Toxin production by Pasteurella multocida isolated from rabbits with atrophic rhinitis.

Naturally acquired turbinate atrophy in rabbits was associated with Pasteurella multocida infection. Several in vitro and in vivo studies were conducted to document toxin production from P multocida isolates and to determine the relation of toxin to atrophic rhinitis in rabbits. Ten isolates of P multocida serotype A:12 were obtained from adult New Zealand White rabbits with noninduced atrophic rhinitis. Specific-pathogen-free rabbits inoculated intranasally with isolates of P multocida developed rhinitis and turbinate atrophy. However, inoculation with filtrates of the same bacteria failed to induce turbinate atrophy. Cytotoxicity was observed in assays, using bovine embryonic turbinate cell cultures with extracts of P multocida, but not in agar overlay cytotoxicity assays, using bovine embryonic turbinate, bovine embryonic lung, or Vero cell cultures, or in a sandwich ELISA, using monoclonal antibodies to purified P multocida toxin. Thus, turbinate atrophy was experimentally reproduced in rabbits with isolates of P multocida, but toxin was only detected in vitro by cell culture assay of P multocida extracts.

Methylation-dependent transcription controls plasmid replication of the CloDF13 cop-1(Ts) mutant.

The CloDF13 cop-1(Ts) mutant expresses a temperature-dependent plasmid copy number. At 42 degrees C the mutant shows a "runaway" behavior, and cells harboring this plasmid are killed. The cop-1(Ts) mutation is a G-to-A transition that disturbs one of the two methylation sites which are located opposite in the stem-loop structure within a region involved in both the initiation of primer synthesis for DNA replication and the termination of the cloacin operon transcript. We demonstrate that the mutation results in an increased primer (RNA II) synthesis resulting from nonconditional enhanced RNA II promoter activity, which at 42 degrees C causes a decrease in the amount of active replication repressor molecules (RNA I) synthesized from the opposite strand. We found that the absence of Dam methylation abolishes the mutant phenotype and that under this condition the high mutant level of RNA II synthesis is reduced, which is accompanied by a restoration of the regulation by RNA I. The role of methylation in the regulation of plasmid replication is discussed.

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13.

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13 has been determined. The plasmid consists of 9957 base pairs (molecular weight 6.64 X 10(6] with a GC content of 54.4%. At this moment 16 identified biological functions can be assigned to the primary structure of the CloDF13 DNA. The functions include those of eight protein encoding genes, two untranslated RNA species, and six DNA sites. We discuss these functions in relation to the structure of CloDF13 DNA. For convenience we have divided the CloDF13 genome into five defined regions: region I (origin of vegetative replication, priming and control of replication, type I incompatibility), region II (cloacin DF13, cloacin immunity, cloacin release, cloacin operon control), region III (double-stranded DNA-phage interaction, type II incompatibility, multimer resolution), region IV (inhibition of male specific RNA phages and transfer of Flac), and region V (mobility proteins, basis of mobility).

Floral tissue of Petunia hybrida (V30) expresses only one member of the chalcone synthase multigene family.

Twenty independent, petal-specific chalcone synthase (CHS) cDNA clones have been isolated from Petunia hybrida variety Violet 30 (V30). Sequence analysis shows that the largest of these clones contains the entire coding sequence. Using this clone in Southern blot analysis reveals the presence of multiple CHS gene copies in the genome of Petunia hybrida V30. Hybridization and sequence analysis of the CHS cDNA clones shows that they are all copied from a single mRNA species. This indicates the presence of only one transcriptionally active CHS gene in petals. Finally we report the identification, cloning and partial characterization of this gene.

Effects of divalent cations and of phospholipase A activity on excretion of cloacin DF13 and lysis of host cells.

Induction of cloacin DF13 synthesis in Escherichia coli harbouring plasmid CloDF13 results in the release of cloacin DF13, inhibition of growth and ultimately in lysis of the host cells. Expression of the pCloDF13-encoded protein H is essential for both the release of cloacin DF13 and the lysis of the cells. The divalent cations Mg2+ and Ca2+ interfered with the mitomycin C-induced protein H-dependent lysis, but hardly affected the release of cloacin DF13. Essentially all of the bacteriocin was released from the cells before a detectable degradation of the peptidoglycan occurred, independent of the presence of mitomycin C. Experiments with phospholipase A mutants revealed that activation of detergent-resistant phospholipase A was essential for the export of cloacin DF13 across the outer membrane and the lysis of induced cells. Transport of cloacin DF13 across the cytoplasmic membrane was mainly dependent on protein H. A revised model for the excretion of cloacin DF13 is presented.

Isolation and characterization of Paracoccus denitrificans mutants with defects in the metabolism of one-carbon compounds.

Mutants deficient in the metabolism of one-carbon compounds have been obtained by treating Paracoccus denitrificans with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were selected without enrichment procedures by newly developed plate screening tests. The obtained mutants were characterized by their growth responses, cytochrome composition, enzyme activities, and immunogenic reaction with antisera against methanol dehydrogenase. By these criteria five mutant classes could be distinguished. Class I mutants are involved in the expression of methanol dehydrogenase. Three mutants of this class have a defect in the structural gene. A double mutant was found with defects in the expression of both methanol dehydrogenase and hydrogenase. Class II mutants have a defect in a regulatory gene involved in the regulation of both methanol dehydrogenase and methylamine dehydrogenase. Class III mutants are deficient in formaldehyde metabolism. A defect may exist in the expression of a second non-NAD-linked formaldehyde dehydrogenase which was postulated to be involved in C1 metabolism. Class IV mutants are deficient in cytochrome c. Mutants of class V have a defect in synthesis of the molybdenum cofactor essential for the function of formate dehydrogenase.

Maintenance of multicopy plasmid Clo DF13 III. Role of plasmid size and copy number in partitioning.

To elucidate the mechanisms that operate in plasmid maintenance, we analysed the stability of different combinations of Clo DF13 derivatives present in the same bacterial cell. From the data described in this paper we conclude: (i) each Clo DF13 plasmid molecule has an equal chance of colonizing daughter cells upon cell division, (ii) the Clo DF13 minimal replicon harbours functions involved in plasmid segregation and incompatibility, (iii) in the case of cells harbouring plasmid replicons which differ in size, the smaller plasmid is gradually lost from the cell population, (iv) in the case of cells habouring plasmid replicons which differ in copy number, the lower copy number plasmid is always lost from the cell population. The effect of plasmid copy number is dominant over the effect of plasmid size.

Maintenance of multicopy plasmid Clo DF13 in E. coli cells: evidence for site-specific recombination at parB.

Certain derivatives of copy-control mutants of plasmid Clo DF13 are not stably inherited in E. coli. These plasmids, predominantly present as multimeric DNA molecules, lack a specific region, designated parB. Here we present the nucleotide sequence of this parB region spanning 328 bp between 46% and 49% on the plasmid genome. parB is a noncoding region with extensive internal symmetry. A recA-independent, site-specific resolution process occurs between two intramolecular parB sites present in direct orientation relative to each other. A gene located in the direct vicinity of parB, gene L, is not essential for parB functioning. However, our genetic data indicate that transcription from the gene L-containing operon into parB is required. We conclude that the efficient maintenance of Clo DF13 cop derivatives containing parB is provided by resolution of mutimeric molecules. Because Clo DF13 wt and cop derivatives have a different response to the deletion of parB we postulate that two different recombination systems, a parB-dependent and a parB-independent system, operate in the efficient maintenance of Clo DF13 plasmids.

Structure and regulation of gene expression of a Clo DF13 plasmid DNA region involved in plasmid segregation and incompatibility.

The bacteriocinogenic plasmid Clo DF13 contains genetic information involved in the accurate partitioning of the plasmid (parA and parB) as well as in incompatibility phenomena (incA, B, C and D). In this paper we report on the primary structure and regulation of gene expression of the 29% - 50% part of Clo DF13, containing the DNA regions incA, incB and parB as well as genes K and L. According to the results of our DNA sequence analysis, mapping of transposon insertions, RNA blotting and S1 mapping experiments, we conclude that: a) genes K and L are transcribed as one operon; transcription of this operon is initiated at a promoter (P2) located at 32.5% and proceeds in a clockwise direction. b) treatment of cells with mitomycin-C, significantly enhances transcription from P2, although this promoter is probably not directly repressed by lexA protein. c) Termination of transcription of this operon occurs between genes K and L, as well as distal to gene L. The possible role of gene products and/or sites, located within the 29-50% DNA region, in plasmid incompatibility and segregation is discussed.

Molecular structure and function of the bacteriocin gene and bacteriocin protein of plasmid Clo DF13.

In this paper we present the complete nucleotide sequence of the bacteriocin gene of plasmid Clo DF13. According to the predicted aminoacid sequence the bacteriocin, cloacin DF13, consists of 561 aminoacids and has a molecular weight of 59,293 D. To obtain insight into the structure and function of specific parts of the cloacin molecule, we constructed a hydration profile and we predicted the secondary structure of the protein. According to our predictions, the N-terminus of cloacin DF13 (corresponding to the first 150-180 aminoacids) is relatively hydrophobic and is rich in glycine residues. The data obtained support previous findings that the N-terminal part of cloacin DF13 is involved in translocation of this protein across the cell membrane. The C-terminal part of the cloacin protein is rich in positively charged aminoacids; this might reflect the RNase activity located within this domain. A comparison of the bacteriocin genes and corresponding proteins of Clo DF13 and Col E1 did not reveal any homology at the level of either the nucleotide or the aminoacid sequence. The codon usage of both genes, however, exhibits striking similarities. The sequence data obtained during this study enabled us to present the nucleotide sequence of the entire cloacin operon. The structure of this operon and the regulation of expression of the genes, located within this operon, is discussed.

Localization and nucleotide sequence of the bom region of Clo DF13.

Mobilization of the non-conjugative plasmid Clo DF13 requires both gene products of a conjugative plasmid and Clo DF13 encoded proteins as well as a cis-acting Clo DF13 DNA region, termed bom (basis of mobility). The bom region was located within a 264 bp fragment around the unique HpaI site. Comparison with the corresponding ColE1 and pBR322 sequences showed similarities with respect to the secondary structure. With respect to a possible relationship between the origin of vegetative replication (oriV) and the origin of transfer (oriT), we found that neither distance nor orientation of the Clo DF13 bom region with respect to oriV had any significant influence on the mobilization frequency. Surprisingly, after cloning of the 264 bp HpaII fragment in a bom- vector, restoration of the bom+ phenotype was only observed in one orientation. From these observations and from the sequence analysis of the bom region we suggest that transcription into this cloned bom fragment is essential for effective mobilization of the plasmid.

The nucleotide sequence of the bacteriocin promoters of plasmids Clo DF13 and Co1 E1: role of lexA repressor and cAMP in the regulation of promoter activity.

Treatment of cells, harbouring the bacteriocinogenic plasmic Clo DF13 with mitomycin-C, which induces the cellular SOS response, results in a significantly increased transcription of the operon encoding the bacteriocin cloacin DF13, the immunity protein and the lysis protein H. The nucleotide sequences of the promoter regions and N-terminal parts of the bacteriocin genes of Clo DF13, Col E1 and the pMB1 derivative pBR324 have been determined. A comparison of these sequences with those of corresponding regions of the lexA, recA and uvrB genes revealed that the promoter regions of the bacteriocin genes studied contain binding sites for the lexA protein, which is the repressor of the E. coli DNA-repair system. Using both, a thermosensitive lexA host strain and a host with pACYC184 into which the lexA gene had been cloned, we were able to demonstrate, that in vivo the lexA protein is involved in the regulation of bacteriocin synthesis. From the data presented, we conclude that bacteriocin synthesis is controlled at least by the lexA repressor. It has been reported that also catabolite repression might play an essential role in the control of bacteriocin synthesis. Computer analysis of the DNA sequence data indicated that the promoter regions of both, the cloacin DF13 and colicin E1 genes contain potential binding sites for the cyclic AMP-cyclic AMP Receptor Protein complex.

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. I. Localisation and mutual interactions of four Clo DF13 incompatibility regions.

The incompatibility properties of the bacteriocinogenic plasmid Clo DF13 have been examined. By using Clo DF13, Clo DF13 deletion, and transposon insertion mutants as well as compatible R plasmids into which Clo DF13 fragments have been cloned, we could identify and localise four different incompatibility regions on the Clo DF13 genome. These regions, designated incA, incB, incC, and incD are located in the following positions: incA about 32%, incB between 45% and 50%; incC about 97% and incD between 1.8% and 9% of the Clo DF13 genome. We studied the contribution of each of the four inc regions, separately and/or in combination with each other, to the incompatibility between two plasmid replicons. Two types of incompatibility can be distinguished: Type I evoked by incD, that overlaps the replication control area of Clo DF13 and type II, caused by incA, B and C. From our observations we present a model for plasmid incompatibility based on a combination of the existing repressor dilution and membrane attachment models.

Mitomycin C-induced synthesis of cloacin DF13 and lethality in cloacinogenic Escherichia coli cells.

Treatment of cloacinogenic cultures with increasing concentrations of mitomycin C induced an increasing synthesis of cloacin DF13 accompanied by a decreasing number of colony-forming cells. Cells grown in the presence of glucose required a 10-fold-higher concentration of mitomycin C for optimal induction of cloacin production than did cells grown with lactate. Release of the cloacin was hampered in glucose-grown cells. Experiments with various CloDF13 insertion and deletion mutants revealed that the transcription of CloDF13 deoxyribonucleic acid sequences adjacent to the cloacin structural gene was essential for mitomycin C-induced lethality.

Identification of mutations affecting replication control of plasmid Clo DF13.

The bacteriocinogenic plasmid Clo DF13, originally isolated from Escherichia cloacae, is stably maintained in Escherichia coli to the extent of about 10 copies per cell. Its replication resembles that of many other small, multicopy plasmids; plasmid-encoded protein is not required but plasmid-specific genetic information is involved in regulation of replication as both conditional and nonconditional copy-number mutants of Clo DF13, and transcomplementable copy-number mutants of plasmid Col E1 have been described. The sequences essential for replication of Col E1 (refs 16, 17) and Clo DF13 (refs 18, 19) have been identified within a region surrounding the replication origin. Initiation of Col E1 replication is preceded by transcription of the origin region, providing the RNA primer at the origin. However, transcription in the opposite direction results in a small transcript of approximately 100 nucleotides (RNA-100) for both Col E1 (refs 21, 22) and Clo DF13 (ref. 23). Data suggest that Col E1 RNA-100 acts as a negative control element for the initiation of replication. We show here that single base transitions in the RNA-100 cistron of Clo DF13 can result in a nonconditional increase in plasmid copy-number. Also, sequence analysis has revealed that a specific base transition in a DNA region, apparently involved in both termination and initiation of transcription towards the replication origin, results in a thermosensitive plasmid copy-number.

Protein H encoded by plasmid Clo DF13 involved in lysis of the bacterial host. I. Localisation of the gene and identification and subcellular localisation of the gene H product.

The gene expression of the Clo DF13 "replication region", located between 1.8% and 12% on the plasmid genome, was studied using newly constructed Clo DF13 insertion and deletion mutants. We were able to detect a Clo DF13 specified protein of 6 kilodaltons (kd) by electrophoretic analysis of plasmid proteins, synthesized in Escherichia coli minicells, on 14-25% gradient polyacrylamide gels. The gene encoding this protein was mapped between 1.8% and 12% on the Clo DF13 genome. The nucleotide sequence of this region, as determined by Stuitje et al. (1980), revealed three open reading frames each potentially coding for a protein of 6 kd. Since these three proteins differ in amino acid composition we could distinguish which of these proteins was actually synthesized, by labeling Clo DF13 proteins with specific 14C-labeled amino acids. We found that gene H, located between 9.3% (bp 744) and 11% (bp 893), encodes the observed protein of 6 kd (denominated protein H). With respect to the subcellular localization we observed that protein H, which contains a large hydrophobic region at its C-terminal part, is predominantly present in the bacterial membrane. Although gene H is located close to the region known to be involved in Clo DF13 replication, its gene product, protein H, is not essential for the plasmid DNA replication process. The possibility of the existence of a comparable protein encoded by the related plasmid Col E1 will be discussed.

Protein H encoded by plasmid Clo DF13 involved in lysis of the bacterial host. II. Functions and regulation of synthesis of the gene H product.

We studied the expression of gene H, located between 9.3% and 11% on the CLo DF13 genome, as well as the functions of the gene product. We found that treatment of bacterial cells with mitomycin-C results in the induced synthesis of three Clo DF13 specified proteins namely cloacin DF13, immunity protein and protein H. Evidence was obtained that the genes encoding these proteins form one, mitomycin-C induceable, operon; the promoter at 32% in front of the cloacin gene is essential for the induced expression. Furthermore we could demontrate that protein H is involved in the lethal effect of mitomycin-C treatment of bacteriocinogenic cells. The data in this paper show that a high concentration of protein H in cells, due either to an induced expression of gene H (mitomycin-C induction) or to a gene dosage effect (Clo DF13 copl Ts copy control mutant), results in the lysis of bacterial cells. The implication of these data are discussed.