Response of the metabolic activity and taxonomic composition of bacterial communities to mosaically varying soil salinity and alkalinity.

Soil salinity and sodicity is a worldwide problem that affects the composition and activity of bacterial communities and results from elevated salt and sodium contents. Depending on the degree of environmental pressure and the combined effect of other factors, haloalkalitolerant and haloalkaliphilic bacterial communities will be selected. These bacteria play a potential role in the maintenance and restoration of salt-affected soils; however, until recently, only a limited number of studies have simultaneously studied the bacterial diversity and activity of saline-sodic soils. Soil samples were collected to analyse and compare the taxonomic composition and metabolic activity of bacteria from four distinct natural plant communities at three soil depths corresponding to a salinity‒sodicity gradient. Bacterial diversity was detected using 16S rRNA gene Illumina MiSeq amplicon sequencing. Community-level physiological profiles (CLPPs) were analysed using the MicroResp™ method. The genus-level bacterial composition and CLPPs differed significantly in soils with different alkaline vegetation. The surface soil samples also significantly differed from the intermediate and deep soil samples. The results showed that the pH, salt content, and Na+ content of the soils were the main edaphic factors influencing both bacterial diversity and activity. With salinity and pH, the proportion of the phylum Gemmatimonadota increased, while the proportions of Actinobacteriota and Acidobacteriota decreased.

Calcium carbonate precipitating extremophilic bacteria in an Alpine ice cave.

Extensive research has provided a wealth of data on prokaryotes in caves and their role in biogeochemical cycles. Ice caves in carbonate rocks, however, remain enigmatic environments with limited knowledge of their microbial taxonomic composition. In this study, bacterial and archaeal communities of the Obstans Ice Cave (Carnic Alps, Southern Austria) were analyzed by next-generation amplicon sequencing and by cultivation of bacterial strains at 10 °C and studying their metabolism. The most abundant bacterial taxa were uncultured Burkholderiaceae and Brevundimonas spp. in the drip water, Flavobacterium, Alkanindiges and Polaromonas spp. in the ice, Pseudonocardia, Blastocatella spp., uncultured Pyrinomonadaceae and Sphingomonadaceae in carbonate precipitates, and uncultured Gemmatimonadaceae and Longimicrobiaceae in clastic cave sediments. These taxa are psychrotolerant/psychrophilic and chemoorganotrophic bacteria. On a medium with Mg2+/Ca2+ = 1 at 21 °C and 10 °C, 65% and 35% of the cultivated strains precipitated carbonates, respectively. The first ~ 200 µm-size crystals appeared 2 and 6 weeks after the start of the cultivation experiments at 21 °C and 10 °C, respectively. The crystal structure of these microbially induced carbonate precipitates and their Mg-content are strongly influenced by the Mg2+/Ca2+ ratio of the culture medium. These results suggest that the high diversity of prokaryotic communities detected in cryogenic subsurface environments actively contributes to carbonate precipitation, despite living at the physical limit of the presence of liquid water.

Habitat-related variability in the morphological and taxonomic diversity of microbial communities in two Hungarian epigenic karst caves.

The physical and chemical characteristics of the bedrock, along with the geological and hydrological conditions of karst caves may influence the taxonomic and functional diversity of prokaryotes. Most studies so far have focused on microbial communities of caves including only a few samples and have ignored the chemical heterogeneity of different habitat types such as sampling sites, dripping water, carbonate precipitates, cave walls, cave sediment and surface soils connected to the caves. The aim of the present study was to compare the morphology, the composition and physiology of the microbiota in caves with similar environmental parameters (temperature, host rock, elemental and mineral composition of speleothems) but located in different epigenic karst systems. Csodabogyós Cave and Baradla Cave (Hungary) were selected for the analysis of bacterial and archaeal communities using electron microscopy, amplicon sequencing, X-ray diffraction, and mass spectroscopic techniques. The microbial communities belonged to the phyla Pseudomonadota, Acidobacteriota, Nitrospirota and Nitrososphaerota, and they showed site-specific variation in composition and diversity. The results indicate that morphological and physiological adaptations provide survival for microorganisms according to the environment. In epigenic karst caves, prokaryotes are prone to increase their adsorption surface, cooperate in biofilms, and implement chemolithoautotrophic growth with different electron-donors and acceptors available in the microhabitats.

Planktonic and epilithic prokaryota community compositions in a large temperate river reflect climate change related seasonal shifts.

In freshwaters, microbial communities are of outstanding importance both from ecological and public health perspectives, however, they are threatened by the impact of global warming. To reveal how different prokaryotic communities in a large temperate river respond to environment conditions related to climate change, the present study provides the first detailed insight into the composition and spatial and year-round temporal variations of planktonic and epilithic prokaryotic community. Microbial diversity was studied using high-throughput next generation amplicon sequencing. Sampling was carried out monthly in the midstream and the littoral zone of the Danube, upstream and downstream from a large urban area. Result demonstrated that river habitats predominantly determine the taxonomic composition of the microbiota; diverse and well-differentiated microbial communities developed in water and epilithon, with higher variance in the latter. The composition of bacterioplankton clearly followed the prolongation of the summer resulting from climate change, while the epilithon community was less responsive. Rising water temperatures was associated with increased abundances of many taxa (such as phylum Actinobacteria, class Gammaproteobacteria and orders Synechococcales, Alteromonadales, Chitinophagales, Pseudomonadales, Rhizobiales and Xanthomonadales), and the composition of the microbiota also reflected changes of several further environmental factors (such as turbidity, TOC, electric conductivity, pH and the concentration of phosphate, sulphate, nitrate, total nitrogen and the dissolved oxygen). The results indicate that shift in microbial community responding to changing environment may be of crucial importance in the decomposition of organic compounds (including pollutants and xenobiotics), the transformation and accumulation of heavy metals and the occurrence of pathogens or antimicrobial resistant organisms.

Variation in Sodic Soil Bacterial Communities Associated with Different Alkali Vegetation Types.

In this study, we examined the effect of salinity and alkalinity on the metabolic potential and taxonomic composition of microbiota inhabiting the sodic soils in different plant communities. The soil samples were collected in the Pannonian steppe (Hungary, Central Europe) under extreme dry and wet weather conditions. The metabolic profiles of microorganisms were analyzed using the MicroResp method, the bacterial diversity was assessed by cultivation and next-generation amplicon sequencing based on the 16S rRNA gene. Catabolic profiles of microbial communities varied primarily according to the alkali vegetation types. Most members of the strain collection were identified as plant associated and halophilic/alkaliphilic species of Micrococcus, Nesterenkonia, Nocardiopsis, Streptomyces (Actinobacteria) and Bacillus, Paenibacillus (Firmicutes) genera. Based on the pyrosequencing data, the relative abundance of the phyla Proteobacteria, Actinobacteria, Acidobacteria, Gemmatimonadetes and Bacteroidetes also changed mainly with the sample types, indicating distinctions within the compositions of bacterial communities according to the sodic soil alkalinity-salinity gradient. The effect of weather extremes was the most pronounced in the relative abundance of the phyla Actinobacteria and Acidobacteria. The type of alkali vegetation caused greater shifts in both the diversity and activity of sodic soil microbial communities than the extreme aridity and moisture.

Denaturing gradient gel electrophoresis and multi-SIR profiles of soil microbial communities from a karst doline at Aggtelek National Park, Hungary.

Soils play an important role in the ecosystem of karstic landscapes both as a buffer zone and as a source of acidity to belowground water. Although the microbiota of karstic soils is known to have a great effect on karstification processes, the activity and composition of these communities are largely unknown. This study gives a comparative analysis of soil microbial profiles from different parts of a doline located at Aggtelek, Hungary. The aim was to reveal the relationships between the vegetation type and genetic fingerprints and substrate utilisation (multi-SIR) profiles of the soil microbiota. Soil samples were collected in early and late springs along a transect in a doline covered with different types of vegetation. Genetic fingerprints of bacterial communities were examined by denaturing gradient gel electrophoresis (DGGE) based on the 16S rRNA gene, along with multi-SIR profiles of the microbial communities measured by the MicroResp method using 15 different carbon sources. Genetic fingerprinting indicated that vegetation cover had a strong effect on the composition of soil bacterial communities. Procrustean analysis showed only a weak connection between DGGE and multi-SIR profiles, probably due to the high functional redundancy of the communities. Seasonality had a significant effect on substrate usage, which can be an important factor to consider in future studies.

In situ modelling of biofilm formation in a hydrothermal spring cave.

Attachment of microorganisms to natural or artificial surfaces and the development of biofilms are complex processes which can be influenced by several factors. Nevertheless, our knowledge on biofilm formation in karstic environment is quite incomplete. The present study aimed to examine biofilm development for a year under controlled conditions in quasi-stagnant water of a hydrothermal spring cave located in the Buda Thermal Karst System (Hungary). Using a model system, we investigated how the structure of the biofilm is formed from the water and also how the growth rate of biofilm development takes place in this environment. Besides scanning electron microscopy, next-generation DNA sequencing was used to reveal the characteristic taxa and major shifts in the composition of the bacterial communities. Dynamic temporal changes were observed in the structure of bacterial communities. Bacterial richness and diversity increased during the biofilm formation, and 9-12 weeks were needed for the maturation. Increasing EPS production was also observed from the 9-12 weeks. The biofilm was different from the water that filled the cave pool, in terms of the taxonomic composition and metabolic potential of microorganisms. In these karstic environments, the formation of mature biofilm appears to take place relatively quickly, in a few months.

Effects of Active Volcanism on Bacterial Communities in the Highest-Altitude Crater Lake of Ojos del Salado (Dry Andes, Altiplano-Atacama Region).

Periglacial and volcanic environments are considered terrestrial analogs of Mars with regard to astrobiological characteristics due to their specific set of extreme features. Ojos del Salado, the highest volcano on Earth (6893 m a.s.l.), is surrounded by several craters, one of which harbors the highest known altitude lake (6480 m a.s.l.), which is influenced by a rare combination of extreme environmental factors, that is, low mean temperature, permafrost, fumarolic activity, acidity, and extreme low organic matter content. To assess the genetic diversity and ecological tolerance of bacteria, samples were taken in February 2016 from the sediments covered with acidic cold (pH 4.88, 3.8°C) and warm (pH 2.08, 40.8°C) water. As a control, a nonvolcanic high-altitude lake (at 5900 m a.s.l.) was also studied by both cultivation-based and next-generation DNA sequencing methods. Isolates from the crater lake showed tolerance toward acidic pH values, unlike isolates from the nonvolcanic lake. Illumina MiSeq sequencing of the 16S rRNA gene exposed simplified, although characteristically different, bacterial communities in the warm and cold water-saturated sediments. In the fumarolic creek sediments, acidophilic iron oxidizers (Ferrithrix, Gallionella) and iron reducers (Acidiphilium) were abundant, and bacteria involved in the sulfur oxidation (Hydrogenobaculum, Thiomonas) and reduction (Desulfosporosinus) were also detected. Therefore, we propose an integrated model that addresses the potential role of bacteria in the sulfur and iron geomicrobiological cycles.

Bacterial Diversity of a High-Altitude Permafrost Thaw Pond Located on Ojos del Salado (Dry Andes, Altiplano-Atacama Region).

Microbial ecology of permafrost, due to its ecological and astrobiological importance, has been in the focus of studies in past decades. Although permafrost is an ancient and stable environment, it is also subjected to current climate changes. Permafrost degradation often results in generation of thaw ponds, a phenomenon not only reported mainly from polar regions but also present in high-altitude permafrost environments. Our knowledge about microbial communities of thaw ponds in these unique, remote mountain habitats is sparse. This study presents the first culture collection and results of the next-generation DNA sequencing (NGS) analysis of bacterial communities inhabiting a high-altitude permafrost thaw pond. In February 2016, a permafrost thaw pond on the Ojos del Salado at 5900 m a.s.l. (meters above sea level) was sampled as part of the Hungarian Dry Andes Research Programme. A culture collection of 125 isolates was established, containing altogether 11 genera belonging to phyla Bacteroidetes, Actinobacteria, and Proteobacteria. Simplified bacterial communities with a high proportion of candidate and hitherto uncultured bacteria were revealed by Illumina MiSeq NGS. Water of the thaw pond was dominated by Bacteroidetes and Proteobacteria, while in the sediment of the lake and permafrost, members of Acidobacteria, Actinobacteria, Bacteroidetes, Patescibacteria, Proteobacteria, and Verrucomicrobia were abundant. This permafrost habitat can be interesting as a potential Mars analog.

Cave bacteria-induced amorphous calcium carbonate formation.

Amorphous calcium carbonate (ACC) is a precursor of crystalline calcium carbonates that plays a key role in biomineralization and polymorph evolution. Here, we show that several bacterial strains isolated from a Hungarian cave produce ACC and their extracellular polymeric substance (EPS) shields ACC from crystallization. The findings demonstrate that bacteria-produced ACC forms in water-rich environment at room temperature and is stable for at least half year, which is in contrast to laboratory-produced ACC that needs to be stored in a desiccator and kept below 10 °C for avoiding crystallization. The ACC-shielding EPS consists of lipids, proteins, carbohydrates and nucleic acids. In particular, we identified large amount of long-chain fatty acid components. We suggest that ACC could be enclosed in a micella-like formula within the EPS that inhibits water infiltration. As the bacterial cells lyse, the covering protective layer disintegrates, water penetrates and the unprotected ACC grains crystallize to calcite. Our study indicates that bacteria are capable of producing ACC, and we estimate its quantity in comparison to calcite presumably varies up to 20% depending on the age of the colony. Since diverse bacterial communities colonize the surface of cave sediments in temperate zone, we presume that ACC is common in these caves and its occurrence is directly linked to bacterial activity and influences the geochemical signals recorded in speleothems.

Deinococcus fonticola sp. nov., isolated from a radioactive thermal spring in Hungary.

A Gram-stain-negative, aerobic, non-motile and coccus-shaped bacterium, designated strain FeSDHB5-19T, was isolated from a biofilm sample collected from a radioactive thermal spring (Budapest, Hungary), after exposure to 5 kGy gamma radiation. A polyphasic approach was used to study the taxonomic properties of strain FeSDHB5-19T, which had highest 16S rRNA gene sequence similarity to Deinococcus antarcticus G3-6-20T (96.5 %). The 16S rRNA gene sequence similarity to type strains of other Deinococcus species were 93.0 % or lower. The DNA G+C content of the draft genome sequence, consisting of 3.9 Mb, was 63.9 mol%. Strain FeSHDB5-19T was found to grow at temperatures of 10-32 °C (optimum, 28 °C) and pH 5-10 (pH 6.5-7.5) and tolerated up to 1.5 % NaCl (w/v) with optimum growth at 0-0.5 % NaCl. The predominant fatty acids (>10 %) were C16 : 0 and C16 : 1ω7c. The cell-wall peptidoglycan type was A3ß l-Orn-Gly1-2. The whole-cell sugars were glucose and low amounts of galactose. Strain FeSDHB5-19T possessed MK-8 as the predominant respiratory quinone, typical of the genus Deinococcus. The polar lipid profile contained unidentified phosphoglycolipids and unidentified glycolipids. The isolate was found to be highly resistant to gamma (D10<8 kGy) and UV (D10~800 J m-2) radiation. According to its genotypic, phenotypic and chemotaxonomic characteristics, strain FeSDHB5-19T represents a novel species in the genus Deinococcus, for which the name Deinococcusfonticola sp. nov. is proposed. The type strain is FeSDHB5-19T (=NCAIM B.02639T=DSM 106917T).

Dual bloom of green algae and purple bacteria in an extremely shallow soda pan.

In April 2014, dual bloom of green algae and purple bacteria occurred in a shallow, alkaline soda pan (Kiskunság National Park, Hungary). The water was only 5 cm deep, in which an upper green layer was clearly separated from a near-sediment purple one. Based on microscopy and DNA-based identification, the upper was inhabited by a dense population of the planktonic green alga, Oocystis submarina Lagerheim, while the deeper layer was formed by purple, bacteriochlorophyll-containing bacteria, predominated by Thiorhodospira and Rhodobaca. Additional bacterial taxa with a presumed capability of anoxygenic phototrophic growth belonged to the genera Loktanella and Porphyrobacter. Comparing the bacterial community of the purple layer with a former blooming event in a nearby soda pan, similar functional but different taxonomic composition was revealed. Members from many dominant bacterial groups were successfully cultivated including potentially new species, which could be the result of the application of newly designed media.

Radioactive environment adapted bacterial communities constituting the biofilms of hydrothermal spring caves (Budapest, Hungary).

The thermal waters of Gellért Hill discharge area of the Buda Thermal Karst System (Hungary) are characterized by high (up to 1000 Bq/L) 222Rn-activity due to the radium-accumulating biogeochemical layers. Samples were taken from these ferruginous and calcareous layers developed on spring cave walls and water surface. Accumulation of potentially toxic metals (e.g. As, Hg, Pb, Sn, Sr, Zn) in the dense extracellular polymeric substance containing bacterial cells and remains was detected by inductively coupled plasma mass spectrometry. The comparison of bacterial phylogenetic diversity of the biofilm samples was performed by high throughput next generation sequencing (NGS). The analysis showed similar sets of mainly unidentified taxa of phyla Chloroflexi, Nitrospirae, Proteobacteria, Planctomycetes; however, large differences were found in their abundance. Cultivation-based method complemented with irradiation assay was performed using 5, 10 and 15 kGy doses of gamma-rays from a 60Co-source to reveal the extreme radiation-resistant bacteria. The phyla Actinobacteria, Firmicutes, Proteobacteria (classes Alpha- Beta- and Gammaproteobacteria), Bacteriodetes and Deinococcus-Thermus were represented among the 452 bacterial strains. The applied irradiation treatments promoted the isolation of 100 different species, involving candidate novel species, as well. The vast majority of the isolates belonged to bacterial taxa previously unknown as radiation-resistant microorganisms. Members of the genera Paracoccus, Marmoricola, Dermacoccus and Kytococcus were identified from the 15 kGy dose irradiated samples. The close relatives of several known radiation-tolerant bacteria were also detected from the biofilm samples, alongside with bacteria capable of detoxification by metal accumulation, adsorption and precipitation in the form of calcium-carbonate which possibly maintain the viability of the habitat. The results suggest the establishment of a unique, extremophilic microbiota in the studied hydrothermal spring caves.

Anaerobacillus alkaliphilus sp. nov., a novel alkaliphilic and moderately halophilic bacterium.

Two alkaliphilic and moderately halophilic bacterial strains B16-10T and Z23-18 characterized by optimal growth at pH 9.0-10.0 and 5 % (w/v) NaCl, were isolated from the rhizosphere soil of the bayonet grass (Bolboschoenus maritimus) in the Kiskunság National Park, Hungary. Cells of both strains stained Gram-positive, were motile straight rods, and formed terminal, ellipsoidal endospores with swollen sporangia. The isolates were facultative anaerobic, catalase positive, oxidase negative. Both strains contained meso-diaminopimelic acid as diagnostic diaminoacid of the peptidoglycan. Menaquinone-7 (MK-7) was the predominant isoprenoid quinone. Anteiso-C15 : 0, C16 : 1ω11c and iso-C14 : 0 were the major cellular fatty acids. The DNA G+C content of both strains was 35.8 mol%. The 16S rRNA gene based phylogenetic analysis revealed that the facultative anaerobic strains B16-10T and Z23-18 showed the highest similarities to the type strains of anaerobic Anaerobacillus isosaccharinicus NB2006T (98.7 and 99.1 %), A. macyae JMM-4T (98.2 and 98.4 %), A. alkalidiazotrophicus MS 6T (97.7 and 98.4 %), A. alkalilacustris Z-0521T (97.5 and 98.3 %) and A. arseniciselenatis DSM 15340T (97.5 and 98.2 %). However, the distinctive phenotypic and genetic results of this study confirmed that strains B16-10T and Z23-18 represent a novel species, for which the name Anaerobacillus alkaliphilus sp. nov. is proposed. The type strain is B16-10T (=DSM 29790T=NCAIM B 02608T).

Unexpected consequences of bombing. Community level response of epiphytic diatoms to environmental stress in a saline bomb crater pond area.

The spatial response of epiphytic diatom communities to environmental stress was studied in a moderately saline wetland area located in the plain of Danube-Tisza Interfluve, Hungary. The area is characterised by World War II bomb crater ponds and can be regarded as an excellent ecological model system where the dispersion of species is slightly limited by distance. To study the effect of environmental variables on the communities, canonical correspondence analysis was applied. Salinity, pH, total suspended solids, total phosphorous and depth proved to be significant environmental drivers in this analysis. The ecological status of the ponds was assessed with Ziemann's halobity index, as the trophity-depending metric cannot be applied to these habitats (due to the naturally high phosphorus content). Ponds in "good" ecological status significantly differed from those appertaining to water quality category of "not-good" ecological status considering characteristic of natural astatic soda pans (e.g. salinity, pH, ammonium, total phosphorous concentration, nitrogen:phosphorous ratio and turbidity). The differences between epiphytic diatom communities inhabiting the ponds were detected using non-parametric multidimensional scaling. The samples formed three groups according to the types of ponds ("transparent", "transitional" and "turbid") based on the width of the macrophyte belt around them. Indicator species related to the ecological status of the ponds and diatom communities contributing to the separation of groups of ponds were identified. One of the indicator species differed from species already described. Light and scanning electron microscopy features and phylogenetic analyses based on three genes (18S and 28S rRNA genes, rbcL) proved that it was a new species of Nitzschia genus, closely related to Nitzschia frustulum and Nitzschia inconspicua. Therefore, description of a new species, Nitzschia reskoi Ács, Duleba, C.E.Wetzel & Ector is proposed. We concluded that the increasing abundance of Nitzschia reskoi was a signal of the degradation of the intermittent saline wetlands.

Biofilm forming bacteria and archaea in thermal karst springs of Gellért Hill discharge area (Hungary).

The Buda Thermal Karst System (BTKS) is an extensive active hypogenic cave system located beneath the residential area of the Hungarian capital. At the river Danube, several thermal springs discharge forming spring caves. To reveal and compare the morphological structure and prokaryotic diversity of reddish-brown biofilms developed on the carbonate rock surfaces of the springs, scanning electron microscopy (SEM), and molecular cloning were applied. Microbial networks formed by filamentous bacteria and other cells with mineral crystals embedded in extracellular polymeric substances were observed in the SEM images. Biofilms were dominated by prokaryotes belonging to phyla Proteobacteria, Chloroflexi and Nitrospirae (Bacteria) and Thaumarchaeota (Archaea) but their abundance showed differences according to the type of the host rock, geographic distance, and different water exchange. In addition, representatives of phyla Acidobacteria, Actinobacteria, Caldithrix, Cyanobacteria, Firmicutes Gemmatimonadetes, and several candidate divisions of Bacteria as well as Crenarchaeota and Euryarchaeota were detected in sample-dependent higher abundance. The results indicate that thermophilic, anaerobic sulfur-, sulfate-, nitrate-, and iron(III)-reducing chemoorganotrophic as well as sulfur-, ammonia-, and nitrite-oxidizing chemolithotrophic prokaryotes can interact in the studied biofilms adapted to the unique and extreme circumstances (e.g., aphotic and nearly anoxic conditions, oligotrophy, and radionuclide accumulation) in the thermal karst springs.

Bacterial diversity in soils of different Hungarian karst areas.

Karst areas have great environmental importance as sources of subsurface water and often maintain very sensitive ecosystems. In recent years, increasing number of microbiological studies focused on the bacterial communities of karst soils. In this study, diversity examinations on two distinct Hungarian karst areas, Aggtelek and Tapolca, were performed using parallel cultivation and molecular cloning methods. The phylogenetic affiliation of bacterial strains and molecular clones was determined based on their 16S rRNA gene sequences. Bacterial isolates were identified as members of the phyla Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes. Besides the taxa identified by cultivation, members of the phyla Chloroflexi, Cyanobacteria, Acidobacteria, Verrucomicrobia, and Gemmatimonadetes were detected by the cloning. The difference in the composition of soil bacterial communities was related to geographic locations and soil types. Both the highest and the lowest bacterial diversities were detected in samples from Aggtelek National Park, characterized by Leptic Luvisol and Rendzic Leptosol soil types. The difference in the composition of bacterial communities between Rendzic Leptosol and Leptic Phaeozem soil types at Tapolca could be the result of human impacts.

Nitrincola alkalilacustris sp. nov. and Nitrincola schmidtii sp. nov., alkaliphilic bacteria isolated from soda pans, and emended description of the genus Nitrincola.

Three alkaliphilic and halotolerant bacterial strains, designated ZV-19T, R4-8T and S4-12, were isolated from the water of soda pans located in the Kiskunság National Park, Hungary. Cells of all three strains were Gram-staining-negative, rod-shaped, motile and non-endospore-forming. They were facultatively anaerobic, and oxidase- and catalase-positive. Their major isoprenoid quinone was Q-8, and their predominant fatty acids were C18 : 1ω7c, C16 : 1ω7c and C16 : 0. The DNA G+C content was 54.5 mol% in strain ZV-19 T and 45.8 mol% in strain R4-8T. The 16S rRNA gene based phylogenetic analysis showed that all three strains were members of the genus Nitrincola (family Oceanospirillaceae, class Gammaproteobacteria). Strain ZV-19T showed 96.6 and 95.5 % sequence similarities and 19±3 and 18±3 % DNA-DNA relatedness to Nitrincolalacisaponensis DSM 16316T and Nitrincolaalkalisediminis JCM 19317T, respectively. Strains R4-8T and S4-12 exhibited 97.9 and 98.6 % sequence matches and 34±4 and 13±8 % DNA-DNA hybridization values with N. lacisaponensis DSM 16316T and N. alkalisediminis JCM 19317T, respectively. According to the phenotypic, chemotaxonomic and phylogenetic data, the strains studied represent two novel species, Nitrincola alkalilacustris sp. nov. with the type strain ZV-19T (=DSM 29817T=NCAIM B 02612T) and Nitrincola schmidtii sp. nov. with the type strain R4-8T (=DSM 100788T=NCAIM B.02626T). An emended description of the genus Nitrincola is also presented.

Nesterenkonia pannonica sp. nov., a novel alkaliphilic and moderately halophilic actinobacterium.

An alkaliphilic and moderately halophilic bacterial strain characterized by optimal growth at pH 9.0-10.0 and with 5-7 % (w/v) NaCl, designated BV-35T, was isolated from water of a soda pan located in Kiskunság National Park, Hungary. Cells of the orange-pigmented colony were Gram-stain-positive, non-motile and non-endospore-forming coccoid rods. The isolate was strictly aerobic, catalase-positive and oxidase-negative. Strain BV-35T displayed a peptidoglycan similar to type A4α, l-Lys-l-Glu (A11.54 according to www.peptidoglycan-types.info) but containing additionally 4-aminobutyric acid. Menaquinone-7 (MK-7) was the predominant isoprenoid quinone, and anteiso-C15 : 0 and anteiso-C17 : 0 were its major cellular fatty acids. The DNA G+C content of strain BV-35T was 65.4 mol%. Based on 16S rRNA gene sequence similarities, the novel isolate showed the closest relationship to Nesterenkonia populi GP 10-3T (97.9 %). The DNA-DNA relatedness between BV-35T and N. populi was 46.7 %. The distinguishing phenotypic and genetic results of this polyphasic study revealed that strain BV-35T represents a novel member of the genus Nesterenkonia, for which the name Nesterenkonia pannonica sp. nov. is proposed. The type strain is BV-35T (=DSM 29786T=NCAIM B 02606T).

Bacillus kiskunsagensis sp. nov., a novel alkaliphilic and moderately halophilic bacterium isolated from soda soil.

An alkaliphilic and moderately halophilic strain characterized by optimal growth at pH 9.0-10.0 and 7 % (w/v) NaCl, and designated B16-24T, was isolated from the rhizosphere soil of the bayonet grass Bolboschoenus maritimus at a soda pond in the Kiskunság National Park, Hungary. Cells of the strain were Gram-staining-positive, non-motile, straight rods, and formed central, ellipsoidal endospores with slightly swollen sporangia. The isolate was facultative anaerobic, catalase positive, oxidase negative, and contained a peptidoglycan of type A1γ based on meso-diaminopimelic acid. Menaquinone-7 (MK-7) was the predominant isoprenoid quinone, and anteiso-C15 : 0 the major cellular fatty acid. The DNA G+C content of strain B16-24T was 36.6 mol%. The 16S rRNA gene-based phylogenetic analysis revealed that the novel isolate had the greatest similarities to the type strains of Bacillus okhensis Kh10-101T (97.8 %), B. akibai 1139T (97.4 %), B. alkalisediminis K1-25T (97.3 %) and B. wakoensis N-1T (97.1 %). The DNA-DNA relatedness of strain B16-24T and the closely related Bacillus species ranged between 24±6 % and 35±3 %. The distinctive phenotypic and genetic results of this study confirmed that strain B16-24T represents a novel species within the genus Bacillus, for which the name Bacillus kiskunsagensis sp. nov. is proposed. The type strain is B16-24T (=DSM 29791T=NCAIM B.02610T).