The pyruvate-lactate axis modulates cardiac hypertrophy and heart failure

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Direct fractionation of genes by preparative electrophoresis of Bacillus subtilis DNA

Discontinuous electrophoresis through agarose has been shown to be a satisfactory method for preparation of biologically active restriction fragments from milligram quantities of DNA. The DNA is obtained in sufficient quantity for: (1) direct use in genetic transformation, (2) the production of multiple-dimensional restriction analyses, or (3) use as a high-resolution hybridization probe.     

Begattung und Eiablage von Eupagurus prideauxi Leach

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Microbial Life at 90 C: the Sulfur Bacteria of Boulder Spring

The physiology of the bacteria living in Boulder Spring (Yellowstone National Park) at 90 to 93 C was studied with radioactive isotope techniques under conditions approximating natural ones. Cover slips were immersed in the spring; after a fairly even, dense coating of bacteria had developed, these cover slips were incubated with radioactive isotopes under various conditions and then counted in a gas flow or liquid scintillation counter. Uptake of labeled compounds was virtually completely inhibited by formaldehyde, hydrochloric acid, and mercuric bichloride, and inhibition was also found with streptomycin and sodium azide. The water of Boulder Spring contains about 3 mug of sulfide per ml. Uptake of labeled compounds occurs only if sulfide or another reduced sulfur compound is present during incubation. The pH optimum for uptake of radioactive compounds by Boulder Spring bacteria is 9.2, a value near that of the natural spring water (8.9). Many experiments with a variety of compounds were performed to determine the temperature optimum for uptake of labeled compounds. The results with all the compounds were generally similar, with broad temperature optima between 80 and 90 C, and with significant uptake in boiling (93 C) but not in superheated water (97 C). The results show that the bacteria of Boulder Spring are able to function at the temperature of their environment, although they function better at temperatures somewhat lower. The fine structure of these bacteria has been studied by allowing bacteria in the spring to colonize glass slides or Mylar strips which were immediately fixed, and the bacteria were then embedded and sectioned. The cell envelope structure of these bacteria is quite different from that of other mesophilic or thermophilic bacteria. There is a very distinct plasma membrane, but no morphologically distinct peptidoglycan layer was seen outside of the plasma membrane. Instead, a rather thick diffuse layer was seen, within which a subunit structure was often distinctly visible, and connections frequently occurred between this outer layer and the plasma membrane. The thick outer layer usually consisted of two parts, the outer part of which was sometimes missing. Within the cells, structures resembling ribosomes were seen, and regions lacking electron density which probably contained deoxyribonucleic acid were also visible.     

Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5-ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase

5-Ketofructose (5-KF) is a promising low-calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane-bound three-subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5-KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5-KF. In a batch fermentation, 129 g l⁻¹ 5-KF were formed from 150 g l⁻¹ fructose within 23 h, corresponding to a space-time yield of 5.6 g l⁻¹ h⁻¹. Besides fructose, also sucrose could be used as substrate for 5-KF production by plasmid-based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5-KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5-KF production.     

Klebsiella pneumoniae genes for citrate lyase and citrate lyase ligase: localization, sequencing, and expression

In the course of studies on anaerobic citrate metabolism in Klebsiella pneumoniae, the DNA region upstream of the gene for the sodium-dependent citrate carrier (dtS) was investigated. Nucleotide sequence analysis revealed a cluster of five new genes that were oriented inversely to citS and probaby form an operon. The genes were named citCDEFG. Based on known protein sequence data, the gene products derived from citD, citE and citF could be identified as the λ-, β-, and α-subunits of citrate lyase, respectively. This enzyme catalyses the cleavage of citrate to oxaloacetate and acetate. The gene product derived from citC (calculated M_r 36476) exhibited no obvious similarity to other proteins. In the presence of acetate and ATP, cell extracts from a citC-expressing Escherichia coli strain were able to reactivate purified citrate lyase from K. pneumoniae that had been inactivated by chemical deacetylation of the prosthetic group. This represents 5-phosphoribosyi-dephospho-acetyl-coenzyme A which is covalently bound to serine-14 of the acyl carrier protein (λ-subunit). CitC was thus identified as acetate:SH-citrate lyase ligase. The function of the gene product derived from citG (M_r 32 645) has not yet been identified. Expression of the CitCDEFG gene cluster in E. coli led to the formation of citrate lyase which was active only in the presence of acetyl-coenzyme A, a compound known to substitute for the prosthetic group. These and other data strongly indicated that the enzyme synthesized in E. coli lacked its prosthetic group. Thus, additional genes besides citCDEFG appear to be required for the formation of holo-citrate lyase.