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Archives of Microbiology (v.175, #4)
No Title by Andrew S. Lang; J. Thomas Beatty (pp. 241-249).
Transduction, bacteriophage-mediated gene transfer, is thought to play an important role in the evolution of prokaryote genomes. Several gene transfer agents that resemble transducing phages have been found in diverse prokaryotes. This mini-review discusses these interesting agents of genetic exchange with a focus on the gene transfer agent (GTA) of Rhodobacter capsulatus, at present the only member of this group for which genetic information exists about the production of transducing particles. Production of GTA results from expression of genes that are similar to phage genes, yet transcription of these genes is dependent upon cellular (two-component) signaling proteins. The significance of these relationships, as well as the finding of GTA gene homologues in the bacterium Rhodopseudomonas palustris, is discussed.
Keywords: GTA Gene transfer Transduction Rhodobacter capsulatus ctrA cckA
No Title by Karl-Heinz van Pée (pp. 250-258).
Halometabolites are compounds that are commonly found in nature and they are produced by many different organisms. Whereas bromometabolites can mainly be found in the marine environment, chlorometabolites are predominately produced by terrestrial organisms; iodo- and fluorocompounds are only produced infrequently. The halogen atoms are incorporated into organic compounds by enzyme-catalyzed reactions with halide ions as the halogen source. For over 40 years haloperoxidases were thought to be responsible for the incorporation of halogen atoms into organic molecules. However, haloperoxidases lack substrate specificity and regioselectivity, and the connection of haloperoxidases with the in vivo formation of halometabolites has never been demonstrated. Recently, molecular genetic investigations showed that, at least in bacteria, a different class of halogenases is involved in halometabolite formation. These halogenases were found to require FADH2, which can be produced from FAD and NADH by unspecific flavin reductases. In addition to FADH2, oxygen and halide ions (chloride and bromide) are necessary for the halogenation reaction. The FADH2-dependent halogenases show substrate specificity and regioselectivity, and their genes have been detected in many halometabolite-producing bacteria, suggesting that this type of halogenating enzymes constitutes the major source for halometabolite formation in bacteria and possibly also in other organisms.
Keywords: Halometabolite Halogenase Chloroperoxidase Antibiotic Pyrrolnitrin Vancomycin Flavin reductase Pyoluteorin Chlorination Bromination
No Title by J. E. Dugas; L. Zurek; B. J. Paster; B. A. Keddie; E. R. Leadbetter (pp. 259-262).
A 16S rDNA sequence cloned directly from whole-gut microbiota of the American cockroach, Periplaneta americana, indicated the presence of a member of the Bacteroides/Flavobacterium group most closely related to the genus Flavobacterium. In an attempt to confirm this finding, we isolated a yellow-pigmented bacterium (strain FR2) from the hindgut of this insect. Strain FR2 was phylogentically and phenotypically most similar to species of Flavobacterium and related bacteria, namely Chryseobacterium indologenes. Fifty-four other yellow-pigmented bacteria isolated during a 1-year study shared the salient phenotypic characteristics of Chryseobacterium spp., and thus were considered the same phenotype. This phenotype's abundance was related to the fiber content of the insect diet, being consistently detected only in cockroaches fed a high-fiber diet (30% crude fiber by weight). The highest population density was in the hindgut, ranging from 2×106 to 1.2×107 colony forming units ml–1 during a 1-year period. The nature of the symbiosis between the FR2 phenotype and P. americana is discussed.
Keywords: Flavobacteria Chryseobacterium Flexirubin Fumarate respiration Gut American cockroach
No Title by Nikhil A. Thomas; Edward D. Chao; Ken F. Jarrell (pp. 263-269).
Archaeal flagellins are made initially as preproteins with short, positively charged leader peptides. Analysis of all available archaeal preflagellin sequences indicates that the –1 position is always held by a glycine while the –2 and –3 positions are almost always held by charged amino acids. To evaluate the importance of these and other amino acids in the leader peptides of archaeal flagellins for processing by a peptidase, Methanococcus voltae mutant FlaB2 preflagellin genes were generated by PCR and the proteins tested in a methanogen preflagellin peptidase assay that detects the removal of the leader peptide from preflagellin. When the –1 position was changed from glycine to other amino acids tested, no cleavage was observed by the peptidase, with the exception of a change to alanine at which poor, partial processing was observed. Amino acid substitutions at the –2 lysine position resulted in a complete loss of processing by the peptidase, while changes at the –3 lysine resulted in partial processing. A mutant preflagellin with a leader peptide shortened from 12 amino acids to 6 amino acids was not processed. When the invariant glycine residue present at position +3 was changed to a valine, no processing of this mutant preflagellin was observed. The identification of critical amino acids in FlaB2 required for proper processing suggests that a specific preflagellin peptidase may cleave archaeal flagellins by recognition of a conserved sequence of amino acids.
Keywords: Archaea Methanococcus voltae Flagellin Flagella Peptidase Leader peptide Mutants
No Title by Oliver Pelz; Antonis Chatzinotas; Nils Andersen; Stefano M. Bernasconi; Christian Hesse; Wolf-Rainer Abraham; Josef Zeyer (pp. 270-281).
Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubated under anoxic denitrifying conditions with [methyl-13C]toluene. These microcosms served as a laboratory model system to evaluate the combination of isotope (13C-labeling of polar-lipid-derived fatty acids) and molecular techniques (16S rRNA-targeting gene probes) to identify the toluene-metabolizing population. After total depletion of toluene, the following bacterial phospholipid fatty acids (PLFA) were 13C-enriched: 16:1ω7c, 16:1ω7t, 16:0, cy17:0, and 18:1ω7c. Pure culture experiments demonstrated that these compounds were also found in PLFA profiles of PHC-degrading Azoarcus spp. (β-Proteobacteria) and related species. The origin of the CO2 evolved in the microcosms was determined by measurements of stable carbon isotope ratios. Toluene represented 11% of the total pool of mineralized substrates in the contaminated sediment and 54% in the pristine sediment. The microbial community in the microcosm incubations was characterized by using DAPI staining and whole-cell hybridization with specific fluorescently labeled 16S rRNA-targeted oligonucleotide probes. Results revealed that 6% of the DAPI-stained cells in the contaminated sediment and 32% in the pristine sediment were PHC-degrading Azoarcus spp. In biotic control microcosms (incubated under denitrifying conditions, no toluene added), Azoarcus spp. cells remained at less than 1% of the DAPI-stained cells. The results show that isotope analysis in combination with whole-cell hybridization is a promising approach to identify and to quantify denitrifying toluene degraders within microbial communities.
Keywords: Petroleum-hydrocarbon-contaminated aquifer Denitrifying β-Proteobacteria Azoarcus Toluene Stable isotopes 13C-labeling Polar lipid fatty acids (PLFA) Whole-cell hybridization
No Title by Axel Niebisch; Michael Bott (pp. 282-294).
In this work, the genes for cytochrome aa 3 oxidase and the cytochrome bc 1 complex in the gram-positive soil bacterium Corynebacterium glutamicum were identified. The monocistronic ctaD gene encoded a 65-kDa protein with all features typical for subunit I of cytochrome aa 3 oxidases. A ctaD deletion mutant lacked the characteristic 600 nm peak in redox difference spectra, and growth in glucose minimal medium was strongly impaired. The genes encoding subunit III of cytochrome aa 3 (ctaE) and the three characteristic subunits of the cytochrome bc 1 complex (qcrABC) were clustered in the order ctaE-qcrCAB. Analysis of the deduced primary structures revealed a number of unusual features: (1) cytochrome c 1 (QcrC, 30 kDa) contained two Cys-X-X-Cys-His motifs for covalent heme attachment, indicating that it is a di-heme c-type cytochrome; (2) the 'Rieske' iron-sulphur protein (QcrA, 45 kDa) contained three putative transmembrane helices in the N-terminal region rather than only one; and (3) cytochrome b (QcrB, 60 kDa) contained, in addition to the conserved part with eight transmembrane helices, a C-terminal extension of about 120 amino acids, which presumably is located in the cytoplasm. Staining of C. glutamicum proteins for covalently bound heme indicated the presence of a single, membrane-bound c-type cytochrome with an apparent molecular mass of about 31 kDa. Since this protein was missing in a qcrCAB deletion mutant, it most likely corresponds to cytochrome c 1. Similar to the ΔctaD mutant, the ΔqcrCAB mutant showed strongly impaired growth in glucose minimal medium, which indicates that the bc 1-aa 3 pathway is the main route of respiration under these conditions.
Keywords: Corynebacterium glutamicum c-Type cytochromes Cytochrome aa3 oxidase Cytochrome bc1 complex Respiratory chain
No Title by Antje Sturmfels; Friedrich Götz; Andreas Peschel (pp. 295-300).
Staphylococcal exoproteins can be divided into two groups. One group comprises proteins bearing only a signal peptide, the other group requires an additional propeptide for secretion. The secretion signals of the propeptide-requiring lipase from Staphylococcus hyicus (Lip) have been frequently used to produce recombinant secretory proteins in the food-grade species Staphylococcus carnosus. However, it has been unclear whether recombinant proteins can be secreted using signal peptides of staphylococcal proteins without propeptide. The human growth hormone protein (hGH) was fused to various staphylococcal secretion signals of proteins without propeptide (Seb, SceA, and SceB) and of proteins requiring a propeptide (lipase, lysostaphin, and glycerol ester hydrolase). Secretory hGH was efficiently produced by S. carnosus after fusion with any propeptide-containing secretion signal, whereas precursor proteins were retained in the cells when only a signal peptide was used. Addition of the first six amino acid residues of mature SceA to the signal peptide did also not lead to secretion of hGH. It was concluded that the properties of the mature protein domains determine whether a propeptide is required for secretion or not. The Lip propeptide could be efficiently removed from hGH after introduction of an enterokinase cleavage site between the two protein domains.
Keywords: Staphylococcus carnosus Staphylococcus aureus Exoprotein Propeptide Human growth hormone hGH Protein secretion Recombinant protein Lipase Lysostaphin
No Title by M. Guadalupe Cabral; Cristina A. Viegas; Isabel Sá-Correia (pp. 301-307).
Acquisition of resistance to lethal concentrations of octanoic acid was induced in cells of Saccharomyces cerevisiae grown in the presence of sublethal concentrations of this lipophilic acid or following rapid exposure (1 h) of unadapted yeast cells to mild stress imposed by the same acid. Experimental evidence indicated that the referred adaptation involved de novo protein synthesis, presumably due to the rapid induction of a plasma membrane transporter which mediates the active efflux of octanoate out of the cell. Rapid exposure of cells to mild ethanol stress also led to increased resistance to lethal concentrations of octanoic acid. This cross-resistance to octanoic-acid-induced death was below the level of resistance induced by mild octanoic acid stress and did not involve induction of the active expulsion of octanoate out of the cell. However, the rapid exposure of yeast cells to octanoic acid or ethanol led to the activation of plasma membrane H+-ATPase. The physiological role of the two stress responses examined during the present study, namely, the active efflux of octanoate specifically induced by octanoic acid and the stimulation of plasma membrane H+-ATPase activity, is discussed.
Keywords: Saccharomyces cerevisiae Octanoic acid Ethanol Acquisition of octanoic acid resistance Cross-protection Octanoate efflux Plasma membrane H+-ATPase
No Title by Ron Ronimus; Edwin de Heus; Andreas Rückert; Hugh Morgan (pp. 308-312).
The full-length gene encoding a 554-amino-acid, active pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila was cloned and sequenced using a combination of degenerate and inverse PCR, and the enzyme expressed to a high level in Escherichia coli. The recombinant enzyme, with a calculated molecular mass of 61 kDa, was purified to near homogeneity and found to be similar to the purified native enzyme for most properties examined. Phylogenetic analysis demonstrated a close relationship between the thermophilic S. thermophila phosphofructokinase and the large β-subunits of the phosphofructokinases from Borrelia burgdorferi and Treponema pallidum.
Keywords: Phosphofructokinase Spirochete Treponema Borrelia Spirochaeta thermophila Pyrophosphate