RESUMO
Bacterial cells communicate with one another using chemical signaling molecules. This phenomenon is termed quorum sensing (QS). QS in Klebsiella pneumoniae is mediated by the synthesis of interspecies autoinducer 2 (AI-2), a furanosyl borate diester molecule. The response of Type 2 QS to environmental cues such as carbon sources, the initial pH of the medium, and boracic acid was investigated in the present study using a Vibrio harveyi BB170 reporter assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The results show that glucose can affect AI-2 synthesis to the greatest extent, and 3.0% glucose can stimulate K. pneumoniae to produce more AI-2, with a four times increase in activity compared with that of the control culture. According to our previous research, Type 2 QS in K. pneumoniae is luxS dependent. Therefore, the close relationship between glucose concentration and luxS transcription level was confirmed with qRT-PCR technology. The results show that the response of QS to a fluctuating glucose concentration was observed as a change in the amount of luxS RNA transcripts. A maximum of luxS transcription appeared during the exponential growth phase when the glucose concentration was 30.0 g/L. At the same time, AI-2 production was also slightly impacted by the low initial pH. It is noteworthy that the addition of boracic acid at microdosage (0.1 g/L) can also induce AI-2 synthesis. Presumably, in K. pneumoniae, the 4,5-dihydroxy-2,3-pentanedione cyclizes by the addition of borate and loss of water, is hydrated, and is converted to the final AI-2 signaling molecule.
Assuntos
Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/metabolismo , Meios de Cultura/química , Meio Ambiente , Regulação Bacteriana da Expressão Gênica/fisiologia , Homosserina/análogos & derivados , Klebsiella pneumoniae/fisiologia , Percepção de Quorum/fisiologia , Ácidos Bóricos/análise , Primers do DNA/genética , Glucose/metabolismo , Homosserina/biossíntese , Concentração de Íons de Hidrogênio , Klebsiella pneumoniae/metabolismo , Lactonas , Pentanos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VibrioRESUMO
Bacterial cells communicate with one another using chemical signaling molecules. The phenomenon is termed quorum sensing. The quorum sensing bacterium Klebsiella pneumoniae secretes a non-homoserine lactone autoinducer in the exponential phase of growth as detected by a Vibrio harveyi reporter assay for autoinducer 2 (AI-2). To further investigate regulation of AI-2 production in K. pneumoniae, the pfs and luxS promoter fusions to an operon luxCDABE reporter were constructed in a low copy number vector, which is derived from pBR322 and pET28a((+)) and allows an examination of transcription of the genes in the pathway for signal synthesis. In this study, comparisons were performed on the cell densities of wild-type and recombinant K. pneumoniae, on the transcription activity of pfs and luxS promoters, and on the synthesis of AI-2 as a function of culture time. The results show that luxS expression is constitutive and the transcription of luxS is tightly correlated to AI-2 production in K. pneumoniae because the peaks of AI-2 production and transcriptional level of luxS appear at the same time point. The close relation of the profiles of luxS transcription and AI-2 production was also confirmed with quantitative reverse transcription-PCR technology. These facts support the idea that the quorum sensing in K. pneumoniae is luxS dependent.