Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea.

The hyperthermophilic anaerobic archaeon Pyrococcus abyssi, which lacks thymidine kinase, incorporates label from extracellular uracil, but not from thymidine, into its DNA. This implies that P. abyssi must synthesize dTMP (thymidylate), an essential precursor for DNA synthesis, de novo. However, iterative similarity searches of the three completed Pyrococcus genomes fail to detect candidate genes for canonical thymidylate synthase ThyA, suggesting the presence of alternative pathways for dTMP synthesis. Indeed, by identifying a novel class of flavin-dependent thymidylate synthases, ThyX, we have recently proven that two distinct pathways for de novo synthesis of dTMP are operational in the microbial world. While both thyX and thyA can be found in hyperthermophilic micro-organisms, the phylogenetic distribution of thyX among hyperthermophiles is wider than that of thyA. In this contribution, we discuss the differences in the distinct mechanisms of dTMP synthesis, with a special emphasis on hyperthermophilic micro-organisms.

In vivo expression of the mannose-resistant fimbriae of Photorhabdus temperata K122 during insect infection.

Photorhabdus temperata K122 is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae: Surface fimbriae are important for the colonization of many pathogenic bacteria, and here we report the nucleotide sequence and analysis of the expression of a 12-kbp fragment encoding the mannose-resistant fimbriae of P. temperata (mrf). The mrf gene cluster contains 11 genes with an organization similar to that of the mrp locus from Proteus mirabilis. mrfI (encoding a putative recombinase) and mrfA (encoding pilin), the first gene in an apparent operon of nine other genes, are expressed from divergent promoters. The mrfI-mrfA intergenic region contains inverted repeats flanking the mrfA promoter. This region was shown to be capable of inversion, consistent with an ON/OFF regulation of the operon. In in vitro liquid cultures, both orientations were detected. Nevertheless, when we analyzed the expression of all of the genes in the mrf locus by semiquantitative reverse transcription-PCR during infection of Galleria mellonella (greater wax moth) larvae, expression of mrfA was not detected until 25 h postinfection, preceding the death of the larvae at 32 h. In contrast, mrfJ (a putative inhibitor of flagellar synthesis) was expressed throughout infection. Expression of mrfI was also detected only late in infection (25 to 30 h), indicating a possible increase in inversion frequency at this stage. In both in vitro liquid cultures and in vivo larval infections, the distal genes of the operon were expressed at substantially lower levels than mrfA. These results indicate the complex regulation of the mrf cluster during infection.