Functional genomics and expression analysis of the Corynebacterium glutamicum fpr2-cysIXHDNYZ gene cluster involved in assimilatory sulphate reduction

Abstract

Background: Corynebacterium glutamicum is a high-GC Gram-positive soil bacterium of great biotechnological importance for the production of amino acids. To facilitate the rational design of sulphur amino acid-producing strains, the pathway for assimilatory sulphate reduction providing the necessary reduced sulfur moieties has to be known. Although this pathway has been well studied in Gram-negative bacteria like Escherichia coli and low-GC Gram-positives like Bacillus subtilis, little is known for the Actinomycetales and other high-GC Gram-positive bacteria.

Results: The genome sequence of C. glutamicum was searched for genes involved in the assimilatory reduction of inorganic sulphur compounds. A cluster of eight candidate genes could be identified by combining sequence similarity searches with a subsequent synteny analysis between C. glutamicum and the closely related C. efficiens. Using mutational analysis, seven of the eight candidate genes, namely cysZ, cysY, cysN, cysD, cysH, cysX, and cysI, were demonstrated to be involved in the reduction of inorganic sulphur compounds. For three of the up to now unknown genes possible functions could be proposed: CysZ is likely to be the sulphate permease, while CysX and CysY are possibly involved in electron transfer and cofactor biosynthesis, respectively. Finally, the candidate gene designated fpr2 influences sulphur utilisation only weakly and might be involved in electron transport for the reduction of sulphite. Real-time RT-PCR experiments revealed that cysIXHDNYZ form an operon and that transcription of the extended cluster fpr2 cysIXHDNYZ is strongly influenced by the availability of inorganic sulphur, as well as L-cysteine. Mapping of the fpr2 and cysIXHDNYZ promoters using RACE-PCR indicated that both promoters overlap with binding-sites of the transcriptional repressor McbR, suggesting an involvement of McbR in the observed regulation. Comparative genomics revealed that large parts of the extended cluster are conserved in 11 of 17 completely sequenced members of the Actinomycetales.

Conclusion: The set of C. glutamicum genes involved in assimilatory sulphate reduction was identified and four novel genes involved in this pathway were found. The high degree of conservation of this cluster among the Actinomycetales supports the hypothesis that a different metabolic pathway for the reduction of inorganic sulphur compounds than that known from the well-studied model organisms E. coli and B. subtilis is used by members of this order, providing the basis for further biochemical studies.

Figure 1

The fpr2cysIXHDNYZ gene cluster in the C. glutamicum genome. Coloured arrows indicate genes that are part of the cluster most probably involved in assimilatory sulphate reduction. Hairpins mark potential rho-independent transcription termination signals predicted by the TransTerm software. Black bars denote binding sites for the transcriptional repressor McbR [15].

Figure 2

Conservation of the C. glutamicum gene cluster possibly involved in assimilatory sulphate reduction in the Actinomycetales. Functions were inferred based on sequence similarity from BLAST searches against the UniProt database. Only those genes are displayed that were found to be clustered (at least two adjacent genes possibly involved in assimilatory sulphate reduction).

Figure 3

Growth of the C. glutamicum wild-type and the ΔcysZ mutant strain in medium with different sulphate concentrations. Wild-type and mutant strain were grown in liquid minimal medium, cell growth was determined by real-time nephelometry. For each time point the mean of 18 measurements is displayed (3 independent cultivations, 6 parallels per cultivation). The wild-type strain is denoted with filled diamonds, open symbols are used to indicate the mutant strain.

Figure 4

Comparison of the mRNA levels of the cys genes of the C. glutamicum wild-type and a cysI transposon mutant. Total RNA was isolated from cells grown in MMS with 1 mM L-cysteine as sulphur source and the relative transcription levels were determined using real-time RT-PCR to quantify the mRNAs of the displayed genes. Small black bars inside the arrows representing the genes indicate the position of the internal fragments amplified in the real-time RT-PCR.

Figure 5

Change of the fpr2cysIXHDNYZ mRNA levels in the presence of different sulphur sources and varying amounts of sulphate. The relative mRNA levels of the fpr2, cysI, and cysZ genes in cells incubated in MMS with either (A) different sulphur sources at 1 mM concentration or (B) sulphate at varying concentrations were compared to that in cells incubated in MMS without additional S-sources using real-time RT-PCR. Small black bars inside the arrows representing the genes indicate the position of the internal fragments used in real-time RT-PCR.

Figure 6

The promoter/operator regions of the C. glutamicum fpr2 and cysI genes. The determined transcriptional start points of the two transcriptional units fpr2 (A) and cysIXHDNYZ (B) are marked as '+1'. Parts of the two potential promoters (-35, -10, +1) are overlined, bases in bold type in these regions indicate bases matching the C. glutamicum σ⁷⁰ consensus promoter [23]. DNA motifs matching the consensus sequence of the McbR binding-site are boxed. Bases in bold italics mark potential ribosome binding-sites, open underlining arrows indicate the annotated starts of genes.

Figure 7

Model of the pathway for assimilatory sulphur reduction in C. glutamicum.. For proteins with gene names given in black, the involvement in the reduction of sulphate has been verified experimentally, for those in grey it has been inferred from circumstantial evidence.