Biosynthesis of intracellular 5-aminolevulinic acid by a newly identified halotolerant <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Of 23 strains of halotolerant (up to 12% w/v NaCl) photosynthetic bacteria isolated from various sources, one isolate, SH5, accumulated intracellular 5-aminolevulinic acid (ALA) at 0.45 μg/g dry cell wt (DCW) growing aerobically in the dark. The strain was identified as Rhodobacter sphaeroides using 16S rDNA sequencing. Biosynthesis of ALA was enhanced to 14 μg/g DCW using modified glutamate/glucose (50 mM) medium with the addition of 10 mM levulinic acid after 24 h cultivation. Addition of 30 μM Fe²⁺ to this medium increased the yield to 226 μg/g DCW.     

Mutant reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> I(L177)H with strongly bound bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>: Structural properties and pigment-protein interactions

Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H _A ^? . The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls P_A and P_B in the P⁺ state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the P_A and P_B macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P⁺Q^?/PQ spectra suggests the 17³-ester group of the bacteriochlorophyll P_A to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll B_A and the bacteriopheophytin H_A. Structural changes are observed in the monomer bacteriochlorophyll B_B binding site in the inactive chromophore branch of the mutant RCs.     

Heterologous expression of the <Emphasis Type="Italic">msp2 gene</Emphasis> from <Emphasis Type="Italic">Marasmius scorodonius</Emphasis>

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca²⁺, and hemin.     

Functional analysis of a large non-conserved region of FlgK (HAP1) from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The single subpolar flagellum of Rhodobacter sphaeroides shows an enlarged hook-filament junction. One of the two proteins that compose this section of the filament is HAP1 _Rs (FlgK _Rs ) it contains a central non-conserved region of 860 amino acids that makes this protein about three times larger than its homologue in Salmonella enterica serovar Typhimurium. We investigated the role of this central portion of the unusually large HAP1 protein of R. sphaeroides by monitoring the effects of serial deletions in flgK _Rs , the gene encoding HAP1 _Rs , on swimming and swarming. Two deletion mutants did not assemble functional flagella, two were paralyzed and five exhibited reduced free-swimming speeds. Some mutants produced unusual swarming patterns on soft agar without or with Ficoll 400. A segment of approximately 200-aa of the central region of HAP1 _Rs that aligns with the variable region of the flagellin sequence from other γ- and β-proteobacteria was also found. Therefore, it is possible that the origin of this large central domain of HAP1 _Rs could be associated with an event of horizontal transfer and subsequent duplications and/or insertions.     

Metabolically Engineered <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> RV strains for Improved Biohydrogen Photoproduction Combined with Disposal of Food Wastes

Three differently metabolically engineered strains, 2 single PHA^- and Hup^- mutants and one double PHA^-/Hup^- mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes. These phenotypes were designed to abolish, singly or in combination, the competition of H₂ photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H₂ recycling by abolishing the uptake hydrogenase enzyme. The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids. With the lactic acid-based synthetic medium, the single Hup^- and the double PHA^-/Hup^- mutants, but not the single PHA^- mutant, exhibited increased rates of H₂ photoproduction, about one third higher than that of the wild-type strain. With the food-waste-derived growth medium, only the single Hup^- mutant showed higher rates of H₂ production, but all 3 mutants sustained a longer-term H₂ photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H₂ evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H₂ photoproduction using actual waste-derived substrates.     

Biosorption and Bioreduction of Trivalent Aurum by Photosynthetic Bacteria <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

Biosorption has been shown to be an eco-friendly approach to remove heavy metal ions. In this study, the photosynthetic bacteria Rhodobacter capsulatus was screened and found to have strong ability to adsorb Au(III). The maximum specific uptake of living cells was over 92.43 mg HAuCl₄/g dry weight of cell in the logarithmic phase. Biosorpion ability would be enhanced by an acidic environment. As the main cations, during biosorption the quantity of Mg²⁺ exchanged was more than Na⁺. Biosorbed Au(III) could be reduced by carotenoid and enzymes embedded and/or excreted by R. capsulatus, which might be the mechanism of photosynthtic bacteria metal tolerance.     

Characteristics of light-harvesting complex II mutant of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> with alterations at the transmembrane helices of β-subunit

The peripheral light-harvesting complex II (LHII) is an important component of the photosynthetic apparatus of Rhodobacter sphaeroides. In this study, genetic, biochemical, and spectroscopic approaches were applied to investigate the spectral properties and functions of LHII in which two amino acid residues Phe32 and Leu42 in the transmembrane helix domain of pucB-encoded β-apoprotein were replaced by Leu and Pro. The mutated LHII complex showed blue shift of absorbance peaks in the near infrared region at ∼801–845 nm in R. sphaeroides. It should be noted that the B800 peak was much lower than that of the native LHII, and transfer energy was efficient from the B800 to the B850 pigments in the LHII complex. The results suggest that the mutated pucB could be expressed in R. sphaeroides, and the functional LHII was assembled into the membrane of R. sphaeroides notwithstanding with the different spectral properties. These mutated residues were indeed critical for the modulation of characteristics and function of LHII complex. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 993–999.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High production of laccase B from <Emphasis Type="Italic">Trametes</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The coding sequence of a laccase isozyme from Trametes sp. AH28-2 was cloned in pPIC9K vector and heterologously overexpressed in the yeast Pichia pastoris strain GS115. In the minimal medium containing 0.3 mM CuSO₄ and 0.6% alanine, the maximum yield of the recombinant laccase rLacB reached 32,000 U/l (1,012 U/mg), slightly higher than that of the native enzyme nLacB (∼30,000 U/l, 1,356 U/mg). The enzymatic properties of rLacB were different from those of nLacB as well. Regardless of the inferior thermal stability, rLacB had much better stability at both neutral and basic pH range compared to nLacB. In addition, the dye decolorization potential of rLacB was similar to that of nLacB.     

Singlet oxygen generation in the reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Singlet oxygen ((1)O(2)) generation in the reaction centers (RCs) of Rhodobacter sphaeroides wild type was characterized by luminescent emission in the near infrared region (time resolved transients and emission spectra) and quantified to have quantum yield of 0.03 +/- 0.005. (1)O(2) emission was measured as a function of temperature, ascorbate, urea and potassium ferricyanide concentrations and as a function of incubation time in H(2)O:D(2)O mixtures. (1)O(2 )was shown to be affected by the RC dynamics and to originate from the reaction of molecular oxygen with two sources of triplets: photoactive dimer formed by singlet-triplet mixing and bacteriopheophytin formed by direct photoexcitation and intersystem crossing.     

Heterologous expression,purification, and characterization of xylose reductase from <Emphasis Type="Italic">Candida shehatae</Emphasis>

The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His₆-tagged fusion protein. The recombinant XR had K_m values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was specific for d-xylese.     

The <Emphasis Type="Italic">Arxula adeninivorans</Emphasis><Emphasis Type="Italic">ATAL</Emphasis> gene encoding transaldolase-gene characterization and biotechnological exploitation

The yeast Arxula adeninivorans provides an attractive expression platform and can be exploited as gene source for biotechnologically interesting proteins. In the following study, a striking example for the combination of both aspects is presented. The transaldolase-encoding A. adeninivorans ATAL gene, including its promoter and terminator elements, was isolated and characterized. The gene includes a coding sequence of 963 bp encoding a putative 321 amino acid protein of 35.0 kDa. The enzyme characteristics analyzed from isolates of native strains and recombinant strains overexpressing the ATAL gene revealed a molecular mass of ca. 140 kDa corresponding to a tetrameric structure, a pH optimum of ca. 5.5, and a temperature optimum of 20°C. The preferred substrates for the enzyme include d-erythrose-4-phosphate and d-fructose-6-phosphate, whereas d-glyceraldehyde is not converted. The ATAL expression level under salt-free conditions was observed to increase in media supplemented with 5% NaCl rendering the ATAL promoter attractive for moderate heterologous gene expression under high-salt conditions. Its suitability was assessed for the expression of a human serum albumin (HSA) reporter gene.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.     

Substitution of Isoleucine L177 by Histidine Affects the Pigment Composition and Properties of the Reaction Center of the Purple Bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Using site-directed mutagenesis, we obtained the mutant of the purple bacterium Rhodobacter sphaeroides with Ile to His substitution at position 177 in the L-subunit of the photosynthetic reaction center (RC). The mutant strain forms stable and photochemically active RC complexes. Relative to the wild type RCs, the spectral and photochemical properties of the mutant RC differ significantly in the absorption regions corresponding to the primary donor P and the monomer bacteriochlorophyll (BChl) absorption. It is shown that the RC I(L177)H contains only three BChl molecules compared to four BChl molecules in the wild type RC. Considering the fact that the properties of both isolated and membrane-associated mutant RCs are similar, we conclude that the loss of a BChl molecule from the mutant RC is caused by the introduced mutation but not by the protein purification procedure. The new mutant missing one BChl molecule but still able to perform light-induced reactions forming the charge-separated state P+QA- appears to be an interesting object to study the mechanisms of the first steps of the primary electron transfer in photosynthesis.     

Characterization of the <Emphasis Type="Italic">AXDH</Emphasis> gene and the encoded xylitol dehydrogenase from the dimorphic yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter – GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 °C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD⁺ or NADH as coenzymes.     

Photoproduction of hydrogen by <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> from thermophilic fermentation effluent

Rhodobacter capsulatus was used for the phototrophic hydrogen production on effluent solution derived from the thermophilic fermentation of Miscanthus hydrolysate by Thermotoga neapolitana. Pretreatments such as centrifugation, dilution, buffer addition, pH adjustment and sterilization were suggested for the effluent before being fed to the photofermentation. Batch-wise experiments showed that R. capsulatus grows and produces hydrogen on the pretreated effluent solution. Moreover, it was found that the hydrogen yield increased from 0.3 to 1.0 L/L_culture by addition of iron to the effluent solution.