Characterization of the l-arabinofuranose-specific GafABCD ABC transporter essential for l-arabinose-dependent growth of the lignocellulose-degrading bacterium Shewanella sp. ANA-3.

Microbes that have evolved to live on lignocellulosic biomass face unique challenges in the effective and efficient use of this material as food. The bacterium Shewanella sp. ANA-3 has the potential to utilize arabinan and arabinoxylan, and uptake of the monosaccharide, l-arabinose, derived from these polymers, is known to be mediated by a single ABC transporter. We demonstrate that the substrate binding protein of this system, GafASw, binds specifically to l-arabinofuranose, which is the rare furanose form of l-arabinose found in lignocellulosic biomass. The structure of GafASw was resolved to 1.7 Å and comparison to Escherichia coli YtfQ (GafAEc) revealed binding site adaptations that confer specificity for furanose over pyranose forms of monosaccharides, while selecting arabinose over another related monosaccharide, galactose. The discovery of a bacterium with a natural predilection for a sugar found abundantly in certain lignocellulosic materials suggests an intimate connection in the enzymatic release and uptake of the sugar, perhaps to prevent other microbes scavenging this nutrient before it mutarotates to l-arabinopyranose. This biological discovery also provides a clear route to engineer more efficient utilization of plant biomass components in industrial biotechnology.

Disorder drives cooperative folding in a multidomain protein.

Many human proteins contain intrinsically disordered regions, and disorder in these proteins can be fundamental to their function-for example, facilitating transient but specific binding, promoting allostery, or allowing efficient posttranslational modification. SasG, a multidomain protein implicated in host colonization and biofilm formation in Staphylococcus aureus, provides another example of how disorder can play an important role. Approximately one-half of the domains in the extracellular repetitive region of SasG are intrinsically unfolded in isolation, but these E domains fold in the context of their neighboring folded G5 domains. We have previously shown that the intrinsic disorder of the E domains mediates long-range cooperativity between nonneighboring G5 domains, allowing SasG to form a long, rod-like, mechanically strong structure. Here, we show that the disorder of the E domains coupled with the remarkable stability of the interdomain interface result in cooperative folding kinetics across long distances. Formation of a small structural nucleus at one end of the molecule results in rapid structure formation over a distance of 10 nm, which is likely to be important for the maintenance of the structural integrity of SasG. Moreover, if this normal folding nucleus is disrupted by mutation, the interdomain interface is sufficiently stable to drive the folding of adjacent E and G5 domains along a parallel folding pathway, thus maintaining cooperative folding.

Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding.

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates.

Association between amino acid turnover and chromosome aneuploidy during human preimplantation embryo development in vitro.

This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during development in vitro. One hundred and eighty-eight fresh and cryopreserved embryos from 59 patients (33.9 +/- 0.6 years) were cultured for 2-5 days. The turnover of 18 amino acids was measured in spent media by high-performance liquid chromatography. Embryos were either fixed for interphase fluorescent in situ hybridization analysis of chromosomes 13, 18, 19, 21, X or Y, or were assayed for mitochondrial activity. Amino acid turnover was different (P < 0.05) between stage-matched fresh and cryopreserved embryos due to blastomere loss following warming. The proportion of embryos with aneuploid cells increased as cell division progressed from pronucleate- (23%) to late cleavage stages (50-70%). Asparagine, glycine and valine turnover was significantly different between uniformly genetically normal and uniformly abnormal embryos on Days 2-3 of culture. By Days 3-4, the profiles of serine, leucine and lysine differed between uniformly euploid versus aneuploid embryos. Gender significantly (P < 0.05) affected the metabolism of tryptophan, leucine and asparagine by cleavage-stage embryos. Pronucleate zygotes had a significantly higher proportion of active:inactive mitochondria compared with cleavage-stage embryos. Furthermore, mitochondrial activity was correlated (P < 0.05) with altered aspartate and glutamine turnover. These results demonstrate the association between the metabolism, cytogenetic composition and health of human embryos in vitro.

DNA damage and metabolic activity in the preimplantation embryo.

BACKGROUND: Embryos with greater viability have a lower or 'quieter' amino acid metabolism than those which arrest. We have hypothesized this is due to non-viable embryos possessing greater cellular/molecular damage and consuming more nutrients, such as amino acids for repair processes. We have tested this proposition by measuring physical damage to DNA in bovine, porcine and human embryos at the blastocyst stage and relating the data to amino acid profiles during embryo development. METHODS: Amino acid profiles of in vitro-derived porcine and bovine blastocysts were measured by high-performance liquid chromatography and the data related retrospectively to DNA damage in each individual blastomere using a modified alkaline comet assay. Amino acid profiles of spare human embryos on Day 2-3 were related to DNA damage at the blastocyst stage. RESULTS: A positive correlation between amino acid turnover and DNA damage was apparent when each embryo was examined individually; a relationship exhibited by all three species. There was no relationship between DNA damage and embryo grade. CONCLUSIONS: Amino acid profiling of single embryos can provide a non-invasive marker of DNA damage at the blastocyst stage. The data are consistent with the quiet embryo hypothesis with viable embryos (lowest DNA damage) having the lowest amino acid turnover. Moreover, these data support the notion that metabolic profiling, in terms of amino acids, might be used to select single embryos for transfer in clinical IVF.

Transport and catabolism of the sialic acids N-glycolylneuraminic acid and 3-keto-3-deoxy-D-glycero-D-galactonononic acid by Escherichia coli K-12.

Escherichia coli can transport and catabolize the common sialic acid, N-acetylneuraminic acid (Neu5Ac), as a sole source of carbon and nitrogen, which is an important mucus-derived carbon source in the mammalian gut. Herein we demonstrate that E. coli can also grow efficiently on the related sialic acids, N-glycolylneuraminic acid (Neu5Gc) and 3-keto-3-deoxy-D-glycero-D-galactonononic acid (KDN), which are transported via the sialic acid transporter NanT and catabolized using the sialic acid aldolase NanA. Catabolism of Neu5Gc uses the same pathway as Neu5Ac, likely producing glycolate instead and acetate during its breakdown and catabolism of KDN requires NanA activity, while other components of the Neu5Ac catabolism pathway are non-essential. We also demonstrate that these two sialic acids can support growth of an E. coli ∆nanT strain expressing sialic acid transporters from two bacterial pathogens, namely the tripartite ATP-independent periplasmic transporter SiaPQM from Haemophilus influenzae and the sodium solute symport transporter STM1128 from Salmonella enterica ssp. Typhimurium, suggesting that the ability to use Neu5Gc and KDN in addition to Neu5Ac is present in a number of human pathogens.

Characterization of a novel sialic acid transporter of the sodium solute symporter (SSS) family and in vivo comparison with known bacterial sialic acid transporters.

The function of sialic acids in the biology of bacterial pathogens is reflected by the diverse range of solute transporters that can recognize these sugar acids. Here, we use an Escherichia coliDeltananT strain to characterize the function of known and proposed bacterial sialic acid transporters. We discover that the STM1128 gene from Salmonella enterica serovar Typhimurium, which encodes a member of the sodium solute symporter family, is able to restore growth on sialic acid to the DeltananT strain and is able to transport [(14)C]-sialic acid. Using the DeltananT genetic background, we performed a direct in vivo comparison of the transport properties of the STM1128 protein with those of sialic acid transporters of the major facilitator superfamily and tripartite ATP-independent periplasmic families, E. coli NanT and Haemophilus influenzae SiaPQM, respectively. This revealed that both STM1128 and SiaPQM are sodium-dependent and, unlike SiaPQM, both STM1128 and NanT are reversible secondary carriers, demonstrating qualitative functional differences in the properties of sialic acid transporters used by bacteria that colonize humans.

Metabolism of human embryos following cryopreservation: implications for the safety and selection of embryos for transfer in clinical IVF.

BACKGROUND: Cryopreservation of supernumerary embryos is routinely performed in human-assisted reproduction, providing a source of embryos which can be thawed for use in subsequent treatment cycles. However, the viability of cryopreserved embryos has traditionally relied on morphological assessment, which is a poor predictor of embryo health since freezing leads to a significant overall reduction in implantation potential, and its long-term efficacy is unknown. This study describes how the post-thaw metabolism of human embryos can be used to predict future development to the blastocyst stage. METHODS: HPLC was used to analyse the post-thaw amino acid metabolism of human embryos from day 2 to day 3 of development. RESULTS: It was possible to predict with 87% accuracy which frozen-thawed embryo would develop to the blastocyst stage. Developmentally competent embryos were more metabolically quiescent than their arresting counterparts. Amino acid turnover was also capable of distinguishing between the developmental potential of the best, Grade I embryos P < 0.05. CONCLUSIONS: The data suggests that cryopreservation in IVF is a safe procedure and that amino acid turnover can be used to select which cryopreserved embryo will develop to the blastocyst stage, irrespective of their post-thaw grade.

Human embryos developing in vitro are susceptible to impaired epithelial junction biogenesis correlating with abnormal metabolic activity.

BACKGROUND: Blastocyst biogenesis occurs over several cell cycles during the preimplantation period comprising the gradual expression and membrane assembly of junctional protein complexes which distinguish the outer epithelial trophectoderm (TE) cells from the inner cell mass (ICM). In the human, TE integrity and the formation of a junctional seal can often be impaired. Embryos likely to result in a successful pregnancy after transfer are mostly selected according to morphological criteria. Recent data suggest that non-invasive measurement of amino acid turnover may be useful to complement such morphological scores. Whether morphological and metabolic criteria can be linked to poor TE differentiation thereby underpinning developmental predictions mechanistically remains unknown. METHODS: We examined TE intercellular junction formation in human embryos by immunofluorescence and confocal microscopy and correlated this process with morphological criteria and amino acid turnover during late cleavage. RESULTS: Our results show that TE differentiation may be compromised by failure of membrane assembly of specific junction constituents. This abnormality relates more closely to metabolic profiles than morphological criteria. CONCLUSION: Our data identify that amino acid turnover can predict TE differentiation. These findings are the first to link two mechanisms, metabolism and junction membrane assembly, which contribute to early embryo development.

Na+, K+, ATPase activity in the human and bovine preimplantation embryo.

Blastocyst formation is associated with a marked increase in ATP production, much of which is thought to be associated with the active transport of ions across the trophectoderm mediated by the sodium pump (Na+, K+, ATPase) resulting in the vectorial transport of water into the blastocoel. In this study, the biochemical activity of the sodium pump was measured directly in single human and bovine embryo extracts by monitoring the conversion of ATP to ADP in the presence and absence of ouabain. ATP and ADP were assayed by HPLC. In both species, there was a transient, significant increase in sodium pump activity while the blastocyst was actively expanding. The oxygen consumption of single human blastocysts was measured in order to estimate the proportion of total ATP used by the Na+, K+, ATPase. The results suggest that approximately 60 and 36% of the ATP produced is used by the sodium pump during blastocoel expansion in the human and bovine blastocyst, respectively.

Non-invasive amino acid turnover predicts human embryo developmental capacity.

BACKGROUND: IVF is limited by low success rates and a confounding high multiple birth rate contributing to prematurity, increased neonatal mortality and child handicap. These problems could be overcome if single embryos of known developmental competence could be selected for transfer on day 2/3 of development, but current methods, which rely on morphological appearance, are poor predictors of viability. METHODS: We have measured non-invasively the depletion/appearance (i.e. turnover) of a physiological mixture of 18 amino acids by single human embryos during in-vitro culture using high performance liquid chromatography. RESULTS: From the time of transfer (day 2/3), embryos with future competence to develop to the blastocyst stage (day 5/6) exhibit amino acid flux patterns distinct from those of embryos with similar morphological appearance which arrest. Significantly, the profiles of Ala, Arg, Gln, Met and Asn flux predict blastocyst potentiality at >95%. The amino acid most consistently depleted throughout development by those embryos which form blastocysts was leucine. Of the amino acids which were produced, the most striking was alanine, which appeared in increasing amounts throughout development. CONCLUSIONS: Non-invasive amino acid profiling has the potential to select developmentally competent single embryos for transfer, thereby increasing the success rate and eliminating multiple births in IVF.