical in the Büdöstapolca wells, while members of Actinobacteria (Plantibacter, Actinobacterium, Microbacterium) and Firmicutes (Planococcus) were characteristic to the Matty and Thermal VI wells. In the pellets pyrite framboid crystals were observed by electron microscopy, which are minerals known to be biologically induced by dissimilatory iron- and sulfur (sulfate)-reducing bacteria.

THE EFFECT OF EASILY DEGRADABLE SUBSTRATE FEEDING ON THE COMMUNITY STRUCTURE OF LABORATORY-SCALE WASTEWATER SLUDGE DIGESTERS

The effect of several easily degradable substrates, such as protein, starch and sunflower oil was investigated on the bacterial community of a laboratory-scale biogas model system. Besides measuring gas yield, Denaturing Gradient Gel Electrophoresis (DGGE), Phospholipids Fatty Acid Analysis (PLFA) for Bacteria and T-RFLP analysis of the mcrA gene for Archaea were used. The community of the examined biogas reactors adapted to the new substrates through a robust physiological reaction followed by moderate community abundance shifts. Gas yield data clearly demonstrated the physiological adaptation to substrate shifts. Statistical analysis of DNA and chemotaxonomic biomarkers revealed community abundance changes. Sequences gained from DGGE bands showed the dominance of the phyla Bacteroidetes and the presence of Firmicutes (Clostridia) and Thermotogae. This was supported by the detection of large amounts of branched 15-carbon non-hydroxy fatty acids in PLFA profiles, as common PLFA markers of the Bacteroidetes group. Minor abundance ratios changes were observed in the case of Archaea in accordance with changes of the fed substrates.

Molecular biological investigations on the bacterial communities of curative well waters of Harkany Spa.

Bacterial communities from the sulfide containing curative well waters of Harkány Spa (Hungary) were investigated by cultivation independent molecular cloning and Denaturing Gradient Gel Electrophoresis (DGGE) methods between 2006 and 2008. The DGGE profiles of the bacterial communities originated from the wells of lukewarm waters showed seasonal similarities and were highly different from the thermal well. From the four clone libraries 22 different eubacterial species or genera were identified by sequence analysis. The majority of the clones of the lukewarm waters belonged to unidentified Epsilon-proteobacteria, Desulfocapsa sp. and Thiothrix spp., while the dominant clones of the thermal water were affiliated with the genus Denitratisoma sp. Most of the identified species and genera were related to bacteria with obligate or facultative chemolithotrophic sulfur metabolism, so the microbes of the curative waters may participate in the sulfur-cycle of the wells.