YjeH Is a Novel Exporter of l-Methionine and Branched-Chain Amino Acids in Escherichia coli

Amino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified in Escherichia coli. In this study, we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of l-methionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter of l-methionine and the three branched-chain amino acids. The export of the four amino acids in the yjeH overexpression strain was competitively inhibited in relation to each other. The expression of yjeH was strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical “5 + 5” transmembrane α-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity of l-methionine export by the overexpression of yjeH in an l-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering.     

Characterization of the Intestinal Absorption of Seven Flavonoids from the Flowers of Trollius chinensis Using the Caco-2 Cell Monolayer Model

The human Caco-2 cell monolayer model was used to investigate the absorption property, mechanism, and structure-property relationship of seven representative flavonoids, namely, orientin, vitexin, 2”-O-β-L-galactopyranosylorientin, 2”-O-β-L-galactopyranosylvitexin, isoswertisin, isoswertiajaponin, and 2”-O-(2”‘-methylbutanoyl)isoswertisin from the flowers of Trollius chinensis. The results showed that these flavonoids were hardly transported through the Caco-2 cell monolayer. The compounds with 7-OCH₃ including isoswertisin, isoswertiajaponin and 2”-O-(2”‘-methylbutanoyl)isoswertisin were absorbed in a passive diffusion manner, and their absorbability was increased in the same order as their polarity. The absorption of the remaining compounds with 7-OH including orientin, vitexin, 2”-O-β-L-galactopyranosylorientin, and 2”-O-β-L-galactopyranosylvitexin involved transporter mediated efflux in addition to passive diffusion. Among the four compounds with 7-OH, those with a free hydroxyl group at C-2” such as orientin and vitexin were the substrates of P-glycoprotein (P-gp) and that with a free hydroxyl group at C-2’ such as 2”-O-β-L-galactopyranosylorientin was the substrate of multidrug resistance protein 2 (MRP2). The results of this study also implied that the absorbability of the flavonoids should be taken into account when estimating the effective components of T. chinensis.     

Toxoplasma gondii Is Dependent on Glutamine and Alters Migratory Profile of Infected Host Bone Marrow Derived Immune Cells through SNAT2 and CXCR4 Pathways

The obligate intracellular parasite, Toxoplasma gondii, disseminates through its host inside infected immune cells. We hypothesize that parasite nutrient requirements lead to manipulation of migratory properties of the immune cell. We demonstrate that 1) T. gondii relies on glutamine for optimal infection, replication and viability, and 2) T. gondii-infected bone marrow-derived dendritic cells (DCs) display both “hypermotility” and “enhanced migration” to an elevated glutamine gradient in vitro. We show that glutamine uptake by the sodium-dependent neutral amino acid transporter 2 (SNAT2) is required for this enhanced migration. SNAT2 transport of glutamine is also a significant factor in the induction of migration by the small cytokine stromal cell-derived factor-1 (SDF-1) in uninfected DCs. Blocking both SNAT2 and C-X-C chemokine receptor 4 (CXCR4; the unique receptor for SDF-1) blocks hypermotility and the enhanced migration in T. gondii-infected DCs. Changes in host cell protein expression following T. gondii infection may explain the altered migratory phenotype; we observed an increase of CD80 and unchanged protein level of CXCR4 in both T. gondii-infected and lipopolysaccharide (LPS)-stimulated DCs. However, unlike activated DCs, SNAT2 expression in the cytosol of infected cells was also unchanged. Thus, our results suggest an important role of glutamine transport via SNAT2 in immune cell migration and a possible interaction between SNAT2 and CXCR4, by which T. gondii manipulates host cell motility.     

The coiled-coil domain of Escherichia coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division

The FtsLB complex is a key regulator of bacterial cell division, existing in either an off state or an on state, which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a “detuned” structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the “Y-model”) in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model (“I-model”). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction.     

A Chimera Carrying the Functional Domain of the Orphan Protein SLC7A14 in the Backbone of SLC7A2 Mediates Trans-stimulated Arginine Transport

In human skin fibroblasts, a lysosomal transport system specific for cationic amino acids has been described and named system c. We asked if SLC7A14 (solute carrier family 7 member A14), an orphan protein assigned to the SLC7 subfamily of cationic amino acid transporters (CATs) due to sequence homology, may represent system c. Fusion proteins between SLC7A14 and enhanced GFP localized to intracellular vesicles, co-staining with the lysosomal marker LysoTracker®. To perform transport studies, we first tried to redirect SLC7A14 to the plasma membrane (by mutating putative lysosomal targeting motifs) but without success. We then created a chimera carrying the backbone of human (h) CAT-2 and the protein domain of SLC7A14 corresponding to the so-called “functional domain” of the hCAT proteins, a protein stretch of 81 amino acids that determines the apparent substrate affinity, sensitivity to trans-stimulation, and (as revealed in this study) pH dependence. The chimera mediated arginine transport and exhibited characteristics similar but not identical to hCAT-2A (the low affinity hCAT-2 isoform). Western blot and microscopic analyses confirmed localization of the chimera in the plasma membrane of Xenopus laevis oocytes. Noticeably, arginine transport by the hCAT-2/SLC7A14 chimera was pH-dependent, trans-stimulated, and inhibited by α-trimethyl-l-lysine, properties assigned to lysosomal transport system c in human skin fibroblasts. Expression analysis showed strong expression of SLC7A14 mRNA in these cells. Taken together, these data strongly suggest that SLC7A14 is a lysosomal transporter for cationic amino acids.     

Developmental regulation of amino acid transport in Neurospora crassa

Conidia of Neurospora crassa exhibit an ability to transport various amino acids against a concentration gradient. The conidial transport system has previously been characterized in terms of kinetics, competitions, and genetic control. This study describes the development of a new and highly active transport capability which is elaborated during the early stages of development but prior to evident germination. It has been named “postconidial” transport activity and represents as much as 20-fold greater initial rates as compared to those observed with conidia. Development of the postconidial transport activity requires protein synthesis and can be partially repressed when the substrate amino acid is present during the developmental preincubation period. A mutant has been utilized which exhibits normal conidial but fails to develop normal postconidial transport activity for any amino acid examined. Although temperature optimum and pH dependence are similar in conidial and postconidial systems, there is evidence that the new activity is not a simple amplification of an existing capability. This is reflected as a change in competition patterns between particular amino acids as development proceeds.     

Stroke Code Improves Intravenous Thrombolysis Administration in Acute Ischemic Stroke

Background and Purpose

Timely intravenous (IV) thrombolysis for acute ischemic stroke is associated with better clinical outcomes. Acute stroke care implemented with “Stroke Code” (SC) may increase IV tissue plasminogen activator (tPA) administration. The present study aimed to investigate the impact of SC on thrombolysis.

Methods

The study period was divided into the “pre-SC era” (January 2006 to July 2010) and “SC era” (August 2010 to July 2013). Demographics, critical times (stroke symptom onset, presentation to the emergency department, neuroimaging, thrombolysis), stroke severity, and clinical outcomes were recorded and compared between the two eras.

Results

During the study period, 5957 patients with acute ischemic stroke were admitted; of these, 1301 (21.8%) arrived at the emergency department within 3 h of stroke onset and 307 (5.2%) received IV-tPA. The number and frequency of IV-tPA treatments for patients with an onset-to-door time of <3 h increased from the pre-SC era (n = 91, 13.9%) to the SC era (n = 216, 33.3%) (P<0.001). SC also improved the efficiency of IV-tPA administration; the median door-to-needle time decreased (88 to 51 min, P<0.001) and the percentage of door-to-needle times ≤60 min increased (14.3% to 71.3%, P<0.001). The SC era group tended to have more patients with good outcome (modified Rankin Scale ≤2) at discharge (49.5 vs. 39.6%, P = 0.11), with no difference in symptomatic hemorrhage events or in-hospital mortality.

Conclusion

The SC protocol increases the percentage of acute ischemic stroke patients receiving IV-tPA and decreases door-to-needle time.     

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.”     

Gas Chromatography-Mass Spectrometry-Based Metabolite Profiling of Salmonella enterica Serovar Typhimurium Differentiates between Biofilm and Planktonic Phenotypes

The aim of this study was to utilize gas chromatography coupled with mass spectrometry (GC-MS) to compare and identify patterns of biochemical change between Salmonella cells grown in planktonic and biofilm phases and Salmonella biofilms of different ages. Our results showed a clear separation between planktonic and biofilm modes of growth. The majority of metabolites contributing to variance between planktonic and biofilm supernatants were identified as amino acids, including alanine, glutamic acid, glycine, and ornithine. Metabolites contributing to variance in intracellular profiles were identified as succinic acid, putrescine, pyroglutamic acid, and N-acetylglutamic acid. Principal-component analysis revealed no significant differences between the various ages of intracellular profiles, which would otherwise allow differentiation of biofilm cells on the basis of age. A shifting pattern across the score plot was illustrated when analyzing extracellular metabolites sampled from different days of biofilm growth, and amino acids were again identified as the metabolites contributing most to variance. An understanding of biofilm-specific metabolic responses to perturbations, especially antibiotics, can lead to the identification of novel drug targets and potential therapies for combating biofilm-associated diseases. We concluded that under the conditions of this study, GC-MS can be successfully applied as a high-throughput technique for “bottom-up” metabolomic biofilm research.     

Deletion of Genes Encoding Arginase Improves Use of “Heavy” Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast

The use of “heavy” isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry in the fission yeast Schizosaccharomyces pombe is hindered by the fact that under normal conditions, arginine is extensively catabolized in vivo, resulting in the appearance of “heavy”-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This “arginine conversion problem” significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when ¹³C₆-arginine (Arg-6) is used for labeling, it is less successful when ¹³C₆ ¹⁵N₄-arginine (Arg-10), a theoretically preferable label, is used. In particular, we find that with this method, “heavy”-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of ¹³C₅ ¹⁵N₂-arginine (Arg-7) in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes car1+ and aru1+ prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC.     

Molecular interactions involved in proton-dependent gating in KcsA potassium channels

The bacterial potassium channel KcsA is gated open by the binding of protons to amino acids on the intracellular side of the channel. We have identified, via channel mutagenesis and x-ray crystallography, two pH-sensing amino acids and a set of nearby residues involved in molecular interactions that influence gating. We found that the minimal mutation of one histidine (H25) and one glutamate (E118) near the cytoplasmic gate completely abolished pH-dependent gating. Mutation of nearby residues either alone or in pairs altered the channel’s response to pH. In addition, mutations of certain pairs of residues dramatically increased the energy barriers between the closed and open states. We proposed a Monod–Wyman–Changeux model for proton binding and pH-dependent gating in KcsA, where H25 is a “strong” sensor displaying a large shift in pK_a between closed and open states, and E118 is a “weak” pH sensor. Modifying model parameters that are involved in either the intrinsic gating equilibrium or the pK_a values of the pH-sensing residues was sufficient to capture the effects of all mutations.     

The decisive step in betaxanthin biosynthesis is a spontaneous reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris “Golden Beet”) showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris “Altamo”) plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.     

The distribution of different classes of nuclear localization signals (NLSs) in diverse organisms and the utilization of the minor NLS-binding site inplantnuclear import factor importin-α

The specific recognition between the import receptor importin-α and the nuclear localization signals (NLSs) is crucial to ensure the selective transport of cargoes into the nucleus. NLSs contain 1 or 2 clusters of positively charged amino acids, which usually bind to the major (monopartite NLSs) or both minor and major NLS-binding sites (bipartite NLSs). In our recent study, we determined the structure of importin-α1a from rice (Oryza sativa), and made 2 observations that suggest an increased utilization of the minor NLS-binding site in this protein. First, unlike the mammalian protein, both the major and minor NLS-binding sites are auto-inhibited in the unliganded rice protein. Second, we showed that NLSs of the “plant-specific” class preferentially bind to the minor NLS-binding site of rice importin-α. Here, we show that a distinct group of “minor site-specific” NLSs also bind to the minor site of the rice protein. We further show a greater enrichment of proteins containing these “plant-specific” and “minor site-specific” NLSs in the rice proteome. However, the analysis of the distribution of different classes of NLSs in diverse eukaryotes shows that in all organisms, the minor site-specific NLSs are much less prevalent than the classical monopartite and bipartite NLSs.     

Evidence for amino Acid-h co-transport in oat coleoptiles

Microelectrode and tracer techniques were used to test for possible amino acid-H⁺ co-transport in coleoptiles of Avena sativa L. cv. “Garry.” The amino acid analogue α-aminoisobutyric acid (AIB) caused transient depolarization of the membrane potential. The absolute magnitude of the maximum depolarization was affected by the same factors that affected AIB transport. Both increased with higher concentrations of AIB, increased with higher acidities in the medium, and were enhanced by indoleacetic acid (which hyperpolarized the membrane potential). AIB transport was reduced as K⁺ concentrations in the medium were increased and by the metabolic inhibitor NaN₃, both of which reduce membrane potentials. Our data fit an amino acid-H⁺ co-transport model in which transport is controlled by both the membrane potential and proton concentration components of the chemical potential difference of protons across the coleoptile cell membrane.     

Engineering a Disulfide Bond in the Lid Hinge Region of Rhizopus chinensis Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t _1/2 value at 60°C and a 7°C increase of T _m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k _cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.     

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC₂₀ values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca²⁺-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl₂ by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca²⁺-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca²⁺ influx through L-type Ca²⁺ channels and intracellular Ca²⁺ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.     

Characterization of an ATP Translocase Identified in the Destructive Plant Pathogen “Candidatus Liberibacter asiaticus”

ATP/ADP translocases transport ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate intracellular bacteria. All bacterial ATP/ADP translocases characterized thus far have been found in endosymbionts of protozoa or pathogens of higher-order animals, including humans. A putative ATP/ADP translocase was uncovered during the genomic sequencing of the intracellular plant pathogen “Candidatus Liberibacter asiaticus,” the causal agent of citrus huanglongbing. Bioinformatic analysis of the protein revealed 12 transmembrane helices and predicted an isoelectric point of 9.4, both of which are characteristic of this family of proteins. The “Ca. Liberibacter asiaticus” gene (nttA) encoding the translocase was subsequently expressed in Escherichia coli and shown to enable E. coli to import ATP directly into the cell. Competition assays with the heterologous E. coli system demonstrated that the translocase was highly specific for ATP and ADP but that other nucleotides, if present in high concentrations, could also be taken up and/or block the ability of the translocase to import ATP. In addition, a protein homologous to NttA was identified in “Ca. Liberibacter solanacearum,” the bacterium associated with potato zebra chip disease. This is the first reported characterization of an ATP translocase from “Ca. Liberibacter asiaticus,” indicating that some intracellular bacteria of plants also have the potential to import ATP directly from their environment.Citrus huanglongbing (HLB), also known as citrus greening, is a disease of citrus that was first reported in China in the early 20th century (33) and identified in the United States in August 2005 in South Florida (22). As it spread rapidly across Florida, HLB has caused substantial economic losses to the citrus industry, and now other citrus-producing states may be in danger as well. The effects of this disease range from mild to severe and include symptoms such as yellow shoots, blotchy mottles on leaves, vein yellowing and corking, lopsided fruit with aborted seeds, early fruit dropping, and limb dieback, which can ultimately lead to the total loss of the infected tree. The disease has been associated with three species of bacteria known as “Candidatus Liberibacter” species. Each of the three “Ca. Liberibacter” species was discovered and named based on its presumptive origin, with “Ca. Liberibacter asiaticus” being found in Asia, “Ca. Liberibacter africanus” in Africa (13), and “Ca. Liberibacter americanus” in South America (24). A fourth species, known as “Ca. Liberibacter solanacearum,” is genetically related, although it is not naturally associated with HLB in citrus plants (16). “Ca. Liberibacter solanacearum” is associated with the emerging zebra chip disease of potatoes and tomatoes (15). “Ca. Liberibacter” species are Gram-negative, fastidious alphaproteobacteria (13) that reside in the sieve tube elements of infected plants (23). The same bacteria found in citrus plants have also been found in two phloem-feeding insects, the Asian citrus psyllid (Diaphorina citri) and the African citrus psyllid (Trioza erytreae), which act as vectors for the disease (for recent reviews, see references 3 and 9). Since insects that carry the pathogen do not have a shortened life span or other adverse effects (12), “Ca. Liberibacter” is thought to act more as an endosymbiont than as a pathogen in insects. There is no known cure for HLB, and current management strategies include elimination of infected trees and methods aimed at vector control. Because of the rapid spread and devastating consequences of infection with “Ca. Liberibacter,” understanding this obligate intracellular pathogen will be critical for the survival of the citrus industry.Recently, the complete genome sequence of “Ca. Liberibacter asiaticus” was obtained via metagenomics (5). Within this “Ca. Liberibacter asiaticus” genome, an open reading frame encoding a putative ATP/ADP translocase was found. Translocases are enzymes that aid in the transport of molecules, in this case adenosine phosphate, across a cell membrane. These adenylate transporters can be placed into one of three groups based upon where they reside. The first group was discovered in mitochondria and is involved in transporting the ATP synthesized in the mitochondrial matrix to the cytosol of the cell (28). The second type of transporter is found in plant plastids (19, 21, 31). In contrast to the mitochondrial transporters, which transport ATP to the cytosol, this set of transporters import ATP from the cytosol. Their function is to provide the stroma with a supply of cytosolic ATP in order to facilitate many of the anabolic reactions that take place there. The third set of transporters was originally discovered in the obligate intracellular bacterium Rickettsia prowazekii (30). Similar to their plastid counterparts, these transporters import ATP from the host cell''s cytosol and translocate it into the bacterial cell. Bacteria that posses this enzyme can act as “energy parasites” and import ATP directly from their hosts.Since its discovery in Rickettsia, the ATP/ADP translocase has been identified in other obligate intracellular parasites of animals, such as Chlamydia psittaci and Lawsonia intracellularis (11, 20), in addition to some protist endosymbionts, such as Caedibacter caryophilus and “Protochlamydia amoebophila” (4, 10). Analyses of the translocase proteins in these bacteria have demonstrated that certain translocase homologs can be used by the cell to import nucleotides other than ATP (2, 4, 10, 26), and thus, the family of proteins has come to be known more generally as nucleotide transporters. In spite of all of the previous research in this area, an ATP/ADP translocase from a bacterial plant pathogen has yet to be characterized. Here, we present the first characterization of a nucleotide transport protein (NttA) from the obligate intracellular plant pathogen “Ca. Liberibacter asiaticus.”     

Cellular senescence, radiation damage to mitochondria, and the compensatory response in ripening pear fruits

A compensatory response, viz. in vivo recovery from radiation damage to mitochondria, occurs in preclimacteric pear fruits (Pyrus communis L.) treated with ionizing radiation. The compensatory response is absent or markedly impaired in senescent fruits irradiated at or near the climacteric peak. Senescent cells failed to recover from harmful effects of radiation on: 1) mitochondrial yield, 2) in vivo incorporation of amino acids into mitochondrial protein, and 3) mitochondrial respiratory control and ADP/O. A diminished response to “split-dose” irradiation and a delayed rate of recovery confirmed the degeneracy and loss of compensatory power with cell age.

A loss of restorative activity, especially in mitochondria that supply the cell with essential energy, may underlie the more obvious signs of cumulative stress that accompany cellular senescence. Use of ionizing radiation as an investigative tool and the molecular implications of radiation damage, recovery, and cellular senescence are discussed.

     

Cloning and Inactivation of a Gene Essential to Inorganic Carbon Transport of Synechocystis PCC6803

A clone (HP-1) which transforms the high CO₂-requiring mutant (RKb) of Synechocystis PCC6803 defective in inorganic carbon transport to the wild-type (WT) phenotype was isolated from a WT genomic library. The clone contained a 5.4 kilobase-pair DNA insert. Complementation tests with subclones derived from HP-1 allowed the mutation in RKb to be located within 141 base-pair nucleotides. Sequencing of nucleotides in this region revealed an open reading frame encoding a hydrophobic protein consists of 80 amino acids. A defined mutant (M9) constructed by inactivating this putative inorganic carbon transport gene, designated ictA, was unable to transport CO₂ and HCO₃⁻ into the intracellular inorganic carbon pool. Cloning and sequence analysis of the respective RKb gene revealed a base substitution which generates a stop codon in the middle of ictA.