Impacts of Groundwater Pumping on Subterranean Microbial Communities in a Deep Aquifer Associated with an Accretionary Prism.

Accretionary prisms are composed mainly of ancient marine sediment scraped from the subducting oceanic plate at convergent plate boundaries. Anoxic groundwater is stored in deep aquifers associated with accretionary prisms and can be collected via deep wells. We investigated how such groundwater pumping affects the microbial community in a deep aquifer. Groundwater samples were collected from a deep well drilled down to 1500 m every six months (five times in total) after completion of deep well construction and the start of groundwater pumping. Next-generation sequencing and clone-library analyses of 16S rRNA genes were used to describe the subterranean microbial communities in the samples. The archaea: the prokaryote ratio in groundwater increased significantly from 1 to 7% (0 and 7 months after initiating groundwater pumping) to 59 to 72% (13, 19, and 26 months after initiating groundwater pumping), and dominant prokaryotes changed from fermentative bacteria to sulfate-reducing archaea. The optimal growth temperature of the sulfate-reducing archaea, estimated based on the guanine-plus-cytosine contents of their 16S rRNA genes, was 48-52 °C, which agreed well with the groundwater temperature at the deep-well outflow. Our results indicated that, in deep aquifers, groundwater pumping enhances groundwater flow, and the supply of sulfate-containing seawater activates the metabolism of thermophilic sulfate-reducing archaea.

Temperature-dependent expression of different guanine-plus-cytosine content 16S rRNA genes in Haloarcula strains of the class Halobacteria.

Haloarcula strains, which are halophilic archaea, harbour two to three copies of 16S rRNA genes (rrsA, rrsB and rrsC) in their genomes. While rrsB and rrsC (rrsBC) show almost identical sequences, rrsA shows 4-6% sequence difference and 1-3% guanine-plus-cytosine content (PGC) difference compared to rrsBC. Based on the strong correlation between the PGC of 16S rRNA genes and the growth temperatures of the prokaryotes, we hypothesised that high-PGCrrsA and low-PGCrrsBC are expressed at high and low temperatures, respectively. To verify the hypothesis, we performed sequence analyses and expression surveys of each 16S rRNA gene in eight Haloarcula strains. The secondary structure prediction of the 16S rRNA via computer simulation showed that the structural stability of 16S rRNAs transcribed from rrsA was higher than that of 16S rRNAs transcribed from rrsBC. We measured expression levels of rrsA and rrsBC under various temperature conditions by reverse-transcriptase quantitative PCR. The expression ratio of high-PGCrrsA to low-PGCrrsBC increased with cultivation temperatures in seven of eight Haloarcula strains. Our results suggest that the transcription of high-PGCrrsA and low-PGCrrsBC may be regulated in response to environmental temperature, and that 16S rRNAs transcribed from high-PGCrrsA function under high temperature conditions close to the maximum growth temperature.

Expression and Function of Different Guanine-Plus-Cytosine Content 16S rRNA Genes in Haloarcula hispanica at Different Temperatures.

The halophilic archaeon Haloarcula hispanica harbors three ribosomal RNA (rRNA) operons (rrnA, rrnB, and rrnC) that contain the 16S rRNA genes rrsA, rrsB, and rrsC, respectively. Although rrsB and rrsC (rrsBC) have almost identical sequences, the rrsA and rrsBC sequences differ by 5.4%, and they differ by 2.5% with respect to guanine-plus-cytosine content (PGC). The strong correlation between the typical growth temperatures of archaea and PGC of their 16S rRNA genes suggests that H. hispanica may harbor different 16S rRNA genes having different PGC to maintain rapid growth in a wide range of temperatures. We therefore performed reverse transcription-coupled quantitative PCR to assess expression levels of rrsA (PGC, 58.9%) and rrsBC (PGC, 56.4-56.5%) at various temperatures. The expression ratio of rrsA to rrsBC increased with culture temperature. Mutants with complete deletions of one or two of the three rRNA operons were constructed and their growth rates at different temperatures compared to that of the wild-type. The growth characteristics of the rRNA operon single-mutant strains were indistinguishable from the wild-type. The rRNA operon double-mutant strains maintained the same temperature range as wild-type but displayed reduced growth rates. In particular, the double-mutant strains grew much slower than wild-type at low temperature related to minimum growth temperature of the wild-type. On the other hand, at physiologically high temperatures the wild-type and the double-mutant strain which harbors only rrnA with high-PGCrrsA grew significantly faster than the double-mutant strain which harbors only rrnC with low-PGCrrsC. These findings suggest the importance of 16S rRNAs transcribed from rrsA with high-PGC in maintaining rapid growth of this halophilic archaeon at raised growth temperatures.

Characterization of Bradyrhizobium Strains Isolated from Different Varieties of Soybean with 16SrDNA RFLP from Agricultural Land of Madhya Pradesh, India.

Madhya Pradesh is the major soybean contributor in India. The taxonomy of nitrogen fixing bacteria forming symbiotic associations with leguminous plants has been deeply changed in recent years. The use of very sensitive and accurate molecular methods has enabled the detection of large rhizobial diversity. Molecular biotyping and characterization the Bradyrhizobium, isolates from eleven varieties of soybean from agricultural field of Sehore district of Madhya Pradesh is done using 16S rDNA typing. Bradyrhizobia were identified genetically by determining the %Guanine plus Cytosine content of the whole genome, followed by 16S rDNA-RFLP analysis % Guanine plus Cytosine content of all the Bradyrhizobium isolates reflects similarity at generic level among all Bradyrhizobial isolates. Restriction Fragment Length Polymorphism (RFLP) further showed a considerable level of genetic diversity among the Bradyrhizobial isolates. PCR-RFLP of 16S rDNA supported existence of two divergent groups among indigenous Bradyrhizobial isolates, at similarity level of 66, and 75 and 74% of similarity within the group. The technique used was helpful in characterizing Bradyrhizobium isolates to be used as inoculants for improving productivity of agricultural land of Madhya Pradesh (India).