RESUMO
The present study examined the potential of intact-cell matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid identification of Burkholderia cepacia complex (Bcc) bacteria using an Applied Biosystems 4700 Proteomics Analyser. Two software packages were used to analyse mass profiles based on densitometric curves and peak positions. The 75 strains examined, represented the nine established Bcc species and some commonly misidentified species, closely related or biochemically similar to Bcc and relevant in the context of cystic fibrosis microbiology. All Bcc strains clustered together, separated from non-Bcc strains. Within Bcc, most Bcc strains grouped in species specific clusters, except for Burkholderia anthina and Burkholderia pyrrocinia strains which constituted a single cluster. The present study demonstrates that MALDI-TOF MS is a powerful approach for the rapid identification of Bcc bacteria.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Complexo Burkholderia cepacia/classificação , Complexo Burkholderia cepacia/citologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Técnicas de Tipagem Bacteriana/instrumentação , Infecções por Burkholderia/microbiologia , Complexo Burkholderia cepacia/genética , Complexo Burkholderia cepacia/isolamento & purificação , Fibrose Cística/microbiologia , Microbiologia Ambiental , Humanos , Proteômica/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Fatores de TempoRESUMO
delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) catalyses, via the protein thiotemplate mechanism, the nonribosomal biosynthesis of the penicillin and cephalosporin precursor tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV). The complete and fully saturated biosynthetic system approaches maximum rate of product generation with increasing ATP concentration. Nonproductive adenylation of ACVS, monitored utilising the ATP-[32P]PP(i) exchange reaction, has revealed substrate inhibition with ATP. The kinetic inhibition pattern provides evidence for the existence of a second nucleotide-binding site with possible implication in the regulatory mechanism. Under suboptimal reaction conditions, in the presence of MgATP(2-), L-Cys and inorganic pyrophosphatase, ACVS forms adenosine(5')tetraphospho(5')adenosine (Ap(4)A) from the reverse reaction of adenylate formation involving a second ATP molecule. The potential location of the second ATP binding site was deduced from sequence comparisons and molecular visualisation in conjunction to data obtained from biochemical analysis.
Assuntos
Trifosfato de Adenosina/metabolismo , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Acremonium/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cefalosporinas/biossíntese , Cinética , Modelos Moleculares , Oligopeptídeos/química , Penicilinas/biossíntese , Conformação ProteicaRESUMO
A previous phylogenetic study on type strains of the genus Micromonospora and Micromonospora species bearing non-validly published names has pointed towards the species status of several of latter strains. Subsequent studies on morphological, cultural, chemotaxonomic, metabolic, and genomic properties, and on whole cell mass spectrometric analyses by matrix adsorbed laser desorption/ionization time-of-flight (MALDI-TOF) confirmed the species status, leading to the proposal of eight new Micromonospora species: Micromonospora citrea sp. nov., type strain DSM 43903T, Micromonospora echinaurantiaca sp. nov., type strain DSM 43904T, Micromonospora echinofusca sp. nov., type strain DSM 43913T, Micromonospora fulviviridis sp. nov., type strain DSM 43906T, Micromonospora inyonensis sp. nov., type strain DSM 46123T, Micromonospora peucetia sp. nov., type strain DSM 43363T, Micromonospora sagamiensis sp. nov., type strain DSM 43912T and Micromonospora viridifaciens sp. nov., type strain DSM 43909T.