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. 2017 Jul 12;17(1):158.
doi: 10.1186/s12866-017-1067-6.

Physiological roles of sigma factor SigD in Corynebacterium glutamicum

Hironori Taniguchi  1   2 , Tobias Busche  2 , Thomas Patschkowski  2   3 , Karsten Niehaus  2   3 , Miroslav Pátek  4 , Jörn Kalinowski  2 , Volker F Wendisch  5   6
Affiliations

Physiological roles of sigma factor SigD in Corynebacterium glutamicum

Hironori Taniguchi et al. BMC Microbiol. .

Abstract

Background: Sigma factors are one of the components of RNA polymerase holoenzymes, and an essential factor of transcription initiation in bacteria. Corynebacterium glutamicum possesses seven genes coding for sigma factors, most of which have been studied to some detail; however, the role of SigD in transcriptional regulation in C. glutamicum has been mostly unknown.

Results: In this work, pleiotropic effects of sigD overexpression at the level of phenotype, transcripts, proteins and metabolites were investigated. Overexpression of sigD decreased the growth rate of C. glutamicum cultures, and induced several physiological effects such as reduced culture foaming, turbid supernatant and cell aggregation. Upon overexpression of sigD, the level of Cmt1 (corynomycolyl transferase) in the supernatant was notably enhanced, and carbohydrate-containing compounds were excreted to the supernatant. The real-time PCR analysis revealed that sigD overexpression increased the expression of genes related to corynomycolic acid synthesis (fadD2, pks), genes encoding corynomycolyl transferases (cop1, cmt1, cmt2, cmt3), L, D-transpeptidase (lppS), a subunit of the major cell wall channel (porH), and the envelope lipid regulation factor (elrF). Furthermore, overexpression of sigD resulted in trehalose dicorynomycolate accumulation in the cell envelope.

Conclusions: This study demonstrated that SigD regulates the synthesis of corynomycolate and related compounds, and expanded the knowledge of regulatory functions of sigma factors in C. glutamicum.

Keywords: Corynebacterium glutamicum; Mycomembrane; SigD; Sigma factor; Trehalose dicorynomycolate.

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Conflict of interest statement

Author’s information

HT currently belongs to Synthetic Bioengineering lab, Dept.of Biotechnology, Graduate School of Engineering, Osaka University (Yamadaoka 2-1, Suita, Osaka, 565-0871, Japan).

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Not applicable.

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Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Maximum growth rates of the ΔsigD strain and the sigD overexpressing strain with different IPTG concentrations. The maximum specific growth rate (h−1) was shown for (a) C. glutamicum WT and ΔsigD, and (b) WT(pVWEx1) and WT(pVWEx1-sigD) with different IPTG concentrations (0, 10, 50, 250, 1000 μM). Error bars represent standard deviations from biological triplicates
Fig. 2
Fig. 2
Influence of sigD overexpression on cell cultures and cell morphology. a Cell cultures with 50 μM of IPTG after 36 h of cultivation are shown. b Supernatant turbidity with different concentrations of IPTG (0 μM, 10 μM, 50 μM) after 36 h is shown. Error bars represent standard deviations from biological triplicates. Microscopic images of the WT strain (c) and the sigD overexpressing strain (d) are shown. Cells in the stationary phase were observed under the microscope with a magnification of 1000. e Distribution of the size of cell aggregates is shown. The size of cell aggregates was analyzed by ImageJ, and the distribution was visualized by the box-and-whisker plot. Lower whisker, lower quantile, median, upper quantile and upper whisker are shown. The cross point indicates mean, and outliners were plotted as individual points
Fig. 3
Fig. 3
1D-SDS PAGE of proteins in the supernatants. a Secreted proteins were analyzed by 12% SDS-PAGE. The molecular sizes of proteins in the marker are shown in kDa. WT(pVWEx1) and WT(pVWEx1-sigD) protein samples were obtained by acetone precipitation of the supernatants. Proteins from 200 μL of the supernatant was loaded on each lane. The intensity of secreted protein bands was quantified for WT(pVWEx1) (b) and for WT(pVWEx1-sigD) (c). The highest intensity of the band was normalized to 100%. The protein bands labeled with numbers were subjected to MALDI-TOF/TOF MS. 1: Psp3 (Cg2061), 2: LppS (Cg2720), 3: Cmt1 (Cg0413), 4: Cmt2 (Cg3186) and Cg2052
Fig. 4
Fig. 4
Relative mRNA abundance of genes upregulated during sigD overexpression. Relative abundance of mRNA of each gene was quantified by real-time PCR. The white and gray columns show the abundance in WT(pVWEx1-sigD) without IPTG (0 μM IPTG), and in WT(pVWEx1-sigD) with 50 μM of IPTG (50 μM IPTG), respectively. Error bars represent standard deviations calculated from biological triplicates. The p-value of mRNA abundance was calculated by Student’s t-test (two-tail, unpaired) between 0 μM and 50 μM of IPTG, and is shown by *, ** and *** for <0.05, <0.01 and <0.001, respectively
Fig. 5
Fig. 5
TLC analysis of lipid crude extracts. a Lipid crude extracts were analyzed by thin layer chromatography. b The intensity of bands was quantified for WT(pVWEx1) and WT(pVWEx1-sigD). The highest intensity of the band was normalized to 100%. The dotted and black lines indicate the intensity profile for WT(pVWEx1) and WT(pVWEx1-sigD), respectively. The mobility of TDCM (trehalose dicorynomycolate) and TMCM (trehalose monocorynomycolate) were determined by their Rf values taken from the previous studies [32, 33]. CHCl3/CH3OH/H2O (30:8:1 v/v) was used as development solvent
Fig. 6
Fig. 6
Summary of effects induced by sigD overexpression in C. glutamicum. Biosynthesis pathway of trehalose dicorynomycolate (TDCM) and trehalose monocorynomycolate (TMCM) starting from acyl-CoA (A-CoA) and fatty acid (FA) is shown. The names of enzymes are shown in red letters and the catalyzing reactions are shown in red lines, only when the expression of corresponding genes were confirmed to be upregulated under sigD overexpression by transcriptome analysis. CA-CoA: carboxylated acyl-CoA, TRE: trehalose, CMk: Keto corynomycolic acid, TMCMk: TMCM keto form
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