Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris

Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant-derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe the genome sequence of R. palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp. The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage pathways and four light harvesting 2 systems. R. palustris encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains. Almost 15% of the genome is devoted to transport. This genome sequence is a starting point to use R. palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations.     

A high throughput screen to identify substrates for the ubiquitin ligase Rsp5

Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.     

Deletion of a single amino acid changes the folding of an apamin hybrid sequence peptide to that of endothelin

The solution conformations of a hybrid sequence peptide related to the bee venom peptide apamin have been determined using two-dimensional ¹H-nmr. Apamin is an 18 amino acid peptide containing a C-terminal helix that is stabilized by two disulfide bonds. The deletion of one residue (K4) of the N-terminal “scaffold” region of the apamin sequence results in a helical peptide, but with a change in the pairing of cysteines to form the disulfide cross links. The new disulfide arrangement is analogous to that of the vasoconstrictor peptide endothelin. Two sets of nmr resonances were observed for the apamin-deletion (AD) peptide, due to cis-trans isomerism at the A4-P5 peptide bond. The cis isomer of the AD peptide contains a tight turn in residues 3–6, which is required for formation of the α-helix in residues 7–15. Nuclear Overhauser effects observed for the trans AD peptide are not consistent with any single unique fold, indicating the presence of conformational averaging when the peptide adopts the trans form. Distance geometry calculations on the cis AD peptide reveal an α-helical structure that appears to be more like that of apamin than the crystal structure of human endothelin, despite the reversal of the disulfide pattern in the AD peptide from that of apamin to that of endothelin.© 1997 John Wiley & Sons, Inc. Biopoly 41 : 451–460, 1997     

Purification and properties of benzoate-coenzyme A ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate.

A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 mumol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg2+ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent Km values of 0.6 to 2 microM for benzoate, 2 to 3 microM for ATP, and 90 to 120 microM for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris.     

A single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an alternative coenzyme

Using site-directed mutagenesis, the NADH-linked lactate dehydrogenase from Bacillus stearothermophilus has been specifically altered at a single residue to shift the coenzyme specificity towards NADPH. The single change is at position 53 in the amino acid sequence where a conserved aspartate has been replaced by a serine. This substitution was made to reduce steric hindrance on binding of the extra phosphate group of NADPH and to remove the negative charge of the aspartate group. The resultant mutant enzyme is 20 times more catalytically efficient than the wild-type enzyme with NADPH.     

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.     

Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins.     

2-Hydroxycyclohexanecarboxyl coenzyme A dehydrogenase, an enzyme characteristic of the anaerobic benzoate degradation pathway used by Rhodopseudomonas palustris

A gene, badH, whose predicted product is a member of the short-chain dehydrogenase/reductase family of enzymes, was recently discovered during studies of anaerobic benzoate degradation by the photoheterotrophic bacterium Rhodopseudomonas palustris. Purified histidine-tagged BadH protein catalyzed the oxidation of 2-hydroxycyclohexanecarboxyl coenzyme A (2-hydroxychc-CoA) to 2-ketocyclohexanecarboxyl-CoA. These compounds are proposed intermediates of a series of three reactions that are shared by the pathways of cyclohexanecarboxylate and benzoate degradation used by R. palustris. The 2-hydroxychc-CoA dehydrogenase activity encoded by badH was dependent on the presence of NAD(+); no activity was detected with NADP(+) as a cofactor. The dehydrogenase activity was not sensitive to oxygen. The enzyme has apparent K(m) values of 10 and 200 microM for 2-hydroxychc-CoA and NAD(+), respectively. Western blot analysis with antisera raised against purified His-BadH identified a 27-kDa protein that was present in benzoate- and cyclohexanecarboxylate-grown but not in succinate-grown R. palustris cell extracts. The active form of the enzyme is a homotetramer. badH was determined to be the first gene in an operon, termed the cyclohexanecarboxylate degradation operon, containing genes required for both benzoate and cyclohexanecarboxylate degradation. A nonpolar R. palustris badH mutant was unable to grow on benzoate or cyclohexanecarboxylate but had wild-type growth rates on succinate. Cells blocked in expression of the entire cyclohexanecarboxylate degradation operon excreted cyclohex-1-ene-1-carboxylate into the growth medium when given benzoate. This confirms that cyclohex-1-ene-1-carboxyl-CoA is an intermediate of anaerobic benzoate degradation by R. palustris. This compound had previously been shown not to be formed by Thauera aromatica, a denitrifying bacterium that degrades benzoate by a pathway that is slightly different from the R. palustris pathway. 2-Hydroxychc-CoA dehydrogenase does not participate in anaerobic benzoate degradation by T. aromatica and thus may serve as a useful indicator of an R. palustris-type benzoate degradation pathway.     

Nucleotide sequence of the genome and complete amino acid sequence of a polyprotein of the tick-borne encephalitis virus

We have cloned and sequenced RNA encoding all virion and nonstructural proteins of tick-borne encephalitis virus (TBEV). Its length is 10,477 bases with a single open reading frame (nucleotides 127-10,363) encoding 3412 amino acids. The 5'- and 3'-noncoding regions have stem- and- loop structure. The polyprotein precursor is proteolytically cleaved, apparently, by a mechanism resembling that proposed for the expression of polyproteins of other flaviviruses, such as yellow fever, West Nile and Kunjin viruses. The deduced TBEV gene order is 5'-C-preM (M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5++ +-3'. The genome and the polyprotein of TBEV and other flaviviruses appears to be structurally similar, although these flaviviruses are transmitted to and from their vertebrate hosts by different carriers, such as ticks or mosquitoes. Analysis of sequence homologies of polyproteins of flaviviruses suggests that TBEV is more closely related to yellow fever virus than to other serological subgroups of flaviviruses (West Nile or Dengue viruses). The hydrophobic profiles of the flaviviruses are highly conservative. Nonstructural proteins NS2A, NS2B, NS4A and NS4B are extremely hydrophobic, suggesting that they are likely to be associated with cellular membranes. Proteins E, NS1, NS3 and NS5 are the most conservative and may be involved in general enzymatic activities related to viral replication and virion assembly.     

A novel single amino acid deletion caspase-8 mutant in cancer cells that lost proapoptotic activity

Caspase-8 is an important initiation caspase that activates the caspase cascade during death receptor-mediated apoptosis. We here report a novel caspase-8 mutant with a naturally occurring deletion of leucine 62 (Delta Leu62casp-8). Delta Leu62casp-8 has a shorter half-life than its wild-type counterpart. Unlike wild-type caspase-8, Delta Leu62casp-8 failed to interact with wild-type caspase-8 or with the adaptor protein FADD. Delta Leu62casp-8 lost its proapoptotic activity in mammalian cells. The leucine 62 therefore is critical for caspase-8 function, and the mutation may be one of the mechanisms through which some types of cancer cells escape from programmed cell death.     

Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity

An extract of human seminal plasma was found to have inhibin-like activity. The active factor was purified to homogeneity by ion exchange chromatography, molecular sieving and high performance liquid chromatography. The purified material has a mass of approximately 5 kDa and is very basic. Amino acid analysis showed the presence of approximately 35 residues while the sequencing data allowed the determination of the N-terminal 31 amino acids. There is a possibility of an additional 2–4 residues at the C-terminus, which could not be determined.     

Use of Rhodopseudomonas palustris P1 stimulated growth by fermented pineapple extract to treat latex rubber sheet wastewater to obtain single cell protein

Latex rubber sheet wastewater (non sterile wastewater: RAW) was treated efficiently using a stimulated Rhodopseudomonas palustris P1 inoculum with added fermented pineapple extract (FPE) under microaerobic light conditions. Optimization of wastewater treatment conditions using a central composite design (CCD) found that a 3 % stimulated P1 inoculum with 0.9 % added FPE and a 4-day retention time (RT) were the most suitable conditions. Calculations from CCD experiments predicted that a chemical oxygen demand (COD) of 3,005 mg/L could be 98 % removed, together with 79 % of suspended solids (SS) and 72 % of total sulfide (TtS). No H₂S was detected, production costs were low and single cell protein (SCP) was a by-product. The results of the verification test had an error of only 4–8 % and confirmed removal of COD (initial COD 2,742 mg/L), SS and TtS at 94 %, 75 % and 66 %, respectively. These values were less than the best set obtained from the CCD experiment (2 % stimulated P1 inoculum, 0.75 % FPE and 4 days RT); upon repeating, this set could reduce 96 % of the COD, 78 % SS and 71 % TtS. The treated wastewater met the standard guidelines for irrigation use and no H₂S was detected. The biomass obtaining after wastewater treatment from the best set consisted mostly of R. palustris P1; the biomass of this set had 65 % protein, 3 % fat, 8 % carbohydrate, 14 % ash and 10 % moisture. The results demonstrated that an inoculum of stimulated P1 grew well in RAW supplemented with FPE and could be considered to be an appropriate technology for effectively treating wastewater, with SCP as a by-product.     

The amino acid sequence of the cytochrome c2 from the phototrophic bacterium Rhodopseudomonas globiformis.

The amino acid sequence of the principal soluble cytochrome c from the phototrophic acidophilic bacterium Rhodopseudomonas (or Rhodopila) globiformis was determined. By the criteria of percentage sequence identity and fewness of internal insertions and deletions it is more similar in sequence to some mitochondrial cytochromes c than to any known bacterial cytochrome. The organism does not have any properties that commend it as being particularly similar to postulated prokaryotic precursors of the mitochondrion. We consider that the relatively high degree of sequence similarity is an instance of convergence, and is an example of the limitations that are imposed on attempts to deduce distant evolutionary relationships from sequence information. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50136 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].     

The amino acid sequence of a Bacillus subtilis phosphoprotein that matches an orfY-tsr coding sequence

Bacillus subtilis contains a 30 kDa protein which was phosphorylated during late vegetative growth and sporulation. The sequence for the N-terminal 16 amino acids was found to be identical to the predicted sequence for the N-terminus of a small open reading frame, orfY, but diverged from the predicted sequence thereafter. The orfY region was resequenced and contained one less adenine residue than previously reported, resulting in an open reading frame from within orfY through the entire coding region for tsr which follows orfY. The predicted orfY-tsr amino acid sequence showed 24% identity to Escherichia coli fructose-1,6-bisphosphate aldolase. Two mutants in the tsr region had 2-5% of wild-type aldolase and the nucleotide sequences showed missense mutations. These results indicate that orfY-tsr encodes aldolase and should be renamed fba1.     

Use of wood vinegar to enhance 5-aminolevulinic acid production by selected Rhodopseudomonas palustris in rubber sheet wastewater for agricultural use

This study aimed to produce inexpensive 5-aminolevulinic acid (ALA) in a non-sterile latex rubber sheet wastewater (RSW) by Rhodopseudomonas palustris TN114 and PP803 for the possibility to use in agricultural purposes by investigating the optimum conditions, and applying of wood vinegar (WV) as an economical source of levulinic acid to enhance ALA content. The Box–Behnken Design experiment was conducted under microaerobic-light conditions for 96 h with TN114, PP803 and their mixed culture (1:1) by varying initial pH, inoculum size (% v/v) and initial chemical oxygen demand (COD, mg/L). Results showed that the optimal condition (pH, % inoculum size, COD) of each set to produce extracellular ALA was found at 7.50, 6.00, 2000 for TN114; 7.50, 7.00, 3000 for PP803; and 7.50, 6.00, 4000 for a mixed culture; and each set achieved COD reduction as high as 63%, 71% and 75%, respectively. Addition of the optimal concentration of WV at mid log phase at 0.63% for TN114, and 1.25% for PP803 and the mixed culture significantly increased the ALA content by 3.7–4.2 times (128, 90 and 131 μM, respectively) compared to their controls. ALA production cost could be reduced approximately 31 times with WV on the basis of the amount of levulinic acid used. Effluent containing ALA for using in agriculture could be achieved by treating the RSW with the selected ALA producer R. palustris strains under the optimized condition with a little WV additive.     

A new type of bacteriophytochrome acts in tandem with a classical bacteriophytochrome to control the antennae synthesis in Rhodopseudomonas palustris

Phytochromes are chromoproteins found in plants and bacteria that switch between two photointerconvertible forms via the photoisomerization of their chromophore. These two forms, Pr and Pfr, absorb red and far-red light, respectively. We have characterized the biophysical and biochemical properties of two bacteriophytochromes, RpBphP2 and RpBphP3, from the photosynthetic bacterium Rhodopseudomonas palustris. Their genes are contiguous and localized near the pucBAd genes encoding the polypeptides of the light harvesting complexes LH4, whose synthesis depends on the light intensity. At variance with all (bacterio)phytochromes studied so far, the light-induced isomerization of the chromophore of RpBphP3 converts the Pr form to a form absorbing at shorter wavelength around 645 nm, designated as Pnr for near red. The quantum yield for the transformation of Pr into Pnr is about 6-fold smaller than for the reverse reaction. Both RpBphP2 and RpBphP3 autophosphorylate in their dark-adapted Pr forms and transfer their phosphate to a common response regulator Rpa3017. Under semiaerobic conditions, LH4 complexes replace specifically the LH2 complexes in wild-type cells illuminated by wavelengths comprised between 680 and 730 nm. In contrast, mutants deleted in each of these two bacteriophytochromes display no variation in the composition of their light harvesting complexes whatever the light intensity. From both the peculiar properties of these bacteriophytochromes and the phenotypes of their deletion mutants, we propose that they operate in tandem to control the synthesis of LH4 complexes by measuring the relative intensities of 645 and 710 nm lights.     

Identification of the substrate specificity-conferring amino acid residues of 4-coumarate:coenzyme A ligase allows the rational design of mutant enzymes with new catalytic properties

4-Coumarate:coenzyme A ligases (4CLs) generally use, in addition to coumarate, caffeate and ferulate as their main substrates. However, the recently cloned Arabidopsis thaliana isoform At4CL2 is exceptional because it has no appreciable activity with ferulate. On the basis of information obtained from the crystal structure of the phenylalanine-activating domain of gramicidin S-synthetase, 10 amino acid residues were identified that may form the substrate binding pocket of 4CL. Among these amino acids, representing the putative "substrate specificity motif," only one residue, Met(293), was not conserved in At4CL2, compared with At4CL1 and At4CL3, two isoforms using ferulate. Substitution of Met(293) or Lys(320), another residue of the putative substrate specificity motif, which in the predicted three-dimensional structure is located in close proximity to Met(293), by smaller amino acids converted At4CL2 to an enzyme capable of using ferulate. The activity with caffeate was not or only moderately affected. Conversely, substitution of Met(293) by bulky aromatic amino acids increased the apparent affinity (K(m)) for caffeate up to 10-fold, whereas single substitutions of Val(294) did not affect substrate use. The results support our structural assumptions and suggest that the amino acid residues 293 and 320 of At4CL2 directly interact with the 3-methoxy group of the phenolic substrate and therefore allow a first insight into the structural principles determining substrate specificity of 4CL.