DNA ligase and the pyridine nucleotide cycle in Salmonella typhimurium.

Bacterial DNA ligases use NAD as an energy source. In this study we addressed two questions about these enzymes. First, what is the physiological consequence of completely removing the NAD-dependent enzyme and replacing it with an ATP-dependent DNA ligase? We constructed Salmonella typhimurium strains in which the endogenous NAD-dependent DNA ligase activity was inactivated by an insertion mutation and the ATP-dependent enzyme from bacteriophage T4 was provided by a cloned phage gene. Such strains were physiologically indistinguishable from the wild type, even under conditions of UV irradiation or treatment with alkylating agents. These results suggest that specific functional interactions between DNA ligase and other replication and repair enzymes may be unimportant under the conditions tested. Second, the importance of DNA ligation as the initiating event of the bacterial pyridine nucleotide cycle was critically assessed in these mutant strains. Surprisingly, our results indicate that DNA ligation makes a minimal contribution to the pyridine nucleotide cycle; the Salmonella strains with only an ATP-dependent ligase had the same NAD turnover rates as the wild-type strain with an NAD-dependent ligase. However, we found that NAD turnover was significantly decreased under anaerobic conditions. We suggest that most intracellular pyridine nucleotide breakdown occurs in a process that protects the cell against oxygen damage but involves a biochemical mechanism other than DNA ligation.     

Primary structure and mapping of the hupA gene of Salmonella typhimurium.

In bacteria, the complex nucleoid structure is folded and maintained by negative superhelical tension and a set of type II DNA-binding proteins, also called histonelike proteins. The most abundant type II DNA-binding protein is HU. Southern blot analysis showed that Salmonella typhimurium contained two HU genes that corresponded to Escherichia coli genes hupA (encoding HU-2 protein) and hupB (encoding HU-1). Salmonella hupA was cloned, and the nucleotide sequence of the gene was determined. Comparison of hupA of E. coli and S. typhimurium revealed that the HU-2 proteins were identical and that there was high conservation of nucleotide sequences outside the coding frames of the genes. A 300-member genomic library of S. typhimurium was constructed by using random transposition of MudP, a specialized chimeric P22-Mu phage that packages chromosomal DNA unidirectionally from its insertion point. Oligonucleotide hybridization against the library identified one MudP insertion that lies within 28 kilobases of hupA; the MudP was 12% linked to purH at 90.5 min on the standard map. Plasmids expressing HU-2 had a surprising phenotype; they caused growth arrest when they were introduced into E. coli strains bearing a himA or hip mutation. These results suggest that IHF and HU have interactive roles in bacteria.     

Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.      

Evidence for the presence of β-lactamase inStreptomyces glaucescens and its inhibition by sodium clavulanate

Streptomyces glaucescens is shown to possess -lactamase activity which is inhibitable by clavulanate. This is important in regard to its use as a cloning host for enzymes of \-lactam biosynthesis.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) was found not to inhibit active sodium transport across the isolated frog skin when added in 10^–3 m concentration to the basal-lateral surface. The same Cd⁺⁺ concentration similarly had no effect on Na⁺ transport across the isolated epithelial cell layer from the frog skin, although this dose of Cd⁺⁺ did inhibit Na⁺ transport across the frog urinary bladder and large intestine. When 10^–3 m Cd⁺⁺ was added to the apical surface of the isolated frog skin or to the isolated epithelial cells from the frog skin, sodium transport was reversibly stimulated, in contrast to the irreversible inhibition noted above. If equimolar cysteine was added with Cd⁺⁺ to the apical surface of the skin, however, active Na⁺ transport was irreversibly inhibited. In conjunction with the inhibition produced by equimolar Cd⁺⁺ and cysteine, isotopic Cd⁺⁺ permeation into the tissue was three times higher when added with cysteine than in the absence of cysteine. Thus, the effects of Cd⁺⁺ on epithelial Na⁺ transport is variable according to the epithelium studied and the presence of potential carrier molecules.     

Capacitance,short-circuit current and osmotic water flow across different regions of the isolated toad skin

Summary The amphibian antidiuretic hormone, arginine vasotocin, stimulated osmotic water flow across isolated skin from the pelvic but not the pectoral skin of the toad, Bufo woodhouseii. Changes in the apical membrane capacitance were not observed for either region of the skin following treatment with arginine vasotocin when there was an osmotic gradient across the tissue. In the absence of an osmotic pressure gradient, the apical membrane capacitance of the pelvic skin increased from 2.8±0.5 to 3.3±0.6 F · cm^-2 after treatment with 5 · 10^-8 M arginine vasotocin. Under these conditions, apical membrane capacitance of the pectoral skin was 1.8±0.1 F · cm^-2 and did not change significantly after arginine vasotocin treatment. The amiloride-sensitive short-circuit current across the pelvic skin was stimulated by arginine vasotocin as was the density of channels in the apical membrane as determined by fluctuation analysis. Values for channel density in the pelvic skin also correlated with apical membrane capacitance and increased from 90 to 273 channels per m² of estimated membrane area following arginine vasotocin treatment. In the pectoral skin the stimulation of short-circuit current following arginine vasotocin treatment was small and an increase in channel density could not be demonstrated. The current through single Na⁺ channels in both regions of the skin did not different either before or after arginine vasotocin treatment.Abbreviations A amiloride - ADH antidiuretic hormone - AVT arginine vasotocin - C capacitance - C _a capacitance of apical membrane - f _c corner frequency - i single-channel current - osmotic water flow - IMP intramembrane particles - I _sc short-circuit current - amiloride-sensitive short-circuit current - M channel density - P _o probability of a channel being open - R channel receptor - R _a apical resistance - R _p paracellular resistance