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A diverse range of heterotrophic bacteria was screened for the presence of carbonic anhydrase (CA) activity, sensitivity to inhibition of growth by acetazolamide (CA inhibitor), and the presence of protein binding monospecific antibody prepared against purified Neisseria sicca CA. CA activity was demonstrated only in strains of N. sicca and N. gonorrhoeae. However, all Neisseria strains, including various isolates of N. meningitidis and N. lactamica, were sensitive to acetazolamide, when grown in air, and showed serological cross-reaction with N. sicca CA. Strains of other genera were resistant to acetazolamide. A number of strains including members of the genera Pseudomonas, Staphylococcus, Streptococcus, Serratia and Proteus also strongly expressed a gene product(s) immunologically related to CA. The presence of CA cross-reacting proteins, which lack hydrase activity, is discussed in relation to the function of the various mammalian CA isoenzymes.

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The metabolism of various substrates by non-growing suspensions of Neisseria sicca was investigated by a flow-microcalorimetric technique. Substrate utilisation showed Michaelis kinetics allowing determination of saturation constants (Km) and maximum specific rates of substrate utilisation (Vmax). Pyruvate, lactate, a number of tricarboxylic acid cycle intermediates, and amino acids (aspartate, glutamate and proline) were rapidly metabolised [Vmax 5-35 mumol (g dry wt cells)-1 min-1]; Km values were between 4 and 20 microM. Glucose, glycerol, acetate and the other amino acids investigated gave only a slight or no increase in power. The pattern of substrate utilisation is discussed in relation to the role of carbonic anhydrase in N. sicca.

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After an initial period of growth in medium made up in D2O, most strains of pneumococcus tested dramatically lost viability, the extent of the loss depending on the strain and on the amount of contaminating H2O in the D2O. This was followed by a recovery period. Once a strain was 'adapted', the ability to grow in D2O-medium without cell death was inherited, even after passage through H2O-medium, indicating the selection of mutants. Cultures that had not reached 'full adaptation' also exhibited cell death if transferred into either D2O-medium or H2O-medium, supporting the conclusion that the presence of hydrogen and deuterium together caused the toxicity. 'Adapted' cells exhibited an increased mutation frequency to a variety of antibiotic resistances, the propensity for this appearing in the death phase of 'adaptation'. The specific transforming activity of DNA preparations from cultures undergoing 'adaptation' decreased before DNA synthesis ceased indicating damage to the DNA. The integration efficiency of a low-efficiency marker also dropped during 'adaptation' before returning to the initial value when measured in a Hex- recipient, but remained constant in a Hex+ recipient, suggesting that the Hex system may be involved in repair of the DNA damage. 'Adapted' organisms showed evidence of possessing higher Hex activity and were also able to repair lesions caused by UV-irradiation better than the wild-type.

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We studied the effect of the presence of homologous transforming DNA on the growth of several transformable strains of Streptococcus pneumoniae and on the frequency of mutation of these strains to various antibiotic resistances. We observed no effect on growth until the strains became competent, when growth was depressed. At the end of the competence period, some strains showed recovery to varying degrees, whereas others showed evidence of cell death. Growth was also depressed by the presence of DNA from Escherichia coli, indicating that recombination was not likely to be the cause of the observed effect. Furthermore, cell death was not caused by the induction of a prophage. Several of the strains showed increased mutation frequencies during the competence period, although treatment with E. coli DNA gave no such effect, indicating that the mutagenesis was due to recombination. We observed no mutagenesis due to UV irradiation of the strains. The possibility that integration of the transforming DNA may produce lesions which induce error-prone repair is discussed. Furthermore, a strain that showed no mutability by transforming DNA, indicating the presence of a more efficient repair system, gave evidence of producing higher amounts of the hex system when competent, and the possible relationship between these properties is discussed.

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The transformation test for the detection of infection by Neisseria gonorrhoeae has been examined using pro gonococci as recipients and DNA preparations from 912 clinical isolates and from 240 direct swab specimens as donors. The reliability of the method was checked with DNA from clinical isolates; 82% of the N. meningitidis from throat swab specimens were capable of transforming the gonococcal recipients, but after identification of the meningococcus by the aminopeptidase profile, the transformation test was then 99.5% positive for the gonococcus with virtually no false-positives. The only other organism to give a positive reaction was N. lactamica, which occurred once in 912 specimens. When applied directly to swab specimens, the reliability of the test was reduced, but this may have been related to variability of the specimen itself. However, 7 of 15 specimens which were microscopically suspected to be gonococci but unculturable were positive; also, 9 out of 38 unculturable specimens that were not even suspected to be gonococci were positive. Hence the test was able to identify the presence of gonococci that were unculturable. The aminopeptidase activities were not sensitive enough to be detected in the direct swab specimens, and neither cys nor leu auxotrophs were suitable as recipients to give a differentiation between N. gonorrhoeae and N. meningitidis. Evidence was obtained which would support the proposition that the transfer of genetic material between N. gonorrhoeae and N. meningitidis may occur.

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Cultures of a variety of pneumococcal strains maintained competence longer and gave higher yields of transformants when incubated in M1 medium compared with NS medium. This was most probably due to the cells remaining competent for longer in M1 medium. Various parameters controlling the development of competence in M1 medium were investigated. The onset of competence was delayed in M1 medium compared with that in NS medium, probably due to the presence of Casamino acids in the former. Competence developed normally over a pH range of 7.3 to 8.3, with cultures inoculated from the same batch of frozen "precultures" showing consistent characteristics. This was not observed when frozen "sensitization" cultures were revived. The average cell chain length increased with the development of competence in all the strains tested and, with the exception of cultures which had entered the stationary growth phase, declined after the culture had lost its competence. The extent of the increase in chain length was dependent upon the pH of the medium.

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Mutants of an aminopterin-resistant strain of pneumococcus possessing four different suppressor genes have been isolated after mutagenesis with 5-BUdR. The suppressed strains exhibit a partial revertant phenotype since the parental aminopterin resistance remained unchanged but the associated sensitivity to an excess concentration of the branched chain amino acids L-isoleucine, L-valine and L-leucine was diminished almost to the level of the wild-type strain C13. The suppressor mutations had therefore dissociated the two properties associated with a mutation in the amiA cistron, namely aminopterin resistance and isoleucine sensitivity. The suppressor genes reduced the sensitivity to isoleucine of a number of amiA mutants, but had no effect on the level of resistance to a number of unrelated genes conferring resistance to other antibacterial substances. The suppressor mutations themselves did not confer resistance to aminopterin. Mapping of the suppressor mutations by recombination analysis and by clonal analysis showed them to be intragenic lying in the region near to the amiA-r19, amiA-r23, amiA-r17 loci.

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