MtnK, methylthioribose kinase, is a starvation-induced protein in Bacillus subtilis

Abstract

Background: Methylthioadenosine, the main by-product of spermidine synthesis, is degraded in Bacillus subtilis as adenine and methylthioribose. The latter is an excellent sulfur source and the precursor of quorum-sensing signalling molecules. Nothing was known about methylthioribose recycling in this organism.

Results: Using trifluoromethylthioribose as a toxic analog to select for resistant mutants, we demonstrate that methylthioribose is first phosphorylated by MtnK, methylthioribose kinase, the product of gene mtnK (formerly ykrT), expressed as an operon with mtnS (formerly ykrS) in an abundant transcript with a S-box leader sequence. Although participating in methylthioribose recycling, the function of mtnS remained elusive. We also show that MtnK synthesis is boosted under starvation condition, in the following decreasing order: carbon-, sulfur- and nitrogen-starvation. We finally show that this enzyme is part of the family Pfam 01633 (choline kinases) which belongs to a large cluster of orthologs comprizing antibiotic aminoglycoside kinases and protein serine/threonine kinases.

Conclusions: The first step of methylthioribose recycling is phosphorylation by MTR kinase, coded by the mtnK (formerly ykrT) gene. Analysis of the neighbourhood of mtnK demonstrates that genes located in its immediate vicinity (now named mtnUVWXYZ, formerly ykrUVWXYZ) are also required for methylthioribose recycling.

Figure 1

Comparison of MtnK with the first eight proteins listed in the choline kinase family Pfam 01633. Identical residues in the family 01633 are indicated by a "=" sign, while conserved residues are indicated by a "+" sign (amino acid families are: ASTPG, MLIV, HKR, WYF, DNEQ, and C). When these same residues are also conserved in MtnK they are colored: red for identical residues, blue for conserved residues.

Figure 2

Alignment of the eight putative MtnK protein present in the genome libraries with B. subtilis MtnK together and with the Pfam 016533 consensus of choline kinases (CHLK_CONS) obtained after Blast search of the WWWDDL at the NCBI. Codes as in Figure 1. BACSU: B. subtilis; BACST: B. stearothermophilus; BACA: B. anthracis; TREDE: Treponema denticola; KLEPN: Klebsiella pneumoniae; MESLO: Mesorhizobium loti; SINME: Sinorhizobium meliloti; ARATH: Arabidopsis thaliana. The sequences were extracted from the databases presented in [14].

Figure 3

Autoradiograph showing the outcome of the MTR kinase assay. The assay was carried out for 90 min at 37°C (see Materials and Methods). Lane 1 corresponds to [•³²P]ATP as standard; lanes 2, 4 and 6: no MTR was added for the reactions with cell-free extracts of wild type, BFS1850 and BFS1850 grown with IPTG, respectively; lanes 3, 5 and 7: reaction performed in the presence of MTR and cell-free extracts of wild type, BFS1850 and BFS1850 grown with IPTG, respectively.

Figure 4

Autoradiograph of the MTR kinase assay with increasing concentrations of substrate. The assay was carried out for 90 min at 37°C with cell-free extracts of wild type (see Materials and Methods). Lane 1 corresponds to [³²P]ATP as standard; lanes 2: no MTR added; lanes 3, 4, 5, 6 and 7: reaction performed with increasing concentrations of MTR (20, 40, 80, 160 and 320 μM, respectively). Saturation is observed at 160 μM.