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J Zeilstra-Ryalls

Publications and source records attributed to J Zeilstra-Ryalls.

10 recordsLinked to original sources
PubMed2001

The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1.

Rhodobacter sphaeroides 2.4.1 is an alpha-3 purple nonsulfur eubacterium with an extensive metabolic repertoire. Under anaerobic conditions, it is able to grow by photosynthesis, respiration and fermentation. Photosynthesis may be photoheterotrophic using organic compounds as both a carbon and a reducing source, or photoautotrophic using carbon dioxide as the sole carbon source and hydrogen as the source of reducing power. In addition, R. sphaeroides can grow both chemoheterotrophically and chemoautotrophically. The structural components of this metabolically diverse organism and their modes of integrated regulation are encoded by a genome of approximately 4.5 Mb in size. The genome comprises two chromosomes CI and CII (2.9 and 0.9 Mb, respectively) and five other replicons. Sequencing of the genome has been carried out by two groups, the Joint Genome Institute, which carried out shotgun-sequencing of the entire genome and The University of Texas-Houston Medical School, which carried out a targeted sequencing strategy of CII. Here we describe our current understanding of the genome when data from both of these groups are combined. Previous work had suggested that the two chromosomes are equal partners sharing responsibilities for fundamental cellular processes. This view has been reinforced by our preliminary analysis of the virtually completed genome sequence. We also have some evidence to suggest that two of the plasmids, pRS241a and pRS241b encode chromosomal type functions and their role may be more than that of accessory elements, perhaps representing replicons in a transition state.

Journal Article↗
PubMed2001

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: effect of a transposon insertion in the hbdA gene.

The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985-993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(+)-beta-hydroxybutyryl-coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1999

Characterization of the rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli.

The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli. Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E. coli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo. Pure HemA was recovered in yields of 5-7 mg x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C. The Km values for HemA are 1.9 mM for glycine and 17 microM for succinyl-CoA, with the enzyme showing a turnover number of 430 h-1. In common with other ALASs the recombinant R. sphaeroides HemA requires pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes.

5-Aminolevulinate Synthetase↗
PubMed1996

In vivo protein folding: suppressor analysis of mutations in the groES cochaperone gene of Escherichia coli.

Our previous work has shown that the Escherichia coli groES14 and groES15 mutations result in reduced GroE chaperone machine function. By selecting for restoration of the ability of those mutant groES alleles to suppress the thermosensitivity of bacteria bearing the dnaA46 mutation, we isolated a number of intra- and extragenic suppressors that increase in vivo GroE chaperone function. One of the intragenic suppressors has been mapped to a segment that codes for the GroES mobile loop, previously shown to be indispensable for proper GroES/GroEL interaction. Two extragenic suppressors have been mapped to a groEL segment, previously identified by mutational analysis as coding for an important functional region of the GroEL protein. Our results should contribute to our eventual understanding of the structure-function relationships of the universally conserved GroE chaperone machine.

Bacterial Proteins↗
PubMed1994

Two classes of extragenic suppressor mutations identify functionally distinct regions of the GroEL chaperone of Escherichia coli.

The GroES and GroEL proteins of Escherichia coli function together as the GroE molecular chaperone machine to (i) prevent denaturation and aggregation and (ii) assist the folding and oligomerization of other proteins without being part of the final structure. Previous genetic and biochemical analyses have determined that this activity requires interactions of the GroES 7-mer with the GroEL 14-mer. Recently, we have identified a region of the GroES protein that interacts with the GroEL protein. To identify those residues of the GroEL protein that interact with GroES, we have exploited the thermosensitive phenotype of strains bearing mutations at one or the other of two GroEL-interacting residues of GroES. We have isolated, cloned, and sequenced six suppressor mutations in groEL, three independent isolates for each groES mutant. Changes of only three different amino acid substitutions in GroEL protein were found among these six groEL suppressor mutations. On the basis of a number of in vivo analyses of the chaperone activity of various combinations of groES mutant alleles and groEL suppressor alleles, we propose that an amino-proximal region of the GroEL protein which includes amino acid residues 174 and 190 interacts with GroES and that a carboxyl-proximal region which includes residue 375 interacts with substrate proteins.

Bacterial Proteins↗
PubMed1993

Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity.

In this work we show that the GroEL (Hsp60 equivalent) chaperone protein can protected purified Escherichia coli RNA polymerase (RNAP) holoenzyme from heat inactivation better than the DnaK (Hsp70 equivalent) chaperone can. In this protection reaction, the GroES protein is not essential, but its presence reduces the amount of GroEL required. GroEL and GroES can also reactivate heat-inactivated RNAP in the presence of ATP. The mutant GroEL673 protein, with or without GroES, is incapable of reactivating heat-inactivated RNAP. GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES. The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK's ability to reactivate heat-inactivated RNAP GroEL673 has also been investigated. We found that the DnaJ protein substantially reduces the levels of DnaK protein needed in this reactivation assay. However, the observed lag in reactivation is diminished only in the additional presence of the GrpE protein. Hence, DnaJ and GrpE are involved in both steps of this reactivation reaction (recognition of substrate and release of chaperone from the substrate-chaperone complex) while, in the case of the GroEL-dependent reaction, GroES is involved only during the release of chaperone from the substrate-chaperone complex.

Bacterial Proteins↗
PubMed1993

Characterization of a functionally important mobile domain of GroES.

Although genetic and biochemical evidence has established that GroES is required for the full function of the molecular chaperone, GroEL, little is known about the molecular details of their interaction. GroES enhances the cooperativity of ATP binding and hydrolysis by GroEL (refs 4, 5) and is necessary for release and folding of several GroEL substrates. Here we report that native GroES has a highly mobile and accessible polypeptide loop whose mobility and accessibility are lost upon formation of the GroES/GroEL complex. In addition, lesions present in eight independently isolated mutant groES alleles map in the mobile loop. Studies with synthetic peptides suggest that the loop binds in a hairpin conformation at a site on GroEL that is distinct from the substrate-binding site. Flexibility may be required in the mobile loops on the GroES seven-mer to allow them to bind simultaneously to sites on seven GroEL subunits, which may themselves be able to adopt different arrangements, and thus to modulate allosterically GroEL/substrate affinity.

Amino Acid Sequence↗
PubMed1993

Sequence analysis and phenotypic characterization of groEL mutations that block lambda and T4 bacteriophage growth.

The groES and groEL genes of Escherichia coli have been shown previously to belong to a single operon under heat shock regulation. Both proteins have been universally conserved in nature, as judged by the presence of similar proteins throughout evolution. The GroEL protein has been shown to bind promiscuously to many unfolded proteins, thus preventing their aggregation. ATP hydrolysis by GroEL results in the release of the bound polypeptides, a process that often requires the action of GroES. In an effort to understand GroEL and GroES structure and function, we have determined the nucleotide changes of nine mutant alleles of groEL. All of these mutant alleles were isolated because they block bacteriophage lambda growth. Our sequencing results demonstrate that (i) many of these alleles are identical, in spite of the fact that they were independently isolated, and (ii) most of the different alleles are clustered in the same region of the gene. One of the mutant alleles was shown to possess two nucleotide alterations in the groEL coding phase, one of which is located in a putative ATP-binding domain. The two nucleotide changes were separated by genetic engineering, and each individual change was shown to exert an effect on bacteriophage growth. But, using genetic analyses, we demonstrate that the restriction on bacterial growth at elevated temperatures is conferred only by the mutation within the putative ATP-binding domain. We have cloned the mutant alleles on multicopy plasmids and overexpressed their products. By testing for the ability of bacteriophage either to propagate or to form colonies at 43 degrees C, we have been able to divide the mutant proteins into those with no activity and those with residual activity under the various conditions tested.

Bacterial Proteins↗