The extracellular secretion of the antibacterial toxin colicin V is mediated via a signal sequence independent process which requires the products of two linked genes: cvaA and cvaB. The nucleotide sequence of cvaB reveals that its product is a member of a subfamily of proteins, involved in the export of diverse molecules, found in both eukaryotes and prokaryotes. This group of proteins, here referred to as the ‘MDR‐like’ subfamily, is characterized by the presence of a hydrophobic region followed by a highly conserved ATP binding fold. By constructing fusions between the structural gene for colicin V, cvaC, and a gene for alkaline phosphatase, phoA, lacking its signal sequence, it was determined that 39 codons in the N‐terminus of cvaC contained the structural information to allow CvaC‐PhoA fusion proteins to be efficiently translocated across the plasma membrane of Escherichia coli in a CvaA/CvaB dependent fashion. This result is consistent with the location of point mutations in the cvaC gene which yielded export deficient colicin V. The presence of the export signal at the N‐terminus of CvaC contrasts with the observed C‐terminal location of the export signal for hemolysin, which also utilizes an MDR‐like protein for its secretion. It was also found that the CvaA component of the colicin V export system shows amino acid sequence similarities with another component involved in hemolysin export, HlyD. The role of the second component in these systems and the possibility that other members of the MDR‐like subfamily will also have corresponding second components are discussed. A third component used in both colicin V and hemolysin extracellular secretion is the E. coli host outer membrane protein, TolC.

Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is the first non-patent report of the enzyme currently used for the manufacture of simvastatin, as well as the intermediate evolved variants. Crystal structures and microsecond molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations dramatically altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF.

In Vibrio fischeri, the autoinducer N-3-oxohexanoyl-L-homoserine lactone (AI-1) governs the cell densitydependent induction of the luminescence operon via the LuxR transcriptional activator. The synthesis of AI-1 from bacterial metabolic intermediates is dependent on luxI. Recently, we found a second V. fischeri autoinducer molecule, N-octanoyl-L-homoserine lactone (AI-2), that in E. coli also activates the luminescence operon via LuxR. A locus independent of luxI was identified as being required for AI-2 synthesis. This 2.7-kb ain (autoinducer) locus was characterized by transposon insertion mutagenesis, deletion and complementation analysis, and DNA sequencing. A single 1,185-bp gene, ainS, was found to be the sole exogenous gene necessary for the synthesis of AI-2 in Escherichia coli. In addition, a V. fischeri ainS mutant produced AI-1 but not AI-2, confirming that in its native species ainS is specific for the synthesis of AI-2. ainS is predicted to encode a 45,580-Da protein which exhibits no similarity to LuxI or to any of the LuxI homologs responsible for the synthesis of N-acyl-L-homoserine lactones in a variety of other bacteria. The existence of two different and unrelated autoinducer synthesis genes suggests the occurrence of convergent evolution in the synthesis of homoserine lactone signaling molecules. The C-terminal half of AinS shows homology to a putative protein in Vibrio harveyi, LuxM, which is required for the synthesis of a V. harveyi bioluminescence autoinducer. Together, AinS and LuxM define a new family of autoinducer synthesis proteins. Furthermore, the predicted product of another gene, ainR, encoded immediately downstream of ainS, shows homology to LuxN, which is similarly encoded downstream of luxM in V. harveyi and proposed to have sensor/regulator functions in the bioluminescence response to the V. harveyi autoinducer. This similarity presents the possibility that AI-2, besides interacting with LuxR, also interacts with AinR under presently unknown conditions.

The colicin V production and immunity genes were isolated from plasmid pColV-K30. A HindIH-to-SalI fragment of 9.4 kilobases was cloned into the compatible vectors pBR322 and pACYC184. Mutants defective in colicin production were generated by TnS insertions and by constructing deletions in vitro. Physical analysis of these mutations identified a 4.4-kilobase region of this DNA which contains all the plasmid genes (cva) needed for the production of colicin V. The colicin V immunity determinant (cvI) iS in a 700-base-pair fragment located within one end of this region. Complementation tests Identified three genes, called cvaA, cvaB, and cvaC, required for colicin production. Analysis of the proteins labeled in minicells harboring various TnS insertions allowed us to identify protein products for the cvaA and cvaC genes. Mutations in cvaA and cvaB eliminated colicin activity in culture supernatants, but not within the cells. Mutations in cvaC, however, eliminated all detectable activity. From these results we conclude that the cvaC gene codes for the structural gene for colicin V, while cvaA and cvaB are apparently needed for the normal export of the colicin.Colicin V is a small, proteinaceous toxin whose activity along with an immunity determinant is encoded on large, low-copy-number plasmids (14). ColV plasmids have been found naturally occuring in many strains of Escherichia coli and other members of the family Enterobacteriaceae. These bacteria also define the activity range for colicin V. Its target for growth inhibition is thought to be the cytoplasmic membrane, where it prevents the formation of membrane potential (32). ColV plasmids are often associated with E. coli invasiveness and pathogenicity (29,30). These plasmids also often carry genes which may enhance the ability of cells to proliferate within the host. Examples of these are the aerobactin iron uptake genes (31) and a gene for increased serum resistance (5). In addition, an enhanced adherence to intestinal epithelial cells has been noted in strains harboring ColV plasmids (11). Colicin V production does not appear to be a virulence determinant, but it has been hypothesized that it may help to selectively maintain these genes (26, 31).The colicin V toxin is distinguished from other colicins by the small size of the active protein (13) and by its constitutive, rather than SOS-inducible, synthesis (15). There is also no evidence that, like many colicins, colicin V accumulates in the cell before its release or has a lysis gene product responsible for its release (25).Frick et al. (13) cloned a 900-base-pair (bp) region of the pColV-B188 plasmid which included the colicin V immunity gene (cvi) and an apparent colicin V structural gene (cva). However, cells harboring this cloned fragment did not produce growth inhibition zones on a lawn of sensitive cells, and culture supematants did not contain assayable amounts of colicin. The killing activity coded by this 900-bp fragment could only be assayed after lysing the cells and appeared to be fourfold-less po...

By leveraging enzyme evolution technologies, the enantioselectivity of a KetoREDuctase (KRED) towards the nearly spatially symmetrical ketone tetrahydrothiophene-3-one was increased from 63% ee to 99.3% ee. The biocatalytic process gives (R)-tetrahydrothiophene-3-ol in one step from a commodity chemical and supplants the original multistep hazardous processes starting from the chiral pool. The biocatalytic process has been successfully scaled to 100 kg.