Identification and characterization of a novel inulin binding module (IBM) from the CFTase of Bacillus macerans CFC1

A novel inulin-binding module (IBM), which was identified from the N-terminal region of the cycloinulinooligosaccharide fructanotransferase (CFTase) in Bacillus macerans CFC1, was characterized using the discrete entity of IBM produced by the recombinant Escherichia coli strains. Deletion analyses located the inulin binding activity in the N-terminal region between 241 and 389 amino acid residues, which was removed from the mature enzyme by processing when secreted from the B. macerans CFC1 cells. IBM bound specifically to polyfructans such as inulin and levan but it did not interact with any of the glycan polymers tested in this study including cellulose, xylan, and starch. Binding studies on the IBM revealed that the equilibrium dissociation constant K(d) and the maximum amount of protein bound [(PC)(max)] were 4.7 microM and 22 microM g(-1), respectively. Together, these results indicate that the IBM of CFTase has a relatively high and specific affinity for inulin. Adsorption of the IBM to inulin was highest at pH 7.0 and lowered slowly with decreasing pH down to 3.0. At pH 7.0, the binding activity was enhanced about twofold by the presence of 1 M MgCl(2). Chemical modification experiments with the aromatic amino acid-specific modifiers implied that tryptophan and tyrosine residues in the IBM are likely to participate in the interaction with the inulin molecules.     

Interaction between succinate dehydrogenase and ubiquinone-binding protein from succinate-ubiquinone reductase

Purified ubiquinone-binding protein in succinate-ubiquinone reductase (QPs) reconstitutes with pure soluble succinate dehydrogenase to form succinate-ubiquinone oxidoreductase upon mixing of the two proteins in phosphate buffer at neutral pH. The maximal reconstitution was found with a weight ratio of succinate dehydrogenase to QPs of about 5, which is fairly close to the calculated value of 6.5, a value obtained by assuming one mole of QPs reacts with one mole of succinate dehydrogenase. Succinate-cytochrome c reductase was reconstituted when succinate dehydrogenase and QPs were added to Complex III or cytochrome b-c₁ III complex (a highly purified ubiquinol-cytochrome c reductase). The reconstituted enzyme possessed kinetic parameters which were identical to those of the native enzyme complex. Interaction between QPs and succinate dehydrogenase resulted in the disappearance of low K_m ferricyanide reductase activity from the latter. Unlike soluble succinate dehydrogenase, the reconstituted enzyme, as well as native succinate-cytochrome c reductase, reduced low concentration ferricyanide only in the presence of excess ubiquinone. The apparent K_m for ubiquinone was 6 μM for reduction of ferricyanide (300 μM) by succinate, which is similar to the K_m when ubiquinone was used as electron acceptor. When 2,6-dichlorophenolindophenol was used as electron acceptor for reconstitution of succinate-ubiquinone reductase very little or no exogeneous ubiquinone was needed to show the maximal activity with QPs made by Method II, indicating that the bound ubiquinone in QPs is enough for enzymatic activity. In addition to restoring the succinate-ubiquinone reductase activity the interaction between QPs and succinate dehydrogenase not only stabilized succinate dehydrogenase but also partially deaggregated QPs. The reconstituted succinate-ubiquinone reductase had a minimal molecular weight of 120000 when the reconstituted system was dispersed in 0.2% Triton X-100. The maximal reconstitution was observed at neutral pH in phosphate buffer, Tris-acetate or Tris-phosphate buffer. Tris-HCl buffer, however, produced a less efficient reconstitution. These results indicate that the interaction between QPs and succinate dehydrogenase may involve some cationic group which has a high affinity for Cl^?. Primary amino groups of QPs are not directly involved in the interaction as the reconstitution showed no significant difference when the amino groups of QPs were alkylated with fluorescamine. The Arrhenius plots of reconstituted succinate-ubiquinone reductase show that the enzyme catalyzes the reaction with an activation energy of 19.7 kcal/mol and 26.6 kcal/mol at temperatures above and below 26°C, respectively. These activation energies are similar to those obtained with native enzyme. The Arrhenius plots of the interaction between QPs and succinate dehydrogenase also have a break point at 26°C. The activation energy for this interaction was calculated to be 11.2 kcal/mol and 6.9 kcal/mol for the temperatures above and below the break-point. The significance of the difference in activation energies between the enzymatic reaction and the reconstitution reaction are further explored in the discussion.     

Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase (complex II)

Succinate:quinone reductase is a membrane-bound enzyme of the citric acid cycle and the respiratory chain. Carboxin is a potent inhibitor of the enzyme of certain organisms. The bacterium Paracoccus denitrificans was found to be sensitive to carboxin in vivo, and mutants that grow in the presence of 3′-methyl carboxin were isolated. Membranes of the mutants showed resistant succinate:quinone reductase activity. The mutation conferring carboxin resistance was identified in four mutants. They contained the same missense mutation in the sdhD gene, which encodes one of two membrane-intrinsic polypeptides of the succinate:quinone reductase complex. The mutation causes an Asp to Gly replacement at position 89 in the SdhD polypeptide. P. denitrificans strains that overproduced wild-type or mutant enzymes were constructed. Enzymic properties of the purified enzymes were analyzed. The apparent K _m for quinone (DPB) and the sensitivity to thenoyltrifluoroacetone was normal for the carboxin-resistant enzyme, but the succinate:quinone reductase activity was lower than for the wild-type enzyme. Mutations conferring carboxin resistance indicate the region on the enzyme where the inhibitor binds. A previously reported His to Leu replacement close to the [3Fe-4S] cluster in the iron-sulfur protein of Ustilago maydis succinate:quinone reductase confers resistance to carboxin and thenoyltrifluoroacetone. The Asp to Gly replacement in the P. denitrificans SdhD polypeptide, identified in this study to confer resistance to carboxin but not to thenoyltrifluoroacetone, is in a predicted cytoplasmic loop connecting two transmembrane segments. It is likely that this loop is located in the neighborhood of the [3Fe-4S] cluster. Received: 18 November 1997 / Accepted: 13 February 1998     

Structural properties of the proteoliposomes catalyzing electron transport from formate to fumarate

The electron-transport chain catalyzing fumarate reduction by formate has recently been reconstituted from the formate dehydrogenase complex and the fumarate reductase complex from Vibro succinogenes, in a liposomal preparation containing vitamin K-1 (Unden, G. and Kröger, A. (1982) Biochim. Biophys. Acta 682, 258–263). We have now investigated the structural properties of this preparation. The preparation was found to consist of a homogeneous population of unilamellar proteoliposomes with an average diameter of about 100 nm and an internal volume of 2–4 ml / g phospholipid. The buoyant density (1.07 g / ml) was consistent with the protein / phospholipid ratio (0.2 g / g) of the preparation. Leakage of glucose from the internal spaces of the proteoliposomes was negligibly slow. Proteoliposomes prepared with either of the enzyme complexes showed peripheral projections mainly on the outer surface, when examined by electron microscopy after negative staining. The size, orientation and surface density of the projections were consistent with those of the enzymes. Most of the substrate and dye-reactive sites (70–90%) of the enzymes in the proteoliposomes were accessible to external non-permeant substrates. The proteoliposomes catalyzing electron transport were formed by freeze-thawing a mixture of liposomes and protein-phospholipid complexes which did not perform electron transport from formate to fumarate. Nearly the entire amount of the enzymes supplied (0.2 g protein / g phospholipid) was incorporated into the liposomes by this procedure. The transformation of liposomes into proteoliposomes was accompanied by exchange of the internal solutes with the external medium.     

Properties of a Wolinella succinogenes mutant lacking periplasmic sulfide dehydrogenase (Sud)

A Δsud deletion mutant of Wolinella succinogenes that lacked the periplasmic sulfide dehydrogenase (Sud) was constructed using homologous recombination. The mutant grew with sulfide and fumarate, indicating that Sud was not a component of the electron transport chain that catalyzed fumarate respiration with sulfide as an electron donor. Likewise, growth with formate and either polysulfide or sulfur was not affected by the deletion. Removal of Sud from wild-type W. succinogenes by spheroplast formation did not decrease the activity of electron transport to polysulfide. The Δpsr deletion mutant that lacks polysulfide reductase (Psr) grew by fumarate respiration with sulfide as an electron donor, indicating that Psr is not required for this activity. Received: 31 August 1995 / Accepted: 25 October 1995     

Pyruvate dehydrogenase complex (PDC) export from the mitochondrial matrix

Studies on mitochondria protein import had revealed in detail molecular mechanisms of how peptides and proteins could be selectively targeted and translocated across membrane bound organelles. The opposite process of mitochondrial export, while known to occur in various aspects of cellular physiology and pathology, is less well understood. Two very recent reports have indicated that a large mitochondrial matrix protein complex, the pyruvate dehydrogenase complex (PDC) (or its component subunits), could be exported to the lysosomes and the nucleus, respectively. In the case of the latter, evidence was presented to suggest that the entire complex of 8–10 MDa could translocate in its entirety from the mitochondrial matrix to the nucleus upon mitogenic or stress stimuli. We discuss these findings in perspective to what is currently known about the processes of transport in and out of the mitochondrion.     

Sequence of the gene for a NAD(P)-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) from a marine methanotroph Methylobacter marinus A45

Abstract A fragment of Methylobacter marinus A45 DNA has been cloned and sequenced, and an open reading frame has been identified that could code for a 46-kDa polypeptide. Comparison of the deduced amino acid sequence of the polypeptide against the protein data bank has revealed strong similarity with a number of alcohol dehydrogenases, with highest similarity towards class III alcohol dehydrogenases, which recently have been shown to be identical to glutathione-dependent formaldehyde dehydrogenases. We were unable to measure appreciable levels of NAD(P)-dependent formaldehyde dehydrogenases or alcohol dehydrogenase activities using aldehydes or primary or secondary alcohols in cell-free extracts from batch cultures of M. marinus A45. However, formaldehyde dehydrogenases activity was detected on zymograms. Our data suggest that, although NAD(P)-linked formaldehyde dehydrogenase or alcohol dehydrogenase activities are undetectable in cell-free extracts of most methylotrophs employing the ribulose monophosphate pathway for formaldehyde assimilation and dissimilation, the gene encoding formaldehyde dehydrogenase is present in M. marinus A45 and may be present in more of these organisms as well.     

Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions

We examined succinic acid production in Escherichia coli AFP111 using dual-phase fermentations, which comprise an initial aerobic growth phase followed by an anaerobic production phase. AFP111 has mutations in the pfl, ldhA, and ptsG genes, and we additionally transformed this strain with the pyc gene (AFP111/pTrc99A-pyc) to provide metabolic flexibility at the pyruvate node. Aerobic fermentations with these two strains were completed to catalog physiological states during aerobic growth that might influence succinate generation in the anaerobic phase. Activities of six key enzymes were also determined for these aerobic fermentations. From these results, six transition times based on physiological states were selected for studying dual-phase fermentations. The final succinate yield and productivity depend greatly on the physiological state of the cells at the time of transition. Using the best transition time, fermentations achieved a final succinic acid concentration of 99.2 g/l with an overall yield of 110% and productivity of 1.3 g/l h. Journal of Industrial Microbiology & Biotechnology (2002) 28, 325–332 DOI: 10.1038/sj/jim/7000250 Received 01 October 2001/ Accepted in revised form 12 March 2002     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Biochemical and molecular characterization of a succinate semialdehyde dehydrogenase involved in the catabolism of 4-hydroxybutyric acid in Ralstonia eutropha

A succinate semialdehyde dehydrogenase gene (gabD) was identified to be disrupted in a transposon-induced mutant of Ralstonia eutropha exhibiting the phenotype 4-hydroxybutyric acid-leaky. The native gabD gene was cloned by colony hybridization using a homologous gabD-specific DNA probe. DNA sequencing revealed an 1452-bp open reading frame, and the deduced amino acid sequence showed strong similarities to NADP(+)-dependent succinate semialdehyde dehydrogenases from Escherichia coli, Rhizobium sp., Homo sapiens and Rattus norvegicus. The gabD gene was heterologously expressed in a recombinant E. coli strain harboring plasmid pSK::EE6.8. Similar to the molecular organization of the gab cluster in E. coli, additional genes encoding enzymes for the degradation of gamma-aminobutyrate are closely related to gabD in R. eutropha. Enzymatic studies indicated the existence of a second NAD(+)-dependent succinate semialdehyde dehydrogenase in R. eutropha.     

Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).     

Flutolanil and carboxin resistance in<Emphasis Type="Italic"> Coprinus cinereus</Emphasis> conferred by a mutation in the cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>560</Subscript> subunit of succinate dehydrogenase complex (Complex II)

A gene that confers resistance to the systemic fungicide flutolanil was isolated from a mutant strain of the basidiomycete Coprinus cinereus. The flutolanil resistance gene was mapped to a chromosome of approximately 3.2 Mb, and a chromosome-specific cosmid library was constructed. Two cosmid clones that were able to transform a wild-type, flutolanil-sensitive, strain of C. cinereus to resistance were isolated from the library. Analysis of a subclone containing the resistance gene revealed the presence of the sdhC gene, which encodes the cytochrome b ₅₆₀ subunit of the succinate dehydrogenase (SDH) complex (Complex II) in the mitochondrial membrane. Comparison between the sdhC gene of a wild-type strain and that of a mutant strain revealed a single point mutation, which results in the replacement of Asn by Lys at position 80. Measurements of succinate-cytochrome c reductase activity in the transformants with mutant sdhC gene(s) suggest that flutolanil resistance of the fungus is caused by a decrease in the affinity of the SDH complex for flutolanil. This sdhC mutation also conferred cross-resistance against another systemic fungicide, carboxin, an anilide that is structurally related to flutolanil. In other organisms carboxin resistance mutations have been found in the genes sdhB and sdhD, but this is the first demonstration that a mutation in sdhC can also confer resistance. The mutant gene cloned in this work can be utilized as a dominant selectable marker in gene manipulation experiments in C. cinereus.Communicated by E. Cerdá-Olmedo     

Enantioselective synthesis of (S)-phenylephrine by recombinant Escherichia coli cells expressing the short-chain dehydrogenase/reductase gene from Serratia quinivorans BCRC 14811

BackgroundAn amino alcohol dehydrogenase gene (RE_AADH) from Rhodococcus erythropolis BCRC 10909 has been used for the conversion of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-phenylephrine [(S)-PE]. However RE_AADH uses NADPH as cofactor, and only limited production of (S)-PE from HPMAE is achieved.MethodsA short-chain dehydrogenase/reductase gene (SQ_SDR) from Serratia quinivorans BCRC 14811 was expressed in Escherichia coli BL21 (DE3) for the conversion of HPMAE to (S)-PE.ResultsThe SQ_SDR enzyme was capable of converting HPMAE to (S)-PE in the presence of NADH and NADPH, with specific activities of 26.5 ± 2.3 U/mg protein and 0.24 ± 0.01 U/mg protein, respectively, at 30 °C and at a pH of 7.0. The E. coli BL21 (DE3), expressing NADH-preferring SQ_SDR, converted HPMAE to (S)-PE with more than 99% enantiomeric excess, a conversion yield of 86.6% and a productivity of 20.2 mmol/l h, which was much higher than our previous report using E. coli NovaBlue expressing NADPH-dependent RE_AADH as the biocatalyst.ConclusionThe SQ_SDR enzyme with its high catalytic activity and strong preference for NADH as a cofactor provided a significant advantage in bioreduction.     

Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves

Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence-specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up-regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence-associated expression was confirmed by Northern analyses or quantitative RealTime-PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence-induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1-like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF-like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1-GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed.     

Function of fumarate reductase in methanogenic bacteria (Methanobacterium)

Methanogenic bacteria contain high activities of fumarate reductase. An interesting hypothesis has recently been advanced that this enzyme, in cooperation with a succinate dehydrogenase, functions in a fumarate-succinate cycle for ATP synthesis. This hypothesis was tested by determining whether [2, 3-³H] succinate loses³H when taken up by growing cells.Methanobacterium thermoautotrophicum was grown on H₂ plus CO₂ in the presence of [U-¹⁴C, 2,3-³H] succinate. The double labelled dicarboxylic acid was found to be incorporated into cell material with the loss of only 30% of tritium. Neither was³H released into H₂O in significant amounts. This finding excludes a catabolic oxidation of succinate to fumarate in the growing cells and thus the operation of a fumaratesuccinate cycle. It is shown that the function of fumarate reductase inM. thermoautotrophicum is to provide the cells with succinate for the synthesis of -ketoglutarate, an intermediate in glutamate, arginine and proline synthesis.     

Tauropine dehydrogenase from the sandwormArabella iricolor (Polychaeta: Errantia): Purification and characterization

This is the first report of the purification of tauropine dehydrogenase (NAD: tauropine oxidoreductase) from a polychaete worm. In the sandwormArabella iricolor Montagu (Polychaet: Errantia), two forms of TaDH were detected: major component (pl = 7.5) and minor one (pI = 6.4). The major TaDH component was purified to homogeneity by means of (NH₄)₂SO₄ precipitation, anion-exchange, affinity, chromatofocusing and hydrophobic chromatography, and characterized. From the molecular mass of 43.7 kDa obtained by rapid gel-filtration and that of 43.5 kDa by SDS-PAGE, the sandworm enzyme appeared to be a monomeric protein. Maximum rates of reduction of pyruvate and oxidation of tauropine were observed at pH 7.0 and 8.5, respectively. Pyruvate and taurine were preferred substrate for the enzyme. Apparent Km values determined using constant co-substrate concentrations were: 35.7 mM, 0.34 mM, and 0.036 mM for taurine, pyruvate and NADH, respectively, in the tauropine synthesizing reaction; and 4.8 mM and 0.051 mM for tauropine and NAD⁺, respectively, in the tauropine oxidizing reaction. The tauropine synthesizing reaction was subject to substrate inhibition by pyruvate: maximum rate was observed at 2.5–3.0 mM (inhibitory range of pyruvate concentration producing half-maximal rate was 26.8 mM).     

Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol

Background

In a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol.

Methods

Triple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol.

Results

In the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol.

Conclusion

This study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol.

General significance

The present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.     

Ancestry of plant MADS-box genes revealed by bryophyte (Physcomitrella patens) homologues

Three MADS-box cDNA clones and two corresponding genomic sequences (gDNAs) have been isolated from the bryophyte Physcomitrella patens and sequenced. Our findings indicate that the genes may be expressed in a tissue- or age-specific manner, and that expression of one of them is regulated by an alternative splicing mechanism. Conceptual translation of the clones reveals that the encoded MADS-domain proteins have the typical plant-domain pattern (MIKC). Additionally, there is a high degree of conservation of intron number and positions between angiosperm MADS-box genes and the moss loci. These observations confirm the homology of moss and higher plant MADS-box genes. We conclude that the MIKC pattern evolved in MADS-box genes after the separation of the plant lineage from that of fungi and animals, and that it must have been present in the common ancestor of mosses, ferns and seed plants. Therefore it evolved at least 400 million yr ago. Phylogenetic analysis of a large subset of the sequenced plant MADS-box genes, incorporating those from P. patens , indicates that the bryophyte genes are not orthologues of spermatophyte genes belonging to any of the well recognized higher plant gene subfamilies. This conclusion accords well with reports that the known fern MADS-box genes also comprise subfamilies distinct from those of higher plants. Therefore we tentatively propose that the gene duplication and diversification events that created the MADS-box gene subfamilies, discernible in extant angiosperm and other spermatophyte groups, occurred after separation of the moss and fern lineages from the lineage which produced the higher plants.     

A system for the inducible secretion of proteins from Bacillus subtilis during logarithmic growth

Abstract A Bacillus subtilis-Escherichia coli shuttle vector was constructed containing the B. subtilis levansucrase gene promoter and region encoding its signal sequence.
A site for the restriction enzyme Nae I was included to facilitate precise translational fusions to the DNA encoding the levansucrase signal sequence. Fusions of TEM β-lactamase to this construct displayed sucrose-inducible expression and secretion of B. subtilis .