Transcriptional control of lipid metabolism by the MarR-like regulator FamR and the global regulator GlxR in the lipophilic axilla isolate Corynebacterium jeikeium K411

Abstract

Corynebacterial fatty acid metabolism has been associated with human body odour, and is therefore discussed as a potential target for the development of new deodorant additives. For this reason, the transcription levels of fad genes associated with lipid metabolism in the axilla isolate Corynebacterium jeikeium were analysed during growth on different lipid sources. The transcription of several fad genes was induced two- to ninefold in the presence of Tween 60, including the acyl-CoA dehydrogenase gene fadE6. DNA affinity chromatography identified the MarR-like protein FamR as candidate regulator of fadE6. DNA band shift assays and in vivo reporter gene fusions confirmed the direct interaction of FamR with the mapped fadE6 promoter region. Moreover, DNA affinity chromatography and DNA band shift assays detected the binding of GlxR to the promoter regions of fadE6 and famR, revealing a hierarchical control of fadE6 transcription by a feed-forward loop. Binding of GlxR and FamR to additional fad gene regions was demonstrated in vitro by DNA band shift assays, resulting in the co-regulation of fadA, fadD, fadE and fadH genes. These results shed first light on the hierarchical transcriptional control of lipid metabolism in C. jeikeium, a pathway associated with the development of human axillary odour.

Figure 1

Differential transcription of fatty acid metabolism genes in C. jeikeium. Real-time RT-PCR was performed with total RNA isolated from cells grown in CJK minimal medium supplemented with 1% of Tween 80 or Tween 60. Transcription levels of fad genes in cells grown in the presence of Tween 60 are displayed in comparison to values detected in cells grown on Tween 80 medium. The experiment combines the results of two biological and two technical replicates. Black lines indicate the thresholds of significance (twofold induction and 0.5-fold repression). Genes investigated are: fadD1-11, acyl-CoA synthetase; fadE2-8, acyl-CoA dehydrogenase; fadA1-3, keto-acyl thiolase; fadB1-2, hydroxyacyl-CoA dehydrogenase; fadH, 2,4-dienoyl-CoA reductase. No mRNA was detected in case of the fadE1 gene.

Figure 2

SDS-PAGE of C. jeikeium K411 proteins enriched by DNA affinity chromatography. A 247 bp DNA fragment covering the 5′ region of fadE6 was used for the detection of DNA-binding proteins. A combined protein extract of cultures grown on different types of Tween was incubated with the DNA-coated magnetic beads. The arrows indicate protein bands that were excised from the gel and identified by MALDI-TOF/TOF mass spectrometry. Data of identified proteins are summarized in tabular form. M, Page Ruler Prestained Protein Ladder (Thermo Scientific); E, eluted protein fraction.

Figure 3

In vitro binding of Jk0257 to the upstream region of fadE6.A. A 765 bp fragment of the fadE6 upstream region was subdivided into three overlapping DNA fragments. DNA band shift assays were carried out with 30 pmol of the purified Jk0257 protein and the fluorescently labelled DNA fragments 1–3. A DNA fragment containing the McbR binding site of the cysI gene served as a negative control. Lanes 1, labelled DNA fragments; lanes 2, labelled DNA fragments and purified Jk0257. Additionally, DNA fragment 3 was shifted with purified GlxR protein. Lane 1, labelled DNA fragment; lane 2, labelled DNA fragment and purified GlxR; lane 3, labelled DNA fragment, purified GlxR, and 0.4 mM cAMP.B. DNA band shift assays with shortened versions of fragment 3. The deduced promoter region of fadE6 is displayed. The −10 and −35 hexamers are shown, in which capital letters indicate conserved nucleotides. The transcription start site (+1) of fadE6 is highlighted by an arrow. The predicted DNA binding site of GlxR is marked with a box. The 193 bp fragment used for the in vivo Gfp reporter assay is marked with a brace.

Figure 4

Analyses of regulatory processes controlling the transcription of jk0257.A. A fragment covering the 5′ region of jk0257 was used for the detection of DNA-binding proteins. Eluted proteins were separated by SDS-PAGE. Arrows indicate protein bands that were excised from the gel and identified by MALDI-TOF/TOF mass spectrometry. Data of identified proteins are summarized in tabular form. M, Page Ruler Prestained Protein Ladder (Thermo Scientific); E, eluted protein fraction.B. EMSA conducted with a fluorescently labelled PCR fragment covering the jk0257 upstream region and purified regulatory proteins (GlxR or Jk0257). The assays were separated on 3% agarose gels. Lanes 1, labelled PCR product; lanes 2, PCR product and regulatory protein; lane 3, PCR product, purified GlxR, and 0.4 mM cAMP.C. Genetic features of the jk0257 upstream region. The transcription start site (+1) was identified by 5′ RACE-PCR. The deduced jk0257 promoter region is highlighted by bold grey letters, of which capital letters indicate conserved bases. The predicted GlxR binding site is marked with a box.D. Regulatory network motif for the control of fadE6 transcription. The motif of the incoherent type 2 allows the coordination of global (S_G) and local signals (S_L). GlxR senses the concentration of its effector molecule cAMP (S_G) in the bacterial cell and represses fadE6 and jk0257 transcription accordingly. The retraction of global control by GlxR enables the local regulator Jk0257 to react to further specific signals (S_L) and adjust fadE6 transcription.

Figure 5

Analyses of regulatory processes controlling the transcription of glxR.A. A DNA fragment covering the 5′ region of glxR was used for the detection of DNA-binding proteins. Eluted proteins were separated by SDS-PAGE. Arrows indicate protein bands that were excised from the gel and identified by MALDI-TOF/TOF mass spectrometry. Asterisks mark protein bands with no significant Mascot Score. Data of identified proteins are summarized in tabular form. M, Page Ruler Prestained Protein Ladder (Thermo Scientific); E, eluted protein fraction.B. EMSA conducted with the purified regulatory GlxR protein and a fluorescently labelled PCR fragment covering the glxR upstream region. The assay was separated on a 3% agarose gel. Lane 1, labelled PCR product; lane 2, PCR product and GlxR; lane 3, PCR product, GlxR and 0.6 mM cAMP.C. Genetic features of the glxR upstream region. The transcription start site (+1) was deduced from 5′ RACE-PCR. The glxR promoter region is highlighted by bold grey letters, of which capital letters indicate conserved bases. The predicted GlxR binding site is marked with a box.

Figure 6

DNA band shift assays with GlxR/Jk0257 and the upstream regions of selected fad genes. EMSA were conducted with the purified regulatory protein and fluorescently labelled PCR products carrying predicted GlxR binding sites. Assays were separated on 3% agarose gels and DNA–protein complexes were visualized by fluorescence imaging. Lanes 1, labelled PCR product; lanes 2, PCR product and regulatory protein; lanes 3, PCR product, GlxR and 0.6 mM cAMP.

Figure 7

Transcriptional control of fad genes by GlxR and Jk0257. The regulatory proteins GlxR and Jk0257 co-regulate the transcription of eight fad genes, including fadE6. Only three genes are not under dual control of GlxR and Jk0257. The fadE7 gene was not assigned to either regulon. Arrows indicate a regulatory interaction; −| indicates repressive control mechanisms. Genes carrying a RamA binding site in their 5′ region are marked with an asterisk.