Application of a very high-throughput digital imaging screen to evolve the enzyme galactose oxidase.

Directed evolution has become an important enabling technology for the development of new enzymes in the chemical and pharmaceutical industries. Some of the most interesting substrates for these enzymes, such as polymers, have poor solubility or form highly viscous solutions and are therefore refractory to traditional high-throughput screens used in directed evolution. We combined digital imaging spectroscopy and a new solid-phase screening method to screen enzyme variants on problematic substrates highly efficiently and show here that the specific activity of the enzyme galactose oxidase can be improved using this technology. One of the variants we isolated, containing the mutation C383S, showed a 16-fold increase in activity, due in part to a 3-fold improvement in K(m). The present methodology should be applicable to the evolution of numerous other enzymes, including polysaccharide-modifying enzymes that could be used for the large-scale synthesis of modified polymers with novel chemical properties.

Extremophiles.

NASA: The authors examine the presence of bacteria in extreme climates and their role in biotechnology. Within the past 30 years, scientists have discovered bacteria in areas long thought to be sterile due to extremes in heat, cold, or pH. Enzymes from these bacteria are used in many areas of industry. Examples discussed include the use of enzymes from thermophilic bacteria for polymerase chain reactions, use of enzymes in detergents, and the use of halophiles to enhance extraction of crude oil. Methods of harvesting extremozymes are discussed.^ieng

An overlap between operons involved in carotenoid and bacteriochlorophyll biosynthesis in Rhodobacter capsulatus.

A new example of superoperonal gene arrangement has been documented in the Rhodobacter capsulatus photosynthetic gene cluster. The promoter for the operon initiated by the bchI gene is embedded within an upstream operon for carotenoid synthesis. The stop codon for the crtA gene, the only gene in the first operon, overlaps the start codon of the downstream bchI gene. As a consequence of this overlap, the promoter(s) for the bch operon must be located within the crtA structural gene. The bchI gene is shown here for the first time to be required for the conversion of protoporphyrin IX to subsequent intermediates in bacteriochlorophyll biosynthesis.

Sequence of the indoleglycerol phosphate synthase (trpC) gene from Rhodobacter capsulatus.

We have isolated, cloned, and sequenced the indoleglycerol phosphate synthase gene (trpC) from Rhodobacter capsulatus. Normalized alignment scores comparing the trpC gene of R. capsulatus with the trpC genes of other bacterial species are reported. An unexpected degree of similarity to the trpC gene of Bacillus subtilis was found.

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.

Genetic analysis of functional differences among distinct ferredoxins in Rhodobacter capsulatus.

Rhodobacter capsulatus has been known to possess two ferredoxins (I and II) with distinct physicochemical and structural properties: ferredoxin I is a 2[4Fe-4S] type and the other is a [3Fe-4S] [4Fe-4S] type. To analyze their possible functional differences, their genes (fdxN and fdxA) were cloned, sequenced, and subjected to interposon mutagenesis experiments. The former gene was adjacent to a gene encoding a chloroplast-type [2Fe-2S] ferredoxin (fdxC). Mutants with inactivated fdxN and/or fdxC were obtained, and they showed virtually no growth under nitrogen-fixing conditions. Complementation experiments confirmed that both fdxN and fdxC were required for nitrogen fixation. On the other hand, we have not been able to disrupt fdxA under the screening conditions surveyed, including conditions that do not require nitrogenase activity for growth, suggesting that ferredoxin II could have an unknown essential role(s). These indicate functional differences among multiple ferredoxins in one bacterium other than in cyanobacterial heterocysts and indispensability of certain ferredoxins in nitrogen fixation other than Rhizobium meliloti FdxN.

Two distinct ferredoxins from Rhodobacter capsulatus: complete amino acid sequences and molecular evolution.

Two distinct ferredoxins were purified from Rhodobacter capsulatus SB1003. Their complete amino acid sequences were determined by a combination of protease digestion, BrCN cleavage and Edman degradation. Ferredoxins I and II were composed of 64 and 111 amino acids, respectively, with molecular weights of 6,728 and 12,549 excluding iron and sulfur atoms. Both contained two Cys clusters in their amino acid sequences. The first cluster of ferredoxin I and the second cluster of ferredoxin II had a sequence, CxxCxxCxxxCP, in common with the ferredoxins found in Clostridia. The second cluster of ferredoxin I had a sequence, CxxCxxxxxxxxCxxxCM, with extra amino acids between the second and third Cys, which has been reported for other photosynthetic bacterial ferredoxins and putative ferredoxins (nif-gene products) from nitrogen-fixing bacteria, and with a unique occurrence of Met. The first cluster of ferredoxin II had a CxxCxxxxCxxxCP sequence, with two additional amino acids between the second and third Cys, a characteristics feature of Azotobacter-[3Fe-4S] [4Fe-4S]-ferredoxin. Ferredoxin II was also similar to Azotobacter-type ferredoxins with an extended carboxyl (C-) terminal sequence compared to the common Clostridium-type. The evolutionary relationship of the two together with a putative one recently found to be encoded in nifENXQ region in this bacterium [Moreno-Vivian et al. (1989) J. Bacteriol. 171, 2591-2598] is discussed.

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.

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.

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.

Discrete catalytic sites for quinone in the ubiquinol-cytochrome c2 oxidoreductase of Rhodopseudomonas capsulata. Evidence from a mutant defective in ubiquinol oxidation.

A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.

Oxygen does not directly regulate carotenoid biosynthesis in Rhodopseudomonas capsulata.

We examined the role of bacteriochlorophyll synthesis on the regulation of carotenoid synthesis in Rhodopseudomonas capsulata. Strains capable of making bacteriochlorophyll accumulated greater amounts of carotenoids under low oxygen than they did under high oxygen. However, strains unable to produce bacteriochlorophyll did not regulate their carotenoid production in response to changes in oxygen tension. This indicates that oxygen does not directly regulate carotenoid production.

Depression of the synthesis of the intermediate and large forms of ribulose-1,5-bisphosphate carboxylase/oxygenase in Rhodopseudomonas capsulata.

Rhodopseudomonas capsulata produces both an intermediate (I) and a large (L) form of ribulose-1,5-bisphosphate carboxylase/oxygenase. Both forms are derepressed under CO2-limiting conditions. The L-form of the enzyme is completely repressed when the culture is grown either photoautotrophically or photoheterotrophically with malate as the electron donor. The L-form is derepressed in the late logarithmic phase of growth when cells are grown photoheterotrophically with butyrate as the electron donor and the NaHCO3 supplement is 0.01%. The level of the I-form is increased about fivefold under latter growth conditions when compared to malate-grown cells. Analytical ultracentrifugation revealed the molecular masses of the I- and L-forms to be 300,000 and 542,000, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the I-form to be composed of only one type subunit with a molecular weight of 64,000. The L-form possessed both large and small subunits with molecular weights of 58,000 and 10,000.

Variation of levels of mRNA coding for antenna and reaction center polypeptides in Rhodopseudomonas capsulata in response to changes in oxygen concentration.

The effect of oxygen tension on the transcription of genes coding for the photosynthetic apparatus of Rhodopseudomonas capsulata was determined by the Southern hybridization technique. Restriction endonuclease digests of the R-prime plasmid pRPS404 and a subcloned fragment thereof served as DNA probes for genetically defined regions. The results showed that transcripts corresponding to the genes for certain pigment-binding polypeptides increase in amount by about 40-fold after a drop in oxygen tension. Transcripts hybridizing to genes involved in bacteriochlorophyll biosynthesis increase to a much lesser extent, and several genes involved in carotenoid biosynthesis are not affected by pO2.

Transcriptional regulation of several genes for bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata in response to oxygen.

Although it has been shown that bacteriochlorophyll synthesis in Rhodopseudomonas capsulata is repressed by oxygen and high light intensity, few details of regulation by these environmental factors are known, primarily owing to a lack of assays for the biosynthetic enzymes. We have examined regulation at the transcriptional level by isolating and studying fusions between the Mu d1(Apr lac) phage and various bch genes. In these strains, the lacZ gene of the phage is under the control of bch gene promoters. We have found that atmospheric oxygen tension (20% O2) reduces the expression of these fusions at least twofold compared with low oxygen tension (2% O2). Therefore, transcription of the bchA, bchB, bchC, bchG, and bchH genes is regulated in response to oxygen.

Isolation and characterization of enhanced fluorescence mutants of Rhodopseudomonas capsulata.

After enrichment by a tetracycline suicide under conditions nonpermissive for the growth of mutants defective in photosynthesis, colonies were screened for enhanced fluorescence in near-infrared light by using high-speed infrared photography. Twenty mutants were isolated, and the chromatophore membranes were analyzed by a new, rapid microprocedure that revealed many different phenotypes among the mutants. The enhanced fluorescence mutants typically possessed a functional light-harvesting II antenna, but showed reduced or absent light-harvesting I. Twelve isolates were also defective in reaction center polypeptides. An R-prime plasmid that bears 50 kilobases of Rhodopseudomonas capsulata DNA coding for components of the photosynthetic apparatus (B. L. Marrs, J. Bacteriol. 146:1003-1012, 1981), pRPS404, complemented all 20 enhanced fluorescence mutants as demonstrated by the quenching of fluorescence in mutants that had received the R-prime plasmid by conjugation. Fluorescence was regained upon loss of the 50-kilobase insert. Complementation of the fluorescent lesions implies that most or all of the genes necessary for the expression of the reaction center and the light-harvesting antennae are carried by the R-prime plasmid and that these genes are actively transcribed in the homologous organism. All 20 mutants are complemented by one of two pBR322 subclones of the R-prime plasmid, pRPSEB2 or pRPSE2. pRPSEB2 bears a 4.5-kilobase fragment of R. capsulata DNA including the rxcA locus, and pRPSE2 is a pBR322 derivative bearing a 7.5-kilobase R. capsulata DNA fragment bearing the rxcB locus. These fragments therefore carry sequences necessary for the normal synthesis of the light-harvesting and reaction center polypeptide complexes.