Tyrosine decarboxylase activity of Enterococcus mundtii: new insights into phenotypic and genetic aspects

Few information is available about the tyraminogenic potential of the species Enterococcus mundtii. In this study, two plant‐derived strains of E. mundtii were selected and investigated to better understand the phenotypic behaviour and the genetic mechanisms involved in tyramine accumulation. Both the strains accumulated tyramine from the beginning of exponential phase of growth, independently on the addition of tyrosine to the medium. The strains accumulated also 2‐phenylethylamine, although with lower efficiency and in greater extent when tyrosine was not added. Accordingly, the tyrosine decarboxylase (tyrDC) gene expression level increased during the exponential phase with tyrosine added, while it remained constant and high without precursor. The genetic organization as well as sequence identity levels of tyrDC and tyrosine permease (tyrP) genes indicated a correlation with those of phylogenetically closer enterococcal species, such as E. faecium, E. hirae and E. durans; however, the gene Na+/H+ antiporter (nhaC) that usually follow tyrP is missing. In addition, BLAST analysis revealed the presence of additional genes encoding for decarboxylase and permease in the genome of several E. mundtii strains. It is speculated the occurrence of a duplication event and the acquisition of different specificity for these enzymes that deserves further investigations.     

Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240

We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K _m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K _m value was 6.80 mM. In the presence of 3 M KHCO₃ and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K _m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.     

Is the egg size determining gene,Esd, on the W chromosome identical with the sex-linked giant egg gene,Ge, in the silkworm?

The presence of the egg size-determining (Esd) gene, which acts as a quantitative gene, on the W chromosome of the silkworm was revealed in a previous study by using two types of triploid females, ZZW and ZWW, (Kawamura, Genetica 76: 195–201). The females with the sex-linked giant egg (Ge) gene deposit eggs as large as those laid by tetraploids. If the Ge mutant is induced by translocation of a fragment of the W carrying Esd onto the Z by chance, the egg size increase in the Ge strain and in tetraploids may be easily explained by the double dose of Esd. The measurement of the length of the Z-W bivalent in oocytes showed that the Z of the Ge strain was much longer than that of the other strains which do not carry the Ge gene. The result suggests that the Ge gene is identical with the Esd on the W chromosome of the silkworm.     

Independent evolutionary origins of functional polyamine biosynthetic enzyme fusions catalysing de novo diamine to triamine formation

We have identified gene fusions of polyamine biosynthetic enzymes S‐adenosylmethionine decarboxylase (AdoMetDC, speD) and aminopropyltransferase (speE) orthologues in diverse bacterial phyla. Both domains are functionally active and we demonstrate the novel de novo synthesis of the triamine spermidine from the diamine putrescine by fusion enzymes from β‐proteobacterium Delftia acidovorans and δ‐proteobacterium Syntrophus aciditrophicus, in a ΔspeDE gene deletion strain of Salmonella enterica sv. Typhimurium. Fusion proteins from marine α‐proteobacterium Candidatus Pelagibacter ubique, actinobacterium Nocardia farcinica, chlorobi species Chloroherpeton thalassium, and β‐proteobacterium D. acidovorans each produce a different profile of non‐native polyamines including sym‐norspermidine when expressed in Escherichia coli. The different aminopropyltransferase activities together with phylogenetic analysis confirm independent evolutionary origins for some fusions. Comparative genomic analysis strongly indicates that gene fusions arose by merger of adjacent open reading frames. Independent fusion events, and horizontal and vertical gene transfer contributed to the scattered phyletic distribution of the gene fusions. Surprisingly, expression of fusion genes in E. coli and S. Typhimurium revealed novel latent spermidine catabolic activity producing non‐native 1,3‐diaminopropane in these species. We have also identified fusions of polyamine biosynthetic enzymes agmatine deiminase and N‐carbamoylputrescine amidohydrolase in archaea, and of S‐adenosylmethionine decarboxylase and ornithine decarboxylase in the single‐celled green alga Micromonas.     

Single-nucleotide polymorphisms in the promoter of the growth hormone-releasing hormone receptor gene are associated with growth and reproduction traits in chickens

Growth hormone‐releasing hormone receptor (GHRHR) plays a critical role in growth hormone (GH) synthesis, release and regulation in animals. The objective of this study was to investigate variations of the chicken GHRHR gene and their associations with growth and reproduction traits in 768 Beijing You chickens. Results revealed three single nucleotide polymorphisms (SNPs) in the promoter region of the gene (g.‐1654A>G, g.‐1411A>G and g.‐142T>C). Association analysis revealed that the novel SNP g.‐1654A>G had significant effects on chicken body weight at 7, 9, 11, 13, 17 weeks of age and the age of first egg as well as egg number at 32, 36 and 40 weeks. Significant association was also observed between g.‐1411A>G and g.‐142T>C with EN24. Moreover, the age of first egg was distinctly related with g.‐142T>C (P < 0.05). Although significant statistical difference was not detected in GHRHR mRNA levels among genotypes of the SNPs (P > 0.05), strong expression variations of the gene were found between the ages 17 and 20 weeks in the population (P < 0.05). These results suggest that the three SNPs in the GHRHR promoter could be used as potential genetic markers to improve the growth and reproductive traits in chickens.     

Individual repeatability in laying behaviour does not support the migratory carry‐over effect hypothesis of egg‐size dimorphism in Eudyptes penguins

Penguins of the genus Eudyptes are unique among birds in that their first‐laid A‐egg is 54–85% the mass of their second‐laid B‐egg. Although the degree of intra‐clutch egg‐size dimorphism varies greatly among the seven species of the genus, obligate brood reduction is typical of each, with most fledged chicks resulting from the larger B‐egg. Many authors have speculated upon why Eudyptes penguins have evolved and maintained a highly dimorphic 2‐egg clutch, and why it is the first‐laid egg that is so much smaller than the second, but only recently has a testable, proximate mechanism been proposed. In most species of Eudyptes penguins females appear to initiate egg‐formation at sea during return migration to breeding colonies. In macaroni penguins E. chrysolophus, females with a shorter pre‐laying interval ashore (and thus presumably greater overlap between migration and egg‐formation) lay more dimorphic eggs, suggesting a physiological conflict may constrain growth of the earlier‐initiated A‐egg. This migratory carry‐over effect hypothesis (MCEH) was tested in eastern rockhopper penguins E. chrysocome filholi on Campbell Island, New Zealand, by recording the arrival and lay dates, body sizes, and egg masses of transponder‐tagged females over two years. Females with longer pre‐laying intervals laid less dimorphic clutches, as predicted by the MCEH. However, repeated measures of individual females revealed that within‐individual variation in egg‐size dimorphism between years was unrelated to within‐individual variation in pre‐laying interval. Egg masses, and to a lesser extent egg‐size dimorphism, were highly repeatable traits related to body size and body mass. These results and a detailed consideration of the MCEH suggest that egg‐size dimorphism in Eudyptes penguins is unlikely to be caused by a migratory carry‐over effect.     

Identification of LmUAP1 as a 20‐hydroxyecdysone response gene in the chitin biosynthesis pathway from the migratory locust,Locusta migratoria

In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N‐acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene LmCHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20‐hydroxyecdysone (20E) is still largely unclear. Here, we showed the developmental expression pattern of LmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference (RNAi) directed toward a common region of the two isoforms of LmEcR (LmEcRcom) reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection‐based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late‐response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi‐based pest control.     

Multilocus Typing of Enterocytozoon bieneusi in Pig Reveals the High Prevalence,Zoonotic Potential,Host Adaptation and Geographical Segregation in China

Enterocytozoon bieneusi is one of the most frequently diagnosed Microsporidia of humans and most animals. However, there is no information on E. bieneusi infection of pigs in Tibet and Henan, China. In this study, 1,190 fecal samples were collected from pigs in Tibet and Henan and screened for the presence of E. bieneusi. The overall prevalence of E. bieneusi infection was 54.2% (645/1,190), with differences in prevalence observed among geographical areas, ages, and pig breeds. Moreover, 10 E. bieneusi genotypes were identified based on internal transcribed spacer region genotyping, including eight known genotypes (EbpC, EbpA, CHG19, CHC5, Henan‐III, I, D, and H) and two novel genotypes (XZP‐I and XZP‐II). Multilocus sequence typing revealed 18, 7, 17, and 13 genotypes at minisatellite/microsatellite loci MS1, MS3, MS4, and MS7, respectively. Strong linkage disequilibrium (LD) and few numbers of recombination events, suggest a clonal structure of the E. bieneusi population examined in this study. The low pairwise genetic distance (F_ST) and gene flow (Nm) values indicated limited gene flow in the E. bieneusi population from different hosts, with phylogenetic, structure, and median‐joining network analyses all indicating the existence of host and geographical isolation. The identification of isolates belonging to nine human‐pathogenic genotypes indicates that pigs play an important role in the dissemination of E. bieneusi, improving our present understanding of E. bieneusi epidemiology in the studied region.     

Gene Cloning of Dihydroxyacetone Reductase from a Methylotrophic Yeast,Hansenula ofunaensis,and its Expression in Escherichia coli HB101 for Production of Optically Active 2‐Pentanol

Optically active alcohols are important building blocks as versatile chiral synthons for asymmetric syntheses of pharmaceuticals and agrochemicals. The aim of this paper is to efficiently prepare chiral 2‐pentanol by means of microorganisms. The gene of dihydroxyacetone reductase (EC 1.1.1.6) from a methylotrophic yeast, Hansenula ofunaensis, was cloned and chiral 2‐pentanol was produced by the recombinant Escherichia coli harboring the gene. The gene encoding the enzyme was cloned from an H. ofunaensis genomic library. In the deduced amino acid sequence of 364 residues, the NAD(H) binding motif and the cysteine residues that correspond to the cysteine ligands in the zinc atom were conserved, as they are in alcohol dehydrogenases from other origins. Dihydroxyacetone reductase was similar to alcohol dehydrogenases of prokaryotes. For the production of chiral compounds, an E. coli HB101 strain was transformed. The H. ofunaensis gene product, dihydroxyacetone reductase, catalyzed the NAD⁺‐dependent oxidation of 2‐pentanol to 2‐pentanone as well as the corresponding reverse reactions, showing specificity towards the secondary alcohol in (R)‐configuration. From 100 mM 2‐pentanone, (R)‐2‐pentanol (98 mM, > 99.9 % enantiometric excess, e.e.) was obtained in a 30‐min reaction with resting cells of the E. coli HB101 strain harboring the expression plasmid, pSG‐HOD1, which possesses the genes of both dihydroxyacetone reductase and glucose dehydrogenase as an NADH reproducing system. The stereospecificity changed during the reduction, depending on the pH. E. coli HB101 was also transformed by the expression plasmid, pSE‐HOD4, in which the gene of glucose dehydrogenase was removed from pSG‐HOD1, and designated as E. coli HB101 (pSE‐HOD4). E. coli HB101 (pSE‐HOD4) oxidized only (R)‐2‐pentanol in 100 mM of the racemate (R:S = 52:48), and the reaction medium was enriched with (S)‐2‐pentanol (48 mM, 98 % e.e.) after 30 min of incubation. The reaction was sufficiently promoted without the other additives. E. coli transformants expressing the gene of this enzyme could be particularly advantageous to the production of optically active 2‐pentanol.     

A cyanogenic glucoside of Trifolium repens deters oviposition by the common grass yellow Eurema mandarina

The common grass yellow Eurema mandarina (Pieridae, Coliadinae) widely inhabits Japan, feeds on various fabaceous plants such as silktree (Albizia julibrissin) and uses d ‐pinitol, a cyclitol omnipresent in Fabaceae, as a primary oviposition stimulant. However, E. mandarina has a clear host preference within the Fabaceae; for example, white clover (Trifolium repens) is a nonhost despite containing d ‐pinitol. The present study aims to identify plant chemicals in white clover that inhibit oviposition of E. mandarina. Females lay very few eggs on T. repens foliage and plastic plant models treated with a methanolic extract of the foliage. The foliage extract is fractionated by successive extraction with chloroform, isobutanol and water. None of these fractions induce egg‐laying responses. The aqueous fraction is further separated into four subfractions (Tr‐3‐1 to Tr‐3‐4) by column chromatography. Among these subfractions, females show high egg‐laying responses to Tr‐3‐1, which is known to contain d ‐pinitol. Interestingly, Tr‐3‐2, when mixed with Tr‐3‐1, significantly decreases egg‐laying responses, indicating that it contains oviposition deterrents. Chemical analyses reveal that two cyanogenic glucosides, linamarin and lotaustralin, are the major constituents of Tr‐3‐2. Authentic linamarin does not elicit egg‐laying responses and significantly inhibits female oviposition when mixed with Tr‐3‐1 at the natural concentration. Although these cyanogenic glucosides are reported to synergistically induce oviposition of a coliadine species Colias erate on white clover, we conclude that linamarin acts as an oviposition deterrent for E. mandarina, restricts its host range and regulates their differential host acceptance.     

Genetic control of α‐farnesene production in apple fruit and its role in fungal pathogenesis

Terpenes are important compounds in plant trophic interactions. A meta‐analysis of GC‐MS data from a diverse range of apple (Malus × domestica) genotypes revealed that apple fruit produces a range of terpene volatiles, with the predominant terpene being the acyclic branched sesquiterpene (E,E)‐α‐farnesene. Four quantitative trait loci (QTLs) for α‐farnesene production in ripe fruit were identified in a segregating ‘Royal Gala’ (RG) × ‘Granny Smith’ (GS) population with one major QTL on linkage group 10 co‐locating with the MdAFS1 (α‐farnesene synthase‐1) gene. Three of the four QTLs were derived from the GS parent, which was consistent with GC‐MS analysis of headspace and solvent‐extracted terpenes showing that cold‐treated GS apples produced higher levels of (E,E)‐α‐farnesene than RG. Transgenic RG fruit downregulated for MdAFS1 expression produced significantly lower levels of (E,E)‐α‐farnesene. To evaluate the role of (E,E)‐α‐farnesene in fungal pathogenesis, MdAFS1 RNA interference transgenic fruit and RG controls were inoculated with three important apple post‐harvest pathogens [Colletotrichum acutatum, Penicillium expansum and Neofabraea alba (synonym Phlyctema vagabunda)]. From results obtained over four seasons, we demonstrate that reduced (E,E)‐α‐farnesene is associated with decreased disease initiation rates of all three pathogens. In each case, the infection rate was significantly reduced 7 days post‐inoculation, although the size of successful lesions was comparable with infections on control fruit. These results indicate that (E,E)‐α‐farnesene production is likely to be an important factor involved in fungal pathogenesis in apple fruit.     

Sex pheromone of the red clover casebearer moth,Coleophora deauratella,an invasive pest of clover in Canada

The red clover casebearer, Coleophora deauratella Lienig & Zeller (Lepidoptera: Coleophoridae), is an invasive pest of Trifolium species (Fabaceae) in Canada. We identified candidate sex pheromone components from female pheromone gland extracts using coupled gas chromatographic–electroantennographic analysis detection. Three compounds elicited an electrophysiological response from antennae and were identified as: (Z)‐7‐dodecenyl acetate, (Z)‐5‐dodecenyl acetate, and (Z)‐7‐dodecen‐1‐ol. Field tests of the candidate pheromone components revealed that males were attracted to a binary mixture of (Z)‐7‐dodecenyl acetate and (Z)‐5‐dodecenyl acetate. Male moth trap capture was greatest in traps baited with lures containing 100:10 or 100:20 ratios of these pheromone components, respectively. Trap capture was reduced when (Z)‐5‐dodecenyl acetate was present below 10 or above 20% of (Z)‐7‐dodecenyl acetate. Equal numbers of male moths were captured in traps baited with 10, 100, and 1 000 μg of the attractive binary mixture. These findings allow for the development of a pheromone‐based monitoring system for this invasive pest of clover in Canada.     

SIS8, a putative mitogen‐activated protein kinase kinase kinase,regulates sugar‐resistant seedling development in Arabidopsis

Sugar signaling pathways have been evolutionarily conserved among eukaryotes and are postulated to help regulate plant growth, development and responses to environmental cues. Forward genetic screens have identified sugar signaling or response mutants. Here we report the identification and characterization of Arabidopsis thaliana sugar insensitive8 (sis8) mutants, which display a sugar‐resistant seedling development phenotype. Unlike many other sugar insensitive mutants, sis8 mutants exhibit wild‐type responses to the inhibitory effects of abscisic acid and paclobutrazol (an inhibitor of gibberellin biosynthesis) on seed germination. Positional cloning of the SIS8 gene revealed that it encodes a putative mitogen‐activated protein kinase kinase kinase (MAPKKK; At1g73660). SIS8mRNA is expressed ubiquitously among Arabidopsis organs. A UDP‐glucosyltransferase, UGT72E1 (At3g50740), was identified as an interacting partner of SIS8 based on a yeast two‐hybrid screen and in planta bimolecular fluorescence complementation. Both SIS8–yellow fluorescent protein (YFP) and UGT72E1–YFP fusion proteins localize to the nucleus when transiently expressed in tobacco leaf cells. T‐DNA insertions in At3g50740 cause a sugar‐insensitive phenotype. These results indicate that SIS8, a putative MAPKKK, is a regulator of sugar response in Arabidopsis and interacts with a UDP‐glucosyltransferase in the nucleus.     

Modification of lipid A structure and activity by the introduction of palmitoyltransferase gene to the acyltransferase‐knockout mutant of Escherichia coli

Lauroyltransferase gene (lpxL), Myristoyltransferase gene (lpxM) and palmitoyltransferase gene (crcA) of Escherichia coli BL21 were independently disrupted by the insertional mutations. The knockout mutant of two transferase genes (lpxL and crcA) produced lipid A with no lauric or palmitic acids and only a little amount of myristic acid. The mutant was susceptible to polymyxin B, but showed comparable growth with the wild‐type strain at 30°C. The palmitoyltransferase gene from E. coli (crcA) or Salmonella (pagP) was amplified by PCR, cloned in pUC119, and transferred into the double‐knockout mutant by transformation. The transformant contained palmitic acid in the lipid A, and recovered resistance to polymyxin B. Mass spectrometric analysis revealed that palmitic acid was linked to the hydroxyl group of 3‐hydroxymyristic acid at C‐2 position of proximal (reducing‐end) glucosamine. LPS from the double‐knockout mutant showed reduced IL‐6‐inducing activity to macrophage‐like line cells compared to that of the wild‐type strain, and the activity was only slightly restored by the introduction of palmitic acid to the lipid A. These results suggested that the introduction of one palmitic acid was enough to recover the integrity of the outer membrane, but not enough for the stimulation of macrophages.

     

Sequence analysis of the gene encoding a novel l-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases

The gene (ddc) encoding a novel enzyme, l-2,4-diaminobutyrate decarboxylase (DABA-DC; EC 4.1.1.-) in Acinetobacter baumannii was sequenced, and an open reading frame of 1,530 nucleotides was detected. The sequence of 20 N-terminal amino acids of purified DABA-DC and of its proteolytic peptide fragments coincided with those deduced from the nucleotide sequence determined. Comparison of the predicted amino acid sequence of the A. baumannii enzyme with those of other pyridoxal 5′-phosphate-dependent decarboxylases revealed significant similarity to the group II amino acid decarboxylases and conservation of the putative pyridoxal 5′-phosphate-binding domain. Received:20 February 1996 / Accepted 15 April 1996     

Association of single nucleotide polymorphism in melatonin receptor 1A gene with egg production traits in Yangzhou geese

In the present study the melatonin receptor 1A gene (MTNR1A) was proposed to be a candidate gene for egg production in Yangzhou geese. A total of 210 goose blood samples were collected to investigate the association of the MTNR1A gene with the number of eggs produced. Using a direct sequencing method, a single nucleotide polymorphism (SNP; g.177G>C) was detected in the 5? regulatory region of the MTNR1A gene (Genbank ss1985399687). Two alleles (G and C) and three genotypes were identified. Association analysis results showed that the g.177G>C SNP significantly affected the level of egg production within a 34‐week egg‐laying period (P < 0.05). Furthermore, the geese with the GG genotype produced significantly more eggs compared to the geese with the CC genotype. Quantitative real‐time PCR analysis showed that the MTNR1A gene was highly expressed in small intestine, granulosa cell and ovary compared to other examined tissues. In addition, the mRNA expression level of MTNR1A in ovary indicated that significantly higher expression levels were recorded for geese with the GG genotype compared to those with the CC genotype. Moreover, a luciferase reporter assay showed that the CC genotype had significantly lower promoter activity than did GG. These results suggest that the identified SNP in the MTNR1A gene may influence the number of eggs produced and mRNA expression levels in Yangzhou geese and could be considered as a useful molecular marker in goose selection and improvement, especially for egg production.