SummaryThe facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA , the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfAdependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild-type L. monocytogenes . These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.
The major pigment produced by Staphylococcus aureus Newman is the deep-yellow carotenoid 4,4'-diaponeurosporene; after prolonged cultivation, this pigment is in part converted to the orange end product staphyloxanthin. From this strain a 3.5-kb DNA fragment was identified which after being cloned into Escherichia coli and Staphylococcus carnosus conferred the ability to produce 4,4'-diaponeurosporene. DNA sequencing of this fragment revealed two open reading frames (ORFs) which are very likely cotranscribed. ORF1 encodes a 254-amino-acid hydrophobic protein, CrtM (M(r), 30,121). The deduced sequence of CrtM exhibits in three domains similarities to the sequences of Saccharomyces cerevisiae and human squalene synthases and phytoene synthases of various bacteria. ORF2 encodes a 448-amino-acid hydrophobic protein, CrtN, with an M(r) of 50,853 whose deduced sequence is similar to those of phytoene desaturases of other bacteria. At the N terminus of CrtN a classical FAD-, NAD(P)-binding domain is found. Spectrophotometry and gas chromatography-mass spectrometry analyses of the carotenoid production of E. coli and S. carnosus clones containing either ORF1 or both ORFs together suggest that ORF1 and ORF2 represent the dehydrosqualene synthase gene (crtM) and the dehydrosqualene desaturase gene (crtN), respectively. The results furthermore suggest that the biosynthesis of 4,4'-diaponeurosporene starts with the condensation of two molecules of farnesyl diphosphate by dehydrosqualene synthase (CrtM); it is shown that the reaction product of this enzyme is dehydrosqualene and not squalene. Dehydrosqualene (4,4'-diapophytoene) is successively dehydrogenated by a desaturase (CrtN) to form the yellow main intermediate 4,4'-diaponeurosporene.
The mpi gene encodes a maize proteinase inhibitor (MPI) protein whose mRNA accumulates in response to mechanical wounding. In this study, mpi gene expression in response to different types of damage was investigated. In mechanically damaged leaves of maize (Zea mays L.), mpi mRNA accumulation was affected by the degree of damage inflicted on the leaf. Consecutive wounds resulted in higher levels of mpi transcripts. The MPI protein was expressed in Escherichia coli and purified. Polyclonal antibodies were then produced and used to study MPI accumulation in insect-wounded and mechanically wounded maize leaves. When larvae of the lepidopteran insect Spodoptera littoralis were fed on maize leaves, MPI accumulated in tissues adjacent to the wound site. The level of inhibitor accumulation was higher in leaves chewed by larvae than in leaves that had been damaged mechanically. Longer feeding periods also resulted in higher levels of MPI accumulation. Additionally, the inhibitory properties of MPI toward mammalian and insect digestive serine proteinases were determined. Contrary to the majority of the plant proteinase inhibitors described, MPI is an inhibitor of mammalian elastase that only weakly inhibits mammalian chymotrypsin. However, both elastase and chymotrypsin-like activities from the larval midgut of S. littoralis were effectively inhibited by MPI. We discuss these results with regard to the function and evolution of plant proteinase inhibitors. The availability of a plant proteinase inhibitor which is able to inhibit the two types of insect digestive proteinase, elastase and chymotrypsin, might be useful for engineering protection against lepidopteran insect pests in transgenic plants.
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