The Periplasmic Tail-Specific Protease, Tsp, Is Essential for Secondary Differentiation in Chlamydia trachomatis.

The obligate intracellular human pathogen Chlamydia trachomatis (Ctr) undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms: the elementary body (EB) and the reticulate body (RB). The EB is the smaller, infectious, nondividing form which initiates infection of a susceptible host cell, whereas the RB is the larger, non-infectious form which replicates within a membrane-bound vesicle called an inclusion. The mechanism(s) which drives differentiation between these developmental forms is poorly understood. Bulk protein turnover is likely required for chlamydial differentiation given the significant differences in the protein repertoires and functions of the EB and RB. We hypothesize that periplasmic protein turnover is also critical for the reorganization of an RB into an EB, referred to as secondary differentiation. Ct441 is a periplasmic protease ortholog of tail-specific proteases (i.e., Tsp, Prc) and is expressed in Ctr during secondary differentiation. We investigated the effect of altering Tsp expression on developmental cycle progression. Through assessment of bacterial morphology and infectious progeny production, we found that both overexpression and CRISPR interference/dCas9 (CRISPRi)-mediated knockdown of Tsp negatively impacted chlamydial development through different mechanisms. We also confirmed that catalytic activity is required for the negative effect of overexpression and confirmed the effect of the mutation in in vitro assays. Electron microscopic assessments during knockdown experiments revealed a defect in EB morphology, directly linking Tsp function to secondary differentiation. These data implicate Ct441/Tsp as a critical factor in secondary differentiation. IMPORTANCE The human pathogen Chlamydia trachomatis is the leading cause of preventable infectious blindness and bacterial sexually transmitted infections worldwide. This pathogen has a unique developmental cycle that alternates between distinct forms. However, the key processes of chlamydial development remain obscure. Uncovering the mechanisms of differentiation between its metabolically and functionally distinct developmental forms may foster the discovery of novel Chlamydia-specific therapeutics and limit development of resistant bacterial populations derived from the clinical use of broad-spectrum antibiotics. In this study, we investigate chlamydial tail-specific protease (Tsp) and its function in chlamydial growth and development. Our work implicates Tsp as essential to chlamydial developmental cycle progression and indicates that Tsp is a potential drug target for Chlamydia infections.

Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis.

Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and cell culture approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated ß-casein. Cell culture experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, these data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here, again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of antichlamydial agents. IMPORTANCE Chlamydia trachomatis is an obligate intracellular pathogen and the world's leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new antichlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for antichlamydial compounds.

The structure of caseinolytic protease subunit ClpP2 reveals a functional model of the caseinolytic protease system from Chlamydia trachomatis.

Chlamydia trachomatis (ct) is the most reported bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Caseinolytic proteases (ClpP) from pathogenic bacteria are attractive antibiotic targets, particularly for bacterial species that form persister colonies with phenotypic resistance against common antibiotics. ClpP functions as a multisubunit proteolytic complex, and bacteria are eradicated when ClpP is disrupted. Although crucial for chlamydial development and the design of agents to treat chlamydia, the structures of ctClpP1 and ctClpP2 have yet to be solved. Here, we report the first crystal structure of full-length ClpP2 as an inactive homotetradecamer in a complex with a candidate antibiotic at 2.66 Å resolution. The structure details the functional domains of the ClpP2 protein subunit and includes the handle domain, which is integral to proteolytic activation. In addition, hydrogen-deuterium exchange mass spectroscopy probed the dynamics of ClpP2, and molecular modeling of ClpP1 predicted an assembly with ClpP2. By leveraging previous enzymatic experiments, we constructed a model of ClpP2 activation and its interaction with the protease subunits ClpP1 and ClpX. The structural information presented will be relevant for future rational drug design against these targets and will lead to a better understanding of ClpP complex formation and activation within this important human pathogen.

Tag-Dependent Substrate Selection of ClpX Underlies Secondary Differentiation of Chlamydia trachomatis.

Despite having a highly reduced genome, Chlamydia trachomatis undergoes a complex developmental cycle in which the bacteria differentiate between the following two functionally and morphologically distinct forms: the infectious, nonreplicative elementary body (EB) and the noninfectious, replicative reticulate body (RB). The transitions between EBs and RBs are not mediated by division events that redistribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation likely require bulk protein turnover. One system for targeted protein degradation is the trans-translation system for ribosomal rescue, where polypeptides stalled during translation are marked with an SsrA tag encoded by a hybrid tRNA-mRNA, tmRNA. ClpX recognizes the SsrA tag, leading to ClpXP-mediated degradation. We hypothesize that ClpX functions in chlamydial differentiation through targeted protein degradation. We found that mutation of a key residue (R230A) within the specific motif in ClpX associated with the recognition of SsrA-tagged substrates resulted in abrogated secondary differentiation while not reducing chlamydial replication or developmental cycle progression as measured by transcripts. Furthermore, inhibition of trans-translation through chemical and targeted genetic approaches also impeded chlamydial development. Knockdown of tmRNA and subsequent complementation with an allele mutated in the SsrA tag closely phenocopied the overexpression of ClpXR230A, thus suggesting that ClpX recognition of SsrA-tagged substrates plays a critical function in secondary differentiation. Taken together, these data provide mechanistic insight into the requirements for transitions between chlamydial developmental forms. IMPORTANCE Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections and preventable infectious blindness. This unique organism undergoes developmental transitions between infectious, nondividing forms and noninfectious, dividing forms. Therefore, the chlamydial developmental cycle is an attractive target for Chlamydia-specific antibiotics, which would minimize effects of broad-spectrum antibiotics on the spread of antibiotic resistance in other organisms. However, the lack of knowledge about chlamydial development on a molecular level impedes the identification of specific, druggable targets. This work describes a mechanism through which both the fundamental processes of trans-translation and proteomic turnover by ClpXP contribute to chlamydial differentiation, a critical facet of chlamydial growth and survival. Given the almost universal presence of trans-translation and ClpX in eubacteria, this mechanism may be conserved in developmental cycles of other bacterial species. Additionally, this study expands the fields of trans-translation and Clp proteases by emphasizing the functional diversity of these systems throughout bacterial evolution.

In Vitro and In Vivo Activity of (Trifluoromethyl)pyridines as Anti-Chlamydia trachomatis Agents.

Chlamydia trachomatis is the leading pathogen in sexually transmitted bacterial infections across the globe. The development of a selective treatment against this pathogen could be an attractive therapeutic option that will reduce the overuse of broad-spectrum antibiotics. Previously, we reported some sulfonylpyridine-based compounds that showed selectivity against C. trachomatis. Here, we describe a set of related compounds that display enhanced anti-chlamydial potency when compared to our early leads. We found that the active molecules are bactericidal and have no impact on Staphylococcus aureus or Escherichia coli strains. Importantly, the molecules were not toxic to mammalian cells. Furthermore, a combination of molecule 20 (the most active molecule) and azithromycin at subinhibitory concentrations acted synergistically to inhibit chlamydial growth. Molecule 20 also eradicated Chlamydia in a 3D infection model and accelerated the recovery of Chlamydia-infected mice. This work presents compounds that could be further developed to be used alone or in combination with existing treatment regimens against chlamydial infections.

Codon-Dependent Transcriptional Changes in Response to Tryptophan Limitation in the Tryptophan Auxotrophic Pathogens Chlamydia trachomatis and Streptococcus pyogenes.

Chlamydia trachomatis and Streptococcus pyogenes are among the most prevalent bacterial pathogens of humans. Interestingly, both pathogens are tryptophan (Trp) auxotrophs and must acquire this essential amino acid from their environment. For Chlamydia, an obligate intracellular bacterium, this means scavenging Trp from the host cell in which they reside. For Streptococcus, a primarily extracellular bacterium, this means scavenging Trp from the local environment. In the course of a natural immune response, both pathogens can be exposed to Trp-limiting conditions through the action of the interferon gamma-inducible IDO1 enzyme, which catabolizes Trp to N-formylkynurenine. How these pathogens respond to Trp starvation is incompletely understood. However, we have previously demonstrated that genes enriched in Trp codons were preferentially transcribed in C. pneumoniae during Trp limitation. Chlamydia, but not Streptococcus, lacks a stringent response, which is a global regulon activated by uncharged tRNAs binding in the A site of the ribosome. We hypothesized that the chlamydial response to Trp limitation is a consequence of lacking a stringent response. To test this, we compared global transcription profiles of C. trachomatis to both wild-type and stringent response mutant strains of Streptococcus during Trp starvation. We observed that both Trp auxotrophs respond with codon-dependent changes in their transcriptional profiles that correlate with Trp codon content but not transcript stability. Importantly, the stringent response had no impact on these transcriptional changes, suggesting an evolutionarily conserved adaptation to Trp starvation. Therefore, we have revealed a novel response of Trp auxotrophic pathogens in response to Trp starvation. IMPORTANCE Chlamydia trachomatis and Streptococcus pyogenes are important pathogens of humans. Interestingly, both are auxotrophic for tryptophan and acquire this essential amino acid from the host environment. However, part of the host defense against pathogens includes the degradation of tryptophan pools. Therefore, Chlamydia and Streptococcus are particularly susceptible to tryptophan starvation. Most model bacteria respond to amino acid starvation by using a global regulon called the stringent response. However, Chlamydia lacks a stringent response. Here, we investigated the chlamydial response to tryptophan starvation and compared it to both wild-type and stringent response mutant strains of S. pyogenes to determine what role a functional stringent response plays during tryptophan starvation in these pathogens. We determined that both of these pathogens respond to tryptophan starvation by increasing transcription of tryptophan codon-rich genes. This effect was not dependent on the stringent response and highlights a previously unrecognized and potentially evolutionarily conserved mechanism for surviving tryptophan starvation.

The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development.

Chlamydia trachomatis is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, the elementary body (EB) and reticulate body (RB), each of which expresses its own specialized repertoire of proteins. Both primary (EB to RB) and secondary (RB to EB) differentiations require protein turnover, and we hypothesize that proteases are critical for mediating differentiation. The Clp protease system is well conserved in bacteria and important for protein turnover. Minimally, the system relies on a serine protease subunit, ClpP, and an AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia, ClpX is encoded within an operon 3' to clpP2 We present evidence that the chlamydial ClpX and ClpP2 orthologs are essential to organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability with observable defects in EB morphology noted. Conversely, overexpression of a catalytically inactive ClpP2 mutant significantly impacted developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in trans by a plasmid copy of clpP2 Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation.IMPORTANCEChlamydia trachomatis is the leading cause of infectious blindness globally and the most reported bacterial sexually transmitted infection both domestically and internationally. Given the economic burden, the lack of an approved vaccine, and the use of broad-spectrum antibiotics for treatment of infections, an understanding of chlamydial growth and development is critical for the advancement of novel targeted antibiotics. The Clp proteins comprise an important and conserved protease system in bacteria. Our work highlights the importance of the chlamydial Clp proteins to this clinically important bacterium. Additionally, our study implicates the Clp system playing an integral role in chlamydial developmental cycle progression, which may help establish models of how Chlamydia spp. and other bacteria progress through their respective developmental cycles. Our work also contributes to a growing body of Clp-specific research that underscores the importance and versatility of this system throughout bacterial evolution and further validates Clp proteins as drug targets.

Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each.

Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX, clpC, two clpP paralogs, and clpB In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive clpP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCEChlamydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

Characterization of Chlamydial Rho and the Role of Rho-Mediated Transcriptional Polarity during Interferon Gamma-Mediated Tryptophan Limitation.

As an obligate intracellular, developmentally regulated bacterium, Chlamydia is sensitive to amino acid fluctuations within its host cell. When human epithelial cells are treated with the cytokine interferon gamma (IFN-γ), the tryptophan (Trp)-degrading enzyme, indoleamine-2,3-dioxygenase, is induced. Chlamydiae within such cells are starved for Trp and enter a state of so-called persistence. Chlamydia lacks the stringent response used by many eubacteria to respond to this stress. Unusually, chlamydial transcription is globally elevated during Trp starvation with transcripts for Trp codon-containing genes disproportionately increased. Yet, the presence of Trp codons destabilized 3' ends of transcripts in operons or large genes. We initially hypothesized that ribosome stalling on Trp codons rendered the 3' ends sensitive to RNase activity. The half-life of chlamydial transcripts containing different numbers of Trp codons was thus measured in untreated and IFN-γ-treated infected cells to determine whether Trp codons influenced the stability of transcripts. However, no effect of Trp codon content was detected. Therefore, we investigated whether Rho-dependent transcription termination could play a role in mediating transcript instability. Rho is expressed as a midcycle gene product, interacts with itself as predicted, and is present in all chlamydial species. Inhibition of Rho via the Rho-specific antibiotic, bicyclomycin, and overexpression of Rho are detrimental to chlamydiae. Finally, when we measured transcript abundance 3' to Trp codons in the presence of bicyclomycin, we observed that transcript abundance increased. These data are the first to demonstrate the importance of Rho in Chlamydia and the role of Rho-dependent transcription polarity during persistence.

Metabolic fates of yolk lipid and individual fatty acids during embryonic development of the coot and moorhen.

There is currently little information regarding the metabolic fates of yolk lipid and individual fatty acids during embryonic development of free-living avian species. Here we report the pattern of lipid utilization during embryonic development of the coot (Fulica atra) and the moorhen (Gallinula chloropus), two related species producing precocial offspring from eggs with a distinctive fatty acid composition and with an incubation period similar to that of the chicken. By the time of hatching, the proportions of the initial yolk lipid that had been transferred to the embryo were 88.2% and 79.8% for the coot and moorhen respectively. During the whole incubation period, 42.9% and 40.0% of the initial yolk lipid of the coot and moorhen respectively were lost from the system due to oxidation for energy, equating to 47.8% and 50.0% respectively of the actual amount of lipid transferred over this time. Thus, the lipid received by the embryos of both species is partitioned almost equally between the alternative fates of energy metabolism and incorporation into tissue lipids. In the coot, this 50:50 split between oxidation and tissue formation was maintained during the hatching process. The proportions of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) in the yolk lipids of these species were 2.5-3.5 times higher than in eggs of domestic poultry. In contrast to the situation in the chicken, there was no preferential uptake of 22:6n-3 from the yolk during coot and moorhen development. The fatty acid compositions of the whole body lipids of the coot and moorhen hatchlings were almost identical to those of the initial yolks indicating that, unlike the chicken, these species display relatively little overall biomagnification of 20:4n-6 and 22:6n-6 during development. It is suggested that the yolk fatty acid profiles of the coot and moorhen are particularly well matched to the requirements of the embryo, reducing the need for selective uptake of 22:6n-3 and for the overall biomagnification of 22:6n-3 and 20:4n-6.

Interspecies variation in yolk selenium concentrations among eggs of free-living birds: The effect of phylogeny.

Birds deposit the trace element selenium (Se) into their eggs because an adequate supply of this micronutrient is essential for embryonic development. Although there is considerable interest in egg Se with regard to topics as diverse as poultry nutrition and environmental pollution, data on the natural levels of Se in eggs of free-living avian species are currently very limited. To address this lack of information, we measured the yolk Se concentrations in eggs of 14 avian species collected in the wild. The concentrations (ng/g wet yolk) varied from 394 to 2238, with a mean value of 1040. Values (means+/-SD) for eggs from the UK, Canada and New Zealand were, respectively, 522+/-192 (3 species), 1194+/-584 (8 species) and 1147+/-200 (3 species). However, analysis by appropriate statistical models indicates that the effect of phylogenetic relatedness among these species is so significant that it removes any effect of geographical location. In particular, species belonging to the order Passeriformes displayed significantly higher yolk Se levels than Non-Passeriforme species. In marked contrast to the free-living species, our previously published data indicate that the Se concentration in egg yolk of the domestic chicken is only about 100 ng/g wet yolk when the birds are maintained on a basal commercial diet without supplementary Se. The results reveal an extensive interspecies variation in yolk Se (across a 6-fold range) for eggs collected from the wild. Nevertheless, the Se concentrations in the yolks of all the free-living species were far higher (4-21-fold) than that achieved in the yolk of the domestic chicken consuming a standard basal diet.

Tissue-specific distribution of carotenoids and vitamin E in tissues of newly hatched chicks from various avian species.

The aim of this study was to evaluate carotenoid and vitamin E distribution in egg and tissues of newly hatched chicks from wild mallard (Anas platyrhynchos), game pheasant (Phasianus colchicus), free-range guinea fowl (Numida meleagris), hen (Gallus domesticus) and domestic duck (Anas platyrhynchos) and intensively housed hens. Carotenoid concentrations in the egg yolk of free-range guinea fowl, pheasant and wild mallard were similar (61.3-79.2 microg/g). Egg yolks from ducks and intensively housed hens were characterised by the lowest carotenoid concentration comprising 11.2-14.8 microg/g. However, carotenoid concentration in eggs from free-range ducks and hens was less than half of that in free-range guinea fowl or pheasant. Depending on carotenoid concentration in the livers of species studied could be placed in the following descending order: free living pheasant>free-range guinea fowl>>free-range hen>>intensively housed hen>wild mallard>>housed duck>free-range duck. The carotenoid concentrations in other tissues of free-range guinea fowl and pheasant were substantially higher than in the other species studied. Egg yolk of housed hens was characterised by the highest alpha- and gamma-tocopherol concentrations. In accordance with the alpha-tocopherol concentration in the egg yolk, the birds can be placed in the following descending order: intensively housed hen>wild mallard>free-living pheasant>free-range duck>free-range hen=free-range guinea fowl>housed duck. The main finding of this work is species- and tissue-specific differences in carotenoid and vitamin E distribution in the various avian species studied.

Timing of incorporation of docosahexaenoic acid into brain and muscle phospholipids during precocial and altricial modes of avian development.

We investigated the possibilities that the proportion of docosahexaenoic acid (DHA) in phospholipids of brain and skeletal muscle at hatch, and the ontogenetic timing of the DHA accretion spurt in these tissues, might serve as indices of neonatal functional maturity that discriminate between precocial and altricial avian developmental modes. Comparison of the fatty acid profiles of the initial and residual yolks of two free-living altricial species, the swallow (Hirundo rustica) and the sparrow (Passer domesticus), reveals that, in contrast to precocial birds, there is no preferential uptake of DHA from the yolk during embryonic development. At hatch, the proportions of DHA in brain phospholipid (wt.% of fatty acids) of the swallow and sparrow, at 8.1% and 5.0%, respectively, are far lower than the values (16.9-19.6%) reported for non-altricial species. This reflects a marked difference in the timing of the brain DHA accretion spurt, which occurs during the first half of the embryonic period of precocial birds, but is largely delayed until after hatching in the altricial species. By the time of fledging, the proportion of DHA in the swallow brain phospholipid has increased to 14.3%. For non-altricial birds, the brain DHA concentration at hatch shows little interspecies variation, despite major differences in yolk DHA content. The proportions of DHA in leg muscle phospholipid of the newly hatched swallow and sparrow, at 2.9% and 2.5%, respectively, are far lower than the value (6.7%) for the precocial chicken. Again, this relates to differences in developmental timing, with muscle DHA accretion occurring in the first half of the chicken's embryonic period, whereas, in the swallow, this increase is delayed until after hatching. By the time of fledging in the swallow, DHA forms 9.3% of muscle phospholipid fatty acids, equivalent to the level attained in chicken muscle at the mid-embryo stage. The results indicate a clear distinction between altricial and non-altricial avian species in the timing of tissue DHA accretion during development, presumably reflecting differences in neonatal functional maturity.

Establishment of the fatty acid profile of the brain of the king penguin (Aptenodytes patagonicus) at hatch: effects of a yolk that is naturally rich in n-3 polyunsaturates.

Because the yolk lipids of the king penguin (Aptenodytes patagonicus) contain the highest concentrations of long-chain n-3 polyunsaturated fatty acids yet reported for an avian species, the consequences for the establishment of the brain's fatty acid profile in the embryo were investigated. To place the results in context, the fatty acid compositions of yolk lipid and brain phospholipid of the king penguin were compared with those from three other species of free-living birds. The proportions of docosahexaenoic acid (22:6n-3; DHA) in the total lipid of the initial yolks for the Canada goose (Branta canadensis), mallard (Anas platyrhynchos), moorhen (Gallinula chloropus), and king penguin were (% w/w of fatty acids) 1.0+/-0.1, 1.9+/-0.2, 3.3+/-0.1, and 5.9+/-0.2, respectively. The respective concentrations of DHA (% w/w of phospholipid fatty acids) in brains of the newly hatched chicks of these same species were 18.5+/-0.2, 19.6+/-0.7, 16.9+/-0.4, and 17.6+/-0.1. Thus, the natural interspecies diversity in yolk fatty acid profiles does not necessarily produce major differences in the DHA content of the developing brain. Only about 1% of the amount of DHA initially present in the yolk was recovered in the brain of the penguin at hatch. There was no preferential uptake of DHA from the yolk during development of the king penguin.