Rosemary A. Dorrington

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Production of D-amino acids from D,L-5-substituted hydantoins by an Agrobacterium tumefaciens strain and isolation of a mutant with inducer-independent expression of hydantoin-hydrolysing activity

Conversion of D,L-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine by Agrobacterium tumefaciens RU-OR involved a racemase, an hydantoinase and an unusual D-selective N-carbamylamino acid amidohydrolase which was active at alkaline pH and was not inhibited by N-carbamyl-L-amino acids. Enzyme activity was induced by growth in media containing 2-thiouracil. A mutant strain (RU-ORL5) was isolated, which expressed both the hydantoinase and N-carbamylamino acid amidohydrolase enzymes in the absence of inducer.

A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing L-amino acids

Abstract Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N -carbamyl amino acid. In certain bacteria where an N -carbamyl amino acid amidohydrolase (NCAAH) is present, the N -carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO 2 . In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding l -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has l -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of l -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.

Dimerization of the yeast eukaryotic translation initiation factor 5A requires hypusine and is RNA dependent.

Post‐translational modification of the highly conserved K51 residue of the Saccharomyces cerevisiae eukaryotic translation initiation factor 5A (eIF5A) to form hypusine, is essential for its many functions including the binding of specific mRNAs. We characterized hypusinated yeast eIF5A by size‐exclusion chromatography and native PAGE, showing that the protein exists as a homodimer. A K51R mutant, which was not functional in vivo eluted as a monomer and inhibition of hypusination abolished dimerization. Furthermore, treatment of dimeric eIF5A with RNase A resulted in disruption of the dimer, leading us to conclude that RNA binding is also required for dimerization of eIF5A. We present a model of dimerization, based on the Neurospora crassa structural analogue, HEX‐1.

Production of enantiomerically pure amino acids: characterisation of South African hydantoinases and hydantoinase-producing bacteria

Abstract Chiral amino acid derivatives can be synthesised by the biocatalytic conversion of substituted hydantoins using microbial enzymes or resting cells: a hydantoinase performs the ring-opening cleavage of the hydantoin to produce an N -carbamylamino acid and N -carbamylamidohydrolase then converts this intermediate to the amino acid, ammonia and CO 2 . The hydantoinases from four locally isolated bacterial strains are currently being characterised in terms of conditions for optimal enzyme activity assay, and to demonstrate the effects of pH, temperature, metal ions, protease inhibitors, surfactants, and anti-oxidants on hydantoinase activity in crude extracts. Typically, pH 8, 50°C, and 0.3 mM Cu 2+ were found to be optimal. Disruption of cells using a detergent or membrane freeze-fracture resulted in increased activities, suggesting that the hydantoinase enzymes may be membrane bound. It was also found that three Pseudomonas strains exhibited higher activities than the Agrobacterium strain, in terms of hydantoin conversion, with % conversion of hydantoins to N -carbamylamino acids from 66% to 2%. Comparisons of hydantoinase and amidohydrolase activity in resting cells and in cell extracts also show marked differences in activity profile for different strains, e.g., strain RU-KM1 exhibited hydantoinase activity in whole cells and cell extracts, but amidohydrolase activity only in cell extracts, while RU-OR showed higher amidohydrolase activity than hydantoinase activity.

Functional domain mapping and subcellular distribution of Dal82p in Saccharomyces cerevisiae.

Previous studies have shown that (i) Dal81p and Dal82p are required for allophanate-induced gene expression inSaccharomyces cerevisiae; (ii) the cis-acting element mediating the induced transcriptional response to allophanate is a dodecanucleotide, UIS ALL; and (iii) Dal82p binds specifically to UIS ALL. Here we show that Dal82p is localized to the nucleus and parallels movement of the DNA through the cell cycle. Deletion analysis of DAL82identified and localized three functional domains. Electrophoretic mobility shift assays identified a peptide (consisting of Dal82p amino acids 1–85) that is sufficient to bind a DNA fragment containingUIS ALL. LexA-tethering experiments demonstrated that Dal82p is capable of mediating transcriptional activation. The activation domain consists of two parts: (i) an absolutely required core region (amino acids 66–99) and (ii) less well defined regions flanking residues 66–99 that are required for full wild-type levels of activation. The Dal82p C terminus contains a predicted coiled-coil motif that down-regulates Dal82p-mediated transcriptional activation.

Genome organization and translation products of Providence virus: insight into a unique tetravirus

Providence virus (PrV) is a member of the family Tetraviridae, a family of small, positive-sense, ssRNA viruses that exclusively infect lepidopteran insects. PrV is the only known tetravirus that replicates in tissue culture. We have analysed the genome and characterized the viral translation products, showing that PrV has a monopartite genome encoding three ORFs: (i) p130, unique to PrV and of unknown function; (ii) p104, which contains a read-through stop signal, producing an N-terminal product of 40 kDa (p40) and (iii) the capsid protein precursor (p81). There are three 2A-like processing sequences: one at the N terminus of p130 (PrV-2A₁) and two more (PrV-2A₂ and PrV-2A₃) at the N terminus of p81. Metabolic radiolabelling identified viral translation products corresponding to all three ORFs in persistently infected cells and showed that the read-through stop in p104 and PrV-2A₃ in p81 are functional in vivo and these results were confirmed by in vitro translation experiments. The RNA-dependent RNA polymerase domain of the PrV replicase is phylogenetically most closely related to members of the families Tombusviridae and Umbraviridae rather than to members of the family Tetraviridae. The unique genome organization, translational control systems and phylogenetic relationship with the replicases of (+ve) plant viruses lead us to propose that PrV represents a novel family of small insect RNA viruses, distinct from current members of the family Tetraviridae.

Mutational analysis of the hydantoin hydrolysis pathway in Pseudomonas putida RU-KM3S

The biocatalytic conversion of 5-mono-substituted hydantoins to the corresponding d-amino acids or l-amino acids involves first the hydrolysis of hydantoin to a N-carbamoylamino acid by an hydantoinase or dihydropyrimidinase, followed by the conversion of the N-carbamoylamino acid to the amino acid by N-carbamylamino acid amidohydrolase (N-carbamoylase). Pseudomonas putida strain RU-KM3S, with high levels of hydantoin-hydrolysing activity, has been shown to exhibit non-stereoselective hydantoinase and l-selective N-carbamoylase activity. This study focused on identifying the hydantoinase and N-carbamoylase-encoding genes in this strain, using transposon mutagenesis and selection for altered growth phenotypes on minimal medium with hydantoin as a nitrogen source. Insertional inactivation of two genes, dhp and bup, encoding a dihydropyrimidinase and β-ureidopropionase, respectively, resulted in loss of hydantoinase and N-carbamoylase activity, indicating that these gene products were responsible for hydantoin hydrolysis in this strain. dhp and bup are linked to an open reading frame encoding a putative transport protein, which probably shares a promoter with bup. Two mutant strains were isolated with increased levels of dihydropyrimidinase but not β-ureidopropionase activity. Transposon mutants in which key elements of the nitrogen regulatory pathway were inactivated were unable to utilize hydantoin or uracil as a nitrogen source. However, these mutations had no effect on either the dihydropyrimidinase or β-ureidopropionase activity. Disruption of the gene encoding dihydrolipoamide succinyltransferase resulted in a significant reduction in the activity of both enzymes, suggesting a role for carbon catabolite repression in the regulation of hydantoin hydrolysis in P. putida RU-KM3S cells.

Over-production of hydantoinase and N-carbamoylamino acid amidohydrolase enzymes by regulatory mutants of Agrobacterium tumefaciens

Abstract. While the hydantoin-hydrolysing enzymes from Agrobacterium strains are used as biocatalysts in the commercial production of D-p-hydroxyphenylglycine, they are now mostly produced in heterologous hosts such as Escherichia coli. This is due to the fact that the activity of these enzymes in the native strains is tightly regulated by growth conditions. Hydantoinase and N-carbamoylamino acid amidohydrolase (NCAAH) activities are induced when cells are grown in the presence of hydantoin or an hydantoin analogue, and in complete medium, enzyme activity can be detected only in early stationary growth phase. In this study, the ability of Agrobacterium tumefaciens RU-OR cells to produce active enzymes was found to be dependent upon the choice of nitrogen source and the presence of inducer, 2-thiouracil, in the growth medium. Growth with (NH4)2SO4 as the nitrogen source repressed the production of both enzymes (nitrogen repression) and also resulted in a rapid, but reversible loss of hydantoinase activity in induced cells (ammonia shock). Mutant strains with inducer-independent production of the enzymes and/or altered response to nitrogen control were isolated. Of greatest importance for industrial application was strain RU-ORPN1F9, in which hydantoinase and NCAAH enzyme activity was inducer-independent and no longer sensitive to nitrogen repression or ammonia shock. Such mutants offer the potential for native enzyme production levels equivalent to those achieved by current heterologous expression systems.

Keeping it in the family: Coevolution of latrunculid sponges and their dominant bacterial symbionts

The Latrunculiidae are a family of cold water sponges known for their production of bioactive pyrroloiminoquinone alkaloids. Previously it was shown that the bacterial community associated with a Tsitsikamma sponge species comprises unusual bacterial taxa and is dominated by a novel Betaproteobacterium. Here, we have characterized the bacterial communities associated with six latrunculid species representing three genera (Tsitsikamma, Cyclacanthia, and Latrunculia) as well as a Mycale species, collected from Algoa Bay on the South African southeast coast. The bacterial communities of all seven sponge species were dominated by a single Betaproteobacterium operational taxonomic unit (OTU0.03), while a second OTU0.03 was dominant in the Mycale sp. The Betaproteobacteria OTUs from the different latrunculid sponges are closely related and their phylogenetic relationship follows that of their hosts. We propose that the latrunculid Betaproteobacteria OTUs are members of a specialized group of sponge symbionts that may have coevolved with their hosts. A single dominant Spirochaetae OTU0.03 was present in the Tsitsikamma and Cyclacanthia sponge species, but absent from the Latrunculia and Mycale sponges. This study sheds new light on the interactions between latrunculid sponges and their bacterial communities and may point to the potential involvement of dominant symbionts in the biosynthesis of the bioactive secondary metabolites.