Péter Papp

The ntrPR Operon of Sinorhizobium meliloti Is Organized and Functions as a Toxin-Antitoxin Module

The chromosomal ntrPR operon of Sinorhizobium meliloti encodes a protein pair that forms a toxin-antitoxin (TA) module, the first characterized functional TA system in Rhizobiaceae. Similarly to other bacterial TA systems, the toxin gene ntrR is preceded by and partially overlaps with the antitoxin gene ntrP. Based on protein homologies, the ntrPR operon belongs to the vapBC family of TA systems. The operon is negatively autoregulated by the NtrPNtrR complex. Promoter binding by NtrP is weak; stable complex formation also requires the presence of NtrR. The N-terminal part of NtrP is responsible for the interaction with promoter DNA, whereas the C-terminal part is required for protein-protein interactions. In the promoter region, a direct repeat sequence was identified as the binding site of the NtrPNtrR complex. NtrR expression resulted in the inhibition of cell growth and colony formation; this effect was counteracted by the presence of the antitoxin NtrP. These results and our earlier observations demonstrating a less effective downregulation of a wide range of symbiotic and metabolic functions in the ntrR mutant under microoxic conditions and an increased symbiotic efficiency with the host plant alfalfa suggest that the ntrPR module contributes to adjusting metabolic levels under symbiosis and other stressful conditions.

Site-Specific Integrative Elements of Rhizobiophage 16-3 Can Integrate into Proline tRNA (CGG) Genes in Different Bacterial Genera

The integrase protein of the Rhizobium meliloti 41 phage 16-3 has been classified as a member of the Int family of tyrosine recombinases. The site-specific recombination system of the phage belongs to the group in which the target site of integration (attB) is within a tRNA gene. Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the 16-3 phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells. Here we report that a plasmid (pSEM167) carrying the attP element and the integrase gene (int) of the phage can integrate into the chromosomes of R. meliloti 1021 and eight other species. In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin. Multiple alignment of the sequences suggested that the location of the att core was different from that expected previously. The minimal attB was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA.

The bacterial attachment site of the temperate Rhizobium phage 16-3 overlaps the 3′ end of a putative proline tRNA gene

Bacteriophage 16-3 inserts its genome into the chromosome of Rhizobium meliloti strain 41 (Rm41) by site-specific recombination. The DNA regions around the bacterial attachment site (attB) and one of the hybrid attachment sites bordering the integrated prophage (attL) were cloned and their nucleotide sequences determined. We demonstrated that the 51 by region, where the phage and bacterial DNA sequences are identical, is active as a target site for phage integration. Furthermore it overlaps the 3′ end of a putative proline tRNA gene. This gene shows 79% similartiy to the corresponding proline tRNA-like genomic target sequence of certain integrative plasmids in Actinomycetes.

Identification of differentially expressed genes in the developing antler of red deer Cervus elaphus

Understanding the molecular mechanisms underlying bone development is a fundamental and fascinating problem in developmental biology, with significant medical implications. Here, we have identified the expression patterns for 36 genes that were characteristic or dominant in the consecutive cell differentiation zones (mesenchyme, precartilage, cartilage) of the tip section of the developing velvet antler of red deer Cervus elaphus. Two major functional groups of these genes clearly outlined: six genes linked to high metabolic demand and other five to tumor biology. Our study demonstrates the advantages of the antler as a source of mesenchymal markers, for distinguishing precartilage and cartilage by different gene expression patterns and for identifying genes involved in the robust bone development, a striking feature of the growing antler. Putative roles for “antler” genes that encode α-tropomyosine (tpm1), transgelin (tagln), annexin 2 (anxa2), phosphatidylethanolamine-binding protein (pebp) and apolipoprotein D (apoD) in intense but still controlled tissue proliferation are discussed.

THE ISOLATED N-TERMINAL DNA BINDING DOMAIN OF THE C REPRESSOR OF BACTERIOPHAGE 16-3 IS FUNCTIONAL IN DNA BINDING IN VIVO AND IN VITRO

SummaryThe 197 amino acid c repressor of the temperateRhizobium meliloti phage16-3 still regulates theOR operator of the phage after removal of its carboxyl terminal region. When cloned in the low-copy-number plasmid pGA46, a severely truncated variant (R1-77), which retains only the first 77 amino acids of the intact protein, repressed in vivo transcription from the phage promoterPR. When theR1-77 repressor was fused toE. coli β-galactosidase, the hybrid protein boundOR operator DNA in vitro. The behavior of fusion proteins derived from a point mutant is consistent with the assignment of DNA binding specificity to the amino-terminal region. Furthermore two repressor alleles bearingts mutations that mapped in theR1-77 region (near a helix-turn-helix motif) were also temperature sensitive for regulation of theOR site, while an 18 by “in frame” deletion mutant, which mapped in the carboxyl terminal segment, regulated theOR operator in wild-type fashion. The carboxyl terminal region of the repressor is however necessary for the control of lysogenic development of16-3.

Binding sites of different geometries for the 16-3 phage repressor

Prokaryotic repressor–operator systems provide exemplars for the sequence-specific interactions between DNA and protein. The crucial atomic contacts of the two macromolecules are attained in a compact, geometrically defined structure of the DNA–protein complex. The pitch of the DNA interface seems an especially sensitive part of this architecture because changes in its length introduce new spacing and rotational relations in one step. We discovered a natural system that may serve as a model for investigating this problem: the repressor of the 16-3 phage of Rhizobium meliloti (helix-turn-helix class protein) possesses inherent ability to accommodate to various DNA twistings. It binds the cognate operators, which are 5′-ACAA-4 bp-TTGT-3′ (OL) and 5′-ACAA-6 bp-TTGT-3′ (OR) and thus differ 2 bp in length, and consequently the two half-sites will be rotated with respect to each other by 72° in the idealized B-DNA (64° by dinucleotide steps calculations). Furthermore, a synthetic intermediate (DNA sequence) 5′-ACAA-5 bp-TTGT-3′ (O5) also binds specifically the repressor. The natural operators and bound repressors can form higher order DNA–protein complexes and perform efficient repression, whereas the synthetic operator-repressor complex cannot do either. The natural operators are bent when complexed with the repressor, whereas the O5 operator does not show bending in electrophoretic mobility assay. Possible structures of the complexes are discussed.

On the site specific recombination of phage 16-3 of Rhizobium meliloti: identification of genetic elements and att recombinations

SummaryThe genome segment carrying the activities int and xis, responsible for integration and excision of phage 16-3, have been identified and cloned. Mutants were isolated, permitting the investigation of int, xis and att sites (attP, attR, attB) in trans arrangements. The efficiency and role of int- and xis-promoted reactions and of homologous recombination in the formation of lysogenic cells are established. The possible use of the cloned int-attP chromosomal segment in the manipulation of Rhizobium meliloti is discussed.

Antler development and coupled osteoporosis in the skeleton of red deer Cervus elaphus: expression dynamics for regulatory and effector genes

Antlers of deer display the fastest and most robust bone development in the animal kingdom. Deposition of the minerals in the cartilage preceding ossification is a specific feature of the developing antler. We have cloned 28 genes which are upregulated in the cartilaginous section (called mineralized cartilage) of the developing (“velvet”) antler of red deer stags, compared to their levels in the fetal cartilage. Fifteen of these genes were further characterized by their expression pattern along the tissue zones (i.e., antler mesenchyme, precartilage, cartilage, bone), and by in situ hybridization of the gene activities at the cellular level. Expression dynamics of genes col1A1, col1A2, col3A1, ibsp, mgp, sparc, runx2, and osteocalcin were monitored and compared in the ossified part of the velvet antler and in the skeleton (in ribs and vertebrae). Expression levels of these genes in the ossified part of the velvet antler exceeded the skeletal levels 10–30-fold or more. Gene expression and comparative sequence analyses of cDNAs and the cognate 5′ cis-regulatory regions in deer, cattle, and human suggested that the genes runx2 and osx have a master regulatory role. GC–MS metabolite analyses of glucose, phosphate, ethanolamine-phosphate, and hydroxyproline utilizations confirmed the high activity of mineralization genes in governing the flow of the minerals from the skeleton to the antler bone. Gene expression patterns and quantitative metabolite data for the robust bone development in the antler are discussed in an integrated manner. We also discuss the potential implication of our findings on the deer genes in human osteoporosis research.

Identification of Tail Genes in the Temperate Phage 16-3 of Sinorhizobium meliloti 41

Genes encoding the tail proteins of the temperate phage 16-3 of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 41 have been identified. First, a new host range gene, designated hII, was localized by using missense mutations. The corresponding protein was shown to be identical to the 85-kDa tail protein by determining its N-terminal sequence. Electron microscopic analysis showed that phage 16-3 possesses an icosahedral head and a long, noncontractile tail characteristic of the Siphoviridae. By using a lysogenic S. meliloti 41 strain, mutants with insertions in the putative tail region of the genome were constructed and virion morphology was examined after induction of the lytic cycle. Insertions in ORF017, ORF018a, ORF020, ORF021, the previously described h gene, and hII resulted in uninfectious head particles lacking tail structures, suggesting that the majority of the genes in this region are essential for tail formation. By using different bacterial mutants, it was also shown that not only the RkpM and RkpY proteins but also the RkpZ protein of the host takes part in the formation of the phage receptor. Results for the host range phage mutants and the receptor mutant bacteria suggest that the HII tail protein interacts with the capsular polysaccharide of the host and that the tail protein encoded by the original h gene recognizes a proteinaceous receptor.

A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16-3 is functional and convertible to suppressor tRNA.

Several temperate bacteriophage utilize chromosomal sequences encoding putative tRNA genes for phage attachment. However, whether these sequences belong to genes which are functional as tRNA is generally not known. In this article, we demonstrate that the attachment site of temperate phage 16‐3 (attB) nests within an active proline tRNA gene in Rhizobium meliloti 41. A loss‐of‐function mutation in this tRNA gene leads to significant delay in switching from lag to exponential growth phase. We converted the putative Rhizobium gene to an active amber suppressor gene which suppressed amber mutant alleles of genes of 16‐3 phage and of Escherichia coli origin in R. meliloti 41 and in Agrobacterium tumefaciens GV2260. Upon lysogenization of R. meliloti by phage 16‐3, the proline tRNA gene retained its structural and functional integrity. Aspects of the co‐evolution of a temperate phage and its bacterium host is discussed. The side product of this work, i.e. construction of amber suppressor tRNA genes in Rhizobium and Agrobacterium, for the first time widens the options of genetic study.