The Role of \u3ci\u3eCellulose Synthase-like D\u3c/i\u3e Genes in Tip Growth of \u3ci\u3ePhyscomitrella patens\u3c/i\u3e

Physcomitrella patens is a non-vascular plant with a relatively small genome and is amongst the few eukaryotic organisms that have a high rate of homologous recombination. This is valuable in biological research because it allows for targeted genetic modification of the organism. In vascular plants like Arabidopsis thaliana, a model organism, Cellulose Synthase-like D (CSLD) genes have been discovered to be important in tip growth. This type of growth is observed in the pollen tubes and root hairs of these plant types. The CSLD genes in Arabidopsis were found to play a crucial role in the growth of root hairs and the production of cellulose or cellulose-like β-1,4-glucan chains in root hair tips. The CSLD genes have also been recognized to be important in pollen tube growth of vascular plants. Physcomitrella patens also contains genes similar to the vascular plant CSLDs, but their functions are not yet fully understood. Within the P. patens genome there are eight genes that make up the CSLD gene family. Additionally, the life cycle of P. patens includes a stage that consists primarily of tip growing cells. This growth stage can be optimized in order to study the role of CSLD genes in tip growth of P. patens. In an effort to further study the roles of the CSLD genes in tip growth of P. patens, we constructed a plasmid that expresses the CSLD1 protein with a green fluorescent protein (GFP) tag. This allowed us to visualize the expression of CSLD1 in living cells using fluorescence microscopy. We also constructed plasmids that were designed to remove specific CSLD genes from the genome and transformed them into wild type or CLSD1 knockout tissue of P. patens. This created single or double knockout mutants that could then be compared to the wild type for changes in the phenotypic characteristics of the plant. These findings will aid in uncovering the roles of the CSLD gene family in P. patens and may provide insight into the functions of these genes in other plants

Tiamulin-Resistant Mutants of the Thermophilic Bacterium \u3cem\u3eThermus thermophilus\u3c/em\u3e

Tiamulin is a semisynthetic pleuromutilin antibiotic that binds to the 50S ribosomal subunit A site and whose (((2-diethylamino)ethyl)thio)-acetic acid tail extends into the P site to interfere with peptide bond formation. We have isolated spontaneous tiamulin-resistant mutants of the thermophilic bacterium Thermus thermophilus, containing either single amino acid substitutions in ribosomal protein uL3 or single base substitutions in the peptidyltransferase active site of 23S rRNA. These mutations are consistent with those found in other organisms and are in close proximity to the crystallographically determined tiamulin binding site. We also conducted a cross-resistance analysis of nine other single-base substitutions in or near the peptidyltransferase active site, previously selected for resistance to structurally unrelated antibiotics. While some of the base substitutions in 23S rRNA are positioned to directly affect tiamulin-ribosome contacts, others are some distance from the tiamulin binding site, indicating an indirect mechanism of resistance. Similarly, amino acid substitutions in uL3 are predicted to act indirectly by destabilizing rRNA conformation in the active site. We interpret these observations in light of the available ribosome X-ray crystal structures. These results provide a more comprehensive profile of tiamulin resistance caused by mutations in the bacterial ribosome

A Survey of Spontaneous Antibiotic-Resistant Mutants of the Halophilic, Thermophilic Bacterium Rhodothermus marinus

Rhodothermus marinus is a halophilic extreme thermophile, with potential as a model organism for studies of the structural basis of antibiotic resistance. In order to facilitate genetic studies of this organism, we have surveyed the antibiotic sensitivity spectrum of R. marinus and identified spontaneous antibiotic-resistant mutants. R. marinus is naturally insensitive to aminoglycosides, aminocylitols and tuberactinomycins that target the 30S ribosomal subunit, but is sensitive to all 50S ribosomal subunit-targeting antibiotics examined, including macrolides, lincosamides, streptogramin B, chloramphenicol, and thiostrepton. It is also sensitive to kirromycin and fusidic acid, which target protein synthesis factors. It is sensitive to rifampicin (RNA polymerase inhibitor) and to the fluoroquinolones ofloxacin and ciprofloxacin (DNA gyrase inhibitors), but insensitive to nalidixic acid. Drug-resistant mutants were identified using rifampicin, thiostrepton, erythromycin, spiramycin, tylosin, lincomycin, and chloramphenicol. The majority of these were found to have mutations that are similar or identical to those previously found in other species, while several novel mutations were identified. This study provides potential selectable markers for genetic manipulations and demonstrates the feasibility of using R. marinus as a model system for studies of ribosome and RNA polymerase structure, function, and evolution

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants

The secondary cell walls of tracheary elements and fibers are rich in cellulose microfibrils that are helically oriented and laterally aggregated. Support cells within the leaf midribs of mosses deposit cellulose-rich secondary cell walls, but their biosynthesis and microfibril organization have not been examined. Although the Cellulose Synthase (CESA) gene families of mosses and seed plants diversified independently, CESA knockout analysis in the moss Physcomitrella patens revealed parallels with Arabidopsis (Arabidopsis thaliana) in CESA functional specialization, with roles for both subfunctionalization and neofunctionalization. The similarities include regulatory uncoupling of the CESAs that synthesize primary and secondary cell walls, a requirement for two or more functionally distinct CESA isoforms for secondary cell wall synthesis, interchangeability of some primary and secondary CESAs, and some CESA redundancy. The cellulose-deficient midribs of ppcesa3/8 knockouts provided negative controls for the structural characterization of stereid secondary cell walls in wild type P. patens. Sum frequency generation spectra collected from midribs were consistent with cellulose microfibril aggregation, and polarization microscopy revealed helical microfibril orientation only in wild type leaves. Thus, stereid secondary walls are structurally distinct from primary cell walls, and they share structural characteristics with the secondary walls of tracheary elements and fibers. We propose a mechanism for the convergent evolution of secondary walls in which the deposition of aggregated and helically oriented microfibrils is coupled to rapid and highly localized cellulose synthesis enabled by regulatory uncoupling from primary wall synthesis

Tiamulin-Resistant Mutants of the Thermophilic Bacterium Thermus thermophilus

Tiamulin is a semisynthetic pleuromutilin antibiotic that binds to the 50S ribosomal subunit A site and whose (((2-diethylamino)ethyl)thio)-acetic acid tail extends into the P site to interfere with peptide bond formation. We have isolated spontaneous tiamulin-resistant mutants of the thermophilic bacterium Thermus thermophilus, containing either single amino acid substitutions in ribosomal protein uL3 or single base substitutions in the peptidyltransferase active site of 23S rRNA. These mutations are consistent with those found in other organisms and are in close proximity to the crystallographically determined tiamulin binding site. We also conducted a cross-resistance analysis of nine other single-base substitutions in or near the peptidyltransferase active site, previously selected for resistance to structurally unrelated antibiotics. While some of the base substitutions in 23S rRNA are positioned to directly affect tiamulin-ribosome contacts, others are some distance from the tiamulin binding site, indicating an indirect mechanism of resistance. Similarly, amino acid substitutions in uL3 are predicted to act indirectly by destabilizing rRNA conformation in the active site. We interpret these observations in light of the available ribosome X-ray crystal structures. These results provide a more comprehensive profile of tiamulin resistance caused by mutations in the bacterial ribosome