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An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria. Such biologically realistic simulations validate the use of the IbM approach to predicting behaviour of bacteriophages in bacterial populations. The applicability of the model for more complex scenarios aimed at predictive modelling of bacterial evolution in a changing environment and the implications for the spread of viruses in a wider context are discussed.

We have developed a novel rule-based computing system of microbial interactions and communications, referred to as COSMIC-Rules, for simulating evolutionary processes within populations of virtual bacteria. The model incorporates three levels: the bacterial genome, the bacterial cell and an environment inhabited by such cells. The virtual environment in COSMIC-Rules can contain multiple substances, whose relative toxicity or nutrient status is specified by the genome of the bacterium. Each substance may be distributed uniformly or in a user-defined manner. The organisms in COSMIC-Rules possess individually-defined physical locations, size, cell division status and genomes. Genes and/or gene systems are represented by abstractions that may summate sometimes complex phenotypes. Central to COSMIC-Rules is a simplified representation of bacterial species, each containing a functional genome including, where desired, extrachromosomal elements such as plasmids and/or bacteriophages. A widely applicable computer representation of biological recognition systems based on bit string matching is essential to the model. This representation permits, for example, the modelling of protein-protein interactions, receptor-ligand interactions and DNA-DNA transactions. COSMIC-Rules is intended to inform studies on bacterial adaptation and evolution, and to predict behaviour of populations of pathogenic bacteria and their viruses. The framework is constructed for parallel execution across a large number of machines and efficiently utilises a 64 processor development cluster. It will run on any Grid system and has successfully tested simulations with millions of bacteria, of multiple species and utilising multiple substrates. The model may be used for large-scale simulations where a genealogical record for individual organisms is required.

COSMIC-rules, an individual-based model for bacterial adaptation and evolution, has been used to study virtual transmission of plasmids within bacterial populations, in an environment varying between supportive and inhibitory. The simulations demonstrate spread of antibiotic resistance (R) plasmids, both compatible and incompatible, by the bacterial gene transfer process of conjugation. This paper describes the behaviour of virtual plasmids, their modes of exchange within bacterial populations and the impact of antibiotics, together with the rules governing plasmid transfer. Three case studies are examined: transfer of an R plasmid within an antibiotic-susceptible population, transfer of two incompatible R plasmids and transfer of two compatible R plasmids. R plasmid transfer confers antibiotic resistance on recipients. For incompatible plasmids, one or other plasmid could be maintained in bacterial cells and only that portion of the population acquiring the appropriate plasmid-encoded resistance survives exposure to the antibiotics. By contrast, the compatible plasmids transfer and mix freely within the bacterial population that survives in its entirety in the presence of the antibiotics. These studies are intended to inform models for examining adaptive evolution in bacteria. They provide proof of principle in simple systems as a platform for predicting the behaviour of bacterial populations in more complex situations, for example in response to changing environments or in multi-species bacterial assemblages.

Ray Paton oversaw the creation of a long lineage of Individual-based Models (IbMs) and this paper discusses the five most successful. All of these concern the development of adaptation, covering both evolutionary time and organism lifetime (somatic time). Of the five models discussed here, the first is based on a plant-herbivore model, the other four are based on a substrate-bacteria model, with the option of antibiotics.

DNA reassociation and hydroxyapatite chromatography were used to isolate high-copy DNA of the grass Zingeria biebersteiniana (2n = 4). In situ hybridization demonstrated that the DNA isolated was enriched for pericentromere-specific repetitive sequences. One abundant pericentromere-specific component is the differentially methylated tandem-repeat family Zbcen1. Other sequences isolated, Zb46 and Zb47A, are dispersed and display similarity to parts of the gypsy- and copia-like retrotransposable elements of other grasses. In situ hybridization with the copia-like sequence Zb47A resulted in dispersed labelling along the chromosome arms, with a significant signal accumulation in the pericentromeric region of all chromosomes. It is concluded that the pericentromeric heterochromatin of Z. biebersteiniana is composed of members of the Zbcen1 tandem repeat family and that these tandem arrays are intermingled with accumulated putative copia-like retrotransposon sequences. An observed Rab1 interphase orientation suggests that the length of the chromosomes rather than the genome size is the determining factor of the Rab1 phenomenon.

Plant chromosome microdissection techniques together with different isolation and amplification methods of microisolated DNA are described. Such isolated DNA was used to 'chromosome paint' B chromosomes of the dicot Brachycome dichromosomatica and the monocot Secale cereale. It is demonstrated that the specific painting of the described chromosomes was possible because of enrichment for chromosome-specific repetitive sequences, rather than the chromosome specific low- and single-copy sequences which are responsible for the painting of mammalian chromosomes. The feasibility of 'chromosome painting' of standard chromosomes in plant species with relatively small or large genomes is discussed.

Microsomal chitinase from yeast and hyphal cells of Candida albicans was activated endogenously by incubation at 30 degrees C and exogenously by trypsin. The putative activating factor of yeast cells was separated from chitinase activity by fractionation of lysed protoplasts on an Iodixanol density gradient. The vacuole fraction contained no significant chitinase activity, but was enriched in chitinase activating factor. Activity of microsomal chitinase increased upon incubation with this, but no other gradient factor. Results suggest that the regulatory system governing microsomal chitinase activity, like that governing chitin synthase, involves a 'vacuolar' activating factor in Candida albicans.

Monoclonal antibodies were produced to Streptomyces lividans spore surface antigens. One particular hybridoma cell line, 43H6, produced a monoclonal antibody that reacted exclusively with Streptomyces cluster group 21 in an enzyme-linked immunosorbent assay (ELISA). Antibody 43H6 was found to be of subclass IgG1, kappa light chain. Western blot (immunoblot) analysis revealed that 43H6 recognized a major outer spore polypeptide of about 37,000 Da. The epitope was stably maintained in S. lividans spores over at least seven sporulation cycles on laboratory medium and for at least 14 weeks in sterile soil systems. The species group specificity of antibody 43H6 was exploited in the development of an immunocapture technique for the isolation of streptomycetes from soil. Magnetic beads coated with antibody 43H6 were mixed with soil samples seeded with S. lividans spores. Spore-bead complexes were recovered using magnets. Treatment of beads with blocking agents and the inclusion of detergents in the recovery system lessened non-specific binding of spores to beads and improved recovery. In buffer solutions decreasing the spore concentration increased the recovery values for a fixed bead concentration. At a spore concentration of 5 x 10(7) ml-1 the recovery was 4.3% whilst at 5 x 10(2) ml-1 it was 76% for a fixed bead concentration of 0.6 mg ml-1. Using a bead concentration of 2 mg per 10 g soil, approximately 30% of the target spore population of 10(6) c.f.u. was recovered from sterile soil and 4% from non-sterile soil. This method offers a rapid means of selectively recovering and concentrating Streptomyces spores from soil samples.

Plasmid constructs pNW1 through pNW6 containing a controllable xylE gene (for catechol 2,3-dioxygenase) were introduced into Streptomyces lividans strains to provide a selectable marker system. xylE functions in S. lividans under the control of bacteriophage lambda promoters lambda pL and lambda pR. Thermoregulated expression of xylE is provided through the lambda repressor cI857. Catechol 2,3-dioxygenase activity was increased 2.8-fold from plasmid construct pNW2 (lambda pL, xylE, cI857) and 9.5- and 7.4-fold from constructs pNW3 (lambda pR, xylE, cI857) and pNW5 (lambda pR, xylE, cI857), respectively, when the temperature was shifted from 28 degrees C to 37 degrees C. The stability of the constructs varied from 4.7% for pNW2 to 99.4% for pNW4 (lambda pL, xylE) over two rounds of sporulation. Marked S. lividans strains released into soil systems retained the XylE phenotype for more than 80 days, depending on the marker plasmid, when examined by a selective plating method. Furthermore, S. lividans harboring plasmid pNW5 was detectable by nucleic acid hybridization at less than 10 CFU g-1 (dry weight) of soil as mycelium and 10(3) CFU g-1 (dry weight) of soil as spores with the xylE marker DNA extracted from soil and amplified by using the polymerase chain reaction.

The growth and survival of strains of Streptomyces lividans and S. violaceolatus in sterile and nonsterile soil was investigated by using inoculated soil microcosms run as batch systems. It was evident that, after an initial short mycelial growth phase of 2 to 3 days, sporulation occurred and inoculants survived as spores. The transfer of a high-copy-number, self-transmissible plasmid, pIJ673, was detected by using intra- and interspecific crosses. The initial detection of transconjugants correlated with the development of the mycelial state of the inoculants (as confirmed by scanning electron microscopy) after 2 days of incubation. Subsequent spread of the plasmid was attributed to spread within existing mycelium followed by sporulation. In natural soil, inoculant numbers remained constant or declined, but plasmid transfer was readily detected.

When E. coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements. Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib). Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb). Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions. Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants. This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants. In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.

The deletion end-points of a number of type I (less than monomeric) plasmid deletants obtained by transforming recA+ or recA- E. coli with linear pBR322 DNA were determined by DNA sequencing. In both monodirectional and bidirectional deletions the recyclization point was normally characterized by recombination between directly repeated sequences of between 4 and 10 bp present on each arm of the linearized pBR322 molecule. Frequently, short tracts of uninterrupted homology involved in recombinational recircularization were embedded in regions of relative non-homology. A model predicting the probability of matching sequences in either end of a linear plasmid molecule is presented. It is proposed that exonucleolytic processing of the exposed termini of linear plasmid molecules generates substrates for subsequent recombinational recyclization and deletion. The activity of host recombination and repair functions in recircularizing linear DNA molecules explains the generation of many of the aberrant recombinant DNA constructs obtained during gene cloning procedures.

Over 20 different plasmid-encoded beta-lactamases have so far been discovered. This paper considers genetic mechanisms by which beta-lactamase genes encoded by plasmids are disseminated across generic boundaries. Particular emphasis is placed on the evolution of plasmids carrying all or part of Tn3 and encoding TEM-1 beta-lactamase in Haemophilus and Neisseria species. Examples of the acquisition of broad host range plasmids carrying Tn3 sequences and of rescue of transposon sequences to indigenous plasmids or to the host chromosome have been found in these two genera. Studies on nonconjugative beta-lactamase plasmids in Neisseria and Haemophilus are consistent with the evolution of a family of plasmids originating from the insertion of Tn3 into an indigenous progenitor plasmid. A series of subsequent insertional and deletional events, most probably occurring as a consequence of genetic transfer, have given rise to the existing group of small, closely related ampicillin-resistance plasmids found currently in these genera. A general model for deletional and other rearrangements caused by recombinational recyclization during the evolution of resistance plasmids is described.

Uptake of gentamicin by a gentamicin-resistant strain of Staphylococcus aureus possessing the aminoglycoside-modifying phosphotransferase enzyme APH(2") was enhanced by the protein synthesis inhibitor, puromycin or by the proton-translocating ATPase inhibitor, N,N'-dicylohexylcarbodiimide. Such enhanced uptake was inhibited by carbonyl cyanide p trifluoromethoxyphenylhydrazone or by valinomycin in the presence of potassium ions, suggesting a role for the transmembrane proton motive force in the process. The accumulated gentamicin did not cause loss of cell viability and exhibited altered chromatographic mobility compared with a control (unmodified) preparation of gentamicin.

The photosynthetically-incompetent mutant V-2 of Rhodopseudomonas spheroides which is incapable of synthesising bacteriochlorophyll was grown aerobically under conditions of both high and low aeration. Potentiometric titration a at 560 nm minus 570 nm revealed the presence of several different components tentatively identified as b-type cytochromes. Two such components of oxidation-reduction midpoint potentials of +390 mV +/- 10 mV and +255 mV +/- 7mV have not previously been detected in membranes of Rps. spheroides. These components have also been resolved by difference spectra at controlled oxidation-reduction potentials and fourth derivative spectra. Neither component appeared to react with CO. With increasing aeration of the culture medium the relative concentration of these two b-type cytochromes diminished, whilst that of the a-type oxidase increased.

Accumulation of gentamicin by gentamicin-susceptible Staphylococcus aureus was examined by using the ionophorous antibiotic, valinomycin and the protontranslocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The effects of these inhibitors on the transmembrane electrical potential (delta psi) were determined by measuring the equilibrium distribution of the tetraphenylphosphonium ion. The results indicate a direct correlation between delta psi and the extent of gentamicin uptake. However, under conditions where a significant delta psi existed across the plasma membrane, uptake of gentamicin was negligible. A threshold delta psi may thus be required to initiate gentamicin uptake. The proposed threshold delta psi appears to vary depending upon the external concentration of gentamicin.