- Volume 129, Issue 5, 1983

The form of cytochrome d in Escherichia coli and Azotobacter vinelandii that shows an absorption maximum at 648 to 652 nm (cytochrome d 650) is generally regarded as the oxidized form of this terminal oxidase. Membranes from E. coli grown under oxygen-limited conditions, when treated with ferricyanide, do not reveal cytochrome d 650, whereas a sharp symmetrical band at 652 nm results from the reaction of the reduced enzyme with O2 at either room temperature or after flash photolysis of the CO-liganded form at 130 C. Electron paramagnetic resonance spectroscopy of cytochrome d 650 trapped at 130 C shows that its spectrum is indistinguishable from the CO-liganded form and does not reveal resonances of high spin ferric haem previously attributed to cytochrome d. An hypothesis is proposed in which cytochrome d 650 is an early intermediate in the reaction of reduced cytochrome d with oxygen and is not the fully-oxidized (ferric) species. An analogy between cytochrome d 650 and oxyhaemoglobin is presented and the hypothesis discussed in relation to earlier work, in which the indirect interconversions of reduced cytochrome d and d 650 have been explained by proposing the existence of an invisible form. It is suggested that this form could be the oxidized enzyme.

Optical changes in d- and b-type cytochromes, following initiation of the reaction of cytochrome oxidase d with O2, have been studied in cells and derived membrane particles from oxygen-limited cultures of Escherichia coli K12. At successively higher temperatures between − 132 and − 88 °C, the first scan after photolysis of the CO-liganded, reduced oxidase in the presence of O2 shows a diminution of cytochrome d 650 (believed to be an early intermediate in the O2 reaction) and a slow increase in absorbance at 675 to 680 nm due to an unidentified chromophore. A similar sequence occurs when a single sample is scanned repetitively at − 91 °C. At higher temperatures, oxidation of at least two spectrally distinct cytochromes b occurs. Selective photolysis of the cytochrome d-CO complex with a He-Ne laser shows that neither of these cytochromes is the CO-binding cytochrome o 436. In all oxidation states examined, no absorbance in the 720 to 860 nm region was observed; it is concluded that both cytochromes d and o 436 lack redox-active copper that has an environment similar to the copper(s) in mitochondrial cytochrome c oxidase.

The amount of cytochrome d 650 (but not the amount of reduced cytochrome o 436) formed after photolysis is directly proportional to the oxygen concentration in the sample at the time of freeze trapping. The results are discussed in relation to the composition and mechanism of action of cytochrome d.

Chlorobium vibrioforme f. sp. thiosulfatophilum was shown to oxidize sulphate via substrate phosphorylation involving adenylylsulphate (APS) reductase, ADP-sulphurylase and adenylate kinase. APS reductase was purified 200-fold and shown to be a flavoprotein of molecular weight 1·8 × 105, containing 1 mol flavin adenine dinucleotide (FAD), 4–6 mol non-haem iron and 6–8 mol labile sulphide (mol enzyme)−1. Substrate inhibition of the enzyme was observed when the AMP concentration was above 2 mm. ADP-sulphurylase purified free of adenylate kinase had an apparent K m value for APS of 0·25 mm, which increased with decreasing concentrations of phosphate.

Assay procedures were developed for a soluble glycerol kinase [apparent K m (glycerol) 9 μm] and a probably membrane-associated, NAD-independent l-glycerol-3-phosphate dehydrogenase [apparent K m (l-glycerol 3-phosphate) 7 mM] present in Streptomyces coelicolor A3(2). Both enzymes were cold sensitive. They were co-ordinately induced (about 35-fold) by addition of glycerol to cultures growing on arabinose as sole carbon source. Induction was rifampicin sensitive. The dehydrogenase was absent from glycerol-sensitive mutants, and both kinase and dehydrogenase were absent from glycerol non-utilizing (but glycerol-resistant) mutants, demonstrating that the two enzymes are part of the major pathway of glycerol catabolism in S. coelicolor. Circumstantial evidence suggested that their inducer is glycerol 3-phosphate rather than glycerol. The enzymes were subject to co-ordinate repression by various carbon sources, of which glucose exerted the strongest effect (a fivefold repression). Previously described mutants resistant to 2-deoxyglucose, shown here to have very low glucose kinase activity, were defective in glucose repression of the glycerol enzymes.

With radioisotopes, it was shown that suspensions of Mycobacterium leprae oxidized glycerol, 6-phosphogluconate, glucose, glucose 6-phosphate, and, at a low rate, gluconate, to CO2. The incubation period in these experiments was usually 20 h, but after 140 h up to five times more glucose and gluconate had been converted to CO2. Studies with differentially labelled glucose indicated that glycolysis and the hexose monophosphate pathway were used for glucose dissimilation.

Key enzymes of glycolysis, the hexose monophosphate pathway and glycerol catabolism were detected in cell-free extracts from purified M. leprae, but phosphoketolase, Entner-Doudoroff pathway activity and gluconate kinase were absent. All these enzymes were present also in host-tissue, but biochemical evidence is presented which indicates that all enzymes detected in extracts from M. leprae were authentic bacterial enzymes. Additionally, they could all be detected in extracts of M. leprae prepared by treatment with NaOH in which host enzymes adsorbed to M. leprae are inactivated.

Photobacterium leiognathi closely resembles Escherichia coli with respect to cell lysis by lysozyme, and the fractionation of outer and cytoplasmic membranes. The two organisms differ in their phospholipid contents and, more significantly, in outer membrane protein compositions.

The development of a protoplast fusion technique for oxytetracycline-producing Streptomyces rimosus strains, and its evaluation for the application for a breeding programme, has been described. Treatment of S. rimosus protoplasts with 40% (w/v) PEG 1550 for 30 min gave optimal numbers of recombinants ranging from 1 to 10% of the total progeny. Therefore, by comparison with conjugation, protoplast fusion increased the frequency of recombination by two to three orders of magnitude. The proportion of multiple crossover classes amongst recombinants was higher, by a factor of ten, after protoplast fusion (13·3%) than after conjugation (1·5%). Participation of less frequent complementary genotype doubled from 9·0% in conjugation to 17·9% in protoplast fusion. Overall, this suggested that the opportunities for crossing over in a fusion of S. rimosus protoplasts were spatially and/or temporally extended leading to a loosening of linkage with a near-random assortment of genotypes in a cross. However, by minimizing the multiple crossover classes and calculating allele frequency gradients, it was shown that the protoplast fusion technique allows arrangement of genetic markers on the S. rimosus chromosome. These are ideal characteristics for the recombination of divergent lines in a strain improvement programme.

An Escherichia coli plasmid, p1949, that is derived from pMB9 and pC194, and unable to replicate in Bacillus subtilis, can give rise to stable CmR transformants of the latter species if it is inserted into the bacterial chromosome. A purified segment of the B. subtilis chromosome, with transforming activity against pheA1 and nic-38 recipients, was used to direct the insertion of p1949 into the B. subtilis chromosome. Insertion of the ligated DNA segments occurred in the region of the chromosome from which the purified phe-nic segment was derived. Many of the properties of the resulting CmR transformants of B. subtilis are consistent with the occurrence of a Campbell recombination mechanism leading to integration. However, certain of these properties are more easily explained if it is proposed that integration occurs by a reciprocal recombination event involving a linear ligation product. Evidence is presented suggesting that the inserted sequences may be tandemly duplicated. This may effectively vitiate the use of p1949 as a convenient means for complementation analysis of recessive mutations in B. subtilis.

pNZ500 is a 1.5 kb cryptic plasmid from a Shigella sonnei isolate. It was introduced into Escherichia coli by cotransformation, where it is maintained at about 30 copies per chromosome equivalent. Hybridization studies show that pNZ500 exhibits a high level of sequence similarity to other 1.5 kb plasmids found in different S. sonnei isolates but shares no homology with larger S. sonnei plasmids. pNZ500 shares a small degree of sequence homology with pBR322 and with pAC184. The homology with pBR322 is restricted to sequences close to the ori-bom region of this plasmid. Nevertheless, pNZ500 maintenance in E. coli is not dependent on DNA polymerase I activity, and does depend on continuing protein synthesis. pNZ500 encodes two polypeptide gene products whose monomer molecular weights are 24500 and 18000. The examination of host cells for the expression of possible plasmid phenotypes revealed no differences between cells bearing pNZ500 and plasmidless cells.

A transposable element, Tn2401, was found in a clinical isolate of Pseudomonas aeruginosa. Tn2401 had a size of 7190 nucleotides and encoded aminoglycoside 3′-phosphotransferase and aminoglycoside 6′-N-acetyltransferase. The sequence encoding the former enzyme was homologous with that of Tn903. Pseudomonas aeruginosa strains harbouring this transposon were resistant to kanamycin, neomycin, lividomycin, ribostamycin, paromomycin, netilmycin, tobramycin, dibekacin, gentamicin, sisomicin, and butirosin.

Adherence of 14C-labelled strains of Actinomyces viscosus to uncoated and saliva-coated silica and hydroxyapatite beads had both loose and firm components, probably reflecting different subpopulations of bacteria within a single culture. Adherence was characterized by the proportion of bacteria available for each type of adherence and a constant (K b) for each combination of bacterial strain and bead surface. Loose adherence, which was greater with silica than with hydroxyapatite beads, always involved many more bacteria than firm adherence. Firm adherence was greater with A. viscosus WVU627 than A. viscosus TF11. The association rate constants (K a) for loose and firm adherence were similar, indicating simultaneous processes, but the dissociation rate constant (K d) was lower for loose adherence than for firm adherence. Removal of loosely adhering bacteria by washing may only reflect their distance from the bead surface. Silica beads were convenient for studying bacterial adherence and formed an acceptable coating of salivary glycoprotein.

Escherichia coli F-17 Sr a human faecal isolate, is resistant to the T-series of bacteriophages (i.e. T2 to T7). A T2-sensitive mutant of E. coli F-17 Sr was isolated following acriflavin treatment. This mutant, E. coli F-17 Sr Ts was found to be sensitive to the entire T-series of phages. E. coli F-17 Sr and E. coli F-17 Sr Ts did not differ quantitatively in total LPS content. However, analysis of LPS revealed that a large fraction of E. coli F-17 Sr Ts was devoid of O-side-chains. This accounted for the sensitivity of this strain to bacteriophages T3, T4, and T7. In addition, E. coli F-17 Sr Ts contained only about half the amount of capsular material contained by E. coli F-17 Sr accounting for the sensitivity of the mutant to bacteriophages T2, T5, and T6. Although the two strains colonized equally well when fed individually to streptomycin-treated mice, when fed simultaneously to streptomycin-treated mice, E. coli F-17 Sr Ts colonized at a level of about 1 × 108 cells (g faeces)−1, whereas E. coli F-17 Sr colonized at only 1 × 104 cells (g faeces)−1. These studies suggest that bacterial cell surface components modulate the large intestine colonizing ability of E. coli F-17 Sr in the mouse large intestine.

Encapsulated and non-encapsulated variants of one strain of gonococcus were compared for their capacity to produce infection in chambers implanted subcutaneously in mice, for their reactions with specific antibody and for their precipitation with wheat germ agglutinin. Only the encapsulated variant could infect implanted chambers. Specific rabbit antiserum raised against the non-encapsulated variant killed only that variant, whereas antibody raised against the encapsulated variant killed both variants.

Saline extracts and lipopolysaccharide preparations of the encapsulated variant differed from those of the non-encapsulated one in their reactions with wheat germ agglutinin and antibody in diffusion and electrophoresis tests. Preparations from infective encapsulated gonococci reacted with wheat germ agglutinin while those from the non-encapsulated variant did not. Immunodiffusion tests showed that lipopolysaccharides from both variants share a common antigenic determinant, but saline extracts and lipopolysaccharides from encapsulated gonococci possess an additional determinant. The significance of these findings is discussed.