Widespread presence of "bacterial-like" PPP phosphatases in eukaryotes

Background

In eukaryotes, PPP (p rotein p hosphatase P) family is one of the two known protein phosphatase families specific for Ser and Thr. The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases.

Results

We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some α-Proteobacteria, including Rhizobiales, Rhodobacterales and Rhodospirillaceae. This group is therefore termed Rhi zobiales / Rh odobacterales / Rh odospirillaceae-l ike ph osphatases, or Rhilphs. Phosphatases of the other group are found in Viridiplantae, Rhodophyta, Trypanosomatidae, Plasmodium and some fungi. They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia, and are termed She wanella-l ike ph osphatases, or Shelphs. Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed A paH-l ike ph osphatases, or Alphs. Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades. In addition, comparison of primary structures revealed a previously unrecognised (I/L/V)D(S/T)G motif, conserved in all bacterial and "bacterial-like" eukaryotic PPPs, but not in "conventional" eukaryotic and archaeal PPPs.

Conclusions

Our findings demonstrate that many eukaryotes possess diverse "bacterial-like" PPP phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined.

     

Biotechnological potential of a rhizosphere <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">aeruginosa</Emphasis> strain producing phenazine-1-carboxylic acid and phenazine-1-carboxamide

Bacterial phenazine metabolites belong to a group of nitrogen-containing heterocyclic compounds with antimicrobial activities. In this study, a rhizosphere Pseudomonas aeruginosa strain PA1201 was isolated and identified through 16S rDNA sequence analysis and fatty acid profiling. PA1201 inhibited the growth of various pathogenic microorganisms, including Rhizotonia solani, Magnaporthe grisea, Fusarium graminearum, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Staphylococcus aureus. High Performance Liquid Chromatography showed that PA1201 produced high levels of phenazine-1-carboxylic acid (PCA), a registered green fungicide ‘Shenqinmycin’ with the fermentation titers of 81.7 mg/L in pigment producing medium (PPM) and 926.9 mg/L in SCG medium containing soybean meal, corn steep liquor and glucose. In addition, PA1201 produced another antifungal metabolite, phenazine-1-carboxaminde (PCN), a derivative of PCA, with the fermentation titers of 18.1 and 489.5 mg/L in PPM and SCG medium respectively. To the best of our knowledge, PA1201 is a rhizosphere originating P. aeruginosa strain that congenitally produces the highest levels of PCA and PCN among currently reported P. aeruginosa isolates, which endows it great biotechnological potential to be transformed to a biopesticide-producing engineering strain.     

Global insight into the distribution of velvet-like B protein in <Emphasis Type="Italic">Cochliobolus</Emphasis> species and implication in pathogenicity and fungicide resistance

The Cochliobolus genus consist of over 55 species among which the 5 most devastating are Cochliobolus carbonum, Cochliobolus heterostrophus, Cochliobolus miyabeanus, Crocus sativus and Cochliobolus lunatus causing damages in sorghum, wheat, rice, maize, cassava and soybean estimated at over 10 billion USD per annum worldwide. The dynamic pathogenicity of Cochliobolus species and the plethora of infected hosts is determined by the evolution of virulence determinants such as the velvet-like B protein (VelB). Nonetheless, the knowledge on the distribution of Cochliobolus VelB and its implication in pathogenicity and fungicide resistance are often lacking. By scanning through the annotated genomes of C. lunatus, C. heterostrophus, C. carbonum, C. victoriae, C. sativus and C. miyabeanus, it is revealed that the numbers of ortholog VelB and cognates vary. By using the phylogenetic approach, it is established that the diversification rates among velvet-domain-containing proteins for phytopathogenic Cochliobolus species could impact differently on their oxidant and fungicide resistance potentials, ability to form appressoria-like structures and infection pegs during infection. This study provides new insights into the pathogenicity evolution of Cochliobolus species at the VelB locus which is relevant for designing effective strategies for durable management of Cochliobolus diseases.     

Elemental composition and flavonol content of <Emphasis Type="Italic">Pentaphylloides fruticosa</Emphasis> (L.) O. Schwarz in connection with plant age

The elemental composition and flavonol content of Pentaphylloides fruticosa (L.) O. Schwartz under the conditions of the Altai mountains are investigated. Some differences in the concentrations of elements and flavonols were detected in the leaves of P. fruticosa plants of different age classes corresponding to two periods of development: pregenerative (immature and virginal plants) and generative (young, middle-aged and old generative individuals). The maximum content of flavonols, in particular quercetin, in the leaves of P. fruticosa corresponds to the young generative age. A very high correlation between the flavonol content and the concentrations of Mn, Zn, Mo, and Se was revealed.     

The Peptide-Mediated Interactions Between Human Osteoclast-Stimulating Factor and Its Partner Proteins in Osteoporosis: Which Binds to Which?

Human osteoclast-stimulating factor (hOSF) is an intracellular protein produced by osteoclasts that induces osteoclast formation and bone resorption in osteoporosis by recruiting multiple signaling complexes with its diverse biological partners through peptide-mediated interactions (PMIs). The protein contains a modular peptide-recognition domain of Src homology 3 (SH3), which can recognize and bind to the polyproline regions of its partner proteins, as well as two N-terminal polyproline segments, which can be recognized and bound by the SH3 domains of its partner proteins. Here, we attempted to elucidate the complicated PMIs between the different SH3 domains and different polyprolines of hOSF and its three known interacting partners, i.e. proto-oncogene tyrosine-protein kinase (c-Src), survival motor neuron (SMN) and Src-associated in mitosis, 68 kD (Sam68). A total of 29 peptide segments containing the SH3-binding motif PXXP were extracted from these partner proteins, which are potential binding sites of hOSF SH3 domain, while the c-Src kinase also possesses a SH3 domain that may recognize and bind the two polyproline peptides at hOSF N-terminus. Structural bioinformatics analysis identified a number of biologically functional PMI candidates between these SH3 domains and these polyproline peptides, which were then tested in vitro using fluorescence spectroscopy assays. Consequently, it is found that (i) hOSF SH3 domain exhibits strong binding potency to two Sam68 peptides ³⁶RQPPLPHR⁴³ (K _d = 13.7 μM) and ⁴²⁵APPARPVK⁴³² (K _d = 3.2 μM) as well as moderate affinity to three SMN peptides ¹⁹³FLPPPPPM²⁰⁰ (K _d = 56.2 μM), ²³⁵PFPSGPPI²⁴² (K _d = 28.4 μM) and ²⁴⁶PPPICPDS²⁵³ (K _d = 74.5 μM), but has only weak or no binding to c-Src peptides. Instead, a proline-rich region at hOSF N-terminal that contains two overlapping peptides (³KPPPKPVK¹⁰ and ⁶PKPVKPGQ¹³) can be bound tightly by c-Src SH3 domain with high and moderate affinity (K _d = 5.8 and 39.6 μM, respectively).     

Genome-wide identification and characterization of the RIO atypical kinase family in plants

Members of the right open reading frame (RIO) atypical kinase family are present in all three domains of life. In eukaryotes, three subfamilies have been identified: RIO1, RIO2, and RIO3. Studies have shown that the yeast and human RIO1 and RIO2 kinases are essential for the biogenesis of small ribosomal subunits. Thus far, RIO3 has been found only in multicellular eukaryotes. In this study, we systematically identified members of the RIO gene family in 37 species representing the major evolutionary lineages in Viridiplantae. A total of 84 RIO genes were identified; among them, 41 were classified as RIO1 and 43 as RIO2. However, no RIO3 gene was found in any of the species examined. Phylogenetic trees constructed for plant RIO1 and RIO2 proteins were generally congruent with the species phylogeny. Subcellular localization analyses showed that the plant RIO proteins were localized mainly in the nucleus and/or cytoplasm. Expression profile analysis of rice, maize, and Arabidopsis RIO genes in different tissues revealed similar expression patterns between RIO1 and RIO2 genes, and their expression levels were high in certain tissues. In addition, the expressions of plant RIO genes were regulated by two drugs: mycophenolic acid and actinomycin D. Function prediction using genome-wide coexpression analysis revealed that most plant RIO genes may be involved in ribosome biogenesis. Our results will be useful for the evolutionary analysis of the ancient RIO kinase family and provide a basis for further functional characterization of RIO genes in plants.     

Towards the mode of action of <Emphasis Type="Italic">Strobilanthes crispus</Emphasis> through integrated computational and experimental analyses

Bioactive sub-fractions from the tropical herbal plant Strobilanthes crispus (S. crispus) has been shown to induce apoptosis of breast cancer cells in vitro and reduce tumor size in vivo by our earlier studies. We have recently isolated five major compounds from S. crispus sub-fraction, namely lutein, β-sitosterol, campesterol, stigmasterol and pheophytin a. In this study, we set out to investigate each compound’s protein targets and mechanism of action through prediction of protein targets via a ligand-based target prediction protocol, Prediction IncluDinG INactives, and radioligand binding assays. The three phytosterol molecules (β-sitosterol, campesterol, stigmasterol) showed enrichment of hormone signaling GO terms [average ratio (AR) <0.01], while the SMAD signaling pathway was associated with pheophytin a (AR < 0.01). GO terms associated with retinoic acid receptor (RAR) and retinoid X receptor (RXR) were distinctly represented by protein targets of lutein (AR < 0.01). All members of the RAR/RXR family of proteins were predicted to be targeted by lutein, a feature that was not present in the other four S. crispus-derived compounds. Radioligand binding assay in vitro validated that lutein showed higher binding affinity with RXRα (IC₅₀: 5.74 µM; K_i: 4.55 µM) than RARα (IC₅₀: 25.1 µM; K_i: 14 µM). Molecular docking analysis demonstrated that lutein could occupy a large hydrophobic cavity of the hRXRα-LBP crystal structure mainly through hydrophobic interactions with leucine and isoleucine residues, and also hydrogen bond between a hydroxyl group of lutein with Glu²³⁹. Our findings suggest that lutein-RXRα interaction might play a role in the anti-breast cancer effects rendered by S. crispus.     

Nucleotide Correspondence between Protein-Coding Sequences of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains 26695 and J99

Comparison of ORFs between H. pylori strains 26695 and J99 showed that transitions (more than 3%) prevail over transversions (less than 1%). The predominance of transitions was explained by the high rates of cytosine replacement by thymine in the coding (3.5–5.3%) and noncoding (2.9–3.9%) DNA strands. The proportion of transversion-type correspondences (A → C, A → T, C → A, C → G, G → C, G → T, T → A, and T → G) did not exceed 0.84%. The highest proportion (28.3%) was observed for correspondences between C and T in ACGT-ATGT, the target site of active methyltransferase of H. pylori J99 (M.Hpy99XI). It was assumed that C → T mutations due to cytosine methylation-deamination are prevalent in H. pylori.     

Zearalenone formation by<Emphasis Type="Underline">Fusarium crookwellense</Emphasis> /Burgess,Nelson and Toussoun/ isolates from Poland and their pathogenicity towards cereals

Fusarium crookwellense /B.N. and T./ isolated from affected cereals in Poland formed zearalenone on wheat grain up to 602 mg/kg. Tested isolates have been found strong to severe pathogens of wheat, rye, triticale and barley seedlings and corn ears with pathogenicity similar to that ofF. culmorum andF. graminearum.     

Infection ability of mycelium and spores of<Emphasis Type="Underline">Microdochium</Emphasis><Emphasis Type="Underline">nivale</Emphasis> (Fr) samuels hallett to<Emphasis Type="Underline">Lolium</Emphasis><Emphasis Type="Underline">Perenne</Emphasis> L.

Mycelium and spores ofMicrodochium nivale /Syn.Fusarium nivale/ were compared according to their ability to infect plants ofLolium perenne. The experiments were carried out according to the “cold chamber” method (Cormack, Lebeau 1956 modified by Pronczuk 1987). Between these two types of inoculum significant differences were found. The spore inoculum did not give any symptoms while the mycelial inoculum incited a severe disease in plants ofLolium perenne during one month of incubation.Under laboratory conditions it was found that the spore cultures ofMicrodochium nivale grew very slowly at 0 – 1°C, whereas their growth at 18 – 20°C was very fast. Growth of the mycelial cultures was not as profoundly affected by temperatures studied as the spore ones.It was concluded that to incite a disease the spore inoculum require longer incubation time than mycelial ones. The mycelial inoculum is more useful for screening of plants for resistsance.     

The variability of critical species ofLotus L. in Iran and in the neighbouring countries I. the circle ofL. corniculatus L. andL. gebelia Vent.

The region of Iran, Iraq, Afghanistan and the neighbouring countries is important for some groups of the speciesLotus L., especially those of the circle ofL. corniculatus L. andL. gebelia Vent. The first group is represented by the speciesL. corniculatus L. with 4 subspecies (3 of which are important for this region), andL. tenuis Waldst. etKit. which here attains the eastern boundary of the continuous area of distribution, and by the eastern speciesL. krylovii Schischk. etSerg. andL. rechingeri Chrtková-?ertová. The second group is represented by the speciesL. gebelia Vent.,L. michauxianus Ser. in DC. andL. libanoticus Boiss. their areas of distribution covering mostly those regions. Most of the species show considerable variability within the species.     

Characterization of aspartate kinase and homoserine dehydrogenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> IWJ001 and systematic investigation of <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine biosynthesis

Previously we have characterized a threonine dehydratase mutant TD^F383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS^{P176S, D426E, L575W} (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK^A279T (encoded by lysC1) and a homoserine dehydrogenase mutant HD^G378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK^A279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HD^G378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD^F383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HD^G378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.     

Karyo-taxonomic study of the genus<Emphasis Type="Italic">Pseudolysimachion</Emphasis> (<Emphasis Type="Italic">Scrophulariaceae</Emphasis>) in the Czech Republic and Slovakia

Flow cytometry was used to determine ploidy levels in the Czech and Slovak taxa of the genusPseudolysimachion (W.D.J. Koch)Opiz (=Veronica auct. p.p.,Scrophulariaceae). In total, 123 populations from the Czech Republic, Slovakia, Ukraine (one locality), Austria (one locality) and Hungary (one locality) were analyzed. InP. maritimum (L.)Á. Löve etD. Löve andP. spicatum (L.)Opiz, two cytotypes were found: diploid (2n=2x=34) and tetraploid (2n=4x=68). In both species the tetraploid cytotype predominated (P. maritimum: 41 tetraploid populations out of 45;P. spicatum: 57 tetraploid populations out of 58). The two cytotypes ofP. maritimum have no taxonomic significance because ploidy level is not obviously correlated with morphology, distribution pattern or ecology. Tetraploid populations ofP. spicatum belong to two morphologically different subspecies, subsp.spicatum and subsp.fischeri Trávní?ek. The diploid cytotype (one population only) should be provisionally classified as a third subspecies ofP. spicatum, which is morphologically similar to the Asian subsp.porphyrianum (Pavlov)Trávní?ek. Only diploid plants (2n=2x=34) ofP. orchideum (Crantz)Wraber were found; all 13 populations that were analyzed belong toP. orchideum s.str. One diploid population sample ofP. spurium subsp.foliosum (Waldst. etKit.)Holub (2n=2x=34) and one tetraploid sample ofP. incanum subsp.pallens (Host)Trávní?ek (2n=4x=68) were also analyzed. In addition, three tetraploid populations of hybrid origin were investigated:P. maritimum ×P. spicatum subsp.spicatum (one population) andP. maritimum ×P. spurium subsp.foliosum (two populations). While hybrid plants ofP. maritimum ×P. spicatum arose from tetraploid parental species, plants ofP. maritimum ×P. spurium probably resulted from a cross between tetraploidP. maritimum and diploidP. spurium. The putative origin and evolutionary importance of polyploids in thePseudolysimachion are discussed.     

Rational design and analysis of an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain for high-efficiency tryptophan production

l-tryptophan (l-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient l-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the l-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG ^fbr ). To further improve the production of l-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of l-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.     

Characterization of an IAA-glucose hydrolase gene <Emphasis Type="Italic">TaTGW6</Emphasis> associated with grain weight in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In rice, the TGW6 gene determines grain weight and encodes a protein with indole-3-acetic acid (IAA)-glucose hydrolase activity. Its homolog in wheat, TaTGW6, is considered as a candidate gene related to grain development. To amplify this gene, we designed primers based on a homologous conserved domain of the rice TGW6 gene. Sequence analysis indicated that TaTGW6 comprises only one exon, with 1656 bp in total and an open reading frame of 1035 bp. Three alleles at TaTGW6 locus detected by the primer pair TG23 were designated as TaTGW6-a, TaTGW6-b and TaTGW6-c, respectively. Compared with TaTGW6-a, TaTGW6-b had a 6-bp InDel at the position 170 downstream of initiation codon, and TaTGW6-c was a null mutant. Both TaTGW6-b and TaTGW6-c could significantly increase grain size and weight other than TaTGW6-a; however, the former two alleles showed a low frequency distribution in modern varieties. TaTGW6 was located on chromosome 4AL using a recombinant inbred line population and a set of Chinese Spring nullisomic-tetrasomic lines. It was linked to the SSR locus Xbarc1047 with a genetic distance of 6.62 cM and explained 15.8–21.0 % of phenotypic variation of grain weight in four environments. Association analysis using a natural population and Chinese wheat mini-core collections additionally validated the relationship of TaTGW6 with grain weight; the gene could explain 7.7–12.4 % of phenotypic variation in three environments. Quantitative real-time PCR revealed that TaTGW6-b showed relatively lower expression than TaTGW6-a in immature grain at 20 and 30 days post-anthesis and in mature grain. The low expression of TaTGW6 generally associated with low IAA content, but with high grain weight. The novel functional marker, designated as TG23, can be used for marker-assisted selection to improve grain weight in wheat and also provides insights into the regulatory mechanism underlying grain weight.     

The causal deletions in the second exon of <Emphasis Type="Italic">An-3</Emphasis> closely associated with awn development and rice yield

Awn is one of important traits during rice domestication. To understand the development of rice awn and the roles it played in rice domestication, we preliminary mapped a major QTL An-3 for awn development using chromosome segment substitution line CSSL138 developed by introgressed genomic fragments of long-awned Guangxi common wild rice (GXCWR, Oryza rufipogon Griff.) into genetic background of short-awned indica cultivar 93–11. An-3 was then fine mapped to a 7-kb region of chromosome 8. An epidermal patterning factor-like protein gene was identified as the single candidate gene corresponding to this QTL. An-3 was showed to be an allele of RAE2 and GAD1, and negatively regulated 1000-grains weight, grain length, and length–width ratio. Comparing with the coding sequences of An-3 from CSSL138, a 2- and 4-bp frame-shift deletions in the second exon were identified in 93–11 and Nipponbare, respectively. Taken together, our results provide valuable natural variation in the alleles of An-3 between common wild rice and cultivated rice, which will be helpful in clarifying the mechanism of awn development and promoting the application of an-3 in genetic improvement of rice yield traits.     

Production of optically pure 2,3-butanediol from <Emphasis Type="Italic">Miscanthus floridulus</Emphasis> hydrolysate using engineered <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strains

2,3-Butanediol (2,3-BD) can be produced by fermentation of natural resources like Miscanthus. Bacillus licheniformis mutants, WX-02ΔbudC and WX-02ΔgldA, were elucidated for the potential to use Miscanthus as a cost-effective biomass to produce optically pure 2,3-BD. Both WX-02ΔbudC and WX-02ΔgldA could efficiently use xylose as well as mixed sugars of glucose and xylose to produce optically pure 2,3-BD. Batch fermentation of M. floridulus hydrolysate could produce 21.6 g/L d-2,3-BD and 23.9 g/L meso-2,3-BD in flask, and 13.8 g/L d-2,3-BD and 13.2 g/L meso-2,3-BD in bioreactor for WX-02ΔbudC and WX-02ΔgldA, respectively. Further fed-batch fermentation of hydrolysate in bioreactor showed both of two strains could produce optically pure 2,3-BD, with 32.2 g/L d-2,3-BD for WX-02ΔbudC and 48.5 g/L meso-2,3-BD for WX-02ΔgldA, respectively. Collectively, WX-02ΔbudC and WX-02ΔgldA can efficiently produce optically pure 2,3-BD with M. floridulus hydrolysate, and these two strains are candidates for industrial production of optical purity of 2,3-BD with M. floridulus hydrolysate.