Selective degradation of phage RNAs by the Csm6 ribonuclease provides robust type III CRISPR immunity in Streptococcus thermophilus.

Type III CRISPR immune systems bind viral or plasmid RNA transcripts and activate Csm3/Cmr4 and Cas10 nucleases to uniquely cleave both invader RNA and DNA, respectively. Additionally, type III effector complexes generate cyclic oligoadenylate (cOA) signaling molecules to activate trans-acting, auxiliary Csm6/Csx1 ribonucleases, previously proposed to be non-specific in their in vivo RNA cleavage preference. Despite extensive in vitro studies, the nuclease requirements of type III systems in their native contexts remain poorly understood. Here we systematically investigated the in vivo roles for immunity of each of the three Streptococcus thermophilus (Sth) type III-A Cas nucleases and cOA signaling by challenging nuclease defective mutant strains with plasmid and phage infections. Our results reveal that RNA cleavage by Csm6 is both sufficient and essential for maintaining wild-type levels of immunity. Importantly, Csm6 RNase activity leads to immunity against even high levels of phage challenge without causing host cell dormancy or death. Transcriptomic analyses during phage infection indicated Csm6-mediated and crRNA-directed preferential cleavage of phage transcripts. Our findings highlight the critical role of Csm6 RNase activity in type III immunity and demonstrate specificity for invader RNA transcripts by Csm6 to ensure host cell survival upon phage infection.

Type III-A CRISPR systems as a versatile gene knockdown technology.

CRISPR-Cas systems are functionally diverse prokaryotic antiviral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis, and S. thermophilus) were each expressed on a single plasmid in E. coli, and the efficiency and specificity of gene knockdown was assessed by northern blot and transcriptomic analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and noncoding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as specific and effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.

A tale of two plasticities: leaf hydraulic conductances and related traits diverge for two tropical epiphytes from contrasting light environments.

We compared the effects of different light environments on leaf hydraulic conductance (Kleaf ) for two congeneric epiphytes, the tank bromeliads Guzmania lingulata (L.) Mez and Guzmania monostachia (L.) Rusby ex Mez. They occur sympatrically at the study site, although G. monostachia is both wider ranging and typically found in higher light. We collected plants from two levels of irradiance and measured Kleaf as well as related morphological and anatomical traits. Leaf xylem conductance (Kxy ) was estimated from tracheid dimensions, and leaf conductance outside the xylem (Kox ) was derived from a leaky cable model. For G. monostachia, but not for G. lingulata, Kleaf and Kxy were significantly higher in high light conditions. Under both light conditions, Kxy and Kox were co-limiting for the two species, and all conductances were in the low range for angiosperms. With respect to hydraulic conductances and a number of related anatomical traits, G. monostachia exhibited greater plasticity than did G. lingulata, which responded to high light chiefly by reducing leaf size. The positive plasticity of leaf hydraulic traits in varying light environments in G. monostachia contrasted with negative plasticity in leaf size for G. lingulata, suggesting that G. monostachia may be better able to respond to forest conditions that are likely to be warmer and more disturbed in the future.

Aquatic microbial habitats within a neotropical rainforest: bromeliads and pH-associated trends in bacterial diversity and composition.

Tank-forming bromeliads, suspended in the rainforest canopy, possess foliage arranged in compact rosettes capable of long-term retention of rainwater. This large and unique aquatic habitat is inhabited by microorganisms involved in the important decomposition of impounded material. Moreover, these communities are likely influenced by environmental factors such as pH, oxygen, and light. Bacterial community composition and diversity was determined for the tanks of several bromeliad species (Aechmea and Werauhia) from northern Costa Rica, which span a range of parameters, including tank morphology and pH. These were compared with a nearby forest soil sample, an artificial tank (amber bottle), and a commercially available species (Aechmea). Bacterial community diversity, as measured by 16S rRNA analysis and tRFLP, showed a significant positive correlation with tank pH. A majority of 16S rRNA bacterial phylotypes found in association with acidic bromeliad tanks of pH < 5.1 were affiliated with the Alphaproteobacteria, Acidobacteria, Planctomycetes, and Bacteroidetes, and were similar to those found in acidic peat bogs, yet distinct from the underlying soil community. In contrast, bromeliads with tank pH > 5.3, including the commercial bromeliad with the highest pH (6.7), were dominated by Betaproteobacteria, Firmicutes, and Bacteroidetes. To empirically determine the effect of pH on bacterial community, the tank pH of a specimen of Aechmea was depressed, in the field, from 6.5 to 4.5, for 62 days. The resulting community changed predictably with decreased abundance of Betaproteobacteria and Firmicutes and a concomitant increase in Alphaproteobacteria and Acidobacteria. Collectively, these results suggest that bromeliad tanks provide important habitats for a diverse microbial community, distinct from the surrounding environment, which are influenced greatly by acid-base conditions. Additionally, total organic carbon (∼46%) and nitrogen (∼2%) of bromeliad-impounded sediment was elevated relative to soil and gene surveys confirmed the presence of both chitinases and nitrogenases, suggesting that bromeliad tanks may provide important habitats for microbes involved in the biological cycling of carbon and nitrogen in tropical forests.

Leaf hydraulic conductance for a tank bromeliad: axial and radial pathways for moving and conserving water.

Epiphytic plants in the Bromeliaceae known as tank bromeliads essentially lack stems and absorptive roots and instead take up water from reservoirs formed by their overlapping leaf bases. For such plants, leaf hydraulic conductance is plant hydraulic conductance. Their simple strap-shaped leaves and parallel venation make them suitable for modeling leaf hydraulic conductance based on vasculature and other anatomical and morphological traits. Plants of the tank bromeliad Guzmania lingulata were investigated in a lowland tropical forest in Costa Rica and a shaded glasshouse in Los Angeles, CA, USA. Stomatal conductance to water vapor and leaf anatomical variables related to hydraulic conductance were measured for both groups. Tracheid diameters and numbers of vascular bundles (veins) were used with the Hagen-Poiseuille equation to calculate axial hydraulic conductance. Measurements of leaf hydraulic conductance using the evaporative flux method were also made for glasshouse plants. Values for axial conductance and leaf hydraulic conductance were used in a model based on leaky cable theory to estimate the conductance of the radial pathway from the vein to the leaf surface and to assess the relative contributions of both axial and radial pathways. In keeping with low stomatal conductance, low stomatal density, low vein density, and narrow tracheid diameters, leaf hydraulic conductance for G. lingulata was quite low in comparison with most other angiosperms. Using the predicted axial conductance in the leaky cable model, the radial resistance across the leaf mesophyll was predicted to predominate; lower, more realistic values of axial conductance resulted in predicted radial resistances that were closer to axial resistance in their impact on total leaf resistance. Tracer dyes suggested that water uptake through the tank region of the leaf was not limiting. Both dye movement and the leaky cable model indicated that the leaf blade of G. lingulata was structurally and hydraulically well-suited to conserve water.

Bromeliad catchments as habitats for methanogenesis in tropical rainforest canopies.

Tropical epiphytic plants within the family Bromeliaceae are unusual in that they possess foliage capable of retaining water and impounded material. This creates an acidic (pH 3.5-6.5) and anaerobic (<1 ppm O(2)) environment suspended in the canopy. Results from a Costa Rican rainforest show that most bromeliads (n = 75/86) greater than ~20 cm in plant height or ~4-5 cm tank depth, showed presence of methanogens within the lower anoxic horizon of the tank. Archaea were dominated by methanogens (77-90% of recovered ribotypes) and community structure, although variable, was generally comprised of a single type, closely related to either hydrogenotrophic Methanoregula or Methanocella, a specific clade of aceticlastic Methanosaeta, or Methanosarcina. Juvenile bromeliads, or those species, such as Guzmania, with shallow tanks, generally did not possess methanogens, as assayed by polymerase chain reaction specific for methanogen 16S rRNA genes, nor did artificial catchments (~100 ml volume), in place 6-12 months prior to sample collection. Methanogens were not detected in soil (n = 20), except in one case, in which the dominant ribotype was different from nearby bromeliads. Recovery of methyl coenzyme M reductase genes supported the occurrence of hydrogenotrophic and aceticlastic methanogens within bromeliad tanks, as well as the trend, via QPCR analysis of mcrA, of increased methanogenic capacity with increased plant height. Methane production rates of up to 300 nmol CH(4) ml tank water(-1) day(-1) were measured in microcosm experiments. These results suggest that bromeliad-associated archaeal communities may play an important role in the cycling of carbon in neotropical forests.