Resistance to Co-Occurring Phages Enables Marine Synechococcus Communities To Coexist with Cyanophages Abundant in Seawater

Recent reports documenting very high viral abundances in seawater have led to increased interest in the role of viruses in aquatic environments and a resurgence of the hypothesis that viruses are significant agents of bacterial mortality. Synechococcus spp., small unicellular cyanobacteria that are important primary producers at the base of the marine food web, were used to assess this hypothesis. We isolated a diverse group of Synechococcus phages that at times reach titers of between 10³ and 10⁴ cyanophages per ml in both inshore and offshore waters. However, despite their diversity and abundance, we present evidence in support of the hypothesis that lytic phages have a negligible effect in regulating the densities of marine Synechococcus populations. Our results indicate that these bacterial communities are dominated by cells resistant to their co-occurring phages and that these viruses are maintained by scavenging on the relatively rare sensitive cells in these communities.     

Early-Life Exposure to Polycyclic Aromatic Hydrocarbons and ADHD Behavior Problems

Importance

Polycyclic aromatic hydrocarbons are widespread urban air pollutants from combustion of fossil fuel and other organic material shown previously to be neurotoxic.

Objective

In a prospective cohort study, we evaluated the relationship between Attention Deficit Hyperactivity Disorder behavior problems and prenatal polycyclic aromatic hydrocarbon exposure, adjusting for postnatal exposure.

Materials and Methods

Children of nonsmoking African-American and Dominican women in New York City were followed from in utero to 9 years. Prenatal polycyclic aromatic hydrocarbon exposure was estimated by levels of polycyclic aromatic hydrocarbon- DNA adducts in maternal and cord blood collected at delivery. Postnatal exposure was estimated by the concentration of urinary polycyclic aromatic hydrocarbon metabolites at ages 3 or 5. Attention Deficit Hyperactivity Disorder behavior problems were assessed using the Child Behavior Checklist and the Conners Parent Rating Scale- Revised.

Results

High prenatal adduct exposure, measured by elevated maternal adducts was significantly associated with all Conners Parent Rating Scale-Revised subscales when the raw scores were analyzed continuously (N = 233). After dichotomizing at the threshold for moderately to markedly atypical symptoms, high maternal adducts were significantly associated with the Conners Parent Rating Scale-Revised DSM-IV Inattentive (OR = 5.06, 95% CI [1.43, 17.93]) and DSM-IV Total (OR = 3.37, 95% CI [1.10, 10.34]) subscales. High maternal adducts were positivity associated with the DSM-oriented Attention Deficit/Hyperactivity Problems scale on the Child Behavior Checklist, albeit not significant. In the smaller sample with cord adducts, the associations between outcomes and high cord adduct exposure were not statistically significant (N = 162).

Conclusion

The results suggest that exposure to polycyclic aromatic hydrocarbons encountered in New York City air may play a role in childhood Attention Deficit Hyperactivity Disorder behavior problems.     

Peroxisomal ATP Import Is Essential for Seedling Development in Arabidopsis thaliana

Several recent proteomic studies of plant peroxisomes indicate that the peroxisomal matrix harbors multiple ATP-dependent enzymes and chaperones. However, it is unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether external ATP fuels the energy-dependent reactions within peroxisomes. The existence of transport proteins that supply plant peroxisomes with energy for fatty acid oxidation and other ATP-dependent processes has not previously been demonstrated. Here, we describe two Arabidopsis thaliana genes that encode peroxisomal adenine nucleotide carriers, PNC1 and PNC2. Both proteins, when fused to enhanced yellow fluorescent protein, are targeted to peroxisomes. Complementation of a yeast mutant deficient in peroxisomal ATP import and in vitro transport assays using recombinant transporter proteins revealed that PNC1 and PNC2 catalyze the counterexchange of ATP with ADP or AMP. Transgenic Arabidopsis lines repressing both PNC genes were generated using ethanol-inducible RNA interference. A detailed analysis of these plants showed that an impaired peroxisomal ATP import inhibits fatty acid breakdown during early seedling growth and other β-oxidation reactions, such as auxin biosynthesis. We show conclusively that PNC1 and PNC2 are essential for supplying peroxisomes with ATP, indicating that no other ATP generating systems exist inside plant peroxisomes.The β-oxidation of fatty acids, a process that exclusively occurs within peroxisomes in plants and yeast, plays an important role in storage oil mobilization to support seedling establishment of oilseed plants, such as Arabidopsis thaliana (Graham and Eastmond, 2002; Baker et al., 2006; Graham, 2008). Upon germination, fatty acids are released from storage oil triacylglycerol (TAG) by lipolysis, degraded via β-oxidation in specialized peroxisomes, termed glyoxysomes, and subsequently converted to sucrose, which drives growth and development until seedlings become photoautotrophic (Graham and Eastmond, 2002; Baker et al., 2006; Graham, 2008). Before the fatty acids can enter β-oxidation, they are imported into peroxisomes by a peroxisomal ATP binding cassette (ABC) transporter, variously known as CTS (COMATOSE), At PXA1 (Arabidopsis peroxisomal ABC transporter), or PED3 (peroxisomal defective 3) and hereafter referred to as CTS (Zolman et al., 2001; Footitt et al., 2002; Hayashi et al., 2002). Subsequently, the imported fatty acids are activated by esterification to CoA. This ATP-dependent reaction within peroxisomes is catalyzed by long-chain acyl-CoA synthetases 6 and 7 (LACS6 and LACS7, respectively), which are named according to their substrate specificity for long-chain fatty acids, which are significant components of seed storage oil in Arabidopsis (Fulda et al., 2002, 2004).In Saccharomyces cerevisiae, two mechanisms exist for import and activation of fatty acids, depending on chain length (Hettema et al., 1996). Long-chain fatty acids (C16 and C18) are converted to acyl-CoA esters in the cytosol prior to transport by the heterodimeric peroxisomal ABC transporter, Pxa1p/Pxa2 (Hettema et al., 1996). By contrast, short- and medium-chain fatty acids (≤C14) that enter the peroxisomes by passive diffusion or by an unknown transport protein are activated within peroxisomes (Hettema et al., 1996). The possibility cannot be excluded, though, that CTS imports the corresponding CoA derivatives, as is the case for the yeast Pxa1p/Pxa2p heterodimer (Hettema et al., 1996; Verleur et al., 1997), implicating a cytosolic activation of the fatty acids, catalyzed by a hitherto unknown enzyme. The actual substrates transported by CTS in Arabidopsis have not yet been experimentally determined (Theodoulou et al., 2006). However, the sucrose-dependent seedling growth phenotype of the lacs6 lacs7 double knockout mutant demonstrated that peroxisomal activation is essential for lipid mobilization to provide energy for early seedling growth (Fulda et al., 2004). The lacs6 lacs7 mutant is impaired in the degradation of fatty acids, leading to growth arrest shortly after germination (Fulda et al., 2004).Besides fatty acid mobilization, β-oxidation is also involved in generation of signaling molecules, such as the phytohormones auxin and fatty acid–derived jasmonic acid (JA) (Zolman et al., 2000; Schaller et al., 2004; Delker et al., 2007). By analogy to fatty acids released from storage oil, the precursors of these signaling molecules require CoA esterification before they can enter β-oxidation (Baker et al., 2006; Goepfert and Poirier, 2007). While the enzymes responsible for ATP-dependent activation of natural auxin (indole butyric acid [IBA]) and proherbicide 2,4-dichlorophenoxybutyric acid (2,4-DB) are currently unknown, several enzymes belonging to the acyl-activating enzyme (AAE) family have been implicated in jasmonate biosynthesis (Schneider et al., 2005; Koo et al., 2006; Kienow et al., 2008). Moreover, several as yet uncharacterized members of the large AAE family carry a putative peroxisome targeting signal (PTS) and thus might be good candidates to activate the additional β-oxidation substrates within peroxisomes (Shockey et al., 2002, 2003).In the case where activation of fatty acids or other substrates takes place within peroxisomes, the question arises as to how these ATP-dependent reactions are supplied with ATP. It is currently unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether they depend on external ATP to supply energy-dependent reactions within peroxisomes. So far, transport proteins that supply plant peroxisomes with energy for fatty acid oxidation have not been characterized. However, in bakers'' yeast, a peroxisomal adenine nucleotide transporter, ANT1, that is required for the ATP-dependent activation of medium-chain fatty acids inside peroxisomes has been characterized (Palmieri et al., 2001).ATP transport proteins play an important role in the distribution of the primary agent coupling endergonic and exergonic reactions in every cellular compartment (Winkler and Neuhaus, 1999). In Arabidopsis and other plants, various adenine nucleotide carriers have been identified at the molecular level. The mitochondrial ADP/ATP carrier mediates the export of ATP that is synthesized in the mitochondrion to provide energy for cellular metabolism (Heimpel et al., 2001; Haferkamp et al., 2002). The plastidial ATP/ADP transporter (nucleotide transporter) is involved in ATP uptake by both chloroplasts and heterotrophic plastids, to enable the nocturnal ATP supply required for chlorophyll biosynthesis (Reiser et al., 2004; Reinhold et al., 2007), as well as by heterotrophic plastids to drive starch biosynthesis (Batz et al., 1992; Tjaden et al., 1998). Yet another ATP/ADP antiporter located in the endoplasmic reticulum (ER) membrane provides energy by importing ATP into the ER for the accumulation of ER-related storage lipids and proteins (Leroch et al., 2008).In this study, we identified two novel peroxisomal adenine nucleotide carrier proteins (PNC1 and PNC2) from Arabidopsis. Colocalization studies demonstrated that these proteins are targeted to peroxisomes. Yeast complementation and in vitro ATP uptake assays showed that both PNC1 and PNC2 catalyze the counterexchange of ATP with AMP. Using an inducible RNA interference (RNAi) repression strategy, we further established several transgenic Arabidopsis lines with reduced expression levels of both PNC1 and PNC2. Our results showed that import of ATP into peroxisomes that is catalyzed by PNC1 and PNC2 is essential for activation of fatty acids during seedling germination and plays a role in other β-oxidation reactions in peroxisomes, such as auxin metabolism. Analysis of PNC1 and PNC2 repression lines further indicates that no other ATP generating systems exist inside plant peroxisomes and that ATP import is the only way to supply the peroxisomal matrix with ATP.     

Proteome analysis of dermal fibroblasts cultured in vitro from human healthy subjects of different ages

Aging is a complex multifactorial process still far from being completely understood. The aim of the present study was to compare the proteome of in vitro cultured dermal fibroblasts from healthy subjects of different ages (i.e. 15 +/- 2, 41 +/- 4 and 82 +/- 3 years old). Proteins of the cell layer were separated by two-dimensional electrophoresis and protein identification was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry; moreover, synthetic gels were qualitatively and quantitatively analyzed by Melanie 3 software. Our study did not reveal any protein typical of any one age group. On the other hand, we observed 38 proteins exhibiting more than three-fold reproducible variations with aging, some (45%) being reduced such as F-actin capping protein alpha1, proteasome subunit alpha type 3, heat shock protein 27, ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial thioredoxin-dependent peroxide reductase, cathepsin B, glutathione S-transferase P, cyclophilin A and calgizzarin. In contrast, T-complex protein 1, probable protein disulfide isomerase ER60, phosphoglycerate kinase 1, Ran-specific GTPase-activating protein, proteasome subunit alpha type 5, triosephosphate isomerase and superoxide dismutase (Mn) increased with age. Furthermore, annexin 1, elongation factor 1beta, proteasome activator complex subunit 1, phosphoglycerate mutase, superoxide dismutase (Cu-Zn) and cofilin, exhibited the highest levels in adult cells; whereas, septin 2 homolog, RNA-binding protein regulatory subunit and ATP synthase D chain revealed the lowest values in adults. The present investigation, underlining the complexity of the aging process, highlights the role of synthetic and degradative pathways in modulating the whole cell machinery and emphasizes that metabolic impairment with age could depend partly on different expression of a number of genes and leading to an imbalance among functional proteins.     

Seed storage oil catabolism: a story of give and take

The transition from seed to seedling is an important step in the life cycle of plants, which is fuelled primarily by the breakdown of triacylglycerol (TAG) in 'oilseed' species. TAG is stored within cytosolic oil bodies, while the pathway for fatty acid β-oxidation resides in the peroxisome. Although the enzymology of fatty acid β-oxidation has been relatively well characterised, the processes by which fatty acids are liberated from oil bodies and enter the peroxisome are less well understood and, together with metabolite, cofactor and co-substrate transporters, represent key targets for future research in order to understand co-ordination of peroxisomal metabolism with that of other subcellular compartments.     

A novel function for a redox-related LEA protein (SAG21/AtLEA5) in root development and biotic stress responses

SAG21/AtLEA5 belongs to the late embryogenesis-associated (LEA) protein family. Although it has been implicated in growth and redox responses, its precise roles remain obscure. To address this problem, we characterized root and shoot development and response to biotic stress in SAG21/AtLEA5 over-expressor (OEX) and antisense (AS) lines. AS lines exhibited earlier flowering and senescence and reduced shoot biomass. Primary root length was reduced in AS lines, as was the number of laterals relative to the primary root. Root hair number was unchanged but root hair length was proportional to SAG21/AtLEA5 expression level, with longer root hairs in OEX lines and shorter root hairs in AS, relative to wild type. Growth of the fungal nectroph, Botrytis cinerea and of a virulent bacterial pathogen (Pseudomonas syringae pv. tomato) was affected by SAG21/AtLEA5 expression; however, growth of an avirulent P.syringae strain was unaffected. A SAG21/AtLEA5-YFP fusion was localized to mitochondria, raising the intriguing possibility that SAG21 interacts with proteins involved in mitochondrial ROS signalling, which in turn, impacts on root development and pathogen responses.     

The Arabidopsis Peroxisomal ABC Transporter,Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.