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Single-cell analysis reveals gene-expression heterogeneity in syntrophic dual-culture of Desulfovibrio vulgaris with Methanosarcina barkeri.

Qi Z, Pei G, Chen L, Zhang W - Sci Rep (2014)

Bottom Line: In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture.The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture.Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Synthetic Microbiology, School of Chemical Engineering &Technology, Tianjin University, Tianjin 300072, P.R. China [2] Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, P.R. China [3] SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), 300072, P.R. China.

ABSTRACT
Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

Show MeSH
Evaluation of three internal reference candidates in monoculture and syntrophic dual-culture.CT is the qPCR quantification cycle, the fractional cycle number where fluorescence increases above the threshold. 13 cells from monoculture and 13 cells from syntrophic dual-culture were used to evaluate the consistency of the internal reference genes. The standard deviations (SD) across single cells were calculated using the OriginPro 8.0 software. Mean Ct values and SD values calculated using 13 single cells from each of the two conditions (i.e., mono- or dual-cultures) were shown above the plots.
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f2: Evaluation of three internal reference candidates in monoculture and syntrophic dual-culture.CT is the qPCR quantification cycle, the fractional cycle number where fluorescence increases above the threshold. 13 cells from monoculture and 13 cells from syntrophic dual-culture were used to evaluate the consistency of the internal reference genes. The standard deviations (SD) across single cells were calculated using the OriginPro 8.0 software. Mean Ct values and SD values calculated using 13 single cells from each of the two conditions (i.e., mono- or dual-cultures) were shown above the plots.

Mentions: In order to ensure that the gene expression across different conditions or analytical platforms is quantitatively comparable, expression measurements need to be normalized against an internal reference gene in RT-PCR analyses2630. Although successful application of several internal reference genes has been demonstrated in bulk-cell-based RT-qPCR analyses in various microbes, so far limited information is available regarding the constant expression of these internal reference genes across single cells31323334. In addition, early studies suggested that they could also be variable among single cells35. In a previous study, Shi et al. (2013) evaluated the expression variation of three possible internal reference genes, the tubulin gene, the 18S rRNA gene, and the actin gene in 12 single cells for a marine diatom Thalassiosira pseudonana, and the result showed that even for the 18S rRNA and tubulin genes, which were widely used as internal reference genes in various bulk-cell-based RT-qPCR analyses, significant cell-cell heterogeneity of their gene expression levels existed in T. pseudonana single cells23. So far no single-cell RT-qPCR analysis has been reported for D. vulgaris, thus no information is available for internal reference gene. As the proper use of the internal reference gene is the key to a successful RT-qPCR gene expression analysis32, we first evaluated the performance of several possible candidates as internal reference genes in single-cell analysis. Based on previous RT-PCR studies in bulk cells of D. vulgaris and other similar species, the 16S ribosomal RNA gene (Dv16SA)1136, the L-lactate dehydrogenase gene (DVU0600, ldh), and the recombinase A gene (DVU1090, recA)3738, were selected as candidates of internal reference genes for further evaluation. Two sets of single cells, each with 13 single cells, were randomly isolated from both D. vulgaris monoculture and dual-culture, respectively, and subjected to gene-expression analysis for the three candidates of internal reference genes. The standard deviations (SD) of the CT values were calculated using the OriginPro 8.0 software. The results showed that the standard deviations (SD) of the CT values were 0.98, 0.52 and 0.66 cycles among 13 single cells from the monoculture, and 0.88, 0.74 and 0.49 cycles among 13 single cells from the dual-culture, for the Dv16SA, DVU0600 and DVU1090 genes, respectively (Fig. 2), and the mean CT values were 29.31, 33.66 and 31.78 cycles among 13 single cells from the monoculture, and 27.60, 33.00 and 32.47 cycles among 13 single cells from the dual-culture, for the Dv16SA, DVU0600 and DVU1090 genes, respectively. Although slightly different between monoculture and dual-cultures, DVU1090 (recA) presented a relatively low variation of SD among single cells and low difference of mean CT values between two growth modes, which is consistent with the previous publications for its stable expression in bulk cells3739, and was thus selected as an internal reference gene for the further RT-qPCR analysis in this study. In various bulk-cell based studies, Dv16SA was often considered to be a good internal reference gene for gene expression analysis for D. vulgaris1134; however, our single-cell analysis showed that significant heterogeneity existed for the Dv16SA gene, and it may not function well as an internal reference gene for single-cell based analysis. The cause and biological significance that certain genes are more variable than others across single cells may worth further investigation.


Single-cell analysis reveals gene-expression heterogeneity in syntrophic dual-culture of Desulfovibrio vulgaris with Methanosarcina barkeri.

Qi Z, Pei G, Chen L, Zhang W - Sci Rep (2014)

Evaluation of three internal reference candidates in monoculture and syntrophic dual-culture.CT is the qPCR quantification cycle, the fractional cycle number where fluorescence increases above the threshold. 13 cells from monoculture and 13 cells from syntrophic dual-culture were used to evaluate the consistency of the internal reference genes. The standard deviations (SD) across single cells were calculated using the OriginPro 8.0 software. Mean Ct values and SD values calculated using 13 single cells from each of the two conditions (i.e., mono- or dual-cultures) were shown above the plots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265775&req=5

f2: Evaluation of three internal reference candidates in monoculture and syntrophic dual-culture.CT is the qPCR quantification cycle, the fractional cycle number where fluorescence increases above the threshold. 13 cells from monoculture and 13 cells from syntrophic dual-culture were used to evaluate the consistency of the internal reference genes. The standard deviations (SD) across single cells were calculated using the OriginPro 8.0 software. Mean Ct values and SD values calculated using 13 single cells from each of the two conditions (i.e., mono- or dual-cultures) were shown above the plots.
Mentions: In order to ensure that the gene expression across different conditions or analytical platforms is quantitatively comparable, expression measurements need to be normalized against an internal reference gene in RT-PCR analyses2630. Although successful application of several internal reference genes has been demonstrated in bulk-cell-based RT-qPCR analyses in various microbes, so far limited information is available regarding the constant expression of these internal reference genes across single cells31323334. In addition, early studies suggested that they could also be variable among single cells35. In a previous study, Shi et al. (2013) evaluated the expression variation of three possible internal reference genes, the tubulin gene, the 18S rRNA gene, and the actin gene in 12 single cells for a marine diatom Thalassiosira pseudonana, and the result showed that even for the 18S rRNA and tubulin genes, which were widely used as internal reference genes in various bulk-cell-based RT-qPCR analyses, significant cell-cell heterogeneity of their gene expression levels existed in T. pseudonana single cells23. So far no single-cell RT-qPCR analysis has been reported for D. vulgaris, thus no information is available for internal reference gene. As the proper use of the internal reference gene is the key to a successful RT-qPCR gene expression analysis32, we first evaluated the performance of several possible candidates as internal reference genes in single-cell analysis. Based on previous RT-PCR studies in bulk cells of D. vulgaris and other similar species, the 16S ribosomal RNA gene (Dv16SA)1136, the L-lactate dehydrogenase gene (DVU0600, ldh), and the recombinase A gene (DVU1090, recA)3738, were selected as candidates of internal reference genes for further evaluation. Two sets of single cells, each with 13 single cells, were randomly isolated from both D. vulgaris monoculture and dual-culture, respectively, and subjected to gene-expression analysis for the three candidates of internal reference genes. The standard deviations (SD) of the CT values were calculated using the OriginPro 8.0 software. The results showed that the standard deviations (SD) of the CT values were 0.98, 0.52 and 0.66 cycles among 13 single cells from the monoculture, and 0.88, 0.74 and 0.49 cycles among 13 single cells from the dual-culture, for the Dv16SA, DVU0600 and DVU1090 genes, respectively (Fig. 2), and the mean CT values were 29.31, 33.66 and 31.78 cycles among 13 single cells from the monoculture, and 27.60, 33.00 and 32.47 cycles among 13 single cells from the dual-culture, for the Dv16SA, DVU0600 and DVU1090 genes, respectively. Although slightly different between monoculture and dual-cultures, DVU1090 (recA) presented a relatively low variation of SD among single cells and low difference of mean CT values between two growth modes, which is consistent with the previous publications for its stable expression in bulk cells3739, and was thus selected as an internal reference gene for the further RT-qPCR analysis in this study. In various bulk-cell based studies, Dv16SA was often considered to be a good internal reference gene for gene expression analysis for D. vulgaris1134; however, our single-cell analysis showed that significant heterogeneity existed for the Dv16SA gene, and it may not function well as an internal reference gene for single-cell based analysis. The cause and biological significance that certain genes are more variable than others across single cells may worth further investigation.

Bottom Line: In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture.The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture.Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Synthetic Microbiology, School of Chemical Engineering &Technology, Tianjin University, Tianjin 300072, P.R. China [2] Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, P.R. China [3] SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), 300072, P.R. China.

ABSTRACT
Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

Show MeSH