Complete genome sequence and lifestyle of black-pigmented Corynebacterium aurimucosum ATCC 700975 (formerly C. nigricans CN-1) isolated from a vaginal swab of a woman with spontaneous abortion

Abstract

Background: Corynebacterium aurimucosum is a slightly yellowish, non-lipophilic, facultative anaerobic member of the genus Corynebacterium and predominantly isolated from human clinical specimens. Unusual black-pigmented variants of C. aurimucosum (originally named as C. nigricans) continue to be recovered from the female urogenital tract and they are associated with complications during pregnancy. C. aurimucosum ATCC 700975 (C. nigricans CN-1) was originally isolated from a vaginal swab of a 34-year-old woman who experienced a spontaneous abortion during month six of pregnancy. For a better understanding of the physiology and lifestyle of this potential urogenital pathogen, the complete genome sequence of C. aurimucosum ATCC 700975 was determined.

Results: Sequencing and assembly of the C. aurimucosum ATCC 700975 genome yielded a circular chromosome of 2,790,189 bp in size and the 29,037-bp plasmid pET44827. Specific gene sets associated with the central metabolism of C. aurimucosum apparently provide enhanced metabolic flexibility and adaptability in aerobic, anaerobic and low-pH environments, including gene clusters for the uptake and degradation of aromatic amines, L-histidine and L-tartrate as well as a gene region for the formation of selenocysteine and its incorporation into formate dehydrogenase. Plasmid pET44827 codes for a non-ribosomal peptide synthetase that plays the pivotal role in the synthesis of the characteristic black pigment of C. aurimucosum ATCC 700975.

Conclusions: The data obtained by the genome project suggest that C. aurimucosum could be both a resident of the human gut and possibly a pathogen in the female genital tract causing complications during pregnancy. Since hitherto all black-pigmented C. aurimucosum strains have been recovered from female genital source, biosynthesis of the pigment is apparently required for colonization by protecting the bacterial cells against the high hydrogen peroxide concentration in the vaginal environment. The location of the corresponding genes on plasmid pET44827 explains why black-pigmented (formerly C. nigricans) and non-pigmented C. aurimucosum strains were isolated from clinical specimens.

Figure 1

The complete genome of the clinical isolate C. aurimucosum ATCC 700975. (A), Plot of the C. aurimucosum ATCC 700975 chromosome. The circles represent from the outside: circle 1, DNA base position; circles 2 and 3, predicted protein-coding sequences transcribed clockwise and anticlockwise, respectively; circles 4 to 7, genes encoding orthologous proteins in C. diphtheriae NCTC 13129, C. jeikeium K411, C. urealyticum DSM7109, and C. kroppenstedtii DSM44385; circle 8, G+C content plotted using a 10-kb window; circle 9, G/C skew plotted using a 10-kb window. The predicted protein-coding sequences are coloured according to their functional classification into the Clusters of Orthologous Groups of proteins [93]. The genomic position of the putative prophages ϕCauri I and ϕCauri II is marked. (B), Fosmid scaffold of the C. aurimucosum ATCC 700975 chromosome. Individual fosmid clones used to build the genomic scaffold by terminal insert sequencing are represented as green segments. (C), Genetic map of plasmid pET44827 detected in C. aurimucosum ATCC 700975. The predicted protein-coding regions are shown by arrows indicating the direction of transcription. A direct repeat region is indicated as green box. Colour code: black, non-ribosomal peptide synthetase (NRPS) gene region; yellow, transposase genes and repetitive sequences; red, genes involved in plasmid replication and maintenance; blue, genes encoding hypothetical proteins.

Figure 2

Distribution of architecture imparting sequences in the chromosome of C. aurimucosum ATCC 700975 and nucleotide sequence of the deduced dif site. (A), Distribution of the architecture imparting sequences G(A/T/C)GGGGGA and (T/C)GGGGGAG on the leading and lagging strands of the C. aurimucosum ATCC 700975 chromosome. The deduced position of the dif locus at around 1.425 Mbp is indicated in the linear representation of the chromosome. The position of the origin of replication (oriC) is marked. (B), Nucleotide sequence of the dif region in the C. aurimucosum ATCC 700975 chromosome. The 28-bp sequence is aligned with the consensus sequence of actinobacterial dif sites [20]. Identical nucleotides of the alignment are marked with asterisks. Matches to non-conserved nucleotides in the dif consensus sequence are specifically marked (:). Abbreviations: V = {A, C, G}; N = {A, C, G, T}.

Figure 3

Synteny between the chromosome of C. aurimucosum ATCC 700975 and those from C. diphtheriae NCTC 13129, C. jeikeium K411, C. urealyticum DSM7109, and C. kroppenstedtii DSM44385. The graphs represent X-Y plots of dots forming syntenic regions between the chromosomes of pathogenic corynebacteria. Each dot represents a predicted C. aurimucosum ATCC 700975 protein having an ortholog in another corynebacterial genome with co-ordinates corresponding to the position of the respective coding region in each genome. Orthologous proteins were detected by reciprocal best BLASTP matches. The genomic position of the putative prophages ϕCauriI and ϕCauriII is marked in the synteny plot with C. diphtheriae NCTC 13129.

Figure 4

Reconstruction of pathways involved in the central metabolism of C. aurimucosum ATCC 700975. The metabolic reconstruction of the carbohydrate metabolism was facilitated by using manually curated pathway maps in conjunction with the bioinformatics tool CARMEN. The resulting reconstruction was visualized with the CellDesigner software (version 3.2). Genes encoding transporters or enzymes involved in carbohydrate uptake and metabolism are shown in yellow boxes. Key metabolites are indicated by purple circles. Abbreviations for metabolites are as follows: α-Keto-G, α-ketoglutarate; β-Glcs, β-glycoside; 1,3PP-Gly, glycerate-1,3-bisphosphate; 2P-Gly, 2-phosphoglycerate; 3P-Gly, 3-phosphoglycerate; 5P-Ri-1PP, 5-phospho-ribose-1-diphosphate; 6P-β-Glcs, 6-phospho-β-glycoside; 6P-Gct, 6-phosphogluconate; 6P-Gll, 6-phosphogluconolactone; Aacetyl-CoA, acetoacetyl-CoA; Acetyl-P, acetyl-phosphate; CDP-ME, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol; CDP-ME2P, 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol; CHM, 5-carboxymethyl-2-hydroxy-muconic acid; CHMS, 5-carboxymethyl-2-hydroxy-muconic semialdehyde; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; Ery-4P, erythrose-4-phosphate; FattyA-CoA, fatty acyl-CoA; Fru-1,6PP, fructose-1,6-bisphosphate; Fru-1P, fructose-1-phosphate; Fru-6P, fructose-6-phosphate; GA-3P, glyceraldehyde-3-phosphate; Gal-1P, galactose-1-phosphate; Glc-1P, glucose-1-phosphate; Glc-6P, glucose-6-phosphate; GlcNAc, N-acetylglucosamine; GlcNAc-6P, N-acetylglucosamine-6-phosphate; Glu-6P, glucosamine-6-phosphate; Glycerol-3P, glycerol-3-phosphate; GPDE, glycerophosphodiester; HHDD, 2-hydroxy-hept-2,4-diene-1,7-dioic acid; HHED, 2,4-dihydroxy-hept-2-ene-1,7-dioic acid; HMBDP, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate; HPC, homoprotocatechuate; IPP, isopentenyl diphosphate; Man-1P, mannose-1-phosphate; Man-6P, mannose-6-phosphate; ManNAc-6P, N-acetyl-mannosamine-6-phosphate; MECDP, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; OHED, 2-oxo-hept-3-ene-1,7-dioic acid; OPET, 5-oxo-pent-3-ene-1,2,5-tricarboxylic acid; PEP, phosphoenolpyruvate; Ribose-5P, ribose-5-phosphate; Sed-7P, sedoheptulose-7-phosphate; Ribu-5P, ribulose-5-phosphate; Suc-CoA, succinyl-CoA; Sucrose-6P, sucrose-6-phosphate; Sucsemiald, succinate semialdehyde; Tre-6P, trehalose-6-phosphate; UDP-Gal, UDP-galactose; UDP-Glc, UDP-glucose; Xyu-5P, xylulose-5-phosphate.

Figure 5

Specific features of the C. aurimucosum ATCC 700975 metabolism detected by the analysis of singletons. (A), Genes and pathway for the catabolism of aromatic amines. Genetic maps of the hpa gene clusters encoding the HPC meta-cleavage pathway in C. aurimucosum and E. coli are presented. Genes with similar colour participate in the same enzymatic step of the pathway. The arrows show the direction of gene transcription. Abbreviations in the biochemistry of the pathway: DHPAL, 3,4-dihydroxyphenylacetaldehyde; HPC, homoprotocatechuate; CHMS, 5-carboxymethyl-2-hydroxy-muconic semialdehyde; CHM, 5-carboxymethyl-2-hydroxy-muconic acid; OPET, 5-oxo-pent-3-ene-1,2,5-tricarboxylic acid; HHDD, 2-hydroxy-hept-2,4-diene-1,7-dioic acid; OHED, 2-oxo-hept-3-ene-1,7-dioic acid; HHED, 2,4-dihydroxy-hept-2-ene-1,7-dioic acid. The enzymes are: HpaX, transport protein; HpaY, monoamine oxidase; PadA, phenylacetaldehyde dehydrogenase; HpaD, HPC 2,3-dioxygenase; HpaE, CHMS dehydrogenase; HpaF, CHM isomerase; HpaG, OPET decarboxylase; HpaH, hydratase; HpaI, HHED aldolase; GabD2, succinic semialdehyde dehydrogenase. (B), Genes and pathway for the catabolism of L-histidine. Genetics maps of the hut gene clusters coding for histidine catabolism in C. aurimucosum and Pseudomonas species. Genes with similar colour participate in the same enzymatic step of the pathway. The enzymes of the hut pathway are: HutH, histidine ammonia-lyase; HutU, urocanate hydratase; HutI, imidazolonepropionase; HutG, formimidoylglutamase. (C), Chromosomal gene region for selenol formate dehydrogenase and selenocysteine synthesis and incorporation in C. aurimucosum ATCC 700975. The UGA (opal) stop used for recoding the fdnG gene is indicated. The selC gene encodes the specific tRNA^Sec.

Figure 6

The putative SpaH-like pilus of C. aurimucosum ATCC 700975. (A), Genes clusters involved in the synthesis of adhesive pili in C. aurimucosum ATCC 700975 and C. diphtheriae NCTC 13129. Colour code: green, sortases required for the assembly of the pilus; red, major pilin; dark blue, minor pilin; light blue, tip protein; grey, protein of unknown function. Specifically marked are: pilin boxes (WPK), E-boxes (Ebox), sorting signals (LPXTG), hydrophobic domains in major pilins (hatched boxes), and charged tails in minor pilins (hatched boxes). (B), Model representation of the corynebacterial SpaH-like pilus. The pilus is covalently anchored to the corynebacterial cell wall [55]. Sorting motifs (S) and pilin motifs (P) used for the pilus assembly, as well as E-boxes (E) and sorting signals used for anchoring of the minor pilins (S') are marked.