Expression of amino acid transporter genes in developmental stages and adult tissues of Antarctic echinoderms

Epithelial cells in the body wall of adult and developmental stages of marine invertebrates absorb dissolved organic material directly from seawater. Despite over a century of study, little is known about the molecular biological mechanisms responsible for this transport process. Previous studies on embryonic and larval Antarctic echinoderms show that amino acid uptake could provide an important supplement of metabolic substrates. In the present study, partial cDNA sequences of 11 putative amino acid transporter genes were isolated from six species of Antarctic echinoderms including the Antarctic sea stars Acodontaster hodgsoni, Diplasterias brucei, Odontaster meridionalis, Odontaster validus, and Perknaster fuscus, and the Antarctic sea urchin Sterechinus neumayeri. Conserved domains of cDNA-deduced amino acid sequences characterized these genes as being members of a family of amino acid transporters (solute carrier family 6). Expression of these genes was detected throughout embryonic and larval development of two species that have contrasting developmental modes (A. hodgsoni: lecithotrophic; O. meridionalis: planktotrophic). In all six species studied, the expression of amino acid transporter genes was detected in tube feet and digestive organs of adult animals, demonstrating that members of a single amino acid transporter gene family are expressed during the entire life history of a marine invertebrate. The identification of these genes is an important step toward developing a mechanistic understanding of amino acid transport capacities in Antarctic marine invertebrates.     

Free amino acids in some sponges

Most invertebrates, particularly those of marine origin, have relatively high concentrations of free amino acids which are considered an important constituent of their osmoregulatory mechanisms [1]. Very little information is available on the free amino acid distribution in Porifera [2,3]. Common amino acids in some sponges were recognised by paper chromatography by Inskip and Cassidy [4] and Ackermann et al. [5,6] included a few sponges in their survey of the occurence of nitrogen compounds in marine invertebrates. More recently Bergquist and Hartman [7] surveyed semiquantitatively the distribution of free amino acids in several sponges. In the present paper we report on the amino acid composition of 12 species of sponges belonging to the class Demospongiae as a part of a study on the metabolites of Porifera [8]. Fresh sponges were extracted with aqueous ethanol. The organic solvent was removed and the aqueous solution, after removal of the ether soluble compounds, was separated into cationic, anionic and neutral fractions by ion-exchange chromatography. The cation fraction was analysed for amino acids using an automatic amino acid analyser. The results, which are presented in Table 1, show that all species of sponges examined have a similar composition in common amino acids. Glycine almost always appears as the dominant protein amino acid, followed by high concentrations of alanine and glutamic acid, whereas relatively lower concentrations of basic amino acids are present. In Axinella cannabina, Chondrosia reniformis, Chondrilla nucula, Cliona viridis and Hymeniacidon sanguinea, glycine represents more than 77% of the total amino acids. The high percentage of free glycine (90.4%) in Chondrosia reniformis is noteworthy. The anionic and the neutral fractions were examined for sulfur-containing amino acids using PC. Taurine (Table 2) was detected in all the Porifera examined; this is in agreement with previous observations [5–7]. N-Methyltaurine was identified in some of the species examined, whereas neither N,N-dimethyltaurine nor N,N,N-trimethyltaurine were found.     

Bacicyclin,a new antibacterial cyclic hexapeptide from Bacillus sp. strain BC028 isolated from Mytilus edulis

A new cyclic hexapeptide, cyclo-(Gly-Leu-Val-IIe-Ala-Phe), named bacicyclin (1), was isolated from a marine Bacillus sp. strain associated with Mytilus edulis. The sequences of the amino acid building blocks of the cyclic peptide and its structure were determined by 1D- and 2D-NMR techniques. Marfey’s analysis showed that the amino acid building blocks had L-configuration in all cases except for alanine and phenylalanine, which had D-configuration. Bacicyclin (1) exhibited antibacterial activity against the clinically relevant strains Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentration values of 8 and 12?µM, respectively. These results demonstrate the potential of marine bacteria as a promising source for the discovery of new antibiotics.     

Isolation and structure of a novel peptide inhibitor of HIV-1 integrase from marine polychaetes

A homogeneous peptide with a mass of 683 Da which inhibits HIV-1 integrase with IC₅₀ 3 × 10^?5 M was separated from aqueous extracts of a marine worm Eunicidae sp. by multistage chromatography purification. The Asp-Leu-Hse-His-Ala-Gln structure was proposed for this peptide according to amino acid analysis, automated amino acid Edman sequences, and TLC with witness homoserine and MS/MS fragmentation. The proposed structure is the first example of a natural peptide containing an amino acid homoserine residue.     

Diversity of radA Genes from Cultured and Uncultured Archaea: Comparative Analysis of Putative RadA Proteins and Their Use as a Phylogenetic Marker

Archaea-specific radA primers were used with PCR to amplify fragments of radA genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15 radA genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species of Crenarcheota. The two most deeply branching archaeal radA genes found had some amino acid deletion and insertion patterns characteristic of bacterial recA genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.     

Ultraviolet radiation negatively affects growth and food quality of the pelagic diatom Skeletonema costatum

There are now compelling observations of the ecological impacts of global change on marine ecosystems. A predicted 10% increase in UVB doses at the Earth's surface has been shown to impair indirectly the reproductive output of marine copepods. Wild females of Calanus helgolandicus fed with UVB-irradiated diatoms produced fewer eggs and unhealthy offspring exhibiting a large proportion of high lethal naupliar deformities. By reproducing the same irradiative stress on the pelagic diatom Skeletonema costatum, we looked for potential modifications of the algal properties (cell size, biovolume, chlorophyll content) and biochemical characteristics (fatty acid and amino acid contents). Our results confirmed that the metabolism of S. costatum is adversely affected by enhanced UVB exposure: cell growth was reduced and the biochemical characteristics, and thus the algal nutritional quality, were significantly altered. UVB irradiation reduced cell division, leading to cell elongation and thus increasing the biovolume. Meanwhile, the amino acid and fatty acid contents did not increase concomitantly to the cell enlargement and were thus diluted in the cell. Because of this dilution, the irradiated S. costatum represents a poorer diet for its potential consumers. Moreover, UVB dramatically affects the relative contribution of certain essential fatty acids such as eicosapentanoic acid (EPA), which are essential for the development of marine invertebrates.     

Development of Defined, Minimal, and Complete Media for the Growth of Hyphomicrobium neptunium

A complete synthetic medium containing 15 amino acids, a minimal synthetic medium (GAMS) containing 4 amino acids, and a supplemented minimal medium (GAMS + calcium pantothenate) have been developed for the cultivation of Hyphomicrobium neptunium ATCC 15444. Depending on the complexity of the synthetic media, generation times were approximately 2 to 3 times longer, and maximum cell densities were 0.3 to 0.9 log₁₀ lower than in ZoBell marine broth 2216. The fates of ¹⁴C-labeled amino acids in GAMS were monitored. Results suggested that H. neptunium was auxotrophic for methionine, utilized glutamic acid as a primary energy source, and readily anabolized and catabolized serine and aspartic acid. Individual amino acid concentrations above 125 mM induced prolonged lag periods, whereas only methionine was not growth limiting at a concentration as low as 2 mM.     

Ecophysiological and Trophic Implications of Light-Stimulated Amino Acid Utilization in Marine Picoplankton

By using microautoradiography, light-stimulated utilization of dissolved amino acids for natural marine phytoplankton assemblages was demonstrated. The <2-μm-size (diameter) picoplankton, known to be a dominant fraction of marine primary production, revealed a widespread capability for this process. Autofluorescent (chlorophyll a-containing) picoplankton and some larger phytoplankton from diverse oceanic locations, as well as isolates of the representative cyanobacterial picoplankton Synechococcus spp. (WH7803, WH8101), showed light-stimulated incorporation of amino acids at trace concentrations. Dark-mediated amino acid utilization was dominated by nonfluorescent bacterial populations. Among autofluorescent picoplankton, light-stimulated exceeded dark-mediated amino acid incorporation by 5 to 75%; light-stimulated amino acid incorporation was only partially blocked by the photosystem II inhibitor 3(3,4-dichloro-phenyl)-1,1-dimethy-lurea (2 × 10^-5 M), suggesting a photoheterotrophic incorporation mechanism. Parallel light versus dark incubations with glucose and mannitol indicated a lack of light-stimulated utilization of these nonnitrogenous compounds. Since marine primary production is frequently nitrogen limited, light-mediated auxotrophic utilization of amino acids and possibly other dissolved organic nitrogen (DON) constituents may represent exploitation of the relatively large DON pool in the face of dissolved inorganic nitrogen depletion. This process (i) increases the efficiency of DON retention at the base of oceanic food webs and (ii) may in part be responsible for relatively high rates of picoplankton production under conditions of chronic dissolved inorganic nitrogen limitation. Picoplanktonic recycling of organic matter via this process has important ramifications with respect to trophic transfer via the “microbial loop.”     

Overexpression and Characterization of an Iron Storage and DNA-Binding Dps Protein from Trichodesmium erythraeum

Although the role of iron in marine productivity has received a great deal of attention, no iron storage protein has been isolated from a marine microorganism previously. We describe an Fe-binding protein belonging to the Dps family (DNA binding protein from starved cells) in the N₂-fixing marine cyanobacterium Trichodesmium erythraeum. A dps gene encoding a protein with significant levels of identity to members of the Dps family was identified in the genome of T. erythraeum. This gene codes for a putative Dps_{T. erythraeurm} protein (Dps_tery) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dps_tery, and we found that Dps_tery, like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps_tery binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps_tery indicated that it has an iron core that resembles that of horse spleen ferritin.     

Gas-liquid chromatography and enzymatic determination of alanopine and strombine in tissues of marine invertebrates

Gas-liquid chromatography (GLC) and enzymatic assays were developed for quantitating the imino acids, alanopine and strombine, alternate products of anaerobic glycolysis (replacing lactate) in the tissues of many marine invertebrates. For GLC analysis, d-strombine (2-methyliminodiacetic acid) and meso-alanopine (2,2′-iminodipropionic acid) were chromatographeo as N-trifluoroacetyl isobutyl esters. Modifications of techniques used for GLC analysis of amino acids were required to overcome steric hindrance in the acylation reaction caused by the presence of imino, rather than amino, groups. Both imino acids were separated from each other and from all amino acids by GLC. Detection limit of the technique was 0.05 μg imino acid. Enzymatic determination of imino acids made use of the alanopine-specific alanopine dehydrogenase (ADH) purified from the periwinkle, Littorina littorea, and the strombine/alanopine utilizing strombine dehydrogenase (SDH) from the clam, Mercenaria mercenaria, with assay conditions: 300 mm hydrazine buffer, pH 9.0, 5 mm NAD, and 0.3 unit ADH or 1.0 unit SDH. Enzymatic determinations of mixtures of alanopine and strombine in tissue samples required a dual analysis using both enzymes. Production of alanopine and strombine during anoxic stress in two species of marine molluscs was quantitated.     

Wound Healing Properties of Collagen from the Bone of Two Marine Fishes

Type I collagen was extracted from the bone of two marine fishes; Magalaspis cordyla and Otolithes ruber and its efficacy in wound healing was studied. The yields of acid soluble (30.5 and 27.6%) and pepsin soluble (45.1 and 48.6%) collagen were respectively as per their dry weight basis. Extracted protein was characterized as type I collagen basing on SDS PAGE, UV?CVis, FTIR spectrometer and amino acid composition. The characterized collagen was cross-linked with glutaraldehyde and found to possess three dimensional pores and acted efficiently in healing the excision wounds made on Wistar rats. Bone is generally considered as waste and discarded in many processing plant. But, the current study proved that it has excellent medicinal importance and reveals a possible way to convert this waste into a medicinally important source.     

Occurrence of beta-Aminoglutaric Acid in Marine Bacteria

beta-Aminoglutaric acid, a nonprotein amino acid isomer of glutamic acid, was found in the free amino acid pool of a marine bacterium, Alteromonas luteoviolacea. It was also found in a mixed culture of fermenting bacteria enriched from an anoxic marine sediment.     

Cloning and functional analysis of putative malonyl-CoA:acyl-carrier protein transacylase gene from the docosahexaenoic acid-producer Schizochytrium sp. TIO1101

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), which transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), is a key enzyme in fatty acid biosynthesis. Schizochytrium sp. TIO1101 is a marine protist with high levels of docosahexaenoic acid accumulation. In this study, the putative fabD gene coding MCAT was isolated from Schizochytrium sp. TIO1101. The Schizochytrium MCAT gene (ScTIOfabD) contained an 1176 bp open reading frame encoding a protein of 391 amino acids. The ScTIOfabD gene exhibited high novelty in nucleotide and amino acid sequence. The highest amino acid identity was only 35 % between ScTIOMCAT and the reported MCATs. Further studies demonstrated that ScTIOMCAT could bind malonyl-CoA directly and transfer malonyl group from malonyl-CoA to the ACP domain in vitro. Phylogenetic analysis suggested that ScTIOMCAT was relative close to MCATs of yeast strains. Overexpression of ScTIOMCAT in Saccharomyces cereviseae significantly increased the MCAT activity, without negative effects on the growth rate of the host strain. In addition, ScTIOMCAT generated 16.8 and 62 % increase in biomass and fatty acid accumulation, respectively, and did not alter the profile of fatty acid. Our results indicated that the novel MCAT gene from Schizochytrium sp. TIO1101 was crucial for fatty acid synthesis and had potential applications for genetic modifications of oil-producing species.     

Initial Evidence for Adaptive Selection on the NADH Subunit Two of Freshwater Dolphins by Analyses of Mitochondrial Genomes

A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the ‘river dolphins’, early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (d_N/d_S>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three ‘river dolphins’ (Families Pontoporidae, Lipotidae and Inidae), once in the riverine Sotalia fluviatilis (but not in its marine sister taxa), once in the riverine Orcaella brevirostris from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the E. coli structure and Sotalia fluviatilis model. In E. coli this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of cetaceans.     

Community-Level Analysis of psbA Gene Sequences and Irgarol Tolerance in Marine Periphyton

This study analyzes psbA gene sequences, predicted D1 protein sequences, species relative abundance, and pollution-induced community tolerance in marine periphyton communities exposed to the antifouling compound Irgarol 1051. The mechanism of action of Irgarol is the inhibition of photosynthetic electron transport at photosystem II by binding to the D1 protein. The metagenome of the communities was used to produce clone libraries containing fragments of the psbA gene encoding the D1 protein. Community tolerance was quantified with a short-term test for the inhibition of photosynthesis. The communities were established in a continuous flow of natural seawater through microcosms with or without added Irgarol. The selection pressure from Irgarol resulted in an altered species composition and an inducted community tolerance to Irgarol. Moreover, there was a very high diversity in the psbA gene sequences in the periphyton, and the composition of psbA and D1 fragments within the communities was dramatically altered by increased Irgarol exposure. Even though tolerance to this type of compound in land plants often depends on a single amino acid substitution (Ser₂₆₄→Gly) in the D1 protein, this was not the case for marine periphyton species. Instead, the tolerance mechanism likely involves increased degradation of D1. When we compared sequences from low and high Irgarol exposure, differences in nonconserved amino acids were found only in the so-called PEST region of D1, which is involved in regulating its degradation. Our results suggest that environmental contamination with Irgarol has led to selection for high-turnover D1 proteins in marine periphyton communities at the west coast of Sweden.     

Motualevic acids and analogs: Synthesis and antimicrobial structure–activity relationships

Synthesis of the marine natural products motualevic acids A, E, and analogs in which modifications have been made to the ω-brominated lipid (E)-14,14-dibromotetra-deca-2,13-dienoic acid or amino acid unit are reported, together with antimicrobial activities against Staphylococcus aureus, methicillin-resistant S. aureus, Enterococcus faecium, and vancomycin-resistant Enterococcus.     

The metabolism of organic acids by a marine pennate diatom

Cocconeis diminuta, a marine benthic diatom, metabolizes acetate and lactate-¹⁴C. In the light, the major product was lipid, whereas in the dark, CO₂ was the major product. Analysis of proteins synthesized in the presence of acetate or lactate showed that radioactivity was incorporated predominantly into the glutamate family of amino acids and those amino acids related directly to the substrate. Light and dark assimilation of substrate was inhibited slightly by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea and 2,4-dinitrophenol. 3-(3′,4′-Dichlorophenyl)-1,1-dimethylurea caused a pattern of metabolism of acetate in the light characteristic of that which occurs in the dark. Monofluoroacetic acid inhibited assimilation considerably in the dark, but less in the light. The level of enzymes of the tricarboxylic acid cycle and NADH-oxidase were found to be about the same as those in other autotrophs. The metabolism of acetate and lactate is discussed in relation to the autotrophic mode of nutrition of Cocconeis diminuta.     

Amino acid receptor sites in the facial taste system of the sea catfish Arius felis

1. The amino acid sensitivity and specificity of the facial taste system of the marine catfish, Arius felis, is characterized electrophysiologically.
2. The facial taste system of Arius felis responded to all 28 amino acids tested, but was highly sensitive to only a few. In general, acidic amino acids and neutral amino acids with short side chains were more effective than imino, basic and neutral amino acids with long side chains.
3. A reciprocal cross-adaptation protocol used to characterize the receptor sites identified at least some relatively independent receptor sites for L-arginine, L-histidine, L-proline, L-alanine, glycine, D-alanine and L-glutamate.
4. Of the 7 amino acids that were indicated to have relatively independent receptor sites, the median electrophysiological threshold for L-alanine, the most stimulatory, and L-proline, the least stimulatory compounds, were 10 nM and 10,000 nM, respectively. The integrated facial taste response did not saturate at test amino acid concentrations up to 10 mM.
5. The generalized depression in responsiveness to test stimuli observed during amino acid adaptation is proposed to be a result of the co-distribution of sensitivity at the level of single taste cells rather than high cross-reactivity of the respective amino acid receptor sites for the test stimuli.

     

Cloning of the Novel Gene Encoding β-Agarase C from a Marine Bacterium, Vibrio sp. Strain PO-303, and Characterization of the Gene Product

The β-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. β-Agarase C was identified as the first β-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.     

Acides aminés de microsporidies parasites de crustacés décapodes marins

The amino acids of four species of Microsporidia parasitizing crustaceans were investigated: three species — Thelohania maenadis, Ormieresia carcini and Ameson pulvis — are parasites of Carcinus mediterraneus; Inodosporus sp. is a parasite of Palaemon serratus. Seventeen protein amino acids were identified of which aspartic acid, glutamic acid and lysine were quantitatively the most important, being quantitatively similar in all four parasites. The relative amounts of the next most abundant amino acids were found to vary and might serve as a systematic criterion at the genus level. For example, phenylalanine is predominant in Ormieresia and leucine in Inodosporus: serine in Thelohania and glycine in Ameson are also predominant, but to a lesser extent. The free amino acids composition shows little qualitative variation among the four genera, but quantitative differences are found in the composition of Microsporidia parasitizing the same host species; this may reflect variations in the amino acid metabolism of the parasite. The urea cycle in Ormeresia is most remarkable in this connection. The free amino acid level in the parasites was generally found to be in inverse proportion to the level in the host; the amino acids which are found to exist at high levels in the parasites correspond to essential amino acids of the Crustacea. Metabolic and adaptative relations are discussed.