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Heterologous expression of sahH reveals that biofilm formation is autoinducer-2 independent in Streptococcus sanguinis, but is associated with an intact AMC

ABSTRACT: 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 ≥ 3; p ≤ 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 ≥ 3; p ≤ 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 ΔluxS, and S. sanguinis SR ΔluxS/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.

ORGANISM(S): Streptococcus sanguinis SK36

PROVIDER: GSE37007 | GEO | 2012/04/04

SECONDARY ACCESSION(S): PRJNA157871

REPOSITORIES: GEO

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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.

Project description:LuxS is an enzyme involved in the activated methyl cycle and the by-product autoinducer 2 (AI-2) was a quorum sensing signal in some species. In our previous study, the functional LuxS in AI-2 production was verified in the porcine respiratory pathogen Actinobacillus pleuropneumoniae. Enhanced biofilm formation and reduced virulence were observed in the luxS mutant. To comprehensively understand the luxS function, in this study, the transcriptional profiles were compared between the A. pleuropneumoniae luxS mutant and its parental strain in four different growth phases using microarray. Many genes associated with infection were differentially expressed. The biofilm formation genes pgaABC in the luxS mutant were up-regulated in early exponential phase, while 8 genes associated with adhesion were down-regulated in late exponential phase. A group of genes involved in iron acquisition and metabolism were regulated in four growth phases. Further investigations on these virulence traits demonstrated that the luxS mutant showed enhanced biofilm formation and reduced adhesion ability and these effects were not due to lack of AI-2. But AI-2 could increase biofilm formation and adhesion of A. pleuropneumoniae independent of LuxS. Growth under iron restricted condition could be controlled by LuxS through AI-2 production. These results revealed pleiotropic roles of LuxS and AI-2 on A. pleuropneumoniae virulence traits. A. pleuropneumoniae strains were cultured in TSB medium supplemented with 10 M-NM-<g/ml of nicotinamide adenine dinucleotide (NAD) and 10% (v/v)filtered cattle serum at 37M-BM-0C. The samples were collected from early exponential phase, middle exponential phase, late exponential phase and stationary phase respectively and the total RNA were extracted using RNA-Solv Reagent (Omega) according to the manufacturerM-bM-^@M-^Ys instructions. For each time point, four biological replicates were combined into two samples. The intensities were normalized and transformed into log2 value.The fold changes >=1.5 or <=-1.5 were selected as differentially expressed genes.

Project description:Investigation of whole genome gene expression levels of P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651, S. mutans UA159 in an 24 h old culture. Additionally, whole genome gene expression level changes of S. mutans UA159 biofilm cells after co-cultivation with S. mitis ATCC 11843 were compared to its single species biofilm growth after 24 h. Aim: Demonstration of the usefulness of a five-species gene expression array. Multiple probes per gene enabled identification of single inter-species cross-hybridizing probes. The deletion of such probes lead almost not to the deletion of the whole gene. This was investigated and confirmed by a two-species biofilm expression analysis: The here described array was used for the identification of genes of S. mutans influenced by the presence of S. mitis. Materials and Methods: P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651,and S. mutans UA159 were grown in CDM/succrose or artificial saliva/galactose in a single-species culture for 24 h anaerobically resulting in biofilm structures or monolayers. Total RNA was isolated and used for microarray analysis. Probes were analysed for the presence of biological false positive signals caused by cross-hybridizing probes of one of the other species presented on the chip. Further, a simple procedure was developed for automatical identification and deletion of false positive signals caused by washing artefacts, resulting in a more reliable outcome. In the case of the S. mutans/S. mitis mixed-species biofilm, both species were cultured together for 24 h like previously described. The found gene regulations were verified by RT-PCR. Results: Experiments with cDNA from 24 h old single-species cultures allowed the identification of cross-species hybridizing probes on the array, which can be eliminated in mixed-species experimental settings without the need to exclude the whole genes from the analysis. Between 69 % and almost 100 % represented genomes on this array were found actively transcribed under the mono-species monolayer and biofilm conditions used here. S. mutans / S. mitis co-culture: Physiological investigations revealed an increase in S. mutans biofilm mass with a decrease in pH-value under the influence of S. mitis, thereby confirming previously published data. A stringent fold change cut-off of 2 (p<0.05) identified 19 S. mutans transcripts with increased abundance, and 11 with decreased abundance compared to a S. mutans mono-species biofilm. Many of the genes have previously been found differentially regulated under general and acid stress, thereby confirming the value of this array. Conclusions: Taken together, this new array allows transcriptome studies on multi-species oral biofilm interactions and could become an important asset in future oral biofilm and inhibitor/therapy studies. The chip study used pooled total RNA recovered from three biologically independent mono-species biofilms or adherent cells/monolayers of P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651, and S. mutans UA159. In the case of gene expression analysis of S. mutans/S.mitis biofilm structures compared to the single species biofilm of S. mutans three separate single and three separate two-species biofilm cultures were analysed. Each chip measured the expression level of all together 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 and with a three-fold technical redundancy.

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Heterologous expression of sahH reveals that biofilm formation is autoinducer-2 independent in Streptococcus sanguinis, but is associated with an intact AMC

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