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Archives of Microbiology (v.173, #3)
No Title by Kathleen Sandman; John Reeve (pp. 165-169).
A decade after the discovery of histones in Archaea, there is now also a biochemical description of the archaeal nucleosome. A tetrameric core of archaeal histones is encircled by ~80 bp of DNA, and nuclease digestions indicate that adjacent archaeal nucleosomes exist in vivo compacting archaeal genomic DNA. Most Eukarya employ a similar structure to organize their chromosomal DNA, the eukaryal nucleosome, with a histone octamer and 146 bp of DNA. Here we compare the properties of both nucleosomes in terms of DNA packaging and the accessibility of the packaged DNA for transcription.
Keywords: Histone Chromatin Nucleosome Genome compaction Chromosome structure Archaea
No Title by Caroline Blumer; Dieter Haas (pp. 170-177).
A few bacterial species are known to produce and excrete hydrogen cyanide (HCN), a potent inhibitor of cytochrome c oxidase and several other metalloenzymes. In the producer strains, HCN does not appear to have a role in primary metabolism and is generally considered a secondary metabolite. HCN synthase of proteobacteria (especially fluorescent pseudomonads) is a membrane-bound flavoenzyme that oxidizes glycine, producing HCN and CO2. The hcnABC structural genes of Pseudomonas fluorescens and P. aeruginosa have sequence similarities with genes encoding various amino acid dehydrogenases/oxidases, in particular with nopaline oxidase of Agrobacterium tumefaciens. Induction of the hcn genes of P. fluorescens by oxygen limitation requires the FNR-like transcriptional regulator ANR, an ANR recognition sequence in the –40 region of the hcn promoter, and nonlimiting amounts of iron. In addition, expression of the hcn genes depends on a regulatory cascade initiated by the GacS/GacA (global control) two-component system. This regulation, which is typical of secondary metabolism, manifests itself during the transition from exponential to stationary growth phase. Cyanide produced by P. fluorescens strain CHA0 has an ecological role in that this metabolite accounts for part of the biocontrol capacity of strain CHA0, which suppresses fungal diseases on plant roots. Cyanide can also be a ligand of hydrogenases in some anaerobic bacteria that have not been described as cyanogenic. However, in this case, as well as in other situations, the physiological function of cyanide is unknown.
Keywords: Hydrogen cyanide Hydrogen cyanide synthase Opine oxidase Anaerobic control Iron regulation Pseudomonas fluorescens Pseudomonas aeruginosa Chromobacterium violaceum Biocontrol Hydrogenase
No Title by Stella M. Trutko; Vasilii K. Akimenko; Nataliya E. Suzina; Lyudmila A. Anisimova; Mikhail G. Shlyapnikov; Boris P. Baskunov; Vitalii I. Duda; Aleksandr M. Boronin (pp. 178-186).
The terminal oxidases of the respiratory chain of seven strains of gram-negative bacteria were shown to be involved in the reduction of tellurite. The rate of tellurite reduction correlated with the intensity of respiration. The inhibitors of terminal oxidases, carbon monoxide and cyanide, inhibited the reduction of tellurite. In Pseudomonas aeruginosa PAO ML4262 and P. aeruginosa PAO ML4262 (pBS10), the respiratory chain was found to contain three types of cytochrome c, one of which (the carbon monoxide-binding cytochrome c) was involved in the reduction of tellurite. Agrobacterium tumefaciens VKM B-1219, P. aeruginosa IBPM B-13, and Escherichia coli G0-102bd++ cells contained oxidases aa 3, bb 3, and bd, respectively. The respiratory chain of other strains contained two oxidases: E. coli DH5α of bb 3- and bd-type, and Erwinia carotovora VKM B-567 of bo 3- and bd-type. All the strains under study reduced tellurite with the formation of tellurium crystallites. Depending on the position of the active center of terminal oxidases in the plasma membrane, the crystallites appeared either in the periplasmic space [P. aeruginosa PAO ML4262 and P. aeruginosa PAO ML4262 (pBS10)], or on the outer surface of the membrane (A. tumefaciens VKM B-1219 and P. aeruginosa IBPM B-13), its inner surface (E. coli G0-102bd ++), or on both surfaces (E. coli DH5α and E. carotovora VKM B-567).
Keywords: Bacteria Tellurite reduction Compartmentalization Tellurium crystallites R plasmids Respiratory chain Inhibitors Terminal oxidases Spectrophotometry Heme
No Title by Akihiro Hara; Mitsuyoshi Ueda; Satoru Misawa; Toru Matsui; Keizo Furuhashi; Atsuo Tanaka (pp. 187-192).
Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order to establish a selectable marker. The resistance gene for hygromycin B has often been used as a selectable marker in yeast transformation. However, C. tropicalis harboring the hygromycin resistance gene (HYG) was as sensitive to hygromycin B as the wild-type strain. Nine CTG codons were found in the ORF of the HYG gene. This codon has been reported to be translated as serine rather than leucine in Candida species. Analysis of the tRNA gene in C. tropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary to the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. In C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not functional due to the nonuniversal usage of the CTG codon. Each of the nine CTG codons in the ORF of the HYG gene was changed to a CTC codon, which is read as leucine, by site-directed mutagenesis. When a plasmid containing the mutated HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygromycin-resistant transformants were successfully obtained. This mutated hygromycin resistance gene may be useful for direct selection of C. tropicalis transformants.
Keywords: Candida tropicalis Hygromycin B Codon usage Site-directed mutagenesis
No Title by Hemalata M. Joshi; F. Robert Tabita (pp. 193-199).
A ribulose-1,5-bisphosphate carboxylase/oxygenase-deficient mutant strain (strain I-19) of Rhodospirillum rubrum was capable of growth under photoheterotrophic conditions in the absence of exogenous electron acceptors. These results suggested that alternative means of removing reducing equivalents have been acquired that allow this strain to remove reducing equivalents in the absence of a functional Calvin-Benson-Bassham reductive pentose phosphate pathway. Previously, the proton-reducing activity of the dinitrogenase complex was implicated in helping to maintain redox balance . However, since considerable amounts of CO2 were still fixed in this strain, the complete profile of enzymes involved in alternative CO2 fixation schemes was assessed. A specific and substantial induction of carbon monoxide dehydrogenase (CO dehydrogenase) synthesis was found in the mutant strain; although none of the other CO2 fixation pathways or enzyme activities were altered. These results suggested that CO dehydrogenase contributes to the photoheterotrophic success of strain I-19. Furthermore, the data implicate interacting and complex regulatory processes required to maintain the proper redox balance of this organism and other nonsulfur purple bacteria.
Keywords: Ribulose 1,5-bisphosphate carboxylase/oxygenase Carbon monoxide dehydrogenase Calvin-Benson-Bassham reductive pentose phosphate pathway CO2 fixation Regulation
No Title by Satoru Suga; Noriyuki Koyama (pp. 200-205).
Exiguobacterium aurantiacum BL77/1 possesses at least two distinct membrane-bound ATPases. One of them was solubilized with decanoyl N-methylglucamide, a non-ionic detergent, and purified by successive chromatography on DEAE-Sepharose and hydroxyapatite. The purified ATPase appears to consist of a single polypeptide component with an apparent molecular mass of 54 kDa. Among the triphosphates of various nucleosides tested, ATP was the best substrate. The enzyme exhibited a K m of 0.5 mM for ATP and a V max of 109 µmol ATP (mg protein)–1 min–1; the optimum pH for activity was near 6.5. The enzyme was sensitive to azide and inactivated by N,N'-dicyclohexylcarbodiimide. Analysis of the inhibition kinetics by N,N'-dicyclohexylcarbodiimide suggested that binding of the drug to a single carboxyl group per ATPase molecule is sufficient for inactivation.
Keywords: ATPase Azide sensitivity Alkaliphile
No Title by Wolfram Reichenbecher; Bodo Philipp; Marc J.-F. Suter; Bernhard Schink (pp. 206-212).
The recently isolated sulfate reducer Desulfovibrio inopinatus oxidizes hydroxyhydroquinone (1,2,4-trihydroxybenzene; HHQ) to 2 mol acetate and 2 mol CO2 (mol substrate)–1, with stoichiometric reduction of sulfate to sulfide. None of the key enzymes of fermentative HHQ degradation, i.e. HHQ-1,2,3,5-tetrahydroxybenzene transhydroxylase or phloroglucinol reductase, were detected in cell-free extracts of D. inopinatus, indicating that this bacterium uses a different pathway for anaerobic HHQ degradation. HHQ was reduced with NADH in cell-free extracts to a nonaromatic compound, which was identified as dihydrohydroxyhydroquinone by its retention time in HPLC separation and by HPLC-mass spectrometry. The compound was identical with the product of chemical reduction of HHQ with sodium borohydride. Dihydrohydroxyhydroquinone was converted stoichiometrically to acetate and to an unknown coproduct. HHQ reduction was an enzymatic activity which was present in the cell-free extract at 0.25–0.30 U (mg protein)–1, with a pH optimum at 7.5. The enzyme was sensitive to sodium chloride, potassium chloride, EDTA, and o-phenanthroline, and exhibited little sensitivity towards sulfhydryl group reagents, such as copper chloride or p-chloromercuribenzoate.
Keywords: Trihydroxybenzenes Hydroxyhydroquinone Phloroglucinol Reductive dearomatization Desulfovibrio sp.
No Title by Caterina Signoretto; Pietro Canepari (pp. 213-219).
Penicillin-binding proteins 5 (PBP5s) of enterococci are structurally and immunologically related proteins that are characterized by their low affinity for penicillin. For this reason, they are mainly involved in penicillin resistance, due essentially to their ability to take over the function of all other PBPs already bound and inhibited by the β-lactam. It has been demonstrated that penicillin resistance in enterococci is acquired either by overproduction of PBP5 or by the presence of specific amino acid sequences in the protein that further decrease the affinity for penicillin. In particular, a specific amino acid box (ANNGA) previously identified in Enterococcus faecium is responsible for the high penicillin resistance displayed by this species. Here, we describe the insertion of the PBP5 amino acid box ANNGA in Enterococcus faecalis, an enterococcal species usually more sensitive to penicillin, by site-directed mutagenesis. Mutagenized PBP5 was re-introduced into a pbp5 mutant of E. faecalis obtained by insertion of transposon Tn916. Data indicate that this amino acid box brings about no reduction in penicillin sensitivity in the recipient E. faecalis strain, but, paradoxically, dramatically lowers the penicillin minimal inhibitory concentration caused by the native PBP5. We deduce that, although enterococcal PBP5s are a family of closely related proteins as far as biological function is concerned, differences exist in their three-dimensional structure that affect penicillin affinity.
Keywords: Penicillin-binding proteins β-Lactam resistance Site-directed mutagenesis Enterococci
No Title by Christina Herzberg; Alexandra Friedrich; Beate Averhoff (pp. 220-228).
Here we describe five tandemly arranged and converging ORFs in Acinetobacter sp. BD413, namely lytB, orfY, orfX, comB, and orfZ, located upstream of the previously identified competence gene comC. The N-termini of the deduced proteins OrfY and ComB exhibit the conserved endopeptidase cleavage motifs of prepilin proteins; the deduced protein ComB is similar to type IV pilins. LytB is similar to the Escherichia coli LytB, which has been implicated in the stringent response. No homologues of OrfX, OrfY and OrfZ could be identified. A mutation in orfY or orfZ led to 100-fold reduced transformation frequencies and a mutation in comB resulted in a noncompetent phenotype. Disruption of lytB did not affect the natural transformation phenotype. Complementation studies clearly demonstrated that comB is involved in natural transformation, whereas the transformation-deficient phenotypes of orfY and orfZ mutants were due to polar effects on comB and comC, respectively. Analyses of the twitching motility phenotype and of the ultrastructure of the noncompetent comB mutant suggested that the competence gene comB is not essential for the biogenesis of type IV pili and expression of the type IV pili-associated property of twitching motility. Transcriptional fusions between comB and a promoter-free lacZ gene were constructed, and analysis of growth-phase-dependent transcription revealed increased expression of comB during prolonged exponential and stationary phases.
Keywords: Acinetobacter sp. BD413 Competence Natural transformation ComB Pilin-like competence factor
No Title by Sonia M. Cavaignac; Stefan J. White; Geoffrey W. de Lisle; Desmond M. Collins (pp. 229-231).
Mycobacterium paratuberculosis causes Johne's disease, a common wasting disease in ruminants. As a first step in studying virulence mechanisms, libraries of random mutants were produced in two M. paratuberculosis strains by using the conditionally replicating shuttle phasmid phAE94 which contains the transposon Tn5367. Two thousand mutants were screened for auxotrophy, carbon source preference, and altered cell wall. Genes interrupted by insertion were identified for seven mutants isolated from the screening process. Two mutants had insertions in putative genes involved in synthesis of the cell wall.
Keywords: Mycobacterium paratuberculosis Paratuberculosis Transposon mutagenesis Congo red Auxotrophy Carbon source Johne's disease