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Archives of Microbiology (v.178, #2)
No Title by Frank Sargent; Ben C. Berks; Tracy Palmer (pp. 77-84).
The Tat protein-export system serves to translocate folded proteins, often containing redox cofactors, across the bacterial inner membrane. Substrate proteins are directed to the Tat apparatus by distinctive N-terminal signal peptides containing a consensus SRRxFLK 'twin-arginine' motif. Here we review recent studies of the Tat system with particular emphasis on the assembly of membrane-bound respiratory complexes. We discuss the connection between Tat targeting and topological organisation of the complexes and consider the role of chaperone proteins in cofactor insertion and Tat targeting. The crystal structure of Escherichia coli formate dehydrogenase-N demonstrates that some Tat substrates are integral membrane proteins. Sequence analysis suggests that one-quarter of all traffic on the E. coli Tat pathway is inner-membrane proteins.
Keywords: Escherichia coli Tat protein-targeting system Twin-arginine signal peptide Formate dehydrogenase Metalloenzyme biosynthesis Molecular chaperones Membrane proteins
No Title by Botho Bowien; Bernhard Kusian (pp. 85-93).
The nutritional versatility of facultative autotrophs requires efficient overall control of their metabolism. Most of these organisms are Proteobacteria that assimilate CO2 via the highly energy-demanding Calvin-Benson-Bassham reductive pentose-phosphate cycle. The enzymes of the cycle are encoded by cbb genes organized in cbb operons differing in size and composition, although conserved features are apparent. Transcription of the operons, which may form regulons, is strictly controlled, being induced during autotrophic but repressed to varying extents during heterotrophic growth of the bacteria. The chemoautotroph Ralstonia eutropha is one of the organisms studied extensively for the mechanisms involved in the expression of cbb gene systems. CbbR is a LysR-type transcriptional regulator and the key activator protein of cbb operons. The cbbR gene is typically located adjacent and in divergent orientation to its cognate operon. The activating function of CbbR seems to be modulated by metabolites signaling the nutritional state of the cell to the cbb system. Phosphoenolpyruvate is such a signal metabolite acting as a negative effector of R. eutropha CbbR, whereas NADPH has been proposed to be a coactivator of the protein in two other chemoautotrophs, Xanthobacter flavus and Hydrogenophilus thermoluteolus. There is evidence for the participation of additional regulators in cbb control. In the photoautotrophs Rhodobacter capsulatus and Rhodobacter sphaeroides, response regulator RegA of the global two-component signal transduction system RegBA serves this function. It is conceivable that specific variants of cbb control systems have evolved to ensure their optimal integration into regulatory networks operating in the diverse autotrophs characterized by different metabolic capabilities.
Keywords: Autotrophic bacteria Calvin-Benson-Bassham cycle cbb genes CO2 assimilation Gene regulation Ralstonia eutropha
No Title by Jiri Volf; Mojmir Sevcik; Hana Havlickova; Frantisek Sisak; Jiri Damborsky; Ivan Rychlik (pp. 94-101).
sdiA in Salmonella enterica serovar Typhimurium encodes a protein belonging to the LuxR family of transcriptional regulators. Initial computer analysis revealed the presence of a fur box 19 bp upstream of the start codon of the sdiA gene and a helix-turn-helix motif in the carboxy-terminal part of the SdiA protein typical for transcriptional regulators. Deletion of the fur box resulted in twofold increase of sdiA transcription. Furthermore, addition of dipyridyl, an iron chelator, to culture media increased sdiA transcription to the level observed in the fur box mutant, confirming that sdiA is suppressed in the presence of iron. When S. enterica sv. Typhimurium was grown in conditioned medium, sdiA transcription was repressed to 30% of that in cells grown in fresh LB broth; this repression was independent of the fur box. Oral infection of mice with the strain lacking the helix-turn-helix domain of SdiA indicated increased virulence of this S. enterica sv. Typhimurium mutant. sdiA, dually controlled by iron concentration and culture-density-derived signals, may therefore play an important role in S. enterica sv. Typhimurium virulence regulation.
Keywords: Salmonella enterica sv. Typhimurium sdiA Quorum sensing Virulence
No Title by Ute Galm; Judith Schimana; Hans-Peter Fiedler; Jürgen Schmidt; Shu-Ming Li; Lutz Heide (pp. 102-114).
The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticus Tü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A1. Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.
Keywords: Streptomyces antibioticus Tü 6040 Simocyclinone D8 Aminocoumarin antibiotics Angucyclinone antibiotics Gene cluster Gene inactivation
No Title by Huei-Fen Lo; Long-Liu Lin; Wen-Ying Chiang; Meng-Chun Chie; Wen-Hwei Hsu; Chen-Tien Chang (pp. 115-123).
The α-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial α-amylases and an additional functionally unknown domain (amino acids 517–613) in the C-terminal region. By sequence comparison, we found that this latter domain contained a sequence motif typical for raw-starch binding. To investigate the functional role of the C-terminal region of the α-amylase of Bacillus sp. strain TS-23, four His6-tagged mutants with extensive deletions in this region were constructed and expressed in Escherichia coli. SDS-PAGE and activity staining analyses showed that the N- and C-terminally truncated α-amylases had molecular masses of approximately 65, 58, 54, and 49 kDa. Progressive loss of raw-starch-binding activity occurred upon removal of C-terminal amino acid residues, indicating the requirement for the entire region in formation of a functional starch-binding domain. Up to 98 amino acids from the C-terminal end of the α-amylase could be deleted without significant effect on the raw-starch hydrolytic activity or thermal stability. Furthermore, the active mutants hydrolyzed raw corn starch to produce maltopentaose as the main product, suggesting that the raw-starch hydrolytic activity of the Bacillus sp. strain TS-23 α-amylase is functional and independent from the starch-binding domain.
Keywords: Amylase Bacillus sp. strain TS-23 Deletion analysis Starch-binding domain
No Title by Dušica Vujaklija; Werner Schröder; Marija Abramić; Peijian Zou; Ivana Leščić; Peter Franke; Jasenka Pigac (pp. 124-130).
An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.
Keywords: GDS(L) lipolytic enzymes Streptomyces lipases Streptomyces rimosus Gram-positive bacteria
No Title by Boris Alexander; Jesper H. Andersen; Raymond P. Cox; Johannes F. Imhoff (pp. 131-140).
The phylogeny of green sulfur bacteria was studied on the basis of gene sequences of the 16S rRNA and of the Fenna-Matthews-Olson (FMO) protein. Representative and type strains (31 strains total) of most of the known species were analyzed. On the basis of fmoA gene sequences from Chlorobium tepidum ATCC 49652T and Chlorobium limicola DSM 249T available from the EMBL database, primers were constructed that allowed sequence analysis of a major part of the fmoA gene. The largely congruent phylogenetic relationship of sequences of the fmoA gene and of 16S rDNA gives considerable support to the phylogeny of green sulfur bacteria previously suggested on the basis of 16S rDNA sequences. Distinct groups of strains were recognized on the basis of 16S rDNA and FMO sequences and supported by characteristic signature amino acids of FMO. Marine strains formed clusters separate from freshwater strains. The resulting phylogenetic grouping and relationship of the green sulfur bacteria do not correlate with their current taxonomic classification.
Keywords: Green sulfur bacteria Chlorobiaceae Chlorobium Nucleotide sequences Ribosomal DNA 16S rDNA Phylogeny fmoA
No Title by Anand Ballal; Ralf Heermann; Kirsten Jung; Michael Gaßel; Shree Apte; Karlheinz Altendorf (pp. 141-148).
The kdpFABC operon, coding for a high-affinity K+-translocating P-type ATPase, is expressed in Escherichia coli as a backup system during K+ starvation or an increase in medium osmolality. Expression of the operon is regulated by the membrane-bound sensor kinase KdpD and the cytosolic response regulator KdpE. From a nitrogen-fixing cyanobacterium, Anabaena sp. strain L-31, a kdpD gene was cloned (GenBank accession no. AF213466) which codes for a KdpD protein (365 amino acids) that lacks both the transmembrane segments and C-terminal transmitter domain and thus is shorter than E. coli KdpD. A chimeric kdpD gene was constructed and expressed in E. coli coding for a protein (Anacoli KdpD), in which the first 365 amino acids of E. coli KdpD were replaced by those from Anabaena KdpD. In everted membrane vesicles, this chimeric Anacoli KdpD protein exhibited activities, such as autophosphorylation, transphosphorylation and ATP-dependent dephosphorylation of E. coli KdpE, which closely resemble those of the E. coli wild-type KdpD. Cells of E. coli synthesizing Anacoli KdpD expressed kdpFABC in response to K+ limitation and osmotic upshock. The data demonstrate that Anabaena KdpD can interact with the E. coli KdpD C-terminal domain resulting in a protein that is functional in vitro as well as in vivo.
Keywords: Response regulator Sensor kinase Kdp-ATPase Signal transduction Osmolality Potassium
No Title by Silke Fiedler; Alexander Steinbüchel; Bernd H. Rehm (pp. 149-160).
In order to investigate the role of the putative epimerase function of the β-oxidation multienzyme complex (FadBA) in the provision of (R)-3-hydroxyacyl-CoA thioesters for medium-chain-length polyhydroxyalkanoate (PHAMCL) biosynthesis, the fadBA Po operon of Pseudomonas oleovorans was cloned and characterized. The fadBA Po operon and a class-II PHA synthase gene of Pseudomonas aeruginosa were heterologously co-expressed in Escherichia coli to determine whether the putative epimerase function of FadBAPo has the ability to provide precursors for PHA accumulation in a non-PHA-accumulating bacterium. Cultivation studies with fatty acids as carbon source revealed that FadBAPo did not mediate PHAMCL biosynthesis in the E. coli wild-type strain harboring a PHA synthase gene. However, PHA accumulation was strongly impaired in a recombinant E. coli fadB mutant, which harbored a PHA synthase gene. These data indicate that in pseudomonads FadBA does not possess the inherent property, based on a putative epimerase function, to provide the (R)-enantiomer of 3-hydroxyacyl-CoA efficiently and that other linking enzymes are required to efficiently channel intermediates of β-oxidation towards PHAMCL biosynthesis. However, the phaJ gene from P. oleovorans and from Pseudomonas putida, both of which encoded a 3-Re enoyl-CoA hydratase, was identified. The co-expression of phaJ Po/Pp with either a class-II PHA synthase gene or the PHA synthase gene from Aeromonas punctata in E. coli revealed that PhaJPo/Pp mediated biosynthesis of either PHAMCL, contributing to about 1% of cellular dry mass, or of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), contributing to 3.6% of cellular dry mass, when grown on decanoate. These data indicate that FadBAPo does not mediate the provision of (R)-3-hydroxyacyl-CoA, which resembles FadBA of non-PHA-accumulating bacteria, and that 3-Re enoyl-CoA hydratases are required to divert intermediates of fatty acid β-oxidation towards PHA biosynthesis in P. oleovorans.
Keywords: Fatty acid β-Oxidation Multienzyme complex 3-Re enoyl-CoA hydratase Pseudomonads Polyhydroxyalkanoate biosynthesis Epimerase
No Title by Carlos Acosta-Durán; Esperanza Martínez-Romero (pp. 161-164).
The Rhizobium species that nodulate the legume tree Gliricidia sepium were analyzed by phenotypic characteristics (including nodule formation in different hosts), PCR-RFLP patterns and sequences of 16S rRNA genes, multilocus enzyme electrophoresis, and plasmid patterns. Strains of Rhizobium tropici type A and B, Sinorhizobium spp., and Rhizobium etli bv. phaseoli were encountered in G. sepium nodules and their presence depended on the site sampled.
Keywords: Rhizobium Symbiosis Legume trees Gliricidia sepium Rhizobium tropici