Morphology of pili determined by the N incompatibility group plasmid N3 and interaction with bacteriophages PR4 and IKe

The IncN plasmid N3 was transferred to bald strains of Salmonella typhimurium LT2 and Escherichia coli K-12. In both cases, transconjugants were found to carry short pili, which were designated N pili. They were very easily detached from cells and readily broken into short pieces. N pili were found to be straight inflexible rods 9.5 nm thick and sharply pointed at the distal end. The N-specific filamentous bacteriophage IKe and the lipid-containing phage PR4 both adsorbed to the pointed tips when mixed with cell-free suspensions of N pili.     

Variant pili produced by mutants of the Flac plasmid

Transfer-proficient Flac mutants with reduced abilities to plate various F-specific phages were isolated, either by selection after mutagenesis, or as revertants of Flac traA mutants. In many of the mutants pilus-related properties were altered, including physical adsorption of R17 phage, the number of pili per cell and the outgrowth/retraction equilibrium. Complementation studies showed that the mutations were in traA, suggesting that specific alterations in the amino-acid sequence of the pilin subunit protein were responsible for the altered pilus properties. Complementation between the Flac traA mutants and the derepressed plasmid R100-1 restored phage sensitivity in some cases, suggesting that the incorporation of both mutant and R100-1 subunits into the pilus structure may result in conformational changes which increase the capacity of the pilus to interact with phages.     

The occurrence of hepatitis-associated antigen in a group of Canadian patients with leukemia

Serial blood samples from 37 Nova Scotian patients with different forms of leukemia and other malignant conditions of bone marrow and lymphoid tissue were tested for the hepatitis-associated antigen (HAA). Ten patients were found to be positive for HAA. Not only is there an increased incidence of HAA in this population group, but when present the antigen is carried for a longer period than in the normal individual. The increased incidence was completely unrelated to the blood transfusion histories of the patients.     

Effect of chlorpromazine on conjugal plasmid transfer and sex pili

The major tranquillizer chlorpromazine (Cpz) inhibited the conjugal transfer of R and F'lac plasmids. The frequency of transfer of R-144 and R-100 plasmids was reduced with 2-3 log by Cpz at a concentration of 50-100 microgram/ml, while the frequency of RM-98 plasmid did not change under the same conditions. Cpz at 100 microgram/ml was an effective inhibitor of the transfer of F'lac plasmid. By means of electron microscopy and plaque assay, 100 microgram/ml Cpz was shown to reduce the adsorption rate of male specific ribonucleic acid phages MS-2 to the sides of F-pili. Common pili and flagellae seemed to be intact, but sex pili probably retracted in the presence of Cpz.     

Phylogeny and compatibility: plasmid classification in the genomics era

Whole genome sequences are present-day bonanzas for taxonomists. Comparative genomics provides a promising perspective to reveal the evolutionary relationship between organisms, but this strategy is not applicable for extrachromosomal elements due to their high recombination frequencies. Classification of plasmids is based on their compatibility, i.e., the ability to coexist within the same cell. Compatibility testing is a laborious experimental discipline of pairwise comparisons developed for a small set of replicons. Thus, novel approaches are urgently required to deal with the exponentially increasing amount of sequence data. In this minireview, a short overview about the functional role and distribution of plasmids as well as a summary of recent strategies to classify the replicons via phylogenetic analyses is given. Our own work essentially bases on genes of the replication module, i.e., the replicase and two conserved partitioning genes and we exemplified this approach for the four different plasmid types from Alphaproteobacteria. It is suitable for a reliable classification of these replicons and allows in silico predictions about their compatibility. The development of a general classification scheme for plasmids from all microbial lineages will ensure a systematic assessment of the upcoming data flood and help to understand the distribution of extrachromosomal elements.     

Determination of very thin pili by the bacterial drug resistance plasmid R485.

The IncX bacterial drug resistance plasmid R485 was found by electron microscopy to determine numerous very thin filaments (designated 485 pili) only 5.0 nm thick. They exhibited a characteristic helical structure (pitch, 4.6 nm), and were able to form large pseudocrystals when detached from the cell. The concomitant transfer of both pili and the sulfonamide resistance determinant of R485 to RecA strains of Escherichia coli confirmed that the pilus determinant was part of the plasmid and had not been mobilized from the chromosome of the host strain. An extensive examination failed to reveal any similar pili on strains carrying the IncX type plasmid R6K.     

R plasmids of a new incompatibility group determine constitutive production of H pili

R plasmids of a new incompatibility group, IncHII, determined the constitutive production of H pili, had high molecular weights, and determined tellurite resistance. They were designated IncHII because, during incompatibility tests, they sometimes eliminated or were eliminated by, previously described IncH plasmids, which they resembled in several respects. Nevertheless, stable and separate coexistence, i.e., compatibility, with plasmids of IncH1, IncH2, and IncH3 was demonstrated. The latter subgroups, members of which are all incompatible with one another, were distinguished on the basis of DNA-DNA hybridization experiments (N. D. F. Grindley, G. O. Humphreys, and E. S. Anderson, 1973, J. Bacteriol. 115, 387–398; A. F. Roussel, and Y. A. Chabbert, 1978, J. Gen. Microbiol. 104, 269–276.); it is proposed that they be called IncHI, the subgroups being HI1, HI2, and HI3.     

Analytical isoelectric focusing of R factor-determined beta-lactamases: correlation with plasmid compatibility.

R factor-determined beta-lactamases have been investigated by analytical isoelectric focusing. The enzymes such as those specified by the R6K and RP4 plasmids (TEM-type enzymes) are notably homogenous in biochemical tests (Hedges et al., 1974), but two subclasses can be distinguished by isoelectric focusing. Three subclasses can be distinguished among the oxacillin-hydrolyzing enzymes, in good agreement with the classification based upon biochemical characteristics (Dale and Smith, 1974). The TEM-type beta-lactamases are promiscuously distributed among plasmids of a wide variety of compatibility groups, whereas the various oxacillin-hydrolyzing enzymes show some degree of correlation with compatibility.     

Characterization and properties of a novel plasmid vector for Bacillus thuringiensis displaying compatibility with host plasmids.

A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker gene from Bacillus subtilis, was constructed for use in B.t. Unlike other vectors which have been reported to be acceptable for B.t., this new B.t. vector was stably maintained in the absence of Er and did not displace host plasmids, some of which carry crystal protein-encoding genes (cry genes). The compatibility of this B.t. vector with native plasmids is highly desirable when introducing new cry genes into a wild-type B.t. strain. When a cryIIIA gene of B.t. tenebrionis was cloned in this vector and introduced into B.t. kurstaki (kur) HD119, cryIIIA was highly expressed without affecting the level of expression of native cry genes. The stability of this vector and its compatibility with native B.t. plasmids were achieved by subcloning only nucleotide sequences required for the vector to replicate in B.t. The origin of replication was first cloned on a 9.6-kb Bg/II fragment from a 75-kb plasmid of B.t. kur HD73 and then localized to a 2.4-kb region within the 9.6-kb fragment. Sequencing of the 2.4-kb region revealed the presence of an open reading frame (ORF), encoding a putative 312-amino acid (aa) protein. The deduced aa sequence of the ORF showed no homology to any published aa sequences. Deletion analysis indicated that the B.t. vector required at least the ORF and up to 300 bp surrounding the ORF, in order to replicate.     

Adhesive properties associated with the Vir plasmid: a transmissible pathogenic characteristic associated with strains of invasive Escherichia coli

Escherichia coli strain S5 (O15:K+:H21) isolated from a septicaemic lamb and previously shown to possess a virulence plasmid, Vir, attached in vitro to calf epithelial tissue from the ileum, oesophagus and trachea in the presence of 0.5% (w/v) D-mannose. The Vir+ recombinant strains 711v and H209av, which had received the Vir plasmid(s) from strain S5, also attached to these epithelia but the parent strains 711 and H209a without the Vir plasmid were non-adhesive. The attachment of the Vir+ strain 711v to intestinal brush borders was inhibited by antiserum to live Vir+ strain H209av but not by antiserum to strain H209a lacking Vir. No adherence occurred with Vir+ organisms grown at 18 degrees C or after heating at 65 degrees C. Adhesion was unaffected by 0.5% (w/v) formaldehyde. Glucosamine, mannosamine, their N-acetyl derivatives and wheat germ lectin each inhibited attachment of Vir+ strain 711v to brush border epithelia.     

The formation and occurrence of polynuclear aromatic hydrocarbons associated with food

Polynuclear aromatic hydrocarbons are common contaminants of processed food, usually at trace levels. These hydrocarbons are products of combustion and pyrolysis, and are present in petroleum and coal, and in products derived from them. Most polynuclear aromatic hydrocarbons are not carcinogenic, but some of them are, and a few are potent inducers of skin and lung tumors in mice. Their carcinogenic properties have not been fully explored, but they seem to be less potent by ingestion or inhalation, and they are known as a group to produce cancer in humans. The most effective carcinogens among them are those with 5 or 6 fused rings, and these tend to be less prevalent in mixtures than the 3- and 4-ring hydrocarbons, most of which are not carcinogenic. Sophisticated analytical methods, using solvent extraction and chromatography have been developed to detect and measure polynuclear aromatic hydrocarbons at levels of 1 in 10(9) (1 part per billion) or less, and these have been applied to the measurement of individual compounds in foods, as well as in products of combustion and pyrolysis. Wood smoke and smoked foods contain the carcinogenic benzo[a]pyrene at levels of 1 ppb, and other hydrocarbons; liquid smoke has lower levels. Crude vegetable oils have higher concentrations, but purified 'deodorized' oils have benzo[a]pyrene levels near 1 ppb. Sausages cooked over burning logs had as much as 200 ppb benzo[a]pyrene. Charcoal-broiled steaks and ground meat had benzo[a]pyrene concentrations up to 50 micrograms/kg, while less fatty pork and chicken had lower concentrations (up to 10 micrograms/kg). It was probable that the rendered fat dripped on to the hot charcoal and pyrolyzed to form quantities of polynuclear aromatic hydrocarbons, which rose with the smoke to deposit on the meat. Therefore, oven cooking or cooking with a heat source above the meat, or segregation of the meat from the smoke resulted in food containing negligible amounts of polynuclear aromatic hydrocarbons. Modifications of cookings practices accordingly would greatly reduce exposure to this group of carcinogens.     

Replication of the promiscuous plasmid pLSI: a region encompassing the minus origin of replication is associated with stable plasmid inheritance

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RS_R.