Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches

Diverse ketosynthase (KS) genes were retrieved from the microbial community associated with the Great Barrier Reef sponge Pseudoceratina clavata. Bacterial isolation and metagenomic approaches were employed. Phylogenetic analysis of 16S rRNA of culturable sponge-associated bacterial communities comprised eight groups over four phyla. Ten KS domains were amplified from four genera of isolates and phylogenetics demonstrated that these KS domains were located in three clusters (actinobacterial, cyanobacterial and trans-AT type). Metagenomic DNA of the sponge microbial community was extracted to explore community KS genes by two approaches: direct amplification of KS domains and construction of fosmid libraries for KS domain screening. Five KS domains were retrieved from polymerase chain reaction (PCR) amplification using sponge metagenome DNA as template and five fosmid clones containing KS domains found using multiplex PCR screening. Analysis of selected polyketide synthase (PKS) from one fosmid showed that the PKS consists of two modules. Open reading frames located up- and downstream of the PKS displayed similarity with membrane synthesis-related proteins such as cardiolipin synthase. Metagenome approaches did not detect KS domains found in sponge isolates. All KS domains from both metagenome approaches formed a single cluster with KS domains originating from metagenomes derived from other sponge species from other geographical regions.

Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP

Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic sub unit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs front that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required mgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator.

Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing

Deterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype, but 90% identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate Defluvicoccus vanus. Fluorescence in situ hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic-aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6.6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33% of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55% of Bacteria) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified Defluvicoccus-related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.

The five families of sucrose-phosphate synthase genes in Saccharum spp. are differentially expressed in leaves and stem

Sucrose-phosphate synthase (SPS) is a key enzyme in the pathway of sucrose synthesis. Five different gene families encoding SPS have been reported in the Poaceae [Castleden CK, Aoki N, Gillespie VJ, MacRae EA, Quick WP, Buchner P, Foyer CH, Furbank RT, Lunn JE (2004) Evolution and function of the sucrose-phosphate synthase gene families in wheat and othergrasses. PlantPhysiology 135, 1753-1764]. Expression of the five families in leaf and stem tissues of Saccharum spp. at different stages of development was determined by quantitative real-time PCR. The type B and C families of SPS genes were predominantly expressed in both immature and mature leaves, whereas the two subfamilies making up the type D family were expressed at similar levels in all tissues examined. In the type A family, expression was lowest in leaves and increased from the meristem region down to internode 7 of the stem.

The identification and characterisation of alleles of sucrose phosphate synthase gene family III in sugarcane

Little is known about the extent of allelic diversity of genes in the complex polyploid, sugarcane. Using sucrose phosphate synthase (SPS) Gene (SPS) Family III as an example, we have amplified and sequenced a 400 nt region from this gene from two sugarcane lines that are parents of a mapping population. Ten single nucleotide polymorphisms (SNPs) were identified within the 400 nt region of which seven were present in both lines. In the elite commercial cultivar Q165(A), 10 sequence haplotypes were identified, with four haplotypes recovered at 9% or greater frequency. Based on SNP presence, two clusters of haplotypes were observed. In IJ76-514, a Saccharum officinarum accession, 8 haplotypes were identified with 4 haplotypes recovered at 13% or greater frequency. Again, two clusters of haplotypes were observed. The results suggest that there may be two SPS Gene Family III genes per genome in sugarcane, each with different numbers of different alleles. This suggestion is supported by sequencing results in an elite parental sorghum line, 403463-2-1, in which 4 haplotypes, corresponding to two broad types, were also identified. Primers were designed to the sugarcane SNPs and screened over bulked DNA from high and low Sucrose-containing progeny from a cross between Q165(A) and IJ76-514. The SNP frequency did not vary in the two bulked DNA samples, suggesting that these SNPs from this SPS gene family are not associated with variation in sucrose content. Using an ecotilling approach, two of the SPS Gene Family III haplotypes were mapped to two different linkage groups in homology group 1 in Q165(A). Both haplotypes mapped near QTLs for increased sucrose content but were not themselves associated with any sugar-related trait.

Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.]

Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.