The Putative Coupling Protein TcpA Interacts with Other pCW3-Encoded Proteins To Form an Essential Part of the Conjugation Complex

Conjugative plasmids encode antibiotic resistance determinants or toxin genes in the anaerobic pathogen Clostridium perfringens. The paradigm conjugative plasmid in this bacterium is pCW3, a 47-kb tetracycline resistance plasmid that encodes the unique tcp transfer locus. The tcp locus consists of 11 genes, intP and tcpA-tcpJ, at least three of which, tcpA, tcpF, and tcpH, are essential for the conjugative transfer of pCW3. In this study we examined protein-protein interactions involving TcpA, the putative coupling protein. Use of a bacterial two-hybrid system identified interactions between TcpA and TcpC, TcpG, and TcpH. This analysis also demonstrated TcpA, TcpC, and TcpG self-interactions, which were confirmed by chemical cross-linking studies. Examination of a series of deletion and site-directed derivatives of TcpA identified the domains and motifs required for these interactions. Based on these results, we have constructed a model for this unique conjugative transfer apparatus.Conjugation systems are important contributors to the dissemination of antibiotic resistance determinants and virulence factors. Extensive analysis of conjugative plasmids from gram-negative bacteria has led to the elucidation of a general mechanism of conjugative transfer (10, 22). In this process, the transferred DNA is processed by components of a relaxosome complex. Specifically, the DNA is nicked at the origin of transfer (oriT) by a relaxase, which remains covalently coupled to the transferred DNA strand. The single-stranded DNA complex then interacts with the coupling protein, a DNA-dependent ATPase that provides the energy to actively pump the DNA through the mating pair formation (Mpf) complex into the recipient cell (36). The coupling protein interacts with both DNA processing proteins and components of the Mpf complex (1, 4, 12, 35, 38). These interactions have been demonstrated using bacterial and yeast two-hybrid approaches as well as gel filtration, pull-down, and coimmunoprecipitation studies.The mechanism of conjugative transfer has yet to be precisely determined for conjugative plasmids from gram-positive bacteria although bioinformatics analysis has identified similar gene arrangements and conservation of gene sequences within the transfer regions encoded on conjugative plasmids identified from strains of streptococcal, staphylococcal, enterococcal, and lactococcal origin (15). It was proposed that gram-positive and gram-negative conjugation systems utilize a similar transfer mechanism (15).In the anaerobic pathogen Clostridium perfringens conjugative plasmids have been shown to encode antibiotic resistance genes or extracellular toxins (3, 8, 9, 18). Although the contribution of conjugation to disease dissemination has not been systematically evaluated, it has been proposed that transfer of the C. perfringens enterotoxin plasmid pCPF4969 to normal flora isolates of C. perfringens may contribute to the severity of disease caused by non-food-borne isolates of C. perfringens (9).The prototype conjugative plasmid in C. perfringens is the 47-kb tetracycline resistance plasmid, pCW3. The complete sequence of pCW3 has been determined, and its unique replication protein and conjugation locus have been identified (8). Bioinformatics analysis of this C. perfringens tcp conjugation locus identified several proteins with limited similarity to proteins encoded within the transfer region of the conjugative transposon, Tn916 (8). The role of the tcp locus in the transfer of pCW3 has been confirmed by isolation of independent tcpA, tcpF, and tcpH mutants and subsequent complementation studies (8, 29). Since the region that encompasses the tcp locus is conserved in all conjugative plasmids from C. perfringens (2, 3, 8, 9, 18, 27) and since divergent tcpA homologues can complement a pCW3tcpA mutant (29), it appears that the conjugative transfer of both antibiotic resistance and toxin plasmids from this bacterium utilizes a common but poorly understood mechanism. Note that the C. perfringens tcp conjugation locus is different from the transfer regions of conjugative plasmids from other gram-positive bacteria.We have recently shown that the essential conjugation protein TcpH, a putative membrane-associated Mpf complex component, is localized to the poles of C. perfringens cells, as is another essential conjugation protein, TcpF (37). TcpH has also been shown to interact with itself and with the pCW3-encoded TcpC protein (37). In this study we have focused on the essential conjugation protein TcpA. Since TcpA encodes an FtsK/SpoIIIE domain found in DNA translocases (8), it is proposed that TcpA is involved in the movement of DNA during conjugative transfer, fulfilling a role equivalent to that of coupling proteins in other conjugation systems. Like such proteins, TcpA encodes two N-terminal transmembrane domains (TMDs) and a C-terminal cytoplasmic region that contains three motifs predicted to be involved in ATP binding and hydrolysis (8). Our previous studies revealed that the conserved motifs, motif I (Walker A box), motif II (Walker B box), and motif III (RAAG box), are essential for the function of TcpA. The C-terminal 61 amino acids (aa), though not essential for TcpA function, were shown to be important for efficient transfer of pCW3, as were the putative TMDs (29).To further investigate pCW3 transfer and the role of TcpA in this process, we have used bacterial two-hybrid analysis to examine protein-protein interactions involving TcpA. Using this system, interactions were observed between TcpA and itself, TcpC, TcpG, and TcpH. In addition, TcpC and TcpG were also found to self-interact. By combining these data with other data generated in this laboratory (37), we have constructed a model for the conjugative transfer of pCW3.     

Single copies of subunits d, oligomycin-sensitivity conferring protein, and b are present in the Saccharomyces cerevisiae mitochondrial ATP synthase

In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology. We have previously demonstrated that intact mtATPase complexes incorporating a Hex6-tagged subunit can be isolated via Ni2+-nitrilotriacetic acid affinity chromatography (Bateson, M., Devenish, R. J., Nagley, P., and Prescott, M. (1996) Anal. Biochem. 238, 14-18). Strains were constructed in which Hex6-tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex6-tagged subunits. The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni2+-nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex6-tagged subunit. As only the Hex6-tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case.     

Subunit gamma-green fluorescent protein fusions are functionally incorporated into mitochondrial F1F0-ATP synthase,arguing against a rigid cap structure at the top of F1

We have investigated the question of the presence of a cap structure located at the top of the F(1) alpha(3)beta(3) hexamer of the yeast mitochondrial F(1)F(0)-ATP synthase complex. Specifically, we sought to determine whether the putative cap has a rigid structure and occludes the central shaft space formed by the alpha(3)beta(3) hexamer or alternatively whether the cap is more flexible permitting access to the central shaft space under certain conditions. Thus, we sought to establish whether subunit gamma, an essential component of the F(1) central stalk housed within the central shaft space and whose N and C termini would both lie beneath a putative cap, could be fused at its C terminus to green fluorescent protein (GFP) without loss of enzyme function. The GFP moiety serves to report on the integrity and location of fusion proteins containing different length polypeptide linkers between GFP and subunit gamma, as well as being a potential occluding structure in itself. Functional incorporation of subunit gamma-GFP fusions into ATP synthase of yeast cells lacking native subunit gamma was demonstrated by the ability of intact complexes to hydrolyze ATP and retain sensitivity to oligomycin. Our conclusion is that the putative cap structure cannot be an inflexible structure, but must be of a more flexible nature consistent with the accommodation of subunit gamma-GFP fusions within functional ATP synthase complexes.     

Evaluating the impact of biodiversity offsetting on native vegetation

Biodiversity offsetting is a globally influential policy mechanism for reconciling trade-offs between development and biodiversity loss. However, there is little robust evidence of its effectiveness. We evaluated the outcomes of a jurisdictional offsetting policy (Victoria, Australia). Offsets under Victoria's Native Vegetation Framework (2002–2013) aimed to prevent loss and degradation of remnant vegetation, and generate gains in vegetation extent and quality. We categorised offsets into those with near-complete baseline woody vegetation cover (“avoided loss”, 2702 ha) and with incomplete cover (“regeneration”, 501 ha), and evaluated impacts on woody vegetation extent from 2008 to 2018. We used two approaches to estimate the counterfactual. First, we used statistical matching on biophysical covariates: a common approach in conservation impact evaluation, but which risks ignoring potentially important psychosocial confounders. Second, we compared changes in offsets with changes in sites that were not offsets for the study duration but were later enrolled as offsets, to partially account for self-selection bias (where landholders enrolling land may have shared characteristics affecting how they manage land). Matching on biophysical covariates, we estimated that regeneration offsets increased woody vegetation extent by 1.9%–3.6%/year more than non-offset sites (138–180 ha from 2008 to 2018) but this effect weakened with the second approach (0.3%–1.9%/year more than non-offset sites; 19–97 ha from 2008 to 2018) and disappeared when a single outlier land parcel was removed. Neither approach detected any impact of avoided loss offsets. We cannot conclusively demonstrate whether the policy goal of ‘net gain’ (NG) was achieved because of data limitations. However, given our evidence that the majority of increases in woody vegetation extent were not additional (would have happened without the scheme), a NG outcome seems unlikely. The results highlight the importance of considering self-selection bias in the design and evaluation of regulatory biodiversity offsetting policy, and the challenges of conducting robust impact evaluations of jurisdictional biodiversity offsetting policies.     

A conference report from "Down Under": Talking autophagy at OzBio2010

OzBio2010 was held at the Melbourne Convention and Exhibition Centre, September 26 to October 1, 2010. This international conference catered to researchers in several fields having complementary interests including biochemistry, molecular biology, cell biology, plant physiology and health-related research. It was held under the auspices of two major international scientific societies, IUBMB and FAOBMB, and the ComBio2010 collective (representing nine professional societies and groupings from Australia). A number of pre-eminent speakers presented at plenary sessions and in a wide array of specialist symposia. One of the plenary sessions and a specialist symposium highlighted autophagy-related topics.     

Topology and proximity relationships of yeast mitochondrial ATP synthase subunit 8 determined by unique introduced cysteine residues.

We have used site-directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase). Unique cysteine residues were introduced at the N or C-terminus of Y8 by site-directed mutagenesis. Expression and targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex. The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol-specific nonpermeant and permeant reagents in both intact and lysed mitochondria. The data indicate that the N-terminus of Y8 is located in the intermembrane space of mitochondria whereas the C-terminus is located within the mitochondrial matrix. The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross-linking reagents, each with one thiol-specific reactive group and one nonspecific, photoactivatible reactive group. These experiments revealed the proximity of the C-terminal domain of Y8 to subunits d and f, and that of the N-terminal domain to subunit f. It is concluded that Y8 possesses a single transmembrane domain which extends across the inner membrane of intact mitochondria. As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observed cross-links, that Y8 may also be part of the stator stalk.