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Type IV secretory systems are a group of bacterial transporters responsible for the transport of proteins and nucleic acids directly into recipient cells. Such systems play key roles in the virulence of some pathogenic organisms and in conjugation-mediated horizontal gene transfer. Many type IV systems require conserved 'coupling proteins,' transmembrane polypeptides that are critical for transporting secreted substrates across the cytoplasmic membrane of the bacterium. In vitro evidence suggests that the functional form of coupling proteins is a homohexameric, ring-shaped complex. Using a library of tagged mutants, we investigated the structural and functional organization of the F plasmid conjugative coupling protein TraD by coimmunoprecipitation, cross-linking, and genetic means. We present direct evidence that coupling proteins form stable oligomeric complexes in the membranes of bacteria and that the formation of some of these complexes requires other F-encoded functions. Our data also show that different regions of TraD play distinct roles in the oligomerization process. We postulate a model for in vivo oligomerization and discuss the probable participation of individual domains of TraD in each step.

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies ~100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.

Taking advantage of a chaperone-like function of MalK, a stable complex of MalF-MalG could be solubilized from the Escherichia coli membrane and purified in high yield in the absence of MalK. This MalF-MalG complex was competent for efficient reassembly of a functional MalFG[K.sub.2] maltose transporter complex both in detergent solution and in proteoliposomes.

Research has been conducted on TraG-like proteins, important for DNA transfer in bacterial conjugation. The biochemical characterization of TraG-like protein family members has been carried out and the results indicate that these proteins tend to form oligomers and bind DNA without sequence specificity.

This paper discusses the use of two separate methods for investigating membrane protein topology in Escherichia coli. Combining gene fusion derived reporter hybrid protein method with that of a trypsin-sensitive 31-codon insertion technique, topology of the F factor TraD protein is presented.

Transposon-mediated insertion mutagenesis studies were undertaken to determine the permissive and non-permissive regions in the MalG component of the maltose transport complex of Escherichia coli. This transport complex consists of two integral membrane proteins, MalG and MalF and two copies of MalK, a peripheral cytoplasmic membrane protein. Insertion mappings reveal the permissive and non-permissive regions of MalG.

A simple method for the production of relatively large insertion mutations in cloned genes has been developed. A set of insertion mutants of the Escherichia coli lac repressor was studied to determine whether the analysis of such mutations can be used to study structure-function relationships in proteins. Findings are in agreement with the previous designation of mutationally tolerant spacer regions of LacI as well as proposed differences in dimerization interactions among regions of the protein core domain.

The effects of insertional mutations on different phenotypes associated with the MalK peripheral membrane protein were analyzed to characterize the assembly and folding of the MalFGK2 maltose transport complex. Insertional mutations of the malK gene in Escherichia coli cells affected the ability of the peripheral protein to associate with maltose permease integral membrane proteins such as MalF or MalG. Furthermore, insertions in specific regions of the MalK affected its different functions during MalFGK2 assembly.

INTRODUCTION The assembly of integral membrane proteins is a fundamental cellular activity that combines the processes of protein export, folding, and oligomerization. Numerous diseases are caused by mutant membrane proteins, [...]

A method simplifying the analysis of membrane protein topology by gene fusions was developed. The procedure involves the construction of gene fusions using the polymerase chain reaction on the C terminus of the hydrophilic region of the membrane protein. The procedure was tested on the Escherichia coli MalG protein, resulting in the confirmation of a topological model constructed by hydropathy analysis.

The nonequilibrium synthesis of Cu2O nanoparticles is accomplished using an engineered derivative of the DNA-binding protein Tral in a room-temperature precursor electrolyte. The results show that a protein with no intrinsic inorganic synthesis activity can be endowed with the ability to control the formation of inorganic nanostructures under thermodynamically unfavorable conditions, reproducing a key feature of biological hard-tissue growth and assembly.

Quorum-sensing (QS) systems allow organisms, such as the pathogen Pseudomonas aeruginosa, to link gene expression with their population density and the diffusion and flow characteristics of their environment. The leading hypotheses about QS systems' biological functions necessitate that QS-controlled gene expression be suppressed until a threshold culture density (the quorum) is reached. Despite a detailed understanding of QS in P. aeruginosa, known regulatory elements do not fully explain how the quorum threshold for gene activation is produced. Here we investigated the mechanism with a screening approach that used random gene activation. These experiments uncovered a regulator without close homologs in other species that produces the quorum expression threshold. Expression of this regulator (named QteE) reduces LasR protein stability without affecting LasR transcription or translation. QteE also independently reduces RhIR levels. Because QteE can block QS when signal levels are high, it could provide a mechanism for individual cells to exert autonomous control over their QS regulons. This unique regulator governs two central QS control points in P. aeruginosa and shapes the expression pattern thought funda- mental to the biological functions of QS. [ABSTRACT FROM AUTHOR]