Specific inhibition of outgrowth of Bacillus subtilis spores by novobiocin.

Spores of a Bacillus subtilis mutant temperature sensitive in deoxyribonucleic acid (DNA) replication proceeded through outgrowth at the nonpermissive temperature to the same extent as the wild-type parent spores. In contrast, the DNA synthesis inhibitor novobiocin completely prevented spore outgrowth while displaying a marginal effect on logarithmic growth during one generation time. Inhibition of outgrowth by novobiocin occurred in the absence of DNA replication, as demonstrated in an experiment with spores of the temperature-sensitive DNA synthesis mutant at the restrictive temperature. Novobiocin inhibited the initial rate of ribonucleic acid synthesis to the same extent in germinated spores and in exponentially growing cells. A novobiocin-resistant mutant underwent normal outgrowth in the presence of novobiocin. Therefore, novobiocin inhibition was independent of its effect on chromosome replication per se.     

Development of Resistance to Novobiocin, Tetracycline, and a Novobiocin-Tetracycline Combination in Staphylococcus aureus Populations

The antibiotic sensitivity of the individual organisms of a bacterial population was determined to study the comparative rates of development of resistance of Staphylococcus aureus to novobiocin, tetracycline, and to a combination of these antibiotics. Serial subculture of S. aureus with the combination of novobiocin-tetracycline (N-T 2.5:1; the ratio in serum of patients dosed with Panalba) showed a significant retardation of resistance outgrowth compared with subculture in the presence of the antibiotics individually. Increase in organisms resistant to novobiocin seen after one N-T subculture was related to the “concentration gap” between novobiocin and tetracycline. Two additional subcultures with N-T caused little or no increase in organisms resistant to novobiocin, tetracycline, or to the combination. The data suggest that the retardation of further development of resistance was the result of tetracycline inhibition of novobiocin-resistant strains and vice versa.     

Coagulase-Negative,Novobiocin-Resistant Staphylococci on the Skin of Animals and Man,on Meat and in Milk

The occurrence of coagulase-negative, novobiocin-resistant staphylococci, i.e. Staphylococcus cohnii, Staphylococcus saprophyticus, Staphylococcus sciuri and Staphylococcus xylosus, on the skin of animals and man has been studied. On cultures from cats, cows, dogs, guinea pigs, mice, rabbits and sheep studied, such organisms were predominant among the coagulase-negative staphylococci. From the skin of the hands of 21 of 38 persons whose professions brought them into contact with animals, e.g. inséminât ors, slaughterhouse workers and veterinarians, coagulase-negative, novobiocin-resistant staphylococci were isolated. This finding contrasted with that regarding 50 persons lacking such contacts, of whom only 1 harboured such bacteria. S. saprophyticus was isolated only from those slaughterers presenting with wounds on their hands. Coagulase-negative, novobiocin-resistant staphylococci were also isolated from every second specimen collected from the surface of meat at a slaughterhouse. No difference in the culture results could be demonstrated from specimens collected before and after cutting-up of the carcasses. Of 26 strains of coagulase-negative, DNase-negative staphylococci isolated from milk with pathological CMT, all but 5 were novobiocin-resistant. Fifteen were classified as S. xylosus, 4 as S. sciuri and 1 as S. cohnii. Of another 15 DNase-positive strains, 3 were resistant to novobiocin. Finally, clinical infections with coagulase-negative, novobiocin-resistant staphylococci in man, e.g. urinary tract infections caused by S. saprophyticus, are considered in relation to possible contagious reservoirs and modes of spread.     

Characterization of the DNA gyrase from the thermoacidophilic archaeon Thermoplasma acidophilum

Thermoplasma acidophilum is sensitive to the antibiotic drug novobiocin, which inhibits DNA gyrase. We characterized DNA gyrases from T. acidophilum strains in vitro. The DNA gyrase from a novobiocin-resistant strain and an engineered mutant were less sensitive to novobiocin. The novobiocin-resistant gyrase genes might serve as T. acidophilum genetic markers.     

Chromosomal map location of the methicillin resistance determinant in Staphylococcus aureus.

Three-factor genetic crosses performed by transformation have shown that the methicillin resistance determinant of Staphylococcus aureus strain DU4916 (the mec-4916 marker) is linked to a novobiocin resistance (Novr) marker (nov-142) and mutational sites affecting pyrimidine (pyr-141), purine (pur-102), and histidine (hisG15) biosynthesis in S. aureus strain 8325. The linkage group thus defined is pyr-141-hisG15-nov-142-pur-102-mec-4916. Phage 80alpha previously propagated on a novobiocin-resistant, methicillin-sensitive (Mecs) 8325 strain was used to infect 21 novobiocin-sensitive, methicillin-resistant clinical isolates (including strain DU4916). Among the novobiocin-resistant transductants so obtained from each recipient, between 1 and 5% were methicillin sensitive (reflecting cotransduction of Novr and Mecs). These results are consistent with the genetic determinant of methicillin resistance having a single chromosomal locus in most, if not all, strains of S. aureus.     

Preferential inhibition of plasmid replication in vivo by altered DNA gyrase activity in Escherichia coli.

The thermosensitive growth phenotype exerted by runaway-mutant plasmids was suppressed by sublethal doses of the DNA gyrase inhibitors novobiocin or nalidixic acid, although the latter drug was less efficient. A novobiocin-resistant gyrB mutant Escherichia coli strain prevented expression of the runaway phenotype at 37 to 42 degrees C in the absence of any drug.     

The Effect of Some Inhibitory Sugars upon the Content of Adenosine Triphosphate in Wheat Roots

The content of adenosine triphosphate (ATP) in roots of -wheat (Triticum aestivum L.) was determined with the fire-fly-luciferase method. The content is decreased by D-mannose, which inhibits root growth, respiration and chloride uptake. In intact seedlings the inhibition of root growth is relieved by other sugars and also by the flavanone naringenin and by 2,4-dinitrophenol. This reversal is combined with an increased content of ATP. The inhibition of chloride uptake by mannose in excised roots is reversed by some other sugars (including D-galactose which is in itself inhibitory to root growth), and also in this case the ATP content is increased. Naringenin and dinitrophenol do not relieve the inhibition of chloride uptake caused by mannose. Nor do they increase the content of ATP in this case. The primary effect of mannose seems to be inhibition of glycolysis whereas the effect upon root growth is secondary. Galactose, which also inhibits root growth, does not inhibit respiration or reduce the ATP content and the primary effect of galactose (and also of 2-deoxy-D-glucose and 2-deoxy-D-galactose) seems to be on the synthesis of cell wall substances.     

Novobiocin resistance marker in Haemophilus influenzae that is not expressed on a plasmid.

The plasmid pNov2, carrying a cloned chromosomal marker conferring resistance to at least 2.5 micrograms of novobiocin per ml, was constructed with a new Haemophilus influenzae cloning vehicle, pDM2. The novobiocin marker of pNov2 was not normally expressed, but in Rec+ cells approximately one in 10(4) cells in a culture of a transformant became novobiocin resistant, a frequency about four orders of magnitude higher than the spontaneous mutation frequency. Variants of such cells that had lost the plasmid were also novobiocin resistant. Since Rec- cultures bearing pNov2 showed novobiocin resistance only at the normal mutation frequency, we concluded that the Rec+ novobiocin-resistant transformants arose because of a rare recombination between plasmid and chromosome in which the chromosome acquired the novobiocin marker from the plasmid. Evidence is presented that novobiocin sensitivity is dominant over this particular novobiocin resistance marker.     

Morphological variation of new Thermoplasma acidophilum isolates from Japanese hot springs.

We isolated 12 strains of Thermoplasma acidophilum from hot springs in Hakone, Japan. T. acidophilum strains showed morphological variation in the crystal-like structure in the cell and the fibrous structure on the cell surface. Two strains tested were sensitive to novobiocin. However, a novobiocin-resistant mutant was obtained by spontaneous mutation.     

Novobiocin-induced elimination of F'lac and mini-F plasmids from Escherichia coli.

Novobiocin eliminated (cured) F'lac and three low-copy-number mini-F plasmids (pML31, pMF21, and pMF45) from Escherichia coli to different extents. F'lac was cured 0 to 3%. pML31, whose replication region is contained on the 9-kilobase f5 EcoRI restriction enzyme fragment of F, was eliminated 10 to 92%. pMF21, deleted of the origin of mini-F replication at 42.6 kilobases on the F map and known to initiate from an origin at 45.1 kilobases, and its closely related derivative pMF45 were cured to the greatest extent (greater than 97%). pMF45 was eliminated from a wild-type bacterial strain but not from an isogenic novobiocin-resistant gyrB mutant strain, indicating involvement of the B subunit of DNA gyrase in the curing phenomenon. The number of bacteria containing pMF45 halved with each generation of growth in the presence of novobiocin, as is predicted for complete inhibition of plasmid DNA replication.     

Urinary tract infections due to Staphylococcus saprophyticus biotype 3.

Staphylococcus saprophyticus biotype 3 (Micrococcus subgroup 3 or M3) has usually been shown to be the second commonset cause of urinary tract infections in European women who are not in hospital. It generally causes pyuria and symptoms as severe as those caused by Escherichia coli. Unlike S. epidermidis it is seldom found as a contaminant in midstream urine specimens, and almost exclusively infects women in their reproductive years. However, S. saprophyticus is seldom differentiated from S. epidermidis in Canadian clinical laboratories. Urinary isolates of S. saprophyticus were presumptively differentiated from other coagulase-negative Micrococcaceae by their resistance to novobiocin as demonstrated by a simple disc susceptibility test that misidentified the infecting organism in only 3.4% of specimens. These novobiocin-resistant, coagulase-negative organisms caused similar proportions of the urinary tract infections in young women in York, England and Vancouver -- 6.6% and 6.9% respectively. In York these organisms were associated with significantly greater pyuria than novobiocin-sensitive organisms or bile-tolerant streptococci but not S. aureus or Enterobacteriaceae. In both communities novobiocin-sensitive, coagulase-negative Micrococcaceae were appreciably more resistant to penicillin than novobiocin-resistant organisms. Thus, differentiating S. saprophyticus from novobiocin-sensitive, coagulase-negative organisms provides information that is clinically useful, particularly for primary care practitioners working in the community or in outpatient clinics.     

D-mannose transport and metabolism in isolated enterocytes

D-mannose transport and metabolism has been studied in enterocytes isolated from chicken small intestine. In the presence of Na(+), the mannose taken up by the cells either remains free, is phosphorylated, is catabolized to H(2)O, or becomes part of membrane components. The mannose remaining free in the cytosol is released when the cells are transferred to an ice bath. The Na(+)-dependent D-mannose transport is electrogenic and inhibited by ouabain and dinitrophenol; its substrate specificity differs from SGLT-1 transporter. The Glut2 transporter inhibitors phloretin and cytochalasin B added following 30-min mannose uptake reduced the previously accumulated D-mannose, whereas these two agents increased the cell to external medium 3-O-methyl-glucose (3-OMG) concentration ratio. D-mannose efflux rate from preloaded D-[2-(3)H]-mannose enterocytes is Na(+)-independent. Phloretin did not affect D-mannose efflux rate, whereas it inhibited that of 3-OMG. Neither mannose uptake nor efflux rate were affected by fructose. It is concluded that part of the mannose taken up by the enterocytes is rapidly metabolized and that enterocytes have two D- mannose transport systems: one is concentrative and Na(+)-dependent and the other is Na(+)-independent and passive.     

Accumulation and depletion of trans octadecenoic acid in rat peripheral nerve phospholipid

Crude extracts from cells of a Streptomyces strain isolated from a palm-grove soil and grown on different carbon sources showed a constitutive glucokinase. Specifically inducible kinase activity for mannose was found in cells grown on mannose or beta-D-mannan. The activity on mannose was due to a highly specific mannokinase (ATP:D-mannose 6-phosphotransferase, EC 2.7.1.7) which has been separated from the glucokinase by Ultrogel AcA 54 gel filtration chromatography. Initial velocity and inhibition product studies were carried out to investigate the reaction pathway. In the absence of products, reciprocal plots intersecting on the abscissas were observed when either mannose or ATP concentration was varied in the presence of several fixed concentrations of the non-varied substrate. Km values for mannose and Mg-ATP complex are 0.33 and 1.1 mM, respectively. In the product inhibition studies, mannose-6-P was observed to be competitive with mannose and non-competitive with Mg-ATP. The reverse was observed when ADP was used as inhibitor. These data are consistent with a random sequential Bi-Bi mechanism with two dead-end ternary complexes.     

Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb

Carbohydrate binding properties of a new plant lectin (GNA) isolated from snowdrop bulbs were studied using the technique of quantitative precipitation, hapten inhibition, and affinity chromatography on immobilized lectin. Purified GNA precipitated highly branched yeast mannans but did not react with most glucans. Hapten inhibition experiments showed that D-mannose is an inhibitor of GNA-mannan interaction but neither N-acetyl-D-mannosamine nor D-glucose is an inhibitor. Hapten inhibition with various sugars showed that GNA requires the presence of equatorial hydroxyl groups at the C-3 and C-4 positions and an axial group at the C-2 position of the D-pyranose ring. A nonreducing terminal D-mannose residue is necessary for the interaction of oligosaccharides, and oligosaccharides with terminal Man(alpha-1-3)Man units showed the highest inhibitory potency (10-30 times greater than D-mannose) among the manno-oligosaccharides tested. The presence of the hydrophobic p-nitrophenyl aglycone increased the affinity of D-mannose only slightly. Immobilized GNA bound yeast mannan but did not bind glycogen. The behavior of glycoproteins with high mannose type glycan chains depended on the density and the structure of their glycan chains. Glycopeptides which carry Man(alpha 1-3)Man units were retarded on the immobilized GNA column whereas those lacking this unit or with hybrid type glycan chains were not retarded on the column.     

Enhancement of macromolecular ligand binding by rabbit alveolar macrophages by mannose oligosaccharides and related compounds

When rabbit alveolar macrophages were incubated with 10 mM D-mannose, binding of macromolecular ligands containing D-mannose, such as bovine serum albumin modified with mannose (Man-BSA), was enhanced more than 100%, but was inhibited at higher concentrations [C.A. Hoppe and Y. C. Lee (1982) J. Biol. Chem. 257, 12831-12834]. This phenomenon was further investigated with ovalbumin-derived glycopeptide, Asn(GlcNA2,Man5), and with a wide variety of synthetic mannose oligosaccharides. The extent of enhancement is related to the fine structure of the oligosaccharide groups, but the results are complicated by concurrent inhibition exerted by these compounds. It appears that the more inhibitory a compound is, the less capable it is of exerting the enhancement effect. Thus, small mannose derivatives such as glycosides, including clustered mannosides based on tris(hydroxymethyl)aminomethane [Y. C. Lee (1978) Carbohydr. Res. 67, 509-514], and most of the biantennary mannose oligosaccharides were found to be effective in enhancing the binding of radiolabeled Man-BSA. Triantennary oligosaccharides, on the other hand, showed only a slight enhancement effect and a much stronger inhibitory effect. The effects of ligand size, valency, as well as the fine structure on enhancement are discussed.     

Morphological changes associated with novobiocin resistance in Bacillus licheniformis.

Spontaneously occurring novobiocin-resistant (Nov) mutants of Bacillus licheniformis ATCC 9945, resistant to low levels of novobiocin (15 mug/ml), were isolated with a frequency of 3 in 106 organisms. Such isolates grew well, but nearly all exhibited consistent plleiotropic alterations in colonial and cell morphologies. One mutant, nov-12, grew as chains of unseparated but clearly distinct daughter cells in the absence of novobiocin in liquid culture. When novobiocin was present, nov-12 grew as very long "filaments" which were, however, septate. Septa formed in the presence of the antibiotic were normal, except that no annular clevage of the septal wall was observed. Septa were also irregularly positioned along the filament. These observations were compared with previous findings on the effects of novobiocin and novobiocin resistance described for other organisms. It was concluded that the primary action of novobiocin might differ in gram-positive and gram-negative organisms. However, when the low-level novobiocin sensitivity, normally associated with gram-positive organisms, was genetically abolished in Nov strains of B. licheniformis they became susceptible to an action of novobiocin more analogous to that found for gram-negative organisms. The morphological alterations associated with the Nov phenotype in this organism, together with observations in other organisms, indicate that novobiocin resistance might be generally useful in the search for mutants of gram-positive organisms with altered cell walls.     

Cell wall composition of novobiocin-resistant pleiotropic mutant staphylococci.

Physically purified cell walls were prepared from selected pleiotropic novobiocin-resistant staphylococcal strains. The quantitative amino acid, amino sugar, and phosphorus contents of these walls are reported. This pleiotype was culturally diagnosed by its inability to support the growth of typing phages, inability to release latent bacteriophage, failure to elaborate coagulase, altered sugar catabolic pattern, and resistance to novobiocin. The strains were divided into two groups on the basis of wall composition. The walls of both groups of strains appeared to possess at least two phosphorus-containing polymers. On group of strains contained elevated levels of phosphorus in the cell walls. The second group contained the novel amino sugar galactosamine in the cell walls. This amino sugar is probably associated with one of the phosphorus-containing wall polymers of this group. On the basis of the data presented, it is suggested that the pleiotropy of these strains is the result of genetic change in the control of the biosynthesis of teichoic acids.     

Novobiocin-resistance sequences from the novobiocin-producing strain Streptomyces niveus

Two distinct DNA sequences expressing novobiocin resistance in Streptomyces lividans were cloned from the novobiocin-producing species Streptomyces niveus. Clone pGL101 (5kb) conferred resistance to 50 micrograms ml-1 novobiocin, whereas clones pGL102 and pGL103, which carry the same 6.5kb insert but in opposite orientations, expressed resistance to 150 micrograms ml-1. The cloned inserts from pGL101 and pGL103 failed to hybridize with each other or with the cloned novobiocin-resistant gyrB sequence from Streptomyces sphaeroides. Both probes hybridized strongly with DNA from the novobiocin-producing species S. niveus and S. sphaeroides but no hybridization (pGL103) or very weak hybridization (pGL101) was detected with DNA from the non-producing species S. lividans, Streptomyces griseus and Streptomyces antibioticus. S. niveus contains at least three novobiocin-resistance determinants with the pGL101 and pGL103 cloned sequences specific for novobiocin-producing strains of Streptomyces.     

Studies on the Germination of Pine Pollen (Pinus mugo) in vitro. III. Inhibition by D-Mannose and Deoxyhexoses

D-Mannose, 2-deoxy-D-glucose, 6-deoxy-D-galactose, and 2-deoxy-D-galactose inhibit germination of pine pollen (Pinus mugo Turra) probably competitively with a metabolizable sugar. Inhibition by D-mannose, 2-deoxy-D-glucose, or 6-deoxy-D-galactose is reversed by transfer of pollen to sucrose medium, if the inhibitors was added before tube growth has started. In contrast, inhibition by 2-deoxy-D-galactose is irreversible except after very short exposures to the inhibitor, in which case the transfer results in reduced growth and germination. Incubation with 2-deoxy-D-glucose, 6-deoxy-n-galactose, or 2-deoxy-D-galactose after tube growth has started, results in irreversible inhibition of growth. If D-mannose is used, growth is resumed if the pollen are transferred to sucrose medium. Addition of D-mannose or lowering of the temperature prior to incubation with the deoxyhexoses protected against the irreversible growth inhibition. Uptake of oxygen and ³²P-labelled phosphate is reduced upon addition of either of the inhibitors.     

Effect of nalidixic acid and novobiocin on pBR322 genetic expression in Escherichia coli minicells.

The effects of two deoxyribonucleic acid (DNA) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pBR322 in Escherichia coli minicells were studied. Quantitative estimates of the synthesis of pBR322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactamase precursor) was increased to as much as 200%. Nalidixic acid affected the synthesis of the tetracycline resistance protein similarly to novobiocin, although to a lesser extent. The effects of nalidixic acid were not observed in a nalidixic-resistant mutant; those induced by novobiocin were only partially suppressed in a novobiocin-resistant mutant. The synthesis of one of the inducible tetracycline-resistant proteins (34,000) coded by plasmid pSC101 was also reduced in nalidixic acid- and novobiocin-treated minicells. These results suggest that the gyrase inhibitors modified the interaction of ribonucleic acid polymerase with some promoters, either by decreasing the supercoiling density of plasmid DNA or by altering the association constant of the gyrase to specific DNA sites.