A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus.

Investigation of the chromosomal region downstream of the lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus revealed the presence of a gene (prtB) encoding a proteinase of 1,946 residues with a predicted molecular mass of 212 kDa. The deduced amino acid sequence showed that PrtB proteinase displays significant homology with the N termini and catalytic domains of lactococcal PrtP cell surface proteinases and is probably synthesized as a preproprotein. However, the presence of a cysteine near the histidine of the PrtB active site suggests that PrtB belongs to the subfamily of cysteine subtilisins. The C-terminal region strongly differs from those of PrtP proteinases by having a high lysine content, an imperfect duplication of 41 residues, and a degenerated sequence compared with the consensus sequence for proteins anchoring in the cell walls of gram-positive bacteria. Finally, the product of the truncated prtM-like gene located immediately upstream of the prtB gene seems too short to be involved in the maturation of PrtB.     

Spatial variation in selection in a plant-pollinator system in the wadis of Sinai,Egypt

We studied an insect-plant pollination system in adjacent steep-sided wadis and a connecting plain in the mountains of southern Sinai (Egypt): this environment creates a strongly divided habitat, which may promote the local differentiation of sub-populations. We tested for spatial differences in phenotypic reproductive characters of the only plant flowering abundantly in early spring, Alkanna orientalis (Boraginaceae), and its major pollinator at that time of year, Anthophora pauperata (Apoidea, Anthophoridae). There were significant morphological differences between sub-populations of Alkanna, mainly between plants from the narrower wadis and those on the interconnecting plain. Flowers on the plain were larger, with wider corollas and more nectar standing crop; these plants retained more flowers on the inflorescence, but received many fewer visits to flowers. There was a significant selection gradient between flower size and maternal fitness (seed set) in the plain, but not elsewhere. Natural selection may have increased resources devoted to attracting insect visitors in response to fewer pollinating visits in the plain. Consistent with this explanation, by experimentally manipulating flower number per plant, we showed that within a wadi having more flowers on a plant secured more visits.     

87Sr/86Sr Chronostratigraphy and dolomitization history of the Seroe Domi Formation,Curaçao (Netherlands Antilles)

Summary The Seroe Domi Formation is a 350 m-thick sequence of Neogene marine limestones and silicilastic sandstones cropping out on the leeward coast of Cura?ao, Netherlands Antilles. Integrated analyses of lithofacies, biostratigraphy, geochemistry and Sr isotope model age analyses indicate that Seroe Domi Formation has experienced three major episodes of limestone diagenesis and dolomitization (Dolomites I, I′, and II) that have taken place after successive Mio-Plio-Pleistocene depositional and subaerial exposure events (Subunits 1, 2, and 3). Subunit 1, the lowermost 30 to 100 m of the Seroe Domi Formation, is composed of interbedded coralgal grainstone gravity flows, pelagic wackestones, and allochthonous blocks deposited in Middle Miocene deep-water (>500 m) fore-reef and carbonate slope environments. Subunit 2, the uppermost 250 m of the Seroe Domi Formation, consists of coralgal packstones with basement-derived siliciclastic sands that were deposted in shallowing fore-reef to reef-front environments during the Late Miocene to Pliocene. Subunit 3 siliciclastic sandstones were deposited during the Early Pleistocene within erosional cavities in the Subunit 2 limestones, and are overlain by Late Pleistocene Quaternary Limestone Terraces. The petrography, distribution and geochemistry of Dolomites I, I′ and II indicate that they were precipitated from seawater-freshwater mixing zone fluid environments. Dolomite rhombs and meteoric calcite cements within biomolds illustrate that the host Seroe Domi Formation limestones were subaerially exposed prior to each dolomitization event. Dolomite I (δ¹⁸O = +1.04 to +2.46% PDB; δ¹³C = −2.55 to −6.79 PDB;⁸⁷Sr/⁸⁶Sr=0.708866 to 0.708915; Zn=0 ppm; Cu=0 ppm) was precipitated from mixtures of seawater with isotopically-depleted freshwater during the late Middle Miocene. Dolomite I′ (δ¹⁸O = +2.08 to +3.55 PDB, δ¹³C = −1.53 to 1.69 PDB,⁸⁷Sr/⁸⁶Sr=0.708981−0.709030; Zn=0 ppm; Cu=0 ppm) was also precipitated from mixtures of seawater with isotopically-depleted freshwater, but during late Late Miocene. In contrast, Dolomite II (δ¹⁸O = +2.69 to +3.51 PDB; δ¹³C = −0.34 to +1.53 PDB;⁸⁷Sr/⁸⁶Sr=0.708954 to 0.709088; Zn=20 ppm; Cu=20 ppm) precipitated from late Early Pliocene mixtures of seawater with isotopically-depleted freshwater that had derived Zn, Cu, and less-radiogenic Sr from basalts comprising the Cura?ao basement.     

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.     

Recurrent and novel LDL receptor gene mutations causing heterozygous familial hypercholesterolemia in La Habana

The molecular basis of familial hypercholesterolemia (FH) in three families of Spanish descent from La Habana was investigated by the candidate gene approach. The Arg3500Gln mutation of apolipoprotein B-100 was not found. Identification of low density lipoprotein receptor (LDLR) gene haplotypes segregating with FH guided the characterisation of three point mutations by automated sequencing. One, a Val408Met missense mutation, a founder mutation in Afrikaner FH patients, was recurrent, being associated with a distinct DNA haplotype. The other two, Glu256Lys and Val776Met missense mutations, were novel and modified highly conserved residues. These mutations were absent in normolipidemic subjects and were associated in heterozygous carriers with twice the cholesterol levels observed in noncarriers. Noticeably, cardiovascular complications were rarely observed in older heterozygotes, even in those with the Afrikaner FH-2 mutation. These findings confirm the molecular heterogeneity of LDLR gene mutations causing FH and the variability of their expression across different populations.     

High level expression of a heterologous protein in Lactobacillus plantarum and its effect on the persistence of the recombinant strain in silage

Summary This paper reports the high level expression of the Staphylococcus aureus cat gene in Lactobacillus plantarum using the expression vector pMTL500F. When the recombinant strain of L. plantarum was grown in pure culture, CAT contributed 1.4% of the total soluble cell protein. The recombinant strain of L. plantarum continued to express a high level of CAT when inoculated into silage, the heterologous protein constituting up to 1.75% of the total soluble cell protein. The recombinant L. plantarum strain was still able to survive and proliferate when inoculated into silage, despite its additional metabolic load.     

Yeast communities associated withDrosophila species and related flies in an eastern oak-pine forest: A comparison with western communities

Summary Intestinal yeast mycobiota were studied in 14 species ofDrosophila and in the drosophilid speciesChymomyza amoena, captured at Pinery Provincial Park, Ontario. Over 56 yeast species, some undescribed, were isolated. These yeast communities were compared with those from two similar surveys conducted in western portions of North America. The community structures were influenced significantly by the habitat rather than phylogeny of the flies. Geographic separation was a factor affecting yeast taxa frequencies in the fly species, but it was largely overshadowed by ecological factors when the communities were described physiologically. The notion that habitats are filled by yeasts which add up to a suitable physiological potential, more or less independently of their taxonomic affinities, was thus confirmed.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase (Xy1D) release a 5-5' ferulic dehydrodimer (diferulic acid) from barley and wheat cell walls.

Diferulate esters strengthen and cross-link primary plant cell walls and help to defend the plant from invading microbes. Phenolics also limit the degradation of plant cell walls by saprophytic microbes and by anaerobic microorganisms in the rumen. We show that incubation of wheat and barley cell walls with ferulic acid esterase from Aspergillus niger (FAE-III) or Pseudomonas fluorescens (Xy1D), together with either xylanase I from Aspergillus niger, Trichoderma viride xylanase, or xylanase from Pseudomonas fluorescens (XylA), leads to release of the ferulate dimer 5-5' diFA [(E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid]. Direct saponification of the cell walls without enzyme treatment released the following five identifiable ferulate dimers (in order of abundance): (Z)-beta-(4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy)-4-hydroxy-3-methoxycinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl) -7-methoxy-2, 3-dihydrobenzofuran-3-carboxylic acid, 5-5' diFA, (E,E)-4, 4'-dihydroxy-3, 5'-dimethoxy-beta, 3'-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl) -6-methoxy-1, 2-dihydronaphthalene-2, 3-dicarboxylic acid. Incubation of the wheat or barley cell walls with xylanase, followed by saponification of the solubilized fraction, yielded 5-5'diFA and, in some cases, certain of the above dimers, depending on the xylanase used. These experiments demonstrate that FAE-III and XYLD specifically release only esters of 5-5'diFA from either xylanase-treated or insoluble fractions of cell walls, even though other esterified dimers were solubilized by preincubation with xylanase. It is also concluded that the esterified dimer content of the xylanase-solubilized fraction depends on the source of the xylanase.