Sequencing bands of ribosomal intergenic spacer analysis fingerprints for characterization and microscale distribution of soil bacterium populations responding to mercury spiking

Two major emerging bands (a 350-bp band and a 650-bp band) within the RISA (ribosomal intergenic spacer analysis) profile of a soil bacterial community spiked with Hg(II) were selected for further identification of the populations involved in the response of the community to the added metal. The bands were cut out from polyacrylamide gels, cloned, characterized by restriction analysis, and sequenced for phylogenetic affiliation of dominant clones. The sequences were the intergenic spacer between the rrs and rrl genes and the first 130 nucleotides of the rrl gene. Comparison of sequences derived from the 350-bp band to The GenBank database permitted us to identify the bacteria as being mostly close relatives to low G+C firmicutes (Clostridium-like genera), while the 650-bp band permitted us to identify the bacteria as being mostly close relatives to beta-proteobacteria (Ralstonia-like genera). Oligonucleotide probes specific for the identified dominant bacteria were designed and hybridized with the RISA profiles derived from the control and spiked communities. These studies confirmed the contribution of these populations to the community response to the metal. Hybridization of the RISA profiles from subcommunities (bacterial pools associated with different soil microenvironments) also permitted to characterize the distribution and the dynamics of these populations at a microscale level following mercury spiking.     

Effects of Spilled Oil on Bacterial Communities of Mediterranean Coastal Anoxic Sediments Chronically Subjected to Oil Hydrocarbon Contamination

The effects of spilled oil on sedimentary bacterial communities were examined in situ at 20 m water depth in a Mediterranean coastal area. Sediment collected at an experimental site chronically subjected to hydrocarbon inputs was reworked into PVC cores with or without a massive addition of crude Arabian light oil (∼20 g kg⁻¹ dry weight). Cores were reinserted into the sediment and incubated in situ at the sampling site (20 m water depth) for 135 and 503 days. The massive oil contamination induced significant shifts in the structure of the indigenous bacterial communities as shown by ribosomal intergenic spacer analysis (RISA). The vertical heterogeneity of the bacterial communities within the sediment was more pronounced in the oiled sediments particularly after 503 days of incubation. Response to oil of the deeper depth communities (8–10 cm) was slower than that of superficial depth communities (0–1 and 2–4 cm). Analysis of the oil composition by gas chromatography revealed a typical microbial alteration of n-alkanes during the experiment. Predominant RISA bands in oiled sediments were affiliated to hydrocarbonoclastic bacteria sequences. In particular, a 395-bp RISA band, which was the dominant band in all the oiled sediments for both incubation times, was closely related to hydrocarbonoclastic sulfate-reducing bacteria (SRB). These bacteria may have contributed to the main fingerprint changes and to the observed biodegradation of n-alkanes. This study provides useful information on bacterial dynamics in anoxic contaminated infralittoral sediments and highlights the need to assess more precisely the contribution of SRB to bioremediation in oil anoxic contaminated areas.     

Shifts in Diversity and Microscale Distribution of the Adapted Bacterial Phenotypes due to Hg(II) Spiking in Soil

In a previous experiment [Ranjard et al. (2000) FEMS Microbiol Ecol 31:107–115], the spatial heterogeneity of a mercury impact on soil bacterial community was revealed by an increase of mercury-resistant (Hg^R) bacterial numbers in the outer fraction and the sand fractions when compared to those in the silt fractions. The objectives of the present study were (i) to investigate whether mercury exposure affects the diversity and the distribution within the various fractions of the Hg^R populations and (ii) to evaluate the contribution of the Hg^R populations to the overall community adaptation. A total of 236 strains isolated before (104 isolates) and 30 days (132 isolates) after spiking were characterized by an amplified ribosomal DNA restriction analysis (ARDRA) and grouped into 30 different genotypes. Whereas almost the same numbers of different genotypes were observed at both sampling times when considering all microenvironments, important changes in their evenness were observed. At the microscale level, we also noticed a heterogeneous distribution of the genotypes. Partial 16S rDNA sequences of each genotype were determined to permit phylogenetic affiliation. Whereas Pseudomonas-like species were dominant in all microenvironments at T = 0, genotypes not detected before spiking such as genotypes closely related to Xanthomonas, Pseudoaminobacter, and Sphingomonas-like species became dominant at T = 30. Similarly, several genotypes close to Bacillus, Streptomyces, and Rhodococcus species were only detected in the sand fractions at T = 0 and could not be detected in these fractions at T = 30. Probes defined within the intergenic spacer between the rrs and rrl genes were designed for the dominant genotypes and were hybridized toward the RISA (Ribosomal Intergenic Spacer Analysis) profiles derived from the T = 0 and T = 30 bacterial communities associated to the unfractionated soil. Results showed that the culturable dominant Hg^R genotypes partially contributed to the adaptation of the whole bacterial community.     

Microbial Succession in a Compost-packed Biofilter Treating Benzene-contaminated Air

Air artificially contaminated with increasing concentrations of benzene was treated in a laboratory scale compost-packed biofilter for 240 days with a removal efficiency of 81–100%. The bacterial community in the packing material (PM) at different heights of the biofilter was analysed every 60 days. Bacterial plate counts and ribosomal intergenic spacer analysis (RISA) of the isolated strains showed that the number of cultivable aerobic heterotrophic bacteria and the species diversity increased with benzene availability. Identification of the isolated species and the main bands in denaturing gradient gel electrophoresis (DGGE) profiles from total compost DNA during the treatment revealed that, at a relatively low volumetric benzene load (1.2≤VBL≤6.4 g m⁻³ _PM h⁻¹), besides low G+C Gram positive bacteria, originally present in the packing compost, bacteroidetes and β- and γ-proteobacteria became detectable in the colonising population. At the VBL value (24.8 g m⁻³ _PM h⁻¹) ensuring the maximum elimination capacity of the biofilter (20.1 g m⁻³ _PM h⁻¹), strains affiliated to the genus Rhodococcus dominated the microflora, followed by β-proteobacteria comprising the genera Bordetella and Neisseria. Under these conditions, more than 35% of the isolated strains were able to grow on benzene as the sole carbon source. Comparison of DGGE and automated RISA profiles of the total community and isolated strains showed that a complex bacterial succession occurred in the reactor in response to the increasing concentrations of the pollutant and that cultivable bacteria played a major role in benzene degradation under the adopted conditions.     

Response of rhizobacterial communities in watermelon to infection with cucumber green mottle mosaic virus as revealed by cultivation-dependent RISA

We investigated the rhizobacterial densities and community structure in watermelon rhizosphere under the infection of cucumber green mottle mosaic virus (CGMMV) by artificial inoculation. Rhizobacterial densities and communities were analysed from healthy and infected plants under aerobic and anaerobic culture techniques. The highest total number of aerobic rhizobacteria was counted to be 2.7 × 10⁸ colony forming units per gram (CFU · g^?1) and anaerobic rhizobacteria was to be 3.2 × 10⁶ CFU · g^?1, in healthy and infected plants, respectively. Cultivation-dependent ribosomal intergenic spacer analysis (RISA) was employed for further analysis on the rhizobacterial community structure. By incorporating the relative abundance of amplicons, the per cent similarity was determined by the similarity coefficients based only upon the absence or presence of DNA bands. The cluster analysis of RISA showed that the community structure of aerobic rhizobacteria exhibited 60% similarity between healthy and infected plant. The highest community structure similarity (50% similarity) of anaerobic rhizobacteria occurred between before planting and infected plant.     

ITS-polymorphism of salt-tolerant and salt-sensitive native isolates of Sinorhizoblum meliloti-symbionts of alfalfa,clover and fenugreek plants

Polymorphism of rrs-rrl sequence of ribosomal operons (intergenic sequence, ITS) was studied among 81 isolates of Sinorhizobium meliloti (AK001–AK210) derived from the collection of alfalfa nodulating bacteria of the Laboratory of genetics of ARRIAM, by using species-specific primers FGPS1490/FGPL132VM. Isolates were obtained from nodules of different species of wild host plants from Medicago, Melilotus and Trigonella genera grown in salinized North-Western region of Kazakhstan. The typical structure of ITS, similar to that of test strain Rm1021, was dominant in native rhizobia population, while in one third of the isolates (33.3%) this sequence was different. Among the latter, the ITS type of strain AK83 (RCAM00182) was dominant. Here, we show for the first time that isolates with reduced level of salt-tolerance had more diverse intergenic sequences of rrn-operons. No phylogenetic separation was observed between isolates grouped on the basis of their tolerance or sensitivity towards 0.6 M NaCl. However, the frequency of divergent ITS types within the two groups of rhizobia depended on the host symbiotic preference observed in natural environment, allowing to speculate about the existence of a chromosome types specific for S. meliloti isolates with differential salt tolerance. In conclusion, we propose that in the area subjected to secondary salinization, which are also the centre of introgressive hybridization of alfalfa, micro-evolutionary processes, affecting rrn-operons and associated with salt adaptation, are also occurring in symbiotic root nodule bacteria populations.     

Molecular Assessment of Inoculated and Indigenous Bacteria in Biofilms from a Pilot-Scale Perchlorate-Reducing Bioreactor

Bioremediation of perchlorate-contaminated groundwater can occur via bacterial reduction of perchlorate to chloride. Although perchlorate reduction has been demonstrated in bacterial pure cultures, little is known about the efficacy of using perchlorate-reducing bacteria as inoculants for bioremediation in the field. A pilot-scale, fixed-bed bioreactor containing plastic support medium was used to treat perchlorate-contaminated groundwater at a site in Southern California. The bioreactor was inoculated with a field-grown suspension of the perchlorate-respiring bacterium Dechlorosoma sp. strain KJ and fed groundwater containing indigenous bacteria and a carbon source amendment. Because the reactor was flushed weekly to remove accumulated biomass, only bacteria capable of growing in biofilms in the reactor were expected to survive. After 26 days of operation, perchlorate was not detected in bioreactor effluent. Perchlorate remained undetected by ion chromatography (detection limit 4 μg L⁻¹) during 6 months of operation, after which the reactor was drained. Plastic medium was subsampled from top, middle, and bottom locations of the reactor for shipment on blue ice and storage at −80°C prior to analysis. Microbial community DNA was extracted from successive washes of thawed biofilm material for PCR-based community profiling by 16S-23S ribosomal intergenic spacer analysis (RISA). No DNA sequences characteristic of strain KJ were recovered from any RISA bands. The most intense bands yielded DNA sequences with high similarities to Dechloromonas spp., a closely related but different genus of perchlorate-respiring bacteria. Additional sequences from RISA profiles indicated presence of representatives of the low G+C gram-positive bacteria and the Cytophaga–Flavobacterium–Bacteroides group. Confocal scanning laser microscopy and fluorescence in situ hybridization (FISH) were also used to examine biofilms using genus-specific 16S ribosomal RNA probes. FISH was more sensitive than RISA profiling in detecting possible survivors from the initial inoculum. FISH revealed that bacteria hybridizing to Dechlorosoma probes constituted <1% of all cells in the biofilms examined, except in the deepest portions where they represented 3–5%. Numbers of bacteria hybridizing to Dechloromonas probes decreased as biofilm depth increased, and they were most abundant at the biofilm surface (23% of all cells). These spatial distribution differences suggested persistence of low numbers of the inoculated strain Dechlorosoma sp. KJ in parts of the biofilm nearest to the plastic medium, concomitant with active colonization or growth by indigenous Dechloromonas spp. in the biofilm exterior. This study demonstrated the feasibility of post hoc analysis of frozen biofilms following completion of field remediation studies.     

Microbial community analysis of field-grown soybeans with different nodulation phenotypes

Microorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod(+) soybean roots. Fusarium solani was stably associated with nodulated (Nod(+) and Nod(++)) roots and less abundant in Nod(-) soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod(-), Nod(+), or Nod(++)). The analysis of root samples indicated that the microbial community in Nod(-) soybeans was more similar to that in Nod(++) soybeans than to that in Nod(+) soybeans.     

Molecular Characterization of Yarrowia lipolytica and Candida zeylanoides Isolated from Poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.     

A soil microscale study to reveal the heterogeneity of Hg(II) impact on indigenous bacteria by quantification of adapted phenotypes and analysis of community DNA fingerprints

The short term impact of 50 μM Hg(II) on soil bacterial community structure was evaluated in different microenvironments of a silt loam soil in order to determine the contribution of bacteria located in these microenvironments to the overall bacterial response to mercury spiking. Microenvironments and associated bacteria, designated as bacterial pools, were obtained by successive soil washes to separate the outer fraction, containing loosely associated bacteria, and the inner fraction, containing bacteria retained into aggregates, followed by a physical fractionation of the inner fraction to separate aggregates according to their size (size fractions). Indirect enumerations of viable heterotrophic (VH) and resistant (Hg(R)) bacteria were performed before and 30 days after mercury spiking. A ribosomal intergenic spacer analysis (RISA), combined with multivariate analysis, was used to compare modifications at the community level in the unfractionated soil and in the microenvironments. The spatial heterogeneity of the mercury impact was revealed by a higher increase of Hg(R) numbers in the outer fraction and in the coarse size fractions. Furthermore, shifts in RISA patterns of total community DNA indicated changes in the composition of the dominant bacterial populations in response to Hg(II) stress in the outer and in the clay size fractions. The heterogeneity of metal impact on indigenous bacteria, observed at a microscale level, is related to both the physical and chemical characteristics of the soil microenvironments governing mercury bioavailability and to the bacterial composition present before spiking.     

Effects of Wildfire and Harvest Disturbances on Forest Soil Bacterial Communities

Wildfires and harvesting are important disturbances to forest ecosystems, but their effects on soil microbial communities are not well characterized and have not previously been compared directly. This study was conducted at sites with similar soil, climatic, and other properties in a spruce-dominated boreal forest near Chisholm, Alberta, Canada. Soil microbial communities were assessed following four treatments: control, harvest, burn, and burn plus timber salvage (burn-salvage). Burn treatments were at sites affected by a large wildfire in May 2001, and the communities were sampled 1 year after the fire. Microbial biomass carbon decreased 18%, 74%, and 53% in the harvest, burn, and burn-salvage treatments, respectively. Microbial biomass nitrogen decreased 25% in the harvest treatment, but increased in the burn treatments, probably because of microbial assimilation of the increased amounts of available NH₄⁺ and NO₃⁻ due to burning. Bacterial community composition was analyzed by nonparametric ordination of molecular fingerprint data of 119 samples from both ribosomal intergenic spacer analysis (RISA) and rRNA gene denaturing gradient gel electrophoresis. On the basis of multiresponse permutation procedures, community composition was significantly different among all treatments, with the greatest differences between the two burned treatments versus the two unburned treatments. The sequencing of DNA bands from RISA fingerprints revealed distinct distributions of bacterial divisions among the treatments. Gamma- and Alphaproteobacteria were highly characteristic of the unburned treatments, while Betaproteobacteria and members of Bacillus were highly characteristic of the burned treatments. Wildfire had distinct and more pronounced effects on the soil microbial community than did harvesting.     

Individualization and estimation of relatedness in crocodilians by DNA fingerprinting with a Bkm-derived probe

Individual-specific DNA fingerprints of crocodilians were obtained by the use of Bkm-2(8) probe. Pedigree analyses of Crocodylus palustris, C. porosus and Caiman crocodilus revealed that the multiple bands (22–23 bands with Aludigest) thus obtained were inherited stably in a Mendelian fashion. Unique fingerprints permitted us to identify individuals, assign parentage, and reconstruct the DNA profile of a missing parent. Average band sharing between unrelated crocodiles was found to be 0.37. Band sharing between animals of known pedigrees increased predictably with relatedness and provided a basis for distinguishing relatives from non-relatives. Similar results obtained in other species/genera, using the same probe, suggest that this approach may be applicable to all species of crocodilians, and could facilitate genetic studies of wild and captive populations.     

Brood size modifications affect plumage bacterial assemblages of European starlings

During reproduction, birds face trade-offs between time and energy devoted to parental effort and traits associated with self-maintenance. We manipulated brood sizes to investigate the effects of such trade-offs on feather bacterial densities and the structure of bacterial assemblages on feathers in adult European starlings, Sturnus vulgaris, and in vitro feather degradation. As predicted by a trade-off between parental effort and self-maintenance, we found that birds with enlarged broods had more free-living bacteria on their feathers than birds with reduced broods. Furthermore, we found a significant interaction between brood manipulation and original brood size on free-living bacterial densities suggesting that the trade-off is mediated by the adults' initial reproductive investment. In contrast, brood size manipulations had no significant effect on densities of attached bacteria. Using ribosomal intergenic spacer analysis (RISA), we demonstrated that brood manipulations significantly modified the structure (band pattern) of feather-degrading bacterial assemblages, but had no significant effect on their richness (number of bands) or the in vitro feather degradation. In vitro feather degradation varied in relation to the premanipulation brood size and positively with the richness of the feather degrading bacterial community. Besides brood manipulation effect, we found that ecological factors and individual traits, such as the age, the nest location or the capture date, shaped bacterial assemblages and feather degradation capacities.     

The complete mitochondrial genome of the endangered Apollo butterfly,Parnassius apollo (Lepidoptera: Papilionidae) and its comparison to other Papilionidae species

The Apollo butterfly, Parnassius apollo is a representative species of the butterfly subfamily Parnassiinae. This charming species is one of the most endangered butterfly species in the world. In this study, we sequenced its complete mitochondrial genome (mitogenome), with the aim of accumulating genetic information for further studies of population genetics and mitogenome evolution in the Papilionidae. The 15,404-bp long mitogenome harbors a typical set of 37 genes and is the largest butterfly mitogenome determined, except for Papilio maraho (16,094 bp). Like many other sequenced lepidopteran species, one tRNA^Trp-like and one tRNA^Leu(UUR)-like sequences were detected in the AT-rich region. A total of 164 bp of non-coding sequences are dispersed in 14 regions throughout the genome. The longest intergenic spacer (68 bp) is located between tRNA^Ser(AGN) and tRNA^Glu, and is the largest spacer at this location among Papilionidae species. This spacer may have resulted from an 8-fold repetition of a TTTCTTCT motif or a 4-fold repetition of a CTTTATTT motif.     

Linking the Physicochemical Properties with the Abundance and Diversity of Rhizospheric Bacterial Population Inhabiting Paddy Soil Based on a Concerted Multivariate Analysis of PCR-DGGE and RISA

To unravel the existence of dominant bacterial population in the paddy fields of Eastern Uttar Pradesh, India and their relation to the prevailing soil physicochemistry using multivariate statistical analyses, a cumulative culture-independent 16S rRNA based Polymerase chain reaction-Denaturing gradient gel electrophoresis (PCR-DGGE) and a 16S-23S ribosomal intergenic spacer analysis (RISA) have been performed. Detrended correspondence analysis (DCA) and principal component analysis (PCA) biplot analyses were used to assess the relation between soil bacterial population and its physicochemistry. DCA analysis exhibited a strong dependence of bacterial existence on the soil physicochemical variables, such as organic matter, total nitrogen, inorganic nutrients, temperatures, and moisture status. Soil dehydrogenase activity (DHA) was assessed to check the metabolic activity of all soil samples which showed a range of 0.012–0.050 nmol TPF g^?1 min^?1 with significant variation (p < 0.01). Out of 96 bands excised, 45 different phylotypes were obtained using both techniques which elucidated the abundance of Cyanobacteria over other soil bacterial population. Scytonema sp., Leptolyngbya sp. and different uncultured cyanobacterial species were the major genera found. Profiling data obtained through PCR-DGGE and RISA were used in alpha diversity and rarefaction curve analysis suggested site 6 (Chandauli) as the most diversity rich site. Thus extensive dataset of weighted and unweighted variables generated through DGGE and RISA coupled with metabolic functioning of soil and multivariate analyses provided an excellent opportunity to map the soil microbial structure in paddy fields and their regulation with existing soil environment.     

Determination of Cyanobacterial Diversity during Algal Blooms in Daechung Reservoir, Korea, on the Basis of cpcBA Intergenic Spacer Region Analysis

The detection and prevention of cyanobacterial blooms are important issues in water quality management. As such, the diversity and community dynamics of cyanobacteria during cyanobacterial bloom in the Daechung Reservoir, Korea, were studied by analyzing the intergenic spacer (IGS) region between phycocyanin subunit genes cpcB and cpcA (cpcBA IGS). To amplify the cpcBA IGS from environmental samples, new PCR primers that could cover a wider range of cyanobacteria than previously known primers were designed. In the samples taken around the bloom peak (2 September 2003), seven groups of cpcBA IGS sequences were detected, and none of the amplified cpcBA IGSs was closely related to the cpcBA IGS from chloroplasts. Apart from the Microcystis-, Aphanizomenon (Anabaena)-, Pseudanabaena-, and Planktothrix (Oscillatoria)-like groups, the three other groups of cpcBA IGS sequences were only distantly related to previously reported sequences (<85% similarity to their closest relatives). The most prominent changes during the bloom were the gradual decrease and eventual disappearance of the Aphanizomenon (Anabaena)-like group before the bloom peak and the gradual increase and sudden disappearance of Planktothrix (Oscillatoria)-like groups right after the bloom peak. The community succession profile obtained based on the cpcBA IGS analysis was also supported by a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA genes.     

Diverse cellulolytic bacteria isolated from the high humus, alkaline-saline chinampa soils

Aiming at learning the functional bacterial community in the high humus content, saline-alkaline soils of chinampas, the cellulolytic bacteria were quantified and 100 bacterial isolates were isolated and characterized in the present study. Analysis of 16S-23S IGS (intergenic spacer) RFLP (restriction fragment length polymorphism) grouped the isolates into 48 IGS types and phylogenetic analysis of 16S rRNA genes identified them into 42 phylospecies within 29 genera and higher taxa belonging to the phyla Actinobacteria, Firmicutes and Proteobacteria, dominated by the genera Arthrobacter, Streptomyces, Bacillus, Pseudomonas, Pseudoxanthomonas and Stenotrophomonas. Among these bacteria, 63 isolates represent 26 novel putative species or higher taxa, while 37 were members of 17 defined species according to the phylogenetic relationships of 16S rRNA gene. Except for the novel species, the cellulolytic activity was not reported previously in 9 of the 17 species. They degraded cellulose in medium at pH?4.5–10.0 or supplied with NaCl up to 9 %. In addition, 84.8 and 71.7 % of them degraded xylan and Avicel, respectively. These results greatly improved the knowledge about the diversity of cellulolytic bacteria and demonstrated that the chinampa soils contain diverse and novel cellulolytic bacteria functioning at a wide range of pH and salinity levels, which might be a valuable biotechnological resource for biotransformation of cellulose.     

High intra-individual sequence variation in the nuclear rDNA LSU-5S intergenic spacer in the Sargassaceae (Fucales, Phaeophyceae)

Of four species of Sargassaceae, representing the genera Carpophyllum, Cystoseira, Landsburgia, and Sargassum, the intergenic spacer of the ribosomal cistron was amplified using a forward primer annealing at the 3′-end of the large subunit (LSU) of the ribosomal cistron and a reverse primer annealing at the 5S rRNA gene. The PCR products were cloned and the DNA sequences of multiple clones were determined. Almost each clone showed a unique DNA sequence. Intra-individual variation of this LSU-5S intergenic spacer was extremely high and was characterized by great length variation and a high number of short tandem repeats. Sequences were unalignable and therefore it was concluded that the LSU-5S intergenic spacer is unsuitable for phylogenetic and phylogeographic studies of sargassacean taxa.     

Influence of Effluent Irrigation on Community Composition and Function of Ammonia-Oxidizing Bacteria in Soil

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the α-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein.     

The Bacteriome of Bat Flies (Nycteribiidae) from the Malagasy Region: a Community Shaped by Host Ecology,Bacterial Transmission Mode,and Host-Vector Specificity

The Nycteribiidae are obligate blood-sucking Diptera (Hippoboscoidea) flies that parasitize bats. Depending on species, these wingless flies exhibit either high specialism or generalism toward their hosts, which may in turn have important consequences in terms of their associated microbial community structure. Bats have been hypothesized to be reservoirs of numerous infectious agents, some of which have recently emerged in human populations. Thus, bat flies may be important in the epidemiology and transmission of some of these bat-borne infectious diseases, acting either directly as arthropod vectors or indirectly by shaping pathogen communities among bat populations. In addition, bat flies commonly have associations with heritable bacterial endosymbionts that inhabit insect cells and depend on maternal transmission through egg cytoplasm to ensure their transmission. Some of these heritable bacteria are likely obligate mutualists required to support bat fly development, but others are facultative symbionts with unknown effects. Here, we present bacterial community profiles that were obtained from seven bat fly species, representing five genera, parasitizing bats from the Malagasy region. The observed bacterial diversity includes Rickettsia, Wolbachia, and several Arsenophonus-like organisms, as well as other members of the Enterobacteriales and a widespread association of Bartonella bacteria from bat flies of all five genera. Using the well-described host specificity of these flies and data on community structure from selected bacterial taxa with either vertical or horizontal transmission, we show that host/vector specificity and transmission mode are important drivers of bacterial community structure.