Purification and properties of RhaR, the positive regulator of the L-rhamnose operons of Escherichia coli

The product of the rhaR gene, which regulates the level of mRNA produced from the four L-rhamnose-inducible promoters of the rhamnose operon, has been hypersynthesized and purified by a two-column procedure. The purified protein is a 33 kDa DNA-binding protein that binds to an inverted repeat structure located within the psr promoter, the promoter for the rhaS and rhaR genes. The equilibrium binding constants and kinetic constants have been determined under a variety of solution conditions. The protein binds with high affinity and its binding is sensitive to salt concentration and the presence of L-rhamnose. The nucleotides and phosphate residues contacted by RhaR were identified by chemical interference assays. All of the contacts are made to one face of the DNA and the symmetrical pattern matches the inverted repeat sequence proposed for the binding site. An unusual property of the binding site is that the two half-sites of the inverted repeat are separated from one another by 17 base-pairs of uncontacted DNA. Significant binding is retained if the 17 base-pairs are extended by insertions of integral turns of DNA, but not by half-integral turns. The complex of RhaR-DNA appears to be sharply bent, approximately 160 degrees.     

基于rhaSR嵌合操纵子的构建及其在大肠杆菌中表达的研究

通过PCR等重组DNA技术,构建了含rhaSR启动子表达调控元件、RhaR基因、报告基因gst(谷胱甘肽-S-转移酶)的两个嵌合操纵子,并插入大肠杆菌表达载体pALEX中构成pALEX-PR1和pALEX-PR2。其中pALEX-PR2的RhaR基因上游为原有的SD序列,而pALEX-PR1的RhaR基因上游则插入了增强的SD序列。把这两个重组表达质粒分别转入大肠杆菌BL21(DE3)中,报告基因gst能够在L-鼠李糖诱导下表达,其表达量是非诱导条件下的4～5倍,且pALEX-PR1的表达量是pALEX-PR2的3.14倍。以上结果表明,gst的表达既受L-鼠李糖诱导,同时又受RhaR的正调控。SDS-PAGE结果显示,GST占大肠杆菌培养物总可溶蛋白的5.41%(W/W),平均1L培养物可获得3.0mg纯化的GST。酶活性分析表明,所构建的嵌合操纵子表达的GST保持了正确的构型且具有很高的活性。     

大肠杆菌鼠李糖调节子

大肠杆菌（Escherichia coil）L-鼠李糖（rha）调节子由三个功能相关的操纵子（operon）组成,位于大肠杆菌染色体基因组中。它编码大肠杆菌吸收和利用L-鼠李糖的蛋白,即一个鼠李糖运输蛋白（RhaT）、三个鼠李糖代谢酶（RhaB、RhaA、RhaD）以及两个调节蛋白（RhaS、RhaR）。三个操纵子均受到L-鼠李糖本身的诱导,同时以调控蛋白RhaS、RhaR和CRP（cAMP受体蛋白）为中介的正调控也参与调节。     

Chlamydomonas reinhardtii mutants abnormal in their responses to phosphorus deprivation.

P-starved plants scavenge inorganic phosphate (Pi) by developing elevated rates of Pi uptake, synthesizing extracellular phosphatases, and secreting organic acids. To elucidate mechanisms controlling these acclimation responses in photosynthetic organisms, we characterized the responses of the green alga Chlamydomonas reinhardtii to P starvation and developed screens for isolating mutants (designated psr [phosphorus-stress response]) abnormal in their responses to environmental levels of Pi. The psr1-1 mutant was identified in a selection for cells that survived exposure to high concentrations of radioactive Pi. psr1-2 and psr2 were isolated as strains with aberrant levels of extracellular phosphatase activity during P-deficient or nutrient-replete growth. The psr1-1 and psr1-2 mutants were phenotypically similar, and the lesions in these strains were recessive and allelic. They exhibited no increase in extracellular phosphatase activity or Pi uptake upon starvation. Furthermore, when placed in medium devoid of P, the psr1 strains lost photosynthetic O2 evolution and stopped growing more rapidly than wild-type cells; they may not be as efficient as wild-type cells at scavenging/accessing P stores. In contrast, psr2 showed elevated extracellular phosphatase activity during growth in nutrient-replete medium, and the mutation was dominant. The mutant phenotypes and the roles of Psr1 and Psr2 in P-limitation responses are discussed.     

Redefining the role of psr in beta-lactam resistance and cell autolysis of Enterococcus hirae

The contribution of penicillin-binding protein 5 (PBP5) and the PBP5 synthesis repressor (Psr) to the beta-lactam resistance, growth, and cell autolysis of wild-type strain ATCC 9790 and resistant strain R40 of Enterococcus hirae was investigated by disruption or substitution of the corresponding pbp5 and psr genes by Campbell-type recombination. The resulting modifications were confirmed by hybridization and PCR. The low susceptibility of E. hirae to beta-lactams was confirmed to be largely dependent on the presence of PBP5. However, against all expectations, inactivation of psr in ATCC 9790 or complementation of R40 cells with psr did not modify the susceptibility to benzylpenicillin or the growth and cell autolysis rates. These results indicated that the psr gene does not seem to be involved in the regulation of PBP5 synthesis and consequently in beta-lactam resistance or in the regulation of cell autolysis in E. hirae.     

Junctions between repetitive DNAs on the PSR chromosome of Nasonia vitripennis: Association of palindromes with recombination

The Paternal-Sex-Ratio (PSR) chromosome of Nasonia vitripennis contains several families of repetitive DNAs that show significant sequence divergence but share two palindromic regions. This study reports on the analysis of junctions between two of these repetitive DNA families (psr2 and psr18). Three lambda clones that hybridized to both repeat families were isolated from PSR-genomic DNA libraries through multiple screenings and analyzed by Southern blots. Analysis of clones showed a region in which the two repeat types are interspersed, flanked by uniform blocks of each repeat type. PCR amplification of genomic DNA confirmed the contiguous arrangement of psr2 and psr18 on PSR and identified an additional junction region between these repeats that was not present in the lambda inserts. We isolated and sequenced 41 clones from the lambda inserts and genomic PCR products containing junction sequences. Sequence analysis showed that all transitions between psr2 and psr18 repeats occurred near one of the two palindromes. Based on the inheritance pattern of PSR, recombination between repeats on this chromosome must be mitotic (rather than meiotic) in origin. The occurrence of exchanges near the palindromes suggests that these sequences enhance recombination between repeat units. Rapid amplification of repetitive DNA may have been an important factor in the evolution of the PSR chromosome. Correspondence to: John H. Werren     

Evidence that the PBP 5 synthesis repressor (psr) of Enterococcus hirae is also involved in the regulation of cell wall composition and other cell wall-related properties.

psr has been reported by M. Ligozzi, F. Pittaluga, and R. Fontana, (J. Bacteriol. 175:2046-2051, 1993) to be a genetic element located just upstream of the structural gene for the low-affinity penicillin-binding protein 5 (PBP 5) in the chromosome of Enterococcus hirae ATCC 9790 and to be involved in the repression of PBP 5 synthesis. By comparing properties of strains of E. hirae that contain a full-length, functional psr with those of strains that possess a truncated form of the gene, we have obtained data that indicate that psr is involved in the regulation of several additional surface-related properties. We observed that cells of strains that possessed a truncated psr were more sensitive to lysozyme-catalyzed protoplast formation, autolyzed more rapidly in 10 mM sodium phosphate (pH 6.8), and, in contrast to strains that possess a functional psr, retained these characteristics after the cultures entered the stationary growth phase. Cellular lytic properties did not correlate with differences in the cellular contents of muramidase-1 or muramidase-2, with the levels of PBP 5 produced, or with the penicillin susceptibilities of the strains. However, a strong correlation was observed with the amounts of rhamnose present in the cell walls of the various strains. All of the strains examined that possessed a truncated form of psr also possessed approximately one-half of the rhamnose content present in the walls of strains that possessed a functional psr. These data suggest that psr is also involved in the regulation of the synthesis of, or covalent linkage to the cell wall peptidoglycan of, a rhamnose-rich polysaccharide. These differences in cell wall composition could be responsible for the observed phenotypic differences. However, the multiple effects of psr suggest that it is part of a global regulatory system that, perhaps independently, affects several cell surface-related properties.     

A phosphate-starvation inducible β-glucosidase gene (psr3.2) isolated from Arabidopsis thaliana is a member of a distinct subfamily of the BGA family

We have previously isolated a phosphate starvation-response (psr) cDNA clone, psr3.1, from Brassica nigra which encodes a -glucosidase. Southern blots of Arabidopsis thaliana genomic DNA probed with the psr3.1 cDNA indicated that this gene exists as a single locus. A genomic library of A. thaliana was screened at high stringency to isolate the corresponding genomic clone. The resultant clone was coined psr3.2 because of its sequence divergence from isolated psr3.1 cDNA clones. Northern blotting with probes derived from the coding region of the genomic clone showed that this gene is expressed at high levels in P_i-starved roots and the enhancement occurred within two days of growth in medium lacking P_i. The expression of this gene is repressed by heat shock and anaerobic conditions, and it is not significantly induced by high salinity, or by nitrogen or sulfur deprivation. Sequence analysis of the genomic clone revealed the existence of 13 exons interrupted by 12 AT-rich introns and it possessed a high homology with the B. nigra psr3.1 as well as various other -glucosidase genes from other species. Sequence similarity and divergence percentages between the deduced amino acid sequences of the psr3 clones and other -glycosidases suggests that they should be included along with two other Brassicaceae genes in a distinct subfamily of the BGA glycosidase gene family. The presence of an endoplasmic reticulum retention signal at the carboxy terminus indicates the likely cellular location of PSR3.2. The possible metabolic and regulatory roles of this enzyme during the P_i-starvation response are discussed.     

Molecular genetic identification of Avena chromosomes related to the group 1 chromosomes of the Triticeae.

A collection of 19 wheat (Triticum aestivum) probes, detecting sequences in the seven homoeologous groups of chromosomes, were hybridized to DNA from the 'Kanota' series of oat monosomic lines (Avena byzantina) to investigate their use for identifying groups of homoeologous oat chromosomes. Three probes from homoeologous group 1 of wheat, psr161, psr162, and psr121, mapped among the set of oat chromosomes 1C, 14, and 17. One homoeologous group 6 probe, psr167, mapped to oat chromosomes 1C and 17. Two oat probes that had previously been shown to map to oat chromosomes 1C, 14, and 17 were then hybridized to DNA from the 'Chinese Spring' wheat ditelosomics. They localized to homoeologous group 1 wheat chromosomes, one to the short arm and one to the long arm. These results reveal that in hexaploid oat there is a group of three chromosomes that correspond at least in part to homoeologous group 1 of wheat. The remaining wheat probes identifying other wheat homoeologous sets did not detect a complete series of homoeologous chromosomes in oat. This was presumably due to the incomplete status of the 'Kanota' monosomic series, chromosomal rearrangement in Avena, weak hybridization signals owing to low probe-target sequence homology, and (or) detection of only two hybridization bands by the wheat probe.