Genetic studies of mutants in a high-affinity methionine transport system in Salmonella typhimurium

Summary A total of 30 metP mutations defective in the high-affinity methionine transport system were linked in P1 transduction to the zaf-1351::Tn10 insertion mutation at min 5–6 on the Salmonella typhimurium chromosome map. The relationship of metP to several other markers in this region was studied. Methionine transport was strongly inhibited by arsenate, suggesting that the metP system belongs to the shock-sensitive category and possesses a periplasmic binding protein. However, other experiments provided less clear cut evidence. Transport activity was only slightly reduced by osmotic shock; a methionine binding activity was detected in shock fluids from the wildtype strain, and although this activity was reduced by 50% in 3 frameshift mutants, mutants without any activity were not found. No differences were detected in the shock fluids of the 30 mutants when examined by SDS-polyacrylamide gel electrophoresis.     

Methionine sulfoxide is transported by high-affinity methionine and glutamine transport systems in Salmonella typhimurium.

Three lines of evidence indicated that methionine sulfoxide is transported by the high-affinity methionine and glutamine transport systems in Salmonella typhimurium. First, methionine-requiring strains (metE) which have mutations affecting both of these transport systems (metP glnP) were unable to use methionine sulfoxide as a source of methionine. These strains could still grow on L-methionine because they possessed a low-affinity system (or systems) which transported L-methionine but not the sulfoxide. A methionine auxotroph with a defect only in the metP system, which was dependent upon the glnP+ system for the transport of methionine sulfoxide, was inhibited by L-glutamine because glutamine inhibited the transport of the sulfoxide by the glnP+ system. Second, a metE metP glnP strain could be transduced at either the metP or glnP genes to restore its ability to grow on methionine sulfoxide. Third, the transport of [14C]methionine sulfoxide was inhibited by methionine and by glutamine in the metP+ glnP+ strain. No transport was detected in the metP glnP double-mutant strain.     

Influence of day length and temperature on number of main stem leaves and time to flowering in lupin

A growth chamber experiment was carried out to investigate the influence of day length and temperature on the development of flowering in eight varieties of the three grain lupin species Lupinus albus (Wat and C3396), L. angustifolius (Gungurru, Polonez and W26) and L. luteus, (Juno, Radames and Teo). The plants were grown at two temperatures, 10°C and 18°C, in combination with five daylength regimes: 10, 14, 18, 24 h day at full light intensity and 10 h full light extended with 8 h low intensity light. Increased daylength decreased days from sowing to flowering in all varieties, but had little effect on thermal time to flowering in most varieties. However, C3396, W26 and Radames had a significantly longer thermal time to flowering at high, non‐vernalising temperature (18°C) at short daylengths. Low light intensity daylength extension did not significantly influence thermal time to flowering. For flower initiation, measured as number of leaves on the main stem three types of response were found. All varieties formed fewer leaves on the main stem at 10°C than at 18°C, although the two thermo‐neutral varieties of L. luteus, Juno and Teo, gave only a small response to temperature and daylength. In Polonez, Gungurru and Wat, low temperature decreased leaf number, but there was only a small response to changes in daylength. Three varieties, C3396, W26 and Radames, showed longer thermal time to flowering at 18°C with short daylengths. This could be explained by a greater number of main stem leaves formed at short daylength at non‐vernalising temperatures. Increased daylength decreased leaf number in these varieties, but never to a smaller number than for plants grown at 10°C. In these varieties, low intensity extension of the daylength had a similar (W26, Radames) or decreased (C3396) effect compared to full light extension. The hastening of time to flowering by long days could be separated into two effects: a high light energy effect hastened development by increasing the rate of leaf appearance in all varieties, while low light energy in thermo‐sensitive varieties was able to substitute for vernalisation by decreasing leaf number.     

The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium.

Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Influence of the GroE molecular chaperone machine on the in vitro refolding of Escherichia coli beta-galactosidase.

We have studied the effect of the components of the GroE molecular chaperone machine on the refolding of the Escherichia coli enzyme beta-galactosidase, a tetrameric protein whose 116-kDa promoters should not completely fit within the central cavity of the GroEL toroid. In the absence of other additives, GroEL formed a weak complex with chemically denatured beta-galactosidase, reduced its propensity to aggregate, and increased the recovery yields of active enzyme twofold without altering its folding pathway. When present together with the chaperonin, ATP--and to a lesser extent AMP-PNP--reduced the recovery yields and led to the resumption of aggregation. The use of the complete chaperonin system (GroEL, GroES, and ATP) eliminated the GroEL-mediated increase in recovery and folding proceeded less efficiently than in buffer alone. This unusual behavior can be explained in terms of a chaperonin "buffering" effect and the different affinities of GroE complexes for denatured beta-galactosidase.     

Mutations in the pterin-4α-carbinolamine dehydratase (PCBD) gene cause a benign form of hyperphenylalaninemia

Four patients with primapterinuria, postulated to be due to pterin-4α-carbinolamine dehydratase (PCD) deficiency, were diagnosed by biochemical and DNA analysis. All four patients presented in the neonatal period with hyperphenylalaninemia, and elevated neopterin and decreased biopterin levels in the urine. These symptoms are common to 6-pyruvoyltetrahydropterin synthase deficiency and thus there is a danger of misdiagnosis. In addition, all four patients had elevated urinary excretion of primapterin (7-biopterin), the only persistent biochemical abnormality. Analysis of fibroblast DNA from the patients identified the following mutations in the PCBD gene: one patient homozygous for the missense mutation E96K and one homozygous for the nonsense mutation Q97X, both in exon 4; one compound heterozygote with the mutations E96K and Q97X; and one patient with two different homozygous mutations: E26X in exon 2 and R87Q in exon 4. In two families, the parents were investigated and found to be obligate heterozygotes for particular mutations. One sibling was found to be unaffected. These results further substantiate the idea that primapterinuria is associated with mutations in the PCBD gene. Received: 4 March 1998 / Accepted: 17 April 1998     

The role of lipopolysaccharide as a receptor for some bacteriophages of Pseudomonas aeruginosa

Typing phages of the Colindale typing set for Pseudomonas aeruginosa have been tested for the use of lipopolysaccharide (LPS) as a receptor. Studies using the reference strains of the International Antigenic Typing Scheme for O-serotypes of P. aeruginosa supported earlier indications that none of the phages were O-specific. Studies of the adsorption of phages to LPS showed that typing phages 16, 44, F8, 68, 109, 352, and 1214 (as well as other phages 2 and H22) were LPS-specific, but were not consistently adsorbed by isolated LPS from all sensitive strains. Water-soluble fractions from LPS did not adsorb phages and did not inhibit their neutralization by whole LPS. No endoglycosidase activity against LPS was detected for any phage. The significance of these results for the roles of LPS in the adsorption process and phage sensitivity are discussed.