Comparison of Transcriptional Heterogeneity of Eight Genes between Batch Desulfovibrio vulgaris Biofilm and Planktonic Culture at a Single-Cell Level

Abstract

Sulfate-reducing bacteria (SRB) biofilm formed on metal surfaces can change the physicochemical properties of metals and cause metal corrosion. To enhance understanding of differential gene expression in Desulfovibrio vulgaris under planktonic and biofilm growth modes, a single-cell based RT-qPCR approach was applied to determine gene expression levels of 8 selected target genes in four sets of the 31 individual cells isolated from each growth condition (i.e., biofilm formed on a mild steel (SS) and planktonic cultures, exponential and stationary phases). The results showed obvious gene-expression heterogeneity for the target genes among D. vulgaris single cells of both biofilm and planktonic cultures. In addition, an increased gene-expression heterogeneity in the D. vulgaris biofilm when compared with the planktonic culture was also observed for seven out of eight selected genes at exponential phase, and six out of eight selected genes at stationary phase, respectively, which may be contributing to the increased complexity in terms of structures and morphology in the biofilm. Moreover, the results showed up-regulation of DVU0281 gene encoding exopolysaccharide biosynthesis protein, and down-regulation of genes involved in energy metabolism (i.e., DVU0434 and DVU0588), stress responses (i.e., DVU2410) and response regulator (i.e., DVU3062) in the D. vulgaris biofilm cells. Finally, the gene (DVU2571) involved in iron transportation was found down-regulated, and two genes (DVU1340 and DVU1397) involved in ferric uptake repressor and iron storage were up-regulated in D. vulgaris biofilm, suggesting their possible roles in maintaining normal metabolism of the D. vulgaris biofilm under environments of high concentration of iron. This study showed that the single-cell based analysis could be a useful approach in deciphering metabolism of microbial biofilms.

Tools for Genomic and Transcriptomic Analysis of Microbes at Single-Cell Level

“…Traditional micropipette method could be easily applied in any laboratory on an inverted microscope with mechanical liquid handling. Although very labor-consuming and low-throughput, approximately in the order of 50 cells/h and person ( Picelli, 2016 ), it is the first choice if only a small number of cells are required for the next step analysis ( Qi et al, 2014 , 2016 ; Wang et al, 2015 ). In addition, commercial robotic manipulation system for automated single-cell selection has also been developed and applied for microbial single-cell analysis ( Anis et al, 2008 ; Merza et al, 2009 ; Gao et al, 2011 ; Banerjee et al, 2014 ), making it possible for relatively high-throughput single-cell isolation.…”

“…However, cells are typically subjected to physical stresses during the sorting, such as fluidic pressure, laser beam, electrostatic charges, voltage fields, and collisions with container surfaces, which could significantly affect the cell physiology and even the recovery rate during the cultivation ( Marie et al, 2017 ). In the case when the sorted cells are used for gene expression or transcriptome analysis, proper RNA protectant needs to be added ( Qi et al, 2014 , 2016 ; Wang et al, 2015 ); while in the case when the sorted cells are used for clonal cultivation, extra efforts to carefully optimize the cultivation conditions are necessary in order to maximize the success rates ( Marie et al, 2017 ). Under the conception of ‘Lab-on-a-chip,’ microfluidic devices have become the most popular method for single-cell isolation.…”

“…For example, methods using fluorescent reporter proteins coupling with high-throughput data acquisition approaches such as flow cytometry have been widely applied for detecting gene expression heterogeneities within the microbial population ( Taniguchi et al, 2010 ; Roberfroid et al, 2016 ). In addition, methods using RT-qPCR for detecting gene expression in single cells have also been reported and successfully applied to several types of microbes for heterogeneity analysis ( Gao et al, 2011 ; Shi et al, 2013 ; Qi et al, 2014 , 2016 ; Wang et al, 2015 ; Thompson et al, 2017 ; Turkarslan et al, 2017 ). However, these methods could only reveal gene expression patterns of a very limited number of genes, while not able to uncover global information in a cell.…”

Unintended Laboratory-Driven Evolution Reveals Genetic Requirements for Biofilm Formation by Desulfovibrio vulgaris Hildenborough

“…Yet, the mechanism of biofilm formation of SRB such as D. vulgaris Hildenborough has not been determined. Studies of SRB biofilm on steel have predominantly focused on the exopolysaccharide fraction of the biofilm matrix, where targeted and genome-wide expression analyses have shown increases in expression of exopolysaccharide biosynthesis proteins in biofilm compared to planktonic cells ( 8 , 9 ). The matrix of D. vulgaris Hildenborough biofilm on glass slides was observed to be predominantly comprised of protein ( 10 ), with a carbohydrate/protein (C/P) ratio of the biofilm biomass of approximately 0.13 µg/µg ( 11 ).…”

Enhanced Biocide Treatments with D-amino Acid Mixtures against a Biofilm Consortium from a Water Cooling Tower

“…Microbiologically influenced corrosion is a major problem in various industrial sectors, such as water utilities, oil and gas, and power generation ( Qi et al, 2016 ; Xu et al, 2017 ; Jia et al, 2017c ). MIC in recirculating cooling water systems causes deterioration of metallic surfaces and reduces the lifetime of the systems ( Ilhan-Sungur and Çotuk, 2010 ).…”