- Volume 131, Issue 1, 1985

The nitro radical-anion of metronidazole has been detected in vivo in the sexually transmitted human parasite, Trichomonas vaginalis, under anaerobic conditions by electron spin resonance spectrometry. Exposure of organisms to oxygen decreased the intensity of the radical signal in both metronidazole-sensitive ATCC strain 30001 and in the metronidazole-resistant strain 85. The sensitive strain still gave radical signals at partial pressures of oxygen (>6 kPa) sufficient to remove all detectable radicals from the resistant strain. This evidence suggests that the resistant strain has defective oxygen scavenging system(s).

Twelve Streptomyces venezuelae mutants blocked in chloramphenicol biosynthesis were isolated. Two of these (Cml-1 and Cml-12) were apparently blocked in the conversion of chorismic acid to p-aminophenylalanine and three (Cml-4, Cml-5 and Cml-8) accumulated p-aminophenyl-alanine and may have been blocked in the hydroxylation reaction that converted this intermediate to p-aminophenylserine. One mutant (Cml-2) accumulated d-threo-1-p-nitro-phenyl-2-propionamido-1,3-propanediol and d-threo-1-p-nitrophenyl-2-isobutyramido-1,3-propanediol, indicating that chlorination of the α-N-acyl group of chloramphenicol was blocked. The remaining six strains did not excrete any detectable chloramphenicol pathway intermediates.

The presence and location of an acetolysis-resistant polymer (sporopollenin) in various fruiting structures of the cellular slime moulds were demonstrated. Macrocysts (giant cells with thick cell walls) formed by a mutant (MF-1) of Dictyostelium mucoroides 7 contained large amounts of sporopollenin in their walls. In contrast, microcysts (round, walled resting cells) of Polysphondylium pallidum ws-320 lacked sporopollenin, at least in a tight networked form. This difference in sporopollenin distribution seems to be at least partly related to a difference in the germination efficiency of the two types of cells. In Acytostelium irregularvsporum fruiting bodies, the acellular stalk was found to consist mainly of sporopollenin, but the spores lacked it in a tight networked form. Spores of Polysphondylium pallidum and P. violaceum contained sporopollenin in the wall region. All the above species belong to the class Dictyostelia. Fruiting bodies of Acrasis rosea, a member of the class Acrasea, lack a tight network of cellulose wall as well as sporopollenin. It is clear that slime mould sporopollenin shows unique distribution patterns depending upon cell type and species. The functional significance of sporopollenin is discussed, with special reference to the late developmental stages of slime mould cells.

A spontaneous change, possibly involving a mating-type switch, occurred during storage of a homokaryotic culture (F2.4) of the basidiomycete Stereum hirsutum and resulted in a dramatic transformation of its cultural and interactive properties. Subcultures grown on malt agar rapidly produced farinaceous hymenial surfaces if exposed to light, and were not receptive to nucleus migration when paired with compatible homokaryons. These properties were transmissible to other homokaryons by direct pairing, the recipients becoming transformed regardless of whether they were sexually or somatically compatible or incompatible with F2.4. With the incompatible sib F2.9, transformation apparently occurred without nuclear transfer.

Progeny monobasidiospore cultures from transformed F2.4 and F2.9 strains consistently belonged to two interaction groups, standard and reversed, the former reacting similarly to the corresponding untransformed strains against a range of sib homokaryons, the latter interacting in exactly the opposite fashion. Reversed-standard pairings resulted in all cases in an unusual ‘quasi-compatible’ reaction, characterized by development of an asymmetrically migrating farinaceous region.

Progeny from transformed F2.9 were dimorphic, being divisible into (a) slow-growing, dense and (b) fast-growing, effuse colony types, the former reverting spontaneously to the latter. In several cases mycelial ‘mounds’ developed on reverted slow, dense colonies. Basidiospore progeny and mycelial subcultures from mounds produced colonies with further mounds. These and further observations indicate pleiotropic effects initiated or mediated by a cytoplasmically transmissible, possibly insertional factor on recognition and developmental processes in S. hirsutum.

Addition of cytidine or guanosine to UV-irradiated cells of a RecA+ strain of Escherichia coli did not produce any effect on the induction of two SOS functions: inhibition of cell division and expression of the umuC gene. Under the same conditions adenine gave a slight increase in the induction of these two responses. In a RecA441 mutant growing at 42°C, both cytidine and guanosine inhibited these SOS functions, whereas adenine produced a large increase in their expression. Moreover, the ATP concentration of the RecA441 mutant at 42°C showed a decrease which occurred earlier in the cells growing in the presence of cytidine or guanosine than in the absence of either compound. Adenine induced an increase of about three times the initial ATP concentration of this mutant at 42°C which dropped quickly after 10 min. Neither cytidine nor guanosine increased the evolution of cellular ATP in UV-irradiated cells of the RecA+ strain, whereas adenine had only a slight positive effect. However, in UV-irradiated RecA+ cells with and without adenine, ATP levels dropped quickly to the initial value after 20 min. These data suggest that the influence of adenine, cytidine and guanosine on the expression of the RecA441 phenotype at 42°C may be due to alteration of the cellular ATP concentration of this mutant.

Thirteen mycobacillin-negative (My–) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. The wild-type producer, three feeble producers and three strictly My– mutants did not accumulate any ninhydrin-positive peptide in the culture medium while the remaining seven My– mutants did accumulate ten such peptides whose amino acid composition indicated that there might be only three different peptides. The N-terminal and C-terminal amino acid residues implicated one of these peptides as a pentapeptide intermediate in mycobacillin synthesis; this was further confirmed by its molecular weight and sequence. Studies on cell-free synthesis showed that only the enzyme system from the wild-type strain synthesized mycobacillin while the defective ones from all the My– mutants synthesized one and the same pentapeptide as found in the culture broth of some of the mutants. Further studies in which the enzymes responsible for mycobacillin synthesis by cell-free extracts were separated into three fractions, A, B and C, showed that seven of the mutants were defective in fraction B whereas the three other mutants had defects in both fractions B and C. Thus the pentapeptide Pro→Asp→Glu→Tyr→Asp appears to be implicated in mycobacillin biosynthesis.

Several plaque-forming phage P1 derivatives carrying DNA rearrangements associated with IS elements are described. They have IS1, IS3 and IS5 inserted in four distinct locations, all of which are non-essential regions for phage P1 propagation. One derivative carries a genome segment, inverted relative to the one in the P1 wild-type genome, between two inverted copies of IS1. The inverted DNA segment spans about 23 kb of the 90 kb long P1 genome and it includes the invertible C segment. This phage is as viable as an isomeric P1 which carries the relevant segment in its original orientation. These results are discussed with regard to the genome organization of phage P1.

A mutation in a strain of Escherichia coli 15 produced ribosomes by an abnormal pathway that caused the accumulation of 47S ribonucleoprotein particles. The mutation was transferred to strains of E. coli K12 by transductions with bacteriophage Plcam and was at about 82 min, between cysE and pyrE and rather closer to the latter. The location and the physiological properties of the mutant suggested that the mutation was in the rpmB,G transcription unit and affected the synthesis of ribosomal proteins L28 and L33.

We have previously constructed a derivative of the broad host range streptococcal plasmid pIP501, a conjugative plasmid designated pVA797, that confers chloramphenicol resistance and contains a unique EcoRI site in a non-essential region of the plasmid molecule. pVA797 (30·7 kb) when cloned in toto as an EcoRI fragment into the positive selection vector pOP203(A2 +) gave a recombinant, pVA904 (37·7 kb), which was able to replicate in Escherichia coli and in streptococcal species. It can be phenotypically monitored in either genus by specific drug resistance markers (chloramphenicol resistance in streptococci, tetracycline resistance in E. coli). pVA904 segregates into E. coli minicells where it specifies the production of at least 13 polypeptides. Many of the polypeptides are missing in minicells containing a transfer-defective, deletion derivative of pVA904. pVA904 is an ideal model replicon for the study of streptococcal conjugation because it is a shuttle plasmid thus enabling manipulation using procedures established for E. coli. Specifically, it should be possible to define the genetic basis of streptococcal conjugation by coupling mutagenesis protocols and minicell protein analyses in E. coli with evaluation of transfer function in streptococci.

Granulomatous reactions induced by lipid extracts from the dermatophyte fungi Fonsecaea pedrosoi, Fonsecaea compactum, Cladosporium carrionii and Phialophora verrucosum, the causal organisms of chromoblastomycosis, were studied. Charcoal particles coated with the lipid extracts were prepared and injected intravenously into mice. Inflammation was characterized by an intense mononuclear cell infiltrate that lodged in the lung from 4 to 8 d after inoculation.

During MgSO4-induced modulation of Bordetella pertussis, adenylate cyclase activity, histamine-sensitizing activity (HSA) and the major cell-envelope polypeptides with M r 28000 and 30000 (X polypeptides) were lost synchronously at a rate which could be accounted for by a simple growth-dilution effect. MgSO4 and other compounds which induced the above phenotypic change caused little inhibition of adenylate cyclase activity. Nicotinic acid was the sole exception and at 4·1 mM-caused 60% inhibition of activity. Lysates of modulated cells, mixed with lysates of unmodulated cells, had no effect on either adenylate cyclase activity or HSA. Protein synthesis was a prerequisite for MgSO4-induced modulation and also for the reversal of this process. Exogenous cAMP and dibutyryl cAMP (5 mm) had no counteracting effect on MgSO4- or nicotinic acid-induced modulation. The concentration of MgSO4 required to induce loss of the X polypeptides (10 to 11 mm) was not altered by promoting adenylate cyclase activity by including an activator in the growth medium. In one culture containing 10 mm-MgSO4 and activator, partial loss of the X polypeptides occurred and yet the extracellular cAMP concentration was twice that of cultures without activator and where full expression of the X polypeptides occurred. [3H]cAMP-binding activity was detected in cell extracts of several strains of B. pertussis, but antiserum against purified Escherichia coli catabote repressor protein gave no reaction with B. pertussis cell extracts. Respiration rates with amino acids were similar for modulated and unmodulated variants and an avirulent strain of B. pertussis. These results are discussed in relation to a possible causal role for adenylate cyclase in modulation of B. pertussis.

The energy-based classification of heterotrophic substrates requires biochemical evaluation because some substrates can be assimilated by a variety of different metabolic pathways. By using the Y ATP-concept it was shown that the classification depends on the yield of ATP and reducing equivalents already generated on the way to the precursor (phosphoglycerate). With carbon-excess substrates a part of the total substrate consumed must be oxidized to completion merely for energy production, whereas with energy-excess substrates more energy is provided on the route to the precursor than is needed for assimilation of the precursor carbon. By means of this approach it was possible to assess experimental growth yields obtained on mixed substrates and to predict the optimum mixing proportion in order to attain the maximum carbon conversion efficiency. The validity of this method was shown for some examples.

The transition of Saccharomyces uvarum from oxidative to oxido-reductive glucose metabolism was characterized by the immediate formation of ethanol and the gradual adaptation of cells to a new physiological state, as expressed by a decrease in mitochondrial cytochrome content and a loss of malate dehydrogenase activity. Shifting the cells from carbon-limited to oxygen-, nitrogen- and iron-limited media in a continuous culture growing oxidatively led to the initiation of the same effects. The results lend support to the concept of a limited respiratory capacity as the basis for the occurrence of oxido-reductive glucose metabolism. Limitation of growth by a nutrient other than glucose results in an imbalance between glucose flux and biosynthetic potential of the cells and causes oxido-reductive glucose breakdown.

The distribution of Cd2+ taken up by Saccharomyces cerevisiae was examined by treatment of the organism with chitosan, which affects cell membrane permeability. In a Cd-sensitive strain, almost all of the Cd2+ bound to insoluble material, while in the Cd-resistant strain the Cd2+ was in the cytosol and bound to protein of low molecular weight (<30000). A Cd-resistant strain was also characterized by the decreased uptake of Cd2+

Saccharomyces cerevisiae Y185, enriched in linoleyl residues and incubated for up to 4 h in derepression buffer, more rapidly acquired general amino-acid permease (GAP) activity, as measured by the rate of accumulation of l-alanine, compared with organisms enriched in oleyl residues. A GAP-less mutant incubated under the same conditions did not acquire further l-alanine-accumulating ability, irrespective of the nature of the fatty-acyl enrichment. During derepression, K T values for the GAP were virtually identical irrespective of the fatty-acyl enrichment, but V max values were greater for linoleyl residue-enriched organisms, particularly after 1 h in derepression buffer. During incubation in derepression buffer, organisms with either fatty-acyl enrichment did not differ in the size of the amino-N pool, the concentration of l-alanine in that pool, rates of protein synthesis and glucose fermentation, or rate and extent of incorporation of label from . Under conditions used to measure rates of l-alanine accumulation, organisms with either enrichment showed no evidence of metabolism of accumulated l-alanine.