- Volume 117, Issue 1, 1980

Cyanide production by Chromobacterium violaceum growing on a l-glutamate/minimal salts medium was stimulated by addition of 2 mm-glycine and 05 mm-l-methionine to the medium. However, when the initial concentration of glycine was 06 mm, a lower level of cyanogenesis was obtained than that with glycine-free medium. Cystathionine, dl-methionine sulphoxide, d-methionine and S-adenosyl-l-homocysteine were effective substitutes for l-methionine, but a wide range of other compounds including betaine and choline were inactive. Cyanogenesis was stimulated when threonine was used as a replacement for glycine, but serine and a range of analogues of glycine were ineffective. In a minimal salts medium containing glucose as the carbon source, glycine or NH4Cl but not methionine could serve as the sole nitrogen source for growth. Growth on glucose plus NH4Cl in the presence of glycine and methionine resulted in greater yields of cyanide than growth on a similar medium in the absence of NH4Cl. In the latter case raising the initial concentration of glycine, to compensate for its utilization as the sole source of nitrogen, had little effect on cyanogenesis. Addition of cyclic AMP to both the NH4Cl-containing and NH4Cl-free media caused a decrease in cyanide formation.

Association patterns and regulatory properties of chorismate mutase, prephenate dehydratase and prephenate dehydrogenase from Pseudomonas aureofaciens ATCC 15926 were studied. Prephenate dehydrogenase (molecular weight 95000) was separated by Sephadex G-100 chromatography from both the chorismate mutase–prephenate dehydratase I complex (molecular weight 75000) and from a second, low molecular weight prephenate dehydratase (prephenate dehydratase II; molecular weight 30000). The chorismate mutase-prephenate dehydratase complex persisted after DEAE-Sephadex A-50 chromatography. With the exception of prephenate dehydratase II, enzyme activities were influenced by end-products. Chorismate mutase was competitively inhibited by l-phenylalanine (K i = 3·5 μ m). Prephenate dehydratase I was inhibited by l-phenylalanine (K i = 8 μ m) and activated by l-tyrosine (K a = 5 μ m). Prephenate dehydrogenase was feedback-inhibited by l-tyrosine. Substrate saturation curves of chorismate mutase and of prephenate dehydratase II were hyperbolic with K m values of 0·31 mm for chorismate and 0·015 mm for prephenate, respectively. The substrate saturation curve of the complexed prephenate dehydratase I was sigmoid; a K m value of 0·18 mm was calculated for prephenate. Chorismate mutase, prephenate dehydratase and prephenate dehydrogenase were not repressed by aromatic amino acids.

The activity in vitro of the bacitracin synthetase of Bacillus licheniformis ATCC 10716 is influenced by divalent metal ions (Mg2+, Mn2+, Fe2+ or Co2+) and by bacitracin. It is possible that complexes between bacitracin and metal ions exert feedback control on the synthetase. An overall control mechanism for bacitracin synthetase may consist of substrate-metal ion complexes and product-metal ion complexes.

The localization of cellulase components in a Pseudomonas sp. isolated from activated sludge was studied. Endoglucanases were found in the culture media during growth on cellulose but not on cellobiose. Gel filtration and electrophoresis indicated that the extracellular enzyme contained two components, EI and EII. During growth on both substrates intracellular endoglucanases and aryl-β-glucosidases were formed. Late-exponential phase organisms from media containing cellulose or cellobiose were subjected to osmotic shock treatment or to spheroplast formation. Osmotic shock fluid contained endoglucanases and aryl-β-glucosidases. Spheroplast formation also released these enzymes. The glucosidases could be separated into aryl-β-glucosidase I and aryl-β-glucosidase II by ion-exchange chromatography. Endoglucanase was located differently on the cell surface depending on whether the growth substrate was cellulose or cellobiose. The cytoplasm contained another endoglucanase and aryl-β-glucosidase I. None of the enzymes hydrolysed avicel, but all except aryl-β-glucosidase III hydrolysed carboxymethylcellulose. All aryl-β-glucosidases, but none of the endoglucanases, hydrolysed cellobiose.

A poly(l,4-α-d-galacturonide) lyase (EC 4.2.2.2) from the culture fluid of Lachnospira multiparus was purified about 20-fold. The optimum pH and temperature for enzyme activity were 8·0 and 40C. The enzyme required Ca2+ and was inhibited by EDTA; it preferred polygalacturonate as substrate, cleaving 1,4-α-glycosidic linkages randomly to form unsaturated galacturonates, mainly the unsaturated digalacturonate. Some properties of the crude and purified enzyme preparations are described. An exopolygalacturonase is also produced by this organism.

Four of the five penicillin-binding proteins in the cytoplasmic membranes of Bacillus megaterium have been purified to protein homogeneity. The method used involved the solubilization of the penicillin-binding proteins from the membranes by treatment with non-ionic detergent, followed by partial separation of the proteins by ion-exchange chromatography on DEAE-Sepharose CL-6B. Each protein was then purified to protein homogeneity by covalent affinity chromatography on ampicillinaffinose. The protein with the lowest molecular weight is a dd-carboxypeptidase. The other three proteins have previously been postulated to be peptidoglycan transpeptidases, endopeptidases or dd-carboxypeptidases in vivo, but it was not possible to demonstrate any of these activities with the purified proteins in various in vitro systems. Possible reasons for the observed lack of enzymic activity in vitro are discussed.

Production of sheathed flagella by Pseudomonas stizolobii strain 0268A was markedly inhibited by growth at 34°C. Flagella produced during growth at 32°C, at which some flagellar inhibition was evident, possessed normal sheath and core components, but at 34°C, irregular ‘tubule’ appendages were sometimes produced in low numbers. Cells rendered aflagellate by growth at 34°C could regenerate normal sheathed flagella at 28°C within half a mean generation time after shift-down, and such regeneration was inhibited by chloramphenicol. The use of the sheathed flagellum of P.stizolobii as an experimental model for studies of biosynthesis and self-assembly in complex organelles is suggested.

The genetic basis for spontaneous resistance to tetracyline (Tet) and penicillin (Pen) in Neisseria gonorrhoeae was investigated. Tet and pen are polygenes which confer small but distinct levels of resistance to Tet and Pen, respectively. Mtr is a multiple-drug resistance polygene which increases resistance to Tet and Pen (as well as to other unrelated antibiotics). Tem is a modifier gene affecting resistance to Tet and Pen. Pem is a modifier gene for Pen resistance.

The following gene combinations code for resistance to five antibiotics: tet, mtr and tem for Tet; pen, mtr, pem and tem for Pen: tet, tem and mtr for doxycycline; pen and pem for ampicillin; pen, pem and mtr for nafcillin.

The genetic map of plasmid RP4 was correlated with its adenine + thymine (AT) map. For this purpose, RP4 DNA was digested with one or both of the restriction enzymes EcoRI and HindIII and the resulting linear RP4 molecules and fragments were partially denatured, examined in the electron microscope and their AT maps were determined using a computer program. From these AT maps the EcoRI and HindIII restriction sites were located on the AT map of RP4. Since the positions of these restriction sites on the genetic map of RP4 are known, the two maps could be compared. They revealed a high AT content for the Tn1 transposon and the kanamycin resistance gene. The tra-1 region is also distinguished by a sharply defined AT-rich region, whereas tra-2 and the tetracycline resistance gene have an AT content which is not distinctly different from the average AT content of RP4.

Escherichia coli ML308 225 was killed and lysed by human serum. Bacterial sensitivity to serum was maximal for organisms in the early-exponential phase of batch culture and declined as growth proceeded. Bacterial killing was dependent upon the complete complement sequence and the subsequent action of serum lysozyme caused bacteriolysis. Purified lipopolysaccharide from this organism bound to serum-sensitive bacteria and inhibited the bactericidal reaction.

The lipid content and composition of four strains of Paracoccidioides brasiliensis were analysed to determine any possible correlation with their virulence for hamsters and mice. Two strains, Pb168 and Pb141, were equal in virulence, Pb9 was slightly virulent and Pb140 was avirulent under the experimental conditions. No correlation was observed between virulence and the total lipid or phospholipid content of the strains. The lipid yield was highest in Pb9 and lowest in Pb168. Polar lipids were highest in Pb9 and least in Pb140. Phosphatidylcholine was the dominant phospholipid in all strains but its percentage was lower in the avirulent strain Pb140. Diphosphatidylglycerol, the least saturated lipid in all strains, was less abundant in Pb140 than in the virulent strains Pb168 and Pb141. In all four strains, neutral lipids constituted the major fraction of total lipids and triglycerides were the predominant individual lipid class, being more abundant in the avirulent and slightly virulent strains than in the virulent strains. The fatty acid profiles of total lipids and individual lipid classes of neutral and polar lipids obtained from the four strains were similar; however, the individual lipid classes showed patterns of preferential distribution of these fatty acids.

Methyl glyoxal inhibits the growth of Escherichia coli in synchronous and asynchronous culture. The inhibition of growth is accompanied by immediate inhibition of protein synthesis and of the initiation of replication of DNA. When methyl glyoxal is added after initiation of a round of replication the elongation of new DNA chains is not inhibited. Cell division is inhibited if methyl glyoxal is added up to about 22 min prior to division. These results support the view that the primary effect of methyl glyoxal is on protein synthesis.

The rate of biosynthesis of nitrogenase polypeptides in Klebsiella pneumoniae was determined in a medium containing NaNO3 or NaNO2. Nitrogenase biosynthesis was completely repressed by NO3 − in a mutant strain, strain SK-25, that is derepressed for nitrogenase biosynthesis in the presence of NH4 +. Chlorate-resistant mutants, derived from strain SK-25, that are defective in NO3 − respiration produced nitrogenase in the presence of NO3 −. Strain SK-561, a chlorate-resistant derivative capable of NO3 − respiration, produced no nitrogenase in the presence of NO3 − or NO2 −. Klebsiella pneumoniae respired under anaerobic conditions utilizing either NO3 − or NO2 − as terminal electron acceptor. A mechanism for the control of nitrogenase biosynthesis is discussed involving the redox control of anaerobic enzyme systems.