- Volume 93, Issue 1, 1976

Summary: The pyruvate carboxylase of Pseudonomas fluorescens was purified 160-fold from cells grown on glucose at 20 °C. The activity of this purified enzyme was not affected by acetyl-coenzyme A or l-aspartate, but was strongly inhibited by ADP, which was competitive towards ATP. Pyruvate gave a broken double reciprocal plot, from which two apparent Km values could be determined, namely 0·08 and 0·21 mm, from the lower and the higher concentration ranges, respectively. The apparent K m for HCO− 3 at pH 6·9, in the presence of the manganese ATP ion (MnATP2−), was 3·1 mm. The enzyme reaction had an optimum pH value of 7·1 or 9·0, depending on the use of MnATP2− or MgATP2−, respectively, as substrate. Free Mg2+ was an activator at pH values below 9·0. The enzyme was strongly activated by monovalent cations; NH+ 4 and K+ were the better activators, with apparent Ka values of 0·7 and 1·6 mm, respectively. Partially purified enzymes from cells grown on glucose at 1 or 20 °C had the same properties, including the thermal stability. In both cases 50% of the enzyme activity was lost after preincubation for 10 min at 46 °C. The molecular weight was estimated to be about 300000 daltons by gel filtration on Sephadex G-200. The regulatory properties and molecular weight are thus similar to those determined for the pyruvate carboxylases from Pseudomonas citronellolis and Azotobacter vinelandii.

Summary: Cytoplasmic ribosomal RNA extracted from Crithidia oncopelti and analysed by gel electrophoresis at 4 °C consisted of two components, with molecular weights (relative to E. coli rRNA) of 1·30 × 106 and 0·83 × 106 daltons, present in equimolar amounts. On heating briefly at 51 followed by rapid cooling, the 1·30 × 106 RNA completely dissociated into two components of molecular weights 0·70 × 106 and 0·56 × 106 (present in equimolar amounts). Fifty per cent dissociation of the molecule occurred at 28. That the integrity of the RNA molecule at low temperatures is maintained by its secondary structure was confirmed by electrophoresis under denaturing conditions (98%, v/v, formamide). To account for these phenomena, latent cleavage of the molecule in vivo is proposed.

Summary: Representative strains of the genus Nocardia, when grown on iron-deficient media, produce intracellular lipid-soluble, iron-binding compounds known as nocobactins. However, strains representing the rhodochrous taxon fail to form such compounds. The formation of the nocobactins is completely repressed in bacteria grown on iron-sufficient media. Procedures for purifying the nocobactins are described. From their various properties (u.v. and visible spectra of the ferri-and desferri-materials, mobility upon thin-layer chromatography, and dissociation patterns in HCl), they can be distinguished from the mycobactins (related compounds from mycobacteria) and divided into three main classes. The classes correspond to the three well-described species of the genus Nocardia: N. asteroides, N. brasiliensis and N. caviae. The correlation of the nocobactin data with previous results of conventional numerical taxonomy is high. Two strains of N. asteroides, however, did not produce nocobactins of the group type.

SUMMARY: A His+ Nif+ Escherichia coli K12, Hfr strain (unf43) was constructed by an intergeneric mating between a Klebsiella pneumoniae donor strain (HF3) and a His-, Hfr E. coli strain (sbi824) which transfers his as an early marker. An F-prime nif plasmid, FN39, carrying genes which correspond to the E. coli chromosomal region, metG gnd his shiA, but excluding purF and aroD, was isolated from UNF43. Translocation of carbenicillin resistance genes from a P-type R-factor, R68, to FN39 increased the stability of his and nif on the derivative F-prime, FN68.

Sedimentation analysis of both F-primes in sucrose gradients revealed four covalently closed circular(CCC) DNA species of molecular weights 279 ± 9,136 ± 3,90 ± 1 and 44 ± 1 megadaltons. It is suggested that the two smallest CCC-DNA species are component replicons of the composite F-primes of molecular weight 136 ± 3 megadaltons, and that the molecules of 279 ± 9 megadaltons are CCC-dimers.

FN68 was transferable in intergeneric matings to Klebsiella aerogenes, K. pneumoniae and Salmonella typhimurium but not to Proteus mirabilis; only carbenicillin resistance and sex factor activity were transferred to Erwinia herbicola. nif genes on FN68 were expressed in a Nif− mutant of K. pneumoniae and also in S. typhimurium, which in conventional tests is naturally non-nitrogen-fixing; expression of the his determinant of FN68 became temperature-sensitive in S. typhimurium.

Summary: Thirty-one tryptophan-requiring mutants of Coprinus lagopus have been assigned by genetic and complementation analyses to four loci designated trp-1, trp-2, trp-3 and trp-4. The trp-1 and trp-3 loci were located in group III and trp-2 in group G of the linkage map. The trp-4 locus showed no linkage to the other trp loci or to markers in three additional linkage groups tested. From auxanographic tests and a study of accumulated biosynthetic intermediates, the enzymes controlled by each locus have been provisionally assigned. The trp-2 and trp-3 loci both appear necessary for anthranilate synthetase activity since mutants accumulated no intermediates. Only the trp-3 mutant could utilize anthranilic acid, therefore the trp-2 locus must also be involved in a subsequent step in the pathway. The trp-4 mutants utilized indole and accumulated anthranilic acid, and hence this locus is involved in the conversion of anthranilic acid to indoleglycerol phosphate. The trp-1 mutants utilized only tryptophan and accumulated indoleglycerol phosphate and anthranilic acid. They are therefore blocked in the final steps of the pathway catalysed by tryptophan synthetase.

Summary: The extent of the deletions of five Tets Tra− mutants of R100-1 was determined by complementation experiments with wild-type and tra mutants of F lac. The presence or absence of the origin of transfer on the mutants was also investigated. Using the results, a tentative map of this region of the R factor was drawn: it was essentially similar to the analogous region of the E. coli K12 F factor, except that tet was located between traJ and traA.

Some of the deletions had removed the promoter for the transfer operon. This allowed detection of the transcription of traC and distal genes from a weak, traJ-independent promoter. This is probably the Is2 promoter, since R100-1 carries an Is2 insertion sequence located immediately to the left of traC in the correct orientation. Since neither the transfer operon promoter nor the Is2 promoter seemed to be required for transcription of traI, it was concluded that, unlike the F factor, this was located in a separate operon.

SUMMARY: The properties of three additional Proteus mirabilis hosts for the high frequency transducing (HFT) phages 5006MHFT k and 5006MHFTak are described. The phages transduce resistance to kanamycin and to ampicillin plus kanamycin, respectively, and were produced by ultraviolet induction of derivatives of P. mirabilis strain PM5006. Strain PM804 could not be shown to adsorb the phages although it yielded a few transductants. All transductants, even those produced at low multiplicities of phage input, were lysogenic and segregated markers at high frequency. Ultraviolet induced phage lysates of these transductants transduced PM804 at higher frequencies and PM5006 at lower frequencies than the original phages. Strain PM804 or its derivatives did not adsorb phage from these lysates. Transmission experiments through PM5006 of phage in the transductant lysates confirmed that PM804 had a host-controlled modification system which modified HFT phage from PM5006. That PM804 also possessed a restriction system was inferred from the greater numbers of transductants obtained with phage which bore a compatible modification pattern. Strain N and a restrictionless mutant of it named NHI, adsorbed the HFT phages avidly and failed to modify their host range. Transduction frequencies of the phage markers were about 10−4/p.f.u. adsorbed to strain NHI and only about tenfold lower to strain N which did not plate the phages. Transductants also had the features of heterogenotes and those obtained at low multiplicities of infection were non-lysogenic. The latter transductants adsorbed homologous phage while NHI transductants also plated the HFT lysates. Strain NHI, lysogenized by the parent phage 5006M, did not adsorb the HFT phages. These findings suggest that the HFT phages were most likely defective for lysogenic conversion to homologous phage non-adsorption. The postulated restriction enzymes of PM804 and strain N could not be shown to be thermolabile.

Summary: DNA-DNA reassociation studies showed that Flavobacterium meningosepticum strains had a genetic relatedness of 91 to 100% with inter-strain duplexes having high thermal stabilities. The only exception, strain NCTC10016, had an average relatedness of only 43% to other strains of F. meningosepticum. The apparent divergence in DNA base sequence of this strain was reflected in the structural differences of some enzymes. There was a gradation of DNA relatedness among the Flavobacterium group II-b strains, but three strains were sufficiently related to constitute a species. Low levels of genetic relatedness were confirmed between F. meningosepticum and strains of Flavobacterium group II-b, group II-f, F. aquatile, F. breve, F. heparinum, F. pectinovorum, F. odoratum and Moraxella saccharolytica. All strains had base compositions in the range 32 to 46% guanine plus cytosine. The genome sizes of representative strains of F. meningosepticum and Flavobacterium group II-b were 2·50 × 109 to 3·52 × 109 daltons, whereas the group II-f strains had smaller genomes of 1·64 × 109 to 1·81 × 109 daltons. The taxonomic implications of these findings are discussed.