Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Feb 5;9(2):e88260.
doi: 10.1371/journal.pone.0088260. eCollection 2014.

Bifidobacteria exhibit LuxS-dependent autoinducer 2 activity and biofilm formation

Zhongke Sun  1 , Xiang He  2 , Vincenzo F Brancaccio  1 , Jing Yuan  2 , Christian U Riedel  1
Affiliations

Bifidobacteria exhibit LuxS-dependent autoinducer 2 activity and biofilm formation

Zhongke Sun et al. PLoS One. .

Abstract

Autoinducer-2 (AI-2) molecules are one class of signalling molecules involved in gene regulation dependent on population density in a mechanism commonly referred to as quorum sensing (QS). AI-2 is produced by the methylthioadenosine/S-adenosyl-homocysteine nucleosidase LuxS. In the present study, we characterise the function of bifidobacterial LuxS proteins to address the question whether these economically important bacteria are able to perform QS communication. All publically available genome sequences of bifidobacteria harbour putative luxS genes. The deduced amino acid sequences are well conserved in the genus and show good homology to the LuxS protein of the prototypical AI-2 producer Vibrio harveyi. The luxS genes of three bifidobacterial strains were successfully expressed in AI-2-negative Escherichia coli DH5α. Supernatants of these recombinant E. coli strains contained significant AI-2 activity. In initial experiments, we failed to detect AI-2 activity in supernatants of bifidobacteria grown in MRSc. High concentration of glucose as well as acidic pH had strong inhibitory effects on AI-2 activity. AI-2 activity could be detected when lower volumes of supernatants were used in the assay. Homologous overexpression of luxS in Bifidobacterium longum NCC2705 increased AI-2 levels in the supernatant. Furthermore, over-expression of luxS or supplementation with AI-2-containing supernatants enhanced biofilm formation of B. longum NCC2705. Collectively, these results suggest that bifidobacteria indeed harbour functional luxS genes that are involved in the production of AI-2-like molecules. To the best of our knowledge, this represents the first report on AI-2 activity produced by bifidobacteria. Self-produced AI-2 activity as well as AI-2-like molecules of other bacteria of the intestinal tract may have a regulatory function in biofilm formation and host colonization by bifidobacteria.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The corresponding author, Christian Riedel, is an Academic Editor to PLoS ONE, and hereby declares that he had no influence on the reviewing process or the decision making whatsoever. Of course he fully adheres to the policies of PLoS ONE on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Alignment of the LuxS sequences of B. longum NCC2705, B. longum E18, B. bifidum S17, B. breve UCC2003, B. adolescentis L2-32, B. lactis AD011, B. dentium Bd1 and V. harveyi BB170.
Amino acids are color-coded according to their polarity and the degree of conservation amongst the sequences is indicated below the alignment on a black-green scale with black indicating low and green high conservation.
Figure 2
Figure 2. AI-2 activity in culture supernatants.
V. harveyi BB170 (Vh), B. bifidum S17 (S17), B. longum E18 (E18) and B. longum NCC2705 (NCC2705) were grown in AB (Vh) or MRSc (bifidobacteria) medium to stationary growth phase. Sterile AB or MRSc medium was used as negative controls. Values are mean ± standard deviation (SD) of four replicates per condition and results of one representative of at least three independent experiments are shown. Data was analysed by pair-wise comparison to the respective medium controls (AB for Vh or MRSc for S17, E18 and NCC2705) using Student's t-test (***: p<0.001).
Figure 3
Figure 3. LuxS expression and AI-2 activity of recombinant E. coli strains.
(A) Coomassie-stained SDS-PAGE of crude extracts of E. coli DH5α harbouring pBAD_LuxSE18 (E18), pBAD_LuxSNCC (NCC2705) or pBAD_LuxSS17 (S17). Expression of luxS genes was induced for 4 h with arabinose at the indicated concentrations. Non-induced cultures (-) served as negative controls. The marker band represents a protein of 15 kDa. (B) AI-2 activity in supernatants of E. coli strains induced with 0.2 µM arabinose were analysed and compared to non-induced cultures. (C) AI-2 activity in culture supernatants of E. coli DH5α pBAD_LuxSS17 induced with different concentrations of arabinose. Results from one representative of at least three independent cultures for each strain are shown. Values in (B) and (C) are mean ± SD of four replicates per supernatant. Data was analysed by pair-wise comparison to the respective negative controls (LB in B; - in C) using Student's t-test (*: p<0.05; ***: p<0.001).
Figure 4
Figure 4. Inhibition of AI-2 activity by acidic pH and high glucose concentrations.
AI-2 activity in supernatant of V. harveyi BB170 adjusted to the indicated pH (A) or spiked with different concentrations of glucose (Glc; B). Values are mean ± SD of one representative supernatant measured in three replicates and similar results were obtained with supernatants of three independent cultures. Data was analysed by pair-wise comparison to the respective positive controls (pos) using Student's t-test (**: p<0.01; ***: p<0.001).
Figure 5
Figure 5. Improved detection of AI-2 activity in bifidobacterial culture supernatants.
AI-2 activity (A) and growth of the reporter strain V. harveyi BB170 (B) using 1%, 2.5% or 10% culture supernatant of either B. bifidum S17 (S17), B. longum E18 (E18) or B. longum NCC2705 (NCC2705) grown in MRSc. Sterile MRSc was used as negative control (neg). Values are mean ± SD of one representative culture per strain measured in four replicates and similar results were obtained with supernatants of three independent cultures. Data in (A) was analysed by pair-wise comparison to the respective negative controls (neg) using Student's t-test (***: p<0.001).
Figure 6
Figure 6. AI-2 activity and biofilm formation of B. longum NCC2705 can be increased by homologous over-expression of LuxSNCC.
(A) AI-2 activity in the culture supernatant of V. harveyi BB170 (Vh), B. longum NCC2705 (WT) or its luxS overexpressing derivative B. longum NCC2705 pMgap_LuxSNCC (OE). (B) Biofilm formation of WT or OE grown in MRSc with (+) or without (−) supplementation with 10% filter-sterilized culture supernatant of OE. Values are mean ± SD of one representative culture per strain measured in four (A) or eight (B) replicates and similar results were obtained with supernatants of three independent cultures. Data was analysed by pair-wise comparison to the wildtype controls (WT) or as indicated by the bars using Student's t-test (**: p<0.01; ***: p<0.001).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Miller MB, Bassler BL (2001) Quorum sensing in bacteria. Annu Rev Microbiol 55: 165–199 10.1146/annurev.micro.55.1.165 - DOI - PubMed
    1. Parveen N, Cornell KA (2011) Methylthioadenosine/S-adenosylhomocysteine nucleosidase, a critical enzyme for bacterial metabolism. Mol Microbiol 79: 7–20 10.1111/j.1365-2958.2010.07455.x - DOI - PMC - PubMed
    1. Vendeville A, Winzer K, Heurlier K, Tang CM, Hardie KR (2005) Making “sense” of metabolism: autoinducer-2, LuxS and pathogenic bacteria. Nat Rev Microbiol 3: 383–396 10.1038/nrmicro1146 - DOI - PubMed
    1. Xavier KB, Bassler BL (2003) LuxS quorum sensing: more than just a numbers game. Curr Opin Microbiol 6: 191–197. - PubMed
    1. Rezzonico F, Duffy B (2008) Lack of genomic evidence of AI-2 receptors suggests a non-quorum sensing role for luxS in most bacteria. BMC Microbiol 8: 154 10.1186/1471-2180-8-154 - DOI - PMC - PubMed

Publication types

MeSH terms

Grants and funding

This study was partially funded by the German Academic Exchange Service/Federal Ministry of Education and Research (grant D/09/04778) to CUR. ZS was supported by a PhD fellowship of the German Academic Exchange Service. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

LinkOut - more resources

-