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Archives of Microbiology (v.182, #4)
Seeing green bacteria in a new light: genomics-enabled studies of the photosynthetic apparatus in green sulfur bacteria and filamentous anoxygenic phototrophic bacteria by Niels-Ulrik Frigaard; Donald A. Bryant (pp. 265-276).
Based upon their photosynthetic nature and the presence of a unique light-harvesting antenna structure, the chlorosome, the photosynthetic green bacteria are defined as a distinctive group in the Bacteria. However, members of the two taxa that comprise this group, the green sulfur bacteria (Chlorobi) and the filamentous anoxygenic phototrophic bacteria (“Chloroflexales”), are otherwise quite different, both physiologically and phylogenetically. This review summarizes how genome sequence information facilitated studies of the biosynthesis and function of the photosynthetic apparatus and the oxidation of inorganic sulfur compounds in two model organisms that represent these taxa, Chlorobium tepidum and Chloroflexus aurantiacus. The genes involved in bacteriochlorophyll (BChl) c and carotenoid biosynthesis in these two organisms were identified by sequence homology with known BChl a and carotenoid biosynthesis enzymes, gene cluster analysis in Cfx. aurantiacus, and gene inactivation studies in Chl. tepidum. Based on these results, BChl a and BChl c biosynthesis is similar in the two organisms, whereas carotenoid biosynthesis differs significantly. In agreement with its facultative anaerobic nature, Cfx. aurantiacus in some cases apparently produces structurally different enzymes for heme and BChl biosynthesis, in which one enzyme functions under anoxic conditions and the other performs the same reaction under oxic conditions. The Chl. tepidum mutants produced with modified BChl c and carotenoid species also allow the functions of these pigments to be studied in vivo.
Keywords: Bacteriochlorophyll aBacteriochlorophyll biosynthesis; Bacteriochlorophyll cCarotenoid biosynthesisChlorobiumChloroflexusChlorosome; Functional genomics; Inorganic sulfur metabolism
Unusual ADP-forming acetyl-coenzyme A synthetases from the mesophilic halophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum by Christopher Bräsen; Peter Schönheit (pp. 277-287).
ADP-forming acetyl-CoA synthetase (ACD), the novel enzyme of acetate formation and energy conservation in archaea (% !AMS LaTeX.tdl!TeX -- AMS-LaTeX!% MathType!MTEF!2!0!+-% feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaaeyqaiaabo% gacaqGLbGaaeiDaiaabMhacaqGSbGaaeylaiaaboeacaqGVbGaaeyq% aiabgUcaRiaabgeacaqGebGaaeiuaiabgUcaRiaabcfadaWgaaWcba% GaaeyAaaqabaGccqWIehcGcaqGHbGaae4yaiaabwgacaqG0bGaaeyy% aiaabshacaqGLbGaey4kaSIaaeyqaiaabsfacaqGqbGaey4kaSIaae% 4qaiaab+gacaqGbbaaaa!53B0!$${ ext{Acetyl - CoA}} + { ext{ADP}} + { ext{P}}_{{ ext{i}}}
ightleftarrows { ext{acetate}} + { ext{ATP}} + { ext{CoA}}$$), has been studied only in few hyperthermophilic euryarchaea. Here, we report the characterization of two ACDs with unique molecular and catalytic features, from the mesophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. ACD from H. marismortui was purified and characterized as a salt-dependent, mesophilic ACD of homodimeric structure (166 kDa). The encoding gene was identified in the partially sequenced genome of H. marismortui and functionally expressed in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization and refolding in the presence of salts. The ACD catalyzed the reversible ADP- and Pi-dependent conversion of acetyl-CoA to acetate. In addition to acetate, propionate, butyrate, and branched-chain acids (isobutyrate, isovalerate) were accepted as substrates, rather than the aromatic acids, phenylacetate and indol-3-acetate. In the genome of P. aerophilum, the ORFs PAE3250 and PAE 3249, which code for α and β subunits of an ACD, overlap each other by 1 bp, indicating a novel gene organization among identified ACDs. The two ORFs were separately expressed in E. coli and the recombinant subunits α (50 kDa) and β (28 kDa) were in-vitro reconstituted to an active heterooligomeric protein of high thermostability. The first crenarchaeal ACD showed the broadest substrate spectrum of all known ACDs, catalyzing the conversion of acetyl-CoA, isobutyryl-CoA, and phenylacetyl-CoA at high rates. In contrast, the conversion of phenylacetyl-CoA in euryarchaeota is catalyzed by specific ACD isoenzymes.
Keywords: ADP-forming acetyl-coenzyme A synthetase; Halophilic and hyperthermophilic archaeaHaloarcula marismortuiPyrobaculum aerophilumGene organization
Diversity of coexisting Planktothrix (Cyanobacteria) chemotypes deduced by mass spectral analysis of microystins and other oligopeptides by Martin Welker; Guntram Christiansen; Hans von Döhren (pp. 288-298).
Cyanobacteria are reported to produce secondary metabolites of which toxic and bioactive peptides are of scientific and public interest. Many peptides are synthesized by the non-ribosomal peptide synthesis pathway and their presence is a stable feature of individual clones. We isolated 18 clonal strains of Planktothrix from a single water sample from lake Maxsee near Berlin and analyzed them by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, HPLC, and PCR for their production of peptides and the presence of microcystin synthetase genes. Microcystins could be detected in seven of the strains with considerable variability of contents and numbers of structural variants. Other known peptides like anabaenopeptins B and E/F, microviridin I, and prenylagaramide B and new variants of known peptide classes like aeruginosins and cyanopeptolins were detected in some strains while lacking in others. The 18 strains represented 15 chemotypes with respect to their peptide patterns. In contrast, all strains were morphologically very similar with respect to cell dimensions and pigmentation. Given the diversity of chemotypes among the randomly selected isolates, an immense diversity of chemotypes in the entire population can be assumed.
Keywords: CyanobacteriaPlanktothrixNon-ribosomal peptides; Diversity; Chemotypes; Matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF mass spectrometry
The glycosylated cell surface protein Rpf2, containing a resuscitation-promoting factor motif, is involved in intercellular communication of Corynebacterium glutamicum by Michael Hartmann; Aiko Barsch; Karsten Niehaus; Alfred Pühler; Andreas Tauch; Jörn Kalinowski (pp. 299-312).
The genome of Corynebacterium glutamicum ATCC 13032 contains two genes, rpf1 and rpf2, encoding proteins with similarities to the essential resuscitation-promoting factor (Rpf) of Micrococcus luteus. Both the Rpf1 (20.4 kDa) and Rpf2 (40.3 kDa) proteins share the so-called Rpf motif, a highly conserved protein domain of approximately 70 amino acids, which is also present in Rpf-like proteins of other gram-positive bacteria with a high G+C content of the chromosomal DNA. Purification of the C. glutamicum Rpf2 protein from concentrated supernatants, SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified modified Rpf2 variants with increased or reduced mobility when compared with the calculated size of Rpf2. A Western blot-based enzyme immunoassay demonstrated glycosylation of the Rpf2 variants with higher molecular masses. Galactose and mannose were identified as two components of the oligosaccharide portion of the Rpf2 glycoprotein by capillary gas chromatography coupled to mass spectrometry. The Rpf2 protein was localized on the surface of C. glutamicum with the use of immuno-fluorescence microscopy. C. glutamicum strains with defined deletions in the rpf1 or rpf2 gene or simultaneous deletions in both rpf genes were constructed, indicating that the rpf genes are neither individually nor collectively essential for C. glutamicum. The C. glutamicum rpf double mutant displayed slower growth and a prolonged lag phase after transfer of long-stored cells into fresh medium. The addition of supernatant from exponentially growing cultures of the rpf double mutant, the wild type or C. glutamicum strains with increased expression of the rpf1 or rpf2 gene significantly reduced the lag phase of long-stored wild-type and rpf single mutant strains, but addition of purified His-tagged Rpf1 or Rpf2 did not. In contrast, the lag phase of the C. glutamicum rpf double mutant was not affected upon addition of these culture supernatants.
Keywords: Corynebacterium glutamicumResuscitation-promoting factor; Intercellular communication; Protein glycosylation
Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this methanogenic archaeon on folic acid or p-aminobenzoic acid by Bärbel Buchenau; Rudolf K. Thauer (pp. 313-325).
Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H4SPT) rather than tetrahydrofolate (H4F) as a pterin C1 carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN, folD, glyA and metF, that are predicted to encode for H4F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N5,N10-methylene-H4F dehydrogenase/N5,N10-methenyl-H4F cyclohydrolase) and GlyA (serine:H4F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli, purified and found to be specific for methylene-H4F and H4F, respectively (apparent Km below 5 μM). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri. The results thus indicate that M. barkeri should contain H4F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p-aminobenzoic acid, a biosynthetic precursor of H4F. From the p-aminobenzoic acid requirement, an intracellular H4F concentration of approximately 5 μM was estimated. Evidence is presented that the p-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H4SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H4SPT and H4F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C1 pools in these methanogens.
Keywords: Tetrahydrofolate; Tetrahydromethanopterin; Tetrahydrosarcinapterinp-Aminobenzoic acid; Folate biosynthesis; Methanopterin biosynthesis; Purine biosynthesis; C1 metabolism; MethanogenesisMethanosarcina barkeri
Nutrient-specific effects in the coordination of cell growth with cell division in continuous cultures of Saccharomyces cerevisiae by Jinbai Guo; Brad A. Bryan; Michael Polymenis (pp. 326-330).
Cell cycle progression of Saccharomyces cerevisiae cells was monitored in continuous cultures limited for glucose or nitrogen. The G1 cell cycle phase, before initiation of DNA replication, did not exclusively expand when growth rate decreased. Especially during nitrogen limitation, non-G1 phases expanded almost as much as G1. In addition, cell size remained constant as a function of growth rate. These results contrast with current views that growth requirements are met before initiation of DNA replication, and suggest that distinct nutrient limitations differentially impinge on cell cycle progression.
Keywords: START; Chemostat; Growth; Cell cycleSaccharomyces cerevisiae
Complexity of phenotypes and symbiotic behaviour of Rhizobium leguminosarum biovar trifolii exopolysaccharide mutants by Jerzy Wielbo; Andrzej Mazur; Jarosław Król; Małgorzata Marczak; Jolanta Kutkowska; Anna Skorupska (pp. 331-336).
Rhizobium leguminosarum biovar trifolii strain TA1 polysaccharide synthesis (pss) mutants in the pssD, pssP, pssT and pssO genes and altered in exopolysaccharide (EPS) synthesis were investigated. EPS-deficient mutants were also changed in lipopolysaccharide structure. All mutants exhibited varied sensitivities to detergents, ethanol and antibiotics, thus indicating changes in bacterial membrane integrity. Using pss mutants marked with the gusA gene, EPS-deficient mutants were found to have abnormalities in nodule development and to provoke severe plant defence reactions. The pss mutants that produced altered quantities of EPS with a changed degree of polymerisation generally occupied the younger developmental zones of the nodules and elicited moderate plant defence reactions.
Keywords: Exopolysaccharide mutants; Detergent sensitivity; Antibiotic resistance; Nodule invasion