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).

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.

The cecropins and attacins are two antibacterial peptides induced uponimmunization in the moth Hyalophora cecropia. They are both synthesized aspreproproteins that are processed and post-translationally modified along the exportpathway.

To study these processing and modification events, the baculovirus expressionvector system was employed as a model system. The strong polyhedrin promoter ofAutographa californica nuclear polyhedrosis virus (AcNPV) was used to driveexpression of different forms of cecropin and attacin. To successfully express thesesmall proteins that are sensitive to proteolysis, Trichoplusia ni larvae and H.cecropia pupae were used instead of Spodoptera frugiperda cell culture. Therecombinant proteins produced by living animals were biologically active, accuratelyprocessed and the cecropins were C-terminally amidated.

The amidation machinery of H. cecropia was further investigated. It was foundthat enzymes needed for amidation are present in small amounts in both fat body andhemolymph of H. cecropia pupae, and that a bacterial infection causes an increase ofthe amidation activity, indicating a coregulation of the immune proteins and theenzymes needed for their processing.

In order to establish that T. ni was a suitable host for heterologous expression of theantibacterial peptides, the endogeneous immune response of the larvae wasinvestigated and it was ascertained that the immune proteins do not have a deleteriouseffect on AcNPV.

The potential of diapausing H. cecropia pupae as expression host was explored byexpressing four different recombinant proteins. It was shown that H. cecropia pupaecompare favourably as expression host to T. ni larvae.

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.

Bacillus thuringiensis (Bt) is a motile, Gram positive, rod-shaped and spore-forminginsect pathogenic bacterium found on plants and in soil. The main insecticidal activityresides in a proteinacious crystal inclusion body (the S-endotoxin), but also the sporesare important for lethality. Bt is subclassified according to flagellar serotype and the 5-endotoxin they produce. Subspecies that lack 6-endotoxin but still kill insects have beenisolated, proving the importance of other virulence factors. This thesis includes theinvestigation of two such virulence factors: Immune inhibitor A (InA), and flagella andtheir constituent proteins, the flageilins. In addition, an effective method to transformvegetative Bt cells with plasmid DNA is described.

InA is a 72 kDa molecular weight, neutral metalloprotease toxic to lepidopteran,coleopteran and dipteran insects. The LD50 dose in Trichoplusia ni is 12 ng/mg oflarval body weight. This thesis presents the cloning and molecular characterization ofInA and the deduced amino acid sequence is compared to that of other neutralmetalloproteases such as elastase, a virulence protease from Pseudomonas aeruginosa.InA was found to be secreted by at least 70% of tested Bt subspecies.

Flagella, and motility as such, were found to be important for Bt invasiveness, butthey may also serve additional functions. The results in this thesis suggest that flageilinsare co-expressed with other virulence determinants such as secretion of lecithinase,ability to adhere to and kill Spodoptera frugiperda cells and ability to avoid and survivethe Hyalophora cecropia host defence systems. Flagellin from Bt var. alesti andgelechiae have been purified, and one complete and one partial alesti flagellin genehave been cloned and sequenced. A striking feature of one of these genes is the presence ofa 355 bp direct repeat.

The aim of this thesis was to study components involved in translational termination. Most of the work is centered around this, but the outcome of some of the experiments, led me onto other paths too.

Firstly, isolation of a mutant which was shown to be in release factor one, was mapped to min 26,5 on the Escherichia coli chromosome. The gene was named prfA and the mutant allele prfAl. The mutated RF1 is most probably impaired in its binding to the ribosome and not in its ability to recognize the termination site. This leads to a slower termination rate and allows a competing ternary complex to come in and read the stop codon. This can be studied as suppression and increased efficiency of UAG and UAA tRNA-suppressors.

By compiling all available E. coli sequences, some determinants for context have been inferred, with their main feature residing in the neighbouring codons, probably the 3' nucleotide. However, some element influencing the termination event, resides outside this immediate neighbourhood on the 5' side. Experiments performed in this thesis, suggest that the 3' determinant can be either U or C for the stop codon UAG, and that the 5' context effect is mediated through the peptidyl-tRNA in the P-site of the ribosome.

Secondly, it is known that ribosomal frameshift occurs at hot spots. As such the stop codon at the end of the frameshift window has been suggested. This hypothesis was tested on some frameshift mutations which are spontaneously suppressed and where the window ends with a UGA codon. If the compensating frameshift occurs at the stop codon it should be possible to decrease its suppression by addition of more release factor. It was shown that overproduction of active RF2 and not RF1 decreases the spontaneous suppression of the three frameshift mutations tested. This indicates that the stop codon can induce frameshift and thus, many experiments estimating frameshift frequency are likely to be overestimates.

Thirdly, a mutant isolated as a misreader, was shown to map in the gene for ribosomal protein S20, rpsT. The misreading characteristic is only part of a pleiotropic phenotype caused by truncated protein S20. Since S20 is one of the primary binding proteins in subunit assembly, the main defect is in formation of 30S subunits and their association with 50S subunits to form 70S ribosomes. It is also shown that two modifications, m62A and m5C, are essential for formation of active ribosomes in the mutant.

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. 

The humoral immune system in the pupa of the giant silk moth Hyalophora cecropia can be induced by an injection of non-pathogenic bacteria. Three families of antibacterial proteins are synthesized, namely lysozyme, the attacins and the cecropins. This thesis deals with the isolation and characterization of cDNA and genomic clones coding for these proteins.Lysozyme shows a high degree of homology with chicken type lysozymes. In the Cecropia population there are probably three allelic forms which can be explained by point mutations. Attacins comprise six forms of molecules which are the products of two related genes. Two cDNA clones have been isolated which correspond to the two main forms, basic and acidic attacins. Comparison of their primary structure revealed up to 76% homology in coding regions suggesting that they must have originated from a common ancestral gene. At least basic attacin is synthesized in a pre-pro form. The heterogeneity found in attacins isolated from hemolymph is probably due to secondary modifications of the two precursor molecules. Cecropin B cDNA clones have been isolated and sequenced. The deduced amino acid sequence corresponds to a precursor molecule which is 62 amino acids long. A glycine terminates the coding region. It was proposed that this residue is the donor of the amide group for the amidation of the C-terminal of the protein. The precursor of cecropin B is therefore processed at both ends. The deduced structure for cecropin B was confirmed by solid phase synthesis, because the natural and the synthetic compounds were indistinguishable.The chromosomal organization of these genes has been investigated by construction of a genomic library and the characterization of genomic clones which include genes coding for the three classes of antibacterial proteins. The transcription unit of a cecropin B gene is analyzed in detail. The gene is approximately 0.9 kb long and is interrupted by a single intron. Indications were found for the presence of multigene families and multiple copy genes.