Search

The interactions of Staphylococcus aureus, Bacillus cereus I, TEM, Klebsiella pneumoniae K1 and Enterobacter cloacae P99 beta-lactamases with the novel penem inhibitor BRL 42715 were investigated kinetically and, in some cases, by electrospray mass spectrometry (e.s.m.s.). All the beta-lactamases were rapidly inactivated by BRL 42715, with second-order rate constants ranging from 0.17 to 6.4 microM-1.s-1. The initial stoichiometry of beta-lactamase inhibition was essentially 1:1, with the exception of the K1 enzyme. In this instance about 20 molecules of BRL 42715 were hydrolysed before the enzyme was completely inhibited. Inhibited beta-lactamases did not readily regain activity in the absence of BRL 42715, the half-lives for regeneration of free enzyme ranging from 5 min for the K1 beta-lactamase to over 2 days for the staphylococcal enzyme. Recovery of activity was incomplete for TEM-1, K1 and P99 beta-lactamases, suggesting partitioning of the inhibited enzymes to give a permanently (or at least very stable) inactivated species. Examination of the interactions of the penem with TEM, B. cereus I and P99 beta-lactamases by e.s.m.s. also showed rapid and stoichiometric binding of the inhibitor. In all cases a mass increase of 264 Da over the native enzyme was observed, corresponding to the molecular mass of BRL 42715, showing that no fragmentation of the penem occurred on reaction with the beta-lactamases.

Azotobacter vinelandii strain UWD has a defective NADH oxidase and forms poly-β-hydroxybutyrate (pHB) during exponential growth as a means of recycling NAD. One of the factors limiting the use of this strain for commercial pHB production is the low yield (

Heating competent Azotobacter vinelandii at 37 or 42 degrees C resulted in a total loss of competence with no loss of viability. The transformation process was relatively insensitive to heating at either temperature once DNase-resistant DNA binding was nearly complete. Although competent and 42 degrees C-treated cells bound equivalent amounts of [32P]DNA in a DNase-resistant state, no donor DNA marker (nif) or radioactivity was detected in the envelope-free cell lysate of heated cells, suggesting that DNA transport across the cell envelope was a heat-sensitive event. Competence was reacquired in a 42 degrees C-treated culture after 2 h of incubation at 30 degrees C by a process which required RNA and protein syntheses. The release of a surface glycoprotein, required for competence, from cells treated at 42 degrees C occurred in an insufficient amount to account for the total loss of competence. Recovery of competence in 42 degrees C-treated cells and further transformation of competent cells were prevented by the exposure of cells to saturating amounts of transforming DNA. Further DNase-resistant DNA binding, however, still occurred, suggesting that there were two types of receptors for DNase-resistant DNA binding to competent A. vinelandii. DNase-resistant DNA binding was dependent on magnesium ions, and at least one receptor type did not discriminate against heterologous DNA.

The spore coats of mature macroconidia of Microsporum gypseum contained greater carbohydrate–protein ester, disulfide, acid-soluble phosphate, and acid-insoluble phosphate content than mycelial walls. Phosphate was a major constituent of the spore coat, accounting for 6.8% of the dry weight. Spore coat phosphates were located internally as acid-insoluble phosphate, or externally on the spore surface as acid-soluble phosphate. Upon spore germination, the carbohydrate–protein ester, acid-insoluble phosphate, and acid-soluble phosphate content of the spore coats decreased, resulting in considerable alteration of spore coat internal and external properties. Germination was initiated by early alkaline protease and β-1,3-glucanase action, followed by ethyl-esterase, phosphodiesterase, and chitinase activities. These hydrolases were compartmentalized in lysosomal vesicles, which appeared to be delivered to the germinating spore in a coordinated manner.

The double antibody sandwich enzyme-linked immunosorbent assay (DAS-EL1SA) was used to determine the competitive abilities of Rhizobium meliloti strains BALSAC and NRG-185 which are used in commercial alfalfa inoculants for eastern and western Canada, respectively. The ratio of NRG-185:BALSAC in the inoculum applied to aseptically grown alfalfa (Medicago sativa L.) was adjusted to 0:100, 1:99, 50:50, 90:10, 99:1, or 100:0. The ratios between nodules containing only the BALSAC strain and nodules containing only strain NRG-185 were similar to their respective inoculum strain ratios. Nodules containing both strains occurred only with the inoculum strain ratio of 50:50. Total plant weights were highest with the two single strain inocula and were decreased 5–19% with dual strain inoculation. The decrease in total plant weight was significantly correlated with decreases in nodule number and nodule weight. The results suggest that R. meliloti strains BALSAC and NRG-185 have equal competitive abilities, but are somewhat antagonistic, resulting in decreased nodulation and plant growth when both are present in an inoculum.

Azotobacter vinelandii cells required 0.5 mM calcium in the iron-limited competence induction medium. This requirement also was fulfilled by strontium, but not by magnesium. Cells pregrown in competence medium lacking calcium rapidly recovered competence with the addition of 0.5 mM calcium, provided they were suspended in the growth supernatant. A 60,000-dalton glycoprotein (pI 5.10) present in competent or incompetent culture supernatants participated in calcium-mediated competence recovery. Cells grown in calcium-limited medium appeared to have leaky cell envelopes and released a diverse array of proteins into the culture supernatant and into distilled water washes of the cells, seven of which appeared to be more dominant in competent cells. Two distilled water washes of cells grown in calcium-limited medium did not prevent calcium-mediated recovery of competence in the culture supernatant. Four to six distilled water washes removed a competence-specific protein (pI 5.19) and prevented calcium-mediated recovery of competence in the culture supernatant.

Azotobacter vinelandii solubilized iron from certain minerals using only dihydroxybenzoic acid, which appeared to be produced constitutively. Solubilization of iron from other minerals required dihydroxybenzoic acid and the siderophore N,N'-bis-(2,3- dihydroxybenzoyl )-L-lysine ( azotochelin ) or these chelators plus the yellow-green fluorescent siderophore azotobactin . In addition to this sequential production of siderophores, cells also demonstrated partial to hyperproduction relative to the iron-limited control. The iron sources which caused partial derepression of the siderophores caused derepression of all the high-molecular-weight iron-repressible outer membrane proteins except a 77,000-molecular-weight protein, which appeared to be coordinated with azotobactin production. Increased siderophore production correlated with increased production of outer membrane proteins with molecular weights of 93,000, 85,000, and 77,000, but an 81,000-molecular-weight iron-repressible protein appeared at a constant level despite the degree of derepression. When iron was readily available, it appeared to complex with a 60,000-molecular-weight protein believed to form a surface layer on the A. vinelandii cell.

Encystment of Azotobacter vinelandii (ATCC 12837) in modified Burk nitrogen-free medium (pH 7.0) containing 0.2 percent beta-hydroxybutyrate occurs optimally in 0.37 to 0.44 mM solutions of calcium ions. Suspension of cells in media deficient in calcium results in abortive encystment characterized by the release of viscous cyst coat material. Mature cysts rupture in ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, suggesting that calcium is a structural component of the cyst coat. Maximal stimulation of encystment by calcium ions occurs prior to the completion of the cyst exine or outer coat. The uronic acid composition of cyst components is dependent on calcium levels in the medium. Uronic acids account for 31.7 percent of the intine (inner coat) and 13 percent of the exine dry weight, and only mannuronic and guluronic acids are present in these fractions. These can be extracted as homo- and heteropolymeric sequence "blocks" characteristic of alginic acids. The polyuronic acid fraction of both the cyst coats contain approximately equal amounts of heteropolymeric (mannuronic acid/guluronic acid) blocks. The exine, however, is richer in polyguluronic acid and the intine is richer in polymannuronic acid. As a result, the mannuronic acid/guluronic acid ratio of the exine is lower than that of the intine. Slimes that form in abortive encystment are rich in polymannuronic acid and have a high mannuronic acid/guluronic acid ratio. A polymannuronic acid 5-epimerase is active in the mature cyst central body and the encystment culture fluid.

Antisera were prepared against five Rhizobium meliloti strains including three strains adapted to Canadian soil and climate conditions and recently released for use in the production of commercial alfalfa (Medicago spp.) inoculants. The antisera were highly cross-reactive, but specificity was obtained by repeated massive adsorptions of the antisera with cells of the cross-reacting strains. The adsorbed antisera were used in microagglutination tests and the enzyme-linked immunosorbent assay (ELISA) to demonstrate the presence of the Canadian strains in their respective commercial peat-base inoculants at 108–109 viable cells/g. A plant infection technique which was also used to evaluate inoculant quality was specific only at the Rhizobium species level and required up to 4 weeks for completion. Serological techniques made it possible to identify and quantitate inoculant strains of R. meliloti while reducing the time required for testing. The agglutination analysis of inoculants was simple, but required regrowth of each colony isolate to generate sufficient cell numbers. The ELISA technique was used directly with colonies picked from plate counts, and the results were available within 5 days, including the time for colony growth on the plate media.

Azotobacter vinelandii (ATCC 12837) became competent to be transformed by exogenous deoxyribonucleic acid towards the end of exponential growth. Competence in wild-type and nitrogenase auxotrophic (nif-) strains was repressed by the addition of ammonium salts or urea to the transformation medium. Transformation of wild-type cells and nif- strains was optimal on nitrogen-free or nitrogen-limiting medium, respectively. Transformation of wild-type cells also was enhanced when the transformation medium had low molydbate content. During the development of competence, nitrogen was growth limiting, whereas carbon (glucose) was in excess. Carbon source shift-down was not effective in inducing competence. Shifting glucose-grown wild-type cells to medium containing 0.2% beta-hydroxybutyrate initiated encystment and also induced competence. The addition of glucose to this medium blocked encystment and early competence induction and reduced the transformation frequency to the basal level. Cyclic adenosine 3',5'-monophosphate induced competence in wild-type nitrogen-fixing cells and increased the transformation frequency 1,000-fold over the basal level. Exogenous cyclic adenosine 3',5'-monophosphate however, did not reverse nitrogen repression of competence in ammonia-grown wild-type or nif- strains.

The penem BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, is a potent inhibitor of a broad range of bacterial beta-lactamases, including the plasmid-mediated TEM, SHV, OXA, and staphylococcal enzymes, as well as the chromosomally mediated enzymes of Bacteroides, Enterobacter, Citrobacter, Serratia, Morganella, Escherichia, Klebsiella, and Proteus species. The concentration of BRL 42715 needed to reduce the initial rate of hydrolysis of most beta-lactamase enzymes by 50% was less than 0.01 micrograms/ml, which was 10- to 100-fold lower than for other beta-lactamase inhibitors. These potent inhibitory activities were reflected in the low concentrations of BRL 42715 needed to potentiate the antibacterial activity of beta-lactamase-susceptible beta-lactams. Concentrations of 0.25 micrograms/ml or less considerably enhanced the activity of amoxicillin against many beta-lactamase-producing strains. The MIC50 (MIC for 50% of strains tested) of amoxicillin for 412 beta-lactamase-producing members of the family Enterobacteriaceae fell from greater than 128 to 2 micrograms/ml in the presence of 1 microgram of BRL 42715 per ml, whereas 5 micrograms of clavulanic acid per ml brought the MIC50 down to 8 micrograms/ml. Among these 412 strains were 73 Citrobacter and Enterobacter strains, and 1 microgram of BRL 42715 per ml reduced the MIC50 of amoxicillin from greater than 128 to 2 micrograms/ml for the 48 cefotaxime-susceptible strains and from greater than 128 to 8 micrograms/ml for the 25 cefotaxime-resistant strains.

A normally asporogenous pleomorphic strain of Microsporum gypseum was induced to sporulate by controlled aeration and dehydration. Aeration of the pleomorphic strain under optimal cultivation conditions caused the initiation of a sporulation cycle with equivalent growth parameters and percentage intracellular water loss as the wild-type strain. Initiation of sporulation was not due to alteration of the medium's nutrient concentration or consistency, concentration of fungal growth by-products, or removal of volatile “staling factors.” Macroconidia formed by the pleomorphic colonies were of characteristic wildtype morphology, but germinated to form typical pleomorphic colonies, indicating that the induced sporulation was strictly phenotypic and reversible. Other asporogenous pleomorphic strains from different dermatophyte genera also were induced to form macroconidia by aeration, suggesting a similarity in sporulation induction in Microsporum sp., Epidermophyton floccosum , and Trichophyton violaceum . Initiation of sporulation by aeration further suggested that the pleomorphic mutation was one which affected the sensitivity of the pleomorphic aerial hyphae to natural sporulation inducers (i.e., decreased humidity) and did not represent a loss in the ability to form fertile macroconidia.

The major outer membrane protein was extracted from Campylobacter coli by Triton X-100/EDTA fractionation of cell envelopes. This heat-modifiable protein was shown to have pore-forming activity in black lipid bilayers. The C. coli porin formed a relatively small cation-selective pore with a mean single-channel conductance of 0.53 +/- 0.16 nS in 1.0 M KCl. There was no evidence of oligomer formation, which suggested that each protein monomer formed a pore. Pore-forming activity of the C. coli porin and similarly prepared Campylobacter jejuni porin was also measured in liposome-swelling assays. These results confirmed the cation selectivity of both pores. The C. coli porin formed a small pore, which hindered the penetration of solutes with a molecular weight of 262, and a larger pore, which hindered the penetration of solutes with a molecular weight of 340, in a protein-concentration-dependent manner. C. jejuni formed one size of pore that was slightly larger than the C. coli pore and just permitted the passage of solutes, with a molecular weight of 340. A review of the literature concerning in vitro screening of antimicrobial agents tended to confirm the low permeability of the C. jejuni outer membrane to hydrophilic antimicrobial agents except when the molecules had molecular weights of less than 360. The porins of C. jejuni and C. coli may contribute to intrinsic resistance to antimicrobial agents, whereas alternative (nonporin) routes of antimicrobial agent uptake may be more important determinants of susceptibility to antimicrobial agents.

Ethylenediamine-soluble glycoproteins were extracted from isolatedMicrosporum gypseumhyphal walls during sporulation and from spore coats before and after germination. This study was carried out to identify a sporulation-specific cell wall protein that possibly served as a substrate for the alkaline protease which initiated the macroconidial germination of this fungus. Analyses revealed that water-insoluble glycoprotein accounted for 10% of the ungerminated spore coat but only for 4 to 5% of the mycelial wall dry weight. This fraction was modified in its amino acid composition during sporulation, and it decreased in protein content during spore germination. Water-soluble glycoprotein, which accounted for approximately 3 to 3.5% of either the spore coat or mycelial wall dry weight, was of similar amino acid composition from both sources and did not decrease in protein content upon spore germination. The water-insoluble glycoprotein was found to be rich in leucine, aspartic acid, glycine, glutamic acid, and phenylalanine residues. The water-soluble glycoprotein was rich in proline, threonine, glycine, serine, glutamic acid, and alanine.

Inorganic phosphate was found to decrease the caseinolytic and ethyl-esterase activities of theMicrosporum gypseumgermination protease. The germination protease possessed exokeratinase (beta-keratinase) activity immediately after release from the fungal spore. After phosphate treatment of the enzyme, the germination protease also possessed endo-keratinase (alpha-keratinase) activity. Phosphate altered the protease's pH optimum from 9.0 to 7.0 and decreased the molecular weight from 33,000 to 16,000. These values were identical to those found for the keratinase. Alpha- and beta-keratinase activities were stimulated in excess of 200-fold by disulfide reducing agents. Natural and suspected keratin degradation products also enhanced keratinase activity. Cell fractionation and in vitro conversion of the alkaline germination protease into a functional keratinase suggested that the subunits comprising the germination protease and the keratinase were of a common origin.

Three types of lysosomes containing either acid protease, alkaline protease, or phosphodiesterase were isolated from a Microsporum gypseum macroconidial homogenate on Ficoll gradients. The acid protease was contained in an assimilative lysosome since its activity was affected by the complexity of the exogenous nitrogen source. Ultracentrifugation and electron microscopy revealed that the alkaline protease-containing vesicles were associated with the spore coat material prior to macroconidial germination. During macroconidial germination, zones of spore coat hydrolysis were seen surrounding these vesicles. Other larger vesicles, believed to contain the phosphodiesterase, were also observed in the spore coat during macroconidial germination.

Germination ofMicrosporum gypseummacroconidia was accompanied by the release of alkaline protease, calcium ions, and inorganic phosphate into the germination fluid. The rate of germination was greatest during the first 2 hr, decreasing thereafter. This decrease in rate was accompanied by a decrease in protease activity, which was caused by an interaction of the enzyme with the inorganic phosphate released from the spores and accumulated in the germination medium after 2 hr. Germination of high spore densities was regulated by the ratio of released phosphate to protease protein, resulting in a constant percentage of germination at both high and low spore densities. A germination-defective mutant strain failed to germinate normally and released excessively high concentrations of phosphate into the germination medium during the initial 2 hr of incubation. Addition of calcium ions to germination mutant macroconidia stabilized spore morphology, prevented protease inactivation, and allowed normal germ-tube outgrowth. The germination of macroconidia appears to be regulated by the release of phosphate ions, which then inhibit the alkaline protease.