Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel.

Newly described phylogenetic lineages within the domain Archaea have recently been found to be significant components of marine picoplankton assemblages. To better understand the ecology of these microorganisms, we investigated the relative abundance, distribution, and phylogenetic composition of Archaea in the Santa Barbara Channel. Significant amounts of archaeal rRNA and rDNA (genes coding for rRNA) were detected in all samples analyzed. The relative abundance of archaeal rRNA as measured by quantitative oligonucleotide hybridization experiments was low in surface waters but reached higher values (20 to 30% of prokaryotic rRNA) at depths below 100 m. Probes were developed for the two major groups of marine Archaea detected. rRNA originating from the euryarchaeal group (group II) was most abundant in surface waters, whereas rRNA from the crenarchaeal group (group I) dominated at depth. Clone libraries of PCR-amplified archaeal rRNA genes were constructed with samples from 0 and 200 m deep. Screening of libraries by hybridization with specific oligonucleotide probes, as well as subsequent sequencing of the cloned genes, indicated that virtually all archaeal rDNA clones recovered belonged to one of the two groups. The recovery of cloned rDNA sequence types in depth profiles exhibited the same trends as were observed in quantitative rRNA hybridization experiments. One representative of each of 18 distinct restriction fragment length polymorphism types was partially sequenced. Recovered sequences spanned most of the previously reported phylogenetic diversity detected in planktonic crenarchaeal and euryarchaeal groups. Several rDNA sequences appeared to be harbored in archaeal types which are widely distributed in marine coastal waters. In total, data suggest that marine planktonic crenarchaea and euryarchaea of temperate coastal habitats thrive in different zones of the water column. The relative rRNA abundance of the crenarchaeal group suggests that its members constitute a significant fraction of the prokaryotic biomass in subsurface coastal waters.     

Structure of a Transducing Mycobacteriophage

Electron micrographs of the transducing phage I3 for Mycobacterium smegmatis strain SN2 revealed a phage with a contractile tail and a head with isometric symmetry and visible capsomeres.     

Characterization of T-Even Bacteriophage Substructures: II. Tail Plates

Tail plates obtained from T4D amber mutants were examined with respect to sedimentation behavior, subunit molecular weights, amino acid composition, isoelectric points, and morphology. Intact plates had an S_20,w of 77S from pH 5 to 9. The only conformational change noted was that below pH 5 tail plates readily dimerized yielding vis-à-vis dimers with an S_20,w of 124S. Dissociated plates consisted of three major proteins with molecular weights of 53 K ± 5, 31 K ± 3, and 17 K ± 2 daltons. The amino acid analyses indicated that plates had a composition distinct from fibers and tubes and were relatively rich in tryptophan. Degradation studies with dimethyl sulfoxide (DMSO) indicated that tail plates had a unique biological structure. After treatment with DMSO, and to some extent without DMSO, or from lysates of defective mutants, tetrad structures were observed in the electron microscope. These structures had an amino acid content and relative amounts of types of subunits similar but not identical to intact plates. It was proposed that plates were composed of nine such tetrads giving rise to a structure with six- and threefold symmetry.     

Functional expression of human and Arabidopsis protein phosphatase 2A in Saccharomyces cerevisiae and isolation of dominant-defective mutants.

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic subunit and two distinct regulatory subunits, A and B. The primary sequence of the catalytic (C) subunit is highly conserved in evolution, and its function has been shown to be essential in yeast, Drosophila and mice. In many eukaryotes, the C subunit is encoded by at least two nearly identical genes, impeding conventional loss-of-function genetic analysis. We report here the development of a functional complementation assay in S. cerevisiae that has allowed us to isolate dominant-defective alleles of human and Arabidopsis C subunit genes. Wild-type human and Arabidopsis C subunit genes can complement the lethal phenotype of S. cerevisiae PP2A-C mutations. Site-directed mutagenesis was used to create two distinct, catalytically impaired C subunit mutants of the human and Arabidopsis genes. In both cases, expression of the mutant subunit in yeast prevented growth, even in the presence of functional C subunit proteins. This dominant growth defect is consistent with a dominant-interfering mode of action. Thus, we have shown that S. cerevisiae provides a rapid system for the functional analysis of heterologous PP2A genes, and that two mutations that abrogate phosphatase activity exhibit dominant-defective phenotypes in S. cerevisiae.     

PINOID Kinase Regulates Root Gravitropism through Modulation of PIN2-Dependent Basipetal Auxin Transport in Arabidopsis

Reversible protein phosphorylation is a key regulatory mechanism governing polar auxin transport. We characterized the auxin transport and gravitropic phenotypes of the pinoid-9 (pid-9) mutant of Arabidopsis (Arabidopsis thaliana) and tested the hypothesis that phosphorylation mediated by PID kinase and dephosphorylation regulated by the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1) protein might antagonistically regulate root auxin transport and gravity response. Basipetal indole-3-acetic acid transport and gravitropism are reduced in pid-9 seedlings, while acropetal transport and lateral root development are unchanged. Treatment of wild-type seedlings with the protein kinase inhibitor staurosporine phenocopies the reduced auxin transport and gravity response of pid-9, while pid-9 is resistant to inhibition by staurosporine. Staurosporine and the phosphatase inhibitor, cantharidin, delay the asymmetric expression of DR5∷revGFP (green fluorescent protein) at the root tip after gravistimulation. Gravity response defects of rcn1 and pid-9 are partially rescued by treatment with staurosporine and cantharidin, respectively. The pid-9 rcn1 double mutant has a more rapid gravitropic response than rcn1. These data are consistent with a reciprocal regulation of gravitropism by RCN1 and PID. Furthermore, the effect of staurosporine is lost in pinformed2 (pin2). Our data suggest that reduced PID kinase function inhibits gravitropism and basipetal indole-3-acetic acid transport. However, in contrast to PID overexpression studies, we observed wild-type asymmetric membrane distribution of the PIN2 protein in both pid-9 and wild-type root tips, although PIN2 accumulates in endomembrane structures in pid-9 roots. Similarly, staurosporine-treated plants expressing a PIN2∷GFP fusion exhibit endomembrane accumulation of PIN2∷GFP, but no changes in membrane asymmetries were detected. Our data suggest that PID plays a limited role in root development; loss of PID activity alters auxin transport and gravitropism without causing an obvious change in cellular polarity.A variety of important growth and developmental processes, including gravity response, embryo and vascular development, and the branching of roots and shoots, are controlled by the directional and regulated transport of auxin in higher plants. Reversible protein phosphorylation is an important regulatory strategy that may modulate auxin transport and dependent processes such as root gravitropism, perhaps through action of the PINOID (PID) kinase (for review, see DeLong et al., 2002; Galvan-Ampudia and Offringa, 2007). PID is an AGC family Ser/Thr kinase (Christensen et al., 2000) and belongs to an AGC kinase clade containing WAG1, WAG2, AGC3-4, and D6PK/AGC1-1 (Santner and Watson, 2006; Galvan-Ampudia and Offringa, 2007; Zourelidou et al., 2009). PID activity has been demonstrated in vitro and in vivo (Christensen et al., 2000; Michniewicz et al., 2007), and several pid mutant alleles exhibit altered auxin transport in the inflorescence and a floral development defect resembling that of auxin transport mutants (Bennett et al., 1995). Overexpression of the PID gene results in profound alterations in root development and responses to auxin transport inhibitors, reduced gravitropism and auxin accumulation at the root tip (Christensen et al., 2000; Benjamins et al., 2001; Michniewicz et al., 2007), as well as enhanced indole-3-acetic acid (IAA) efflux in tobacco (Nicotiana tabacum) cell cultures (Lee and Cho, 2006) and altered PINFORMED1 (PIN1), PIN2, and PIN4 localization patterns (Friml et al., 2004; Michniewicz et al., 2007), consistent with PID being a positive regulator of IAA efflux. However, the effects of pid loss-of-function mutations on auxin transport activities and gravitropic responses in roots have not yet been reported (Robert and Offringa, 2008).In contrast, auxin transport and gravitropism defects of a mutant with reduced protein phosphatase activity have been characterized in detail. The roots curl in naphthylphthalamic acid1 (rcn1) mutation, which ablates the function of a protein phosphatase 2A regulatory subunit, causes reduced PP2A activity in vivo and in vitro (Deruère et al., 1999). Roots and hypocotyls of rcn1 seedlings have elevated basipetal auxin transport (Deruère et al., 1999; Rashotte et al., 2001; Muday et al., 2006), and rcn1 roots exhibit a significant delay in gravitropism, consistent with altered auxin transport (Rashotte et al., 2001; Shin et al., 2005). These data indicate that PP2A is a negative regulator of basipetal transport and suggest that if PID-dependent phosphorylation regulates root auxin transport and gravitropism, then it may act in opposition to PP2A-dependent dephosphorylation.In roots, auxin transport is complex, with distinct sets of influx and efflux carriers that define tissue-specific and opposing directional polarities (for review, see Leyser, 2006). IAA moves acropetally, from the shoot toward the root apex, through the central cylinder (Tsurumi and Ohwaki, 1978), and basipetally, from the root apex toward the base, through the outer layer of cells (for review, see Muday and DeLong, 2001). When plants are reoriented relative to the gravity vector, auxin becomes asymmetrically distributed across the root tip, as a result of a process termed lateral auxin transport (for review, see Muday and Rahman, 2008). Several carriers that mediate root basipetal IAA transport have been clearly defined and include the influx carrier AUXIN-INSENSITIVE1 (AUX1; Marchant et al., 1999; Swarup et al., 2004; Yang et al., 2006) and efflux carriers of two classes, PIN2 (Chen et al., 1998; Müller et al., 1998; Rashotte et al., 2000) and ATP-BINDING CASSETTE TYPE B TRANSPORTER4/MULTIDRUG-RESISTANT4/P-GLYCOPROTEIN4 (ABCB4/MDR4/PGP4; Geisler et al., 2005; Terasaka et al., 2005; Lewis et al., 2007). Lateral transport at the root tip may be mediated by PIN3, an efflux carrier with a gravity-dependent localization pattern (Friml et al., 2002; Harrison and Masson, 2007).Gravitropic curvature of Arabidopsis (Arabidopsis thaliana) roots requires changes in IAA transport at the root tip (for review, see Muday and Rahman, 2008). Auxin transport inhibitors (Rashotte et al., 2000) and mutations in genes encoding basipetal transporters, including aux1 (Bennett et al., 1996), pin2/agr1 (Chen et al., 1998; Müller et al., 1998), and abcb4/mdr4/pgp4 (Lin and Wang, 2005; Lewis et al., 2007), alter gravitropism. Auxin-inducible reporters exhibit asymmetric expression across the root tip prior to differential growth, and this asymmetry is abolished by treatment with auxin transport inhibitors that prevent gravitropic curvature (Rashotte et al., 2001; Ottenschläger et al., 2003). Additionally, the pin3 mutant exhibits slightly reduced rates of gravitropic curvature (Harrison and Masson, 2007), and PIN3 is expressed in the columella cells, which are the site of gravity perception (Blancaflor et al., 1998; Friml et al., 2002). The PIN3 protein relocates to membranes on the lower side of columella cells after gravitropic reorientation, consistent with a role in facilitating asymmetric IAA transport at the root tip (Friml et al., 2002; Harrison and Masson, 2007).The available data suggest a model in which PID and RCN1 antagonistically regulate basipetal transport and gravitropic response in root tips (Fig. 1). In this model, the regions with the highest IAA concentrations in the epidermal and cortical cell layers are indicated by shading, and the arrows indicate the direction and relative amounts of basipetal auxin transport. Our previous work suggests that elevated basipetal IAA transport in rcn1 roots impairs gravitropic response, presumably due to the inability of roots either to form or to perceive a lateral auxin gradient in the context of a stronger polar IAA transport stream (Rashotte et al., 2001). Enhanced basipetal transport may increase the initial auxin concentration along the upper side of the root, impeding the establishment or perception of a gradient in rcn1 and cantharidin-treated wild-type roots (Fig. 1, right). Based on the published pid inflorescence transport data (Bennett et al., 1995), we hypothesize that pid seedling roots and staurosporine-treated wild-type roots have reduced basipetal auxin transport (Fig. 1, left). Upon reorientation of roots relative to the gravity vector, the reduced basipetal IAA transport in pid may lead to slower establishment of an auxin gradient across the root. This model then predicts that cantharidin treatment of pid-9 or staurosporine treatment of rcn1 seedlings would enhance or restore gravitropism in these mutants. Similarly, a double mutant might be expected to exhibit a corrected gravitropic response relative to the single mutants.Open in a separate windowFigure 1.Auxin transport defects in pid-9 and rcn1 mutants alter auxin redistribution after reorientation relative to the gravity vector. This model predicts that differences in basipetal auxin transport activities of wild-type, pid-9, and rcn1 roots will affect the formation of lateral auxin gradients. The shaded area in each root represents the region of highest IAA concentration in epidermal and cortical cells, with darker shading in the central columella cells, believed to be the auxin maxima. The direction and amount of basipetal IAA transport are indicated by arrows. The region of differential growth during gravitropic bending is indicated by the shaded rectangle. If auxin transport is reduced (as shown in the pid-9 mutant or in staurosporine-treated seedlings), this would lead to a slower formation of an auxin gradient in root tips. The rcn1 mutation (or treatment with cantharidin) has already been shown to lead to increased basipetal transport and a reduced rate of gravitropic bending, consistent with altered formation or perception of an auxin gradient. The antagonistic effects of kinase and phosphatase inhibition are predicted to lead to normal gravity responses in the pid-9 rcn1 double mutant as well as in pid-9 and rcn1 single mutants treated with the “reciprocal” inhibitor.The experiments described here were designed to test this model by examining gravitropism and root basipetal IAA transport in pid and staurosporine-treated seedlings. We investigated the regulation of gravity response by PID kinase and RCN1-dependent PP2A activities and observed antagonistic interactions between the rcn1 and pid-9 loss-of-function phenotypes that are consistent with reciprocal kinase/phosphatase regulation. We found that loss of kinase activity in the pid mutant and in staurosporine-treated wild-type plants inhibits basipetal auxin transport and the dependent physiological process of root gravitropism. Our results suggest that staurosporine acts to regulate these processes through inhibition of PID kinase and that PID effects are PIN2 dependent. In both wild-type and pid-9 roots, we observed polar membrane distribution of the PIN2 protein; unlike wild-type roots, though, pid-9 roots exhibited modest accumulation of PIN2 in endomembrane structures. Similarly, we detected asymmetric distribution and endomembrane accumulation of PIN2∷GFP in staurosporine-treated roots. Our data suggest that PID plays a limited role in root development; loss of PID activity alters PIN2 trafficking, auxin transport, and gravitropism without causing an obvious loss of cellular polarity. Together, these experiments provide insight into phosphorylation-mediated control of the gravity response and auxin transport in Arabidopsis roots.     

The importance of indirect cues for white-browed sparrow-weaver (Plocepasser mahali) risk assessment

Both direct cues that provide information about the actual presence of a predator and indirect environmental cues that provide information about the probability of encountering a predator may be used by animals assessing predation risk, but relatively few studies manipulate both simultaneously to study their relative importance. We conducted two experiments to study the foraging decisions of white-browed sparrow-weavers (Plocepasser mahali). The first experiment manipulated both direct and indirect cues in a feeding array by simultaneously placing feeding stations at different distances from humans (to manipulate direct risk) and from protective cover (to manipulate indirect risk). Weaver foraging was influenced more by indirect risk than by direct risk. The second experiment aimed to determine if weaver’s indirect risk assessment was sensitive to variation in benefits. We set two feeding stations at different distances from cover but the same distance from the human observers and systematically increased the amount of food at the station farther from cover. Weavers far from cover initially foraged at higher rates than those close to cover, but the addition of food reduced the foraging rate. Together, our results illustrate that weaver foraging decisions are sensitive to variation in risk and that indirect cues are relatively more important than direct cues.     

The ecological consequences of environmentally induced phenotypic changes

Population dynamics and species persistence are often mediated by species traits. Yet many important traits, like body size, can be set by resource availability and predation risk. Environmentally induced changes in resource levels or predation risk may thus have downstream ecological consequences. Here, we assess whether quantity and type of resources affect the phenotype, the population dynamics, and the susceptibility to predation of a mixotrophic protist through experiments and a model. We show that cell shape, but not size, changes with resource levels and type, and is linked to carrying capacity, thus affecting population dynamics. Also, these changes lead to differential susceptibility to predation, with direct consequences for predator‐prey dynamics. We describe important links between environmental changes, traits, population dynamics and ecological interactions, that underscore the need to further understand how trait‐mediated interactions may respond to environmental shifts in resource levels in an increasingly changing world.     

Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype

Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%–86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size.     

Triplex formation by a psoralen-conjugated oligodeoxyribonucleotide containing the base analog 8-oxo-adenine.

Oligodeoxyribonucleotides containing thymidine and 8-oxo-2'-deoxyadenosine can form pyr.pur.pyr type triplexes with double-stranded DNA. Unlike triplexes whose third strands contain thymidine and deoxycytidine, the stability of these triplexes is independent of pH. We have prepared d-ps-TAAATAAATTTTTAT-L [I(A)], where A is 8-oxo-2'-deoxyadenosine, ps is 4'-hydroxymethyl-4,5',8- trimethylpsoralen and L is a 6-amino-2-(hydroxymethyl)hexyl linker. The oligomer is designed to interact with a homopurine sequence in the promoter region of the human gene coding for the 92 kDa form of collagenase type IV. Oligomer I(A) and oligomer I(C), which contains 2'-deoxycytidine in place of 8-oxo-2'-deoxycytidine, both form stable triplexes at pH 6.2, but only I(A) forms a stable triplex with a model duplex DNA target at pH 7.5, as determined by UV melting experiments. Triplex formation is stabilized by the presence of the psoralen group. Upon irradiation both I(A) and I(C) form photoadducts with the DNA target at pH 6.2, but only I(A) forms a photoadduct at pH 7.5. In these photoreactions oligomer I(A) appears to selectively form a photoadduct with a C in the purine-rich strand of the duplex target. Although a T residue is present in the pyrimidine-rich strand of the target at the duplex/triplex junction, essentially no adduct formation takes place with this strand, nor is interstrand cross-linking observed. The extent of photoadduct formation decreases with increasing temperature, behavior which is consistent with the UV melting curve of the triplex. A tetramethylrhodamine derivative of I(A) was prepared and found to cross-link less extensively than I(A) itself. Oligomer I(A) is completely resistant to hydrolysis when incubated for 24h in the presence of 10% fetal bovine serum at 37 degree C, although it is hydrolyzed by S1 nuclease. The properties of oligomer I(A) suggest that 8-oxo- containing oligomers may find utility as antigene oligonucleotide reagents.