The clones from mucoid colonies were transferred to E. coli DH5α by triparental conjugation, and then reintroduced into strain Rm11105 to confirm the associated mucoid colony phenotype on YM agar. Five of these clones, designated Proteases inhibitor pCX92, pCX9M1, pCX9M3, pCX9M4, and pCX9M5, were found to exhibit unique BamH1 restriction patterns. PHB accumulation was confirmed in the transconjugants of all clones by PHB assay (Table 2) and by transmission electron

microscopy for the first clone isolated, pCX92 (Fig. 1). The differentiation of mucoid from dry colony phenotype on YM agar required close inspection, and the possibility of missing complemented colonies was a concern. We found that incorporation of 0.5 μg mL−1 Nile red into the YM agar (YM-NR) resulted in bright pink staining of PHB-producing colonies, with no staining of the colonies that did not produce PHB. Examination under long-wave UV light enhanced the fluorescence, but it was not necessary to differentiate NU7441 chemical structure between the PHB mutant and the wild-type colonies. The exoY∷Tn5 mutant Rm7055, in which the extracellular polysaccharide succinoglycan is not produced, formed colonies that were not mucoid on YM-NR. These dry colonies fluoresced brightly under UV illumination. Strain Rm11476, containing both exoY∷Tn5 and phaC∷Tn5-233 mutations, was constructed by transduction. On YM-NR, this

strain formed dry colonies that did not stain or fluoresce. This was found to be the best genetic background for the detection of PHB-accumulating clones, especially on densely populated plates, and was used to screen for complementing subclones of the originally isolated cosmid clones. BamH1 fragments were subcloned from the cosmid clones pCX92, pCXM4, 17-DMAG (Alvespimycin) HCl and pCXM5 individually into pBBR1MCS-5. Complementing subclones were identified after en masse conjugation

of transformants from E. coli DH5α into strain Rm11105 or Rm11476, screening transconjugants on YM-NR as described above. These subclones were subjected to in vitro mutagenesis with EZ∷TN 〈KAN-2〉 transposon to localize the complementing regions. Complete DNA sequences of the complementing BamH1 fragments were determined, facilitated by sequencing from the EZ∷TN 〈KAN-2〉 transposon insertions using transposon-specific primers, and from the ends of subcloned fragments using vector-specific primers. Thus, pMS1 carries a 16 456-bp fragment from pCX92, pMS2 carries a 5255-bp fragment from pCX9M4, and pMS3 carries a 5015-bp fragment from pCX9M5. In each case, analysis of the sequence confirmed the presence of phaC genes. The complete 33 810-bp sequence of pCX92 insert DNA was determined from a shotgun library prepared by cloning a partial Sau3A1 digest into vector pTZ19R. The identities of the nearest orthologs from a cultured organism and the predicted functions are presented in Table 3, with the relative gene orientations illustrated in Fig. 2.