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S Maloy

Publications and source records attributed to S Maloy.

28 records · Page 2Linked to original sources
PubMed1991

Dissecting the molecular mechanism of ion-solute cotransport: substrate specificity mutations in the putP gene affect the kinetics of proline transport.

Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of the putP gene, suggesting that they may replace amino acids at the active site of the enzyme. If putP substrate specificity mutations directly after the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of three putP substrate specificity mutants. One class of mutation increases the Km over 120 fold but only decreases the Vmax fourfold. Such Km mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases the Vmax 80-fold without changing the Km. Vmax mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both the Km and Vmax of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na+/proline symport.

Amino Acid Transport Systems, Neutral↗
PubMed1991

Regulation of proline utilization in Salmonella typhimurium: a membrane-associated dehydrogenase binds DNA in vitro.

The PutA protein is a membrane-associated enzyme that catalyzes the degradation of proline to glutamate. Genetic evidence suggests that in the absence of proline, the PutA protein also represses transcription of the putA and putP genes. To directly determine whether PutA protein binds to the put control region, we analyzed gel retardation of put control region DNA by purified PutA protein in vitro. The put control region is 420 bp. Purified PutA protein bound specifically to several nonoverlapping fragments of control region DNA, indicating the presence of multiple binding sites in the control region. Electrophoretic abnormalities and behavior of circularly permuted fragments of control region DNA indicate that it contains a region of intrinsically curved DNA. To determine whether the multiple binding sites or the DNA curvature are important in vivo, two types of deletions were constructed: (i) deletions that removed sequences predicted to contribute to DNA curvature as well as potential operator sites and (ii) deletions that removed only potential operator sites. Both types of deletions increased expression of the put genes but were still induced by proline, indicating that multiple cis elements are involved in repression. These data suggest a model for put repression that invokes the formation of a complex between PutA protein molecules bound at different sites in the control region, brought into proximity by a loop of curved DNA.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1991

Regulation of proline utilization in enteric bacteria: cloning and characterization of the Klebsiella put control region.

Enteric bacteria can grow on proline as the sole nitrogen and carbon source. Expression of the proline utilization (put) operon in Klebsiella strains and Escherichia coli is responsive to nitrogen regulation. In contrast, Salmonella typhimurium cannot activate put operon expression when growing in medium with glucose as a carbon source and proline as the sole nitrogen source. To compare nitrogen regulatory sites in the control regions of the put operons in these three closely related genera, we cloned the Klebsiella put operon onto a plasmid. The putA and putP genes were localized on the plasmid by transposon mutagenesis. The DNA sequence of the put control region was determined and compared with those of the put control regions from S. typhimurium and E. coli. The overall size and organization of the put control region were very similar in all three bacteria. However, no obvious ntr regulatory sites were found in this region, and transcription of the put genes started at the same sites during growth with limiting or excess nitrogen. These results strongly suggested that the Klebsiella put operon may not be directly regulated by the ntr system.

Base Sequence↗
PubMed1990

Regulation of proline utilization in Salmonella typhimurium: how do cells avoid a futile cycle?

Salmonella typhimurium can degrade proline for use as a carbon, nitrogen, or energy source. To determine whether a futile cycle occurs which degrades the proline accumulated by proline biosynthesis, we studied the expression and enzymatic activity of the proline utilization (put) pathway under conditions which increase the concentration of the intracellular proline pools: catabolism of the dipeptide glycyl-proline, overproduction of proline due to a mutation which prevents feedback inhibition of proline biosynthesis, and accumulation of proline due to osmotic stress. The results indicate that: (i) internal proline induces the put genes, but only when accumulated to concentrations greater than the normal proline biosynthetic pool; and (ii) degradation of proline pools accumulated under high osmotic pressure is limited because proline oxidase is directly inhibited under these conditions.

Biological Transport↗
PubMed1990

Phylogenetic placement of the Spirosomaceae.

Comparative analysis of 16S rRNA sequences shows that the family Spirosomaceae belongs within the eubacterial phylum defined by the flavobacteria and bacteriodes. Its constituent genera, Spirosoma, Flectobacillus, and Runella form a monophyletic grouping therein. The phylogenetic assignment is based not only upon evolutionary distance analysis, but also upon sequence signatures and higher order structural synapomorphies in 16S rRNA. Another genus peripherally associated with the Spirosomaceae, Ancylobacter ("Microcyclus"), does not cluster with the flavobacteria and their relatives, but rather belongs to the alpha subdivision of the purple bacteria.

Bacteria↗
PubMed1990

Activation of a new proline transport system in Salmonella typhimurium.

Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes.

Biological Transport↗