Strain RB Research Articles

HIV-1 group P infection: towards a dead-end infection?

HIV/1 group P (HIV-1/P) is the last HIV/1 group discovered and, to date, constitutes only two strains. To obtain new insight into this divergent group, we screened for new infections by developing specific tools, and analysed phenotypic and genotypic properties of the prototypic strain RBF168. In addition, the follow-up of the unique infected patient monitored so far has raised the knowledge of the natural history of this infection and its therapeutic management. We developed an HIV-1/P specific seromolecular strategy and screened over 29 498 specimen samples. Infectivity and evolution of the gag-30 position, considered as marker of adaptation to human, were explored by successive passages of RBF168 strain onto human peripheral blood mononuclear cells. Natural history and immunovirological responses to combined antiretroviral therapy (cART) were analysed based on CD4+ cells and plasmatic viral load evolution. No new infection was detected. Infectivity of RBF168 was found lower, relative to other main HIV groups and the conservative methionine found in the gag-30 position revealed a lack of adaptation to human. The follow-up of the patient during the 5-year ART-free period, showed a relative stability of CD4+ cell count with a mean of 326 cells/μl. Initiation of cART led to rapid RNA undetectability with a significant increase of CD4+ cells, reaching 687 cells/μl after 8 years. Our results showed that HIV-1/P strains remain extremely rare and could be less adapted and pathogenic than other HIV strains. These data lead to the hypothesis that HIV-1/P infection could evolve towards, or even already corresponds to, a dead-end infection.

Effects of culture conditions of Pseudomonas aeruginosa strain RB on the synthesis of CdSe nanoparticles

Cadmium selenide (CdSe) was synthesized by Pseudomonas aeruginosa strain RB in a culture containing lactic acid as a carbon source, 1 mM selenite, and 1 mM cadmium under various conditions. High purity (1.02-1.16 of the atomic ratio of Se to Cd) and efficient synthesis of biogenic CdSe nanoparticles were observed at 25-30°C, 0.05-10 g L(-1) NaCl, and neutral pH conditions compared with other tested conditions. However, the size and shape of synthesized CdSe nanoparticles were not changed by changing culture conditions. The contents of S and Se in the particles respectively increased under alkaline and weak acidic conditions. Furthermore, high temperature (>37°C), high salinity (>10 g L(-1) NaCl), and alkaline pH affected the CdSe-synthesizing rate by strain RB. This report is the first optimizing the culture conditions for synthesizing biogenic CdSe nanoparticles in a batch processing.

Draft Genome Sequence of Pseudomonas aeruginosa Strain RB, a Bacterium Capable of Synthesizing Cadmium Selenide Nanoparticles.

Pseudomonas aeruginosa strain RB is a bacterium capable of synthesizing cadmium selenide (CdSe) nanoparticles and was isolated from a soil sample. Here, we present the draft genome sequence of P. aeruginosa strain RB. To the best of our knowledge, this is the first report of a draft genome of a CdSe-synthesizing bacterium.

Isolation of a selenite-reducing and cadmium-resistant bacterium Pseudomonas sp. strain RB for microbial synthesis of CdSe nanoparticles

Bacteria capable of synthesizing CdSe from selenite and cadmium ion were enriched from a soil sample. After repeated transfer of the soil-derived bacterial cultures to a new medium containing selenite and cadmium ion 42 times (during 360 days), an enrichment culture that can simultaneously remove selenite and cadmium ion (1 mM each) from the liquid phase was obtained. The culture's color became reddish-brown, indicating CdSe nanoparticle production, as confirmed by energy-dispersive x-ray spectra (EDS). As a result of isolation operations, the bacterium that was the most responsible for synthesizing CdSe, named Pseudomonas sp. RB, was obtained. Transmission electron microscopy and EDS revealed that this strain accumulated nanoparticles (10-20 nm) consisting of selenium and cadmium inside and on the cells when cultivated in the same medium for the enrichment culture. This report is the first describing isolation of a selenite-reducing and cadmium-resistant bacterium. It is useful for CdSe nanoparticle synthesis in the simple one-vessel operation.

A Novel Rhodobacter sphaeroides Expression System for Real-Time Evaluation of Heterologous Protein Expression Levels

Heterologous protein expression levels can not be evaluated in real-time by experimental procedures commonly used for most expression systems during host cell culture. Rb. sphaeroides has provided an ideal system for studying both photosynthesis and membrane development and exhibited potential as a novel expression system. We constructed the puc1BA and puc2BA mutant strain Rb. sphaeroides CQU68 and used it as a novel expression system to heterologously express proteins fused to β-subunit of light-harvesting 2 complexes (LH2). The presence of LH2 with β-subunit fusion proteins was spectrally detected by the LH2 typical absorption at ~800 nm and ~850 nm, and the formation of these complexes were further confirmed by SDS-PAGE and Western blot analysis. The expression levels of heterologous protein measured by SDS-PAGE and Western blot turned out to be higher as the typical spectral peak heights increase. These findings suggested that the production of the heterologous protein could be rapidly detected through the LH2 absorption at ~800 nm and ~850 nm. Moreover, the typical absorption could be used as a monitor for rapid and real-time evaluation of heterologous protein expression levels.

Reengineering of a Corynebacterium glutamicum L-arginine and L-citrulline producer.

Toward the creation of a robust and efficient producer of L-arginine and L-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92(up), and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (Delta argR), the production was further increased. The best mutation set, Delta argR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical L-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus allowing significantly higher productivity of arginine/citrulline even at the suboptimal 38 degrees C.

Sequence Comparison Between the Fusion Protein of Human and Bovine Espiratory Syncytial Viruses

The nucleotide sequence was determined for the fusion (F) protein-coding mRNA of the bovine respiratory syncytial virus (strain RB 94) and the amino acid sequence of the F protein was deduced for comparison with the sequence of human respiratory syncytial virus subtypes A and B (RSS-2 and 18537 strains). The human and bovine RS virus F proteins (excluding the cleaved signal peptide) share 83 to 84% homology. The greatest divergence occurred within the F2 subunit in the region preceding the cleavage activation site.

Production and utilization of monoclonal antibodies against Pseudomonas cepacia.

To obtain monoclonal antibodies (MABs) specific to Pseudomonas cepacia (Pc) strain RB 425, the live cells (L) and the fixed cells (F) by glutaraldehyde were injected to mice, respectively. Sixteen and 8 hybridoma lines were established from L-immunized and F-immunized mouse, respectively. Three MABs from L-hybridoma lines and another 3 MABs from F-hybridoma lines were used for the specific detection and analyses of the epitopes on Pc. Although some of the 6 MABs of ascitic fluids showed almost the same titers as rabbit antisera in indirect ELI-SA, they had a limited ability to precipitate and agglutinate the L cells. The reactivity of the 6 MABs was tested with L, F, heated L (HL) and heated F (HF) cells in indirect ELISA. Three out of 6 MABs were heterospecific antibodies that reacted more strongly with HL cells than did with each immunogen. Three types of epitopes were classified by the reactivity of MABs with L and F cells. The specificity of MABs was detected by indirect ELISA. The live cells of 5 Pc strains, 6 pseudomonads species, and other bacterial genera were studied for their antigenicity. Five epitopes were distinguished by the reaction patterns of the MABs, and the specificity of the MABs was different in the 5 strains of Pc. Furthermore, 2 MABs from L-hybridoma lines showed a wide specificity in their reaction with pseudomonads, while the others reacted only with the strains of Pc.

The molecular basis of pathogenicity in Haemophilus influenzae: comparative virulence of genetically-related capsular transformants and correlation with changes at the capsulation locus cap

Serotype b strains of Haemophilus influenzae are strikingly more highly associated with episodes of invasive, life-threatening infection in young children than are strains of other serotypes, but the role that the capsule itself plays in determining this virulence has not been dissected away from that of possibly linked virulence determinants such as lipopolysaccharide (LPS). Using DNA from clinical isolates of all six serotypes (a-f) and a genetically-defined capsule-deficient recipient strain Rb −: 02, we constructed a series of capsular transformants otherwise identical with respect to outer-membrane protein and LPS subtype, biotype, and electrotype. Cloned DNA was also used to create type a and b transformants isogenic outside the capsulation locus to provide the most rigorous test to determine whether capsule alone modulates pathogenicity. Capsular transformants showed the same spectrum of virulence in an infant rat bacteremia/meningitis assay as wild-type strains, thus implicating the capsule polysaccharide as an independent determinant of virulence. Experiments in intact and splenectomised rats identified a critical role for type b capsule in enabling organisms to evade splenic clearance.

Influence of verticillium wilt resistant and susceptible potato genotypes on populations of antagonistic rhizosphere and rhizoplane bacteria and free nitrogen fixers

Verticillium wilt resistant A 66 107-51 and susceptible Russet Burbank potatoes differentially influenced populations of rhizosphere and rhizoplane bacteria. Although differences in total bacteria between the two genotypes were not significantly different, selective differences were evident. These included increases in bacteria antagonistic in vitro toward Verticillium dahliae strain RB 5, and bacteria capable of fixing nitrogen that were more commonly associated with the wilt-resistant potato. Bacteria that were antagonistic to V. dahliae were predominantly Bacillus spp. Other antagonists were species of Pseudomonas, Gluconobacter, Flavobacterium, and Streptomyces. Nitrogen-fixing bacteria were Azotobacter and Azomonas spp. The suppression of Verticillium wilt in Russet Burbank during the growing season following the planting of A 66 107-51 may, in part, be explained by the above findings.

Use of somatic cell genetics to study chromosomes contributing to antigen plus I recognition by T cell hybridomas.

Keyhole limpet hemocyanin (KLH)/I region-specific T cell hybridomas have been prepared by fusing KLH/I-specific T cell blasts from mice with single pairs of metacentric chromosomes to the inducible, interleukin 2 (IL-2)-secreting T cell hybridoma FS6-14.13.AG2.1. T cell hybridomas with KLH/I receptors were identified by their ability to secrete IL-2 in response to KLH and the appropriate antigen-presenting cells. After cloning and subcloning, KLH/I reactivity was correlated with the presence or absence of metacentric chromosomes derived from the KLH/I-specific T cell blast parent. Hybridomas were identified that had lost all chromosomes 4 and 6 or 16 and 17 derived from their normal T cell parent, but retained the ability to respond to KLH/I. This suggested that products of genes on these chromosomes did not contribute to the specific portions of T cell Ag/I receptors. These gene products would include, of course, kappa and lambda chains and H-2. We did not obtain any T cell hybridomas that had lost both metacentric (8.12) chromosomes derived from T cells of the Robertsonian mouse strain Rb(8.12)5, so we could not draw any conclusions about the contributions of products of genes on these chromosomes. T cell hybridomas with KLH/I reactivity were found that contained only one metacentric (8.12) chromosome derived from this strain. Moreover, a T cell hybridoma was found that retained both metacentric (8.12) chromosomes from its normal T cell parent, but had lost KLH/I reactivity. These results suggested that neither two chromosomes 8 nor two chromosomes 12 were required for antigen/I reactivity in normal T cells and that antigen/I reactivity was controlled, at least in part, by genes mapping on chromosomes other than 8 or 12.

Laboratory mice carrying three pairs of Robertsonian translocations: establishment of a strain and analysis of meiotic segregation.

The evolution of a 2n = 34 combined Robertsonian translocation strain Rb(5.19)1Wh/(6.15)1Ald/(8.17)HEM is described. Homozygotes had three large ring translocation bivalents at M I and regular meiotic disjunction at M II. Male and female F<sub>1·</sub> heterozygotes had similar frequencies of abnormal meiotic disjunction at M II, but aneuploid embryos were more frequent in (F<sub>1</sub><i>♂ </i>X NIH GP ♀) crosses (25.6%) than (F<sub>1</sub> ♀ X GP <i>♂</i>) crosses (13.5%) at 10 days’ gestation. It could not be determined whether this discrepancy was genetic in origin or due to limitations in the techniques of M II analysis. Rb(5.19), as well as Rb(6.15) or (8.17), contributed to the aneuploidy, although previous M II analysis of singly heterozygous Rb(5.19)/+ males demonstrated comparatively regular meiotic disjunction. Trisomic embryos were not detected beyond 10 days in five (F<sub>1</sub> ♀ X GP <i>♂</i>) crosses, suggesting that their survival in F<sub>1</sub> females is limited. The ratios of the different types of balanced embryos from the (F<sub>1</sub> X GP) crosses indicated random meiotic segregation of the three translocation chromosomes in triple heterozygotes. Analysis of complementary groups of balanced embryos from the same crosses also demonstrated lack of preferential segregation of the translocations; embryos with translocations and those with their corresponding homologous acrocentrics were equally frequent. Triploid embryos (3.3 %) were also detected in (F<sub>1</sub><i>♂ </i>X NIH GP ♀) crosses. The translocations and Y chromosome served as paternal markers, permitting its origin to be determined in two of three cases (dispermy). One growth-retarded triploid had a distinctive anomaly: failure of lens induction and lack of optic cup development. The combined strain provided a complex of markers for determining polyploidy mechanisms, as well as a common genetic background for comparing behavior of the three translocations at M I.

Host specificity of DNA in Haemophilus influenzae: the restriction and modification systems in strains Rb and Rf.

Summary: Haemophilus influenzae Rf possesses two distinct host specificity systems F1 and F2 each of which is able to restrict and modify Haemophilus phage HP1c1, while strain Rb possesses only one system, B. Among restriction-deficient mutants isolated from strain Rf, the r-m+ as well as r-m- phenotypes for these two systems were obtained after either one or two mutational steps. The F1 system was introduced into H. influenzae Rd by genetic transformation to show that the D1 and F1 systems are not allelic.

Transmission of Toxoplasma gondii across the guinea-pig placenta.

6 guinea-pigs were inoculated intraperitoneally at 4-54 days of pregnancy with 60 or 100 Toxoplasma cysts in mouse-brain saline suspension. All foetuses were infected. 3 sows died on 17-19th post-inoculation day, with signs of meningo-encephalitis, and displayed focal lymphocytic inflammation and, in 2, Toxoplasma rosette formation in the cerebral hemisphere. 6 Sows previoulsy inoculated with 100-200 cysts were followed in later pregnancy: 5 of 17 foetuses were found to be infected when delivered-a further 4, and 3 carneous moles, were not examined. 4 non-pregnant sows died at 12-14 days after inoculation with 100-200 cysts, with similar clinical signs to those in the first group. Antibody titres of 256 were found at 2½ weeks, rising to a maximum of 16 384 at 6 weeks. Levels were consistently falling by 12-14 weeks. Transmission across the placenta could not wholly be prevented by the presence of antibody except in high titre, and the important factor was concluded to be the parasitaemia. The strain RB is midway in virulence in guinea-pigs between strains RH and 76.