Biomedical subjects

J T Beatty

Publications and source records attributed to J T Beatty.

At least 37 records · Page 2Linked to original sources

PubMed1998

Genetic complementation and kinetic analyses of Rhodobacter capsulatus ORF1696 mutants indicate that the ORF1696 protein enhances assembly of the light-harvesting I complex.

Rhodobacter capsulatus ORF1696 mutant strains were created by insertion of antibiotic resistance cartridges at different sites within the ORF1696 gene in a strain that lacks the light-harvesting II (LHII) complex. Steady-state absorption spectroscopy profiles and the kinetics of the light-harvesting I (LHI) complex assembly and decay were used to evaluate the function of the ORF1696 protein in various strains. All of the mutant strains were found to be deficient in the LHI complex, including one (deltaNae) with a disruption located 13 codons before the 3' end of the gene. A 5'-proximal disruption after the 31st codon of ORF1696 resulted in a mutant strain (deltaMun) with a novel absorption spectrum. The two strains with more 3' disruptions (deltaStu and deltaNae) were restored nearly to the parental strain phenotype when trans complemented with a plasmid expressing the ORF1696 gene, but deltaMun was not. The absorption spectrum of deltaMun resembled that of a strain which had a polar mutation in ORF1696. We suggest that a rho-dependent transcription termination site exists between the MunI and proximal StuI sites of ORF1696. A comparison of LHI complex assembly kinetics showed that assembly occurred 2.6-fold faster in the parental strain than in strain deltaStu. In contrast, LHI complex decay occurred 1.7-fold faster in the ORF1696 parental strain than in deltaStu. These results indicate that the ORF1696 protein has a major effect on LHI complex assembly, and models of ORF1696 function are proposed.

Bacterial Proteins↗

PubMed1998

Aerobic anoxygenic phototrophic bacteria.

The aerobic anoxygenic phototrophic bacteria are a relatively recently discovered bacterial group. Although taxonomically and phylogenetically heterogeneous, these bacteria share the following distinguishing features: the presence of bacteriochlorophyll a incorporated into reaction center and light-harvesting complexes, low levels of the photosynthetic unit in cells, an abundance of carotenoids, a strong inhibition by light of bacteriochlorophyll synthesis, and the inability to grow photosynthetically under anaerobic conditions. Aerobic anoxygenic phototrophic bacteria are classified in two marine (Erythrobacter and Roseobacter) and six freshwater (Acidiphilium, Erythromicrobium, Erythromonas, Porphyrobacter, Roseococcus, and Sandaracinobacter) genera, which phylogenetically belong to the alpha-1, alpha-3, and alpha-4 subclasses of the class Proteobacteria. Despite this phylogenetic information, the evolution and ancestry of their photosynthetic properties are unclear. We discuss several current proposals for the evolutionary origin of aerobic phototrophic bacteria. The closest phylogenetic relatives of aerobic phototrophic bacteria include facultatively anaerobic purple nonsulfur phototrophic bacteria. Since these two bacterial groups share many properties, yet have significant differences, we compare and contrast their physiology, with an emphasis on morphology and photosynthetic and other metabolic processes.

Aerobiosis↗

PubMed1997

Reorganization of the genus Erythromicrobium: description of "Erythromicrobium sibiricum" as Sandaracinobacter sibiricus gen. nov., sp. nov., and of "Erythromicrobium ursincola" as Erythromonas ursincola gen. nov., sp. nov.

The results of investigations on the morphology, physiology, pigment composition, light-harvesting antenna and reaction center organization, and electron carriers of five Erythromicrobium representatives, and on phylogenetic relations among them, are summarized. On the basis of clear phenotypic differences and distinct phylogenetic positions shown by 16S ribosomal DNA analysis, the tentative species "Erythromicrobium sibiricum" and "Erythromicrobium ursincola" are formally described as the type species of two new genera: Sandaracinobacter sibiricus gen. nov., sp. nov., and Erythromonas ursincola gen. nov., sp. nov., respectively. The genus Erythromicrobium is at present composed of the type species, E. ramosum, and two species, "E. hydrolyticum" and "E. ezovicum," whose nomenclature is yet to be validated. All species studied group within the alpha-4 subclass of Proteobacteria.

Absorptiometry, Photon↗

PubMed1997

Cloning, sequencing, and oxygen regulation of the Rhodobacter capsulatus alpha-ketoglutarate dehydrogenase operon.

The Rhodobacter capsulatus sucA, sucB, and lpd genes, which encode the alpha-ketoglutarate dehydrogenase (E1o), the dihydrolipoamide succinyltransferase (E2o), and the dihydrolipoamide dehydrogenase (E3) components of the alpha-ketoglutarate dehydrogenase complex (KGD), respectively, were cloned, sequenced, and used for regulatory analyses. The KGD enzymatic activity was greater in cells grown under aerobic, respiratory growth conditions than under anaerobic, photosynthetic conditions. Similarly, the sucA gene was transcribed differentially, leading to a greater accumulation of sucA mRNAs under respiratory growth conditions than under photosynthetic conditions, although differential rates of mRNA decay could also contribute to the different amounts of sucA mRNAs under these two growth conditions. The sucA promoter was located about 4 kb upstream of the 5' end of the sucA gene, and transcripts greater than 9.5 kb hybridized to a sucA probe, suggesting the presence of an operon that produces a polycistronic mRNA. Thus, these genes seem to be expressed as an unstable primary transcript, and we speculate that posttranscriptional processes control the stoichiometry of KGD proteins.

Acyltransferases↗

PubMed1996

Purple sulfur bacteria control the growth of aerobic heterotrophic bacterioplankton in a meromictic salt lake.

In meromictic Mahoney Lake, British Columbia, Canada, the heterotrophic bacterial production in the mixolimnion exceeded concomitant primary production by a factor of 7. Bacterial growth rates were correlated neither to primary production nor to the amount of chlorophyll a. Both results indicate an uncoupling of bacteria and phytoplankton. In the chemocline of the lake, an extremely dense population of the purple sulfur bacterium Amoebobacter purpureus is present year round. We investigated whether anoxygenic phototrophs are significant for the growth of aerobic bacterioplankton in the overlaying water. Bacterial growth rates in the mixolimnion were limited by inorganic phosphorus or nitrogen most of the time, and the biomass of heterotrophic bacteria did not increase until, in autumn, 86% of the cells of A. purpureus appeared in the mixolimnion because of their reduced buoyant density. The increase in heterotrophic bacterial biomass, soluble phosphorus concentrations below the detection limit, and an extraordinarily high activity of alkaline phosphatase in the mixolimnion indicate a rapid liberation of organically bound phosphorus from A. purpureus cells accompanied by a simultaneous incorporation into heterotrophic bacterioplankton. High concentrations of allochthonously derived dissolved organic carbon (mean, 60 mg of C(middot)liter(sup-1)) were measured in the lake water. In Mahoney Lake, liberation of phosphorus from upwelling purple sulfur bacteria and degradation of allochthonous dissolved organic carbon as an additional carbon source render heterotrophic bacterial production largely independent of the photosynthesis of phytoplankton. A recycling of inorganic nutrients via phototrophic bacteria also appears to be relevant in other lakes with anoxic bottom waters.

Journal Article↗

PubMed1996

Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II.

A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC'::pho'A gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yield approximately 1/20th as much activity. Replacement of pho'A with lac'Z in nine of the fusions confirmed the topology, as beta-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membrane-spanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus delta pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.

Alkaline Phosphatase↗

PubMed1996

Directed mutagenesis of the Rhodobacter capsulatus puhA gene and orf 214: pleiotropic effects on photosynthetic reaction center and light-harvesting 1 complexes.

Rhodobacter capsulatus puhA mutant strains containing either a nonpolar, translationally in-frame deletion or a polar insertion of an antibiotic resistance cartridge were constructed and evaluated for their photosynthetic growth properties, absorption spectroscopy profiles, and chromatophore protein compositions. Both types of mutants were found to be incapable of photosynthetic growth and deficient in the reaction center (RC) and light-harvesting 1 (LH1) complexes. The translationally in-frame puhA deletion strains were restored to the parental strain phenotypes by complementation with a plasmid containing the puhA gene, whereas the polar puhA mutants were not. Analogous nonpolar and polar disruptions of orf 214 (located immediately 3' of the puhA gene) were made, and the resultant mutant strains were evaluated as described above. The strain containing the nonpolar deletion of orf 214 exhibited severely impaired photosynthetic growth properties and had greatly reduced levels of the RC and LH1 complexes. Complementation of this strain with a plasmid that expressed orf 214 from the nifHDK promoter restored photosynthetic growth capability, as well as the RC and LH1 complexes. The polar disruption of orf 214 yielded cells that were incapable of photosynthetic growth and had even lower levels of the RC and LH1 complexes, and complementation in trans with orf 214 only marginally improved these deficiencies. These results indicate that orf 214 and at least one additional gene located 3' of orf 214 are required to obtain the RC and LH1 complexes, and transcription read-through from the puhA superoperon is necessary for optimal expression of these new photosynthesis genes.

Amino Acid Sequence↗

PubMed1995

Mutation of the Ser2 codon of the light-harvesting B870 alpha polypeptide of Rhodobacter capsulatus partially suppresses the pufX phenotype.

The exact function of the pufX gene product of Rhodobacter capsulatus is uncertain, but deletion of the pufX gene renders cells incapable of phototrophic growth on a minimal medium, and photosynthetic electron transfer is impaired in vitro. However, suppressor mutants that are able to grow phototropically are readily isolated. Two such suppressor mutants were characterized as to their phototrophic growth properties, their fluorescence at different incident light intensities, the integrity of their chromatophores, and their abilities to generate a transmembrane potential. We found that the photosynthetic apparatus in the suppressor mutants was less stable than that of the pseudo-wild-type and primary mutant strains and that the suppressor mutants used light energy less efficiently than the pseudo-wild-type strain. Therefore, the suppressor strains are more precisely designated partial suppressor mutants. The locations and sequences of the suppressor mutations were determined, and both were found to change the second codon of the pufA gene. It is hypothesized that the serine residue specified by this codon is important in interactions between the B870 alpha protein and other membrane-bound polypeptides and that suppressor mutations at this position partially compensate for loss of the PufX protein. A model is proposed for the function of the PufX protein.

Amino Acid Sequence↗

PubMed1994

Directed mutational analysis of bacteriochlorophyll a biosynthesis in Rhodobacter capsulatus.

Previous studies have established that most if not all of the genes required for synthesis of the Rhodobacter capsulatus essential photosystem are clustered on a 46 kb region of the chromosome known as the photosynthesis gene cluster. This region has recently been sequenced in its entirety by Hearst and co-workers, revealing the existence of 23 open reading frames, many of which are thought to be involved in the synthesis of bacteriochlorophyll. In this study we have undertaken a systematic directed mutational analysis of 12 open reading frames in the photosynthesis gene cluster to evaluate whether individual open reading frames have a role in photopigment biosynthesis. The results of this analysis demonstrate that mutations constructed in seven open reading frames resulted in a loss of bacteriochlorophyll biosynthesis, concomitant with the accumulation of specific intermediates in the Mg-tetrapyrrole biosynthetic pathway. One mutation was observed to result in partial disruption of bacteriochlorophyll biosynthesis, leading to the accumulation of bacteriochlorophyll as well as an intermediate in the biosynthetic pathway. We also observed that disruptions constructed in four open reading frames had no discernible effect on the synthesis of photopigments. The results of this analysis are discussed with regard to our current understanding of the role of each of these open reading frames in the synthesis of the R. capsulatus photosystem.

Bacterial Proteins↗

PubMed1993

The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation.

All photosynthetic organisms, with the exception of several species of photosynthetic bacteria, are thought to contain the sulfolipid 6-sulfo-alpha-D-quinovosyldiacylglycerol. The association of this lipid with photosynthetic membranes has led to the assumption that it plays some role in photosynthesis. Stable null mutants of the photosynthetic bacterium Rhodobacter sphaeroides completely lacking sulfolipid were obtained by disruption of the sqdB gene. The ratios of the various components of the photosynthetic electron transport chain, as well as the electron transfer rates during cyclic electron transport, were not altered in the mutants, when grown under optimal conditions. Growth rates of wild type and mutants were identical under a variety of growth conditions, with the exception of phosphate limitation, which resulted in reduced growth of the mutants. Phosphate limitation of the wild type caused a significant reduction in the amount of all phospholipids and an increased amount of sulfolipid. By contrast, the sulfolipid-deficient mutant had reduced levels of phosphatidylcholine and phosphatidylethanolamine but maintained a normal level of phosphatidylglycerol. In addition, two unidentified lipids lacking phosphorus accumulated in the membranes of both wild-type and mutant strains under phosphate limitation. We conclude that sulfolipid plays no significant unique role in photoheterotrophic growth or photosynthetic electron transport in R. sphaeroides but may function as a surrogate for phospholipids, particularly phosphatidylglycerol, under phosphate-limiting conditions.

Bacterial Chromatophores↗

PubMed1993

Rhodobacter capsulatus puc operon: promoter location, transcript sizes and effects of deletions on photosynthetic growth.

Gene deletions of the puc operon of Rhodobacter capsulatus showed that the pucC and pucE genes, but not pucD, were required for formation of wild-type levels of the LHII complex. Deletion of pucC or pucE also impaired photosynthetic growth. The effects of pucC deletion were suppressed by secondary mutations that mapped outside the puc operon. Fusion of a lac'Z gene to pucE' showed that most of pucE transcription originated from upstream of pucB. RNA blot analysis revealed a 2.4 kb transcript that hybridized to probes specific for the pucBA, pucC and pucDE regions, indicating that some puc operon messages extend from just before the pucB gene to just after the pucE gene.

Bacterial Proteins↗

PubMed1992

Derivation of a mathematical expression useful for the construction of complete genomic libraries.

We present and derive a formula that is useful for the design and evaluation of gene cloning experiments in which a complete gene library of the entire genome of an organism is desired. The formula n = ln(1-phi f)/ln(1-f) (in which n is the number of recombinant clones required to ensure a probability, phi, of obtaining at least one of each of all possible gene sequences, and f is the fraction of the genome contained in an average-sized DNA fragment) applies to construction of libraries, in which at least one copy of all the genetic information of a genome is required. The use of this formula for quantitative evaluation of genomic libraries should give greater assurance that a given library would be complete.

Cloning, Molecular↗

PubMed1992

Pleiotropic effects of pufX gene deletion on the structure and function of the photosynthetic apparatus of Rhodobacter capsulatus.

By deletion of the pufX gene of Rhodobacter capsulatus from a plasmid carrying the puf operon and complementation of a chromosomal puf operon deletion, we created pufX mutants and used them to characterize possible functions of the pufX gene product. The pufX mutants were incapable of photosynthetic growth in a minimal medium, or in a rich medium at low light intensities, although second-site mutations suppressed this phenotype. Measurements made in vitro with intact and solubilized chromatophore preparations indicated that the individual complexes of the photosynthetic unit seemed to function normally, but electron transfer from the reaction center to the cytochrome b/c1 complex was impaired. The structures of the photosynthetic apparatus of pseudo-wild type and mutant strains were evaluated using absorption spectroscopy and electron microscopy. The pufX mutants had intracytoplasmic membrane invaginations about 50% larger in diameter than those of the pseudo-wild type and higher levels of B870 light-harvesting complex. It is concluded that the PufX protein plays an important role in the structure of the functional photosynthetic unit, and its absence results in loss of efficient electron transfer from the QB site of the reaction center to the Qz site of the cytochrome b/c1 complex.

Base Sequence↗

PubMed1991

Overlapping mRNA transcripts of photosynthesis gene operons in Rhodobacter capsulatus.

The crtEF, bchCA, and puf operons of the facultative phototrophic bacterium Rhodobacter capsulatus encode gene products that are necessary for the formation of various components of the photosynthetic apparatus. The crtEF operon encodes two enzymes involved in the biosynthesis of carotenoids, the bchCA operon codes for two enzymes of the bacteriochlorophyll biosynthetic pathway, and the puf operon encodes four pigment-binding polypeptides as well as two polypeptides with less well understood functions. These three operons are adjacent to one another on the chromosome and are transcribed in the same direction. We present the results of RNA blotting and S1 nuclease protection end-mapping experiments which provide direct evidence that the mRNA transcripts of these three operons overlap. Therefore, it is likely that the crtEF, bchCA, and puf operons can be expressed as a single transcriptional unit, although RNA polymerase may initiate transcription at any of several promoters.

Base Sequence↗

PubMed1991

Functional significance of overlapping transcripts of crtEF, bchCA, and puf photosynthesis gene operons in Rhodobacter capsulatus.

The Rhodobacter capsulatus crtEF, bchCA, and puf operons each encode products that function together to produce photosynthetic pigment-protein complexes. The crtEF operon encodes two enzymes of the carotenoid biosynthetic pathway, the bchCA operon encodes two enzymes of the bacteriochlorophyll biosynthetic pathway, and the puf operon contains six genes, four of which are known to code for proteins that bind pigment cofactors and position them in the intracytoplasmic membrane. These operons are adjacent on the chromosome and are transcribed in the same direction. Although each of these operons can be expressed from its own promoter, it is possible for transcription to begin at the crtEF promoter and continue through the bchCA and puf operons. We herein present the results of experiments which demonstrate that this overlapping transcriptional arrangement is important for establishment of normal levels of transcripts of the bchCA and puf operons and that read-through transcription from the bchCA operon contributes significantly to the expression of a pufB'::lac'Z gene fusion. Growth studies of crtF and bchC interposon mutants show that this read-through transcription is necessary for efficient transition from respiratory to photosynthetic growth modes.

Blotting, Northern↗

PubMed1989

Promoter mapping and nucleotide sequence of the bchC bacteriochlorophyll biosynthesis gene from Rhodobacter capsulatus.

Because there are not yet direct assays for most of the proteins required for differentiation of Rhodobacter capsulatus cytoplasmic membrane into photosynthetically competent intracytoplasmic membrane, a molecular inquiry into the mechanism and regulation of this process is difficult. We have, therefore, chosen to isolate R. capsulatus photosynthesis genes by creating in-frame fusions to lac'Z vectors, and selecting for those that direct appropriately regulated levels of beta-galactosidase in R. capsulatus. One lacZ fusion isolate was used to identify an open reading frame (ORF) of unknown function and flanking sequences that promoted initiation of transcription. The chromosomal copy of this ORF was mutated by insertion of a kanamycin-resistant cartridge into the cloned fragment and substitution for the chromosomal copy by homologous recombination. The phenotype of the resultant mutant cells showed that the ORF encodes 2-desacetyl-2-hydroxyethyl bacteriochlorophyllide a dehydrogenase, an enzyme that catalyzes the penultimate step in bacteriochlorophyll a biosynthesis. The nucleotide sequence of this bchC gene and its 5' regulatory region were determined. The deduced amino acid sequence shows that the bchC gene encodes a 33-kDa protein that is less hydrophobic than integral membrane proteins of R. capsulatus, although there are hydrophobic segments that could in principle interact with a lipid membrane. Results of S1 nuclease protection and primer extension experiments show that a 5' mRNA end is positioned within the cloned segment, and that this 5' end maps to sequences with significant sequence similarity to the previously characterized puf operon promoter region.

Amino Acid Sequence↗

PubMed1989

Formation of functional inter-species hybrid photosynthetic complexes in Rhodobacter capsulatus.

A Rhodobacter capsulatus mutant strain deficient in all pigment-binding peptides and hence incapable of photosynthetic growth was genetically complemented with a plasmid-borne copy of the Rhodobacter sphaeroides puf operon. Hybrid reaction centers composed of R. sphaeroides L and M and R. capsulatus H subunits assembled in vivo, and host cells were photosynthetically competent. Light-harvesting complex B875, also encoded by the R. sphaeroides puf operon, was present along with the hybrid reaction center. These cells emitted fluorescence, however, indicating an impairment in energy transduction.

Bacteria↗

PubMed1989

Expression of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) in Escherichia coli: export and processing of the apoprotein.

The expression of cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) was examined in Escherichia coli transformed with either of two plasmids, pJ8 and pJ81. The former has an 840 bp insert of D. vulgaris DNA, containing the structural gene for cytochrome c3 (387 bp) and its promoter region. Plasmid pJ81 was generated from pJ8 by deoxyoligonucleotide-directed mutagenesis to direct the synthesis of a protein with an altered signal peptidase cleavage site [Ala(-1)----Asp(-1)]. Synthesis of the 14 kDa precursor, which was partly processed to the 12 kDa mature protein, was observed in cells of E. coli TG2(pJ8) by SDS gel electrophoresis and Western blotting. Analysis of spheroplasts revealed that the processed polypeptide was present in the periplasm while the precursor was found only in the membrane/cytoplasmic fraction. No processing was observed in E. coli TG2(pJ81) cells, due to the mutation of the signal peptide cleavage site. No insertion of haem into the E. coli product could be detected in E. coli TG2(pJ8) cells by post-electrophoretic protohaem fluorescence analysis. The sensitivity of the cytochrome c3 synthesized in E. coli TG2(pJ8) to digestion by chymotrypsin also indicated that the apoprotein was formed. The results indicate that E. coli is capable of synthesizing and exporting the cytochrome c3 polypeptide, but fails to insert the haems.

Blotting, Western↗