- Volume 133, Issue 3, 1987

Two bacterial strains excreting a yellow pigment were isolated from human urine and identified as Serratia marcescens. The pigment was produced in the late exponential and early stationary phases of growth. Minimal media supplemented with tyrosine, phenylalanine, 3,4-dihydroxyphenylacetate or tryptophan, as well as complex media, induced pigment production. UV-visible spectra of the extracted pigment had peaks characteristic of 2-hydroxy-5-carboxymethyl-muconate semialdehyde, produced from meta-cleavage of 3,4-dihydroxyphenylacetate by the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15). This enzyme was active when the bacteria were grown under conditions promoting pigment production. The kinetics and factors affecting pigment production are also reported.

Immunocytochemical studies using antisera to whole human choriogonadotropin (hCG), to its α- and β-subunits and to the COOH-terminal peptide of hCGβ, and two monoclonal antibodies to hCGβ, demonstrated expression of hCG-like material, its individual subunits and/or fragments in nine bacterial strains. Seven of these were isolated from patients with cancer and were definitely identified as Streptococcus faecalis (three strains), Staphylococcus haemolyticus (two strains) and Staphylococcus epidermidis and Escherichia coli (single strains). The other two strains were cell-wall-deficient (CWD) variants, one identified as Streptococcus bovis, isolated from the blood of a patient with a fever of unknown origin and a possible brain abscess. The other was a Gram-negative diphtheroid isolated from the urine of a pregnant woman, which during the period of study reverted to a Gram-positive Corynebacterium identified as a ‘C. ulcerans’ strain and expressed the hCG-like factor only during its phase as Gram-negative diphtheroid. Electron microscopy of these nine strains (including negative controls of strains of the same species subjected to the same immunocytochemical analyses and under identical cultural conditions) revealed morphological alterations in the bacterial cell walls and cytoplasmic material and/or bizarre forms of reproduction in six of the nine strains expressing hCG-like material including the two CWD variants. Collectively, these results provided evidence that (1) hCG-producing bacteria isolated from patients with overt cancer are not a new and unique species as claimed by others, and (2) there is a close resemblance between the bacterial protein and the human trophoblastic hormone, based on immunochemical recognition of different parts of the hCG molecule. The morphological changes observed by electron microscopy may indicate that some of the bacteria expressing hCG-like material are revertants of CWD variants.

SUMMARY: Bacillus stearothermophilus grows within the temperature range 40–70 °C in a complex medium that contains 115 μm-Ca2+ and 95 μm-Mg2+. The addition of Ca2+ to a final concentration ranging from 2·5 to 10 mm stimulated growth at suboptimal and supraoptimal temperatures, extending growth above 70°C, but had no effect on growth within the optimal temperature range. Mg2+ (2·5 mm) also stimulated growth although to a lesser extent. Furthermore, 10 mm-Mg2+ inhibited growth at temperatures higher than 65°C. This inhibitory effect was relieved by the addition of 2·5 mm-Ca2+. Sr2+ (10 mm), which often behaves as a Ca2+ analogue in biological systems, strongly inhibited growth and produced gross morphological alterations in the cells. The inhibitory effect of Sr2+ could also be relieved by addition of Ca2+.

SUMMARY: An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N′-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0·34 m and 1 mm, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0·34 m-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.

SUMMARY: In glucose-grown cells of Saccharomyces cerevisiae, collected during the stationary phase of growth, the addition of K+ to the external medium reversed glucose-induced internal acidification in 2 min. However, in ethanol-grown cells external K+ did not reverse the effect of glucose even after 20 min. The presence or absence of external K+ did not alter the modification of trehalase and fructose-1,6-bisphosphatase induced by glucose. It is concluded that transient acidification may be sufficient to cause the associated transient increase in cAMP.

Cells of a strain of a Citrobacter sp., pre-grown in cadmium-free continuous culture, accumulated cadmium extensively when resuspended in a buffer that contained Cd2+ and glycerol 2-phosphate. The accumulated compound was identified by X-ray microanalysis and magic angle spinning NMR analysis as cell-bound cadmium phosphate, probably CdHPO4. Its accumulation is consistent with the activity of a phosphatase, induced during pre-growth, that continues to function in the resuspended cells to liberate HPO2− 4 from glycerol 2-phosphate. This anion then combines with Cd2+ to form insoluble cell-bound CdHPO4.

A cell division mutant of Escherichia coli K12 lysA, the temperature sensitive ftsZ strain, was pulse-labelled with [3H]diaminopimelic acid (DAP) during growth in minimal salts medium both at the permissive (28 °C) and restrictive (42 °C) temperature. In contrast to other known cell division mutants, ftsZ filaments obtained during growth at 42 °C show no sign of persisting or newly initiated constrictions. The location of the incorporated DAP in dividing cells and in filaments was analysed with an improved autoradiographic method in which preparations of well-spread sacculi are covered with a dry emulsion. From the populations of sacculi complete distributions were obtained, which compared well with those of the intact cells. The grain-density distributions of cells dividing at 28 °C showed that the rate of surface synthesis was strongly increased at the site of constriction at the expense of the activity in the lateral wall, suggesting a redistribution of surface synthesis activity. In individual filaments elongating at 42 °C no indication for the existence of narrow or broad growth zones was found, suggesting a dispersed mode of lateral wall synthesis. These observations are in accordance with theoretical predictions on the rate of surface synthesis during the constriction period in cells which elongate at a constant diameter.

Yeast cells of Candida albicans1001 produced glucan-hydrolysing activity, most of which was due to an exo-1,3-β-glucanase. The enzyme was periplasmically located; it could be found in culture medium samples, and was secreted by protoplasts when cultured under regeneration conditions. In contrast to most yeast exoglucanases, this enzyme was practically inactive against p-nitrophenyl-β-d-glucoside, hydrolysis of this substrate being carried out by a β-glucosidase located inside the cytoplasmic membrane and not secreted to the external medium. Supernatant fluids from cell-free extracts reached their maximum glucanase level after several days at 0 °C, suggesting that the active enzyme was formed from an inactive precursor. Glucanase activity substantially decreased and sometimes disappeared from the cells when the yeast-to-mycelium transition was induced, but a significant (though lesser) reduction was also observed in yeast cells incubated in the same medium under conditions (temperature, cell concentration) that did not lead to formation of hyphae. It is suggested that C. albicansexo-1,3-β-glucanase may not be necessary for mycelial growth.

Cell-free extracts and culture medium from Saccharomyces cerevisiae S288C contained the two glucan hydrolases that are known to be periplasmically located in vegetative cells of this yeast. These were an endo-1,3-β-glucanase and a much more abundant exo-1,3-β-glucanase. Cell-free extracts of strains carrying two different mutant alleles of the EXB1 gene were totally deficient in the latter enzyme, whereas the former appeared in multiple heterogeneous forms, probably due to incomplete glycosylation. In contrast, protoplast lysates of wild-type and exb1 mutant strains were identical in their complement of glucanases, which consisted of two enzymes clearly distinguishable from the periplasmic glucanases. One was an exo-1,3-β-glucanase, which was active on pustulan, p-nitrophenyl β-d-glucoside, salicin and cellobiose, and was of higher M r than periplasmic exoglucanase. The other was a hydrolase acting on 1,3-β-glucan and 1,6-β-glucan but not the simple glucosides, which was not retained by DEAE-Biogel. Wild-type and exb1 mutant protoplasts secreted portions of the glucanases detected in protoplast lysates, when cultured in osmotically stabilized regeneration medium; the former also secreted the periplasmic exo-1,3-β-glucanase but the latter consistently failed to secrete it. It is concluded that the classically known glucanases of S. cerevisiae, located in the periplasmic space, must be formed as active enzymes, upon secretion, from inactive cytoplasmic precursors. On the other hand, the two new protoplast glucanases could be secreted by an alternative route that assures their incorporation into the wall structure, thus making their release into the culture medium more difficult.

To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay beween lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant strains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (⩽ 1 mm). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0·5 m-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.

Growth of Escherichia coli K12 cultivated in minimal medium was strongly inhibited by 2 mm-cyanate. This inhibition could be specifically reversed by arginine. Citrulline (but not ornithine, N-α-acetylornithine or N-acetylglutamate) could also restore a normal growth rate. Since growth inhibition by cyanate was followed by an accumulation of ornithine within the cell it was concluded that cyanate specifically inhibits the formation of citrulline from ornithine. The effect of cyanate on the growth of defined strains was consistent with a specific inhibition of carbamoylphosphate synthase. A kinetic study of carbamoylphosphate synthase and ornithine carbamoyltransferase in vitro supported this conclusion. Since carbamoylphosphate is probably the only source of endogenous cyanate it is postulated that carbamoylphosphate synthase activity can be regulated by cyanate resulting from the dissociation of carbamoylphosphate in metabolic circumstances leading to its overproduction.

The antibiotic moenomycin is a valuable biochemical tool for studying the metabolism of peptidoglycan and the autolytic system in Escherichia coli, since as a specific inhibitor of peptidoglycan polymerases it can efficiently promote cell lysis. In liquid media the bacteriolytic effect on E. coli K12 was dependent on the concentration of moenomycin, on growth phase and on growth rate. Before lysis cells underwent major morphological alterations. In sucrose-containing medium complete transformation to osmotically sensitive spheroplasts was easily achieved by addition of moenomycin. The minimum inhibitory concentration of the antibiotic varied with the strain of E. coli and was highly dependent on the growth medium. A tritiated derivative of moenomycin, [3H]decahydromoenomycin A, was prepared and found to have the same inhibiting efficiency. Its binding to E. coli membranes and membrane proteins was investigated. The absence of irreversible binding suggested that moenomycin might be a competitive inhibitor of the peptidoglycan polymerases. Spontaneous moenomycin resistant variants were isolated at a frequency of about 10−9.

Chlamydiae have amino acid requirements for growth in tissue culture as defined by those amino acids whose individual omission from the growth medium prevents chlamydial multiplication. We have tested the hypothesis that this inhibition of growth arises as a result of antagonism between particular amino acids such that inhibition occurs when the concentration of one amino acid is reduced in the presence of the antagonist amino acid at high concentration. Using the Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), in the presence of cycloheximide, the requirement for valine was abrogated by the simultaneous omission of isoleucine, that for phenylalanine by simultaneous omission of tryptophan and that for leucine by simultaneous omission of isoleucine plus valine. The antagonism shown between leucine and isoleucine plus valine appears to be unique among bacteria. In the absence of cycloheximide, GPIC had an additional need for tryptophan, tyrosine and isoleucine; these amino acid requirements were shown for both infected McCoy, HeLa and BHK cells. The results are consistent with a mechanism for regulation of parasite growth which depends on the balance of amino acid concentrations in the extracellular environment.

Zn2+ uptake by Saccharomyces cerevisiae was biphasic. The first phase was independent of metabolic energy, consisting of adsorption to the cell surface, and followed a Freundlich isotherm. The second phase was dependent on metabolic energy, ATPase activity and the transmembrane proton gradient, and consisted of uptake into the cell. Energy-dependent uptake showed Michaelis-Menten kinetics with a K m of 3·7μm-Zn2+ and a V max of 1·6 nmol min-1 per 107 cells at Zn2+ concentrations below 80 μm but deviated at higher concentrations. K+ and Mg2+ inhibited energy-dependent Zn2+ uptake while Na+ and Ca2+ did not. The effect of heavy metals was complex and included both inhibition and stimulation of Zn2+ uptake. K+ efflux accompanied Zn2+ uptake at all Zn2+ concentrations but there was no simple stoichiometric relationship between the two. Toxic effects of Zn2+ such as inhibition of H+ efflux and K+ uptake and reduction of viability were observed at all Zn2+ concentrations and toxicity appeared to be a major factor in K+ efflux. Toxicity also affected the kinetics of Zn2+ uptake, being a major cause of deviation from Michaelis-Menten kinetics. Zn2+ was compartmented within the cell: 56 % of the total intracellular pool was in the soluble vacuolar fraction, 39 % was bound to insoluble components and only 5 % was found in the cytosol. Isolated yeast vacuoles possessed an ATP-dependent Zn2+ uptake system whose properties were consistent with a Zn2+/H+ antiport.

The monobactam aztreonam and the cephalosporin ceftazidime, β-lactam antibiotics that possess the same side chain R1, showed unusual effects on exponentially growing pneumococci compared to other β-lactams. Both antibiotics did not induce lysis even at concentrations up to 2 mg ml−1, values well above the respective MICs. However, morphological alterations and growth inhibition of the cells were observed at much lower concentrations. Binding to penicillin-binding proteins (PBPs) in vitro could be monitored directly by using anti-aztreonam antiserum and the Western blot technique. Both antibiotics showed high affinity for PBP 3, but had an extremely low affinity for PBP 2b. It is suggested that the failure to bind to PBP 2b is responsible for the failure to induce lysis in pneumococci.

The production of iturin A by Bacillus subtilis was studied with respect to the composition of the culture medium. Increasing phosphate concentrations did not modify the antibiotic yield. Fructose, sucrose and mannitol were better carbon sources than glucose for antibiotic production. The nature of the nitrogen source was an important factor in the production of antibiotic. Among the amino acids which are components of iturin A, l-asparagine was the best substrate for the biosynthesis of iturin A; l-glutamine and l-serine were rather poor substrates while l-proline and d-tyrosine gave no antibiotic. Ammonium salts permitted good synthesis of antibiotic but the addition of calcium ions to the culture medium inhibited the excretion of antibiotic from the cells.

The long-chain components of 75 strains of mycobacteria, cultivated from Mycobacterium leprae-infected or non-infected armadillos, and of eight clinical and 15 environmental isolates of M. gordonae, were compared. Four major groups could be distinguished based on the presence of 10-methyloctadecanoic (tuberculostearic) and 2-methyl 3-hydroxyeicosanoic acids and secondary alcohols (2-octadecanol and 2-eicosanol). Some heterogeneity was found in strains assigned to M. gordonae: the characteristic absence of tuberculostearic acid and secondary alcohols and the presence of the branched C14 and the hydroxylated C20 acids were seen in only 34 of the 49 strains studied. Three strains were identified as M. malmoense, one as M. kansasii, ten as belonging to the M. avium–M. intracellulare–M. scrofulaceum complex and eight as belonging to new groups of armadillo-derived mycobacteria (ADM 1, ADM 2 and ADM 3) by conventional bacteriological tests and fatty acid compositions, though M. malmoense was heterogeneous in its fatty acids composition. Four strains, identified as M. avium by conventional tests, differed from this species by their fatty acid compositions. Thirteen strains showed some similarity to M. simiae and ten strains differed from all other known mycobacteria.