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An aminoglycoside-resistance-encoding gene (kamC) has been isolated from the sporaricin producer, Saccharopolyspora (Sac.) hirsuta, and expressed both in Streptomyces lividans and Escherichia coli. The pattern of resistance conferred by this gene was identical to that given by another gene (kamB) previously isolated from Streptomyces tenebrarius. In accordance with the known action of the kamB product, the Sac. hirsuta determinant also encodes a methyltransferase that modifies 16S rRNA, thereby rendering ribosomes refractory to certain aminoglycosides. The nucleotide sequences of both genes have been determined and comparison of the deduced amino acid sequences reveals a high degree of similarity.

The mechanism of xylose isomerase (EC 5.3.1.5) has been studied with X-ray crystallography. Four refined crystal structures are reported at 3-A resolution: native enzyme, enzyme + glucose, enzyme + glucose + Mg2+, and enzyme + glucose + Mn2+. One of these structures (E.G.Mg) was determined in a crystal mounted in a flow cell. The other structures were equilibrium experiments carried out by soaking crystals in substrate containing solution. These structures and other studies suggest that, contrary to expectation, xylose isomerase may not use the generally expected base-catalyzed enolization mechanism. A mechanism involving a hydride shift is consistent with the structures presented here and warrants further investigation. Additional evidence in support of a hydride shift comes from comparing xylose isomerase with triosephosphate isomerase which is known to catalyze an analogous reaction via an enediol intermediate. Evidence is presented that suggests that aldose-ketose isomerases can be divided into two groups. Phospho sugar isomerases generally do not require a metal ion for activity and show exchange of substrate protons with solvent. In contrast, simple sugar isomerases all require a metal ion and show very low solvent exchange. These observations are rationalized on the basis of the need for stereospecific sugar binding.

A mutant strain of pneumococcus which fails to discriminate against low-efficiency markers during transformation by homospecific pneumococcal donor DNA retains the wild-type capacity to discriminate against heterospecific (streptococcal) donor DNA. We conclude that discrimination against heterospecific DNA must differ from that against low-efficiency markers by the kind or number of elements being recognized.

A method of transformation on solid medium especially adapted for pneumococcus has been developed. Under specific conditions, all colonies that are allowed to grow in the presence of transforming DNA for six hours give rise to transformed bacteria. Combined with replica plating this technique has been used to isolate mutants modified with regard to recombination. Most of the mutants found are transformation-defective and show a large diversity in their response to ultraviolet light. Some of these mutants have lost their ability to take up transforming DNA. One shows a reduced yield of transformants for a given quantity of DNA taken up. Mutants that manifest altered behavior with regard to marker efficiencies have also been isolated. One of these exhibits a decrease in the transformation efficiency of only the high efficiency markers and two mutants show a decrease in the transformation efficiency of the low efficiency markers.

Clinical isolates of Streptococcus pneumoniae resistant to chloramphenicol were observed in France for the first time in 1973. During a 4-year survey, these strains were found to represent 6% of a total of 564 isolates of S. pneumoniae in a general hospital and to belong to 13 different serotypes. One such strain, referred to as BM 6001, was shown to inactivate chloramphenicol, and the process was found to be inducible. The inactivated products were demonstrated to be O -acetoxy esters of chloramphenicol. The synthesis of an inducible chloramphenicol acetyltransferase was shown to be responsible for the inactivation of the drug. The resistant strain was able to transfer the chloramphenicol marker by transformation to competent strains of pneumococci at a frequency of 1% of that observed for control chromosomal markers. The loss of resistance was enhanced by ethidium bromide treatment, but no chloramphenicol-resistant mutant was isolated by mutagenesis of a “cured” clone or naturally susceptible isolates. All attempts to isolate plasmid deoxyribonucleic acid as covalently closed circular molecules from strain BM 6001 have been unsuccessful, but epidemiological evidence and the fact that the genes specifying chloramphenicol acetyltransferase synthesis are usually located on plasmids suggest that this marker may be plasmid-borne in S. pneumoniae .

The distribution of integration efficiencies of independent mutations spontaneously occurring in the amiA locus was determined in two strains of pneumococcus. Strain Cl 3 integrates genetic markers with different efficiencies during transformation, whereas strain 401, isogenic with strain Cl 3 , does not discriminate between markers and integrates all markers with the same high efficiency. The discriminating strain Cl 3 gives rise to spontaneous mutations in the locus amiA , which fall into four classes with respect to their individual integration efficiencies. Mutations with a low efficiency of integration are equal in number to mutations with a high efficiency. Mutations from the two other classes corresponding to intermediate and very high efficiencies are about five times less frequent. The same four classes were also found among amiA mutants spontaneously occurring in strain 401. However, the two distributions of integration efficiencies of amiA mutants arisen either in strain Cl 3 or strain 401 are significantly different. The number of spontaneous amiA mutants, estimated by two methods, was found to be higher in strain 401 than in strain Cl 3 . The increase of the mutation rate in strain 401 could be accounted for by the excess of mutations falling in the two less-efficient classes observed in this strain. The discriminating process which acts during transformation presumably also intervenes in the appearance of spontaneous mutations.

Transformation of the pneumococcus mutant 401 by DNA's bearing the standard reference marker and several other markers belonging to two unlinked loci has shown that differences in the integration efficiencies of these markers were considerably reduced in this strain compared to the wild-type strain Cl 3. The sensitivities of mutant 401 to ultraviolet light and to X-ray irradiation are the same as those of Cl 3. However, in 401 all the markers tested are more resistant to inactivation as shown by transformation of 401 and Cl 3 by ultraviolet-irradiated DNA. The increase in resistance is greater for low efficiency (LE) markers than for high efficiency (HE) markers.—The decreased discrimination between LE and HE markers in strain 401 is not due to a mechanism related to modification of markers in the transforming DNA by the recipient cells, nor are the proteins inducing competence of the cells responsible for the differences in the integration efficiencies of various markers.—Genetic studies of the fate of recombinants as well as the measure of the amount of DNA taken up have shown that all the markers are integrated in strain 401 by the same recombination process, that specific to high efficiency markers.

Fifty-two strains of pneumococci were tested for sensitivity to lysis by pneumococcus phages ω2, ω3, and ω8. Nine noncapsulated strains derived from seven different wildtype strains were all lysed by the three phages. Two strains carrying the Cs capsule were as sensitive as their parent noncapsulated strains. Forty-one other capsulated strains were insensitive to lysis by any of the phages. Although capsulation apparently severely limits adsorption of phage, capsulated cells can release phage; infection by ω3 of a type 3 pneumococcus temporarily denuded of its capsule by enzymatic action resulted in a burst of progeny phage at a time when the host cell had regained full capsulation.