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.

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...

Summary Erwinia herbicola strain Eh1087 produces the broad‐spectrum phenazine antibiotic D ‐alanylgriseoluteic acid (AGA). In this report, a cluster of 16 ehp ( E rwinia h erbicola p henazine) plasmid genes required for the production of AGA by Eh1087 is described. The extent of the gene cluster was revealed by the isolation of 82 different Eh1087 AGA − mutants, all found to possess single mini‐Tn 5lacZ2 insertions within a 14 kbp DNA region. Additional transposon insertions that did not affect antibiotic production by Eh1087 were created to define the boundaries of the gene cluster. The size and location of genes between these boundaries were derived from a combination of DNA sequence analyses, minicell protein analyses and the correlation between mutation position and the production of coloured AGA intermediates by many ehp mutants. Precursor‐feeding and complementation experiments resulted in 15 ehp genes being assigned to one of four functional groups according to their role in the synthesis of AGA. Group 1 is required for the synthesis of the phenazine nucleus in the form of antibiotic precursor one (AP1, phenazine‐1,6‐dicarboxylic acid). Group 2 is responsible for conversion of AP1 to AP2, which is subsequently modified to AP3 (griseoluteic acid) and exported by the group 3 gene products. Group 4 catalyses the addition of D ‐alanine to AP3 to create AGA, independently of groups 1, 2 and 3. A gene that is divergently transcribed from the 15 AGA synthesis ehp genes confers resistance to AGA.

SummaryWe report the isolation of insertional mutations to the pstC and pstA genes of the phosphate-specific transport ( pst ) operon that results in loss of biofilm formation by Pseudomonas aureofaciens PA147-2. Consistent with the known roles of the Pst system in Escherichia coli and Pseudomonas aeruginosa, both P. aureofaciens pst mutants were demonstrated to have defects in inorganic phosphate (P i ) transport and repression of Pho regulon expression. Subsequently, biofilm formation by the wild type was shown to require a threshold concentration of extracellular P i . The two-component regulatory pair PhoR/PhoB is responsible for upregulation of Pho regulon expression in response to P i -limiting environments. By generating phoR mutants that were unable to express the Pho regulon, we were able to restore biofilm formation by P. aureofaciens in P i -limiting conditions. This result suggests that gene(s) within the Pho regulon act to regulate biofilm formation negatively in low-P i environments, and that phoR mutations uncouple PA147-2 from such regulatory constraints. Furthermore, the inability of pst mutants to repress Pho regulon expression accounts for their inability to form biofilms in non-limiting P i environments. Preliminary evidence suggests that the Pst system is also required for antifungal activity by PA147-2. During phenotypic analysis of pst mutants, we also uncovered novelties in relation to P i assimilation and Pho regulon control in P. aureofaciens.

A mini-TnlO-kan insertion mutation identified a gene in the chromosome of Escherichia coli required for colicin V production from plasmid pColV-K30. With the complete restriction map of E. coli, the mutation was rapidly mapped to 50.0 min, within the purF operon. Sequence analysis showed that the insertion occurred in a gene with no previously known function which is located directly upstream of purF. We designated this gene cvpA for colicin V production. The mutant requires adenine for growth, probably because of a polar effect on purF expression. However, an adenine auxotroph showed no defect in colicin V production, suggesting that the cvpA mutation is responsible for the effect on colicin V production. Two possible models of cvpAl allele function are discussed.Often, plasmid-borne genes require chromosomally coded molecules for their expression (7). By searching for mutations in the Escherichia coli chromosome that affect the expression of genes from such extrachromosomal elements, specific host-plasmid interactions can be identified and characterized. Therefore, a general search was initiated to identify chromosomal mutations in E. coli that reduce colicin V production from plasmid pColV-K30.Colicin V is a small extracellular protein toxin which kills sensitive cells by disrupting their membrane potential (26). Colicin V is produced from large, low-copy plasmids and requires four plasmid genes for synthesis, export, and immunity (5, 6). The cvaC gene is the structural gene for colicin V, and cvaA and cvaB are required for processing and export of the toxin through the inner and outer membranes; cvi confers immunity to the host cell. There are several stages at which host factors could play a role in colicin V production, and mutations that alter any of these functions should result in lowered levels of extracellular colicin V.In addition to studying the role of host factors in colicin V production, we also wanted to demonstrate the speed and ease with which mutations generated with transposon insertions can be physically and genetically mapped on the E. coli chromosome. Previously, approximate map positions of insertions could be assigned on the basis of interrupted mating experiments and P1 transductions (10, 13), but considerable work was required to map insertion mutations precisely. In this study, we used physical maps of a cloned insertion mutation to quickly locate the cvpA gene within the E. coli chromosome by using the complete physical restriction map of the E. coli chromosome (9). MATERIALS AND METHODSBacterial strains and plasmids. The strains and plasmids used in this study, their relevant characteristics, and their sources are listed in derivative which contains a TnJO inserted into a region nonessential for colicin V production or plasmid replication.Media and culture conditions. Liquid and solid media were prepared as described by Miller (14), and antibiotics were used at the following concentrations: ampicillin, 150 ,ug/ml; kanamycin, 25 ,ug/ml; nalidixic acid, 20 pg/ml; streptomycin, 100 ,u...