We describe a rapid procedure for extracting purified chromosomal DNA from Pseudomonas putida, and some minor modifications needed for use in other organisms, including Gram‐positive strains. The technique is rapid and generally produces between 1 and 5 μg of DNA from 1 ml of liquid culture. The DNA is highly purified and can be readily cut with small quantities of restriction endo‐nucleases, cloned into plasmid vectors and used as a substrate for hybridization with labelled DNA probes. Genetical analysis and cloning of genomic DNA from environmentally important organisms like Ps. putida has become increasingly important. Bacterial chromosomal DNA is usually prepared from large volumes of liquid culture, and involves time‐consuming steps necessary to purify the DNA sufficiently for use in cloning experiments (e. g. Marmur 1961). We have developed a method for preparing small quantities of genomic DNA, which involves whole cell lysis and purification by precipitation and centrifugation. Loss of DNA occurs by shearing, but the yield of DNA is sufficient, and the quality is high. The method has been used to prepare DNA from a variety of organisms and is particularly applicable where the preparation and analysis of DNA from a large number of isolates is required.

Neisseria gonorrhoeae strains P9-2 (Pens) and KW2 (PenR) were grown in chemostats of nonferrous design at constant growth rate, pH and dissolved oxygen tension. Iron limitation (pmax 0.1 h-l) was imposed by omitting iron salts from the defined medium and titrating increasing concentrations of the non-metabolizable iron chelators ovotransferrin and Desferal, to progressively decrease the growth yield. Metabolic activity during iron limitation was very high, with a qGlc which was 2-or 11-fold greater than during cystine-or glucose-limited growth, respectively. More aspartate and isoleucine were metabolized during cystine-limited growth, while more glutamate, proline and serine were metabolized during glucose-or iron-limited growth. Significant concentrations of alanine or valine were excreted during cystine-or glucoselimited growth, respectively. Iron-limited growth of an initial inoculum of non-piliated, transparent colony-forming (P-0-) gonococci resulted in the selection of 100% piliated bacteria. Initial inocula of P+ 0-gonococci retained this phenotype for over 100 generations. Iron-limited gonococci were extremely virulent in the guinea-pig subcutaneous chamber model and inocula of even 12 bacteria grew rapidly and persisted. By contrast, cystine-limited (ironreplete) gonococci retained piliation but did not survive in the chambers. Transition from ironlimited to glucose-limited growth resulted in marked loss of piliation but the bacteria remained virulent. Loss of virulence did not correlate with susceptibility to killing by normal human serum, nor with changes in the content or composition of lipooligosaccharide, which contained 2.9,3.7,4.3 and 4-8 kDa moieties. Additional proteins were detectable in Sarkosyl-purified outer membranes of iron-limited gonococci but several proteins with molecular masses similar to those described in the literature for iron-restricted gonococci were detectable in cystine-or glucose-limited bacteria. I N T R O D U C T I O NA versatile physiology (Keevil et al., 1986) or rapid antigenic variation in pili (P+), proteins I1 (O+), or other outer-membrane proteins (OMPs) (Heckels, 1984) might explain the ability of Neisseria gonorrhoeae to colonize and persist at anatomically distinct sites despite the host immune response. One important environmental factor which pathogens frequently encounter in Man is iron deprivation. Iron is an essential nutrient for the majority of bacteria, particularly aerobes. Potential pathogens must therefore acquire this nutrient in competition with the host, since many body fluids contain iron-binding proteins (Weinberg, 1978 ;Griffiths, 1983 ;Brown & Williams, 1985). For example, the mucosal surfaces invaded by N. gonorrhoeae secrete t Present address: Department of Microbiology, University of Bristol, Bristol BS8 ITD, UK.$ Present address: Department of Applied Biology, UWIST, Cardiff CF1 3NU, UK.Abbreviations: KDO, 2-keto-3-deoxyoctonate (3-deoxy-~-rnanno-2-otulosonate); MIRP, major iron-regulated protein; OMP, outer-membrane protein. 0001-4838 0 19...

Summary. Clinical isolates of Neisseria gonorrhoeae harbouring resistance (R) plasmids of mol. wts 4-4 x lo6 (Asian) or 3.2 x lo6 (African) were grown in prolonged glucose-limited continuous culture to determine the segregation efficiency of each type of plasmid and their expression of penicillinase activity in the absence of antibiotic selective pressure. One strain contained the African plasmid and cryptic and conjugative plasmids, which were all retained after 96 generations in the chemostat. By contrast, the other strain lost all plasmids after 100 generations. Both strains showed increased sensitivity to a range of antibiotics, particularly to the penicillins. Loss of penicillinase activity as minimal inhibitory concentration decreased was confirmed for both strains by assaying the enzyme spectrophotometrically. Activity decreased with the number of generations and none was detectable at the time of complete plasmid loss. This decrease was apparently due to individual bacteria ceasing to produce enzyme rather than a gradual decline in production by the whole population. The sensitivities to a broad range of antibiotics also generally increased during glucose-limited growth, but one strain became more resistant to clindamycin and the other to tetracycline.

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Neisseria gonorrhoeae strain P9-2 was grown in iron-limited or replete continuous culture at a dilution rate of 0.05 h-1, in the presence and absence of Staphylococcus epidermidis. Gonococci maintained expression of pili (P+) and the transparent colony phenotype in pure culture during transitions of iron- and cystine-limited growth. They competed well with staphylococci during iron-limited co-culture and comprised greater than 95% of the population. Transition to cystine-limited growth allowed the staphylococcus to predominate but the gonococcus did not wash out. Furthermore, the gonococcal opaque colony phenotype (O+), indicating synthesis of outer membrane proteins II, was now expressed. Restoration of iron limitation returned the co-culture to its original composition but with P+O+ gonococci dominating. These results suggest that environments might exist in Man where gonococci can compete successfully with normal indigenous bacteria during infection.