- Volume 98, Issue 2, 1977

Summary: A new method has been developed for measuring peptide transport in aminoacid auxotrophs of Escherichia coli by following induction of β-galactosidase. Appearance of the enzyme was determined after addition of inducer and peptides to amino-acid starved bacteria. For a given number of lysine equivalents, the rate and the extent of enzyme synthesis were the same for lysine and lysyl peptides; similar results were found for glycine and glycyl peptides. Saturation constants for peptide transport were determined from the exogenous peptide concentration that gave half maximal rates of enzyme synthesis. The saturation constants, studies with mutants defective in peptide transport, and detection of competition between peptides for uptake, all endorsed earlier conclusions from growth tests about the structural specificities for peptide transport. The new method is quicker, more sensitive and more informative than growth tests.

Summary: During growth of Escherichia coli strain SPA o in the presence of methionine, an intermediate accumulates in the medium. This intermediate reacts with 2,4-dinitro-phenylhydrazine, and can be degraded to ethylene either enzymically or photo-chemically, the latter being stimulated by the addition of a flavin. The pH optimum for the photochemical degradation of this intermediate and 2-keto-4-methylthio-butyric acid (KMBA) is pH 3 whereas the optimum for methional is pH 6. The enzyme which converts the intermediate to ethylene also converts KMBA to ethylene and has many of the properties of a peroxidase including inhibition by catalase, cyanide, azide and anaerobiosis. The enzyme which synthesizes the intermediate is not known but requires oxygen and pyridoxal phosphate. A pathway for ethylene biosynthesis is proposed in which methionine is converted to KMBA which can be degraded either by peroxidase or in a flavin-mediated photochemical reaction. Its relevance to the properties of other ethylene-producing bacteria and to the proposed pathway of ethylene release by higher plants is discussed.

Summary: Coprinus cinereus stipes contain glycogen and protein inclusions. The amount of glycogen decreases during stipe elongation: it is at a maximum in stage III and falls to a minimum by stage V. Protein inclusions develop in stage II, reach their greatest size in stage III, and are degraded during stipe elongation in stages IV and V. The morphology of the glycogen differs in the two strains studied. Glycogen and insoluble protein are not evenly distributed throughout the stipe: glycogen is concentrated at the base while insoluble protein is concentrated at the top. During elongation the breakdown of both of these is greatest at the top of the stipe.

Summary: The flagellate stage of the rumen phycomycete Neocallimastix frontalis invades and germinates on plant material in the rumen and in vitro, preferentially invading the lemmas, paleas, awns and flower bracts in members of the plant family Gramineae, and flower bracts in certain of the Papilionaceae. The principal sites of invasion were the stomata and damaged tissue, through which penetration of the plant tissue by the rhizoid of the germinating cell occurred. Subsequent growth of the vegetative stage occurred with the uptake of 14C from 14C-labelled plant tissue. Host diets rich in seed-head material normally supported higher population densities of N. frontalis in the rumen even though the dry matter digestibilities of these diets were lower.

SUMMARY: Synthesis of glutamine synthetase (GS) in anaerobic batch cultures of Escherichia coli was repressed when excess NH4 + was available, but derepressed during growth with a poor nitrogen source. In wild-type bacteria there was only a weak inverse correlation between the activities of GS and glutamate dehydrogenase (GDH) during growth in various media. No positive correlations were found between the activities of GS and nitrite reductase, or between GS and cytochrome C 552: both of these proteins were synthesized normally by mutants that contained no active GS. Although activities of GS and GDH were low in two mutants that are unable to synthesize cytochrome C 552 or reduce nitrite because of defects in the nir A gene, the nir A defect was separated from the GS and GDH defects by transduction with bacteriophage PI. Attempts to show that catabolite repression of proline oxidase synthesis could be relieved during NH4+ starvation also failed. It is, therefore, unlikely that nitrite reduction or proline oxidation by E. coli are under positive control by GS protein. The regulation of the synthesis of enzymes for the utilization of secondary nitrogen sources in E. coli is, therefore, different from that in Klebsiella aerogenes, but is similar to that in Salmonella typhimurium.

SUMMARY: Derivatives of Salmonella typhimurium carrying F prime or P prime plasmids with Klebsiella nif and his genes had specific nitrogenase activities similar to Klebsiella in selective conditions, even to showing ‘hyperinduction’ under argon. No evidence was obtained for catabolite repression of normal nif expression but dibutyl cyclic AMP often augmented ‘hyperinduction’. In non-selective conditions the Klebsiella his nif determinants were rapidly lost from the plasmids; the low levels of nif expression and temperature-sensitive his expression previously reported were probably due to ready loss of his nif in the test conditions used.

SUMMARY: Mutants with defective lipopolysaccharides (LPSs) were isolated from Pseudomonas aeruginosa PACIR (Habs serogroup 3) by selection for resistance to aeruginocin from P. aeruginosa P16. Carbenicillin-sensitive mutants were isolated from P. aeruginosa PACI but not all had defective LPSs. Rough colonial morphology and resistance to bacteriophage 119X appeared to be independent of LPS composition.

The LPSs from five mutants were analysed and compared with that of the parent strain. Separation of partially-degraded polysaccharides from LPS from PACI on Sephadex G75 yielded two different high molecular weight fractions and a phosphorylated low molecular weight fraction (L). The mutant LPSs lacked most or all of the high molecular weight fractions but retained some low molecular weight material. That from PACI and two of the mutants was separated by elution from Biogel P6 into two fractions. One, L2, was the core polysaccharide while the other, LI, contained short antigenic side-chains attached to the core like the semi-rough (SR) LPSs of the Enterobacteriaceae. The two mutants which gave the LI fraction reacted with Habs 3 and PACI antisera as did the parent strain. The other three mutants were unreactiye and their LPSs contained core components only. One appeared to have a complete core while the other two lacked rhamnose and rhamnose plus glucose respectively. Thus there may be four types of LPS in PACI: one contains unsubstituted core polysaccharide and yields L2 on acid hydrolysis, another has short antigenic side-chains of the SR type and yields the LI fraction, while the two high molecular weight fractions are derived from core polysaccharides with different side-chains.

Summary: Streptomyces coelicolor A3(2) and S. lividans 66, which lack chloramphenicol acetyltransferase, gave rise to chloramphenicol-sensitive (Cmls) variants spontaneously at frequencies of 0·5 to 2%. The fertility type of S. coelicolor in respect of the scP1 plasmid (SCP1+, SCP1− or NF) had no effect on chloramphenicol sensitivity or on the frequency at which Cmls variants arose.

Cmls isolates spontaneously reverted to CmlR at frequencies one to three orders of magnitude lower than the frequency with which Cmls strains arose from CmlR CmlR revertants obtained spontaneously from Cmls clones again produced Cmls isolates at the normal frequency of several per cent. Therefore, Cmls and CmlR are reversible phenotypes.

In crosses between marked Cml r and Cml s S. coelicolor strains, transfer of chloramphenicol resistance into the sensitive strain apparently occurred independently of chromosomal recombination. Mapping experiments excluded the possibility that segregation of a chromosomal locus determines Cml r versus Cml s phenotype. In crosses between scP1− strains, fertility was not significantly different in Cml R × Cml s , Cml r × Cml r and Cml s × Cml s combinations.

Covalently closed circular DNA from Cml s and Cml r strains of S. coelicolor was indistinguishable in molecular weight and restriction endonuclease cleavage pattern.

It is suggested that chloramphenicol resistance in S. coelicolor a3(2) is affected by some kind of transposable genetic element that may be capable of extra-chromosomal existence.

Summary: A mutant of A. tumefaciens strain B6S3, carrying the R factor RP4, was able to transfer its TI plasmid to various avirulent Agrobacterium strains and to a strain of Rhizobium. Strains carrying the TI(B6S3) plasmid were selected by their ability to utilize octopine. The isolates were able to induce tumours and exclude phage AP1. The tumours induced on Kalanchoë daigremontiana were rough and contained octopine. It was concluded that octopine utilization, phage exclusion, induction of rough tumours and synthesis of octopine in the tumours are determined by the TI(B6S3) plasmid. Tumorigenicity was determined this plasmid both in Agro-bacteria and in Rhizobium, suggesting that all the genes necessary for tumour induction are plasmid-borne.

Summary: Eleven mutants resistant to a toxic analogue of asparagine, aspartic hydroxamate, have been isolated; they are allelic and map at the ahrA locus. These mutations result in low or non-detectable asparaginase activity. ahrA mutations are recessive for asparaginase activity and aspartic hydroxamate resistance. The ahrA locus is in linkage group VIII and is loosely linked with abaA, palB, uZ9 and chaA. Asparaginase activity was measured the by formation of aspartic hydroxamate from asparagine and hydroxylamine. The K m values of asparaginase for asparagine and hydroxylamine are 0·6 and 8·3 mM respectively. Minimum asparaginase activity is present in cells grown on ammonium or glutamine. Maximum asparaginase activity is present in wild-type cells grown on ammonium and then held in nitrogen-free medium for 3 h. Derepression from this ammonium repression requires protein synthesis. A number of different types of ammonium-repressed and of ammonium-derepressed mutants have abnormal regulation of asparaginase activity.

Summary: Diploid strains of Coprinus lagopus grown on medium supplemented with griseofulvin (20 μg ml−1) produced fast-growing sectors of two types after 7 to 28 days incubation: these were diploid somatic recombinants and haploids. A dikaryon grown on the same medium broke down to give one haploid component only; resolution of this dikaryon and a near-isogenic diploid resulted in selection for sectors carrying a gene for resistance to griseofulvin. The use of this antibiotic is suggested as a tool for routine haploidization in C. lagopus.

Summary: Nitrate reductase (nar) A, B and E mutants of Escherichia coli with plasmids carrying Klebsiella pneumoniae nitrogen fixation (nif) genes reduced acetylene independently of added molybdate, but nar D mutants showed pleiotropic dependence on the concentration of added molybdate for expression of both nar and nif. No complementation of nar mutations by nif occurred; nitrite but not nitrate repressed nif in nar hosts. Derepression of nif occurred in molybdenum-deficient nar D (nif) strains since nitrogenase peptides were present. nifB mutants, thought to have a lesion in the pathway of molybdenum to nitrogenase, as well as nif deletion mutants, had normal nitrate reductase activity.

Summary: RNA isolated from exponential-phase cultures of A. nidulans was used to titrate denatured DNA over a wide range of RNA: DNA ratios. The following results were obtained: (i) 0·6% of A. nidulans DNA was complementary to purified rRNA and 0·062% was complementary to tRNA. (ii) Under the growth conditions employed (35 °C mean generation time 4 h), unstable RNA accounted for 40% of the rapidly labelled RNA fraction but only 2% of the randomly labelled RNA fraction. The half-life of the unstable RNA was estimated to be 3% of the mean generation time. (iii) A wide variation in the abundance of unstable RNA species was observed; more than 80% of the labelled RNA in both rapidly and randomly labelled unstable RNA fractions was homologous to only 10% of the DNA that was actively transcribed (i.e. 1% of the total DNA). In turn, the actively transcribed DNA comprised only 10% of the total DNA since virtually all the unstable and readily hybridizable RNA fraction (> 99%) from rapidly and randomly labelled RNA would form stable hybrid with it. This indicated that the remaining fraction of the DNA (90%) was infrequently transcribed.

Summary: A mutant strain of Escherichia coli k12, r2721, has been shown to differ from its parent strain, S491, in four associated phenotypic characters as a result of a single mutation. This strain did not give recombinants with DNA transduced by bacterio-phage P1 or bacteriophage Mu, nor transformants after exposure to R factor DNA: lysates of bacteriophage P1 grown on this strain did not appear to contain any transducing particles when tested on normal recipients. Moreover, the reversion rates, both spontaneous and ultraviolet-induced, for two auxotrophic markers were reduced. The frequency of revertants was at least two orders of magnitude lower in cultures of r2721 than in cultures of s491. Many of the rare revertants for one or other of the auxotrophic markers were found to have regained normal reversion frequencies for the other marker and for the capacity to be transduced. In all other respects, recombination in r2721 appeared normal, the frequency of chromosomal mobilization by an F’ factor was unaffected and normal yields of recombinants were obtained from matings with Hfr strains. The only circumstance in which transduction of r2721 was observed was when the capacity to ferment galactose was selected and P1 had been grown on a strain carrying λdgal when, presumably, integration was effected by the phage-coded gene products. The mutation has been located on the E. coli chromosome map between ton A and pro and has been given the symbol tdi (transduction inhibition). Double mutants, (tdi recA) and (tdi recB), have been isolated and show no unexpected properties.

SUMMARY: Six strains of Haemophilus species, pathogenic to chickens, required 1·0 to 1·5% (w/v) NaCl for optimum growth. The requirement was for Na+ rather than NaCl. A sodium salt buffer influenced the optimum NaCl requirement and enhanced growth. Each strain required a different concentration of NADH for an optimum rate of growth.