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Archives of Microbiology (v.176, #3)
No Title by Thomas Nyström (pp. 159-164).
A number of regulatory networks are functionally integrated in starving cells of Escherichia coli to reduce oxidation of target macromolecules and to enhance the cell's ability to withstand environmental insults. However, despite the fact that starving wild-type E. coli cells enhance their capacity to manage oxidative stress, the proteins of these cells become increasingly oxidized and the cells gradually lose their ability to reproduce. Indeed, it has been argued that starved and growth-arrested bacterial cells show the same signs of senescence as aging cells of higher organisms and that free radicals may be involved in the gradual loss of bacterial culturability observed in a stationary phase culture. Another model suggests that the apparent loss of viability of starved cells is a programmed and adaptive response in which the cells enter a reversible non-culturable state; the theory of the formation of viable but non-culturable cells. Recent data concerning the physiology and biochemistry of starved E. coli cells favor the model that starvation-induced loss of culturability is the result of stochastic deterioration rather than a programmed and adaptive phenomenon, and these data will be reviewed here.
Keywords: Escherichia coli Starvation Senescence Viable but non-culturable Protein oxidation Mistranslation
No Title by Bram A. van de Pas; Jan Gerritse; Willem M. de Vos; Gosse Schraa; Alfons J. Stams (pp. 165-169).
Desulfitobacterium strain PCE1 is able to use tetrachloroethene and chloroaromatics as terminal electron acceptors for growth. Cell extracts of Desulfitobacterium strain PCE1 grown with tetrachloroethene as electron acceptor showed no dehalogenase activity with 3-chloro-4-hydroxyphenylacetate (Cl-OH-phenylacetate) and other ortho-chlorophenolic compounds in an in vitro assay. Extracts of cells that were grown with Cl-OH-phenylacetate as electron acceptor dechlorinated tetrachloroethene at 10% of the dechlorination rate of Cl-OH-phenylacetate. In both cell extracts dechlorination was inhibited by the addition of 1-iodopropane and dinitrogen oxide, inhibitors of cobalamin-containing enzymes. The enzymes responsible for tetrachloroethene and Cl-OH-phenylacetate dechlorination were partially purified. A 100-fold enriched fraction of chlorophenol reductive dehalogenase was obtained that mainly contained a protein with a subunit size of 48 kDa. The characteristics of this enzyme are similar to that of the chlorophenol reductive dehalogenase of D. dehalogenans. After partial purification of the tetrachloroethene reductive dehalogenase, a fraction was obtained that also contained a 48-kDa protein, but the N-terminal sequence showed no similarity with that of the chlorophenol reductive dehalogenase sequence or with the N-terminal amino acid sequence of tetra- and trichloroethene reductive dehalogenase of Desulfitobacterium strain TCE1. These results provide strong evidence that two different enzymes are responsible for tetrachloroethene and chlorophenol dechlorination in Desulfitobacterium strain PCE1. Furthermore, the characterization of partially purified tetrachloroethene reductive dehalogenase indicated that this enzyme is a novel type of reductive dehalogenase.
Keywords: Anaerobic respiration Halorespiration Reductive dehalogenase Desulfitobacterium strain PCE1 Reductase Cobalamin Tetrachloroethene Chlorophenol
No Title by Dimitry Sorokin; Tat'jana Tourova; Markus C. Schmid; Michael Wagner; Hans-Peter Koops; Gijs J. Kuenen; Mike Jetten (pp. 170-177).
Five mixed samples prepared from the surface sediments of 20 north-east Mongolian soda lakes with total salt contents from 5 to 360 g/l and pH values from 9.7 to 10.5 were used to enrich for alkaliphilic ammonia-oxidizing bacteria. Successful enrichments at pH 10 were achieved on carbonate mineral medium containing 0.6 M total Na+ and ≤4 mM NH4Cl. Five isolates (ANs1–ANs5) of ammonia-oxidizing bacteria capable of growth at pH 10 were obtained from the colonies developed on bilayered gradient plates. The cells were motile and coccoid, with well-developed intracytoplasmic membranes (ICPM) and carboxysomes. At pH 10.0, ammonia was toxic for growth at concentrations higher than 5 mM NH4Cl. The bacteria were able to grow within the salinity range of 0.1–1.0 M of total Na+ (optimum 0.3 M). In media containing 0.3–0.6 M total Na+, optimal growth in batch cultures occurred in the presence of a bicarbonate/carbonate buffer system within the pH range 8.5–9.5, with the highest pH limit at pH 10.5. At pH lower than 8.0, growth was slower, most probably due to decreasing free ammonia. The pH profile of the respiratory activity was broader, with limits at 6.5–7.0 and 11.0 and an optimum at 9.5–10.0. In pH-controlled, NH3-limited continuous culture, isolate ANs5 grew up to pH 11.3, which is the highest pH limit known for ammonia-oxidizing bacteria so far. This showed the existence of extremely alkali-tolerant ammonia-oxidizing bacteria in the soda lakes. Comparative 16S rDNA sequence analysis of the five isolates demonstrated that they possess identical 16S rDNA genes and that they are closely related to Nitrosomonas halophila (sequence similarity 99.3%), a member of the β-subclass of the Proteobacteria. This affiliation was confirmed by comparative sequence analysis of the amoA gene, encoding the active-site subunit of the ammonia-monoxygenase, of one of the isolates. DNA-DNA hybridization data further supported that the soda lake isolates are very similar to each other and represent an alkali-tolerant subpopulation of N. halophila whose species description is herewith amended.
Keywords: Alkalitolerant Ammonia-oxidizing bacteria Nitrification Soda lakes Nitrosomonas
No Title by Jindřich Volc; Elena Kubátová; Geoffrey Daniel; Petr Sedmera; Dietmar Haltrich (pp. 178-186).
Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O2 as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml–1 in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K m=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.
Keywords: Pyranose dehydrogenase Pyranose oxidase 2-Dehydro-D-glucose 2-Keto-D-glucose 3-Dehydro-D-glucose 3-Keto-D-glucose Aldoketoses Macrolepiota rhacodes Basidiomycete fungi Ferricenium
No Title by Anja Wiese; Christoph Syldatk; Ralf Mattes; Josef Altenbuchner (pp. 187-196).
Arthrobacter aurescens DSM 3747 hydrolyzes stereospecifically 5′-monosubstituted hydantoins to α-amino acids. The genes involved in hydantoin utilization (hyu) were isolated on an 8.7-kb DNA fragment, and by DNA sequence analysis eight ORFs were identified. The hyu gene cluster includes four genes: hyuP encoding a putative transport protein, the hydantoin racemase gene hyuA, the hydantoinase gene hyuH, and the carbamoylase gene hyuC. The four genes are transcribed in the same direction. Upstream of hyuP and in opposite orientation to the hyu genes, three ORFs were found showing similarities to cytochrome P450 monooxygenase (ORF1, incomplete), to membrane proteins (ORF2), and to ferredoxin (ORF3). ORF8 was found downstream of hyuC and again in opposite orientation to the hyu genes. The gene product of ORF8 displayed similarities to the LacI/GalR family of transcriptional regulators. Reverse transcriptase PCR experiments and Northern blot analysis revealed that the genes hyuPAHC are coexpressed in A. aurescens after induction with 3-N-CH3-IMH. The expression of the hyu operon was not regulated by the putative regulator ORF8 as shown by gene disruption and mobility-shift experiments.
Keywords: hyu operon Amino acid production 5-Substituted hydantoin Hydantoin racemase Hydantoinase Carbamoylase RT-PCR
No Title by Anne Fjellbirkeland; Per G. Kruger; Vahid Bemanian; Bente T. Høgh; Colin J. Murrell; Harald B. Jensen (pp. 197-203).
A protein with an apparent molecular mass of 46 kDa was detected as the major polypeptide in the culture medium of the biotechnologically important methanotrophic bacterium Methylococcus capsulatus (Bath). The protein cross-reacted with polyclonal antibodies raised against the outer-membrane-associated protein MopE. The antiserum was used to identify a positive clone from a λgt11 library. The nucleotide sequence determined for the clone demonstrated that MopE and the secreted protein are encoded by the same gene, and that the secreted protein represents an N-terminally truncated form of MopE. By using monospecific antibodies against MopE in immunogold electron microscopy, the protein was localized at the cell surface and cell periphery. The mopE gene was expressed in Escherichia coli. The MopE protein synthesized was found in the periplasmic space of E. coli. No protein with sequence similarity over the entire length of MopE was detected in the databases, but some sequence similarity to the copper-repressible CorA protein of the methanotroph Methylomicrobium albus (Berson and Lidstrom 1997) was observed for the C-terminal region of MopE.
Keywords: Secreted protein Methanotroph Methylococcus capsulatus
No Title by Ulla Rasmussen; Mette M. Svenning (pp. 204-210).
The genetic diversity of ten symbiotic Nostoc strains isolated from different Gunnera species was investigated. The strains were analyzed using molecular methods with different taxonomic resolutions, including restriction fragment length polymorphisms (RFLP) of the PCR-amplified 16S ribosomal gene and the 16S-23S internal transcribed spacer (ITS) region combined with computer-assisted analyses. The functional gene hetR, assigned to heterocyst differentiation, was used for denaturing gradient gel electrophoresis. A high genetic diversity was observed among the isolates even in the conserved gene coding for the small ribosomal unit. No correlation was observed between clustering of cyanobacteria and the host species of Gunnera.
Keywords: Cyanobacteria Diversity Phylogeny Denaturing gradient gel electrophoresis (DGGE) Internal transcribed spacer (ITS) 16S rRNA RFLP
No Title by Gili Barel; Edouard Jurkevitch (pp. 211-216).
Host-independent (H-I) mutants of the obligate bacterial parasite Bdellovibrio bacteriovorus were isolated from wild-type strain 109J. Seven H-I mutants differed in morphological features such as cell length (2–30 µm) and shape (short or long spirals or rod-like), plaque size, and pigmentation (from almost colorless to bright orange). The mutants exhibited widely different growth capabilities in rich medium, with biomass doubling times and final biomass varying by a factor of two or more. Growth was always enhanced by the addition of host cell extract or divalent cations to the growth medium, but the effect varied widely between the mutants. Analysis of the hit region, mutations in which were previously proposed to be associated with the H-I phenotype, revealed that changes in the nucleotide sequence in this region occurred only in three of the seven mutants.
Keywords: Bdellovibrio Bacterial parasite Host-independent mutant hit
No Title by Philipp Krubasik; Shinichi Takaichi; Takashi Maoka; Miki Kobayashi; Kazumori Masamoto; Gerhard Sandmann (pp. 217-223).
Carotenogenic mutants of Corynebacterium glutamicum were analyzed for their carotenoid content. Mutant MV10 accumulated the same carotenoids as the wild-type, decaprenoxanthin, decaprenoxanthin monoglucoside, and (2R,6R,2′R,6′R)-decaprenoxanthin di-(β-D)-glucoside, but in three-fold higher amounts. In addition, decaprenoxanthin diglucoside fatty acid esters and the intermediates nonaprene, 2-(3-methyl-2-butenyl)-ε,ψ-carotene, and sarcinene, 2,2′-bis(3-methyl-2-butenyl)-ε,ε-carotene were identified as minor carotenoids. The pink mutants MV40 and MV60 synthesized only lycopene. From another pink mutant, MV70, novel C50-carotenoids were isolated. By NMR and mass spectroscopy, nonaflavuxanthin, 2-(4-hydroxy-3-methyl-2-butenyl)-1,16-didehydro-1,2-dihydro-ψ,ψ-carotene, and flavuxanthin, 2,2′-bis(4-hydroxy-3-methyl-2-butenyl)-1,16,1′,16′-tetradehydro-1,2,1′,2′-tetrahydro-ψ,ψ-carotene, were identified. The identification of these intermediates revealed the detailed pathway for the formation of decaprenoxanthin derivatives in Corynebacterium glutamicum.
Keywords: C50-carotenoid biosynthesis Corynebacterium glutamicum Carotenogenic mutant Decaprenoxanthin diglucoside fatty acid ester Novel C50-carotenoid intermediate
No Title by Ingo Janausch; Ok Kim; Gottfried Unden (pp. 224-230).
Quintuple mutants of Escherichia coli deficient in the C4-dicarboxylate carriers of aerobic and anaerobic metabolism (DctA, DcuA, DcuB, DcuC, and the DcuC homolog DcuD, or the citrate/succinate antiporter CitT) showed only poor growth on succinate (or other C4-dicarboxylates) under oxic conditions. At acidic pH (pH 6) the mutants regained aerobic growth on succinate, but not on fumarate. Succinate uptake by the mutants could not be saturated at physiological succinate concentrations (≤5 mM), in contrast to the wild-type, which had a K m for succinate of 50 µM and a V max of 35 U/g dry weight at pH 6. At high substrate concentrations, the mutants showed transport activities (32 U/g dry weight) comparable to that of the wild-type. In the wild-type using DctA as the carrier, succinate uptake had a pH optimum of 6, whereas succinate uptake in the mutants was maximal at pH 5. In the mutants succinate uptake was inhibited competitively by monocarboxylic acids. Diffusion of succinate or fumarate across phospholipid membranes (liposomes) was orders of magnitude slower than the transport in the wild-type or the mutants. The data suggest that mutants deficient in DctA, DcuA, DcuB, DcuC, DcuD (or CitT) contain a carrier, possibly a monocarboxylate carrier, which is able to transport succinate, but not fumarate, at acidic pH, when succinate is present as a monoanion. Succinate uptake by this carrier was inhibited by addition of an uncoupler. Growth by fumarate respiration (requiring fumarate/succinate antiport) was also lost in the quintuple mutants, and growth was not restored at pH 6. In contrast, the efflux of succinate produced during glucose fermentation was not affected in the mutants, demonstrating that, for succinate efflux, a carrier different from, or in addition to, the known Dcu and CitT carriers is used.
Keywords: C4-dicarboxylate transport DctA Dcu carrier Succinate Fumarate Escherichia coli
No Title by Mária Takács; Gábor Rákhely; Kornél L. Kovács (pp. 231-235).
The hypCD genes, encoding the counterparts of mesophilic proteins involved in the maturation of [NiFe] hydrogenases, were isolated from the hyperthermophilic archaeon Thermococcus litoralis. The deduced gene products showed 30–40% identity to the corresponding mesophilic proteins. HypC and HypD were synthesized by the T7 expression system. Heterologous complementation experiments were done in Escherichia coli and Ralstonia eutropha strains lacking functionally active hypC and hypD genes. Only the cytoplasmic hydrogenase of R. eutropha could be processed by HypD from T. litoralis. This was the first demonstration of mesophilic hydrogenase processing using a hyperthermophilic archaeal accessory protein to produce an active enzyme.
Keywords: Hyperthermophile Thermococcus litoralis Archaea [NiFe] hydrogenase Accessory genes hypC hypD Transcription initiation