Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional and metabolomic consequences of luxS inactivation reveal a metabolic rather than quorum sensing role for LuxS in Lactobacillus reuteri 100-23

ABSTRACT: Autoinducer-2 (AI-2)-mediated quorum sensing has been extensively studied in relation to the regulation of microbial behaviour. There are however two potential roles for the AI-2 synthase (LuxS). The first is in the production of AI-2 and the second is as an enzyme in the activated methyl cycle where it catalyses the conversion of S-ribosylhomocysteine to homocysteine. The by-product of the reaction catalysed by LuxS is (S)-4,5-dihydroxy-2,3-pentanedione (DPD), which spontaneously forms the furanones known collectively as AI-2. The mammalian gastrointestinal tract contains a complex collection of bacterial species so a method of interspecies communication might influence community structure and function. Lactobacillus reuteri 100-23 is an autochthonous inhabitant of the rodent forestomach where it adheres to the non-secretory epithelium of the forestomach forming a biofilm. Microarray comparisons of gene expression profiles of the L. reuteri 100-23 wild type and a luxS mutant under different culture conditions revealed altered transcription of genes encoding proteins involved in cysteine biosynthesis, the oxidative stress response and cell wall proteins. Metabolomic analysis revealed that the luxS mutation affected cellular levels of fermentation products, fatty acids and amino acids. Cell density-dependent changes (log phase versus stationary phase growth) in gene transcription were not detected. Overall, the results indicated that AI-2 was unlikely to be involved in gene regulation in L. reuteri 100-23 in a classical quorum-sensing type manner. Analysis of the microarray data was obtained from two or more independent biological replicates.

ORGANISM(S): Lactobacillus reuteri 100-23

SUBMITTER: Charlotte Wilson

PROVIDER: E-GEOD-32520 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In a transcriptome based trail, we figured out that the deletion of luxS has a massive influence to cell growth and metabolism of Streptococcus sanguinis SK36. The biofilm defective luxS deletion mutant was comlemented by transgenic sahH from Pseudomonas aeruginosa. Thus 209 of 216 influenced genes of the luxS mutant compared to the isogenic wildype were restored in their expression (fold change of n M-bM-^IM-% 3; p M-bM-^IM-$ 0.05), including genes involved in cell division processes, stress response and catabolite control. Phenotypically, the reduced biofilm depth of S. sanguinis SR DELTAluxS was elevated to wild type level by the complementation with sahH. Neither the addition of physiological concentrated artificial autoinducer 2 (AI-2) to the culture of S. sanguinis SR DELTAluxS nor the presence of the wild type strain in a trans-well assay had a cumulative effect on biofilm thickness of the luxS mutant. Furthermore, we identified 9 genes in the sahH complemented luxS mutant, which were regulated directly by AI-2 (fold change M-bM-^IM-% 3; p M-bM-^IM-$ 0.05), amongst them genes involved in competence development. So we concluded that AI-2 has no influence on biofilm growth, but regulative functions in competence development in S. sanguinis. Further, we figured out the suitability of transgenic sahH for the complementation of the interrupted Pfs/LuxS pathway without restoration of AI-2 release acquiring an essential tool for further investigations on AI-2. Aim: Is the presence of AI-2 necessary for the biofilm formation of S. sanguinis SK36? What is the impact of a deletion of luxS, is a disrupted activated methionine cycle the reason for the hampered biofilm depth causes by its inactivation? Materials and Methods: S. sanguinis SK36, S. sanguinis SR M-NM-^TluxS, and S. sanguinis SR M-NM-^TluxS/sahH were grown in CDM/sucrose for 24 h anaerobically; biofilm depth was measured by safranine and crystal violet stain. Biofilm morphology was investigated by scanning electron microscopy. The AI-2 release was monitored hourly to identify the time period of its release. For transcriptome analysis total RNA was isolated after 8 hours of growth and used for microarray analysis. The chip study used total RNA recovered from S. sanguinis SK36, its luxS mutant, and the sahH complementated luxS mutant. Gene expression analysis was done with three independent replicates, each. Chip content: 10186 genes (1883 genes of P. gingivalis W83, 1964 genes of F. nucleatum DSMZ 25586, 2244 genes of S. sanguinis SK36, 2168 genes of A. actinomycetemcomitans HK1651, 1927 genes of S. mutans UA159) with up to thirteen 60-mer probes per gene, with three-fold technical redundancy and additionally 3510 random sequence probe. In this study only the S. sanguinis and RANDOM probes were used for analysis.

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Transcriptional and metabolomic consequences of luxS inactivation reveal a metabolic rather than quorum sensing role for LuxS in Lactobacillus reuteri 100-23

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