- Volume 92, Issue 2, 1976

SUMMARY: Amino acids liberated by peptidase hydrolysis of di-and oligopeptides by Pseudo-monas putida were measured by trinitrobenzenesulphonate assay and high voltage electrophoresis or paper chromatography followed by ninhydrin spray. Intact bacteria or periplasmic contents released by lysozyme treatment did not hydrolyse peptides. Subcellular fractionation showed that glycylmethionine peptidase activity was cytoplasmic. This enzyme had a Km of 2 mm, and was stimulated fivefold by 1 mm-Co2+. Crude peptidase extract did not cleave peptides with d-residues, acylated N-terminal amino groups or N-methylated peptide bonds but otherwise showed a wide specificity. Di-or tripeptides with blocked C-terminus were hydro-lysed. Leucylleucine (12 mm) and leucylglycylglycine (10 mm) did not compete with glycylmethionine (1·mm) and glycylmethionylglycine (1·0 mm), respectively, for hydrolysis. Pseudomonas maltophilia also contained peptidase activity (0·84 μmol amino acid released from glycylmethionylglycine/min/mg protein). Peptidases of both P. putida and P. maltophilia were constitutive.

SUMMARY: The pathway of glucose metabolism in Pseudomonas aeruginosa was regulated by the availability of glucose and related compounds. On changing from an ammonium limitation to a glucose limitation, the organism responded by adjusting its metabolism substantially from the extracellular direct oxidative pathway to the intracellular phosphorylative route. This change was achieved by repression of the transport systems for gluconate and 2-oxogluconate and of the associated enzymes for 2-oxogluconate metabolism and gluconate kinase, while increasing the levels of glucose transport, hexokinase and glucose 6-phosphate dehydrogenase. The role of gluconate, produced by the action of glucose dehydrogenase, as a major inhibitory factor for glucose transport, and the possible significance of these regulatory mechanisms to the organism in its natural environment, are discussed.

Summary:Repression of biosynthetic enzyme synthesis in Pseudomonas putida is incomplete even when the bacteria are growing in a nutritionally complex environment. The synthesis of four of the enzymes of the arginine biosynthetic pathway (N-acetyl-α-glutamokinase/N-acetylglutamate-γ-semialdehyde dehydrogenase, ornithine carbamoyltransferase and acetylornithine-δ-transaminase) could be repressed and derepressed, but the maximum difference observed between repressed and derepressed levels for any enzyme of the pathway was only 5-fold (for orni-thine carbamoyltransferase). No repression of five enzymes of the pyrimidine biosynthetic pathway (aspartate carbamoyltransferase, dihydro-orotase, dihydro-orotate dehydrogenase, orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase) could be detected on addition of pyrimidines to minimal asparagine cultures of P. putida A90, but a 1·5-to 2-fold degree of derepression was found following pyrimidine starvation of pyrimidine auxotrophic mutants of P. putida A90. Aspartate carbamoyltransferase in crude extracts of P. putida A90 was inhibited in vitro by (in order of efficiency) pyrophosphate, CTP, UTP and ATP, at limiting but not at saturating concentrations of carbamoyl phosphate.

Extracellular cyclic AMP-phosphodiesterase accelerates the development of aggregation competence in Dictyostelium discoideum when present during the pre-aggregation stage. The effect on development appears to depend only on hydrolysis of extracellular cyclic AMP and not on other properties of the phosphodi-esterase molecule. Extracellular cyclic AMP-phosphodiesterase, as a promoter of differentiation, acts mainly throughout the first half of interphase. Our evidence supports the proposal that cyclic AMP oscillations control the rate and possibly the initiation of development. Since extracellular cyclic AMP-phosphodiesterase acts from the beginning of interphase cyclic AMP oscillations may also occur from early interphase, at least in the presence of this enzyme. This would imply that the cyclic AMP oscillator is a determinant, but not a product, of the developmental programme.

Summary: His+ hybrids from a cross between a Salmonella typhimurium donor and an Escherichia coli O8 recipient expressed E. coli O8 specificity and in addition Salmonella O4,12-specificity. This indicated that the recipients had received the his-linked donor rfb cluster determining the synthesis of S. typhimurium O-specific repeat units and that the rfb genes of both mating partners are functional in these hybrids. Chemical analyses showed that the hybrids contained an E. coli O8 lipopolysaccharide (0 antigen) and a S. typhimurium specific lipopolysaccharide with only one O-specific repeat unit (SR antigen).

O8-negative mutants selected from the O8-positive hybrids retained the Salmonella O-specificity and represent semi-rough (SR) forms, because the rfc gene(s) determining the polymerization of repeat units has not been transferred. Attempts to introduce the S. typhimurium rfc locus into E. coli O8 remained unsuccessful.

Crosses between a S. typhi donor and E. coli O8 gave rise to smooth (S) and SR His+ recombinants exhibiting only S. typhi O-specificity. The smooth recombinants are assumed to have obtained the his-linked rfb cluster and in addition the rfc gene(s) of the donor. The exchange of the rfb region of such smooth recombinants by that of a S. typhimurium donor led to smooth hybrids with O4,(5), 12-specificity.

The phenotypically smooth recombinants exhibited concomitantly S-and SR-lipopolysaccharides of S. typhi and S. typhimurium O-specificity, respectively.

Summary:High frequency transducing (HFT) phages 5006MHFT k: and 5006MHFT ak for kanamycin or ampicillin plus kanamycin resistance, derived from Proteus mirabilis strains pm5006(R394) and pm5006(R394) respectively, transduced (at low multiplicities of infection, m.o.i.) antibiotic resistance and prototrophy to pm5006 leu-I at high frequency. Simultaneous transduction of these markers occurred at very much lower frequencies. The latter result was correlated with the proportion of multiply-infected bacteria which, due to the great transducing potential of the phage, could register as transductants. Each HFT lysate was thus heterogenous with regard to high frequency transducing phage. Apart from the additional antibiotic resistance marker carried by one phage, no other difference between the two lysates was detected. High segregation frequencies of antibiotic-resistant or proto-trophic transductants indicated transduction by lysogenization. Although antibiotic-sensitive segregants of antibiotic-resistant prototrophic transductants occurred at high frequency, no auxotrophic segregants of these transductants were found. This suggests transduction by a double cross-over event in the leucine region. Most transductants, even at low m.o.i., were lysogenically converted to homologous phage non-adsorption as a result of interaction between the transducing phage genome and the resident cryptic prophage. They could, however, be retransduced by appropriate phage lysates; thus, lysogenic conversion to non-adsorption was not absolute. Some prototrophic transductants were non-lysogenic although their segregants liberated low-titre phage. The latter anomaly, and the fact that the leucine marker and antibiotic resistance were not cotransduced, are explained by the mode of integration of the phage into the host chromosome in relation to the resident cryptic prophage and the leucine region.

Summary:Transfer of antibiotic resistance between Bacteroides organisms and E. coli in mixed culture under optimal anaerobic conditions was attempted. Donor strains used were E. coli with R factors of a number of compatibility groups and Bacteroides with unusually high antibiotic resistance. Recipient strains included E. coli strains with characteristics favouring conjugal transfer. Although control experiments verified that conjugal transfer of derepressed R factors could occur at a high frequency between E. coli in an anaerobic environment, transfer of antibiotic resistance between E. coli and Bacteroides was never demonstrated.

Summary: Two auxotrophic strains of Bacillus subtilis 168 served as recipients for DNA extracted from various wild-type strains of B. subtilis and wild-type species of the genus Bacillus. Depending upon the DNA source, heterologous transformations of the linked try-his-tyr loci were either as efficient as those observed with donor DNA obtained from the wild-type B. subtilis 168 strain or were undetectable. The order and relative distances of the three gene loci were the same for all active DNA preparations. Similar results were obtained in heterologous transformations of the linked leu-ilv loci, except that DNA preparations from the Bacillus globigii and B. subtilis var. niger species exhibited a reduced but detectable frequency of transformation. With the latter preparations a marked polarity of integration favouring the leu + gene was observed, an effect not seen with homologous DNA. Six independent hybrid lines were obtained from transformation of B. subtilis leu− ilv − with DNA from B. subtilis var. niger leu + ilv +. DNA extracted from these lines fell into two classes on the basis of activity in transforming the parental recipient strain: (i) indistinguishable from homologous DNA, and (ii) intermediate between homologous DNA and DNA from the original donor strain. With either class, polarity of integration was no longer observed in the leu-ilv region. The intermediate type of hybrid demonstrates that at least some of the inefficiency of heterospecific transformation must be due to heterology in nucleotide sequence between the different species at the leu-ilv loci.

SUMMARY: Endocytotic activity of Acanthamoeba trophozoites attenuates once the cells enter stationary phase in liquid culture. Phagocytosis, monitored by the ingestion of polystyrene latex beads, essentially ceases and the uptake of [3H]inulin, known to be mediated by pinocytosis, is reduced by about half. The reduced pinocytotic activity of stationary-phase cells remains sensitive to respiratory inhibitors. Preincubation of stationary-phase cells in fresh growth medium for 1·5 h before the injtation of endocytosis has no effect on phagocytosis and only marginally increzses pinocytosis. This impairment of ingestion, particularly of pinocytosis, may account for the reduced contractile vacuole activity known to characterize stationary-phase cells of this organism. The unequal responses of phagocytosis and pinocytosis to the onset of stationary-phase growth suggest that they are independent processes subject to different controls.

Summary:The growth of Streptococcus faecium unh564p and its production of triterpenoid carotenoids under a variety of culture conditions were examined. Total extractable cell lipid and carotenoid levels increased with culture age and paralleled the growth curve of the bacterium. Variations of the medium glucose concentration produced significant changes in both cell growth and carotenoid production, with the xanthophyll content decreasing at high glucose concentrations. Carotenoid degradation products were found in highly aerated cultures although a high glucose concentration appeared to have a sparing effect on oxidative degradation. Culture age appeared to have little effect on carotene: xanthophyll ratios. The significance of the production of total and individual carotenoids under the various culture conditions is discussed and related to a postulated scheme of triterpenoid carotenoid biosynthesis in the organism.

A requirement of choline for the growth of Entodinium caudatum in a simplified culture medium has been demonstrated. Ethanolamine, N-methylethanolamine, or N-dimethylethanolamine were ineffective as substitutes. In the rumen, the normal environment of this organism, levels of free choline were virtually zero even after ingestion of pasture containing phosphatidylcholine which was rapidly catabolized. Free [ Me-14C]choline is very rapidly cleared from rumen fluid, a little being incorporated into the phosphatidylcholine of protozoa, but the clearance also occurs in animals with defaunated rumens. It is suggested that E. caudatum obtains choline for growth mainly from plant membrane material which it has ingested, rather than from the free base in the rumen liquor.