Virioplankton community structure along a salinity gradient in a solar saltern

The virioplankton community structure along a salinity gradient from near seawater (40 per thousand ) to saturated sodium chloride brine (370 per thousand ) in a solar saltern was investigated by pulsed-field gel electrophoresis. Viral populations with genome sizes varying from 10 kb to 533 kb were detected. The viral community structure changed along the salinity gradient. Cluster analysis of the viral genome-banding pattern resulted in two main clusters. The virioplankton diversity within the samples with salinity from 40 per thousand to 150 per thousand was on the same cluster of a cladogram. The other group consisted of virioplankton from samples with salinity above 220 per thousand. The virioplankton diversity in the different samples was calculated using the Shannon index. The diversity index demonstrated an increase in diversity in the samples along the gradient from 40 per thousand to 150 per thousand salinity, followed by a decrease in the diversity index along the rest of the salinity gradient. These results demonstrate how viral diversity changes from habitats that are considered one of the most common (seawater) to habitats that are extreme in salt concentrations (saturated sodium brine). The diversity index was highest in the environments that lie in between the most extreme and the most common.     

Changes in archaeal,bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern

Microbial communities inhabiting a multipond solar saltern were analysed and compared using SSU rRNA polymerase chain reaction (PCR)-based fingerprintings carried out in parallel by four laboratories. A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied for Bacteria, Archaea and Eukarya, and laboratories applied their own techniques and protocols on the same set of samples. Members of all three domains were retrieved from all salt concentrations. Three fingerprinting techniques were used: denaturing gradient gel electrophoresis (DGGE), ribosomal internal spacer analysis (RISA), and terminal-restriction fragments length polymorphism (T-RFLP). In addition, each laboratory used its own biomass collection method and DNA extraction protocols. Prokaryotes were addressed using DGGE and RISA with different 'domain-specific' primers sets. Eukaryotes were analysed by one laboratory using DGGE and T-RFLP, but targeting the same 18S rDNA site. Fingerprints were compared through cluster analysis and non-metric multidimensional scaling plots. This exercise allowed fast comparison of microbial assemblages and determined to what extent the picture provided by each laboratory was similar to those of others. Formation of two main, salinity-based groups of samples in prokaryotes (4-15% and 22-37% salinity) was consistent for all the laboratories. When other clusters appeared, this was a result of the particular technique and the protocol used in each case, but more affected by the primers set used. Eukaryotic microorganisms changed more from pond to pond; 4-5% and 8-37% salinity were but the two main groups detected. Archaea showed the lowest number of bands whereas Eukarya showed the highest number of operational taxonomic units (OTUs) in the initial ponds. Artefacts appeared in the DGGE from ponds with extremely low microbial richness. On the other hand, different 16S rDNA fragments with the same restriction or internal transcribed spacer (ITS) length were the main limitations for T-RFLP and RISA analyses, respectively, in ponds with the highest OTUs richness. However, although the particular taxonomic composition could vary among protocols, the general structure of the microbial assemblages was maintained.     

Prokaryotic diversity of a Tunisian multipond solar saltern

16S rRNA gene clone libraries were separately constructed from three ponds with different salt concentrations, M2 (15%), TS38 (25%) and S5 (32%), located within a multipond solar saltern of Sfax. The 16S rRNA genes from 216 bacterial clones and 156 archaeal clones were sequenced and phylogenetically analyzed. 44 operational taxonomic units (OTUs) were generated for Bacteria and 67 for Archaea. Phylogenetic groups within the bacterial domain were restricted to Bacteroidetes and Proteobacteria, with the exception that one cyanobacterial OTU was found in the TS38 pond. 85.7, 26.6 and 25.0% of the bacterial OTUs from M2, TS38 and S5 ponds, respectively, are novel. All archaeal 16S rRNA gene sequences were exclusively affiliated with Euryarchaeota. 75.0, 60.0 and 66.7% of the OTUs from, respectively, M2, TS38 and S5 ponds are novel. The result showed that the Tunisian multipond solar saltern harbored novel prokaryotic diversity that has never been reported before for solar salterns. In addition, diversity measurement indicated a decrease of bacterial diversity and an increase of archaeal diversity with rising salinity gradient, which was in agreement with the previous observation for thalassohaline systems. Comparative analysis showed that prokaryotic diversity of Tunisian saltern was higher than that of other salterns previously studied. A. Sghir and E. Ammar have equally contributed to this work.     

Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern

Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined.     

Composition changes of phototrophic microbial communities along the salinity gradient in the solar saltern evaporation ponds of Eilat,Israel

There are several conflicting hypothesis that deal with the influence of flooding in the natural river–floodplain systems. According to the Flood Pulse Concept, the flood pulses are not considered to be a disturbance, while some recent studies have proven that floods can be a disturbance factor of phytoplankton development. In order to test whether flooding acts as a disturbance factor in the shallow Danubian floodplain lake (Lake Sakadaš), phytoplankton dynamics was investigated during two different hydrological years—extremely dry (2003) without flooding and usually flooded (2004). A total of 18 phytoplankton functional groups were established. The sequence of phytoplankton seasonality can be summarized P/D → E (W1, W2) → C/P (only in potamophase) → S2/H1/S_N/S1 → W1/W2 → P/D. The canonical correspondence analysis (CCA) demonstrated that the water level was a significant environmental variable in 2004. Due to the higher total biomass of Bacillariophyceae established under potamophase conditions, floodings in the early spring seem to be a stimulating factor for phytoplankton development. On the other hand, the flood pulses in May and June had dilution effects on nutrients, so that a significantly lower phytoplankton biomass was established indicating that flooding pulses can be regarded as a disturbance event. Such conditions supported diatom development (D, P, C species) and prolonged its dominance in the total phytoplankton biomass. A long-lasting Cyanoprokaryota bloom (various filamentous species—S1, S2, S_N and H1 representatives) with very high biomass characterized the limnophase (dry conditions) in summer and autumn of both years. In-lake variables (lake morphology, internal loadings of nutrients from sediments, light conditions) seem to be important for the appearance of Cyanoprokaryota bloom. The equilibrium phase was found during the Cyanoprokaryota bloom only in the extremely dry year. This study showed that depending on the time scale occurrence, flood pulses can be a stimulating or a disturbance factor for phytoplankton development in Lake Sakadaš. Handling editor: J. Padisak     

5S rRNA fingerprints of marine bacteria, halophilic archaea and natural prokaryotic assemblages along a salinity gradient

Natural prokaryotic assemblages from two multi-pond solar salterns and pure cultures of both marine bacteria and halophilic archaea were analyzed and compared by electrophoretic analysis of 5S rRNAs. A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied. The culture-independent, PCR-free, fingerprinting analysis covered two objectives: (i) to compare natural assemblages among them and with results previously obtained through a PCR-dependent approach and (ii) to estimate the in situ relevance of those prokaryotic groups obtained with classical culture methodologies. Natural assemblages were analyzed through cluster analysis of quantitative 5S rRNA band patterns. The resulting groups were in accordance with environmental parameters (i.e., NaCl concentration) and with the clustering obtained after a PCR-dependent approach, showing the formation of three salinity-based groups of samples (<10%, 10-25% and >25% salinity). Similarities between the laboratory strains tested and dominant community members were studied by comparing 5S rRNA band patterns. The lack of match obtained after cluster analysis indicated that the prokaryotic populations relevant in the ponds below 25% salinity were neither Flavobacteria nor haloarchaeal strains belonging to the genera Halococcus, Haloarcula and Halobacterium. Members of Proteobacteria and Gram-positive bacteria were found to match bands in these samples. The 5S rRNA fingerprint from the dominant community members in the ponds above 30% salinity did not fit any of the cultured halophilic archaea studied, in agreement with earlier PCR results. This is consistent with a greater bias introduced by culture-dependent methods than by those based on PCR, especially for archaeal populations.     

PCR amplification of 16S rDNA sequences in Fe-rich sediment of coal refuse drainage

Acid drainage from coal mines or coal refuse, when treated in collection ponds, results in the formation of Fe-rich sediments. The Fe in these sediments inhibits the PCR and hinders identification of bacteria using non-culture 16S rDNA molecular methods. We describe a technique that employs EDTA to dissolve and remove Fe from these sediments, yielding DNA that can be amplified by PCR. The technique allows the identification of bacterial DNA sequences and increases our understanding of the bacterial community in Fe-rich sediments.     

PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.     

Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria

The 16S rDNA genes of an apparently pure culture of a psychrophilic and strict barophilic bacterium (WHB 46) were studied by PCR-mediated amplification and cloning into phage M13 mp18. Sequence analysis of five individual clones revealed the presence of two different 16S rDNA types. The homology value of 90% indicates that culture WHB 46 is actually composed of two closely related species (WHB 46-1 and 46-2). Both strains are members of the γ-subdivision of proteobacteria. Analysis of a sixth clone (WHB 46-1/2) leads to the conclusion that it represents a 16S rDNA hybrid molecule assembled during the PCR reaction. This hypothesis was confirmed by secondary structure analysis of the chimeric rDNA. The appearance of such hybrid molecules point to a potential risk in studies on the diversity of bacterial populations by analysis of rDNA pattern via PCR-mediated amplification because they suggest the existence of organisms that do not actually exist in the sample investigated.     

PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

The sequence of the major portion of aBacillus cycloheptanicus strain SCH^T 16S rRNA gene is reported. This sequence suggests thatB. cycloheptanicus is genetically quite distinct from traditionalBacillus strains (e.g.,B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.The EMBL accession number for the 16S rRNA sequence is X51928.     

Spatial and seasonal prokaryotic community dynamics in ponds of increasing salinity of Sfax solar saltern in Tunisia

The spatial and seasonal dynamics of the halophilic prokaryotic community was investigated in five ponds from Sfax solar saltern (Tunisia), covering a salinity gradient ranging from 20 to 36%. Fluorescence in situ hybridization indicated that, above 24% salinity, the prokaryotic community shifted from bacterial to archaeal dominance with a remarkable increase in the proportion of detected cells. Denaturing gradient gel electrophoresis (DGGE) profiles were rather similar in all the samples analyzed, except in the lowest salinity pond (around 20% salt) where several specific archaeal and bacterial phylotypes were detected. In spite of previous studies on these salterns, DGGE analysis unveiled the presence of microorganisms not previously described in these ponds, such as Archaea related to Natronomonas or bacteria related to Alkalimnicola, as well as many new sequences of Bacteroidetes. Some phylotypes, such as those related to Haloquadratum or to some Bacteroidetes, displayed a strong dependence of salinity and/or magnesium concentrations, which in the case of Haloquadratum could be related to the presence of ecotypes. Seasonal variability in the prokaryotic community composition was focused on two ponds with the lowest (20%) and the highest salinity (36%). In contrast to the crystallized pond, where comparable profiles between autumn 2007 and summer 2008 were obtained, the non-crystallized pond showed pronounced seasonal changes and a sharp succession of “species” during the year. Canonical correspondence analysis of biological and physicochemical parameters indicated that temperature was a strong factor structuring the prokaryotic community in the non-crystallizer pond, that had salinities ranging from 20 to 23.8% during the year.     

Bacterial diversity in two coastal lagoons deduced from 16S rDNA PCR amplification and partial sequencing

Abstract: Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allows the study of microbial diversity in natural ecosystems without the need for cultivation. In this study this methodology has been applied to two coastal lagoons. Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prévost lagoon (located on the French Mediterranean coast) is more eutrophic than that of the Arcachon Bay (French Atlantic coast). Analysis of partial 16S rRNA gene sequences revealed that, in both environments, a relatively large number of clones related to Cytophaga/Flexibacter/Bacteroides as well as to α-Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied, in fact for most clones maximum similarity was below 95% for the approx. 200 bases sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Bacteria were obtained from Prévost lagoon; however only five branches were represented by the data from Arcachon. These findings indicated a higher bacterial phylogenetic diversity in the Prévost lagoon.     

用PCR及RFLP方法分析支原体与植原体的同源性

将猪鼻支原体和泡桐丛枝病植原体的16S rDNA进行PCR扩增,分别得到一条1 kb左右的扩增片段。PCR扩增产物用限制性内切酶EcoRⅠ、HindⅢ、BamHⅠ、SalⅠ和SmaⅠ进行RFLP(限制性片段长度多态性)分析,发现用RFLP分析猪鼻支原体和泡桐丛枝病植原体16S rDNA序列同源性的相关系数为0.72。     

Vibrio communities along a salinity gradient within a marine saltern hypersaline environment (Saline di Tarquinia,Italy)

Vibrio species are ubiquitous in a number of different aquatic environments and promptly adapting to environmental changes due to high genome plasticity. The presence of these bacteria in marine salterns, in relation to a salinity gradient has been not investigated yet. Moreover, it is not clear if these hypersaline environments could represent a reservoir for Vibrio spp. This work investigated, through a metagenetic approach, the distribution of Vibrio (over 2 years) in different ponds along the salinity gradient within the ‘Saline di Tarquinia’ salterns, considering also the adjacent coastal waters and an isolated brine storage basin (BSB). Vibrio occurrence was higher in the sea than in the ponds and BSB, where it usually represented a rare taxon (abundance <1%). In the sea, it showed abundances in-between 1%–2.6% in 8 months out of 24. Four OTUs were assigned to the Vibrio genus; except for one that was more abundant in BSB, the others were much higher in the sea. Redundancy analysis (RDA) suggested a different distribution of the OTUs in relation to water temperature and salinity. Vibrio was found, even with low abundances, at the highest salinities also, suggesting the salterns as a possible reservoir for the bacterium.     

Analyses of microbial activity in biomass-recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products

The relationship between mixed microbial community structure and physiology when grown under substrate-limited conditions was investigated using continuous-flow bioreactors with 100% biomass recycle. Community structure was analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR and RT-PCR amplified V3 region of 16S rDNA and 16S rRNA templates, respectively. Comparisons were made of communities exposed to different types of transient conditions (e.g., long- and short-term starvation, increasing nutrients). With progressively more stringent substrate limitation over time, the specific content of community RNA declined by more than 10-fold and closely followed the decline in specific growth rate. In contrast, the DNA content was variable (up to 3-fold differences) and did not follow the same trend. Cluster analysis of the presence or absence of individual bands indicated that the fingerprints generated by the two templates were different, and community response was first observed in the rRNA fraction. However, both the rDNA and rRNA fingerprints provided a picture of temporal population dynamics. Dice similarity coefficients gave a quantitative measure of the differences and changes between the communities. In comparison, standard cultivation techniques yielded only a quarter of the phylotypes detected by DGGE, but included the most dominant population based on rRNA. Nucleotide-sequence analyses of the almost complete 16S rRNA genes of these isolates place them in the same group of organisms that is typically cultivated from environmental samples: alpha, beta, and gamma Proteobacteria and the high GC and the low GC Gram-positive divisions.     

Detection of Phytoplasma from deferent infected hosts in Jordan based on PCR amplification of the 16S rDNA sequences

Sixteen leaf samples, both healthy and Phytoplasma diseased, were collected from different plants such as grape, peach, almond, tomato, paper, squash, apple and pear in northern Jordan. Extracted DNA from diseased grape, peach, almond, tomato, paper and squash plus from infected periwinkle (Catharanthus roseus) samples were amplified with the Phytoplasma universal 16S rDNA sequences primer pairs. Extracted DNA samples from healthy and diseased apple and pear plants were not amplified with the same primer pairs. All the PCR-amplified DNA samples show a common band with size of 558 bp, indicating Phytoplasma pathogens as a disease-causative agent for grape, peach, almond, tomato, paper and squash plants. The restriction fragment length polymorphisms of Alu1 enzyme for the amplified 16S rDNA sequences shows the same DNA fragment patterns indicating no or a limited diversity among the DNA of the detected Phytoplasma pathogens.     

Simultaneous PCR amplification of two barley 5S rDNA sequence types in the same PCR reaction

Many researchers are currently using PCR technology to amplify individual members of multigene families, including 5S rDNA, for sequencing and related purposes. When members of the family differ in length, analyses would be facilitated if different units could be simultaneously and efficiently amplified. In the present paper we describe conditions that can be used to amplify simultaneously both the “long” and “short” 5S rDNA repeats found in barley (Hordeum vulgare L.).