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C E Bauer

Publications and source records attributed to C E Bauer.

At least 37 records · Page 2Linked to original sources
PubMed1998

Analysis of the puc operon promoter from Rhodobacter capsulatus.

Expression of the Rhodobacter capsulatus puc operon, which codes for structural polypeptides of the light-harvesting-II peripheral antenna complex, is highly regulated in response to alterations in oxygen tension and light intensity. To obtain an understanding of the puc promoter region we report the high-resolution 5' mapping of the puc mRNA transcriptional start site and DNA sequence analysis of the puc upstream regulatory sequence (pucURS). A sigma70-type promoter sequence was identified (pucP1) which has a high degree of sequence similarity with carotenoid and bacteriochlorophyll biosynthesis promoters. Inspection of the DNA sequence also indicated the presence of two CrtJ and four integration host factor (IHF) binding sites. Transcriptional fusions of the pucURS fused to lacZ also confirmed that puc promoter activity is regulated by the transcriptional regulators IHF, CrtJ, and RegA. Gel retardation analysis using cell extracts indicates that mutations in IHF and RegA disrupt protein binding to DNA fragments containing the pucURS.

Base Sequence↗
PubMed1998

Isolation of Rhodospirillum centenum mutants defective in phototactic colony motility by transposon mutagenesis.

The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria.

Chemotaxis↗
PubMed1997

DNA binding characteristics of CrtJ. A redox-responding repressor of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression in Rhodobacter capsulatus.

Previous genetic analysis indicated that the photosynthesis gene cluster from Rhodobacter capsulatus coded for the transcription factor, CrtJ, that is responsible for aerobic repression of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression. In this study, we have heterologously overexpressed and purified CrtJ to homogeneity and shown by gel mobility shift assays that CrtJ is biologically active. DNase I footprint analysis confirms molecular genetic studies by showing that CrtJ binds to conserved palindromic sequences that overlap the -10 and -35 promoter regions of the bchC operon. Graphs of the percentage of DNA bound versus protein concentration show sigmoidal curves, which is highly indicative of cooperative binding of CrtJ to the two palindromic sites. A binding constant for interaction of CrtJ with the palindrome that spans the -10 region was calculated to be 4.8 x 10(-9) M, whereas affinity for the palindrome that spans the -35 region was found to be 2.9 x 10(-9) M. Binding of CrtJ to the bchC promoter region was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-fold higher binding affinity under oxidizing versus reducing conditions.

Bacterial Proteins↗
PubMed1997

Characterization of chlorophyll a and bacteriochlorophyll a synthases by heterologous expression in Escherichia coli.

Genes coding for putative chlorophyll a synthase (chlG) from Synechocystis sp. PCC 6803 and bacteriochlorophyll a synthase (bchG) from Rhodobacter capsulatus were amplified by the polymerase chain reaction and cloned into T7 RNA polymerase-based expression plasmids. In vitro enzymatic assays indicated that heterologous expression of the chlG and bchG gene products in Escherichia coli conferred chlorophyll a and bacteriochlorophyll a synthase activity, respectively. Chlorophyll a synthase utilized chlorophyllide a, but not bacteriochlorophyllide a, as a substrate, whereas bacteriochlorophyll a synthase utilized bacteriochlorophyllide a, but not chlorophyllide a. Both enzymes were also observed to exhibit a marked preference for phytyl diphosphate over geranylgeranyl diphosphate.

Bacteriochlorophylls↗
PubMed1997

Analysis of a chemotaxis operon from Rhodospirillum centenum.

A chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum has been cloned, sequenced, and analyzed for the control of transcription during swimmer-to-swarm cell differentiation. The first gene of the operon (cheAY) codes for a large 108-kDa polypeptide with an amino-terminal domain that is homologous to CheA and a carboxyl terminus that is homologous to CheY. cheAY is followed by cheW, an additional homolog of cheY, cheB, and cheR. Sequence analysis indicated that all of the che genes are tightly compacted with the same transcriptional polarity, suggesting that they are organized in an operon. Cotranscription of the che genes was confirmed by demonstrating through Western blot analysis that insertion of a polar spectinomycin resistance gene in cheAY results in loss of cheR expression. The promoter for the che operon was mapped by primer extension analysis as well as by the construction of promoter reporter plasmids that include several deletion intervals. This analysis indicated that the R. centenum che operon utilizes two promoters; one exhibits a sigma 70-like sequence motif, and the other exhibits a sigma 54-like motif. Expression of the che operon is shown to be relatively constant for swimmer cells which contain a single flagellum and for swarm cells that contain multiple lateral flagella.

Amino Acid Sequence↗
PubMed1997

Chemosensory and photosensory perception in purple photosynthetic bacteria utilize common signal transduction components.

The chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum contains five open reading frames (ORFs) that have significant sequence homology to chemotaxis genes from other bacteria. To elucidate the functions of each ORF, we have made various mutations in the gene cluster and analyzed their phenotypic defects. Deletion of the entire che operon (delta che), as well as nonpolar disruptions of cheAY, cheW, and cheR, resulted in a smooth-swimming phenotype, whereas disruption of cheB resulted in a locked tumbly phenotype. Each of these mutants was defective in chemotactic response. Interestingly, disruption of cheY resulted in a slight increase in the frequency of tumbling/reversal with no obvious defects in chemotactic response. In contrast to observations with Escherichia coli and several other bacteria, we found that all of the che mutant cells were capable of differentiating into hyperflagellated swarmer cells when plated on a solid agar surface. When viewed microscopically, the smooth-swimming che mutants exhibited active surface motility but were unable to respond to a step-down in light intensity. Both positive and negative phototactic responses were abolished in all che mutants, including the cheY mutant. These results indicate that eubacterial photosensory perception is mediated by light-generated signals that are transmitted through the chemotaxis signal transduction cascade.

Bacterial Proteins↗
PubMed1997

Genetic analysis of chlorophyll biosynthesis.

During this decade, there have been major advancements in the understanding of genetic loci involved in synthesis of the family of Mg-tetrapyrroles known as chlorophylls and bacteriochlorophylls. Molecular genetic analysis of Mg-tetrapyrrole biosynthesis was initiated by the performance of detailed sequence and mutational analysis of the photosynthesis gene cluster from Rhodobacter capsulatus. These studies provided the first detailed understanding of genes involved in bacteriochlorophyll a biosynthesis. In the short time since these studies were initiated, most of the chlorophyll biosynthesis genes have been identified by virtue of their ability to complement bacteriochlorophyll a biosynthesis mutants as well as by sequence homology comparisons. This review is centered on a discussion of our current understanding of bacterial, algal, and plant genes that code for enzymes in the Mg-branch of the tetrapyrrole biosynthetic pathway that are responsible for synthesis of chlorophylls and bacteriochlorophylls.

Chlorophyll↗
PubMed1996

Cloning and characterization of the chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by complementation of a bacteriochlorophyll biosynthesis mutant of Rhodobacter capsulatus.

A bacteriochlorophyll a biosynthesis mutant of the purple photosynthetic bacterium Rhodobacter capsulatus was functionally complemented with a cosmid genomic library from Synechocystis sp. PCC 6803. The complemented R. capsulatus strain contains a defined mutation in the bchM gene that codes for Mg-protoporphyrin IX methyltransferase, the enzyme which converts Mg-protoporphyrin IX to Mg-protoporphyrin IX methylester using S-adenosyl-L-methionine as a cofactor. Since chlorophyll biosynthesis also requires the same methylation reaction, the Synechocystis genome should similarly code for a Mg-protoporphyrin IX methyltransferase. Sequence analysis of the complementing Synechocystis cosmid indicates that it contains an open reading frame exhibiting 29% sequence identity to BchM. In addition, expression of the Synechocystis gene in the R. capsulatus bchM mutant via the strong R. capsulatus puc promoter was shown to support nearly wild-type levels of bacteriochlorophyll a synthesis. To our knowledge, the Synechocystis sequence thus represents the first chlorophyll biosynthesis gene homolog of bchM. The complementing Synechocystis cosmid was also shown to code for a gene product that is a member of a highly conserved family of RNA binding proteins, the function of which in cyanobacteria remains undetermined.

Amino Acid Sequence↗
PubMed1995

A prokaryotic origin for light-dependent chlorophyll biosynthesis of plants.

Flowering plants require light for chlorophyll synthesis. Early studies indicated that the dependence on light for greening stemmed in part from the light-dependent reduction of the chlorophyll intermediate protochlorophyllide to the product chlorophyllide. Light-dependent reduction of protochlorophyllide by flowering plants is contrasted by the ability of nonflowering plants, algae, and photosynthetic bacteria to reduce protochlorophyllide and, hence, synthesize (bacterio) chlorophyll in the dark. In this report, we functionally complemented a light-independent protochlorophyllide reductase mutant of the eubacterium Rhodobacter capsulatus with an expression library composed of genomic DNA from the cyanobacterium Synechocystis sp. PCC 6803. The complemented R. capsulatus strain is capable of synthesizing bacteriochlorophyll in the light, thereby indicating that a chlorophyll biosynthesis enzyme can function in the bacteriochlorophyll biosynthetic pathway. However, under dark growth conditions the complemented R. capsulatus strain fails to synthesize bacteriochlorophyll and instead accumulates protochlorophyllide. Sequence analysis demonstrates that the complementing Synechocystis genomic DNA fragment exhibits a high degree of sequence identity (53-56%) with light-dependent protochlorophyllide reductase enzymes found in plants. The observation that a plant-type, light-dependent protochlorophyllide reductase enzyme exists in a cyanobacterium indicates that light-dependent protochlorophyllide reductase evolved before the advent of eukaryotic photosynthesis. As such, this enzyme did not arise to fulfill a function necessitated either by the endosymbiotic evolution of the chloroplast or by multicellularity; rather, it evolved to fulfill a fundamentally cell-autonomous role.

Amino Acid Sequence↗
PubMed1995

Altered monovinyl and divinyl protochlorophyllide pools in bchJ mutants of Rhodobacter capsulatus. Possible monovinyl substrate discrimination of light-independent protochlorophyllide reductase.

In land plants in particular, it has been well established that chlorophyll intermediates, Mg-protoporphyrin, Mg-protoporphyrin monomethylester, protochlorophyllide, and chlorophyllide occur as monovinyl and divinyl forms. The pool of monovinyl and divinyl intermediates differ according to species, age of tissue, and light regime. In this study, we investigated the monovinyl and divinyl characteristics of protochlorophyllide and chlorophyllide in the purple non-sulfur photosynthetic eubacterium Rhodobacter capsulatus. Our results indicate that mutations in genes known to completely block the reduction of protochlorophyllide to chlorophyllide (such as bchN, bchB, and bchL mutants), accumulate a pool of monovinyl and divinyl forms of protochlorophyllide just as observed in plants. However, we also observed that directed insertion and deletion mutations in bchJ, a gene located in the photosynthesis gene cluster, affected the ratio of monovinyl and divinyl protochlorophyllide. Specifically, bchJ-disrupted strains accumulate reduced levels of bacteriochlorophyll concomitant with the accumulation of divinyl protochlorophyllide. Mutants of bchJ in combination with a second mutation in bchL still produce a mixed pool of monovinyl and divinyl protochlorophyllide; however, the ratio of monovinyl to divinyl protochlorophyllide is skewed in favor of divinyl protochlorophyllide. These results thus identify bchJ as the first sequenced gene that affects the divinyl to monovinyl ratio of photopigment intermediates in any photosynthetic organism. In addition, the results of our study also suggest that light-independent protochlorophyllide reductase is discriminatory for a monovinyl substrate.

Base Sequence↗
PubMed1995

Expression of rat liver AdoHcy hydrolase in a Rhodobacter capsulatus ahcY mutant restores pigment formation and photosynthetic growth.

An amino acid alignment of fourteen S-adenosylhomocysteine hydrolases shows that sequences from six photosynthetic species and one species possibly derived from algae have an internal 36 to 41 amino acid sequence that is not present in hydrolase sequences from seven non-photosynthetic species. In the photosynthetic eubacterium Rhodobacter capsulatus, the StLB1 strain has a disrupted hydrolase gene, and hydrolase activity is not detectable. Photopigment synthesis and photosynthetic growth are significantly reduced in the StLB1 strain. Introduction of rat hydrolase cDNA into the StLB1 strain restored hydrolase activity, photopigment synthesis and photosynthetic growth. The results show that the 36 amino acid sequence of Rhodobacter capsulatus S-adenosylhomocysteine hydrolase does not have a photosynthesis specific function.

Adenosylhomocysteinase↗
PubMed1995

Isolation and in vitro phosphorylation of sensory transduction components controlling anaerobic induction of light harvesting and reaction center gene expression in Rhodobacter capsulatus.

Anaerobic induction of light harvesting and reaction center gene expression involves two transacting factors termed RegA and RegB. Sequence and mutational analysis has indicated that RegA and RegB constitute cognate components of a prokaryotic sensory transduction cascade with RegB comprising a membrane-spanning sensor kinase and RegA a cytosolic response regulator. In this study we have purified RegA, as well as a truncated portion of RegB (RegB') and undertaken an in vitro analysis of autophosphorylation and phosphotransfer activities. Incubation of RegB' with [gamma-32P]ATP and MgCl2 resulted in phosphorylation of RegB' (RegB' approximately P) over a 20-min incubation period. Incubation of RegB' approximately P with RegA resulted in rapid transfer of the phosphate from RegB' to RegA. In analogy to other characterized prokaryotic sensory transduction components, mutational and chemical stability studies also indicate that RegB' is autophosphorylated at a conserved histidine and that RegA accepts the phosphate from RegB at a conserved aspartate.

Bacterial Proteins↗
PubMed1995

Macroscopic phototactic behavior of the purple photosynthetic bacterium Rhodospirillum centenum.

Most photosynthetic microorganisms have the capability of photosensing light quality and intensity. Movement of motile photosynthetic microorganisms to locales that offer optimal physical and chemical conditions for light-dependent growth provides obvious selective advantages. Among phototrophs, many cyanobacteria and algae migrate towards or away from the direction of light, a process termed phototaxis. In contrast, anoxygenic photosynthetic bacteria are believed to respond to changes in light intensity in a manner that is not related to the direction of light, a process that is often described by the term "photophobic". We recently reported that "swarm colonies" of the purple photosynthetic bacterium Rhodospirillum centenum are capable of macroscopically visible phototactic behavior. In the present study we further characterize the phototactic behavior of R. centenum swarm colonies and provide an action spectrum that delineates regions of the spectrum that elicit positive and negative phototaxis.

Cell Division↗
PubMed1995

Characterization of an aerobic repressor that coordinately regulates bacteriochlorophyll, carotenoid, and light harvesting-II expression in Rhodobacter capsulatus.

For most species of purple photosynthetic bacteria, the presence of molecular oxygen represses synthesis of carotenoids and bacteriochlorophyll. In this study we characterize a strain of Rhodobacter capsulatus, DB469, which contains a genomic disruption of an open reading frame in the photosynthesis gene cluster termed ORF469. Characterization of the steady-state level of bacteriochlorophyll synthesis demonstrates that disruption of ORF469 results in a 2.5-fold increase in aerobic synthesis of bacteriochlorophyll over that observed with the parent strain. Utilizing reporter plasmids that contain transcriptional fusions of lacZ to various carotenoid and bacteriochlorophyll biosynthesis genes, we also demonstrate that disruption of ORF469 leads to an approximate twofold increase in bacteriochlorophyll and carotenoid gene expression under anaerobic growth conditions. Similar analysis with reporter plasmids that contain translational fusions of lacZ to the puf, puh, and puc operons demonstrates that disruption of ORF469 leads to elevated levels of aerobic transcription of light harvesting-II genes (puc), without affecting light harvesting-I or reaction center gene expression (puf and puh, respectively). Gel mobility analysis demonstrates that DB469 cells lack a DNA-binding protein that interacts with a palindromic sequence in the bchC promoter region. The results of this study indicate that ORF469 codes for a DNA-binding protein that acts as an aerobic repressor of promoters for bacteriochlorophyll, carotenoid, and light harvesting-II gene expression.

Aerobiosis↗
PubMed1995

Cloning and characterization of senC, a gene involved in both aerobic respiration and photosynthesis gene expression in Rhodobacter capsulatus.

The purple nonsulfur photosynthetic eubacterium Rhodobacter capsulatus is a versatile organism that can obtain cellular energy by several means, including the capture of light energy for photosynthesis as well as the use of light-independent respiration, in which molecular oxygen serves as a terminal electron acceptor. In this study, we have identified and characterized a novel gene, senC, mutations in which affect respiration as well as the induction of photosynthesis gene expression. The protein coded by senC exhibits 33% sequence identity to the yeast nucleus-encoded protein SCO1, which is thought to be a mitochondrion-associated cytochrome c oxidase assembly factor. Like yeast SCO1, SenC is required for optimal cytochrome c oxidase activity in aerobically grown R. capsulatus cells. We further show that senC is required for maximal induction from the puf and puh operons, which encode the structural polypeptides of the light-harvesting and reaction center complexes.

Amino Acid Sequence↗
PubMed1994

Molecular genetic analysis of terminal steps in bacteriochlorophyll a biosynthesis: characterization of a Rhodobacter capsulatus strain that synthesizes geranylgeraniol-esterified bacteriochlorophyll a.

Site-directed mutational analysis of the Rhodobacter capsulatus photosynthesis gene cluster was undertaken in order to identify and characterize genetic loci involved in bacteriochlorophyll a biosynthesis. A mutant in orf304 was shown to accumulate the tetrapyrrole intermediate "bacteriochlorophyllide a" which is a tetrapyrrole that has a bacteriochlorophyll a ring structure without the presence of an esterifying alcohol. A mutant in orf391 is shown to synthesize bacteriochlorophyll a that is esterified with geranylgeraniol rather than the normal phytol. This latter result provides the first genetic confirmation that esterification of bacteriochlorophyllide a initially involves the addition of a geranylgeraniol group followed by sequential reduction of the geranylgeraniol moiety to phytol which is the end product of the pathway. An R. capsulatus strain synthesizing geranylgeraniol-esterified bacteriochlorophyll is shown to exhibit severely impaired photosynthetic growth capability. This is despite our observation that synthesis of geranylgeraniol-esterified bacteriochlorophyll does not affect the energy transfer rate from light harvesting to reaction center complexes nor the electron transfer function as measured by the yield of electron transfer to the primary and secondary quinones, the charge recombination rate from the quinones, and the rate of cytochrome c2 oxidation. We conclude that the observed reduction of the photosynthetic growth rate observed for R. capsulatus strains that synthesize geranylgeraniol-esterified bacteriochlorophyll is primarily a consequence of the reduced steady-state level of the photosystem.

Bacteriochlorophylls↗