Genetic modifications to temperate Enterococcus faecalis phage Ef11 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection.

Ef11 is a temperate bacteriophage originally isolated by induction from a lysogenic Enterococcus faecalis strain recovered from an infected root canal, and the Ef11 prophage is widely disseminated among strains of E. faecalis. Because E. faecalis has emerged as a significant opportunistic human pathogen, we were interested in examining the genes and regulatory sequences predicted to be critical in the establishment/maintenance of lysogeny by Ef11 as a first step in the construction of the genome of a virulent, highly lytic phage that could be used in treating serious E. faecalis infections. Passage of Ef11 in E. faecalis JH2-2 yielded a variant that produced large, extensively spreading plaques in lawns of indicator cells, and elevated phage titres in broth cultures. Genetic analysis of the cloned virus producing the large plaques revealed that the variant was a recombinant between Ef11 and a defective FL1C-like prophage located in the E. faecalis JH2-2 chromosome. The recombinant possessed five ORFs of the defective FL1C-like prophage in place of six ORFs of the Ef11 genome. Deletion of the putative lysogeny gene module (ORFs 31-36) and replacement of the putative cro promoter from the recombinant phage genome with a nisin-inducible promoter resulted in no loss of virus infectivity. The genetic construct incorporating all the aforementioned Ef11 genomic modifications resulted in the generation of a variant that was incapable of lysogeny and insensitive to repressor, rendering it virulent and highly lytic, with a notably extended host range.

Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis.

The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.

Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440.

Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

Pseudomonas syringae Hrp type III secretion system and effector proteins.

Pseudomonas syringae is a member of an important group of Gram-negative bacterial pathogens of plants and animals that depend on a type III secretion system to inject virulence effector proteins into host cells. In P. syringae, hrp/hrc genes encode the Hrp (type III secretion) system, and avirulence (avr) and Hrp-dependent outer protein (hop) genes encode effector proteins. The hrp/hrc genes of P. syringae pv syringae 61, P. syringae pv syringae B728a, and P. syringae pv tomato DC3000 are flanked by an exchangeable effector locus and a conserved effector locus in a tripartite mosaic Hrp pathogenicity island (Pai) that is linked to a tRNA(Leu) gene found also in Pseudomonas aeruginosa but without linkage to Hrp system genes. Cosmid pHIR11 carries a portion of the strain 61 Hrp pathogenicity island that is sufficient to direct Escherichia coli and Pseudomonas fluorescens to inject HopPsyA into tobacco cells, thereby eliciting a hypersensitive response normally triggered only by plant pathogens. Large deletions in strain DC3000 revealed that the conserved effector locus is essential for pathogenicity but the exchangeable effector locus has only a minor role in growth in tomato. P. syringae secretes HopPsyA and AvrPto in culture in a Hrp-dependent manner at pH and temperature conditions associated with pathogenesis. AvrPto is also secreted by Yersinia enterocolitica. The secretion of AvrPto depends on the first 15 codons, which are also sufficient to direct the secretion of an Npt reporter from Y. enterocolitica, indicating that a universal targeting signal is recognized by the type III secretion systems of both plant and animal pathogens.

Reciprocal secretion of proteins by the bacterial type III machines of plant and animal pathogens suggests universal recognition of mRNA targeting signals.

Bacterial pathogens of both animals and plants use type III secretion machines to inject virulence proteins into host cells. Although many components of the secretion machinery are conserved among different bacterial species, the substrates for their type III pathways are not. The Yersinia type III machinery recognizes some secretion substrates via a signal that is encoded within the first 15 codons of yop mRNA. These signals can be altered by frameshift mutations without affecting secretion of the encoded polypeptides, suggesting a mechanism whereby translation of yop mRNA is coupled to the translocation of newly synthesized polypeptide. We report that the type III machinery of Erwinia chrysanthemi cloned in Escherichia coli recognizes the secretion signals of yopE and yopQ. Pseudomonas syringae AvrB and AvrPto, two proteins exported by the recombinant Erwinia machine, can also be secreted by the Yersinia type III pathway. Mapping AvrPto sequences sufficient for the secretion of reporter fusions in Yersinia revealed the presence of an mRNA secretion signal. We propose that 11 conserved components of type III secretion machines may recognize signals that couple mRNA translation to polypeptide secretion.

The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner.

We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways from Pseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22 degrees C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel the P. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name of hrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic.

Immunization of mice with a TolA-like surface protein of Trypanosoma cruzi generates CD4(+) T-cell-dependent parasiticidal activity.

The gene family encoding a trypomastigote-specific protein restricted to the part of the flagellum in contact with the cell body of the trypomastigote form of Trypanosoma cruzi has been isolated, characterized, and expressed in a baculovirus expression system. The gene family contains three tandemly repeated members that have 97 to 100% sequence identity. The predicted protein encoded by the gene family has both significant amino acid sequence identity and other physical and biological features in common with the TolA proteins of Escherichia coli and Pseudomonas aeruginosa. Based on these similarities, we have designated this gene family tolT. Immunization of mice with recombinant TolT generates a population of CD4(+) T lymphocytes that recognize T. cruzi-infected macrophages, resulting in the production of gamma interferon (IFN-gamma), which leads to NO production and a 50 to 60% reduction in parasite numbers compared to that seen with infected macrophages incubated with naive T cells. This population of T cells also produces both IFN-gamma and interleukin 2 (IL-2) but not IL-4 or IL-5 when incubated with spleen cells stimulated with TolT antigen, indicating that they are of the T-helper 1 type. T cells from mice chronically infected with T. cruzi also produce significant levels of IFN-gamma when cocultured with macrophages and either TolT protein or paraflagellar rod protein, indicating that both of these flagellar proteins produce positive T-cell responses in mice chronically infected with T. cruzi.

Cloning of a surface membrane glycoprotein specific for the infective form of Trypanosoma cruzi having adhesive properties to laminin.

Trypomastigotes of Trypanosoma cruzi express a set of surface glycoproteins known, collectively, as Tc-85. A monoclonal antibody to these proteins, named H1A10, inhibits (50-90%) in vitro parasite interiorization into host cells, thus implicating these glycoproteins in the infection process. Two DNA inserts, a genomic DNA fragment and a full-length cDNA encoding the H1A10 epitope, have now been cloned and characterized. Results show that both have high sequence identity with all reported members of the gp85/trans-sialidase gene family, although the H1A10 epitope exists only in the Tc-85 subset of the family. The epitope has been mapped by competition of antibody binding to a Tc-85 recombinant protein with peptides having sequences predicted by the Tc-85 DNA sequence, which contains also putative N-glycosylation sites and COOH-terminal glycosylphosphatidylinositol anchor insertion sites, as expected, since an N-glycan chain and a glycosylphosphatidylinositol anchor have been characterized previously in the Tc-85 subset. The protein encoded by the full-length cDNA insert binds to cells and in vitro to laminin, but not to gelatin or fibronectin, in a saturable manner. For the first time it was possible to assign a defined ligand to a sequenced glycoprotein belonging to the gp85 family. This fact, together with the reported binding of family members to cell surfaces, reinforces the hypothesis that this family encodes glycoproteins with similar sequences but differing enough as to bind to different ligands and thus forming a family of adhesion glycoproteins enabling the parasite to overcome the barriers interposed by cell membranes, extracellular matrices, and basal laminae.

Evidence for four distinct major protein components in the paraflagellar rod of Trypanosoma cruzi.

The major structural proteins present in the paraflagellar rod of Trypanosoma cruzi migrate on SDS-polyacrylamide gels as two distinct electrophoretic bands. The gene encoding a protein present in the faster migrating band, designated PAR 2, has been identified previously. Here we report the isolation and partial characterization of three genes, designated par 1, par 3, and par 4, that encode proteins present in the two paraflagellar rod protein bands. Peptide-specific polyclonal antibodies and monoclonal antibodies against the four proteins encoded by these genes shows that PAR 1 and PAR 3 are present only in the slower migrating paraflagellar rod band, and that PAR 2 and PAR 4 are present only in the faster migrating band. Analysis of the nucleotide sequence of these genes and the amino acid sequence of the conceptual proteins encoded by them indicates that par 2 shares high sequence similarity with par 3 and both are members of a common gene family, of which par 1 may be a distant member. Analysis of gene copy number and steady-state RNA levels suggest that the close stoichiometric ratio of the four PAR proteins is likely maintained by homeostatic regulation of RNA levels rather than gene dosage.

A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture.

The Hrp (type III protein secretion) system is essential for the plant parasitic ability of Pseudomonas syringae and most Gram-negative bacterial plant pathogens. AvrB and AvrPto are two P. syringae proteins that have biological activity when produced via heterologous gene expression inside plant cells or when produced by Hrp+ bacteria. Avr-like proteins, presumably injected by the Hrp system on bacterial contact with plant cells, appear to underlie pathogenic interactions, but none has been observed outside of the bacterial cytoplasm, and identifying novel genes encoding them is tedious and uncertain without a phenotype in culture. Here we describe a cloned Hrp secretion system that functions heterologously in Escherichia coli to secrete AvrB and AvrPto in culture and to promote AvrB and AvrPto biological activity in inoculated plants. The hrp gene cluster, carried on cosmid pCPP2156, was cloned from Erwinia chrysanthemi, a pathogen that differs from P. syringae in being host promiscuous. E. coli DH5alpha carrying pCPP2156, but not related Hrp-deficient cosmids, elicited a hypersensitive response in Nicotiana clevelandii only when also expressing avrB in trans. The use of pAVRB-FLAG2 and pAVRPTO-FLAG, which produce Avr proteins with a C-terminal FLAG-epitope fusion, enabled immunoblot detection of the secretion of these proteins to E. coli(pCPP2156) culture media. Secretion was Hrp dependent, occurred without leakage of a cytoplasmic marker, and did not occur with E. coli(pHIR11), which encodes a functional P. syringae Hrp system. E. coli(pCPP2156) will promote investigation of Avr protein secretion and systematic prospecting for the effector proteins underlying bacterial plant pathogenicity.

Translationally repressive RNA structures monitored in vivo using temperate DNA bacteriophages.

The RNA challenge phage system enables genetic selection of proteins with RNA-binding activity in bacteria. These phages are modified versions of the temperate DNA bacteriophage P22 in which post-transcriptional regulatory events control the developmental fate of the phage. The system was originally developed to identify novel RNA ligands that display reduced affinity for the R17/MS2 coat protein, as well as to select for suppressor coat proteins that recognize mutant RNA ligands. During the course of evaluating whether the HIV-1 Rev protein could direct lysogen development for bacteriophage derivatives that encode Rev response element (RRE) RNA sequences, two examples of RRE RNA ligands that interfere with challenge phage development were identified. In the phage examples described, RRE RNA secondary structure prevents Ant protein biosynthesis and lytic development. Phage lysogen formation occurs efficiently in recipient cells, independent of the expression status of the Rev protein or trans-acting competitor RRE RNA ligands. These studies provide the first example whereby RNA challenge phages may be applied to study RNA folding events and RNA structural interactions in an in vivo context.

Site-specific phosphorylation of the human immunodeficiency virus type-1 Rev protein accelerates formation of an efficient RNA-binding conformation.

Phosphorylation is important in the regulation of many cellular processes, yet the precise role of protein phosphorylation for many RNA-binding protein substrates remains obscure. In this report, we demonstrate that phosphorylation of a recombinant human immunodeficiency virus type-1 Rev protein promotes rapid formation of an efficient RNA-binding state. The apparent dissociation constant for ligand binding is enhanced 7-fold for the protein following phosphorylation; however, phosphate addition leads to a 1. 6-fold decrease in RNA ligand-protein complex stability. RNA ligand binding stimulates slow formation of an equally competent binding state for the unphosphorylated protein, indicating that the addition of phosphate or ligand binding promotes a similar conformational change in Rev. Phosphorylation directly alters the conformation of Rev, as revealed by modification experiments that monitor the solvent accessibility of cysteines in the protein. These biochemical properties are attributed to the addition of phosphate at one of two serine residues (Ser-54 or Ser-56) that lie within the multimerization domain adjacent to the RNA-binding helix. Glutaraldehyde-mediated cross-linking experiments revealed that phosphorylation of Rev does not affect Rev multimerization activity. The Rev protein from the less pathogenic HIV-2 isolate lacks this phosphorylation site in the amino acid sequence; thus, the described biochemical properties of the phosphorylated protein may contribute to Rev activity and possibly to HIV-1 virulence during natural infection.

Direct genetic selection of two classes of R17/MS2 coat proteins with altered capsid assembly properties and expanded RNA-binding activities.

RNA challenge phages are derivatives of bacteriophage P22 that enable direct genetic selection for a specific RNA-protein interaction. The bacteriophage P22 R17 encodes a wild-type R17 operator site and undergoes lysogenic development following infection of susceptible bacterial strains that express the R17/MS2 coat protein. A P22 R17 derivative with an OcRNA site (P22 R17 [A(-10)U]) develops lytically following infection of these strains. RNA challenge phages can be used to isolate second-site coat protein suppressors that recognize an OcRNA sequence by selecting for lysogens with a P22 R17 [Oc] phage derivative. The bacteriophage derivative P22 R17 [A(-10)U] was used in one such scheme to isolate two classes of genes that encode R17 coat proteins with altered capsid assembly properties and expanded RNA-binding characteristics. These mutations map outside the RNA-binding surface and include amino acid substitutions that interfere with interactions between coat protein dimers in the formation of the stable phage capsid. One class of mutants encodes substitutions at the highly conserved first and second positions of the mature coat protein. N-terminal sequence analysis of these mutants reveals that coat proteins with substitutions only at position 1 are defective in post-translational processing of the initiator methionine. All selected proteins possess expanded RNA-binding properties since they direct efficient lysogen formation for P22 R17 and P22 R17 [A(-10)U]; however, bacterial strains that express the protein mutants remain sensitive to lytic infection by other P22 R17 [Oc] bacteriophages. The described selection strategy provides a novel genetic approach to dissecting protein structure within RNA-binding proteins.

Functional recognition of fragmented operator sites by R17/MS2 coat protein, a translational repressor.

The R17/MS2 coat protein serves as a translational repressor of replicase by binding to a 19 nt RNA hairpin containing the Shine-Dalgarno sequence and the initiation codon of the replicase gene. We have explored the structural features of the RNA operator site that are necessary for efficient translational repression by the R17/MS2 coat protein in vivo . The R17/MS2 coat protein efficiently directs lysogen formation for P22 R17 , a bacteriophage P22 derivative that carries the R17/MS2 RNA operator site within the P22 phage ant mRNA. Phages were constructed that contain fragmented operator sites such that the Shine-Dalgarno sequence and the initiation codon of the affected gene are not located within the RNA hairpin. The wild-type coat protein directs efficient lysogen formation for P22 phages that carry several fragmented RNA operator sites, including one in which the Shine-Dalgarno sequence is positioned 4 nt outside the coat protein binding site. Neither the wild-type R17/MS2 coat protein nor super-repressor mutants induce lysogen formation for a P22 phage encoding an RNA hairpin at a distance of 9 nt from the Shine-Dalgarno sequence, implying that a discrete region of biological repression is defined by the coat protein-RNA hairpin interaction. The assembly of RNA species into capsid structures is not an efficient means whereby the coat protein achieves translational repression of target mRNA transcripts. The R17/MS2 coat protein exerts translational regulation that extends considerably beyond the natural biological RNA operator site structure; however, the coat protein still mediates repression in these constructs by preventing ribosome access to linear sequence determinants of the translational initiation region by the formation of a stable RNA secondary structure. An efficient translational regulatory mechanism in bacteria appears to reside in the ability of proteins to regulate RNA folding states for host cell and phage mRNAs.

Improved method for selecting RNA-binding activities in vivo.

RNA challenge phages are modified versions of bacteriophage P22 that allow one to select directly for a specific RNA-protein interaction in vivo. The original construction method for generating a bacteriophage that encodes a specific RNA target requires two homologous recombination reactions between plasmids and phages in bacteria. An improved method is described that enables one to readily construct RNA challenge phages through a single homologous recombination reaction in vivo. We have applied the new method to construct a derivative of P22R17, an RNA challenge phage that undergoes lysogenic development in bacterial cells that express the bacteriophage R17/MS2 coat protein.

Male-specific lethal 2, a dosage compensation gene of Drosophila, undergoes sex-specific regulation and encodes a protein with a RING finger and a metallothionein-like cysteine cluster.

In Drosophila the equalization of X-linked gene products between males and females, i.e. dosage compensation, is the result of a 2-fold hypertranscription of most of these genes in males. At least four regulatory genes are required for this process. Three of these genes, maleless (mle), male-specific lethal 1 (msl-1) and male-specific lethal 3 (msl-3), have been cloned and their products have been shown to interact and to bind to numerous sites on the X chromosome of males, but not of females. Although binding to the X chromosome is negatively correlated with the function of the master regulatory gene Sex lethal (Sxl), the mechanisms that restrict this binding to males and to the X chromosome are not yet understood. We have cloned the last of the known autosomal genes involved in dosage compensation, male-specific lethal 2 (msl-2), and characterized its product. The encoded protein (MSL-2) consists of 769 amino acid residues and has a RING finger (C3HC4 zinc finger) and a metallothionein-like domain with eight conserved and two non-conserved cysteines. In addition, it contains a positively and a negatively charged amino acid residue cluster and a coiled coil domain that may be involved in protein-protein interactions. Males produce a msl-2 transcript that is shorter than in females, due to differential splicing of an intron of 132 bases in the untranslated leader. Using an antiserum against MSL-2 we have shown that the protein is expressed at a detectable level only in males, where it is physically associated with the X chromosome. Our observations suggest that MSL-2 may be the target of the master regulatory gene Sxl and provide the basic elements of a working hypothesis on the function of MSL-2 in mediating the 2-fold increase in transcription that is characteristic of dosage compensation.

Identification of immunodominant epitopes in Trypanosoma cruzi trypomastigote surface antigen-1 protein that mask protective epitopes.

The gene that encodes trypomastigote surface Ag-1 (TSA-1), a major surface Ag of the bloodstream trypomastigote stage of Trypanosoma cruzi, was expressed in a baculovirus expression system. To determine the epitope(s) in TSA-1 that was recognized during T. cruzi infection and after immunization with TSA-1, subregions of the TSA-1 gene were expressed in a bacterial expression system. As seen by Western blotting, both mice and rabbits immunized with recombinant TSA-1 protein, as well as T. cruzi-infected mice, developed strong immune responses to the carboxyl-proximal region of TSA-1, but show no reaction to the amino-proximal portion of TSA-1. When mice were immunized with either recombinant TSA-1 protein or the carboxyl-proximal region of TSA-1, they did not survive challenge with 10(3) bloodstream trypomastigotes. However, 70% of the mice immunized with the amino-proximal portion of TSA-1 survived challenge with 10(3) bloodstream trypomastigotes. Thus, the immune responses elicited by recombinant TSA-1 or the carboxyl-proximal portion of TSA-1 are nonprotective during T. cruzi infection. In contrast, vaccination with the amino proximal region of TSA-1 elicits a protective immune response. These results suggest that responses to immunodominant epitope(s) within the carboxyl-proximal portion of TSA-1 mask epitopes within the amino-proximal portion that are capable of stimulating host-protective immune responses. It is suggested that immunodominant regions in surface molecules such as TSA-1 may provide a mechanism for the parasite to evade the host immune response by directing the response away from epitopes that have the potential to elicit a reaction that is damaging to the parasite.

Expression and evolution of members of the Trypanosoma cruzi trypomastigote surface antigen multigene family.

The trypomastigote specific surface antigens of Trypanosoma cruzi are encoded by a supergene family which includes the TSA family. The TSA family is characterized by the presence of a 27-bp tandem repeat array in the coding region. Here, we report the characterization and analysis of the three TSA family members in the Esmeraldo strain of the parasite. In this strain 2 distinct telomeric members are expressed abundantly as 3.7-kb mRNAs, while the remaining member is located at an internal chromosomal site and is expressed at less than 2% of the level seen for the telomeric members. Based on hybridization to DNA separated by PFGE, 3 chromosomes of sizes 1.8 Mb, 0.98 Mb, and 0.90 Mb each contain one of the telomeric members. In addition, the two smaller chromosomes also contain the single internal member. Since both chromosomes contain similar TSA family members, and vary only slightly in size, we suggest that they are homologues. Comparisons of the nucleotide sequences of the different members of the family show that the internal gene differs from the telomeric genes primarily in sequences found 3' of the repeat array. These comparisons also reveal that the three genes are analogous, supporting the hypothesis that short segments between the family members are exchanged by gene conversion events. We propose that similar conversion events between members of different gene families may generate some of the diversity found within the supergene family.