Targeting the gram-negative bacteria peptidoglycan synthase MraY as a new approach for monoclonal antibody anti-bacterial activity.

The use of antibiotics to target bacteria is a well-validated approach for controlling infections in animals and humans. Peptidoglycan biosynthesis is a crucial process in bacteria, and the conserved peptidoglycan synthase MraY is an attractive target for drug design. However, due to the lack of detailed MraY structural information, antibiotics targeting MraY have not yet been developed. In the present study, 2 hydrophilic regions of MraY from Escherichia coli were expressed as a fusion protein and used to raise a monoclonal antibody in mice. We confirmed that the MraY amino acid sequence PESHFSKRGTPT forms the core epitope recognized by the monoclonal antibody M-H11. Furthermore, our results show that M-H11 effectively controls Escherichia coli BL21 (DE3) plysS infection, both in vitro and in vivo. Our results may be of great value in the search for novel approaches used to control bacterial infections.

DTDP-rhamnosyl transferase RfbF, is a newfound receptor-related regulatory protein for phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3.

Bacteriophages and their hosts are continuously engaged in evolutionary competition. Here we isolated a lytic phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3. We firstly described the phage receptor was regulated by DTDP-rhamnosyl transferase RfbF, encoded within the rfb cluster that was responsible for the biosynthesis of the O antigens. The deletion of DTDP-rhamnosyl transferase RfbF of wild type O:3 strain caused failure in phiYe-F10 adsorption; however, the mutation strain retained agglutination with O:3 antiserum; and complementation of its mutant converted its sensitivity to phiYe-F10. Therefore, DTDP-rhamnosyl transferase RfbF was responsible for the phage infection but did not affect recognition of Y. enterocolitica O:3 antiserum. Further, the deletions in the putative O-antigen biosynthesis protein precursor and outer membrane protein had no effect on sensitivity to phiYe-F10 infection. However, adsorption of phages onto mutant HNF10-ΔO-antigen took longer time than onto the WT, suggesting that deletion of the putative O-antigen biosynthesis protein precursor reduced the infection efficiency.

Involvement of lipoprotein PpiA of Streptococcus gordonii in evasion of phagocytosis by macrophages.

Streptococcus gordonii is a commensal gram-positive bacterium that resides in the human oral cavity, and is one of the most common causes of infective endocarditis (IE). Bacterial surface molecules play an important role in establishing IE, and several S. gordonii proteins have been implicated in binding to host cells during the establishment of IE. In this study, we identified a putative lipoprotein, peptidyl-prolyl cis/trans isomerase (PpiA), and clarified its role in evasion of phagocytosis by macrophages. Attenuation of the gene encoding prolipoprotein diacylglyceryl transferase (Lgt) altered the localization of PpiA from the cell surface to the culture supernatant, indicating that PpiA is lipid-anchored in the cell membrane by Lgt. Both human and murine macrophages showed higher phagocytic activity towards ppiA and lgt mutants than the wild-type, indicating that the presence of PpiA suppresses phagocytosis of S. gordonii. Human macrophages treated with dextran sulfate had significantly impaired phagocytosis of S. gordonii, suggesting that class A scavenger receptors in human macrophages are involved in the phagocytosis of S. gordonii. These results provide evidence that S. gordonii lipoprotein PpiA plays an important role in inhibiting phagocytic engulfment and in evasion of the host immune response.

PSGL-1 function in immunity and steady state homeostasis.

The substantial importance of P-selectin glycoprotein ligand 1 (PSGL-1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL-1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin-PSGL-1 interaction requires branched O-glycan extensions on specific PSGL-1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O-glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL-1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues of the branched O-glycans on PSGL-1 and provide the molecular interactions responsible for the velcro-like catch bonds that support leukocyte rolling. Such binding of PSGL-1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL-1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T-cell homing to secondary lymphoid organs. PSGL-1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O-glycan modifications of PSGL-1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL-1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T-cell traffic. The multi-level influence of PSGL-1 on cell traffic in both inflammatory and steady state settings is therefore substantially determined by the orchestrated addition of O-glycans. However, central as specific O-glycosylation is to PSGL-1 function, in vivo regulation of PSGL-1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL-1 glycosylation and to update understanding of PSGL-1 functional scope.

CD22 and Siglec-G: B-cell inhibitory receptors with distinct functions.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are sialic acid-binding proteins, which are expressed on many cell types of the immune system. B cells express two members of the Siglec family, CD22 (Siglec-2) and Siglec-G, both of which have been shown to inhibit B-cell signaling. CD22 recruits the tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) to immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and inhibits B-cell receptor (BCR)-induced Ca2+ signaling on normal B cells. CD22 interacts specifically with ligands carrying alpha2-6-linked sialic acids. Interaction with these ligands in cis regulates the association of CD22 with the BCR and thereby modulates the inhibitory function of CD22. Interaction of CD22 to ligands in trans can regulate both B-cell migration as well as the BCR signaling threshold. Siglec-G is a recently identified protein with an inhibitory function restricted to a B-cell subset, the B1 cells. Siglec-G inhibits Ca2+ signaling specifically in these cells. In addition, it controls the cellular expansion and antibody secretion of B1 cells. Thus, both Siglecs modulate BCR signaling on different B-cell populations in a mutually exclusive fashion.

Glycosylation in immune cell trafficking.

Leukocyte recruitment encompasses cell adhesion and activation steps that enable circulating leukocytes to roll, arrest, and firmly adhere on the endothelial surface before they extravasate into distinct tissue locations. This complex sequence of events relies on adhesive interactions between surface structures on leukocytes and endothelial cells and also on signals generated during the cell-cell contacts. Cell surface glycans play a crucial role in leukocyte recruitment. Several glycosyltransferases such as alpha1,3 fucosyltransferases, alpha2,3 sialyltransferases, core 2 N-acetylglucosaminlytransferases, beta1,4 galactosyltransferases, and polypeptide N-acetylgalactosaminyltransferases have been implicated in the generation of functional selectin ligands that mediate leukocyte rolling via binding to selectins. Recent evidence also suggests a role of alpha2,3 sialylated carbohydrate determinants in triggering chemokine-mediated leukocyte arrest and influencing beta1 integrin function. The recent discovery of galectin- and siglec-dependent processes further emphasizes the significant role of glycans for the successful recruitment of leukocytes into tissues. Advancing the knowledge on glycan function into appropriate pathology models is likely to suggest interesting new therapeutic strategies in the treatment of immune- and inflammation-mediated diseases.

Regulation of Notch signaling during T- and B-cell development by O-fucose glycans.

Notch signaling is required for the development of all T cells and marginal zone (MZ) B cells. Specific roles in T- and B-cell differentiation have been identified for different Notch receptors, the canonical Delta-like (Dll) and Jagged (Jag) Notch ligands, and downstream effectors of Notch signaling. Notch receptors and ligands are post-translationally modified by the addition of glycans to extracellular domain epidermal growth factor-like (EGF) repeats. The O-fucose glycans of Notch cell-autonomously modulate Notch-ligand interactions and the strength of Notch signaling. These glycans are initiated by protein O-fucosyltransferase 1 (Pofut1), and elongated by the transfer of N-acetylglucosamine (GlcNAc) to the fucose by beta1,3GlcNAc-transferases termed lunatic, manic, or radical fringe. This review discusses T- and B-cell development from progenitors deficient in O-fucose glycans. The combined data show that Lfng and Mfng regulate T-cell development by enhancing the interactions of Notch1 in T-cell progenitors with Dll4 on thymic epithelial cells. In the spleen, Lfng and Mfng cooperate to modify Notch2 in MZ B progenitors, enhancing their interaction with Dll1 on endothelial cells and regulating MZ B-cell production. Removal of O-fucose affects Notch signaling in myelopoiesis and lymphopoiesis, and the O-fucose glycan in the Notch1 ligand-binding domain is required for optimal T-cell development.

T-cell growth, cell surface organization, and the galectin-glycoprotein lattice.

Basal, activation, and arrest signaling in T cells determines survival, coordinates responses to pathogens, and, when dysregulated, leads to loss of self-tolerance and autoimmunity. At the T-cell surface, transmembrane glycoproteins interact with galectins via their N-glycans, forming a molecular lattice that regulates membrane localization, clustering, and endocytosis of surface receptors. Galectin-T-cell receptor (TCR) binding prevents ligand-independent TCR signaling via Lck by blocking spontaneous clustering and CD4-Lck recruitment to TCR, and in turn F-actin transfer of TCR/CD4-Lck complexes to membrane microdomains. Peptide-major histocompatibility complexes overcome galectin-TCR binding to promote TCR clustering and signaling by Lck at the immune synapse. Galectin also localizes the tyrosine phosphatase CD45 to microdomains and the immune synapse, suppressing basal and activation signaling by Lck. Following activation, membrane turnover increases and galectin binding to cytotoxic T-lymphocyte antigen-4 (CTLA-4) enhances surface expression by inhibiting endocytosis, thereby promoting growth arrest. Galectins bind surface glycoproteins in proportion to the branching and number of N-glycans per protein, the latter an encoded feature of protein sequence. N-glycan branching is conditional to the activity of Golgi N-acetylglucosaminyl transferases I, II, IV and V (Mgat1, 2, 4, and 5) and metabolic supply of their donor substrate UDP-GlcNAc. Genetic and metabolic control of N-glycan branching co-regulate homeostatic set-points for basal, activation, and arrest signaling in T cells and, when disturbed, result in T-cell hyperactivity and autoimmunity.

Lipoproteins are critical TLR2 activating toxins in group B streptococcal sepsis.

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Deltalgt and the Deltalsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Deltalgt and Deltalsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Deltalgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Deltalgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.

A monoclonal antibody against a carbohydrate epitope in lipopolysaccharide differentiates Chlamydophila psittaci from Chlamydophila pecorum, Chlamydophila pneumoniae, and Chlamydia trachomatis.

Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid (Kdo) of the sequence Kdo(2-->8)[Kdo(2-->4)] Kdo(2-->4)Kdo. After immunization with the synthetic neoglycoconjugate antigen Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4) Kdo-BSA, we obtained the mouse monoclonal antibody (mAb) S69-4 which was able to differentiate C. psittaci from Chlamydophila pecorum, C. pneumoniae, and C. trachomatis in double labeling experiments of infected cell monolayers and by enzyme-linked immunosorbent assay (ELISA). The epitope specificity of mAb S69-4 was determined by binding and inhibition assays using bacteria, LPS, and natural or synthetic Kdo oligosaccharides as free ligands or conjugated to BSA. The mAb bound preferentially Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4)Kdo(2-->4) with a K(d) of 10 microM, as determined by surface plasmon resonance (SPR) for the monovalent interaction using mAb or single chain Fv. Cross-reactivity was observed with Kdo(2-->4)Kdo(2-->4) Kdo but not with Kdo(2-->8)Kdo(2-->4)Kdo, Kdo disaccharides in 2-->4- or 2-->8-linkage, or Kdo monosaccharide. MAb S69-4 was able to detect LPS on thin-layer chromatography (TLC) plates in amounts of <10 ng by immunostaining. Due to the high sensitivity achieved in this assay, the antibody also detected in vitro products of cloned Kdo transferases of Chlamydia. The antibody can therefore be used in medical and veterinarian diagnostics, general microbiology, analytical biochemistry, and studies of chlamydial LPS biosynthesis. Further contribution to the general understanding of carbohydrate-binding antibodies was obtained by a comparison of the primary structure of mAb S69-4 to that of mAb S45-18 of which the crystal structure in complex with its ligand has been elucidated recently (Nguyen et al., 2003, Nat. Struct. Biol., 10, 1019-1025).

Characterization of the histo-blood group O(2) gene and its protein product.

BACKGROUND AND OBJECTIVES: This study aimed to show the full sequence and function of the O(2) allele, and investigate whether it accounts for the incompatible expression of A antigens in gastric carcinomas of blood group O persons. MATERIALS AND METHODS: By PCR, we determined the ABO genotype of group O subjects (76 gastric carcinoma patients and 165 blood donors). Two expression constructs, encoding either the putative soluble or full-length O(2) protein, were used to transfect Sf9 cells. The expression and the activity of the O(2) protein were analysed by immunohistochemistry and enzymatic assays, respectively. RESULTS: No significant difference was detectable between the O(2) allele frequency in gastric carcinoma patients (3.9%) and blood donors (4.2%). Sequencing analysis of the O(2) allele revealed an intact reading frame identical to that of A transferase except for four nucleotide substitutions. O(2)-transfected Sf9 cells and gastric carcinomas genotyped as O(1)O(2) both expressed a protein recognized by anti-A/B transferase monoclonal antibodies. In enzymatic assays, the O(2) protein failed to show measurable A transferase activity. CONCLUSION: The O(2) allele has an intact reading frame encoding a protein immunologically related to A/B transferases and enzymatically inactive. Further, our data gave no indication that the O(2) allele is related to the phenomenon of incompatible A antigen expression in gastric cancer.

Autoepitope mapping and reactivity of autoantibodies to the dihydrolipoamide dehydrogenase-binding protein (E3BP) and the glycine cleavage proteins in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial antibodies (AMA) directed primarily against the E2 subunits of the pyruvate dehydrogenase complex, the branched chain 2-oxo-acid dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, as well as the dihydrolipoamide dehydrogenase-binding protein (E3BP) of pyruvate dehydrogenase complex. The autoantibody response to each E2 subunit is directed to the lipoic acid binding domain. However, hitherto, the epitope recognized by autoantibodies to E3BP has not been mapped. In this study, we have taken advantage of the recently available full-length human E3BP complementary DNA (cDNA) to map this epitope. In addition, another lipoic binding protein, the H-protein of the glycine cleavage complex, was also studied as a potential autoantigen recognized by AMA. Firstly, the sequence corresponding to the lipoic domain of E3BP (E3BP-LD) was amplified by polymerase chain reaction and recombinant protein and then purified. Immunoreactivity of 45 PBC sera (and 52 control sera) against the purified recombinant E3BP-LD was analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Secondly, reactivity of PBC sera was similarly analyzed by immunoblotting against H-protein. It is interesting that preabsorption of patient sera with the lipoic acid binding domain of E3BP completely removed all reactivity with the entire protein by immunoblotting analysis, suggesting that autoantibodies to E3BP are directed solely to its lipoic acid binding domain. Fifty-three percent of PBC sera reacted with E3BP-LD, with the majority of the response being of the immunoglobulin G (IgG) isotype (95%). Surprisingly, there was little IgM response to the E3BP-LD suggesting that the immune response was secondary because of determinant spreading. In contrast, H-protein does not appear to possess (or expose) autoepitopes recognized by PBC sera. This observation is consistent with structural data on this moiety.

Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.

Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. The final specific activity of the purified enzyme was 1.12 micromol of NADH oxidized per min per mg of protein. The molecular weight of the native enzyme was determined to be 140,000. Sodium dodecyl sulfate-gel electrophoresis revealed a subunit of molecular weight 73,000. The optimum temperature and pH were 30 degrees C and 7.0, respectively. The enzyme was inactivated very rapidly at 70 degrees C. The enzyme required Mg2+ and thiamine pyrophosphate for maximal activity. Xylulose 5-phosphate was found to be the best substrate when formaldehyde was used as a glycoaldehyde acceptor. Erythrose 4-phosphate, glycolaldehyde, and formaldehyde were found to act as excellent substrates when xylulose 5-phosphate was used as a glycoaldehyde donor. The Kms for formaldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 microM, respectively. The enzyme produced dihydroxyacetone from formaldehyde and xylulose 5-phosphate. The enzyme was found to be expressed only in cells grown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.

Evaluation of an aroA mutant Salmonella typhimurium vaccine in chickens using modified semisolid Rappaport Vassiliadis medium to monitor faecal shedding.

In groups of chickens vaccinated orally or intramuscularly with a live aroA mutant Salmonella typhimurium vaccine strain and then experimentally inoculated with 10(8) CFU of wild type S. typhimurium or 10(9) CFU of S. enteritidis, faecal shedding of the vaccine and wild type strains was monitored by the buffered peptone water-modified semisolid Rappaport Vassiliadis medium method, which detected less than 10(2) CFU per gram of faeces. The vaccine strain was shed in the faeces for up to 26 days. Vaccination failed to reduce the faecal shedding of wild type S. typhimurium or S. enteritidis. The variation in the shedding patterns of chickens within each group was greater than between treatment groups.

Effects of seed-specific expression of a cytokinin biosynthetic gene on canola and tobacco phenotypes.

The Agrobacterium tumefaciens isopentenyl transferase gene (ipt), a cytokinin biosynthetic gene, was placed under the control of 1.9 kb of promoter sequence from the 2S albumin AT2S1 gene isolated from an Arabidopsis thaliana library. The construct was introduced into canola (Brassica napus) and tobacco (Nicotiana tabacum). ipt transcripts were followed during embryo development of transgenic plants by northern hybridizations. The phenotype of transformed plants from the T1 generation was analysed and we observed an increased branching of inflorescences in tobacco and canola plants expressing the ipt gene. Comparing with controls, the average number of capsules and siliques in AT2S1-ipt plants was 82.6 and 24.8% higher, respectively. This result was correlated with an increase in cytokinin levels in transgenic plants, as revealed by RIA. Indeed, cytokinin contents of T1 AT2S1-ipt B. napus seeds were found 2.2-fold higher than cytokinin contents of control seeds, and T1 AT2S1-ipt N. tabacum capsules contained 2.6-fold more cytokinins than control capsules. In tobacco, the average seed weight per capsule was lower in AT2S1-ipt plants while the seed number per silique and the average seed weight were not modified in canola carrying this construct. The average seed yield per plant was not significantly increased in AT2S1-ipt tobacco or canola plants.

Cross protective immunity conferred by a marker-free aroA mutant of Pasteurella multocida.

The aroA gene from Pasteurella multocida serotype A:1 (X-73) was cloned by complementation of the Escherichia coli aroA mutant AB2829 with a DNA library constructed in pUC18. The cloned aroA gene was inactivated by deletion of a 300 bp internal sequence and reintroduced by homologous recombination into the chromosome of X-73 and P-1059 (serotype A:3) using a Pasteurella-E. coli shuttle vector pPBA1100. By subjecting the transformed cells to repeated subculturing in the presence of antibiotic selection coupled with auxotrophic enrichment, marker-free aroA mutants of X-73 and of P-1059 were isolated and designated PMP1 and PMP3, respectively. PMP1 and PMP3 were highly attenuated and capable of conferring complete protection against subsequent lethal challenge infection in a mouse model. Moreover, PMP3-immunized mice were protected against heterologous challenge infection with serotype A:1 or A:4.

Estudio histoquímico de los cambios en la expresión de glicanos durante una gingivitis experimental en humanos / A histochemical study of glycan expression changes in a human experimental gingivitis

En tres voluntarios sanos se les indujo un modelo de gingivitis en la hemiarcada superior derecha (14 días sin ninguna medida de higiene bucal); la hemiarcada superior izquierda sirvió como control. Se tuvo el propósito de demostrar los patrones de expresión de diferentes residuos glicosídicos usando un grupo de ocho lectinas biotiniladas en encía sana y enferma. Se tomaron biopsias por congelación en los días 0, 7 y 14 del experimento y se procesaron con la técnica histoquímica de la inmunoperoxidasa. Fue posible determinar la adhesión selectiva de las lectinas a las diferentes estructuras de los tejidos epitelial y conectivo de la encía y se observó un cambio en la expresión de residuos sacáridos a medida que avanzaba la inflamación experimental, posiblemente atribuible a la creación de criptoepítopes mediante el clivaje del azúcar terminal de los glicanos, a la posible acción de una transferasa y a la especificidad de las lectinas por el grupo sanguíneo del paciente al que pertenecía el tejido bajo examen

Cloning, characterization, and heterologous expression of cDNAs for farnesyl diphosphate synthase from the guayule rubber plant reveals that this prenyltransferase occurs in rubber particles.

Two farnesyl diphosphate synthase (FPS) cDNA's from a guayule stembark library were isolated and characterized. Both encode M(r) 39,000 proteins containing 432 amino acids that differ slightly in their deduced molecular weights and isoelectric points. They both contain the DDXXD motifs that are characteristic of prenyltransferases, and both isoforms show high homology to other plant FPS sequences but less overall homology to FPS sequences from nonplant sources. The two isoforms differ by 5% in their amino acid sequence. When expressed in Escherichia coli, each guayule isoform exhibits high specific activity that produces farnesyl diphosphate as the major isoprenoid and small amounts of geranyl diphosphate. Biochemical and immunological evidence also indicates that FPS is associated with guayule rubber particles. Antibodies to chicken FPS cross-react with both guayule isoforms expressed in E. coli and recognize a low abundance M(r) 39,000 protein in rubber particles purified from guayule stembark. Guayule FPS sequences show high homology to peptide fragments of the prenyltransferase associated with rubber particles from Hevea brasiliensis, suggesting that this enzyme may be important for rubber biosynthesis in both species.

An aroA mutant of Yersinia pestis is attenuated in guinea-pigs, but virulent in mice.

This study describes a PCR-based approach for the production of a rationally attenuated mutant of Yersinia pestis. Degenerate primers were used to amplify a fragment encoding 91.45% of the aroA gene of Y. pestis MP6 which was cloned into pUC18. The remainder of the gene was isolated by inverse PCR. The gene was sequenced and a restriction map was generated. The Y. pestis aroA gene had 75.9% identity with the aroA gene of Yersinia enterocolitica. The cloned gene was inactivated in vitro and reintroduced into Y. pestis strain GB using the suicide vector pGP704. A stable aro-defective mutant. Y. pestis GB aroA, was isolated and its virulence was examined in vivo. The mutant was attenuated in guinea-pigs and capable of inducing a protective immune response against challenge with the virulent Y. pestis strain GB. Unusually for an aro-defective mutant, the Y. pestis aroA mutant was virulent in mice, with a median dose which induced morbidity of death similar to that of the wild-type, although time to death was significantly prolonged.