György Barabás

N-Alkane uptake and utilisation by Streptomyces strains.

Streptomyces strains isolated from the Kuwait Burgan oil field were defined as S. griseoflavus, S. parvus, and S. plicatus utilised n-hexadecane, n-octadecane (purified fractions of mineral oil), kerosene, and crude oil as sole carbon and energy sources. The strains were incubated with n-alkanes and increase of the fatty acid content with chain length equivalent to the employed n-alkanes was observed. Signal transducing GTP-binding proteins (GBPs) play an important role in n-alkane uptake in streptomycetes. Specific activators of GBPs increased the uptake of hydrocarbons. Using the hydrophobic fluorescent dye diphenylhexatrien (DPH) as a probe, it was found that the microviscosity of the hydrophobic inner region of the cellular membrane is significantly lower in hydrocarbon utilisers than in non-utilisers. This difference probably reflects differences in the fatty acid composition of the strains. When cultures were grown in n-alkane containing media, electron microscopy revealed that the hydrocarbon utilisers showed less-electron dense areas as inclusions in the cytoplasm. Soil samples inoculated with Streptomyces strains eliminated hydrocarbons much faster than those not containing these strains, serving as control. When inorganic medium was supplied with n-hexadecane-1-14C as sole carbon and energy source, radioactive CO2 was detected. Since streptomycetes have not been used until now for oil elimination, though they are known as abundant soil bacteria tolerating extreme conditions, their possible use for bioremediation of hydrocarbon contaminated soils is discussed.

Age-dependent changes in transmembrane signalling: Identification of G proteins in human lymphocytes and polymorphonuclear leukocytes

In human neutrophils (PMNLs) we found that in the elderly IP3 formation was significantly decreased compared to that of young subjects. For FMLP receptor binding affinity and number no measurable differences occurred upon ageing, studying both the low or the high affinity receptors. The amount of ADP-ribosylated G proteins, catalysed by pertussis toxin (PT) or cholera toxin (CT), was significantly increased in PMNLs of the elderly. In lymphocytes, the PT-catalysed ADP ribosylation of G proteins was also increased with ageing, while the CT-catalysed ribosylation was decreased. The autoradiogram of [32P]ADP-ribosylated proteins by CT in lymphocytes of young individuals showed a major polypeptide of 40,000 M(r). In contrast, in lymphocytes of the elderly, the major polypeptide was 45,000 M(r). In PMNLs, CT labelled quite strongly the 45,000 M(r) band, mainly in the elderly. When PT was used, no age-related pattern changes could be demonstrated, while differences could be observed between the two types of cells. The use of antiserum P680 (G alpha common) showed no age-related pattern changes, while the intensity of the labelled proteins varies with age and cell type. The antiserum U46 (Go alpha) could identify in lymphocytes of young subjects two polypeptides 68,000 and 41,000 M(r). The prominent polypeptide in lymphocytes of the elderly was the 70,000 M(r) and no other polypeptides could be recognized. In PMNLs of young subjects the U46 and serum identified a range of species. In PMNLs of the elderly all these bands were weakly labelled. The present data indicate changes in the pattern and the quantity of G proteins in lymphocytes and PMNLs of elderly subjects.

The possible role of ADP-ribosylation in sporulation and streptomycin production by Streptomyces griseus

Mutants resistant to 3-aminobenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained from Streptomyces griseus IFO 13189, a streptomycin-producing strain. One (strain no. 4), which had significantly reduced ADP-ribosyltransferase activity, was analysed in detail. Mutant 4 displayed a conditional phenotype with respect to cultivation temperature. At 30 degrees C, it exhibited severely reduced ability to produce aerial mycelium (on solid medium) and submerged spores and streptomycin (in liquid culture), but this ability was fully restored at 25 degrees C. The mutant produced A-factor normally, regardless of cultivation temperature, and exhibited normal ability to accumulate ppGpp intracellularly. SDS-PAGE analyses of cellular proteins labelled by [32P]NAD revealed that an ADP-ribosylated protein with a molecular size of 44 kDa, which appeared in sporulating cultures of the parent strain, was missing from the mutant grown at the non-permissive temperature (30 degrees C). Genetic analysis showed that the aba mutation conferring resistance to 3-aminobenzamide was tightly linked to the altered phenotype. Failure to ADP-ribosylate certain cellular protein(s), presumably due to the aba mutation, may be responsible for impaired differentiation in this mutant.

Investigation of β-lactamases in clinical isolates of Staphylococcus aureus for further explanation of borderline methicillin resistance

Background: Borderline methicillin resistance in Staphylococcus aureus is due to β-lactamase overproduction and/or specific methicillinases. Methods: β-Lactamase activity in culture supernatants and in cytoplasmic membrane fractions was estimated by bioassay and by SDS-PAGE combined with nitrocefin assay. Results: During the investigation of borderline methicillin-resistant Staphylococcus aureus (BORSA) strains VU94 and 822 two β-lactamases were detected in the membranes, with molecular weights of 13 and 30 kDa. The latter could be found in the culture supernatants, too. In the presence of globomycin, this enzyme disappeared from the membrane, and the oxacillin-hydrolyzing activity of the membrane decreased to the level of susceptible strains. Both β-lactamases were detected in the methicillin-resistant Staphylococcus aureus strain studied, but the susceptible strains possessed only the first enzyme. Conclusions: The 30-kDa β-lactamase proved to be a methicillinase, and it can be one of the main causes of the borderline phenotype of BORSA strains. The other enzyme is one of the smallest β-lactamases published to date.

Investigation of Oxacillin-Hydrolyzing Beta-Lactamase in Borderline Methicillin-Resistant Clinical Isolates of Staphylococcus aureus

Background: Mechanisms of borderline resistance of Staphylococcus aureus to penicillinase-resistant penicillins (PRPs) may include hyperproduction of classical penicillinase and/or production of β-lactamase hydrolyzing also PRPs. Methods: β-Lactamase activity of whole cells and purified enzymes was estimated spectrophotometrically and in isolated cytoplasmic membranes by bioassay with Bacillus subtilis as test strain. Results: Out of 53 clinical isolates of S. aureus, 18 showed oxacillin MIC values from 0.5 to 2 µg/ml, which were reduced by sulbactam and/or clavulanic acid in the case of four isolates producing large quantities of inducible, type A β-lactamase. Cytoplasmic membranes isolated from these strains showed oxacillin-hydrolyzing activity. One of these strains was grown also in the presence of globomycin, an antibiotic known to interfere with the anchorage of membrane lipoproteins; this treatment eliminated the oxacillin-hydrolyzing activity. Conclusions: The resistance in these strains was due to a membrane-bound lipoprotein with oxacillin-hydrolyzing activity.

The possible role of ADP ribosylation in physiological regulation of sporulation in Streptomyces griseus

The role of ADP ribosylation of proteins in the physiological regulation of sporulation in Streptomyces griseus was studied. We report here that both the activity of NAD+: arginine ADP-ribosyltransferase (ADPRT) and the pattern of ADP-ribosylated proteins showed characteristic changes during the life cycle in S. griseus 2682. Analysis off ADP-ribosylated proteins revealed that in a nonsporulating mutant of the parental wild-type (wt) strain (Bld7 mutant), both the activity of ADPRT and the pattern of ADP-ribosylated proteins were different from those of the parental strain. Addition of 3-aminobenzamide (3AB), the most potent inhibitor of ADPRT, inhibited sporulation of S. griseus 2682 and the A-factor (AF)-induced sporulation of S. griseus Bld7, but in both cases the inhibitory effect of 3AB was strictly age-dependent. Using [alpha-32P]GTP, we have demonstrated the presence of GTP-binding proteins in purified cell membranes of S. griseus 2682 and S. griseus Bld7. The same GTP-binding proteins were observed in Bld7 and the wt. AF stimulated the basal GTPase activity of cell membranes of S. griseus 2682 in a concentration-dependent manner, suggesting that GTP-binding proteins might be involved in the AF-induced sporulation process.

Evidence of a role for NAD+-glycohydrolase and ADP-ribosyltransferase in growth and differentiation of Streptomyces griseus NRRL B-2682: inhibition by m-aminophenylboronic acid

m-Aminophenylboronic acid (APBA) inhibited the germination, growth and sporulation of Streptomyces griseus NRRL B-2682 in an age- and concentration-dependent manner in submerged and solid cultures. When added to cells or cell extracts it irreversibly inhibited NAD+-glycohydrolase and ADP-ribosyltransferase activity. ADP-ribosyltransferase was more sensitive, but inhibition was not complete, even in the presence of 10 mM APBA. The in vivo effects of the inhibitor correlated with its in vitro effect on ADP-ribosylation and on the profile of ADP-ribosylated endogenous proteins. The physiological importance of ADP-ribosyltransferase was supported by the observation that APBA strongly inhibited the growth of a non-sporulating and NAD+- glycohydrolase-negative mutant of the parental strain. The resistance of S. griseus NRRL B-2682 strains able to grow in the presence of APBA was due to permeability factors. A comparison of the ADP-ribosylated protein profiles of S. griseus NRRL B-2682 grown under various conditions showed similarities, but also specific differences. The results suggest that the ADP-ribosyltransferase of S. griseus NRRL B-2682 is an indispensable enzyme for growth and differentiation of the strain. It may regulate the activity of key enzymes or developmental proteins by responding to intra- and extracellular conditions.

Analysis and identification of ADP-ribosylated proteins of Streptomyces coelicolor M145

Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD+ to acceptor proteins catalyzed by ADP-ribosyltransferases. Using m-aminophenylboronate affinity chromatography, 2D-gel electrophoresis, in-gel digestion and MALDI-TOF analysis we have identified eight in vitro ADP-ribosylated proteins in Streptomyces coelicolor, which can be classified into three categories: (i) secreted proteins; (ii) metabolic enzymes using NAD+/NADH or NADP+/NADPH as coenzymes; and (iii) other proteins. The secreted proteins could be classified into two functional categories: SCO2008 and SC05477 encode members of the family of periplasmic extracellular solute-binding proteins, and SCO6108 and SC01968 are secreted hydrolases. Dehydrogenases are encoded by SC04824 and SC04771. The other targets are GlnA (glutamine synthetase I., SC02198) and SpaA (starvation-sensing protein encoded by SC07629). SCO2008 protein and GlnA had been identified as ADP-ribosylated proteins in previous studies. With these results we provided experimental support for a previous suggestion that ADP-ribosylation may regulate membrane transport and localization of periplasmic proteins. Since ADP-ribosylation results in inactivation of the target protein, ADP-ribosylation of dehydrogenases might modulate crucial primary metabolic pathways in Streptomyces. Several of the proteins identified here could provide a strong connection between protein ADP-ribosylation and the regulation of morphological differentiation in S. coelicolor.

Membrane-bound and extracellular β-lactamase production with developmental regulation in Streptomyces griseus NRRL B-2682

A new type of beta-lactamase has been isolated and characterized in Streptomyces griseus NRRL B-2682. The enzyme has membrane-bound and extracellular forms. Biochemical characterization of some of the properties of the enzyme showed that it belongs to the class A group of penicillinases. Comparison of the membrane-bound and extracellular forms of the beta-lactamases suggests that they seem to be differently processed forms of the same enzyme. The N-terminal amino acid sequence of the extracellular form of the beta-lactamase showed a high degree of similarity to a D-aminopeptidase of another Streptomyces griseus strain. Secretion of the beta-lactamase was affected by the differentiation state of the strain since in spontaneous non-sporulating mutants only the membrane-bound form was present. In accordance with this when sporulation of the wild-type strain was inhibited it failed to secrete extracellular beta-lactamase. Addition of globomycin to the non-sporulating cells liberated the enzyme from the membrane, indicating that the protein is processed normally by signal peptidase II and a glyceride-thioether group, together with a fatty acid amide-linkage, is responsible for the attachment of the enzyme to the cellular membrane. Under sporulation-repressed conditions addition of peptidoglycan fragments and analogues or inhibition of cell wall biosynthesis by penicillin-G induced beta-lactamase secretion and also restored sporulation both in solid and submerged cultures. These results confirm that beta-lactamase secretion is tightly coupled to the sporulation process in S. griseus.

Effect of aminoglycoside antibiotics on the autolytic enzyme of Streptomyces griseus

The isolated cell wall of Streptomyces griseus 52–1 strain labelled with fluorescein isothiocyanate (FITC) and containing wall-bound autolytic enzyme was lysed as a function of different cations. The autolysis was accelerated by aminoglycoside antibiotics (streptomycin and the structurally closely related neomycin) which have a polycationic character. Since this strain is a streptomycin producer it is suggested that streptomycin may have a regulatory function on autolysis.