We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli.

pulE, one of 14 genes specifically required for pullulanase secretion in Klebsiella oxytoca, codes for a putative nucleotide-binding protein. Subcellular fractionation indicated that the majority of PulE in Escherichia coli cells expressing all 14 secretion genes is mainly associated with the cytoplasmic membrane through both hydrophobic and non-hydrophobic interactions. Mutational analysis revealed that one of the two regions of PulE that are conserved in many nucleotide-binding proteins (Walker box A) is essential for pullulanase secretion. Likewise, mutations that removed aspartate residues from each of two regions immediately downstream from the Walker box A also reduced secretion. These aspartate-rich regions are highly conserved in all 16 known PulE homologues but not in any other nucleotide-binding proteins. Altogether, these results indicate that PulE might belong to a new family of nucleotide-binding proteins. The protein could not be cross-linked to the photoactivatable ATP analogue azido-ATP, however. Most pulE point or deletion mutations which prevented pullulanase secretion exhibited transdominance when expressed at high levels in cells producing wild-type PulE protein. Evidence presented suggests that PulE might be a homodimer.

An integration vector for use in Methanococcus voltae was constructed, based on the Escherichia coli vector pUC18. It carries the structural gene for puromycin transacetylase from Streptomyces alboniger, which is flanked by expression signals of M. voltae structural genes and hisA gene sequences of this bacterium. Transformed M. voltae cells are puromycin resistant. Several types of integration of the vector into the chromosome were found. Only one case was due to nonhomologous recombination. The integrated sequences were stable under selective pressure but were slowly lost in some cases in the absence of the selective drug. The vector could be excised from M. voltae chromosomal DNA, recircularized and transformed back into E. coli.