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The ActA protein of Listeria monocytogenes is an essential virulence factor and is required for intracellular bacterial motility and cell-to-cell spread. plcB, cotranscribed with actA, encodes a broad-specificity phospholipase C that contributes to lysis of host cell vacuoles and cell-to-cell spread. Construction of a transcriptional fusion between actA-plcB and the green fluorescent protein gene of Aequorea victoria has facilitated the detailed examination of patterns of actA/plcB expression within infected tissue culture cells. actA/plcB expression began approximately 30 min postinfection and was dependent upon entry of L. monocytogenes into the host cytosol. L. monocytogenes Deltahly mutants, which are unable to escape from host cell vacuoles, did not express actA/plcB at detectable levels within infected tissue culture cells; however, complementation of the hly defect allowed entry of the bacteria into the host cytoplasm and subsequent actA/plcB expression. These results emphasize the ability of L. monocytogenes to sense the different host cell compartment environments encountered during the course of infection and to regulate virulence gene expression in response.

We have developed an affinity column to study the interaction of LexA repressor and other substrates with the activated form of RecA protein. Nucleoprotein complexes of RecA protein, (dT)25-30, and adenosine 5'-[gamma-S]thio-triphosphate were formed in solution and bound to RecA protein-agarose columns. These "activated"-RecA nucleoprotein complexes were retained by strong hydrophobic interactions. Purified LexA protein bound tightly to these activated RecA columns, whereas the LexA protein bound poorly to RecA-agarose alone. Once bound, LexA protein underwent specific proteolysis, and the fragments were released from the complex. The mutant LexA protein, LexA-SA119, which cannot carry out self-cleavage or RecA-mediated cleavage in solution, bound efficiently to the activated RecA column but was not cleaved, indicating that these columns can be used to identify residues involved in RecA-LexA binding. As an example of this use, nucleoprotein complexes were prepared using the RecA430 protein. In vivo the recA430 mutation blocks induction of the SOS response. LexA protein was not efficiently retained on the immobilized RecA430 complexes, suggesting that Gly-204 is required for efficient repressor binding. These results show that activated RecA affinity columns can be used to investigate the binding and cleaving properties of mutationally altered RecA and LexA proteins. Additionally, these activated RecA columns have been used to investigate binding interactions of phage lambda repressor, as well as the UmuC protein, which is required for chemical mutagenesis.

We have analyzed the nature of RecA protein-RecA protein interactions using an affinity column prepared by coupling RecA protein to an agarose support. When radiolabeled soluble proteins from Escherichia coli are applied to this column, only the labeled RecA protein from the extract was selectively retained and bound tightly to the affinity column. Efficient binding of purified 35S-labeled RecA protein required Mg2+, and high salt did not interfere with the binding of RecA protein to the column. Complete removal of the bound enzyme from the affinity column required treatment with guanidine HCl (5 M) or urea (8 M). These and other properties suggest that hydrophobic interactions contribute significantly to RecA protein subunit recognition in solution. Using a series of truncated RecA proteins synthesized in vitro, we have obtained evidence that at least some of the sequences involved in protein recognition are localized within the first 90 amino-terminal residues of the protein. Based on the observation that RecA proteins from three heterologous bacteria are specifically retained on the E. coli RecA affinity column, it is likely that this binding domain is highly conserved and is required for interaction and association of RecA protein monomers. Stable ternary complexes of RecA protein and single-stranded DNA were formed in the presence of the nonhydrolyzable ATP analog adenosine 5′-O-(thiotriphosphate) and applied to the affinity columns. Most of the complexes formed with M13 DNA could be eluted in high salt, whereas a substantial fraction of those formed with the oligonucleotide (dT)25-30 remained bound in high salt and were quantitatively eluted with guanidine HCl (5 M). The different binding properties of these RecA protein-DNA complexes likely reflect differences in the availability of a hydrophobic surface on RecA protein when it is bound to long polynucleotides compared to short oligonucleotides.

Prolonged incubation of native bovine brain calmodulin with S-adenosyl-L-[methyl-3H]methionine and protein carboxyl methyltransferase results in maximal methylation of only 1-2% of the calmodulin molecules. In contrast, calmodulin which has been subjected to a prior alkaline treatment (0.1 M NH4OH, 37 degrees C for 3 h) can be methylated to a level of 30-50%. This treatment is known to be effective in deamidating certain asparagine residues which lie in unstable sequences, particularly -Asn-Gly- sequences. Bovine brain calmodulin has three such sequences (Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255, 962-975). The enhancement of methylation by alkaline treatment is substantially reduced if calmodulin is first reacted with bis-(I,I-trifluoroacetoxy)iodobenzene, a reagent which converts the carboxamide group of asparagine and glutamine residues to the corresponding primary amines. Thus, protein carboxyl methyltransferase selectively modifies an abnormal form of calmodulin that is probably deamidated. These findings suggest that protein carboxyl methylation may play a role in the disposition of abnormal proteins which contain atypical, isomerized aspartyl residues arising via spontaneous deamidation of unstable asparagines.