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Archives of Microbiology (v.175, #3)
No Title by Shinichi Takaichi; Takashi Maoka; Satoshi Hanada; Johannes F. Imhoff (pp. 161-167).
The carotenoids in Halorhodospira abdelmalekii and Halorhodospira halochloris were analyzed by spectroscopic methods. The carotenoid composition of the two species was almost the same. Both species contained substantial amounts of unusual carotenoid glycoside fatty acid esters, which have been found for the first time in phototrophic purple bacteria. Methoxy-hydroxylycopene glucoside was a major component, and dihydroxylycopene diglucoside and dihydroxylycopene diglucoside diester were also found. Lycopene, rhodopin, and 3,4,3′,4′-tetrahydrospirilloxanthin were present in very small amounts. Methoxy, glucosyl, and glucosyl ester groups were observed as substituents at the positions of the two original hydroxyl groups of dihydroxylycopene and made up approximately 20, 50, and 20%, respectively, of the total end groups (100%). The fatty acid components of the three carotenoid glucoside esters were the same (C12:0 and C14:1) and were rare in the cellular lipids of the two species.
Keywords: Carotenoid Carotenoid glucoside ester Carotenoid glycoside ester Halorhodospira abdelmalekii Halorhodospira halochloris Lycopene Purple sulfur bacteria
No Title by Marco Candela; Elisa Zaccherini; Davide Zannoni (pp. 168-177).
Membrane fragments isolated from the aerobic phototrophic bacterium Roseobacter denitrificans were examined. Ninety-five percent of the total NADH-dependent oxidative activity was inhibited either by antimycin A or myxothiazol, two specific inhibitors of the cytochrome bc 1 complex, which indicates that the respiratory electron transport chain is linear. In agreement with this finding, light-induced oxygen uptake, an electron transport activity catalyzed by the "alternative quinol oxidase pathway" in membranes of several facultative phototrophic species, was barely detectable in membranes of Rsb. denitrificans. Redox titrations at 561–575 nm, 552–540 nm, and 602–630 nm indicated the presence of three b-type cytochromes (Em,7 of +244±8, +24±3, –163±11 mV), four c-type cytochromes (Em,7 of +280±10, +210±5, +125±8, and 20±3 mV) and two a-type cytochromes (Em,7 of +335±15, +218±18 mV). The latter two a-type hemes were shown to be involved in cytochrome c oxidase activity, which was inhibited by both cyanide (I50=2 µM) and azide (I50=1 mM), while a soluble cytochrome c (c 551, Em,7=+217±2 mV) was shown to be the physiological electron carrier connecting the bc 1 complex to the cytochrome c oxidase. A comparison of the ATP synthesis generated by continuous light in membranes of Rsb. denitrificans and Rhodobacter capsulatus showed that in both bacterial species photophosphorylation requires a membrane redox poise at the equilibrium (Eh≥+80≤+140 mV), close to the oxidation-reduction potential of the ubiquinone pool. These data, taken together, suggest that, although the photosynthetic apparatus of Rsb. denitrificans is functionally similar to that of typical anoxygenic phototrophs, e.g. Rba. capsulatus, the in vivo requirement of a suitable redox state at the ubiquinone pool level restricts the growth capacity of Rsb. denitrificans to oxic conditions.
Keywords: ATP synthesis Cytochromes Inhibitors Respiratory chain Roseobacter denitrificans
No Title by Adnan Hasona; William T. Self; Keelnatham T. Shanmugam (pp. 178-188).
Regulation of transcription of the Escherichia coli moe operon, which codes for proteins connecting molybdate metabolism, molybdopterin synthesis, and apomolybdoenzyme synthesis, was investigated. Expression of the moe operon was independent of genes coding for molybdate transport and Mo-cofactor biosynthesis. Expression of moeA-lacZ increased during anaerobic growth (2.5-fold over the aerobic value) and in the presence of nitrate and trimethylamine N-oxide (3.5- and 1.5-fold, respectively). The nitrate-dependent increase in moe expression required the NarL protein, while the anaerobiosis-dependent increase in moeA-lacZ expression required Arc proteins. ArcA-phosphate and not ArcA bound to the DNA upstream of moe, shifted the electrophoretic mobility of moe promoter DNA, and protected the DNA from DNase I hydrolysis. Nitrate-independent transcription of moeA-lacZ was repressed by the FNR protein, which also protected moe operator DNA from DNase I hydrolysis. These results show that ArcA-phosphate and FNR have opposite effects on the transcriptional regulation of the moe operon, and the combined action of the two redox regulators modulate the level of Mo-cofactor in the cell. Apparently, the control of synthesis of Mo-cofactor and the apomolybdoenzymes nitrate reductase and trimethylamine N-oxide reductase are coupled at the level of the moe operon.
Keywords: Molybdate moe operon Regulation Nitrate ArcA FNR Escherichia coli
No Title by José A. Pérez; Javier Rodríguez; Teresa Ruiz; Luis Rodríguez (pp. 189-197).
The Pichia anomala invertase gene (INV1) was introduced at different copy numbers into a sucrose-nonfermenting mutant of Saccharomyces cerevisiae and expressed from its own promoter sequences. The level reached in the production of invertase by the transformants (up to 540 units/1010 cells) was in agreement with the INV1 gene dosage. Two forms of multimeric active and glycosylated invertase displaying different subcellular locations and molecular masses could be detected in the transformants. One was found to be present in the culture medium and in the periplasm, and the other could only be detected inside the cell. Each of the two heterologous forms of invertase was shown to be an oligomer composed of identical subunits. The difference found in the apparent molecular masses of their monomers (81.5 and 78.3 kDa, respectively) seems to be due to the size of their N-linked oligosaccharide chains (on average 2.4 and 1.9 kDa, respectively), since the number of sugar chains (9) and the molecular mass of the protein moiety (60.5 kDa) are identical in both forms. The shorter size of their oligosaccharides must also be the reason for the lower apparent molecular masses of the heterologous invertases when compared with the enzyme purified from P. anomala. The hypoglycosylated invertase accumulated within the cells of the transformants to an unusual level (up to 130 units/1010 cells). Such accumulation of active enzyme inside the cells, as well as its underglycosylation, could be due to intrinsic properties of the P. anomala invertase that are determined by the particular primary structure of its protein moiety.
Keywords: Pichia anomala Saccharomyces cerevisiae Heterologous gene expression Hypoglycosylation Glycoproteins Invertase
No Title by Konrad Egli; Urs Fanger; Pedro J.J. Alvarez; Hansruedi Siegrist; Jan R. van der Meer; Alexander J.B. Zehnder (pp. 198-207).
Anaerobic ammonium oxidation with nitrite to N2 (anammox) is a recently discovered microbial reaction with interesting potential for nitrogen removal from wastewater. We enriched an anammox culture from a rotating disk contactor (near Kölliken, Switzerland) that was used to treat ammonium-rich leachate with low organic carbon content. This enrichment led to a relative population size of 88% anammox bacteria. The microorganism carrying out the anammox reaction was identified by analysis of the 16S rDNA sequence and by fluorescence in situ hybridization (FISH) with 16S-rRNA-targeting probes. The percentage sequence identity between the 16S rDNA sequences of the Kölliken anammox organism and the archetype anammox strain Candidatus Brocadia anammoxidans was 90.9%, but between 98.5 and 98.9% with Candidatus Kuenenia stuttgartiensis, an organism identified in biofilms by molecular methods. The Kölliken culture catalyzed the anaerobic oxidation of ammonium with nitrite in a manner seemingly identical to that of Candidatus B. anammoxidans, but exhibited higher tolerance to phosphate (up to 20 mM) and to nitrite (up to 13 mM) and was active at lower cell densities. Anammox activity was observed only between pH 6.5 and 9, with an optimum at pH 8 and a temperature optimum at 37 °C. Hydroxylamine and hydrazine, which are intermediates of the anammox reaction of Candidatus B. anammoxidans, were utilized by the Kölliken organisms, and approximately 15% of the nitrite utilized during autotrophic growth was converted to nitrate. Electron microscopy showed a protein-rich region in the center of the cells surrounded by a doughnut-shaped region containing ribosomes and DNA. This doughnut-shape region was observed with FISH as having a higher fluorescence intensity. Similar to Candidatus B. anammoxidans, the Kölliken anammox organism typically formed homogenous clusters containing up to several hundred cells within an extracellular matrix.
Keywords: Anaerobic ammonium oxidation Anammox Identification Planctomycete Fluorescence in situ hybridization Electron microscopy Physiology Wastewater Intermediates
No Title by Jodi Switzer Blum; John F. Stolz; Aharon Oren; Ronald S. Oremland (pp. 208-219).
We isolated an obligately anaerobic halophilic bacterium from the Dead Sea that grew by respiration of selenate. The isolate, designated strain DSSe-1, was a gram-negative, non-motile rod. It oxidized glycerol or glucose to acetate+CO2 with concomitant reduction of selenate to selenite plus elemental selenium. Other electron acceptors that supported anaerobic growth on glycerol were nitrate and trimethylamine-N-oxide; nitrite, arsenate, fumarate, dimethylsulfoxide, thiosulfate, elemental sulfur, sulfite or sulfate could not serve as electron acceptors. Growth on glycerol in the presence of nitrate occurred over a salinity range from 100 to 240 g/l, with an optimum at 210 g/l. Analysis of the 16S rRNA gene sequence suggests that strain DSSe-1 belongs to the order Halanaerobiales, an order of halophilic anaerobes with a fermentative or homoacetogenic metabolism, in which anaerobic respiratory metabolism has never been documented. The highest 16S rRNA sequence similarity (90%) was found with Acetohalobium arabaticum (X89077). On the basis of physiological properties as well as the relatively low homology of 16S rRNA from strain DSSe-1 with known genera, classification in a new genus within the order Halanaerobiales, family Halobacteroidaceae is warranted. We propose the name Selenihalanaerobacter shriftii. Type strain is strain DSSe-1 (ATCC accession number BAA-73).
Keywords: Halophile Anaerobe Selenihalanaerobacter shriftii gen. nov. sp. nov. Dead Sea Selenate reduction Nitrate reduction
No Title by Fu-Pang Lin; Woan-Yuan Juang; Keng-Hao Chang; Hsing-Chen Chen (pp. 220-225).
A G561 mutant of the Aeromonas caviae chitinase ChiA was made by PCR site-directed deletion mutagenesis in order to study the role of the 304 C-terminal amino acid residues of ChiA in the enzymatic hydrolysis of chitin. The recombinant ChiAG561 encoded on a 1.6-kb DNA fragment of A. caviae chiA was expressed in a heterologous Escherichia coli host using the pET20b(+) expression system. The His-Tag-affinity-purified recombinant ChiAG561 had a calculated molecular mass of 63,595 Da, which was consistent with the 67,000 Da estimated by SDS-PAGE. The G561 deletion mutant enzyme had the same optimum pH (6.5) as the full-length ChiA and a lower optimum temperature (37 °C instead of 42.5 °C). Biochemical properties of the recombinant ChiAG561 suggested that deletion of the 304 C-terminal amino acid residues of ChiA did not significantly affect ChiA enzyme activity. However, compared to the full-length ChiA, the mutant chitinase had a ten-fold higher relative activity with 4-methylumbelliferyl-N-N′-N′′-triacetylchitotriose [4-MU-(GlcNAc)3] as a substrate, and higher rates of hydrolysis with both chitin and colloidal chitin substrates. Results obtained from this study suggest that the active region of A. caviae ChiA is located in the region before G561 of the protein molecule.
Keywords: Aeromonas caviae Chitinase Site-directed deletion mutagenesis
No Title by Juan B. Arellano; Carles M. Borrego; Asunción Martínez-Planells; Librado Jesús Garcia-Gil (pp. 226-233).
The effects of inhibition of carotenoid biosynthesis by 2-hydroxybiphenyl on the photosynthetic growth, pigment composition and chlorosome structure of Chlorobium phaeobacteroides strain CL1401 were examined. At a concentration of 20 µg 2-hydroxybiphenyl ·ml–1, carotenoid synthesis was largely inhibited (85%), but the photosynthetic growth rate was almost unaffected (µ control=0.00525±0.00007 h–1 and µ HBP-treated=0.00505±0.0005 h–1). Cells grown in the presence of the inhibitor were 5 µm–70 µm long, while control cells were between 2–5 µm long. Moreover, 2-hydroxybiphenyl-treated cells contained fewer, unevenly distributed chlorosomes per µm of cytoplasmic membrane with an irregular arrangement (2.5±1.5 vs of 9.1±1.9). This was concomitant to the 83% decrease in the content of bacteriochlorophyll (BChl) e in 2-hydroxybiphenyl-treated cells. Electron microscopy revealed that the shape of carotenoid-depleted chlorosomes changed from ellipsoidal to spherical, although the mean volume was similar to that of control chlorosomes. SDS-PAGE analysis of the chlorosome polypeptide composition showed that the amount of CsmA protein decreased by 60% in carotenoid-depleted chlorosomes. This was paralleled by a decrease in the baseplate BChl a content. The data suggest that carotenoids are close to the chlorosomal baseplate, where they carry out both structural and photoprotective functions.
Keywords: Baseplate Bacteriochlorophyll, Carotenoid Chlorosome CsmA Green sulfur bacteria Isorenieratene
No Title by Patricia E. Marini; Carlos A. Perez; Diego de Mendoza (pp. 234-237).
Acetyl-CoA carboxylase catalyses the synthesis of malonyl-CoA, the first intermediate in fatty acid (hence phospholipid) synthesis. The accBC operon from Bacillus subtilis codes for two subunits of acetyl-CoA carboxylase, biotin carboxyl-carrier and biotin carboxylase. In this work, the 5′-end of the accBC mRNA was determined by primer-extension, and transcriptional fusion analysis of the accBC promoter was carried out under a variety of growth conditions. A direct correlation between the levels of transcription of the accBC genes and the rate of cellular growth is reported. Consistent results were also obtained in nutritional upshift and downshift experiments.
Keywords: Growth rate regulation Fatty acids Acetyl CoA carboxylase Biotin carboxyl carrier protein