The qmeA (ts) mutation of Escherichia coli is localized in the fabI gene, which encodes enoyl-ACP reductase.

The phenotypes of temperature-sensitive qmeA and fabI mutants of Escherichia coli appear to be very similar. Furthermore, the qmeA mutation could be complemented by the fabI gene on a plasmid, and the fabI allele derived from the qmeA mutant strain harbours a nucleotide substitution identical to that from a previously characterized fabI mutant. These results show that the qmeA gene is, in fact, identical to the fabI gene, which encodes enoyl-ACP reductase, involved in fatty acid elongation.

Characterization of an Escherichia coli rotA mutant, affected in periplasmic peptidyl-prolyl cis/trans isomerase.

The rotA gene of Escherichia coli encodes a peptidyl-prolyl cis/trans isomerase (PPIase), which is supposed to catalyse protein folding in the periplasm. To investigate the importance of the enzyme, the rotA gene was cloned and a chromosomal deletion mutant was created. The rotA mutant was normally viable. No residual PPIase activity could be detected in the periplasmic fraction of the mutant. Comparison of the patterns of periplasmic and outer membrane proteins by SDS-PAGE revealed no differences in protein composition between the rotA mutant and its parental strain. Similarly, the kinetics of periplasmic protein folding and outer membrane protein assembly appeared unaffected by the rotA mutation. Our results show that the periplasmic PPIase of E. coli is not essential and that the protein does not play an important role in protein folding.

The assembly pathway of outer membrane protein PhoE of Escherichia coli.

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.

Sequence analysis of members of the human Ig VH4 gene family derived from a single VH locus. Identification of novel germ-line members.

We have generated a mouse x human heterohybridoma that contains a single copy of chromosome 14 and, thus, a haploid set of Ig VH genes. This cell line was used to investigate the germ-line content and nucleotide sequences of members of the VH4 gene family in a polymerase chain reaction-based approach. The analysis of 58 full-length sequences revealed the presence of 12 different germ-line VH4 genes, each of which is potentially functional. These germ-line VH4 genes were compared with the nucleotide sequences of published VH4 genes. Three VH4 genes were 100% identical to previously published sequences and belong to a group of VH4 genes that are strongly conserved and highly prevalent in the human population. Three VH4 genes in our collection displayed greater than 99.3% sequence identity with reported germ-line VH4 sequences and likely represent allelic counterparts of these genes. Six genes displayed less than 97.2% sequence identity with published VH4 genes and were identified as novel members of the human VH4 gene family or more distantly related alleles of known VH4 genes. Collectively, these data suggest that, overall, the human VH4 gene family may be more diverse than hitherto assumed, whereas a number of individual members are nonpolymorphic and extremely well conserved.