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We investigated the effect of different nitrogen (N) sources on exopolysaccharide (EPS) production and composition by Frankia strain CcI3, a N 2-fixing actinomycete that forms root nodules with Casuarina species. Frankia cells grown in the absence of NH 4Cl (i.e., under N 2-fixing conditions) produced 1.7-fold more EPS, with lower galactose (45.1 vs. 54.7 mol%) and higher mannose (17.3 vs. 9.7 mol%) contents than those grown in the presence of NH 4Cl as a combined N-source. In the absence of the combined N-source, terminally linked and branched residue contents were nearly twice as high with 32.8 vs. 15.1 mol% and 15.1 vs. 8.7 mol%, respectively, than in its presence, while the content of linearly linked residues was lower with 52.1 mol% compared to 76.2 mol%. To find out clues for the altered EPS production at the transcriptional level, we performed whole-gene expression profiling using quantitative reverse transcription PCR and microarray technology. The transcription profiles of Frankia strain CcI3 grown in the absence of NH 4Cl revealed up to 2 orders of magnitude higher transcription of nitrogen fixation-related genes compared to those of CcI3 cells grown in the presence of NH 4Cl. Unexpectedly, microarray data did not provide evidence for transcriptional regulation as a mechanism for differences in EPS production. These findings indicate effects of nitrogen fixation on the production and composition of EPS in Frankia strain CcI3 and suggest posttranscriptional regulation of enhanced EPS production in the absence of the combined N-source. [ABSTRACT FROM AUTHOR]

The diversity of Salmonella was analysed in biofilm and water samples from the spring and slough arms of Spring Lake, the headwaters of the San Marcos River, Texas, during the drought of 2011, with only one potential run-off event at the beginning of the study. Salmonellae were detected in semiselective enrichment cultures by end-point PCR during the entire sampling period of (11 sampling events during 2 months). From the spring arm site, 73% of the biofilms and 41% of the water samples were positive for salmonellae, while only 9% of the biofilms and 23% of the water samples were positive from the slough arm site. Salmonellae could be isolated from all positive samples, with higher diversity in biofilms compared with water samples and more strains obtained from the spring arm than from the slough arm. Differences between sites were generally caused by less frequently detected isolates, while the majority of isolates that were present in both biofilms and water from both sites was represented by three strains only. Quantification attempts by quantitative PCR directly in samples without prior enrichment did not result in a reliable detection of salmonellae, suggesting that numbers in all samples were below the detection limit. [ABSTRACT FROM AUTHOR]

Sediments and water from the spring and slough arm of Spring Lake, the pristine headwaters of the San Marcos River, Texas, were analyzed for Salmonellae by culture and molecular techniques before and after three major precipitation events, each with intermediate dry periods. Polymerase chain reaction (PCR)-assisted analyses of enrichment cultures detected Salmonellae in samples after all three precipitation events, but failed to detect them immediately prior to the rainfall events. Detection among individual locations differed with respect to the precipitation event analyzed, and strains isolated were highly variable with respect to serovars. These results demonstrate that rainwater associated effects, most likely surface runoff, provide an avenue for short-term pollution of aquatic systems with Salmonellae that do not, however, appear to establish for the long-term in water nor sediments. [ABSTRACT FROM AUTHOR]

Abstract: Comparative sequence analysis of almost complete 16S rRNA genes of members of the Desulfobacteriaceae retrieved from two gene clone libraries of uncultured bacteria of the chemocline of Lake Cadagno, Switzerland, resulted in the molecular identification of nine sequences, with a tight cluster of five sequences that represented at least three different populations of bacteria with homology values of 95% and 93% to their closest cultured relatives Desulfomonile tiedjei and Desulfomonile limimaris, respectively. In situ hybridization with probes DsmA455 targeting two subpopulations and DsmB455 targeting one subpopulation, detected bacteria with a peculiar morphology previously described as “morphotype R”. The individual probes detected about the same number of cells in all samples and together added up to represent all cells of “morphotype R” suggesting that the basic ecophysiological requirements of the subpopulations might be similar. In the monimolimnion, “morphotype R” cells accounted for up to 29% of all Bacteria and entirely represented the Desulfobacteriaceae, the most prominent sulfate-reducing bacteria. In the sediment, “morphotype R” was similarly prominent in the upper cm only where it represented all Desulfobacteriaceae and up to 50% of all Bacteria. Numbers and importance within the Desulfobacteriaceae and Bacteria declined significantly with depth in sediments suggesting potential effects of changing environmental conditions on the fate of “morphotype R”. [Copyright &y& Elsevier]

Investigates the culturable bacteria from the anoxic bulk soil of rice paddy microcosms. Use of molecular and activation techniques; Use of serial liquid dilution cultures to determine population sizes; Identification of microorganisms obtained from the terminal and subterminal positive dilution steps; RNA gene sequences of the isolates.

The interaction between the salt marsh plant Spartina patens, arbuscular mycorrhizal fungi (AMF) and bacteria in salt marsh sediment was examined in a long-term arbuscular mycorrhizas (AM) suppression study by applying the systemic fungicide benomyl to field-collected sediment cores with and without S. patens plants. Microbial populations were sampled four times corresponding to major plant phenological stages (dormancy, vegetative growth, reproduction, and senescence) previously linked to changes in microbial populations under field conditions. Benomyl-treatment of soil cores significantly suppressed AM colonization on S. patens, keeping values relatively consistent throughout the growing season (11.5%) whereas plants in non-treated cores experienced seasonal increases and declines in AM colonization (26.6% during vegetative growth to 11.5% during dormancy). Soil physicochemical parameters were not affected by benomyl application. In unvegetated cores, no benomyl- or seasonal effects were displayed by cell numbers and specific biomass of DAPI-stained organisms, members of the domain bacteria and here especially members of the α-, β-, γ- and δ-subdivisions of proteobacteria that were the most abundant bacterial groups. In vegetated cores, the microbial community as well as specific bacterial populations were at least twice as large in terms of number and biomass than in samples from unvegetated cores with significant seasonal changes for DAPI-stained cells, for members of the domain bacteria and for members of the α- and γ-subdivisions of proteobacteria. In benomyl-treated cores, the population of γ-subdivision of proteobacteria was significantly smaller than in non-treated cores, and a positive association was found between this bacterial group and root length colonized by AM suggesting that AM-suppression can affect populations of specific soil bacterial populations in salt marsh sediment. Benomyl-treatment had no effect on the diversity of N-fixing bacteria as evidenced by PCR-RFLP analysis, but seasonal changes were noted in vegetated cores with populations during active plant growth substantially different from populations during dormancy and senescence. [Copyright &y& Elsevier]

Actinorhizal plants form nitrogen-fixing root nodules in symbiosis with soil-dwelling actinobacteria within the genus Frankia, and specific Frankia taxonomic clusters nodulate plants in corresponding host infection groups. In same-soil microcosms, we observed that some host species were nodulated (Alnus glutinosa, Alnus cordata, Shepherdia argentea, Casuarina equisetifolia) while others were not (Alnus viridis, Hippophaë rhamnoides). Nodule populations were represented by eight different sequences of nifH gene fragments. Two of these sequences characterized frankiae in S. argentea nodules, and three others characterized frankiae in A. glutinosa nodules. Frankiae in A. cordata nodules were represented by five sequences, one of which was also found in nodules from A. glutinosa and C. equisetifolia, while another was detected in nodules from A. glutinosa. Quantitative PCR assays showed that vegetation generally increased the abundance of frankiae in soil, independently of the target gene (i.e., nifH or the 23S rRNA gene). Targeted Illumina sequencing of Frankia-specific nifH gene fragments detected 24 unique sequences from rhizosphere soils, 4 of which were also found in nodules, while the remaining 4 sequences in nodules were not found in soils. Seven of the 24 sequences from soils represented >90% of the reads obtained in most samples; the 2 most abundant sequences from soils were not found in root nodules, and only 2 of the sequences from soils were detected in nodules. These results demonstrate large differences between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils. [ABSTRACT FROM AUTHOR]

Strains CN4 T , CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus . The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N 2 -fixing vesicles and do not fix nitrogen in vitro . The peptidoglycan of strain CN4 T contains meso -diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H 4 ) and MK-9(H 6 ) are the predominant menaquinones (>15 %), and iso-C 16 : 0 and C 17 : 1 ω8 c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4 T is most closely related to Frankia saprophytica CN 3 T . An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3 T , Frankia asymbiotica M16386 T and Frankia inefficax EuI1c T with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4 T and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4 T , CN6, CN7 and CNm7 had dDDH (98.6-99.6 %) and ANI values that grouped them as representing a single species. CN4 T has a 10.76 Mb genome. CN4 T was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4 T (=DSM 114740 T = LMG 32595 T ) to a novel species, with CN4 T as type strain, for which the name Frankia nepalensis sp. nov. is proposed.

Frankia strain Ag45/Mut15 T was isolated from a root nodule of Alnus glutinosa growing in a swamp at lake Grossensee, Germany. The strain forms root nodules on A. glutinosa , in which it produces hyphae and clusters of N 2 -fixing vesicles. N 2 -fixing vesicles are also produced in nitrogen-free growth medium, in addition to hyphae and sporangia. The whole-cell hydrolysates of strain Ag45/Mut15 T contained meso -diaminopimelic acid in the peptidoglycan and ribose, xylose, mannose, glucose, galactose and a trace of rhamnose as cell-wall sugars. The major polar lipids were phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol and glyco-phospholipid. The predominant (>20 %) menaquinones were MK-9(H 6 ) and MK-9(H 4 ). The major fatty acid profile (>10 %) consisted of iso-C 16:0 , C 17 : 1  ω8 c and C 17 : 0 . Pairwise 16S rRNA gene distances showed that strain Ag45/Mut15 T was most closely related to Frankia torreyi CpI1 T and Candidatus Frankia nodulisporulans with 16S rRNA gene similarity values of 0.001335 substitutions per site. An multilocus sequence analysis phylogeny based on atpD , dnaA , ftsZ , pgk and rpoB amino acid sequences positioned the strain within cluster 1 of Alnus - and Myrica -nodulating species, close to Candidatus F. nodulisporulans AgTrS T and F. canadensis ARgP5 T . The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the studied strain Ag45/Mut15 T and all validly named Frankia species were below the defined threshold for prokaryotic species demarcation. Candidatus F. nodulisporulans AgTrS T , which cannot be cultivated in vitro , was found to be the closest phylogenetic neighbour to strain strain Ag45/Mut15 T with dDDH and ANI values of 61.8 and 97 %, respectively. Strain Ag45/Mut15 T was not able to sporulate in nodule tissues like strain AgTrS T .Phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain Ag45/Mut15 T (=DSM 114737 T =LMG 326O1 T ) to a novel species, with Ag45/Mut15 T as type strain, for which the name Frankia umida sp. nov. is proposed.

The genetic diversity of Frankia populations in soil and in root nodules of sympatrically grown Alnus taxa was evaluated by rep-polymerase chain reaction (PCR) and nifH gene sequence analyses. Rep-PCR analyses of uncultured Frankia populations in root nodules of 12 Alnus taxa (n=10 nodules each) growing sympatrically in the Morton Arboretum near Chicago revealed identical patterns for nodules from each Alnus taxon, including replicate trees of the same host taxon, and low diversity overall with only three profiles retrieved. One profile was retrieved from all nodules of nine taxa (Alnus incana subsp. incana, Alnus japonica, Alnus glutinosa, Alnus incana subsp. tenuifolia, Alnus incana subsp. rugosa, Alnus rhombifolia, Alnus mandshurica, Alnus maritima, and Alnus serrulata), the second was found in all nodules of two plant taxa (A. incana subsp. hirsuta and A. glutinosa var. pyramidalis), and the third was unique for all Frankia populations in nodules of A. incana subsp. rugosa var. americana. Comparative sequence analyses of nifH gene fragments in nodules representing these three profiles assigned these frankiae to different subgroups within the Alnus host infection group. None of these sequences, however, represented frankiae detectable in soil as determined by sequence analysis of 73 clones from a Frankia-specific nifH gene clone library. Additional analyses of nodule populations from selected alders growing on different soils demonstrated the presence of different Frankia populations in nodules for each soil, with populations showing identical sequences in nodules from the same soil, but differences between plant taxa. These results suggest that soil environmental conditions and host plant genotype both have a role in the selection of Frankia strains by a host plant for root nodule formation, and that this selection is not merely a function of the abundance of a Frankia strain in soil.

The potential role of host plant species in the selection of symbiotic, nitrogen-fixing Frankia strains belonging to the Elaeagnus host infection group was assessed in bioassays with two Morella, three Elaeagnus, and one Shepherdia species as capture plants, inoculated with soil slurries made with soil collected from a mixed pine/grassland area in central Wisconsin, USA. Comparative sequence analysis of nifH gene fragments amplified from homogenates of at least 20 individual lobes of root nodules harvested from capture plants of each species confirmed the more promiscuous character of Morella cerifera and Morella pensylvanica that formed nodules with frankiae of the Alnus and the Elaeagnus host infection groups, while frankiae in nodules formed on Elaeagnus umbellata, Elaeagnus angustifolia, Elaeagnus commutata, and Shepherdia argentea generally belonged to the Elaeagnus host infection group. Diversity of frankiae of the Elaeagnus host infection groups was larger in nodules on both Morella species than in nodules formed on the other plant species. None of the plants, however, captured the entire diversity of nodule-forming frankiae. The distribution of clusters of Frankia populations and their abundance in nodules was unique for each of the plant species, with only one cluster being ubiquitous and most abundant while the remaining clusters were only present in nodules of one (six clusters) or two (two clusters) host plant species. These results demonstrate large effects of the host plant species in the selection of Frankia strains from soil for potential nodule formation and thus the significant effect of the choice of capture plant species in bioassays on diversity estimates in soil.

Zinc sorption on sheathed Leptothrix discophora bacterium, the isolated extracellular polymeric substances (EPS) sheath, and Mn oxide-coated bacteria was investigated with macroscopic and spectroscopic techniques. Complexation with L. discophora was dominated by the outer membrane phosphoryl groups of the phospholipid bilayer while sorption to isolated EPS was dominated by carboxyl groups. Precipitation of nanoparticulate Mn oxide coatings on the cell surface increased site capacity by over twenty times with significant increase in metal sorption. XAS analysis of Zn sorption in the coated system showed Mn oxide phase contributions of 18 to 43% through mononuclear inner-sphere complexes. The coordination environments in coprecipitation samples were identical to those of sorption samples, indicating that, even in coprecipitation, Zn is not incorporated into the Mn oxide structure. Rather, through enzymatic oxidation by L. discophora, Mn(II) is oxidized and precipitated onto the biofilm providing a large surface for metal sequestration. The nanoparticulate Mn oxide coating exhibited significant microporosity (75%) suggesting contributions from intraparticle diffusion. Transient studies conducted over 7 months revealed a 170% increase in Zn loading. However, the intraparticle diffusivity of 10(-19) cm(2) s(-1) is two orders of magnitude smaller than that for abiotic Mn oxide which we attribute to morphological changes such as reduced pore sizes in the nanoparticulate oxide. Our results demonstrate that the cell-bound Mn oxide particles can sorb significant amounts of Zn over long periods of time representing an important surface for sequestration of metal contaminants.

A nested PCR protocol was used to analyze five endemic salamander species from Central Texas for the presence of the emerging pathogen, chytrid fungus (Batrachochytrium dendrobatidis). Chytrid fungus was detected from samples of each of the five species sampled: with low abundance, in the Texas salamander (Eurycea neotenes) (1 positive out of 16 individuals tested; 1/16), the Blanco River Springs salamander (E. pterophila) (1/20), the threatened San Marcos salamander (E. nana) (1/17), and the endangered Barton Springs salamander (E. sosorum) (1/7); much higher abundance was obtained for the Jollyville Plateau salamander (E. tonkawae) (6/14), which has recently been petitioned for addition to the USA endangered species list. With one exception, sequences of PCR products were identical to the 5.8S rRNA gene, and nearly so for the flanking internal transcribed spacer (ITS) regions of B. dendrobatidis which confirmed the detection of chytrid fungus, and thus demonstrated the presence of this pathogen in populations of endangered species in Central Texas. These confirmations were obtained from nonconsumptive tail clippings which confirms the applicability of historically collected samples from other studies in the examination of the fungus across time.

The interactions among Spartina patens and sediment microbial populations and the interactions among Phragmites australis and sediment microbial populations were studied at monotypic sites in Piermont Marsh, a salt marsh of the Hudson River north of New York, N.Y., at key times during the growing season. Arbuscular mycorrhizal fungi (AMF) effectively colonized S. patens but not P. australis, and there were seasonal increases and decreases that coincided with plant growth and senescence (17 and 6% of the S. patens root length were colonized, respectively). In sediment samples from the Spartina site, the microbial community and specific bacterial populations were at least twice as large in terms of number and biomass as the microbial community and specific bacterial populations in sediment samples from the Phragmites site, and peak values occurred during reproduction. Members of the domain Bacteria, especially members of the alpha-, gamma-, and delta-subdivisions of the Proteobacteria, were the most abundant organisms at both sites throughout the growing season. The populations were generally more dynamic in samples from the Spartina site than in samples from the Phragmites site. No differences between the two sites and no differences during the growing season were observed when restriction fragment length polymorphism analyses of nifH amplicons were performed in an attempt to detect shifts in the diversity of nitrogen-fixing bacteria. Differences were observed only in the patterns generated by PCR or reverse transcription-PCR for samples from the Spartina site, suggesting that there were differences in the overall and active populations of nitrogen-fixing bacteria. Regression analyses indicated that there was a positive interaction between members of the delta-subdivision of the Proteobacteria and root biomass but not between members of the delta-subdivision of the Proteobacteria and macroorganic matter at both sites. In samples from the Spartina site, there were indications that there were bacterium-fungus interactions since populations of members of the alpha-subdivision of the Proteobacteria were negatively associated with AMF colonization and populations of members of the gamma-subdivision of the Proteobacteria were positively associated with AMF colonization.