Investigation of hydrogen bonds in proton transfer complexes derived from the reaction of 2- and 4-(N,N-dimethylamino)pyridines with chloranilic acid.

New complexes of 2-(N,N-dimethylamino)pyridine with chloranilic acid (2-DMAP + CLA) and 4-(N,N-dimethylamino)pyridine with chloranilic acid (4-DMAP + CLA) were synthesized and characterized by single crystal X-ray diffraction, infrared spectroscopy, thermal analysis methods and 1H, 13C and 15N NMR spectroscopy. The NMR spectroscopies were carried out in both, DMSO solution and in the solid state (CPMAS NMR). The 2-DMAP + CLA and 4-DMAP + CLA complexes crystallize in centrosymmetric P-1 and P21/c space group, respectively. In both complexes, the phenomenon of proton transfer is observed, which results in the formation of strong N+-H···O- hydrogen bonds. Thermal decompositions of 2-DMAP + CLA and 4-DMAP + CLA complexes were studied by thermogravimetric analysis. Temperature dependent IR spectra revealed that methyl groups of 4-DMAP + CLA perform fast stochastic reorientational motion at room temperature which is slowed on cooling while in 2-DMAP + CLA reonrientational motion of CH3 groups is much slower due to steric effects.

Positive effect of reduced aeration rate on secretion of alpha-amylase and neutral proteases during pressurised fermentation of thermophilic Bacillus caldolyticus.

The thermophilic microorganism Bacillus caldolyticus was incubated in laboratory scale stirred bioreactors under pressurised conditions at different aeration rates. Increased amounts of CO2/bicarbonate were solubilised under the chosen conditions. A reduction in aeration rate from 1 vvm to 0.1 vvm resulted in accumulation of CO2 and bicarbonate up to 126 mg l(-1) and 733 mg l(-1), respectively and also increased secretion of α-amylase and neutral proteases (increases of 123% and 52%, respectively). In this paper, the effect of reduced aeration rate on CO2/bicarbonate concentration and enzyme activities is presented. The selected fermentation conditions are closely related to those prevalent in large scale bioreactors and may offer the possibility of achieving high enzyme yields at reduced aeration costs on an industrial scale.

Solid-state NMR study of Schiff base derivatives of 2-hydroxynaphthaldehyde. Deuterium isotope effects on 15N chemical shifts in the solid state.

Schiff base derivatives of 2-hydroxynaphthylaldehyde were studied by means of 13C and 15N cross-polarization magic angle spinning NMR spectroscopy and deuterium isotope effects on 15N chemical shifts, deltaN(D), in the solid state. DeltaN(D) in the solid state provided evidenced for the presence of a dynamic proton transfer equilibrium in the solid state at the room temperature.

The 15N and 13C solid state NMR study of intramolecular hydrogen bond in some Schiff's bases.

A series of 11 Schiff's bases derived from substituted salicylaldehyde and aliphatic amines has been studied in the solid state by 15N and 13C cross-polarization magic angle spinning (CPMAS) nuclear magnetic resonance (NMR). 15N CPMAS is especially useful for investigation of the tautomerism in the compounds considered, owing to the large difference in the nitrogen chemical shifts of OH and NH tautomers. In the solid state, three of the compounds examined were shown by 15N NMR to exist as OH tautomeric forms, and the remaining eight as the corresponding NH forms. This was confirmed by 13C CPMAS. The results reported were compared with those obtained in CDCI3 solutions.

A multinuclear magnetic resonance study of intramolecular hydrogen bonding in some Schiff and Schiff-Mannich bases in solution and in the solid state.

Two Schiff bases, N,N'-bis(5-bromosalicylidene)-1,2-diaminoethane, BS, and 7-[(1-[5-bromo-2-hydroxyphenyl] methylidene)amino]-4-methylcoumarin, Sc, and two appropriate Schiff-Mannich bases, N,N'-bis[5-bromo-3-[(diethylamino)methyl]salicylidene]-1,2-diaminoethane, BSM, and 7-[(1-[5-bromo-3-[(diethylamino)methyl]-2-hydroxyphenyl] methylidene)amino]-4-methylcoumarin, SMc, capable of intramolecular hydrogen bonding have been investigated by multinuclear magnetic resonance methods in both solid and liquid phases. In all of the compounds under investigation tautomeric equilibrium involving an intramolecular hydrogen bond has been found. The Schiff-Mannich bases, which can form two different kinds of H bonds at room temperature, form relatively weak H bonds with the imino nitrogen atoms. At low temperatures the tautomeric proton exchange becomes slow on the NMR time scale and both hydrogen-bonded forms can be observed by 1H, 13C, and 15N NMR methods. In the solid state the tautomeric process is frozen and only one H-bonded form is present. On the basis of 13C and 15N CPMAS NMR spectra this is identified as the form with hydrogen bonds involving the imino groups. This conclusion is in good agreement with previous results obtained by X-ray diffraction methods. The investigated Schiff bases (BS and Sc) form relatively weak H bonds. The proton position in the hydrogen bridge, estimated from 15N and 13C chemical shifts, is very similar in both the solution and solid phases. In chloroform solution the observed tautomeric equilibria are almost insensitive to a temperature change within the range 223 to 303 K.

Identification and characterization of two entry exclusion genes of the promiscuous IncP plasmid R18.

Two entry exclusion genes (designated eexA and eexB) from the promiscuous IncP alpha plasmid R18 have been isolated by molecular cloning. They are located between coordinates 26.6-27.4 kb and 27.4-27.6 kb, respectively and are transcribed clockwise on the conventional R18 map. The product of the eexA gene has an apparent molecular mass of 28 kDa and its N-terminus contains a putative signal sequence for protein export. A recombinant plasmid containing R18 eex genes exerted Eex activity towards another promiscuous IncP alpha plasmid, R702, about 50 times more strongly than plasmid R18 itself. Analysis of the DNA sequence revealed no similarity to the eex genes of the F plasmid of Escherichia coli. R18 eexA includes a potential korB binding site and is followed by a potential transcription terminator. A Tn7 insertion at coordinate 20.0 kb of R18 resulted in a host range mutant pM01185, which leads to loss of Eex activity and of conjugative transfer of the plasmid into some bacterial species.

Identification of a seventh operon on plasmid RK2 regulated by the korA gene product.

Broad-host-range IncP plasmids possess a series of operons involved in plasmid maintenance, whose expression is coordinated by a series of regulators, most of which are encoded in a central regulatory operon. The nucleotide sequence of a new monocistronic operon located between coordinates 55.0 and 56.0 kb on the genome of the IncP alpha plasmids RK2 and RP4 is presented. The operon encodes a 34 kDa protein which has a net negative charge. Transcription of the operon, designated by us kfrA (korF-regulated), is repressed not only by the product of the previously described korA gene but also by the product of a gene which we have designated korF and which has not been described previously. The korF gene is encoded downstream from korB within the key korA/korB regulatory operon. We propose that K or F binds to a novel inverted repeat overlapping the promoter for the kfrA operon.

Genetic analysis of insertion mutations of the promiscuous IncP-1 plasmid R18 mapping near oriT which affect its host range.

Transposon Tn7 insertion mutations of the promiscuous IncP-1 plasmid R18 which affect its conjugational transmissibility from Pseudomonas aeruginosa to Escherichia coli C, a strain of E. coli K12, Salmonella typhimurium and P. maltophilia have been mapped physically. They map to coordinate 53.5 kb in the Tral region of the plasmid. An 800-bp fragment mapping between R18 coordinates 52.85 and 53.65 kb, which complemented the host range defect of the mutants when tested with E. coli C as recipient, has been identified. However, complementation occurred only when the 800-bp cloned fragment was provided in the E. coli C recipient but not when situated in the P. aeruginosa donor. It is concluded that a trans-acting gene product of R18 is required, in the transcipient, for conjugative DNA metabolism during, or immediately following, the conjugational transfer of this plasmid between certain donor and recipient hosts.

[Bacterial penicillin-binding proteins as specific targets of beta-lactam-antibiotics and as factors of resistance to antibiotics (author's transl)]. / Penicillinbindeproteine der Bakterien als spezifische Wirkorte der beta-Laktam-Antibiotika und als Faktoren der Antibiotikaresistenz.

Bacteria contain several isofunctional, beta-lactam sensitive membrane enzymes engaged in the synthesis of cell wall peptidoglycan (peptidoglycan-DD-carboxypeptidases, -transpeptidases, -endopeptidases) as members of sets of even more numerous membrane proteins with specific, high binding-affinity for beta-lactam antibiotics (penicillin-binding proteins, PBPs). Effective inhibition of bacterial growth by beta-lactam antibiotics requires simultaneous inactivation of the essential functions of several PBPs by formation of stable enzyme-antibiotic complexes. Failure to achieve permanent inactivation of all essential targets by a given beta-lactam appears to be another cause of bacterial beta-lactam resistance, in addition to known resistance mechanisms based on action of beta-lactamases and on screening off targets from antibiotic by a penetration barrier. Different groups of beta-lactam antibiotics vary characteristically in their affinity for specific essential PBPs. Combined application of two beta-lactams which complement each other in the inactivation to essential targets is a possibility to overcome resistance of single antibiotics.

Differentiation of mycoplasmatales from bacterial protoplast L-forms by assay for penicillin binding proteins.

Membrane proteins with the specific ability for binding penicillin with high affinity (penicillin binding proteins) were found to be present in two strains of the cell wall-less protoplast L-form of P. Mirabilis and were absent from different species of Mycoplasma and from Acholeplasma laidlawii. Thus, the assay for penicillin binding proteins appeared to be suitable for the differentiation of the cell wall-less procaryotes. The absence of penicillin binding proteins from the mycoplasmatales further confirmed the unrelatedness of this group to the bacteria.

Purification of two DD-carboxypeptidases/transpeptidases with different penicillin sensitivities from Proteus mirabilis.

Two membrane-bound enzymes of Proteus mirabilis with the dual functions of peptidoglycan DD-carboxypeptidase and transpeptidase (named DD-carboxypeptidase/transpeptidase H and L) were isolated and purified by selective solubilization with the nonionic detergent Genapol X-100, affinity chromatography on matrix-bound ampicillin, and preparative isoelectric focusing in the presence of detergent. Purified enzymes H and L were, respectively, penicillin-binding proteins 4 and 5 among seven major penicillin-binding proteins present in P. mirabilis. The enzymes differed in the following properties. Enzyme H had an Mr of 49,000; isoelectric point at pH 8.2; high sensitivity to benzylpenicillin and permanent inactivation because of high stability of the enzyme-antibiotic complex EI* (half-life 300 min); fragmentation of benzylpenicillin with formation of phenylacetylglycine during the slow decay of EI*; it functioned as an endopeptidase on peptide-crosslinked side chains of peptidoglycan. Enzyme L had an Mr of 43 000; isoelectric point at pH 5.9; low sensitivity to benzylpenicillin and low stability of EI* (half-life 7.2 min) with rapid recovery of enzyme activity; no function as an endopeptidase. The properties of enzyme L were identical with those of the single active DD-carboxypeptidase found previously in the spheroplast L-form of P. mirabilis grown in the presence of benzylpenicillin. We conclude that the partial penicillin resistance of P. mirabilis, with growth as L-form and synthesis of peptide-crosslinked peptidoglycan, depends on the continuing fuction of enzyme L as a DD-carboxypeptidase and transpeptidase in the presence of the antibiotic.

Interaction between penicillin and the DD-carboxypeptidase of the unstable L-form of Proteus mirabilis strain 19.

Binding of penicillin to the DD-carboxypeptidase of the unstable spheroplast L-form of Proteus mirabilis results in the rapid formation of a modified enzyme-inhibitor complex which in turn undergoes rapid decay into reactivated enzyme and an antibiotically inactive penicillin degradation product. Major antibiotic metabolites recovered from such interactions were benzylpenicilloic acid and phenoxymethylpenicilloic acid from benzylpenicillin and phenoxymethylpenicillin, respectively, suggesting a second enzymic function of the DD-carboxypeptidase as a penicillinase of low efficiency. Statistical analyses made with the help of a linear regression program show that the enzyme interacts with the substrate UDP-N-acetylmuramoyl-L-alanyl-D-gamma-glutamyl-(L)-meso-2,6-diaminopimelyl-(L)-D-alanyl-D-alanine and either benzympenicillin or carbenicillin in a non-competitive manner.

Purification of the membrane-bound DD-carboxypeptidase of the unstable spheroplast L-form of Proteus mirabilis by affinity chromatography. Non-competitive inhibition of the enzyme by penicillins and low stability of the enzyme-inhibitor complex.

Membrane-bound DD-carboxypeptidase of the unstable L-form of Proteus mirabilis was solubilized by the non-ionic detergent Genapol X-100 and purified to protein homogeneity by affinity chromatography on ampicillin bound to succinyl-aminododecyl-cellulose. The purified enzyme with a molecular weight of 43000 is inhibited non-competitively by penicillin G and carbenicillin, indicating a function of the penicillins as allosteric inhibitors. Sensitivity of the enzyme to penicillins is only moderate with a Ki of 1 muM for penicillin G. Breakdown of.the enzyme-inhibitor complex EI with different penicillins occurs rapidly with reappearance of active DD-carboxypeptidase. The half-life of EI with penicillin G is 5.5 min at 30 degrees C and 3.5 min at 37 degrees C, 10--1000-fold shorter than EI half-lives of DD-carboxypeptidases in several other bacteria. The low stability of the enzyme-inhibitor complex and the moderate penicillin sensitivity appear to be the basis for the continued activity of DD-carboxypeptidase during growth of the L-form and synthesis of peptidoglycan in the presence of high concentrations of penicillin.