Studies of hypersaline ecosystems often yield novel organisms and contribute to our understanding of extreme environments. Soils and sediments from La Sal del Rey, a previously uncharacterized, hypersaline lake located in southern Texas, USA, were surveyed to characterize the structure and diversity of their microbial communities. Samples were collected along a transect that spanned vegetated uplands, exposed lakebed sediments, and water-logged locations, capturing a wide range of environments and physical and chemical gradients. Community quantitative PCR (qPCR) was used in combination with tag-encoded pyrosequencing, 16S rRNA gene cloning, and Sanger sequencing to characterize the lake's soil and sediment microbial communities. Further, we used multivariate statistics to identify the relationships shared between sequence diversity and heterogeneity in the soil environment. The overall microbial communities were surprisingly diverse, harboring a wide variety of taxa, and sharing significant correlations with site water content, phosphorus and total organic carbon concentrations, and pH. Some individual populations, especially of Archaea, also correlated with sodium concentration and electrical conductivity salinity. Across the transect, Bacteria were numerically dominant relative to Archaea, and among them, three phyla-the Proteobacteria, Bacteroidetes, and Firmicutes-accounted for the majority of taxa detected. Although these taxa were detected with similar abundances to those described in other hypersaline ecosystems, the greater depth of sequencing achieved here resulted in the detection of taxa not described previously in hypersaline sediments. The results of this study provide new information regarding a previously uncharacterized ecosystem and show the value of highthroughput sequencing in the study of complex ecosystems.

Burkholderia cenocepacia strain K56-2, a representative of the Burkholderia cepacia complex, is part of the epidemic and clinically problematic ET12 lineage. The strain produced plant tissue watersoaking (ptw) on onion tissue, which is a plant disease-associated trait. Using plasposon mutagenesis, mutants in the ptw phenotype were generated. The translated sequence of a disrupted gene (ptwD4) from a ptw-negative mutant showed homology to VirD4-like proteins. Analysis of the region proximal to the transfer gene homolog identified a gene cluster located on the 92-kb resident plasmid that showed homology to type IV secretion systems. The role of ptwD4, ptwC, ptwB4, and ptwB10 in the expression of ptw activity was determined by conducting site-directed mutagenesis. The ptw phenotype was not expressed by K56-2 derivatives with a disruption in ptwD4, ptwB4, or ptwB10 but was observed in a derivative with a disruption in ptwC. Complementation of ptw-negative K56-2 derivatives in trans resulted in complete restoration of the ptw phenotype. In addition, analysis of culture supernatants revealed that the putative ptw effector(s) was a secreted, heat-stable protein(s) that caused plasmolysis of plant protoplasts. A second chromosomally encoded type IV secretion system with complete homology to the VirB-VirD system was identified in K56-2. Site-directed mutagenesis of key secretory genes in the VirB-VirD system did not affect expression of the ptw phenotype. Our findings indicate that in strain K56-2, the plasmid-encoded Ptw type IV secretion system is responsible for the secretion of a plant cytotoxic protein(s).

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