- Volume 136, Issue 1, 1990

Calmodulin (CaM)-interacting proteins of Rhodosporidium toruloides, a basidiomycetous yeast, were studied. CaM-binding proteins isolated from the soluble cell extract of vegetative cells and gamete cells (mating-pheromone-treated cells) by CaM-Sepharose affinity chromatography were analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis to compare the molecular species of CaM-binding proteins of the two cell types. The major CaM-binding proteins of the vegetative cells had molecular masses of 103 and 96 kDa. In the pheromone-treated cells, in addition to the bands seen in the vegetative cells, prominent bands of 40 and 38 kDa and minor bands of 92 and 60 kDa were present. The bands of CaM-binding proteins present in both cell types were more intense in the gamete cells. CaM-binding proteins of total cell extracts were also detected on acrylamide gels by a gel overlay method using 125I-CaM as the probe. The results obtained by the two procedures suggested that various CaM-binding proteins are present in the yeast cell, and the expression of many CaM-binding protein genes is altered by the mating pheromone. Ca2+- and CaM-dependent protein kinase activity was detected in the CaM-affinity-purified proteins. After incubation under conditions for protein phosphorylation by the kinase, the enzyme no longer required the activators, indicating that the enzyme activity is modulated by endogenous phosphorylation. A protein of 60 kDa was labelled by endogenous phosphorylation in a Ca2+/CaM-dependent manner, indicating that the labelled protein may be the Ca2+/CaM-dependent protein kinase or else be involved in its regulation.

31P NMR spectra were obtained from perchloric acid (PCA) and KOH extracts of Phytophthora palmivora mycelium. Signals indicating the presence of large amounts of short-chain polyphosphate were observed in the spectra of PCA extracts of mycelia grown under both low (0·1 mm) and high (10 mm) phosphate conditions. The mean chain length of polyphosphate was calculated from the relative areas of signals arising from terminal and internal P nuclei in the polyphosphate chain. The small amount of polyphosphate evident in the KOH extract had an average chain length similar to PCA-soluble polyphosphate. 32P tracer studies indicated that phosphorus in the PCA fraction accounted for between 50 and 60% of total phosphorus, the bulk of the remainder being divided between the lipid and KOH extracts. The presence of the fungicide phosphorous acid markedly reduced the average chain length of acid-soluble polyphosphate. This reduction occurred both under low-phosphate conditions, in which treatment with phosphorous acid retards growth, and under high-phosphate conditions, in which no significant growth retardation is observed. Treatment with phosphorous acid perturbed phosphorus distribution and lipid composition under low-phosphate conditions.

Klebsiella pneumoniae synthesized only b-type and d-type cytochromes under the wide range of growth conditions tested, and reaction with CO revealed two potential oxidases. The o-type oxidase was produced only in the presence of O2 and appeared to be repressed by glucose. The d-type oxidase was, by contrast, produced only in the absence of measurable O2 (< 1μm), and was the only oxidase expressed in nitrogen-fixing conditions. It was extracted from the membrane, purified and shown to be similar to that from E. coli in being a heterodimer (subunits of M r 52000 and 35000), in containing two distinguishable b haems and haem d (one or two molecules per molecule of oxidase), and in being able to react with O2 to give a stableoxygenated intermediate. The purified d-type cytochrome oxidase had a very high affinity for O2 (K m 20 nm; measured by the spectral properties of leghaemoglobin). It is proposed that this provides a role for this oxidase in lowering the O2 concentration to allow nitrogenase synthesis and function, and to provide a terminal oxidase to permit electron-transport-coupled ATP synthesis which supports the increase in efficiency of nitrogen fixation observed under microaerobic conditions.

Methylobacterium extorquens AM1 contains a novel c-type cytochrome, called cytochrome c-553, previously thought to be a precursor of the electron acceptor (cytochrome c L) for methanol dehydrogenase. Its amino acid composition and serological characteristics show that it has no structural relationship to cytochrome c L. It usually comprises less than 5% of the total c-type cytochromes. In a moxD mutant, which contains neither methanol dehydrogenase nor cytochrome c L, it comprises 30% of the soluble cytochrome and it has been purified and characterized from that mutant. Cytochrome c-553 is large (M r 23000), acidic and monohaem, with a redox potential of 194 mV. It reacts rapidly and completely with CO but is not autoxidizable. It is not autoreducible, and it is not an electron acceptor from methanol dehydrogenase or methylamine dehydrogenase, nor an important electron donor to the oxidase. It is able to accept electrons from cytochrome c L and to donate electrons to cytochrome c H. It is present in the soluble fraction (presumably periplasmic) and membrane fraction of wild-type bacteria during growth on a wide range of growth substrates, but its function in these bacteria or in the moxD mutant has not been determined.

Mycobacterium microti incorporated a wide range of exogenously supplied pyrimidines into its nucleic acids. M. avium incorporated a relatively narrow range of pyrimidines but both M. avium and M. microti when recovered after growth in vivo incorporated a slightly wider range of pyrimidines than the same strains grown in vitro. M. microti and M. leprae could not take up uridine nucleotides directly but could utilize the pyrimidines by hydrolysing them to uridine and then taking up the uridine. Pyrimidine biosynthesis, judged by the ability to incorporate carbon from CO2 or aspartate into pyrimidines was readily detected in non-growing suspensions of M. microti and M. avium harvested from Dubos medium, which does not contain pyrimidines. The biosynthetic activity was diminished in mycobacteria grown in vivo when there is likely to be a source of pyrimidines which they might use. Relative activities for pyrimidine biosynthesis de novo in M. microti were 100 for cells isolated from Dubos medium, 6 for cells isolated from Dubos medium containing the pyrimidine cytidine and 11 from cells recovered after growth in mice. In contrast, relative activities for a scavenging reaction, uracil incorporation, were 100, 71 and 59, respectively. Three key enzymes in the pathway of pyrimidine biosynthesis de novo were detected in M. microti and M. avium. Two, dihydroorotate synthase and orotate phosphoribosyltransferase appeared to be constitutive in M. microti and M. avium. Aspartate transcarbamoylase activity was higher in these mycobacteria grown in vivo than in Dubos medium but it was repressed in M. microti or M. avium grown in Dubos medium in the presence of 50 μm-pyrimidine. Aspartate transcarbamoylase was strongly inhibited by the feedback inhibitors ATP, CTP and UTP. Enzymes for scavenging pyrimidines were detected at low specific activities in all mycobacteria studied. Activities of phosphoribosyltransferases, enzymes that convert bases directly to nucleotides, were not related to the ability of intact mycobacteria to take up pyrimidine bases while activities of pyrimidine nucleoside kinases were generally related to the ability of intact mycobacteria to take up nucleosides. Phosphoribosyltransferase activity for uracil, cytosine, orotic acid and - in organisms grown in Dubos medium with 50 μM-uridine - thymine, as well as kinases for uridine, deoxyuridine, cytidine and thymidine were detected in M. microti. However, M. avium only contained uracil and orotate phosphoribosyltransferase, uridine, cytidine and thymidine kinase, and additionally deoxyuridine kinase when grown axenically with 50 μm-uracil, reflecting its more limited abilities in pyrimidine scavenging.

Aspartokinase activity was detected in extracts from Mycobacterium leprae (recovered from armadillo liver) and in Mycobacterium avium grown axenically and in vivo. Homoserine dehydrogenase activity was only detected in M leprae and in M. avium grown axenically. Activities, when detected, were 50 to 70% lower in M. leprae or M. aviun grown in vivo than in axenically grown M. avium. In these two pathogenic mycobacteria, aspartokinase and homoserine dehydrogenase are subject to feedback inhibition by methionine - an additional regulator over those observed for the enzymes from Mycobacterium smegmatis. Intact mycobacterium incorporated carbon from [U-14C]aspartate into the aspartate family of amino acids (threonine, isoleucine, methionine and lysine) though the rate of incorporation in M. avium grown in vivo was about half that in M. avium grown axenically.

Fatty acid synthetase activity in extracts of Mycobacterium leprae was equivalent to 1·7 pmol malonyl-CoA incorporated into fatty acid min-1 (mg protein)-1. This activity - if representative of living M. leprae organisms - is insufficient to enable them to synthesize their lipid requirements rapidly enough to support growth. The major activity for scavenging fatty acids in extracts of Mycobacterium microti and Mycobacterium avium, as well as in extracts of M. leprae, was acetyl-CoA-dependent fatty acyl-CoA ‘elongase’. This activity was about four times higher in M. avium and M. microti grown in a medium which contained lipids, or when grown in mice, than in medium without added lipids. In contrast, the de novo fatty acid synthetase activity was repressed in M. avium and M. microti when grown in medium that contained lipids, or when grown in mice. These results are consistent with the hypothesis that mycobacteria grown in vivo preferentially scavenge lipids from the host cells, and suggest that a source of lipid should be included in media for attempted axenic isolation of M. leprae.

A nonlinear technique to calculate parameters for segregational instability and differences in cellular growth-rate for plasmid-bearing micro-organisms growing in batch or continuous culture is presented. This method is compared with an approximate technique based upon linear regression. The accuracy and sensitivity of the results are evaluated by use of simulated data and biological data taken from experiments with Pseudomonas aeruginosa(pGSS15) and Escherichia coli(pHSG415). It is demonstrated that the nonlinear analysis gives results which are significantly more accurate and which show much better agreement with the data. Consequently, the new analysis leads to quite different conclusions with regard to the nature of the instability of the plasmid-bearing strain. This method offers an opportunity to study the genetic and physiological aspects of plasmid instability and so aid the design and optimization of cloning vectors.

It has been reported that transposition of Tn3 is temperature-sensitive. The effect of temperature on the transposition of other class II bacterial transposable elements is reported here: Tn21, Tn501, Tn1721, Tn2501 and Tn3926 all also display temperature-sensitivity of transposition. The temperature at which the highest transposition frequency was observed varied between room temperature and 30°C.

Segments of DNA were deleted from recombinant cosmid DNAs during propagation in Escherichia coli hosts in liquid culture. DNAs of more than 1000 cosmids propagated in various E. coli hosts were analysed by agarose gel electrophoresis (AGE). The effects of vectors, insert DNAs and host genetic characters on the formation of deletions were examined. The probability of deletion and the pattern of deletion bands observed by AGE differed from clone to clone, and after extensive culture the deletion band patterns remained almost constant during further culture. Most recombinant clones eventually showed deletion during prolonged liquid culture. Mutations in the recA gene of E. coli hosts, including a deletion mutation, did not prevent deletion. Most deletions occurred in the insert portions of cosmid DNAs. Nucleotide sequence analysis of six deletion junctions in test cosmid cMB15 demonstrated that deletions occurred between two short complete direct repeats of about 4–10 bp, irrespective of whether the cosmid was propagated in a recA host or a rec + host. Some deletions occurred at the same sites either in a recA host or a rec + host. These results suggest that the deletion events are mainly mediated by a recA-independent recombination system(s) of E. coli host cells.

The gene for the osteolytic toxin of Pasteurella multocida has been cloned into a plasmid vector and expressed off its own promoter in Escherichia coli. Particular restriction endonucleases failed to cut the gene and regions flanking it, suggesting an A + T base ratio significantly greater than the remaining genome of P. multocida. Cloned toxin was indistinguishable from the native toxin with respect to molecular mass, antigenicity and toxicity in different tests. A single intraperitoneal injection of toxin purified from the recombinant E. coli reproduced in gnotobiotic pigs the pathological changes characteristic of atrophic rhinitis. The recombinant E. coli produced at least 10 times as much toxin as P. multocida.

The structure and location of nopaline catabolism (noc) genes were examined in various Agrobacterium tumefaciens strains by plasmid transfer and by Southern hybridization analysis. In a pathogenic strain, PyTS3, the noc genes were identified both on the Ti-plasmid, pTiPyTS3, and on the cryptic plasmid, pCr140. Plasmid pCr140 was transmitted to another A. tumefaciens strain at a higher frequency than was pTiPyTS3. Southern hybridization analysis, using Ti-plasmid-derived noc genes as a probe, indicated that the noc genes of pTiPyTS3 differed structurally from those of pCr140. Three non-pathogenic strains possessed plasmids that hybridized to the probe, and all showed identical patterns of hybridization after restriction enzyme cleavage. However, this pattern was different from those of pTiPyTS3 and pCr140. Only pCr140 showed partial hybridization to the probe. A nonpathogenic strain, PyON8, that grew on both nopaline and octopine, showed no hybridization of its plasmid or chromosome to the probe. Since nopaline-utilizing transconjugants were not obtained from PyON8, the noc genes in PyON8 may be chromosomally located. These results indicate that A. tumefaciens contains diverse noc genes which differ in both organization and cellular localization.

Two populations of Coxiella burnetii were isolated from fibroblast tissue cultures and examined for their ability to synthesize DNA when incubated in a defined medium. Both the populations released by mechanical lysis of heavily infected host cells, as well as those recovered from the tissue culture medium, incorporated H3 32PO4 into DNA. Incorporation occurred at pH 4·5 but not at pH 7·0, and proceeded for 12–15 h. When incorporation of [3H]thymidine was studied, only the organisms obtained by mechanical lysis of host cells were active. Those which had been released by natural means into the tissue culture medium, and then recovered for study, did not incorporate precursor thymidine but were extremely active in protein biosynthesis. In mechanically released organisms, thymidine incorporation was inhibited immediately by rifamycin (40 μm) and hydroxyurea (10 mm), but it was not affected by chloramphenicol (310 μm) until 4 h after addition of the drug. Incorporation of H3 32PO4 by both populations of organisms was also inhibited by rifamycin, chloramphenicol and hydroxyurea, but the time sequence of inhibition differed. Southern hybridization utilizing 32P-labelled DNA suggested that both populations synthesized authentic chromosomal DNA sequences, as well as QpH1 plasmid DNA, during acid activation of metabolism.

Some morphological, physiological, enzymic and genetic characteristics of mycelial and plasmodioid phenotypes of Neurospora crassa, obtained by vegetative selection of a single fz; sg; os-1 (‘slime’-like) segregant of a cross ‘slime’ × wild-type, were studied. The mycelial phenotype of the segregant, albeit altered in morphology, was otherwise fully normal for export and regulation of invertase and β-glucosidase. In contrast, both enzymes were oversecreted and were resistant to catabolic regulation in the stable ‘slime’ phenotype, derived from the same isolate. These results suggested that the enzymic defects of ‘slime’ appeared gradually, concomitantly with the loss of ability to construct a cell wall, as a consequence of the process of vegetative selection required to produce stable ‘slime’ from mycelium-forming ‘slime’-like segregants. This idea was reinforced by the isolation of an intermediate phenotype, which exhibited mycelial/spheroplast dimorphism conditioned by the osmolarity of the culture medium. Crosses of the two extreme phenotypes of the fz; sg; os-1 segregant (the mycelial and the stable ‘slime’) failed to demonstrate any significant genetic difference between them.

Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min. The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose. In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination. Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose. The remaining glycogen was degraded upon subsequent germination of the zoospores. B. emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein. On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved. Upon incubation of fractions from the two peaks or of a crude extract in the presence of [2-32P]fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained. The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column. PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained. Phosphofructo 1-kinase and fructose-1,6-bisphosphatase from B. emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2,6-bisphosphate. The physiological significance of these properties is discussed. A simple method for the determination of trehalose is also reported.

Bacteroids isolated from alfalfa nodules induced by Rhizobium meliloti 102F34 transported glycine betaine at a constant rate for up to 30 min. Addition of sodium salts greatly increased the uptake activity, whereas other salts or non-electrolytes had less effect. The apparent K m for glycine betaine uptake was 8·3 μm and V was about 0·84 nmol min-1 (mg protein)-1 in the presence of 200 mm-NaCl which gave maximum stimulation of the transport. Supplementing bacteroid suspensions with various energy-yielding substrates, or ATP, did not increase glycine betaine uptake rates. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the respiratory inhibitor potassium cyanide strongly inhibited glycine betaine uptake, but arsenate was totally inactive. Glycine betaine transport showed considerable structural specificity: choline, proline betaine, γ-butyrobetaine and trigonelline did not competitively inhibit the system, although choline and proline betaine were transported by bacteroids. Both a high-affinity activity and a low-affinity activity were found for choline uptake. These osmoprotective compounds might have a significant role in the maintenance of nitrogenase activity in bacteroids subjected to salt stress.

Three strains of Streptococcus salivarius including a recent clinical isolate were found to possess Ca2+-dependent fructosyltransferase (FTF) activity. The extracellular FTF activity of cells grown on sucrose increased as much as 9-fold compared with cells grown on either glucose, fructose or galactose. This increase in activity was due not to induction of FTF by sucrose, but to the release of the cell-bound form of the enzyme. Studies with washed cells of S. salivarius ATCC 25975 showed that the extent of release of the cell-bound FTF activity was dependent upon the sucrose concentration up to 4 mm, at which concentration maximum release (95%) of cell-bound FTF occurred. Several lines of evidence suggested that either substrate binding or de novo synthesis of fructan is required for the release of the cell-bound FTF activity.