Regulation of alcohol-oxidizing capacity in chemostat cultures of Acetobacter pasteurianus

Acetobacter pasteurianus LMG 1635 was studied for its potential application in the enantioselective oxidation of alcohols. Batch cultivation led to accumulation of acetic acid and loss of viability. These problems did not occur in carbon-limited chemostat cultures (dilution rate = 0.05 h^–1) grown on mineral medium supplemented with ethanol, L-lactate or acetate. Nevertheless, biomass yields were extremely low in comparison to values reported for other bacteria. Cells exhibited high oxidation rates with ethanol and racemic glycidol (2,3-epoxy-1-propanol). Ethanol- and glycidol-dependent oxygen-uptake capacities of ethanol-limited cultures were higher than those of cultures grown on lactate or acetate. On all three carbon sources, A. pasteurianus expressed NAD-dependent and dye-linked ethanol dehydrogenase activity. Glycidol oxidation was strictly dye-linked. In contrast to the NAD-dependent ethanol dehydrogenase, the activity of dye-linked alcohol dehydrogenase depended on the carbon source and was highest in ethanol-grown cells. Cell suspensions from chemostat cultures could be stored at 4°C for over 30 days without significant loss of ethanol- and glycidol-oxidizing activity. It is concluded that ethanol-limited cultivation provides an attractive system for production of A. pasteurianus biomass with a high and stable alcohol-oxidizing activity.     

Evidence for at least eight Fanconi anemia genes.

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder with diverse clinical symptoms including progressive bone marrow failure and increased cancer risk. FA cells are hypersensitive to crosslinking agents, which has been exploited to assess genetic heterogeneity through complementation analysis. Five complementation groups (FA-A through FA-E) have so far been distinguished among the first 20 FA patients analyzed. Complementation groups in FA are likely to represent distinct disease genes, two of which (FAC and FAA) have been cloned. Following the identification of the first FA-E patient, additional patients were identified whose cell lines complemented groups A-D. To assess their possible assignment to the E group, we introduced selection markers into the original FA-E cell line and analyzed fusion hybrids with three cell lines classified as non-ABCD. All hybrids were complemented for cross-linker sensitivity, indicating nonidentity with group E. We then marked the three non-ABCDE cell lines and examined all possible hybrid combinations for complementation, which indicated that each individual cell line represented a separate complementation group. These results thus define three new groups, FA-F, FA-G, and FA-H, providing evidence for a minimum of eight distinct FA genes.     

The Low Biomass Yields of the Acetic Acid Bacterium Acetobacter pasteurianus Are Due to a Low Stoichiometry of Respiration-Coupled Proton Translocation

Growth energetics of the acetic acid bacterium Acetobacter pasteurianus were studied with aerobic, ethanol-limited chemostat cultures. In these cultures, production of acetate was negligible. Carbon limitation and energy limitation were also evident from the observation that biomass concentrations in the cultures were proportional to the concentration of ethanol in the reservoir media. Nevertheless, low concentrations of a few organic metabolites (glycolate, citrate, and mannitol) were detected in culture supernatants. From a series of chemostat cultures grown at different dilution rates, the maintenance energy requirements for ethanol and oxygen were estimated at 4.1 mmol of ethanol (middot) g of biomass(sup-1) (middot) h(sup-1) and 11.7 mmol of O(inf2) (middot) g of biomass(sup-1) (middot) h(sup-1), respectively. When biomass yields were corrected for these maintenance requirements, the Y(infmax) values on ethanol and oxygen were 13.1 g of biomass (middot) mol of ethanol(sup-1) and 5.6 g of biomass (middot) mol of O(inf2)(sup-1), respectively. These biomass yields are very low in comparison with those of other microorganisms grown under comparable conditions. To investigate whether the low growth efficiency of A. pasteurianus might be due to a low gain of metabolic energy from respiratory dissimilation, (symbl)H(sup+)/O stoichiometries were estimated during acetate oxidation by cell suspensions. These experiments indicated an (symbl)H(sup+)/O stoichiometry for acetate oxidation of 1.9 (plusmn) 0.1 mol of H(sup+)/mol of O. Theoretical calculations of growth energetics showed that this low (symbl)H(sup+)/O ratio adequately explained the low biomass yield of A. pasteurianus in ethanol-limited cultures.     

The human alpha-amylase multigene family consists of haplotypes with variable numbers of genes

Polymorphic amylase protein patterns have suggested the presence in the human genome of various haplotypes encoding these allozymes. To investigate the genomic organization of the human alpha-amylase genes, we isolated the pertinent genes from a cosmid library constructed of DNA from an individual expressing three different salivary amylase allozymes. From the restriction maps of the overlapping cosmids and a comparison of these maps with the restriction enzyme patterns of DNA from the donor and family members, we were able to identify two haplotypes consisting of very different numbers of salivary amylase genes. The short haplotype contains two pancreatic genes (AMY2A and AMY2B) and one salivary amylase gene (AMY1C), arranged in the order 2B-2A-1C, encompassing a total length of approximately 100 kb. The long haplotype spans about 300 kb and contains six additional genes arranged in two repeats, each one consisting of two salivary amylase genes (AMY1A and AMY1B) and a pseudogene lacking the first three exons (AMYP1). The order of the amylase genes within the repeat is 1A-1B-P1. All genes are in a head-to-tail orientation except AMY1B, which has the reverse orientation with respect to the other genes. Analysis of somatic cell hybrids confirmed the presence of these short and long haplotypes. Furthermore, we present evidence for the existence of additional haplotypes in the human population and propose a general model for the evolution of the human alpha-amylase multigene family. A general designation 2B-2A-(1A-1B-P)n-1C can describe these haplotypes, n being 0 and 2 for the short and the long haplotypes presented in this paper, respectively.     

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

Analysis of the rat JE gene promoter identifies an AP-1 binding site essential for basal expression but not for TPA induction.

We have cloned the immediate-early serum-reponsive JE gene from the rat in order to study the regulation of this gene. We show that sequences of the JE promoter region confer serum-inducibility to a reporter gene. Analysis of the promoter in transient assays reveals that: i) the -141/-88 region is required for the response to the phorbol ester TPA, ii) the -70/-38 region is essential for basal activity. This latter region harbors the sequence TGACTCC, which resembles the consensus site for AP-1 binding, TGACTCA. DNA-protein binding assays indicate that the JE AP-1 site and the consensus AP-1 site have an overlapping but not identical binding spectrum for AP-1 proteins. Our data suggest that the inability of some AP-1 sites to respond to TPA is caused by subtle differences in affinity for AP-1 proteins.     

Evolution of the human alpha-amylase multigene family through unequal, homologous, and inter- and intrachromosomal crossovers

Human amylase haplotypes differ from each other by different numbers of a long direct repeat unit of approximately 100 kb, encompassing two complete salivary amylase genes and one amylase pseudogene lacking the first three exons. The two salivary genes are part of a 75-kb-long inverted repeat. Two short sequences, hybridizing with a probe containing exons 1-3, were found in the central part of the inverted repeat. Sequencing showed that these fragments, designated r, contain exon 3 sequences. We present evidence that these r-fragments and the pseudogene most likely are remnants of the same ancestral pancreatic gene. We determined the orientation of the exon 3 sequences present in the r-fragment and show that an inversion can explain their origination. Hybridization studies, using random fragments from the intergenic region of the AMY gene cluster as probes, enabled us to detect more extended homologous regions in this cluster than were found previously on the basis of restriction maps only. Together, these results allow us to present a model for the evolution of the human amylase multigene family by a number of consecutive events involving inter- and intrachromosomal crossovers.     

Use of a bioelectrochemical cell for the synthesis of (bio)chemicals

A bioelectrochemical cell containing either d-glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) or xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) plus dichlorophenol-indophenol as electron acceptor in one half-cell, and chloroperoxidase (chloride:hydrogen-peroxide oxidoreductase, EC 1.11.1.10) in the other half-cell is described. Due to a combination of chemical, biochemical and electrochemical reactions, electricity and specific (bio)chemicals can be produced in the cell simultaneously and in both compartments. Furthermore, the oxidases in a bioelectrochemical cell are not inactivated by H₂O₂ and as a result the operational lifetimes of the oxidases were increased about five-fold.     

No predictive value of GC phenotypes for HIV infection and progression to AIDS

Summary Linkage data on human factor H (HF) and 22 other human genetic markers are presented. Close linkage at 0<0.10 can be ruled out for a series of marker systems (Rh, PGM1, ACP1, Jk, Tf, Gc, MNSs, ME2, HLA, GLO1, ORM, Gt, PI, Hp, GPT). Strong evidence for linkage was obtained for peptidase A (PEPA) with lods >3.0 at =0.10 in males and at =0.20 for the sexes combined. From this result the HF locus can be provisionally assigned tochromosome 18.