Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2

A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a "tesB-like" acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.     

Microcin J25 uptake: His5 of the MccJ25 lariat ring is involved in interaction with the inner membrane MccJ25 transporter protein SbmA

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 L-amino acid residues (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G). E. coli RNA polymerase (RNAP) is the intracellular target of MccJ25. MccJ25 enters cells after binding to specific membrane transporters: FhuA in the outer membrane and SbmA in the inner membrane. Here, we studied MccJ25 mutants carrying a substitution of His5 by Lys, Arg, or Ala. The inhibitory effects on cellular growth and in vitro RNAP activity were determined for each mutant microcin. The results show that all mutants inhibited RNAP in vitro. However, the mutants were defective in their ability to inhibit cellular growth. Experiments in which the FhuA protein was bypassed showed that substitutions of MccJ25 His5 affected the SbmA-dependent transport. Our results thus suggest that MccJ25 His5 located in the lariat ring is involved, directly or indirectly, in specific interaction with SbmA and is not required for MccJ25 inhibition of RNAP.     

Inhibition of RecA protein function by the RdgC protein from Escherichia coli

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.     

Regulation of growth differentiation factor 9 expression in oocytes in vivo: a key role of the E-box

Growth differentiation factor 9 (GDF9) is preferentially expressed in oocytes and is essential for female fertility. To identify regulatory elements that confer high-level expression of GDF9 in the ovary but repression in other tissues, we generated transgenic mice in which regions of the Gdf9 locus were fused to reporter genes. Two transgenes (-10.7/+5.6mGdf9-GFP) and (-3.3/+5.6mGdf9-GFP) that contained sequences either 10.7 or 3.3 kb upstream and 5.6 kb downstream of the Gdf9 initiation codon demonstrated expression specifically in oocytes, thereby mimicking endogenous Gdf9 expression. In contrast, transgenes -10.7mGdf9-Luc and -3.3mGdf9-Luc, which lacked the downstream 5.6-kb region, demonstrated reporter expression not only in oocytes but also high expression in male germ cells. This suggests that the downstream 5.6-kb sequence contains a testis-specific repressor element and that 3.3 kb of 5'-flanking sequence contains all the cis-acting elements for directing high expression of Gdf9 to female (and male) germ cells. To define sequences responsible for oocyte expression of Gdf9, we analyzed sequences of Gdf9 genes from 16 mammalian species. The approximately 400 proximal base pairs upstream of these Gdf9 genes are highly conserved and contain a perfectly conserved E-box (CAGCTG) sequence. When this 400-bp region was placed upstream of a luciferase reporter (-0.4mGdf9-Luc), oocyte-specific expression was observed. However, a similar transgene construct (-0.4MUT-mGdf9-Luc) with a mutation in the E-box abolished oocyte expression. Likewise, the presence of an E-box mutation in a longer construct (-3.3MUT-mGdf9-Luc) abolished expression in the ovary but not in the testis. These observations indicate that the E-box is a key regulatory sequence for Gdf9 expression in the ovary.     

Fourier transform infrared spectroscopy provides a fingerprint for the tetramer and for the aggregates of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular beta-sheet structure followed by an increase in nonnative, probably antiparallel beta-sheet contacts (bands at 1,616 and 1,686 cm(-1)) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12 kbar), TTR loses only part of its beta-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of beta-sheets does not change much upon fibrillization; however, the alignment of beta-sheets is altered, resulting in the formation of distinct beta-sheet contacts (band at 1,625 cm(-1)). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the beta-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37 degrees C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.     

An accumulation of tandem DNA repeats on the Y chromosome in Silene latifolia during early stages of sex chromosome evolution

Sex chromosomes in mammals are about 300 million years old and typically have a highly degenerated Y chromosome. The sex chromosomes in the dioecious plant Silene latifolia in contrast, represent an early stage of evolution in which functional X–Y gene pairs are still frequent. In this study, we characterize a novel tandem repeat called TRAYC, which has accumulated on the Y chromosome in S. latifolia. Its presence demonstrates that processes of satellite accumulation are at work even in this early stage of sex chromosome evolution. The presence of TRAYC in other species of the Elisanthe section suggests that this repeat had spread after the sex chromosomes evolved but before speciation within this section. TRAYC possesses a palindromic character and a strong potential to form secondary structures, which could play a role in satellite evolution. TRAYC accumulation is most prominent near the centromere of the Y chromosome. We propose a role for the centromere as a starting point for the cessation of recombination between the X and Y chromosomes.     

Characterization of an equine mannose-binding lectin and its roles in disease

The mannose-binding lectin (MBL), a pattern recognition serum protein, participates in the innate immune system of mammals as an opsonin. In humans, MBL plays a key role in first-line host defense against infection during the lag period prior to the development of a specific immune response. MBL also activates complement via the lectin pathway that requires a MBL-associated serine protease-2 (MASP-2). Homologues of human MBL (hMBL) have been identified in a variety of mammals, fish, and primitive animals such as ascidians. In this study, we report that equine MBL (eMBL) has properties that are similar to hMBL. In addition, we found low levels of MBL:MASP activity in sick horses compared to healthy horses. These results suggest that eMBL is involved in the immune response of the horse and that low MBL:MASP activity could be used to monitor immune function and clinical outcome.     

Large-scale chemoenzymatic synthesis of blood group and tumor-associated poly-N-acetyllactosamine antigens

Poly-N-acetyllactosamines (pLNs) are common terminal sugars of many N- and O-linked glycan structures present in glycoproteins and glycolipids. Utilizing various glycosyltransferases, we developed new and efficient chemoenzymatic methods for the synthesis of pLNs in gram-scale. Specifically, the use of sialyltransferases and fucosyltransferases enabled us to synthesize and purify 24 blood group and tumor-associated pLN derivatives with alpha-(2-->3)- and alpha-(2-->6)-linked sialic acid, as well as with alpha-(1-->2)- and alpha-(1-->3)-linked fucose. All synthesized derivatives were linked to a short 2-azidoethyl spacer for further modification.     

Detection of a serine proteinase gene in Acanthamoeba genotype T6 (Amoebozoa: Lobosea)

Cytopathic proteins are assumed to contribute to the pathogenicity of Acanthamoeba spp. due to their degrading capacity that is required for tissue invasion. In this study, a serine proteinase gene was demonstrated in a highly virulent Acanthamoeba keratitis causing strain with genotype T6. This gene was detected in both, the genomic DNA and the cDNA by PCR and subsequent sequencing. The gene fragment comprises about 500 bp and exhibits high sequence similarity to the serine proteinases of Acanthamoeba strains with genotype T4 and T12. The detection of a serine proteinase in this Acanthamoeba T6 strain is significant, because while T4 is the most common genotype among pathogenic Acanthamoeba strains and also T12 is known to be associated with disease, this is the only virulent Acanthamoeba T6 strain known to date. Obviously, this serine proteinase represents a common tool in pathogenic processes during Acanthamoeba infection.     

RNA editing sites in plant mitochondria can share cis-elements

RNA editing in flowering plant mitochondria alters numerous C nucleotides in a given mRNA molecule to U residues. To investigate whether neighbouring editing sites can influence each other we analyzed in vitro RNA editing of two sites spaced 30 nt apart. Deletion and competition experiments show that these two sites carry independent essential specificity determinants in the respective upstream 20-30 nucleotides. However, deletion of a an upstream sequence region promoting editing of the upstream site concomitantly decreases RNA editing of the second site 50-70 nucleotides downstream. This result suggests that supporting cis-/trans-interactions can be effective over larger distances and can affect more than one editing event.