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  • 1.
    Eskils, Katarina
    Stockholm University, Faculty of Science, Department of Microbiology.
    Microbial ecology and virulence gene studies of the insect pathogen Bacillus thuringiensis2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Bacillus thuringiensis (Bt) is a spore-forming bacterium used as a bio-pesticide due to its insect toxicity. Bt was used as a model organism in a risk evaluation project. Prevalence, survival and spreading, as well as genome stability, putative virulence genes, and gene transfer were examined. Field release of a marked Bt subsp. israelensis (Bti) strain resulted in a minor, transient increase of Bt-like bacteria.

    The bacterial population structure returned to as before release after seven weeks (paper I). Physical chromosome maps were established for Bt subsp. gelechiae, alesti and kurstaki using pulsed-field gel electrophoresis (PFGE). Co-expressed virulence genes are scattered on the chromosome and are not located in operons or pathogenicity islands (paper II). Bt subsp. alesti and kurstaki have identical chromosome maps although their serotypes differ. Sequences homologous to Bacillus cereus enterotoxin genes were found on both chromosomes and on a plasmid in Bt subsp. alesti/kurstaki (paper III). A 5.2 kb region from Bt subsp. alesti was cloned and sequenced, and found to contain three flagellin genes constituting a fla-operon, a putative flagellar motor switch protein gene fliM, and a transglycosylase-like protein gene tlp.

    Deletion mutants in the region are avirulent and do not express previously identified virulence factors (paper IV). Chromosomal genes were transferred by transduction to Bt soil isolate strains, using a bacteriophage originating from soil (paper V).

  • 2.
    Faxén, Margareta
    Stockholm University, Faculty of Science, Department of Microbiology.
    Studies of translation in E. coli1993Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A cis-acting translational mutation in the beginning of the glnS gene was analyzed; this mutation creates a extra base pair to a proposed extended ribosome binding site. When increasing the complementarity to this extended ribosome binding site, it was demonstrated that the expression increased; correspondingly, when complementarity was decreasd the expression went down. It was also shown that the presence of rare codons near the beginning of the gene can affect expression.

    Studies of a S13 mutant (rpsM413) revealed that the mutant allele is associated with slow growth rate and slow elongation rate. The 30S subunit showed a reduced sedimentation coefficient but was able to form apparently normal 70S ribosomes.

    Investigations to see whether ribosomal mutants are able to affect proofreading in vivo found that the missense and nonsense suppressors are affected by rpsD mutations in a rather unpredictable manner. It was shown that the ribosome allele, the nature of the suppressor tRNA, the codon context, and the structure of the anticodon loop are the determinig factors. rpsL mutations decrease the nonsense suppression, in accordance with their restrictive effect on translational error formation.

    Streptomycin, which is known to increase translational error in vitro, did not increase efficiency of nonsense suppressor tRNAs in strains with normal or rpsL ribosomes. It did so only in combination with one rpsL mutation which is associated with streptomycin pseudodependence.

    The presence or absence of the modification ms2i6A37 in the tRNA was found to be a determining factor in the functional response to the ribosomal mutations. These effects strongly indicate that tRNA is directly involved in this phenomena.

    The role of ppGpp was investigated, and the results suggest that it affects transcription efficiency, but not translation. ppGpp seems to play a role in the tuning of the coupling between transcription and translation.

  • 3.
    Lundström, Annika
    Stockholm University, Faculty of Science, Department of Microbiology.
    Identification and characterisation of five innate immune genes in the cabbage looper Trichoplusia ni2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    When insects are infected with bacteria, they respond by producing a range of different antibacterial peptides and by activating protease cascades leading to coagulation and melanisation. Cellular defence mechanisms are also involved in killing of microorganisms through phagocytosis and encapsulation.

    We have found several genes that are upregulated upon bacterial infection in the cabbage looper, Trichoplusia ni, by using the differential display method. Attacin and gloverin are antibacterial proteins previously known from other insect species. Both are glycine-rich proteins that act by disrupting the permeability barrier of the cell membrane.

    An azurocidin homologue without serine protease activity is expressed exclusively in the insect gut. It is induced by injection of bacteria and by feeding larvae with bacteria. Two amino acid substitutions, serine to glutamate and histidine to serine, in the catalytic triad explain the lack of protease activity.

    Another protein induced by a bacterial infection is a 3-dehydroecdysone 3b-reductase-like protein (DERH). This protein was detected in integument and hemolymph of T. ni larvae. In addition, it is developmentally regulated. A possible function for DERH is to convert 3-dehydroecdysone into ecdysone, regulating the titer of biologically active ecdysteroids in the larvae.

    Furthermore, a peptidoglycan recognition protein (PGRP) is upregulated in T. ni by bacterial infection. PGRP is a peptidoglycan- and bacteria binding protein, possibly involved in immune recognition. Homologues were also cloned from mouse and man. The mouse PGRP also binds peptidoglycan, suggesting a functional conservation of the protein from insects to humans.

  • 4.
    Sun, Shao-Cong
    Stockholm University, Faculty of Science, Department of Microbiology.
    Molecular studies of the humoral immune response in Hyalophora cecropia: structure, function and transcriptional regulation of three classes of immunoresponsive genes1992Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Insects have an efficient humoral immune system, an important part of which is constituted by the antibacterial proteins, produced upon bacterial infection or wounding. In this thesis, the molecular mechanism of this immune response was investigated in the giant silkmoth Hyalophora cecropia, where a set of bacteriainducible proteins (immune proteins) had previously been characterized. These included three classes of antibacterial factors, cecropins, attacins and a lysozyme, and a functionally unknown protein named P4.

    cDNA sequencing revealed that P4, now named hemolin, belongs to the immunoglobulin superfamily. It binds to the surface of bacteria and is most likely involved in the recognition of non-self. Unlike the real immunoglobulins that are specified by multiple sets of gene segments, hemolin is encoded by a single gene. The genes coding for two other classes of immune proteins, the attacins and the lysozyme, were also characterized. Two attacin genes, clustered with two attacin pseudogenes, and a single lysozyme gene are present on the genome of Cecropia.

    Expression studies revealed that the immune genes are normally silent or, in the case of the hemolin gene, expressed at a very low level. Upon injection of bacteria or other agents such as lipopolysaccharides and phorbol esters, they are quickly induced for transcription. The induction kinetics for all the sequenced immune genes, except one, are very similar. Sequence analyses showed that these genes contained a similar decameric sequence in their promoter regions. Interestingly, this sequence element is highly homologous to the binding motifs of the mammalian nuclear factor kB (NF-kB), which is involved in the induction of quite a number of immune related genes. Furthermore, the xB-like sequences of the Cecropia immune genes are specifically bound by a nuclear protein that can be detected only in the bacteria-induced pupae but not in the normal pupae. Subsequent investigations showed that this DNA binding protein, named Cecropia immunoresponsive factor (CIF), is strongly induced by bacteria and all the other known inducers of the immune genes. The induction kinetics of CIF is well correlated to the immune gene transcription, indicating its importance in the induction of the immune genes.

    Finally, CIF and NF-kB share certain similarities in their DNA binding specificity, pattern of induction and migration rate on native gels. The purified CIF contains a single polypeptide chain of 65 kDa, which is identical in size to one of the NF-kB subunits. 

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