Is the CvaA protein, encoded within the colicin V export gene cvaA, required for colicin V transport?

The antibacterial peptide toxin colicin V is exported from Escherichia coli cells by a signal sequence-independent, ABC export system. Export requires at least three proteins-membrane fusion protein CvaA, ABC export protein CvaB, and outer membrane protein TolC. The cvaA gene also encodes a second protein, CvaA, initiated from an in-frame translational re-start within the cvaA coding sequence. To determine whether the internally encoded CvaA protein also functions in the export pathway, the putative start codons for CvaA were mutagenized, while maintaining CvaA function. Elimination of CvaA translation caused no change in colicin V export levels, indicating that the CvaA protein is not required in the secretion pathway.

Topology analysis of the colicin V export protein CvaA in Escherichia coli.

The antibacterial protein toxin colicin V is secreted from Escherichia coli cells by a dedicated export system that is a member of the multicomponent ATP-binding cassette (ABC) transporter family. At least three proteins, CvaA, CvaB, and TolC, are required for secretion via this signal sequence-independent pathway. In this study, the subcellular location and transmembrane organization of membrane fusion protein CvaA were investigated. First, a series of CvaA-alkaline phosphatase (AP) protein fusions was constructed. Inner and outer membrane fractionations of cells bearing these fusions indicated that CvaA is inner membrane associated. To localize the fusion junctions, the relative activities of the fusion proteins, i.e., the amounts of phosphatase activity normalized to the rate of synthesis of each protein, as well as the stability of each fusion, were determined. These results indicated that all of the fusion junctions occur on the same side of the inner membrane. In addition, the relative activities were compared with that of native AP, and the protease accessibility of the AP moieties in spheroplasts and whole cells was analyzed. The results of these experiments suggested that the fusion junctions occur within periplasmic regions of CvA. We conclude that CvaA is an inner membrane protein with a single transmembrane domain near its N terminus; the large C-terminal region extends into the periplasm. This study demonstrates the application of AP fusion analysis to elucidate the topology of a membrane-associated protein having only a single transmembrane domain.

Functional complementation between bacterial MDR-like export systems: colicin V, alpha-hemolysin, and Erwinia protease.

The antibacterial protein Colicin V (ColV) is secreted from gram-negative bacteria by a signal sequence-independent pathway. The proteins that mediate the export of ColV share sequence similarities with components from other signal sequence-independent export systems such as those for alpha-hemolysin (Hly) and Erwinia protease (Prt). We report here that the intact HlyBD export system can export active ColV from Escherichia coli strains lacking the ColV export proteins CvaA and CvaB. The individual Hly export genes complement mutations in their respective ColV homologs, but do so at a lower efficiency. When CvaA or CvaB is expressed along with the intact HlyBD exporter, the Cva export protein interferes with export of ColV through the HlyBD system. Gene fusions and point mutations in the ColV structural gene were used to define signals in ColV recognized by the Hly exporter. An export signal in ColV recognized by HlyBD is localized to the amino-terminal 57 amino acids of the protein. In addition, mutations in the ColV export signal differentially affect export through CvaAB and HlyBD, suggesting differences in signal specificity between the Cva and Hly systems. The three Erwinia protease export proteins can also export active ColV, and interference is seen when CvaA or CvaB is expressed along with the intact Prt exporter. Functional complementation is not reciprocal; alpha-hemolysin is not exported through either the ColV system or the Prt system.

Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.

New positive and negative regulators for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

The biosynthesis of most amino acids in Saccharomyces cerevisiae is coregulated. Starvation for a single amino acid results in the derepression of amino acid biosynthetic enzymes in many unrelated pathways. This phenomenon, known as general control, is mediated by both positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the identification and characterization of several new regulatory genes for this system, GCN6, GCN7, GCN8, GCN9, and GCD5. A mutation in the negative regulator GCD5 was isolated on the basis of its suppression of a gcn2 mutation. The effect of gcd5 is a posttranscriptional increase in histidine biosynthetic enzyme activity. Suppressors of gcd5 which are deficient in derepression were in turn isolated. Eight such mutations, defining four new positive regulatory genes (GCN6 through GCN9), were obtained. These mutations are recessive, confer sensitivity to multiple amino acid analogs, and result in decreased mRNA levels for genes under general control. The GCN6 and GCN7 gene products were shown to be positive regulators for transcription of the GCN4 gene, the most direct-acting positive regulator thus far identified. The interaction of GCN6 and GCN7 with GCN4 is fundamentally different from that of previously isolated GCN genes. It should also be noted that these gcn selections gave a completely different nonoverlapping set of mutations from earlier selections which relied on analog sensitivity. Thus, we may have identified a new class of GCN genes which are functionally distinct from GCN1 through GCN5.

A new negative control gene for amino acid biosynthesis in Saccharomyces cerevisiae.

Enzyme levels in multiple amino acid biosynthetic pathways in yeast are coregulated. This control is effected largely at the transcriptional level by a number of regulatory genes. We report the isolation and characterization of a new negative regulatory gene, GCD4, for this general control system. GCD4 mutations are recessive and define a single Medelian gene on chromosome III. A gcd4 mutation results in resistance to different amino acid analogs and elevated, but fully inducible, mRNA levels of genes under general control. Epistasis analysis indicates that GCD4 acts more directly than the positive regulators GCN1, GCN2, GCN3 and GCN5, but less directly than GCN4, on the transcription of the amino acid biosynthetic genes. These data imply that GCD4 is a negative regulator of the positive effector, GCN4. Although GCD4 occupies the same position relative to the GCN genes as other GCD genes, it produces a unique phenotype. These results illustrate the diversity of function of negative regulators in general control.