Isozyme and morphological characteristics of the anaerobic fungus Piromyces mae isolated from the duodenum, rumen and faeces of sheep

Abstract Isolates of anaerobic fungi obtained from the rumen, duodenum and faeces of sheep were identified as Piromyces mae based on their morphological characteristics observed using light microscopy. There was no significant morphological variation among the isolates of P. mae from the rumen, duodenum and faeces. Isozymes of 12 isolates of P. mae (one each from the rumen, duodenum and faeces from 4 different sheep) were analysed by PAGE. A total of 12 isozymes were studied and 5 isozyme loci were successfully typed. They were malic enzyme, malate dehydrogenase, shikimate dehydrogenase, α-esterase and β-esterase. All the isolates of P. mae regardless of whether they were from the rumen, duodenum or faeces or from different animals produced very similar isozyme banding patterns for each of the enzyme systems. The similar isozyme profiles of the isolates indicate that they are of the same species although they exist in different regions of the alimentary tract.     

苹果酸降解相关基因在酿酒酵母中的表达

微生物降酸是现代葡萄酒酿造重要工艺。将裂殖酵母苹果酸通透酶基因(mae1)和苹果酸酶基因(mae2)克隆到酿酒酵母中,构建了苹果酸酒精酵母;将mae1基因和乳酸乳球菌的苹果酸乳酸酶基因(mleS)克隆到酿酒酵母中,构建了苹果酸乳酸酵母。构建的酵母重组子能够有效地分解发酵基质中的苹果酸。     

Didymograptus bifidus and the trans-Atlantic correlation of the Lower Ordovician

Bergström, S. M. & Cooper, R. A.: Didymograptus bi'us and the trans-Atlantic correlation of the Lower Ordovician.
The co-occurrence of Didymograptus bifidus (Hall) and Prioniodus mae Lindstrom, and other conodonts of the middle Arenigian P. mae Zone in the upper part of the Marathon Limestone, Texas, provides the first non-graptolitic evidence that at least part of the D. bifidus Zone, as recognized in the reference area for the standard graptolite zonal succession of the Ordovician of North America, is as old as the BritisK D. nitidus Zone. This has important implications for the correlation of Early Ordovician rocks within North America and across the Atlantic. A regional review of the available evidence from conodonts and shelly megafossils indicates that the D. bifidus—D. artus graptolite assemblage widely used for identification of the D. bifidus Zone in North America ranges in age from the middle Arenigian to, probably, the lower Llanvirnian, and hence is of relatively limited use for precise local and regional correlations. It is also noted that the base of the North American Middle Ordovician falls within hograptus 'caduceus' Zone strata that apparently correspond to an interval in the middle to upper part of the British D. hirundo Zone.     

Mapping candidate genes for Drosophila melanogaster resistance to the parasitoid wasp Leptopilina boulardi

Drosophila melanogaster resistance against the parasitoid wasp Leptopilina boulardi is under the control of a single gene (Rlb), with two alleles, the resistant one being dominant. Using strains bearing deletions, we previously demonstrated that the 55E2-E6; 55F3 region on chromosome 2R is involved in the resistance phenomenon. In this paper, we first restricted the Rlb containing region by mapping at the molecular level the breakpoints of the Df(2R)Pc66, Df(2R)P34 and Df(2R)Pc4 deficiencies, using both chromosomal in situ hybridization and Southern analyses. The resistance gene was localized in a 100 kb fragment, predicted to contain about 10 different genes. Male recombination genetic experiments were then performed, leading to identification of two possible candidates for the Rlb gene. Potential involvement of one of this genes, edl/mae, is discussed.     

Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli.

The citrate utilization determinant from a large 200-kilobase (kb) naturally occurring plasmid was previously cloned into the PstI site of plasmid vector pBR325 creating the Cit+ tetracycline resistance plasmid pWR61 (15 kb). Tn5 insertion mutagenesis analysis of plasmid pWR61 limited the segment responsible for citrate utilization to a 4.8-kb region bordered by EcoRI and PstI restriction nuclease sites. The 4.8-kb fragment was cloned into phage M13, and the DNA sequence was determined by the dideoxyribonucleotide method. Within this sequence was a 1,296-base-pair open reading frame with a preceding ribosomal binding site. The 431-amino-acid polypeptide that could be translated from this open reading frame would be highly hydrophobic. A second long open reading frame with the potential of encoding a 379-amino-acid polypeptide preceded the larger open reading frame. Portions of the 4.8-kb fragment were further subcloned with restriction endonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BglII fragment (which includes the coding region for the 431-amino-acid polypeptide, but only the distal 2/3 of the reading frame for the 379-amino-acid polypeptide). Citrate utilization results from a citrate transport activity encoded by the plasmid. With the 4.8-kb fragment (as with larger fragments) the citrate transport activity was inducible by growth on citrate. On transfer from glucose, succinate, malate, or glycerol medium to citrate medium, the Cit+ Escherichia coli strains showed a delay of 36 to 48 h before growth.     

Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley

Aluminium (Al) toxicity is an important limitation to barley (Hordeum vulgare L.) on acid soil. Al-resistant cultivars of barley detoxify Al externally by secreting citrate from the roots. To link the genetics and physiology of Al resistance in barley, genes controlling Al resistance and Al-activated secretion of citrate were mapped. An analysis of Al-induced root growth inhibition from 100 F2 seedlings derived from an Al-resistant cultivar (Murasakimochi) and an Al-sensitive cultivar (Morex) showed that a gene associated with Al resistance is localized on chromosome 4H, tightly linked to microsatellite marker Bmag353. Quantitative trait locus (QTL) analysis from 59 F4 seedlings derived from an F3 plant heterozygous at the region of Al resistance on chromosome 4H showed that a gene responsible for the Al-activated secretion of citrate was also tightly linked to microsatellite marker Bmag353. This QTL explained more than 50% of the phenotypic variation in citrate secretion in this population. These results indicate that the gene controlling Al resistance on barley chromosome 4H is identical to that for Al-activated secretion of citrate and that the secretion of citrate is one of the mechanisms of Al resistance in barley. The identification of the microsatellite marker associated with both Al resistance and citrate secretion provides a valuable tool for marker-assisted selection of Al-resistant lines.     

Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase.

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.     

The citrate carrier CitS probed by single-molecule fluorescence spectroscopy

A prominent region of the Na(+)-dependent citrate carrier (CitS) from Klebsiella pneumoniae is the highly conserved loop X-XI, which contains a putative citrate binding site. To monitor potential conformational changes within this region by single-molecule fluorescence spectroscopy, the target cysteines C398 and C414 of the single-Cys mutants (CitS-sC398, CitS-sC414) were selectively labeled with the thiol-reactive fluorophores AlexaFluor 546/568 C(5) maleimide (AF(546), AF(568)). While both single-cysteine mutants were catalytically active citrate carriers, labeling with the fluorophore was only tolerated at C398. Upon citrate addition to the functional protein fluorophore conjugate CitS-sC398-AF(546), complete fluorescence quenching of the majority of molecules was observed, indicating a citrate-induced conformational change of the fluorophore-containing domain of CitS. This quenching was specific for the physiological substrate citrate and therefore most likely reflecting a conformational change in the citrate transport mechanism. Single-molecule studies with dual-labeled CitS-sC398-AF(546/568) and dual-color detection provided strong evidence for a homodimeric association of CitS.     

Binding of exogenous DNA to marine sediments and the effect of DNA/sediment binding on natural transformation of Pseudomonas stutzeri strain ZoBell in sediment columns

A new species of strictly anaerobic chytridiomycete was isolated from dried faeces of the Saharian ass that had been stored for up to 150 days. Because of its monocentric thallus and uniflagellate zoospores it belongs to the genus Piromyces. It exhibits a high affinity for P. mae and P. dumbonica but differs from them in its morphological and ultrastructural characteristics. Its flagellar apparatus is similar to that of all previously reported fungi.     

Differential recognition of citrate and a metal-citrate complex by the bacterial chemoreceptor Tcp

The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metal-citrate sensing, and the mutated residues are located in or near the alpha1-alpha2 and alpha3-alpha4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn(67), which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn(67) and allosteric switching.     

Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase

Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate. The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified. A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe. The complete nucleotide sequence of the 1.5-kilobase cDNA was determined. The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase. The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides. A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified. To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078]. The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met. A glutamate-requiring (citrate synthase deficient), recA- E. coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS. pT7-7PCS complemented the E. coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A de novo synthesis citrate transporter, Vigna umbellata multidrug and toxic compound extrusion, implicates in Al-activated citrate efflux in rice bean (Vigna umbellata) root apex

Al-activated organic acid anion efflux from roots is an important Al resistance mechanism in plants. We have conducted homologous cloning and isolated Vigna umbellata multidrug and toxic compound extrusion (VuMATE), a gene encoding a de novo citrate transporter from rice bean. Al treatment up-regulated VuMATE expression in the root apex, but neither in the mature root region nor in the leaf. The degree of up-regulation of VuMATE was both partially Al concentration and time dependent, consistent with the delay in the onset of the Al-induced citrate efflux in rice bean roots. While La(3+) moderately induced VuMATE expression, Cd(2+) and Cu(2+) did not induce the expression. Electrophysiological analysis of Xenopus oocytes expressing VuMATE indicated this transporter can mediate significant anion efflux across the plasma membrane. [(14) C]citrate efflux experiments in oocytes demonstrated that VuMATE is a H(+) -dependent citrate transporter. In addition, expression of VuMATE in transgenic tomato resulted in increased Al resistance, which correlated with an enhanced citrate efflux. Taken together, these findings suggest that VuMATE is a functional homolog of the known citrate transporters in sorghum, barley, maize and Arabidopsis. The similarities and differences of all the known citrate transporters associated with Al stress in the MATE family are also discussed.     

Nucleotide sequence and expression in Escherichia coli of the Lactococcus lactis citrate permease gene.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.     

Density gradient and differential centrifugation methods for chloroplast purification and enzyme localization in leaf tissue

Intact chloroplasts, isolated by differential-centrifugation and sucrose density-gradient methods, have been used to study the degree of apparent artifactual adsorption of citrate synthase (EC 4.1.3.7) to the organelles. Unfractionated homogenates layered directly on to sucrose density gradients gave elution profiles showing definite citrate synthase activity in the intact and broken plastid regions, along with the major mitochondrial peak. Nonreversible triose-phosphate dehydrogenase (EC 1.2.1.9), a cytosolic marker, showed no activity in any particulate region of the gradient. Crude chloroplast pellets and twice washed (resedimented and resuspended) chloroplasts layered on to the gradient gave progressively reduced citrate synthase activity in the plastid regions. In addition, the peak in the mitochondrial region of the gradient was virtually eliminated when washed chloroplasts were fractionated on the gradient. Differences in protein binding behavior on the chloroplasts may necessitate the inclusion of a washing step in chloroplast purification procedures. Moreover, repeated sedimentation and resuspension can also be a useful procedure to reduce mitochondrial contamination of chloroplast preparations.Work supported by the Rubber Research Institute of Malaysia     

Threonine-509 is a determinant of apparent affinity for both substrate and cations in the human Na+/dicarboxylate cotransporter

The Na+/dicarboxylate cotransporter (NaDC1) is involved in the absorption of citric acid cycle intermediates from the lumen of the renal proximal tubule and small intestine. The NaDC1 orthologues from human (h) and rabbit (rb) exhibit differences in citrate and cation transport properties. The citrate Km and sodium KNa values are much larger in human than rabbit NaDC1. Our previous study showed that transmembrane helices (TM) 7, 10, and 11 and associated loop regions contain the amino acids that are important in determining the differences in apparent citrate affinity, whereas TM10 and 11 determine differences in apparent sodium affinity. Chimera R10 (hNaDC1 with a substitution of TM10 and associated loop from rbNaDC1) contains only four amino acid differences between rb and hNaDC1. This chimera has similar apparent affinity for succinate and sodium as the wild-type rbNaDC1, and an intermediate Km for citrate. To identify individual residues in the TM10 region that determine functional differences between rb and hNaDC1, four mutants were made in which the rabbit sequence was substituted for that of the hNaDC1. Mutants with a serine or threonine at position 509 (or 512 in rbNaDC1) in TM10 have partial changes in Km for citrate and succinate but larger changes in apparent affinity for cations and substrate specificity for four-carbon dicarboxylates. The results show that the serine or threonine at position 509 (h) or 512 (rb) is the most important determinant of functional differences in apparent affinity for substrate and cations. Furthermore, the results suggest that the cation and substrate binding sites are located in close proximity to one another in NaDC1.