Biomedical subjects

C E Bauer

Publications and source records attributed to C E Bauer.

At least 19 recordsLinked to original sources

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↗

PubMed1992

Mutational and nucleotide sequence analysis of S-adenosyl-L-homocysteine hydrolase from Rhodobacter capsulatus.

The genetic locus ahcY, encoding the enzyme S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) from the bacterium Rhodobacter capsulatus, has been mapped by mutational analysis to within a cluster of genes involved in regulating the induction and maintenance of the bacterial photosynthetic apparatus. Sequence analysis demonstrates that ahcY encodes a 51-kDa polypeptide that displays 64% sequence identity to its human homolog. Insertion mutants in ahcY lack detectable S-adenosyl-L-homocysteine hydrolase activity and, as a consequence, S-adenosyl-L-homocysteine accumulates in the cells, resulting in a 16-fold decrease in the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine as compared to wild-type cells. The ahcY disrupted strain fails to grow in minimal medium; however, growth is restored in minimal medium supplemented with methionine or homocysteine or in a complex medium, thereby indicating that the hydrolysis of S-adenosyl-L-homocysteine plays a key role in the metabolism of sulfur-containing amino acids. The ahcY mutant, when grown in supplemented medium, synthesizes significantly reduced levels of bacteriochlorophyll, indicating that modulation of the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine may be an important factor in regulating bacteriochlorophyll biosynthesis.

Adenosylhomocysteinase↗

PubMed1992

Regulatory factors controlling photosynthetic reaction center and light-harvesting gene expression in Rhodobacter capsulatus.

Most species of photosynthetic bacteria synthesize their photosynthetic apparatus only under conditions of reduced oxygen tension. To a large extent, this phenomenon is dependent upon anaerobic induction of photosynthesis gene expression. Here we report an example of a regulatory gene, regA, that is involved in transactivating anaerobic expression of the photosynthetic apparatus. We show that RegA is itself responsible for differential induction of light-harvesting and reaction center gene expression relative to operons for photopigment biosynthesis. Surprisingly, strains disrupted for regA were found to retain normal photosynthetic growth capabilities under high light intensities. We further show that photosynthetic growth in the absence of transactivating structural gene expression is a consequence of the superoperonal organization of the photosynthetic gene cluster.

Amino Acid Sequence↗

PubMed1992

Association of tetrapyrrole intermediates in the bacteriochlorophyll a biosynthetic pathway with the major outer-membrane porin protein of Rhodobacter capsulatus.

Rhodobacter capsulatus regulates synthesis of bacteriochlorophyll a in response to changes in oxygen partial pressure and light intensity. One early model proposed that this regulation involved a carrier polypeptide that functions to tether tetrapyrrole intermediates to the membrane. In the present study we isolated tetrapyrrole intermediates accumulated in three strains of R. capsulatus that contain mutations which block bacteriochlorophyll a biosynthesis at different steps of the magnesium branch of the pathway. Each of the tetrapyrrole intermediates was shown to be associated with the same 32 kDa polypeptide, as indicated by similar electrophoretic mobility and antigenic cross-reactivity with polyclonal antisera. The 32 kDa pigment-associated protein was further found to have an electrophoretic mobility, antigenic cross-reactivity and N-terminal sequence identical with those of the previously characterized major outer-membrane porin protein of R. capsulatus.

Amino Acid Sequence↗

PubMed1992

Light-independent chlorophyll biosynthesis: involvement of the chloroplast gene chlL (frxC).

The Chlamydomonas reinhardtii chloroplast gene chlL (frxC) is shown to be involved in the light-independent conversion of protochlorophyllide to chlorophyllide. The polypeptide encoded by chlL contains a striking 53% amino acid sequence identity with the bacteriochlorophyll (bch) biosynthesis bchL gene product in the photosynthetic bacterium Rhodobacter capsulatus. In a previous analysis, we demonstrated that bchL was involved in light-independent protochlorophyllide reduction, thereby implicating chlL in light-independent protochlorophyllide reduction in photosynthetic eukaryotes. To perform a functional/mutational analysis of chlL, we utilized particle gun-mediated transformation to disrupt the structural sequence of chlL at its endogenous locus in the chloroplast genome of Chlamydomonas. Transformants for which the multicopy chloroplast genome was homoplasmic for the disrupted chlL allele exhibit a "yellow-in-the-dark" phenotype that we demonstrated to be a result of the dark accumulation of protochlorophyllide. The presence of a chlL homolog in distantly related bacteria and nonflowering land plants, which are thought to be capable of synthesizing chlorophyll in the dark, was also demonstrated by cross-hybridization analysis. In contrast, we observed no cross-hybridization of a probe of chlL to DNA samples from representative angiosperms that require light for chlorophyll synthesis, in support of our conclusion that chlL is involved in light-independent chlorophyll biosynthesis. The role of chlL in protochlorophyllide reduction as well as recent evidence that both light-independent and light-dependent protochlorophyllide reductases may be of bacterial origin are discussed.

Amino Acid Sequence↗

PubMed1992

Conservation of the photosynthesis gene cluster in Rhodospirillum centenum.

Intraspecies and intergenus complementation analysis were utilized to demonstrate that photosynthesis genes are clustered in distantly related purple photosynthetic bacteria. Specifically, we show that the linkage order for genes involved in bacteriochlorophyll and carotenoid biosynthesis in Rhodospirillum centenum are arranged essentially as in Rhodobacter capsulatus and Rhodobacter sphaeroides. In addition, the location and relative distance observed between the puf and puh operons which encode for light harvesting and reaction-centre structural genes are also conserved between these species. Conservation of the photosynthesis gene cluster implies either that there are structural or regulatory constraints that limit rearrangement of the photosynthesis gene cluster or that there may have been lateral transfer of the photosynthesis gene cluster among different species of phototrophic bacteria.

Bacteriochlorophylls↗

PubMed1991

The superoperonal organization of genes for pigment biosynthesis and reaction center proteins is a conserved feature in Rhodobacter capsulatus: analysis of overlapping bchB and puhA transcripts.

Most of the essential biosynthetic and structural genes involved in bacterial photosynthesis are clustered in a 46 kb region of the Rhodobacter capsulatus genome. Previous analyses have demonstrated that the puf operon, which encodes light harvesting and reaction center structural genes as well as a regulatory gene for bacteriochlorophyll biosynthesis, is expressed from a complex set of overlapping transcripts. Differential initiation and processing of these transcripts is thought to be involved in regulating expression of puf-encoded genes. In this study we demonstrate that the puh operon, which is located 39 kb away from the puf operon, also contains overlapping transcripts. One large 11 kb puhA transcript is shown to be a product of read-through from an upstream operon (bchB) which encodes numerous bacteriochlorophyll biosynthesis genes. A second 1.1 kb mRNA is shown to be derived from the 11 kb bchB transcript by processing and a third, highly expressed, 0.95 kb transcript is shown to be initiated from a promoter located within the distal gene of the bchB operon. The occurrence of overlapping transcripts for the puf and puh operons was further shown to influence development of the photochemical apparatus during conditions of environmental shifts in oxygen tension. Evidence for the occurrence of a "superoperonal" organization of overlapping operons in several different species of purple photosynthetic bacteria is discussed.

Amino Acid Sequence↗

PubMed1991

Genetic analysis of photosynthesis in Rhodospirillum centenum.

A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.

Bacteriochlorophylls↗

PubMed1991

Attenuated effect of oxygen on photopigment synthesis in Rhodospirillum centenum.

Rhodospirillum centenum resembles typical nonsulfur photosynthetic bacteria in a number of respects, including its ability to grow either anaerobically as a phototroph or aerobically as a heterotroph. We demonstrate, however, that R. centenum is unusual in its ability to synthesize a functional photosynthetic apparatus regardless of the presence of molecular oxygen. Aerobically expressed photopigments were shown to be functionally active, as demonstrated by the ability of heterotrophically grown cells to grow photosynthetically, without a lag, when suddenly placed under anaerobic conditions. An R. centenum mutant that has acquired the ability to repress synthesis of photopigments in the presence of oxygen was also characterized. Both the wild type and the oxygen-repressed mutant of R. centenum were found to exhibit high light intensity repression of photopigment biosynthesis. The latter result suggests that R. centenum contains separate regulatory circuits for controlling synthesis of its photochemical apparatus by light intensity and oxygen.

Light↗

PubMed1990

Rhodobacter capsulatus genes involved in early steps of the bacteriochlorophyll biosynthetic pathway.

Three open reading frames in the Rhodobacter capsulatus photosynthesis gene cluster, designated F0, F108, and F1025, were disrupted by site-directed mutagenesis. Mutants bearing insertions in these reading frames were defective in converting protoporphyrin IX to magnesium-protoporphyrin monomethyl ester, protochlorophyllide to chlorophyllide a, and magnesium-protoporphyrin monomethyl ester to protochlorophyllide, respectively. These results demonstrate that the genes examined most likely encode enzyme subunits that catalyze steps common to plant and bacterial tetrapyrrole photopigment biosynthetic pathways. The open reading frames were found to be part of a large 11-kilobase operon that encodes numerous genes involved in early steps of the bacteriochlorophyll a biosynthetic pathway.

Amino Acid Sequence↗

PubMed1989

Genetic evidence for superoperonal organization of genes for photosynthetic pigments and pigment-binding proteins in Rhodobacter capsulatus.

Three adjacent operons, each concerned with photosynthesis in Rhodobacter capsulatus, have been shown by genetic means to be cotranscribable. In the course of describing the characteristics of the bchCA operon, which encodes two enzymes essential for bacteriochlorophyll synthesis, we found that the expression of the bchCA genes is influenced by readthrough from the upstream crtE and crtF genes. The crtE and crtF genes encode enzymes required for carotenoid biosynthesis and function as an operon. Furthermore, the distal structural gene of the bchCA operon, bchA, contains within it both the major oxygen-regulated promotor (Ppuf1) and the constitutive (Ppuf2) promotor for the puf operon. Since these three operons, crtEF, bchCA, and puf, are all transcribed in the same direction, it appears that polymerases traversing the downstream regions may start at any of several promoters. This pattern of transcription, which is unusual among bacteria, demonstrates that the activities of individual operons in a superoperonal cluster may be affected by their positions within the cluster.

Amino Acid Sequence↗

PubMed1989

The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

Attachment Sites, Microbiological↗

PubMed1988

Transcription of the Rhodobacter capsulatus nifHDK operon is modulated by the nitrogen source. Construction of plasmid expression vectors based on the nifHDK promoter.

We characterized the Rhodobacter capsulatus nifHDK promoter by nucleotide sequencing and nuclease S1 analysis of mRNA-protected DNA probes. Comparison of this promoter to nifP and ntrP promoters from other species reveals extensive homology to the canonical nifP consensus sequence. Using lac fusions we have demonstrated that transcription of the nifHDK operon is totally repressed when the growth medium is supplemented with ammonia, becomes fully derepressed in ammonia-free medium, and proceeds at intermediate levels when other nitrogen sources are used. Based on this information, we constructed plasmid expression vectors in which the rates of transcription from cloned DNA fragments are determined by the nitrogen source used in the growth medium.

Base Sequence↗

PubMed1988

Analysis of the Rhodobacter capsulatus puf operon. Location of the oxygen-regulated promoter region and the identification of an additional puf-encoded gene.

In an attempt to identify features of an oxygen-regulated promoter, we have determined the location of transcription initiation for the puf operon. The position for the oxygen-regulated promoter was demonstrated by several independent means to be located 699 base pairs (bp) upstream from the pufB structural gene. DNA sequence analysis of the promoter region demonstrates the presence of a 26-base pair region of dyad symmetry followed by a sequence containing homology to promoters which use the RNA polymerase sigma 60 subunit (ntrA) for recognition of DNA. In addition to the oxygen-regulated promoter, a region responsible for low-level constitutive expression of the puf operon was shown to initiate transcription 511 bp upstream from the pufB gene. In contrast to the oxygen-regulated promoter, this second promoter contains no obvious secondary structure nor sequence homology to ntrA-dependent promoters. DNA sequence analysis demonstrates the existence of an additional open reading frame (designated as pufQ) that is located between the promoters and the pufB structural gene. A translational fusion of pufQ to lacZ was used to demonstrate that pufQ is efficiently translated and regulated in a manner analogous to a translational fusion of pufM to lacZ. Finally, we also demonstrate that puf operon transcription initiation and regulation does not involve any puf-encoded gene products.

Amino Acid Sequence↗

PubMed1988

Rhodobacter capsulatus puf operon encodes a regulatory protein (PufQ) for bacteriochlorophyll biosynthesis.

Biosynthesis of the photochemical apparatus by purple nonsulfur photosynthetic bacteria is known to be inhibited by molecular oxygen and high light intensity. Polypeptides that bind bacteriochlorophyll (BChl) to form the light-harvesting I (LH-I) and reaction-center (RC) complexes are encoded by a single transcriptional unit termed the puf operon. In this investigation we demonstrate that the first structural gene in the puf operon (pufQ) of Rhodobacter capsulatus encodes a protein that is required for BChl biosynthesis and that there exists a linear relationship between the amount of pufQ expression and the level of BChl synthesis. Protein sequence similarity exists between PufQ and the region of RC polypeptides that are known to bind BChl and quinone. These observations suggest that pufQ may regulate BChl biosynthesis by a "carrier polypeptide" mechanism as originally proposed by Lascelles.

Amino Acid Sequence↗

PubMed1987

Role of homology in site-specific recombination of bacteriophage lambda: evidence against joining of cohesive ends.

Bacteriophage lambda integration and excision take place at specific loci called attachment sites. Earlier work has shown that efficient recombination requires the identical sequence to be present in both attachment sites throughout the seven-base-pair region between the points of strand exchange. A plausible model for the role of homology postulates that Int, the site-specific recombinase, makes double-strand breaks at attachment sites such that each broken end has a short single-strand protrusion. Recombination would then depend upon the capacity of these protrusions to form Watson-Crick helices--i.e., to anneal--a process that might require perfect complementarity between the cohesive ends. To test this model, we have studied Int-promoted crosses in which one attachment site is a heteroduplex. Specifically, we constructed sites in which the seven-base-pair region between the points of strand exchange contains one or more noncomplementary pairs. The double-strand break and annealing mechanism predicts that crosses with these heteroduplex sites should yield one completed recombinant and one broken site. We find that such nonreciprocal recombination is uncommon and that the typical outcome of crosses involving a heteroduplex site is a reciprocal recombinant in which both products are resealed. Moreover, the occasional appearance of nonreciprocal products can be explained by our finding that Int can cleave heteroduplex attachment sites after recombination is completed. Taken together, our data strongly indicate that bacteriophage lambda recombination does not proceed by the homology-dependent annealing of cohesive ends; acceptable alternatives for the role of homology are discussed.

Bacteriophage lambda↗

PubMed1986

Mutational analysis of integrase arm-type binding sites of bacteriophage lambda. Integration and excision involve distinct interactions of integrase with arm-type sites.

Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome (attP) and the Escherichia coli genome (attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int "arm-type" binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm-type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm-type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm-type sites.

Attachment Sites, Microbiological↗

PubMed1985

Identification and characterization of mutants affecting transcription termination at the threonine operon attenuator.

Mutations that map in or delete the attenuator of the threonine (thr) operon of Escherichia coli were isolated and characterized. These mutations disrupt or delete the transcription termination structure encoded by the attenuator leading to increased transcriptional readthrough into the thr operon structural genes. Most of the base substitutions and single base-pair insertions and deletions map in the G + C-rich region of dyad symmetry in the attenuator and decrease the calculated stabilities of the attenuator RNA secondary structures to similar extents (from -30.8 kcal/mol to approximately -21 kcal/mol). Most of the mutants showed a three- to fourfold increase in homoserine dehydrogenase (thrA gene product) synthesis relative to the wild-type parent strain. The mutation in one mutant (thrL153 + G) lowered the calculated stability of the RNA secondary structure only slightly (from -30.8 to 27.8 kcal/mol) but the mutant still exhibited high levels of homoserine dehydrogenase synthesis. In addition, three base substitution mutants (thrL135U, thrL139A and thrL156U) showed only slightly (1.5 to 2-fold) elevated levels of homoserine dehydrogenase activity, even though the calculated stabilities of the attenuator RNA secondary structures were reduced as much as most of the other mutants. Two of the mutations (thrL135U and thrL156U) mapped in the G + C-rich-A + T-rich junction of the attenuator. The third mutation (thrL139A) creates an A X C pair in the center of the G + C-rich region of the attenuator stem. The results obtained for these mutants show that the stability of the RNA secondary structure does not always correlate with the efficiency of transcription termination. Finally, analysis of the base changes in the substitution mutations showed that the mutational changes do not appear to be random.

Base Sequence↗