Mutant Host Research Articles

Isolation of Poly-3-Hydroxybutyrate Metabolism Genes from Complex Microbial Communities by Phenotypic Complementation of Bacterial Mutants

The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate D-3-hydroxybutyrate due to absence of the enzyme D-3-hydroxybutyrate dehydrogenase activity. Clones that conferred D-3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored D-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful.

Selective rather than inductive mechanisms favour specific replacement of Purkinje cells by embryonic cerebellar cells transplanted to the cerebellum of adult Purkinje cell degeneration (pcd) mutant mice

Cell replacement after neuronal degeneration in the adult CNS depends on the availability of specific cues to direct specification, differentiation and integration of newly born neurons into mature circuits. Following recent reports indicating that neurogenic signals may be reactivated in the adult injured CNS, here we asked whether such signals are expressed in the cerebellum after Purkinje cell degeneration. Thus, we compared the fate of embryonic cerebellar cells transplanted to the cerebella of adult wild-type and Purkinje cell degeneration (pcd) mutant mice. Donor cells were dissected from beta-actin-enhanced green fluorescent protein (EGFP) transgenic mice and transplanted as a single cell suspension. In both hosts, grafted cells generated all major cerebellar phenotypes, with a precise localization in the recipient cortex or white matter. Nevertheless, the phenotypic distributions showed striking quantitative differences. Most notably, in the pcd cerebellum there was a higher amount of Purkinje cells, while other phenotypes were less frequent. Analysis of cell proliferation by 5-bromo-2'-deoxyuridine (BrDU) incorporation revealed that in both hosts mitotic activity was strongly reduced shortly after transplantation, and virtually all donor Purkinje cells were actually generated before grafting. Together, these results indicate that some compensatory mechanisms operate in the pcd environment. However, the very low mitotic rate of transplanted cells suggests that the adult cerebellum, either wild-type or mutant, does not provide instructive neurogenic cues to direct the specification of uncommitted progenitors. Rather, specific replacement in mutant hosts is achieved through selective mechanisms that favour the survival and integration of donor Purkinje cells at the expense of other phenotypes.

The clock gene period in the medfly Ceratitis capitata

We have isolated the clock gene period (per) from the medfly Ceratitis capitata, one of the most economically important insect pest species. The overall pattern of conserved, non-conserved and functional domains that are observed within dipteran and lepidopteran per orthologues is preserved within the coding sequence. Expression analysis from fly heads revealed a daily oscillation in per mRNA in both light : dark cycles and in constant darkness. However PER protein levels from head extracts did not show any significant evidence for cycling in either of these two conditions. When the Ceratitis per transgene under the control of the Drosophila per promoter and 3'UTR was introduced into Drosophila per -null mutant hosts, the transformants revealed a low level of rescue of behavioural rhythmicity. Nevertheless, the behaviour of the rhythmic transformants showed some similarities to that of ceratitis, suggesting that Ceratitis per carries species-specific information that can evidently affect the Drosophila host's downstream rhythmic behaviour.

Genetic Evidence that GTP Is Required for Transposition of IS 903 and Tn 552 in Escherichia coli

Surprisingly little is known about the role of host factors in regulating transposition, despite the potentially deleterious rearrangements caused by the movement of transposons. An extensive mutant screen was therefore conducted to identify Escherichia coli host factors that regulate transposition. An E. coli mutant library was screened using a papillation assay that allows detection of IS903 transposition events by the formation of blue papillae on a colony. Several host mutants were identified that exhibited a unique papillation pattern: a predominant ring of papillae just inside the edge of the colony, implying that transposition was triggered within these cells based on their spatial location within the colony. These mutants were found to be in pur genes, whose products are involved in the purine biosynthetic pathway. The transposition ring phenotype was also observed with Tn552, but not Tn10, establishing that this was not unique to IS903 and that it was not an artifact of the assay. Further genetic analyses of purine biosynthetic mutants indicated that the ring of transposition was consistent with a GTP requirement for IS903 and Tn552 transposition. Together, our observations suggest that transposition occurs during late stages of colony growth and that transposition occurs inside the colony edge in response to both a gradient of exogenous purines across the colony and the developmental stage of the cells.

Design of Ligand Binding to an Engineered Protein Cavity Using Virtual Screening and Thermal Up-shift Evaluation

Proteins could be used to carry and deliver small compounds. As a tool for designing ligand binding sites in protein cores, a three-step virtual screening method is presented that has been optimised using existing data on T4 lysozyme complexes and tested in a newly engineered cavity in flavodoxin. The method can pinpoint, in large databases, ligands of specific protein cavities. In the first step, physico-chemical filters are used to screen the library and discard a majority of compounds. In the second step, a flexible, fast docking procedure is used to score and select a smaller number of compounds as potential binders. In the third step, a finer method is used to dock promising molecules of the hit list into the protein cavity, and an optimised free energy function allows discarding the few false positives by calculating the affinity of the modelled complexes. To demonstrate the portability of the method, several cavities have been designed and engineered in the flavodoxin from Anabaena PCC 7119, and the W66F/L44A double mutant has been selected as a suitable host protein. The NCI database has then been screened for potential binders, and the binding to the engineered cavity of five promising compounds and three tentative non-binders has been experimentally tested by thermal up-shift assays and spectroscopic titrations. The five tentative binders (some apolar and some polar), unlike the three tentative non-binders, are shown to bind to the host mutant and, importantly, not to bind to the wild type protein. The three-step virtual screening method developed can thus be used to identify ligands of buried protein cavities. We anticipate that the method could also be used, in a reverse manner, to identify natural or engineerable protein cavities for the hosting of ligands of interest.

Biogenesis, structure, and immune-suppressive effects of virus-like particles of a Drosophila parasitoid, Leptopilina victoriae

Drosophila melanogaster larvae are attacked by virulent strains of parasitoid wasps. Females of Leptopilina heterotoma produce virus-like particles (VLPs) that efficiently destroy lamellocytes, a major larval immune effector cell type. We report here that L. victoriae, a closely related wasp species, also produces VLPs that trigger immune suppression responses in fly hosts. We compare the ability of immune suppression of the two parasitoids using a mutant host strain hopscotch Tumorous-lethal ( hop Tum-l ). hop Tum-l larvae have two defects of hematopoietic origin: overproliferation of hemocytes and constitutive encapsulation of self-tissue by lamellocytes. The encapsulation phenotype is suppressed weakly by L. victoriae and strongly by L. heterotoma. In vitro studies on hop Tum-l lamellocytes show that VLP-containing fluid from either wasp species induces lamellocyte lysis, but with different kinetics. Previously undocumented precursors of L. victoriae VLPs are synthesized in the long gland and are first visible within canals connecting secretory cells to the long gland lumen. VLP assembly occurs in the lumen. VLPs show multiple electron-dense projections surrounding a central core. Maturing particles appear segmented, singly or in arrays, embedded in the reservoir matrix. In sections, mature particles are pentagonal or hexagonal; the polygon vertices extending into spikes. Our results suggest that L. victoriae is likely to promote immune suppression by an active mechanism that is mediated by VLPs, similar to that used by L. heterotoma.

Impaired Processing of DNA Photoproducts and Ultraviolet Hypermutability With Loss of p16INK4a or p19ARF

Reduced DNA repair has been linked to an increased risk of cutaneous malignant melanoma, but insights into the molecular mechanisms of that link are scarce. The INK4a/ARF (CDKN2a) locus, which codes for the p16(INK4a) and p19ARF proteins, is often mutated in sporadic and familial malignant melanoma, but it has not been directly associated with reduced DNA repair. We transfected unirradiated mouse fibroblast cells with UV-treated DNA to measure DNA repair in normal, p16INK4a mutant, p19ARF mutant, or double mutant mouse host cells. Loss of either p16(INK4a) or p19ARF reduced the ability of the cells to process UV-induced DNA damage, independent of cell cycle effects incurred by the loss. These results may further explain why INK4a/ARF mutations predispose to malignant melanoma, a UV-induced tumor.

The regulated long-term delivery of therapeutic proteins by using antigen-specific B lymphocytes

Memory lymphocytes are important mediators of the immune response. These cells are long-lived and undergo clonal expansion upon reexposure to specific antigen, differentiating into effector cells that secrete Ig or cytokines while maintaining a residual pool of memory T and B lymphocytes. Here, the ability of antigen-specific lymphocytes to undergo repeated cycles of antigen-driven clonal expansion and contraction is exploited in a therapeutic protocol aimed at regulating protein delivery. The principle of this strategy is to introduce genes encoding proteins of therapeutic interest into a small number of antigen-specific B lymphocytes. Output of therapeutic protein can then be regulated in vivo by manipulating the size of the responder population by antigen challenge. To evaluate whether such an approach is feasible, we developed a mouse model system in which Emu- and Iglambda-based vectors were used to express human erythropoietin (hEPO) gene in B lymphocytes. These mice were then immunized with the model antigen phycoerythrin (PE), and immune splenocytes (or purified PE-specific B lymphocytes) were adoptively transferred to normal or mutant (EPO-deficient) hosts. High levels of hEPO were detected in the serum of adoptively transferred normal mice after PE administration, and this responsiveness was maintained for several months. Similarly, in EPO-deficient anemic recipients, antigen-driven hEPO expression was shown to restore hematocrit levels to normal. These results show that antigen-mediated regulation of memory lymphocytes can be used as a strategy for delivering therapeutic proteins in vivo.

MosA, a Protein Implicated in Rhizopine Biosynthesis in Sinorhizobium meliloti L5-30, is a Dihydrodipicolinate Synthase

MosA is a gene product encoded on a pSym megaplasmid of Sinorhizobium meliloti L5-30. The gene is part of an operon reported to be essential for the synthesis of the rhizopine 3- O-methyl- scyllo-inosamine. MosA has been assigned the function of an O-methyltransferase. However, the reported sequence of this protein is very much like that of dihydrodipicolinate synthase (DHDPS), except for a 40 amino acid residue C-terminal domain. This similarity contradicts accepted ideas regarding structure–function relationships of enzymes. We have cloned and overexpressed the recombinant gene in Escherichia coli, and discovered that the reported sequence contains an error resulting in a frame-shift. The correct sequence contains a new stop codon, truncating the C-terminal 41 amino acid residues of the reported sequence. The expressed protein, bearing an N-terminal polyhistidine tag, catalyzes the condensation of pyruvate and aspartate β-semialdehyde efficiently, suggesting that this activity is not a side-reaction, but an activity for which this enzyme has evolved. Electro-spray mass spectrometry experiments and inhibition by l-lysine are consistent with the enzyme being a DHDPS. E. coli AT997, a mutant host normally requiring exogenous diaminopimelate for growth, could be complemented by transformation with a plasmid bearing the gene encoding MosA. A role for this enzyme in rhizopine synthesis cannot be ruled out, but is called into question.

One-eyed pinhead regulates cell motility independent of Squint/Cyclops signaling

In vertebrates, EGF-CFC factors are essential for Nodal signaling. Here, we show that the zygotic function of one-eyed pinhead, the zebrafish EGF-CFC factor, is necessary for cell movement throughout the blastoderm of the early embryo. During the blastula and gastrula stages, mutant cells are more cohesive and migrate slower than wild-type cells. Chimeric analysis reveals that these early motility defects are cell-autonomous; later, one-eyed pinhead mutant cells have a cell-autonomous tendency to acquire ectodermal rather than mesendodermal fates. Moreover, wild-type cells transplanted into the axial region of mutant hosts tend to form isolated aggregates of notochord tissue adjacent to the mutant notochord. Upon misexpressing the Nodal-like ligand Activin in whole embryos, which rescues aspects of the mutant phenotype, cell behavior retains the one-eyed pinhead motility phenotype. However, in squint;cyclops double mutants, which lack Nodal function and possess a more severe phenotype than zygotic one-eyed pinhead mutants, cells of the dorsal margin exhibit a marked tendency to widely disperse rather than cohere together. Elsewhere in the double mutants, for cells of the blastoderm and for rare cells of the gastrula that involute into the hypoblast, motility appears wild-type. Notably, cells at the animal pole, which are not under direct regulation by the Nodal pathway, behave normal in squint;cyclops mutants but exhibit defective motility in one-eyed pinhead mutants. We conclude that, in addition to a role in Nodal signaling, One-eyed pinhead is required for aspects of cell movement, possibly by regulating cell adhesion.

Homogeneity of the morphological groups of bacteriophages infecting Bacteroides fragilis strain HSP40 and strain RYC2056.

Bacteriophages infecting Bacteroides fragilis strains RYC2056 and HSP40 have been proposed as indicators of water quality. To accomplish this function, homogeneity of the group of phages detected by these strains is necessary to ensure that the final results are not due to the different kinetics of inactivation of the phages. To evaluate homogeneity, we observed by electron microscopy bacteriophages isolated from sewage with two Bacteroides fragilis strains (HSP40 and RYC2056). A predominant group of phages was observed, Siphoviridae with slightly curved tails. Detection of other minority groups, which could be present in the sample, was done with neutralization experiments by using antiserum against the majority group and with host mutants resistant to infection with the predominant phage. Although two other minority groups were observed, results showed that bacteriophages infecting B. fragilis strain HSP40 and strain RYC2056 form a homogeneous group, Siphoviridae with slightly curved tails being the most predominant in sewage.

Cooperative action of the NIT2 and NIT4 transcription factors upon gene expression in Neurospora crassa.

In Neurospora crassa, the nit-3 gene, which encodes nitrate reductase, an enzyme required for the utilization of inorganic nitrate, is subject to a high degree of genetic and metabolic regulation as a member of the nitrogen control circuit. The nit-3 gene promoter contains binding sites for a globally acting protein NIT2 and a pathway-specific protein NIT4. Expression of the nit-3 gene absolutely requires both the NIT2 and NIT4 transcription factors and only occurs under conditions of nitrogen source derepression and nitrate induction. In the sulfur control circuit, the cys-14 gene encodes sulfate permease II, which facilitates the assimilation of sulfate. Expression of cys-14 is strongly regulated by only a single positive-acting factor, CYS3. It was of interest to determine whether NIT2 or NIT4 alone was capable of turning on the expression of cys-14, since this structural gene is normally controlled by only one regulatory protein. NIT2- and/or NIT4-binding elements were introduced into the promoter of a wild-type cys-14 gene and these constructs were transformed into a cys-13(-) cys-14(-) mutant strain and into a nit-2(-) mutant host. We examined whether any of these cys-14 genes in these transformants could now be controlled as a nitrogen-regulated gene. Sulfate permease assays revealed that both NIT2 and NIT4 were required for cys-14 expression upon nitrate induction, while neither alone activated any detectable cys-14 expression. We thus conclude that neither NIT2 nor NIT4 is capable alone of activating gene expression in this context, but together they can cooperate to elicit strong activation.

Cellular Immune Response to Parasite Infection in the Drosophila Lymph Gland Is Developmentally Regulated

The mechanisms by which an organism becomes immune competent during its development are largely unknown. When infected by eggs of parasitic wasps, Drosophila larvae mount a complex cellular immune reaction in which specialized host blood cells, lamellocytes and crystal cells, are activated and recruited to build a capsule around the parasite egg to block its development. Here, we report that parasitization by the wasp Leptopilina boulardi leads to a dramatic increase in the number of both lamellocytes and crystal cells in the Drosophila larval lymph gland. Furthermore, a limited burst of mitosis follows shortly after infection, suggesting that both cell division and differentiation of lymph gland hemocytes are required for encapsulation. These changes, observed in the lymph glands of third-instar, but never of second-instar hosts, are almost always accompanied by dispersal of the anterior lobes themselves. To confirm a link between host development and immune competence, we infected mutant hosts in which development is blocked during larval or late larval stages. We found that, in genetic backgrounds where ecdysone levels are low (ecdysoneless) or ecdysone signaling is blocked (nonpupariating allele of the transcription factor broad), the encapsulation response is severely compromised. In the third-instar ecdysoneless hosts, postinfection mitotic amplification in the lymph glands is absent and there is a reduction in crystal cell maturation and postinfection circulating lamellocyte concentration. These results suggest that an ecdysone-activated pathway potentiates precursors of effector cell types to respond to parasitization by proliferation and differentiation. We propose that, by affecting a specific pool of hematopoietic precursors, this pathway thus confers immune capacity to third-instar larvae.

Non-cell autonomous requirement for thebloodlessgene in primitive hematopoiesis of zebrafish

Vertebrate hematopoiesis occurs in two distinct phases, primitive (embryonic) and definitive (adult). Genes that are required specifically for the definitive program, or for both phases of hematopoiesis, have been described. However, a specific regulator of primitive hematopoiesis has yet to be reported. The zebrafish bloodless (bls) mutation causes absence of embryonic erythrocytes in a dominant but incompletely penetrant manner. Primitive macrophages appear to develop normally in bls mutants. Although the thymic epithelium forms normally in bls mutants, lymphoid precursors are absent. Nonetheless, the bloodless mutants can progress through embryogenesis, where red cells begin to accumulate after 5 days post-fertilization (dpf). Lymphocytes also begin to populate the thymic organs by 7.5 dpf. Expression analysis of hematopoietic genes suggests that formation of primitive hematopoietic precursors is deficient in bls mutants and those few blood precursors that are specified fail to differentiate and undergo apoptosis. Overexpression of scl, but not bmp4 or gata1, can lead to partial rescue of embryonic blood cells in bls. Cell transplantation experiments show that cells derived from bls mutant donors can differentiate into blood cells in a wild-type host, but wild-type donor cells fail to form blood in the mutant host. These observations demonstrate that the bls gene product is uniquely required in a non-cell autonomous manner for primitive hematopoiesis, potentially acting via regulation of scl.

NhaG Na+H+ Antiporter Gene of Bacillus subtilis ATCC9372, Which Is Missing in the Complete Genome Sequence of Strain 168, and Properties of the Antiporter

We cloned a gene which enabled Escherichia coli mutant host cells lacking all of the major Na(+)/H(+) antiporters to grow in the presence of 0.2 M NaCl from chromosomal DNA of Bacillus subtilis ATCC9372. An Na(+)/H(+) antiport activity was observed with membrane vesicles prepared from E. coli cells possessing the cloned gene, but not with vesicles from the host cells. Lithium ion was also a substrate for the antiporter. We sequenced the cloned DNA and found one open reading frame (designated nhaG) preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence. The deduced amino acid sequence of NhaG suggested that it consisted of 524 residues and that the calculated molecular mass was 58.1 kDa. None of the bacterial Na(+)/H(+) antiporters so far reported, except NhaP of Pseudomonas aeruginosa and SynNhaP (NhaS1) of Synechocystis sp., showed significant sequence similarity with the NhaG. However, the NhaP, the SynNhaP, animal NHEs (Na(+)/H(+) exchangers), and some hypothetical Na(+)/H(+) antiporters of several organisms showed significant sequence similarities with the NhaG. Interestingly, the entire DNA region corresponding to the nhaG gene is missing in the reported complete genome sequence of B. subtilis strain 168. We detected a band that hybridized with the nhaG DNA in chromosomal DNA from B. subtilis ATCC9372 but not with that from strain 168. The missing DNA region (1,774 base pairs) is sandwiched by two identical sequences, TTTTCTT.

The pdx genetic marker adjacent to the chloramphenicol biosynthesis gene cluster in Streptomyces venezuelae ISP5230: functional characterization.

The pdx-4 mutation in Streptomyces venezuelae ISP5230 confers a growth requirement for pyridoxal (pdx) and is a marker for the genetically mapped cluster of genes associated with chloramphenicol biosynthesis. A gene regulating salvage synthesis of vitamin B6 cofactors in S. venezuelae was cloned by transforming a pdx-4 mutant host with the plasmid vector pDQ101 carrying a library of wild-type genomic DNA fragments, and by selecting for complementation of the host's pdx requirement. However, the corresponding replicative plasmid could not be isolated. Southern hybridizations and transduction analysis indicated that the complementing plasmid had integrated into the chromosome; after excision by a second crossover, the plasmid failed to propagate. To avoid loss of the recombinant vector, a pdx-dependent Streptomyces lividans mutant, KAA1, with a phenotype matching that of S. venezuelae pdx-4, was isolated for use as the cloning host. Introduction of pIJ702 carrying an S. venezuelae genomic library into S. lividans KAA1, and selection of prototrophic transformants, led to the isolation of a stable recombinant vector containing a 2.5 kb S. venezuelae DNA fragment that complemented requirements for pdx in both S. venezuelae and S. lividans mutants. Sequence analysis of the cloned DNA located an intact ORF with a deduced amino acid sequence that, in its central and C-terminal regions resembled type-I aminotransferases. The N-terminal region of the cloned DNA fragment aligned closely with distinctive helix-turn-helix motifs found near the N termini of GntR family transcriptional regulators. The overall deduced amino acid sequence of the cloned DNA showed 73% end-to-end identity to a putative GntR-type regulator cloned in cosmid 6D7 from the Streptomyces coelicolor A3(2) genome. This location is close to that of pdxA, the first pdx marker in S. coelicolor A3(2) identified and mapped genetically in Sir David Hopwood's laboratory. The S. venezuelae gene and S. coelicolor pdxA are postulated to be homologues regulating vitamin B6 coenzyme synthesis from pdx.

Characterization of an oxygen-dependent inducible promoter, thenar promoter ofEscherichia coli, to utilize in metabolic engineering

The nar promoters, whose transcription is maximally induced under microaerobic conditions in the presence of nitrate ion, were characterized in fed-batch culture to determine whether they can be used for metabolic engineering, by which overall production of valuable chemicals can be increased. For this purpose, we tested whether the expression level of a reporter gene, the lacZ gene from the nar promoter, could be maintained constant throughout the induction period by manipulation of dissolved oxygen (DO) levels at a given nitrate ion concentration. First, E. coli was grown under aerobic conditions (DO 80%) to absorbance at 600 nm (OD(600)) of 35, then the nar promoter was induced by reduction of DO to different levels, combined with different frequencies and duration of alternating microaerobic and aerobic conditions throughout the entire induction period. For a wild-type nar promoter (pMW61) in a mutant host E. coli with a mutation in the narG gene on the chromosome of the host (RK5265), it was possible to maintain production of beta-galactosidase activity per cell (specific beta-galactosidase activity) at a constant rate at 5000, 10,000, 15,000, and 20,000 Miller units, using different combinations of nitrate ion concentrations (0.1%, 0.5%, and 1%) and DO levels. In addition, it was possible to maintain production of specific beta-galactosidase activity at a constant rate at about 10,000 Miller units in the absence of nitrate ion when a nitrate-independent nar promoter (pMW618) in the narL(-) mutant of the W3110 E. coli strain (W3110narL(-)) was used. Based on these results, we conclude that the nar promoter system provides a convenient expression system for metabolic engineering as well as for maximal production of recombinant proteins under conditions of fed-batch culture.

The Yersinia Protein Kinase A Is a Host Factor Inducible RhoA/Rac-binding Virulence Factor

The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F 1F o ATP synthase

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli F 1F o ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b. The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.

Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment.

Toxoplasma gondii is an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of this pervasive cell recognition is not understood. We demonstrate here that binding to the substratum and to host cells is partially mediated by interaction with sulfated glycosaminoglycans (GAGs). Addition of excess soluble GAGs blocked parasite attachment to serum-coated glass, thereby preventing gliding motility of extracellular parasites. Similarly, excess soluble GAGs decreased the attachment of parasites to human host cells from a variety of lineages, including monocytic, fibroblast, endothelial, epithelial, and macrophage cells. The inhibition of parasite attachment by GAGs was observed with heparin and heparan sulfate and also with chondroitin sulfates, indicating that the ligands for attachment are capable of recognizing a broad range of GAGs. The importance of sulfated proteoglycan recognition was further supported by the demonstration that GAG-deficient mutant host cells, and wild-type cells treated enzymatically to remove GAGs, were partially resistant to parasite invasion. Collectively, these studies reveal that sulfated proteoglycans are one determinant used for substrate and cell recognition by Toxoplasma. The widespread distribution of these receptors may contribute to the broad host and tissue ranges of this highly successful intracellular parasite.