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Archives of Microbiology (v.185, #4)
ISPa20 advances the individual evolution of Pseudomonas aeruginosa clone C subclone C13 strains isolated from cystic fibrosis patients by insertional mutagenesis and genomic rearrangements by Andreas U. Kresse; Helmut Blöcker; Ute Römling (pp. 245-254).
Pseudomonas aeruginosa clone C strains, which chronically colonize the lungs of cystic fibrosis patients reorganize their genome structure. In this study, a novel member of the IS3 subfamily of IS elements, ISPa20, was detected which was specific for clone C subclone C13 strains. ISPa20, which was present in high copy number, mediated events of genomic reorganization. ISPa20 was inserted into P. aeruginosa backbone genes leading to adaptation to the cystic fibrosis lung habitat and into DNA acquired through horizontal gene transfer. Further on, large chromosomal inversions were mediated by ISPa20. In contrast to strains of other subclonal linages high rates of genomic rearrangements of subclone C13 strains were observed in vitro. The acquisition of mobile elements by P. aeruginosa clone C strains in the lungs of cystic fibrosis patients supports the chronic colonization by insertional mutagenesis and chromosome restructuring leading to microevolution within clone C that reflects macroevolution observed on the species level.
Keywords: Clone C; Cystic fibrosis; IS element; Large chromosomal inversion; Pseudomonas aeruginosa
Inactivation of the putative tetracycline resistance gene HP1165 in Helicobacter pylori led to loss of inducible tetracycline resistance by Yuhuan Li; H. Kathleen Dannelly (pp. 255-262).
Tetracycline has been used with other antibiotics in treatment of Helicobacter pylori infection. However, tetracycline resistance has developed in H. pylori clinical isolates, rendering treatment failure. Mutations in 16S rRNA genes have been reported to mediate tetracycline resistance in some isolates. The diversity of tetracycline resistance cases suggests multiple genes are involved. HP1165, a putative tetracycline resistance gene in H. pylori 26695, displays 49.8% identity to the tetracycline efflux gene tetA (P) from Clostridium perfringens. To determine the function of the HP1165 gene in H. pylori, the tetracycline resistance phenotype was investigated, transcription of HP1165 was examined by RT-PCR, and a ΔHP1165 mutant was generated by insertion of the pBCα3 plasmid. The results showed that strains harboring HP1165 were induced to intermediate level resistance in the laboratory (minimum inhibitory concentration=4–6 μg/ml). No mutation was found at or near the tetracycline binding sites of the 16S rRNA gene. The gene was transcribed both in the induced tetracycline resistant and wild type strains, indicating translational or posttranslational control of gene function. Mutation of HP1165 gene resulted in increased tetracycline susceptibility and loss of inducible tetracycline resistance, suggesting that the HP1165 gene is involved in the inducible tetracycline resistance in H. pylori.
Keywords: Tetracycline resistance; Inducible tetracycline resistance; Helicobacter pylori ; HP1165
Mutagenesis of conserved charged amino acids in SLH domains of Thermoanaerobacterium thermosulfurigenes EM1 affects attachment to cell wall sacculi by Antje May; Tünde Pusztahelyi; Nadine Hoffmann; Ralf-Jörg Fischer; Hubert Bahl (pp. 263-269).
SLH domains (for surface layer homology) are involved in the attachment of proteins to bacterial cell walls. The data presented here assign the conserved TRAE motif within SLH domains a key role for the binding. The charged amino acids arginine (R) or/and glutamic acid (E) were replaced via site-directed mutagenesis by different amino acids. Effects were visualized in an in vitro binding assay using native cell wall sacculi of Thermoanaerobacterium thermosulfurigenes EM1 and different variants of an SLH protein which consisted of the triplicate SLH domain of xylanase XynA of this bacterium and which was purified after expression in Escherichia coli. The results indicated (1) that the TRAE motif is critical for the binding function of SLH domains, (2) that a functional TRAE motif is necessary in all three domains, (3) that a least one (preferentially positively) charged amino acid in the TRAE motif is required for the functionality of the SLH domain, and (4) that the position of the negatively and positively charged amino acids is important. The finding that the cell wall of T. thermosulfurigenes EM1 contains pyruvate (4 μg mg−1) is in agreement with the hypothesis that pyruvylated secondary cell wall polymers function as ligand for SLH domains.
Keywords: Gram-positive; S-layer (surface layer) homologous domain; S-layer protein; Cell wall polymer; Extracellular enzymes
The rubrerythrin-like protein Hsp21 of Clostridium acetobutylicum is a general stress protein by Falk Hillmann; Ralf-Jörg Fischer; Hubert Bahl (pp. 270-276).
The small heat shock protein Hsp21 of Clostridium acetobutylicum was recently identified as a rubrerythrin-like protein with a rubredoxin-like FeS4 domain at the N-terminus and a ferritin-like diiron domain at the C-terminus. Here, we report that the two identical tandem genes rbr3A and rbr3B, which encode the heat shock protein Hsp21, show the transcription pattern of general stress genes. Northern blot analysis indicated that the transcription of the rbr3AB operon is induced by various environmental stress conditions: in addition to heat and oxidative stress, an increase of the pH of the growth medium from 4.5 to 6.2, addition of the salt NaCl (400 mM) or of the solvent butanol (3.5% v/v), and lowering the incubation temperature from 37 to 25°C resulted in transiently increased transcript levels. The promoter region deduced from the 5′ end of the mRNA has only limited similarity to the consensus promoter sequence of Gram-positive bacteria. A conserved inverted repeat between this promoter and the initiation codon is proposed to have a regulatory role. Although C. acetobutylicum is regarded as a strictly anaerobic bacterium, live/dead staining demonstrated that it can survive exposure to air or H2O2 and other stressors to various extents.
Keywords: General stress; Heat shock; Anaerobic; Clostridium ; Flavoprotein
FTIR spectroscopy as a potential tool to analyse structural modifications during morphogenesis of Candida albicans by Isabelle Adt; Dominique Toubas; Jean-Michel Pinon; Michel Manfait; Ganesh D. Sockalingum (pp. 277-285).
Candida albicans is a polymorphic organism that grows under certain conditions as blastospores, hyphae or pseudohyphae. The potentials of FTIR spectroscopy for assessing structural differences in C. albicans blastospores and hyphae were investigated. The main observed differences were localised in the polysaccharide (950–1,185 cm−1), protein (1,480–1,720 cm−1), and the fatty acids (2,840–3,000 cm−1) regions. Quantitative evaluation of differences between hyphae and blastospores by curve-fitting of these regions indicate that these modifications could be due to both changes in structure and content of components of the cell wall such as β-glucans, mannoproteins, and lipids. Furthermore, glycogen consumption could be involved during hyphae elongation. Thus, FTIR spectroscopy can be an interesting tool to investigate differences in structure and in content between blastospores and hyphae. We also demonstrate through this study that differentiation of C. albicans clinical strains using hyphae is feasible, as this has been previously shown with blastospores. This preliminary work on identification of C. albicans using hyphae is a prelude to a larger clinical study for early typing within 7 h from a pure culture.
Keywords: C . albicans ; Hyphae; FTIR Spectroscopy; Structural analysis; Discrimination
Carbon substrate utilization, antibiotic sensitivity, and numerical taxonomy of bacterial isolates from the Great Salt Plains of Oklahoma by Brandon R. Litzner; Todd M. Caton; Mark A. Schneegurt (pp. 286-296).
The current work extends the phenotypic characterization of a bacterial culture collection from the Great Salt Plains of Oklahoma. This barren expanse of mud flats is typically crusted with thalassohaline salt evaporites. The initial account of the aerobic heterotrophic bacteria from the Great Salt Plains described 105 halotolerant isolates that represented 47 phylotypes. Extensive phenotypic analyses were performed on 76 isolates representing 37 unique phylotypes. The current report extends these observations for 60 of the isolates by measuring a wider set of phenotypic characteristics. Utilization patterns for 45 carbon substrates were used to assign the isolates into seven coherent phenons, along with several singletons and a group of isolates that did not grow on single carbon substrates. Most of the isolates were able to utilize nearly all of the nitrogen sources tested, with nitrate being the least utilized. Little antibiotic resistance was seen in the collection as a whole; however, certain phenons were enriched for antibiotic-resistant organisms. A total of 81 phenotypic characteristics were used to generate dendrograms. The numerical taxonomy trees essentially agreed with those generated using 16S rRNA gene sequences. The pattern of carbon substrate utilization showed substantial changes at different salinities that may have relevance to the variable salinities microbes experience at the Salt Plains over time.
3-Methylglutaconyl-CoA hydratase from Acinetobacter sp by Matthias Mack; Michael Liesert; Johannes Zschocke; Verena Peters; Dietmar Linder; Wolfgang Buckel (pp. 297-306).
Acinetobacter strain IVS-B aerobically grows on isovalerate as sole carbon and energy source. Isovalerate is metabolised via isovaleryl-CoA, an intermediate of the oxidative (S)-leucine degradation pathway. A 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18) was purified 65-fold to apparent homogeneity from cell-free extracts of isovalerate-grown cells of Acinetobacter strain IVS-B. The enzyme was found to be a homotetramer (115.2 kDa) composed of four identical subunits of 28.8 kDa not containing any cofactors. The enzyme was shown to catalyse the hydration of (E)-glutaconyl-CoA (k cat=18 s−1, K m=40 μM) and the dehydration of (S)-3-hydroxyglutaryl-CoA (k cat=13 s−1, K m=52 μM), albeit with somewhat lower catalytic efficiencies as compared to the 3-methyl derivatives, 3-methylglutaconyl-CoA (k cat=138 s−1, K m=14 μM) and (S)-3-hydroxy-3-methylglutaryl-CoA (k cat=60 s−1, K m=36 μM). Thus, the mechanistically simple syn-addition of water to the (E)-isomer of 3-methylglutaconyl-CoA of the leucine degradative pathway leading to the common intermediate (S)-3-hydroxy-3-methylglutaryl-CoA was assigned as the major physiological role to this enzyme. The amino acid sequence of 3-methylglutaconyl-CoA hydratase from Acinetobacter sp. was found to be related to over 100 prokaryotic enoyl-CoA hydratases (up to 50% identity), possibly all being 3-methylglutaconyl-CoA hydratases.
Keywords: Hydration/dehydration reactions; (S)-Leucine degradation; 3-Methylglutaconyl-CoA hydratase; Acinetobacter sp.
Wood stimulates the demethoxylation of [O14CH3]-labeled lignin model compounds by the white-rot fungi Phanerochaete chrysosporium and Phlebia radiata by Outi Niemenmaa; Antti Uusi-Rauva; Annele Hatakka (pp. 307-315).
Mineralization of polymeric wood lignin and its substructures is a result of complex reactions involving oxidizing and reducing enzymes and radicals. The degradation of methoxyl groups is an essential part of this process. The presence of wood greatly stimulates the demethoxylation of a non-phenolic lignin model compound (a [O14CH3]-labeled β-O-4 dimer) by the lignin-degrading white-rot fungi Phlebia radiata and Phanerochaete chrysosporium. When grown on wood, both fungi produced up to 47 and 40% 14CO2 of the applied 14C activity, respectively, under air and oxygen in 8 weeks. Without wood, the demethoxylation of the dimer by both fungi was lower, varying between 0.5 and 35%. Addition of nutrient nitrogen together with glucose decreased demethoxylation when the fungi were grown on spruce wood under air. Because the evolution of 14CO2 in the absence of wood was poor, the fungi may have preferably used wood as a carbon and nitrogen source. The amount of fungal mycelium, as determined by the ergosterol assay, did not show connection to demethoxylation. P. radiata also showed a high demethoxylation of [O14CH3]-labeled vanillic acid in the presence of birch wood. The degradation of lignin and lignin-related substances should be studied in the presence of wood, the natural substrate for white-rot fungi.
Keywords: White-rot fungi; Phanerochaete chrysosporium ; Phlebia radiata ; Demethoxylation; Lignin-related dimer; Vanillic acid; Wood; Ergosterol
WdChs1p, a class II chitin synthase, is more responsible than WdChs2p (Class I) for normal yeast reproductive growth in the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis by Li Zheng; Leonel Mendoza; Zheng Wang; Hongbo Liu; Changwon Park; Sarah Kauffman; Jeffrey M. Becker; Paul J. Szaniszlo (pp. 316-329).
The chitin synthase gene WdCHS1 was isolated from a partial genomic DNA library of the pathogenic polymorphic fungus Wangiella dermatitidis. Sequencing showed that WdCHS1 encoded a class II chitin synthase composed of 988 amino acids. Disruption of WdCHS1 produced strains that were hyperpigmented in rich media, grew as yeast at wild-type rates at both 25 and 37°C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Δ mutants showed that yeast cells often failed to separate, tended to be enriched with chitin in septal regions and, sometimes, were enlarged with multiple nuclei, had broader mother cell–daughter bud regions and had other cell wall defects seen considerably less often than in the wild type or wdchs2Δ strains. Disruption of WdCHS1 and WdCHS2 in the same background revealed that WdChs1p had functions synergistic to those of WdChs2p, because mutants devoid of both isozymes produced growth that was very abnormal at 25°C and was not viable at 37°C unless osmotically stabilized. These results suggested that WdChs1p was more responsible than WdChs2p for normal yeast cell reproductive growth because strains with defects in the latter exhibited no morphological abnormalities, whereas those with defects in WdChs1p were frequently impaired in one or more yeast developmental processes.