RESUMO
A polysaccharide was isolated from the opportunistic human pathogen Providencia alcalifaciens O45:H26 by extraction with aqueous phenol and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional ROESY and H-detected (1)H,(13)C HSQC experiments. The polysaccharide contains N-acetylglucosamine and N-acetylmuramic acid (D-GlcpNAc3Rlac) amidated with L-alanine and has the following structure: â4)-ß-D-GlcpNAc-(1â4)-ß-D-GlcpNAc3(Rlac-L-Ala)-(1â. The polysaccharide possesses a remarkable structural similarity to the bacterial cell wall peptidoglycan. It is not unique to the strain studied but is common to strains of at least four P. alcalifaciens O-serogroups (O3, O24, O38, and O45). No evidence was obtained that the polysaccharide is associated with the LPS, and hence it might represent a bacterial capsule component.
Assuntos
Cápsulas Bacterianas/química , Antígenos O/química , Peptidoglicano/química , Providencia/imunologia , Acetilglucosamina/análise , Alanina/análise , Sequência de Carboidratos , Parede Celular/química , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Ácidos Murâmicos/análise , Peptidoglicano/isolamento & purificaçãoRESUMO
The HaeIV restriction endonuclease (ENase) belongs to a distinct class of ENases, characterized by its ability to cleave double-stranded DNA on both sides of its recognition sequence, excising a short DNA fragment that includes the recognition sequence. The gene encoding the HaeIV ENase was cloned from Haemophilus aegyptius into pUC19 using a previously described system that does not need the knowledge that a particular ENase is produced by a bacterial strain. DNA sequence analysis of the insert contained on this plasmid identified a single open reading frame (ORF), with the predicted protein having an apparent molecular mass of approximately 110 kDa. The protein encoded by this ORF was purified to homogeneity from Escherichia coli strain ER1944 carrying the haeIVRM gene on a recombinant plasmid under the control of the inducible ara promoter. The protein possessed both ENase and methyltransferase (MTase) activities. Amino acid sequence analysis was able to identify several conserved motifs found in DNA MTases, located in the middle of the protein. The enzyme recognizes the interrupted palindromic sequence 5' GAPyNNNNNPuTC 3', cleaving double-stranded DNA on both strands upstream and downstream of the recognition sequence, releasing an approximately 33 bp fragment. The ENase possessed an absolute requirement only for Mg(+2). ATP had no influence on ENase or MTase activities. The ENase made the first strand cleavage randomly on either side of the recognition sequence, but the second cleavage occurred more slowly. The MTase activity modified symmetrically located adenine residues on both strands within the recognition sequence yielding N6-methyl adenine. Furthermore, the MTase was active as a dimer.
Assuntos
Enzimas de Restrição do DNA/metabolismo , Haemophilus/enzimologia , Peptídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sequência Conservada/genética , DNA/química , DNA/genética , DNA/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/isolamento & purificação , Metilases de Modificação do DNA/metabolismo , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/isolamento & purificação , Dimerização , Genes Bacterianos/genética , Haemophilus/genética , Cinética , Magnésio/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The action of beta-lactam antibiotics such as ampicillin and benzylpenicillin on cells of Listeria monocytogenes appears to be bacteriostatic. However, after approximately two hours in the presence of 10 x MIC of benzylpenicillin the cells begin to rapidly lose viability without undergoing lysis. In this report we present the results of studies on the biosynthesis of murein in L. monocytogenes cells during the first 120 min of their exposure to benzylpenicillin as measured by the continuous and pulse incorporation of the precursor N-acetyl-D-[1-3H]glucosamine. The turnover of the murein sacculus in the presence of penicillin as well as the lack of discernible changes in the molecular structure of the murein synthesised in the presence of benzylpenicillin is also discussed.
Assuntos
Listeria monocytogenes/efeitos dos fármacos , Penicilina G/farmacologia , Penicilinas/farmacologia , Peptidoglicano/química , Peptidoglicano/metabolismo , Acetilglucosamina/metabolismo , Glucosamina/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Peptidoglicano/biossíntese , Trítio/metabolismoRESUMO
Physical maps constructed by the localization of the cleavage site of several restriction endonucleases have shown that the genomes of the Haemophilus bacteriophages S2 and HP1c1 exist in variant forms which differ in the molecular organization of the genomes. At least three regions of different organization of the bacteriophage chromosomes have been identified. The different types of molecular organization can be detected both in the DNA isolated from the mature phage particles and after integration of the phage DNA into the bacterial chromosome.
Assuntos
Bacteriófagos/genética , Genes Bacterianos , Genes Virais , Haemophilus influenzae/genética , Sequência de Bases , Cromossomos Bacterianos/fisiologia , Enzimas de Restrição do DNA , Variação Genética , Hibridização de Ácido NucleicoRESUMO
Haemophilus influenzae Rd9 lysogenic for temperate bacteriophage N3 was found to be virtually nontransformable and nontransfectable. This inhibition of transformation and transfection was due partly to the decreased capacity of competent lysogenic cells for irreversible binding of deoxyribonucleic acid (DNA) and partly to some events taking place after adsorption of the DNA. The unadsorbed DNA was not degraded by the competent lysogenic cells.
