The cold-induced two-component system CBO0366/CBO0365 regulates metabolic pathways with novel roles in group I Clostridium botulinum ATCC 3502 cold tolerance

Abstract

The two-component system CBO0366/CBO0365 was recently demonstrated to have a role in cold tolerance of group I Clostridium botulinum ATCC 3502. The mechanisms under its control, ultimately resulting in increased sensitivity to low temperature, are unknown. A transcriptomic analysis with DNA microarrays was performed to identify the differences in global gene expression patterns of the wild-type ATCC 3502 and a derivative mutant with insertionally inactivated cbo0365 at 37 and 15°C. Altogether, 150 or 141 chromosomal coding sequences (CDSs) were found to be differently expressed in the cbo0365 mutant at 37 or 15°C, respectively, and thus considered to be under the direct or indirect transcriptional control of the response regulator CBO0365. Of the differentially expressed CDSs, expression of 141 CDSs was similarly affected at both temperatures investigated, suggesting that the putative CBO0365 regulon was practically not affected by temperature. The regulon involved genes related to acetone-butanol-ethanol (ABE) fermentation, motility, arsenic resistance, and phosphate uptake and transport. Deteriorated growth at 17°C was observed for mutants with disrupted ABE fermentation pathway components (crt, bcd, bdh, and ctfA), arsenic detoxifying machinery components (arsC and arsR), or phosphate uptake mechanism components (phoT), suggesting roles for these mechanisms in cold tolerance of group I C. botulinum. Electrophoretic mobility shift assays showed recombinant CBO0365 to bind to the promoter regions of crt, arsR, and phoT, as well as to the promoter region of its own operon, suggesting direct DNA-binding transcriptional activation or repression as a means for CBO0365 in regulating these operons. The results provide insight to the mechanisms group I C. botulinum utilizes in coping with cold.

FIG 1

Confirmation of DNA microarray results with quantitative reverse transcription-PCR (RT-qPCR). Correlation of log₂ fold changes in expression of cbo0751, cbo0753, cbo1407, cbo2226, cbo2227, cbo2525, cbo2847, cbo3199, and cbo3202 between C. botulinum ATCC 3502 cbo0365 mutant and the wild-type strain 1 h after a temperature downshift from 37 to 15°C observed in the DNA microarray (x axis) and RT-qPCR (y axis) experiments.

FIG 2

Mutants of several genes under putative regulation of CBO0365 and within related metabolic pathways show impaired growth at low temperature. (A to D) Average growth of C. botulinum ATCC 3502 wild type (WT) and mutants with insertionally inactivated cbo3199 (bcd) and cbo3202 (crt) encoding two central enzymes of the ABE fermentation pathway (A), cbo1407 (bdh) and cbo2847 (ctfA) encoding components of the ABE pathway (B), arsenical resistance operon components cbo0751 (arsC) and cbo0753 (arsR) (C), and cbo2525 (phoT) encoding a phosphate ABC transporter (D) at 17°C. Error bars denote the minimum and maximum values of five biological replicates.

FIG 3

Putative ABE fermentation pathway of C. botulinum ATCC 3502 and effect of inactivation of related components on cold tolerance. The genes putatively encoding the pathway components are inferred from homology to C. acetobutylicum ATCC 824. No homologues for C. acetobutylicum acetoacetate decarboxylase adc were discovered in the ATCC 3502 genome, suggesting inability of C. botulinum ATCC 3502 to produce acetone. Acidogenic reactions are presented with dashed, and solventogenic reactions with solid arrows. Bold arrows represent the central reactions of the pathway. The existence of reactions presented with red arrows is uncertain in C. botulinum ATCC 3502 due to missing acetone production pathway genes. Insertional inactivation of genes crt, bcd, ctfA, and bdh (blue background) resulted in deteriorated growth at 17°C.

FIG 4

RT-PCR was performed to show the transcriptional arrangement of the cbo0365 locus. PCR primer pairs were targeted to cbo0363-cbo0364 (A), cbo0364 alone (B), cbo0365 alone (C), and cbo0365-cbo0366 (D). Templates included cDNA from ATCC 3502 wild-type culture RNA synthesized with a primer targeted to the 3′ end of cbo0366 (lanes 1), no-RT control of RNA from ATCC 3502 wild-type culture (lanes 2), positive control; genomic DNA of ATCC 3502 wild type (lanes 3), and water (lanes 4). M, DNA molecular weight marker VIII (Roche Applied Science).

FIG 5

Phosphorylated CBO0365 binds in vitro to promoter regions of cbo0364, cbo3202 (crt), cbo0753 (arsR), and cbo2525 (phoT). (A to D) EMSA results obtained with 0 to 4 μM phosphorylated CBO0365 protein showing binding to double-stranded biotin-labeled DNA probes of putative promoter regions of cbo0364 (A), cbo3202 (B), cbo0753 (C), and cbo2525 (D). (E and F) No binding was observed to a short fragment directly upstream of cbo0753 (E) or to a negative-control fragment from the coding region of 16S rrn (F). No DNA-binding activity was observed for nonphosphorylated CBO0365 (A to E). Specificity was confirmed with addition of 200-fold molar excess of nonlabeled competitor probe.

FIG 6

Mutants of cbo0365, cbo0751 (arsC), and cbo0753 (arsR) show impaired resistance to sodium arsenite. (A and B) Average growth of C. botulinum ATCC 3502 wild type (WT) and mutants with insertionally inactivated cbo0365 or the arsenical resistance operon components cbo0751 (arsC) and cbo0753 (arsR) at 37°C in the presence of 0.1 mM sodium arsenite (A) and of C. botulinum ATCC 3502 wild type (WT) and a mutant with insertionally inactivated cbo0365 at 20°C in the presence of 0.1 mM sodium arsenite (B). Error bars denote the minimum and maximum values of three (A) or five (B) biological replicates.