In Escherichia coli secreted proteins must be maintained in an export-competent state before translocation across the cytoplasmic membrane. This function is carried out by a group of proteins called chaperones. SecB is the major chaperone that interacts with precursor proteins before their secretion. We report results indicating that the DnaK and DnaJ heat shock proteins are also involved in the export of several proteins, most likely by acting as their chaperones. Translocation of alkaline phosphatase, a SecB-independent protein, was inhibited in dnaK-and dnaJ-mutant strains, suggesting that export of this protein probably involves DnaK and DnaJ. In addition, DnaK and DnaJ play a critical role in strains lacking SecB. They are required both for viability and for the residual processing of the SecB-dependent proteins LamB and maltose-binding protein (MBP) seen in secB null strains. Furthermore, overproduction of DnaK and DnaJ permits strains lacking SecB to grow in rich medium and accelerates the processing of LamB and MBP. These results suggest that under conditions where SecB becomes limiting, DnaK and DnaJ probably substitute for SecB and facilitate protein export. This provides the cell with a mechanism to overcome a temporary imbalance in the secretion process caused by an abrupt expansion in the pool of precursor proteins.

The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ∼ 100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P araBAD promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome.

We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At

The DnaK, DnaJ, and GrpE heat shock proteins are required for motility of Escherichia coli. Cells deleted for dnaK or dnaJ, or with some mutations in the dnaK or grpE gene, are nonmotile, lack flagella, exhibit a 10- to 20-fold decrease in the rate of synthesis of flagellin, and show reduced rates of transcription of both the flhD master operon (encoding FlhD and FlhC) and the fliA operon (encoding sigma F). Genetic studies suggest that DnaK and DnaJ define a regulatory pathway affecting flhD and fliA synthesis that is independent of cyclic AMP-catabolite gene activator protein or the chemotaxis system.

Evidence is presented that alanine racemase activity in E. coli K12 is due to two distinct gene products. The predominant isozyme is inducible by either alanine stereoisomer and repressible by glucose. The gene dadX coding for its structure is located by the dadA gene determining the structure of D-amino acid dehydrogenase. The regulatory site for the expression of both genes, dadR, is located on the other side of dadA. The orientation of the dad operon established by multiple-point crosses and deletion mapping is as follows: fadR ...dadRAX ...hemA. The dadX alanine racemase activity is unusually refractory to changes of incubation temperature. It differs strikingly from that of the other isozyme, probably the product of the alr gene. The latter isozyme shows a typical dependence upon incubation temperature. The synthesis of alr alanine racemase is constitutive in respect of both alanine and glucose. In dadX mutants, in which alanine racemase activity equals only 15% of that in wild-type cells grown in the absence of an inducer or catabolite repressor, the dad operon cannot be induced by D-alanine. We presume, therefore, that L-alanine is involved more directly than D-alanine in dad operon regulation.