Expression of genes encoding F(1)-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis

We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F(1) domain of the membrane-bound (F(1)F(0)) H(+)-ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F(1)-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F(1)-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.     

Differential ability of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains to colonize the roots of pea plants

Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)(-1) over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.     

Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Cytopathies involving mitochondrial complex II

Complex II (succinate-ubiquinone oxidoreductase) is the smallest complex in the respiratory chain and contains four nuclear-encoded subunits SdhA, SdhB, SdhC, and SdhD. It functions both as a respiratory chain component and an essential enzyme of the TCA cycle. Electrons derived from succinate can thus be directly transferred to the ubiquinone pool. Major insights into the workings of complex II have been provided by crystal structures of closely related bacterial enzymes, which have also been genetically manipulated to answer questions of structure-function not approachable using the mammalian system. This information, together with that accrued over the years on bovine complex II and by recent advances in understanding in vivo synthesis of the non-heme iron co-factors of the enzyme, is allowing better recognition of improper functioning of human complex II in diseased states. The discussion in this review is thus limited to cytopathies arising because the enzyme itself is defective or depleted by lack of iron-sulfur clusters. There is a clear dichotomy of effects. Enzyme depletion and mutations in SDHA compromise TCA activity and energy production, whereas mutations in SDHB, SDHC, and SDHD induce paraganglioma. SDHC and SDHD are the first tumor suppressor genes of mitochondrial proteins.     

Deletion of p21 (WAF-1/Cip1) does not induce systemic autoimmunity in female BXSB mice

Cell cycle, apoptosis, and replicative senescence are all influenced by the cyclin-dependent kinase inhibitor, p21. It was previously reported that deletion of p21 in 129/Sv x C57BL/6 mixed genetic background mice induced a severe lupus-like disease, almost exclusively in females. However, we did not confirm this finding in an independently derived stock of 129/Sv x C57BL/6 p21(-/-) mice. To further address this discrepancy, we examined the effects of p21 deletion in BXSB female mice that develop late-life, mild lupus-like disease. Survival, polyclonal Igs, anti-chromatin Abs, and kidney histopathology in these mice were unremarkable and identical to wild-type littermates for up to 14 mo of age. We conclude that p21 deficiency does not promote autoimmunity even in females of a predisposed strain. The findings indicate that the use of mixed background 129/Sv x C57BL/6 mice to study effects of gene deletions in systemic autoimmunity may be confounded by the genetic heterogeneity of this cross. We suggest that studies addressing gene deletion effects in systemic autoimmunity should use sufficiently backcrossed mice to attain genetic homogeneity, include wild-type littermate controls, and preferentially use congenic inbred strains with late-life lupus predisposition to emulate the polygenic nature of this disease.     

Breakdown of CTL tolerance to self HLA-B*2705 induced by exposure to Chlamydia trachomatis

There is a strong association between seronegative arthritis and HLA B27, but it is still unresolved whether the contribution of B27 to disease pathogenesis is solely as a restriction element for an arthritogenic peptide, or whether B27 itself serves as an autoantigen. This study uses transgenic rats to address the question as to whether exposure to an arthritogenic pathogen can alter tolerance to B27. Unlike their nontransgenic counterparts, B27-transgenic rats are tolerant of B27 immunization using either B27(+) splenocytes or plasmid DNA and do not develop anti-B27 CTL. However, if splenocytes from such immunized animals are exposed to Chlamydia in vitro, CTL are generated that lyse B27(+) targets. No killing was seen with targets transfected with control B7, B14, B40, or B44. This phenomenon was not observed with immunization by nontransgenic splenocytes, or HLA-A2 DNA alone. Using targets expressing mutated B27, we show that the epitope for autoreactive CTL recognition of B27 involves the Lys(70) amino acid residue in the alpha(1) domain of the MHC class I molecule. The generation of CTL with specificity for B27 under these conditions demonstrates that tolerance to B27 can be subverted by CHLAMYDIA: This indicates a dynamic interrelationship between the pathogen and B27, which may have important implications for B27-related spondyloarthropathies triggered by intracellular bacteria.     

Rapid Concerted Evolution via Gene Conversion at the <Emphasis Type="BoldItalic">Drosophila hsp70</Emphasis> Genes

We analyzed nucleotide variation in the hsp70 genes of Drosophila melanogaster (five genes) and D. simulans (four genes) to characterize the homogenizing and diversifying roles of gene conversion in their evolution. Gene conversion within and between the 87A7 and 87C1 gene clusters homogenize the hsp70 coding regions; in both D. melanogaster and D. simulans, same-cluster paralogues are virtually identical, and large intercluster conversion tracts diminish 87A7/87C1 divergence. Same-cluster paralogues share many polymorphisms, consistent with frequent intracluster conversion. Shared polymorphism is highly biased toward silent variation; homogenizing conversion interacts with purifying selection. In contrast to the coding regions, some hsp70 flanking regions show conversion-mediated diversification. Strong reductions of nucleotide variability and linkage disequilibria among conversion-mediated sites in hsp70Ab and hsp70Bb alleles sampled from a single natural population are consistent with a selective sweep. Comparison of the D. melanogaster and D. simulans hsp70 genes reveals whole-family fixed differences, consistent with rapid propagation of novel mutations among duplicate genes. These results suggest that the homogenizing and diversifying roles of conversion interact to drive dynamic concerted evolution of the hsp70 genes. Received: 25 June 2001 / Accepted: 10 October 2001