galK-based suicide vector mediated allelic exchange in Mycobacterium abscessus.

Mycobacterium abscessus is a fast-growing environmental organism and an important emerging pathogen. It is highly resistant to many antibiotics and undergoes a smooth to rough colony morphology change that appears to be important for pathogenesis. Smooth environmental strains have a glycopeptidolipid (GPL) on the surface, while certain types of clinical strains are often rough and lack this GPL, due to mutations in biosynthetic genes or the mmpL4b transporter gene. We report here the development and evaluation of an allelic exchange system for unmarked alleles in M. abscessus ATCC19977, using a suicide vector bearing the E. coli galK gene and 2-deoxygalactose counterselection. We describe here two variant galK suicide vectors, and demonstrate their utility in constructing a variety of mutants with deletion alleles of the mmpL4b GPL transporter gene, the mbtH GPL biosynthesis gene, the known ß-lactamase gene MAB_2875 and a putative ß-lactamase gene, MAB_2833. We also show that a novel allele of the E. coli aacC4 gene, conferring apramycin resistance (aacC41), can be used as a selectable marker in M. abscessus ATCC19977 at single copy.

Regulation of NAB2 mRNA 3'-end formation requires the core exosome and the Trf4p component of the TRAMP complex.

The nuclear exosome functions in a variety of pathways catalyzing formation of mature RNA 3'-ends or the destruction of aberrant RNA transcripts. The RNA 3'-end formation activity of the exosome appeared restricted to small noncoding RNAs. However, the nuclear exosome controls the level of the mRNA encoding the poly(A)-binding protein Nab2p in a manner requiring an A(26) sequence in the mRNA 3' untranslated regions (UTR), and the activities of Nab2p and the exosome-associated exoribonuclease Rrp6p. Here we show that the A(26) sequence inhibits normal 3'-end processing of NAB2 mRNA in vivo and in vitro, and makes formation of the mature 3'-end dependent on trimming of the transcript by the core exosome and the Trf4p component of the TRAMP complex from a downstream site. The detection of mature, polyadenylated transcripts ending at, or within, the A(26) sequence indicates that exosome trimming sometimes gives way to polyadenylation of the mRNA. Alternatively, Rrp6p and the TRAMP-associated Mtr4p degrade these transcripts thereby limiting the amount of Nab2p in the cell. These findings suggest that NAB2 mRNA 3'-end formation requires the exosome and TRAMP complex, and that competition between polyadenylation and Rrp6p-dependent degradation controls the level of this mRNA.