- Volume 130, Issue 1, 1984

The major phospholipids of the anaerobic rumen phycomycete Piromonas communis were phosphatidylethanolamine (38%), phosphatidylcholine (26%) and phosphatidylinositol (13%); no sphingolipids, glycolipids, plasmalogens or phosphonyl lipids were detected. Free fatty acids, triacylglycerols, 1:2 diacylglycerols and a variable amount of 1:3 diacylglycerol were identified, as were minor amounts of squalene and a triterpenol which is probably tetrahymanol. Approximately half the fatty acids were straight chain, even 12 to 24 carbon, saturated acids, the remainder being even 16 to 24 carbon, mono-unsaturated fatty acids. The double bonds in all except the 16 carbon acid were in the ω9 position. The unsaturation is introduced by a ∆9 desaturase which uses stearic acid as substrate and which does not use oxygen as a terminal electron acceptor. 14C from acetate and glucose was incorporated into the fatty acids of all complex lipids, as were lauric, myristic, palmitic, stearic and oleic acids. [14C]Choline was incorporated into phosphatidylcholine and [14C]ethanolamine into phosphatidylethanolamine and phosphatidylcholine. Label from [14C]serine was recovered in phosphatidylserine and phosphatidylethanolamine, but was not detected in phosphatidylcholine.

Metabolism of 14C-labelled methoxyl groups in vanillic and syringic acid to 14CO2 by various white-rot, brown-rot and soft-rot fungi was investigated in the presence of different amounts of glucose and nitrogen. In general, methoxyl metabolism in these fungi was repressed by 1% (w/v) glucose and stimulated by nitrogen. The soft-rot fungus Petriellidium boydii was the exception and metabolized the methoxyl group of vanillic acid without glucose repression. Generally, brown-rot and soft-rot fungi metabolized the methoxyl groups of syringate faster than the vanillate methoxyl group.

A phosphoenolpyruvate: dihydroxyacetone phosphotransferase was induced in Escherichia coli grown on dihydroxyacetone as sole carbon source or in its presence. This is the first example of a triose which can be acted upon by the membrane complex to provide a central intermediate in glycolysis. The presence of this system explains the ability of a mutant, in which the ATP-dependent glycerol kinase is genetically replaced by a glycerol : NAD 2-oxidoreductase, to grow on glycerol.

A mutant stain, Brevibacterium 19, defective for the enzyme nitrile hydratase but retaining all the amidase activity of the wild-type Brevibacterium R312, was isolated. This is genetic evidence in favour of the hypothesis that all the nitrile-hydratase activity of the wild-type was due to a single enzyme, the structural gene of which was lost in the mutant strain 19.

The specific activities and K m of the nitrile hydratase were determined for 12 different substrates. The affinity of the enzyme increased as the number of hydrogen-bonding positions of the substrate increased, and decreased with more spatial crowding of the hydrocarbon chain. The specific activity of the enzyme for a substrate was enhanced by the nucleophilic and hydrophilic properties of the hydrocarbon side chain of that substrate.

Two proteinases have been purified from cell extracts of the cellular slime mould Dictyostelium discoideum and have been identified as proteinase E and proteinase B by electrophoretic analysis on polyacrylamide gels containing haemoglobin. Both were probably glycoproteins, each consisting of a single polypeptide chain with apparent molecular weights of 58000 (proteinase E) and 30000 (proteinase B) as indicated by SDS-PAGE. A higher molecular weight of 38000 was suggested for proteinase B by gel filtration. On isoelectric focusing multiple forms of proteinase E were revealed with isoelectric points ranging from 3·05 to 3·35. There was only a single form of proteinase B with an isoelectric point at pH 3·55. Both enzymes hydrolysed proteins at low pH and activated trypsinogen at pH 3·5. Proteinase B had much lower activity than proteinase E and required a reducing agent such as DTT for maximum activity. The pH optimum for activity of proteinase E towards hide powder azure was 2·5. Proteinase B, but not proteinase E, also hydrolysed a number of derivatives of basic amino acids and related peptide derivatives. N-Benzoyl-α-dl-arginine-2-naphthylamide (Bz-Arg-NNap) was used as substrate during purification, N-benzoyl-l-prolyl-l-phenylalanyl-L-arginine p-nitroanilide (Bz-Pro-Phe-Arg-Nan) was used for proteinase B characterization. Activity was optimal at pH 6 and was dependent on a reducing agent. Inhibitor studies indicated that proteinase E was probably a pepstatin-insensitive aspartic proteinase which was also inhibited by mercurials through binding to a cysteine residue not involved in the catalytic mechanism. Proteinase B was inactivated by a range of typical cysteine proteinase inhibitors. Proteinase B may be identical to proteinase I previously purified from D. discoideum. Overall the properties of both proteinases were consistent with their being involved in general protein degradation within the lysosomes of D. discoideum.

The adaptation of cells of Rhodopseudomonas sphaeroides f. sp. denitrificans for growth under denitrifying conditions in the light has been studied. The presence of nitrate in photosynthetically grown bacterial cultures resulted in a drastic reduction of carotenoid and bacteriochlorophyll contents as well as a loss of one of the polypeptides of the light harvesting complex, resulting in colour changes. Denitrifying cells had high activities of nitrate, nitrite and nitrous oxide reductases. The polypeptides corresponding to subunits of these enzymes were separated by PAGE. Synthesis of these enzymes was studied by pulse-chase labelling techniques. Nitrate and nitrite reductases are constitutive enzymes and it is likely that copies of mRNA for synthesis of these enzymes are ‘long lived’ in the cells.

Cadmium accumulation by a Citrobacter sp. growing in the presence of the metal occurred as a sharp peak during the mid-exponential phase of growth, but cultures showed considerable inhibition of growth compared to cadmium-free controls. This problem was overcome by pregrowing the cells in cadmium-free medium and subsequently exposing them to the metal in the resting state, under which conditions higher concentrations of cadmium were tolerated and metal uptake was enhanced. This ability was retained when the cells were immobilized and then challenged with a flow containing Cd2+; 65% of the metal presented was removed from solution. The influence on uptake of the composition of the exposure buffer and of various cell treatments were investigated and the results are discussed with respect to the anticipated speciation of the cadmium presented to the cells and also with respect to the probable mechanism of metal uptake. This is thought to occur through the activity of a cell-bound phosphatase, induced during pre-growth by the provision of glycerol 2-phosphate as sole phosphorus source. Continued enzyme function in resting cells would then precipitate the metal as cell-bound cadmium phosphate.

The modified deazaguanosine derivative queuosine (Q) is found in the first position of the anticodon in the Q-family of tRNAs of eubacteria and eukaryotes. The Q-base, queuine, is inserted into tRNA in exchange for guanine. Myxamoebae of Dictyostelium discoideum cannot synthesize queuine de novo, but obtain it from bacteria, their natural food supply. When grown on bacteria, D. discoideum is almost fully modified with respect to Q, whereas the corresponding tRNAs from axenic strains, due to queuine limitation in axenic media, contain submolar amounts of Q. Queuine does not affect growth rate, but accelerates development and stimulates spore germination. To establish whether changes might be caused early in the developmental cycle by an insufficient supply of queuine, vegetative amoebae of the strain AX-2 were induced by starvation to develop in suspension culture. Q deficiency in tRNA was rectified by the addition of queuine to the starvation buffer at a concentration of 10−7 m. During the first 12 h, when the cells acquired aggregation competence, restoration of queuine caused (i) an increase in binding of cAMP to its surface receptors, especially to the low-affinity binding site, and (ii) characteristic changes in the time course of the synthesis of distinct proteins in response to queuine as judged from the pattern of labelled proteins separated by two-dimensional gel electrophoresis.

The patterns of polypeptides synthesized by two co-isogenic monokaryons and the derived dikaryon of the basidiomycete Schizophyllum commune, growing in surface culture, were compared by two-dimensional PAGE after labelling with [35S]sulphate. Synthesis of most of the polypeptides (400 analysed) was common but at day 3 some 30 polypeptides were synthesized in the dikaryon which were not seen in the monokaryons. At this time the dikaryon had formed hyphal aggregates. Ten additional novel polypeptides were being synthesized in the primordia arising from these aggregates at day 4.

In surface culture, monokaryons and dikaryon excreted 4 to 8% of the labelled proteins (over 80 different polypeptides) into the medium compared with only 1 to 2% when cultivation was done in liquid shaken cultures. The polypeptides specifically secreted by the dikaryon at the aggregation stage consisted of seven small polypeptides with molecular weights ranging from 10 to 14 × 103, and two polypeptides with molecular weights 18 × 103 and 26 × 103, respectively. Such differences in excreted proteins between monokaryons and dikaryon were not observed when the mycelia were grown in liquid shaken cultures, indicating a relationship with fruit-body formation.

A comparison of labelled proteins in isolated hyphal walls revealed six polypeptides specifically associated with the walls of monokaryons and dikaryon irrespective of the cultivation method. Among the polypeptides newly appearing in the dikaryon at aggregation two were associated with the walls and co-migrated with the polypeptides of molecular weights 18 × 103 and 26 × 103 excreted into the medium at this stage of development.

A gas-vacuolate strain of Methanosarcina barkeri formed protoplasts in substrate-depleted cultures and gas vesicles were isolated from the protoplasts. Vacuolate protoplasts were separated from unvacuolate ones by flotation and the protoplast membrane was removed by Tween 20, liberating the gas vesicles. The gas vesicles were purified by flotation after initial passage through a 0·45 μm filter to remove contaminating material. Gas vesicle membranes were purified by isopycnic gradient centrifugation and were shown by electron microscopy to have a rib spacing of 4·8 nm.

The insertion pattern of flagellation of Centipeda periodontii was determined from electron micrographs of negatively stained cells treated with various detergents to remove their outer cell wall layer(s) selectively. The best results were obtained with 0·2% sodium dodecyl sulphate and a treatment time of 1–2 min. Detergent-treated cells displayed a unique helical pattern of flagellation which we designate as helicotrichous (Gk n. helix, helix + Gk n. thrix, hair). Flagella originated from an electron-dense region approximately 180 nm wide, which spiralled around the cell body. The length of the insertion path averaged 2·6 μm per 360° turn; its periodicity was 2·5 μm. An average of 19 flagella were inserted per 1 μm length of the electrondense region. The effects of cell division and branching on flagellation were also examined.

Conidia of the endoparasitic nematophagous fungus Meria coniospora adhere to the sensory organs of the nematode Panagrellus redivivus leading to a complete loss of the nematode’s ability to be attracted to fungi. Sialic acids located on the head region of the nematode seem to be involved in this interaction. Treatment of the nematode with sialidase reduced attraction towards fungi, and treatment with the sialic acid-specific lectin, limulin, reduced both nematode attraction and conidial adhesion. Other lectins tested (WGA, SBL, HPL, Con A) gave no effect on either attraction or adhesion. Treatment of conidia with trypsin or glutaraldehyde reduced conidial adhesion, indicating the presence of a fungal sialic acid-specific carbohydrate-binding protein. The results show the importance of sialic acids in nematode chemotaxis and also in adhesion of conidia of M. coniospora. Sialic acid, therefore, seems to be a link between attraction and adhesion in this system.

The addition of Fe(III) to anoxic lake sediments decreased the quantity of volatile fatty acids which accumulated. Similarly, the addition of several substrates to the sediments stimulated the rate of reduction of the naturally occurring Fe(III). Of the substrates used, malate caused the greatest stimulation, and subsequently a malate-fermenting Vibrio was isolated from the sediment. This organism also reduced NO− 3 and Mn(IV), the addition of which to the growth medium significantly lowered the rate of Fe(II) formation. The presence of Fe(III) in the medium increased the molar growth yield by 28% and this did not appear to be associated with the presence of particulate matter or changes in pH and E h. The addition of Fe(III) to the growth medium also produced a minor shift in the fermentation end-products, with a decrease in the quantity of ethanol formed and a concomitant increase in volatile fatty acids, both of which were of the same order as the amount of Fe(III) reduced. It would appear, therefore, that the presence of Fe(III) might permit a slight diversion of metabolism to more energetically favourable end products. The scale of iron reduction, both in the field and the laboratory, was very small compared with the reducing potential of the available substrates.

Roseburia cecicola strain GM is a motile obligate anaerobe that was isolated from mouse caecal mucosa. Twenty-five strains of motility mutants were obtained from populations of strain GM (wild-type) that had been exposed to UV light. Unlike GM cells, mutant bacteria were either non-motile and non-flagellated (Fla−) or migrated slowly or atypically in semi-solid medium. Strain GM and two mutant strains, SLS (Fla−) and WES (atypically motile), were used in mouse colonization experiments. In separate experiments, each strain colonized (4·8 × 109 to 1·5 × 1010 c.f.u. per g caecum) the caecum of germfree mice inoculated intragastrically with pure cultures of the bacteria. In mice mono-associated with either mutant strain, bacteria which were non-motile or atypically motile predominated in their caeca (< 99% of total bacteria recovered). In mice mono-associated with motile cells of strain GM, mutant strains which had lost wild-type motility became predominant in the caecal populations (97% of total bacteria recovered at 48 to 70 days after inoculation). Mice mono-associated with either strain SLS or strain GM were colonized by one strain each of Escherichia coli, Candida pintolopesii, a Bacteroides sp., and a Clostridium sp. Most (99%) of the R. cecicola cells recovered from the caeca of these animals had typical wild-type motility. Motility, although not essential for R. cecicola to colonize germfree mice, is apparently advantageous to this bacterium when other micro-organisms are present with it in the mouse caecum. Motility may thus be essential for R. cecicola to colonize conventional laboratory mice.

Three DNA-dependent ATPases(γ phosphohydrolases)can be isolated from Bacillus subtilis cells. We studied these enzymes in a number of mutants deficient in recombination or repair functions(rec, uvr)and in competent cells. The recA mutant studied had lower ATPase II activity, while competent cells had higher ATPase I activity, in comparison with the parental strain not brought to competence.

Plasmids pUB110 and pBC16 were introduced by protoplast transformation into the entomocidal bacterium Bacillus sphaericus 1593. Transformants expressed the antibiotic resistance determinants present on the plasmid and exhibited sporulation frequencies and larvicidal toxicities which were equivalent to those characteristic of the parent strain. These transformations constitute the first report of genetic transfer in B. sphaericus.

We have isolated a strain of Escherichia coli from chicken caeca which produces two E colicins and colicin M. This strain has seven plasmids, five of which have been transferred to E. coli K12. Two E. coli K12 derivatives which produce the two E colicins separately have been tested against seven standard E colicin producing strains which define seven different immunity groups. Our results indicate that these new E colicins define two further immunity groups, E8 and E9.

Adhesion of the obligate intracellular bacterium Chlamydia trachomatis to host cells is associated with a flux of Ca2+ across the cell membrane, and infection is enhanced by treatment of host cells with Ca2+ ionophore. The possibility that Ca2+ might interact with host cell Ca2+ regulatory proteins to promote chlamydial infection was investigated. Treatment of HeLa 229 cells with the calmodulin inhibitors pimozide, trifluoperazine, chlorpromazine, promethazine or haloperidol reduced chlamydial infectivity as measured by inclusion counting or the specific incorporation of [3H]threonine. The inhibitory effect was reversible, dose-related and shown to be associated with impairment of chlamydial adhesion and uptake by the host cells. This effect was clearly distinguished from the delayed maturation of chlamydiae due to continuous exposure to calmodulin inhibitors which may result from a decrease in the availability of high energy compounds from the host cells necessary for chlamydial growth. The possible mechanisms for calmodulin-mediated chlamydial endocytosis are discussed.

Incubation of serum-sensitive [3H]thymidine labelled Escherichia coli PC2166 (RSF1030) and E. coli AM1281(pBR322) harbouring small plasmids (mol. wt 5·5 × 106 and 2·6 × 106) in serum resulted in killing of 99·9% of the bacteria within 15 min and in the release of 85% of the radioactivity into the medium after 1 h incubation. The fate of chromosomal and plasmid DNA during incubation of the bacteria in serum was analysed by measurement of the amount of DNA-associated radioactivity, by TCA precipitation, by agarose gel electrophoresis and by the capacity of DNA to transform competent acceptor bacteria. Chromosomal DNA and high molecular weight plasmid DNA were rapidly degraded after 1 h incubation of bacteria in serum. However, low molecular weight plasmid DNA was virtually unaffected and remained physicochemically as well as biologically intact during up to 4 h of incubation of bacteria in serum.

In a comprehensive survey of the carbohydrate accumulation profiles of more than 70 strains of cyanobacteria three organic osmotica (glucosylglycerol, sucrose and trehalose) have been identified in both freshwater and marine isolates under conditions of osmotic stress. While the trend was towards glucosylglycerol accumulation in marine strains and sucrose accumulation in freshwater forms, there were no absolute differences between cyanobacteria isolated from each habitat. There was also no clear link between genus and the type of carbohydrate accumulated.