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Archives of Microbiology (v.173, #1)

Nickel transport systems in microorganisms by T. Eitinger; M. -A. Mandrand-Berthelot (pp. 1-9).

The transition metal Ni is an essential cofactor for a number of enzymatic reactions in both prokaryotes and eukaryotes. Molecular analyses have revealed the existence of two major types of high-affinity Ni²⁺ transporters in bacteria. The Nik system of Escherichia coli is a member of the ABC transporter family and provides Ni²⁺ ion for the anaerobic biosynthesis of hydrogenases. The periplasmic binding protein of the transporter, NikA, is likely to play a dual role. It acts as the primary binder in the uptake process and is also involved in negative chemotaxis to escape Ni overload. Expression of the nik operon is controlled by the Ni-responsive repressor NikR, which shows functional similarity to the ferric ion uptake regulator Fur. The second type of Ni²⁺ transporter is represented by HoxN of Ralstonia eutropha, the prototype of a novel family of transition metal permeases. Members of this family have been identified in gram-negative and gram-positive bacteria and recently also in a fission yeast. They transport Ni²⁺ with very high affinity, but differ with regard to specificity. Site-directed mutagenesis experiments have identified residues that are essential for transport. Besides these uptake systems, different types of metal export systems, which prevent microorganisms from the toxic effects of Ni²⁺ at elevated intracellular concentrations, have also been described.

Keywords: Key words Metal ion transport; ABC transporter; Permease family; Metalloregulatory protein; Nickel-containing enzymes; Metal resistance

Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae by H. Salimnia; A. Radia; S. Bernatchez; T. J. Beveridge; J. R. Dillon (pp. 10-20).

We cloned the cell division gene ftsZ of the gram-negative coccus Neisseria gonorrhoeae (Ng) strain CH811, characterized it genetically and phenotypically, and studied its localization in N. gonorrhoeae and Escherichia coli (Ec). The 1,179-bp ORF of ftsZ _Ng encodes a protein with a predicted molecular mass of 41.5 kDa. Protein sequence alignments indicate that FtsZ_Ng is similar to other FtsZ proteins and contains the conserved GTP binding motif. FtsZ homologues were identified in several N. gonorrhoeae strains and in Neisseria lactamica, Neisseria sicca, Neisseria polysaccharae and Neisseria cinerea either by Western blot or by PCR-Southern blot analysis. Attempts to inactivate the ftsZ _Ng on the chromosome failed, indicating that it is essential for gonococcal growth. FtsZ_Ng was synthesized in an in vitro transcription/translation system and was shown to be 43 kDa, the same size as in Western blots. Expression of the ftsZ _Ng gene from nongonococcal promoters resulted in a filamentous phenotype in E. coli. Under controlled expression, the FtsZ_Ng-GFP fusion protein localized at the mid-cell division site in E. coli. E. coli expressing high levels of the FtsZ_Ng-GFP fusion protein formed filaments and exhibited different fluorescent structures including helices, spiral tubules extending from pole to pole, and regularly spaced dots or bands that did not localize ¶at the middle of the cell. Expression of the FtsZ_Ng-GFP fusion protein in N. gonorrhoeae resulted in abnormal cell division as shown by electron microscopy. FtsZ_Ng-GFP fusions were also expressed in a gonococcal background using a unique shuttle vector.

Keywords: Key words Bacteria; Cloning; FtsZ; Cell division; ¶Neisseria gonorrhoeae; Localization; Expression; Green fluorescent protein

P45, an extracellular 45 kDa protein of Listeria monocytogenes with similarity to protein p60 and exhibiting peptidoglycan lytic activity by K. Schubert; A. M. Bichlmaier; E. Mager; K. Wolff; G. Ruhland; F. Fiedler (pp. 21-28).

A monoclonal antibody obtained by immunization of mice with heat-killed cells of Listeria monocytogenes serotype 4d showed reactivity towards a protein (P45) from L. monocytogenes with an apparent molecular mass of 45 kDa. This protein was detected in the culture supernatant and at the cell surface of L. monocytogenes. Proteins cross-reacting with the monoclonal antibody were present in all Listeria strains investigated, except L. grayi. The structural gene was cloned in Escherichia coli and sequenced. Translation of the gene starts at a TTG initiation codon. The gene was found to code for a protein of 402 amino acid residues with a predicted molecular mass of 42.7 kDa. It has a signal peptide of 27 amino acid residues, resulting in a molecular mass for the mature polypeptide of 39.9 kDa. Protein database searches showed that this protein has 55% similarity and 38% identity to protein p60 of L. monocytogenes and exhibits significant sequence similarities to p54 from Enterococcus faecium and Usp45 from Lactococcus lactis. P45 was shown to have peptidoglycan lytic activity and the encoding gene was named spl (secreted protein with lytic property).

Keywords: Key wordsListeria monocytogenes; Secreted protein; P45; spl gene

Primary structure of cytochrome c′ of Methylococcus capsulatus Bath: evidence of a phylogenetic link between P460 and c′-type cytochromes by D. J. Bergmann; J. A. Zahn; A. A. DiSpirito (pp. 29-34).

Cytochrome c′ of Methylococcus capsulatus Bath is involved in electron flow from the enzyme responsible for hydroxylamine oxidation, cytochrome P460, to cytochrome c ₅₅₅. This cytochrome is spectrally similar to other cytochromes c′ but is larger (16,000 Da) and has a lower midpoint potential (–205 mV). By a combination of Edman degradation, mass spectroscopy, and gene sequencing, we have obtained the primary structure of cytochrome c′ from M. capsulatus Bath. The cytochrome shows low sequence similarity to other cytochromes c′, only residues R12, Y53, G56, and the C-terminal heme-binding region (GXXCXXCHXXXK) being conserved. In contrast, cytochrome c′ from M. capsulatus Bath shows considerable sequence similarity to cytochromes P460 from M. capsulatus Bath (31% identity) and from Nitrosomonas europaea (18% identity). This suggests that P460-type cytochromes may have originated from a c′-type cytochrome which developed a covalent cross-link between a lysine residue and the c′-heme.

Keywords: Key words Methanotroph; Methylotroph; Cytochrome ¶c′Methylococcus capsulatus Bath; Hydroxylamine ¶oxidation; Peptide mapping

Initial in vitro characterisation of phosphonopyruvate hydrolase, a novel phosphate starvation-independent, carbon-phosphorus bond cleavage enzyme in Burkholderia cepacia Pal6 by N. G. Ternan; J. T. G. Hamilton; J. P. Quinn (pp. 35-41).

A novel, inducible carbon-phosphorus bond cleavage enzyme, phosphonopyruvate hydrolase, was detected in cell-free extracts of Burkholderia cepacia Pal6, an environmental isolate capable of mineralising l-phosphonoalanine as carbon, nitrogen and phosphorus source. The activity was induced only in the presence of phosphonoalanine, did not require phosphate starvation for induction and was uniquely specific for phosphonopyruvate, producing equimolar quantities of pyruvate and inorganic phosphate. The native enzyme had a molecular mass of some 232 kDa and showed activation by metal ions in the order Co²⁺ > Ni²⁺ > Mg²⁺ > Zn²⁺ > Fe²⁺ > Cu²⁺. Temperature and pH optima in crude cell extracts were ¶50 °C and 7.5, respectively, and activity was inhibited by EDTA, phosphite, sulfite, mercaptoethanol and sodium azide. Phosphonopyruvate hydrolase is the third bacterial C-P bond cleavage enzyme reported to date that proceeds via a hydrolytic mechanism.

Keywords: Key words Phosphonopyruvate; Hydrolase; C-P bond; Inducible; Phosphonoalanine; Organophosphonates; Deregulated; pho regulon

Biochemical controls of citrate synthase in chickpea bacteroids by C. A. Tabrett; L. Copeland (pp. 42-48).

Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear. The K _m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by NADH was linear competitive with respect to acetyl-CoA (K _is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K _is = 56 ± 3.8 μM and K _ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD⁺ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely to be relieved by NAD⁺ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle.

Keywords: Key words Nitrogen fixation; Citrate synthase; Mesorhizobium ciceri; Cicer arietinum; Chickpea; Carbon metabolism; Bacteroids

Two new arsenate/sulfate-reducing bacteria: mechanisms of arsenate reduction by J. M. Macy; J. M. Santini; B. V. Pauling; A. H. O’Neill; L. I. Sly (pp. 49-57).

Two sulfate-reducing bacteria, which also reduce arsenate, were isolated; both organisms oxidized lactate incompletely to acetate. When using lactate as the electron donor, one of these organisms, Desulfomicrobium strain Ben-RB, rapidly reduced (doubling time = 8 h) 5.1 mM arsenate at the same time it reduced sulfate (9.6 mM). Sulfate reduction was not inhibited by the presence of arsenate. Arsenate could act as the terminal electron acceptor in minimal medium (doubling time = 9 h) in the absence of sulfate. Arsenate was reduced by a membrane-bound enzyme that is either a c-type cytochrome or is associated with such a cytochrome; benzyl-viologen-dependent arsenate reductase activity was greater in cells grown with arsenate/sulfate than in cells grown with sulfate only. The second organism, Desulfovibrio strain Ben-RA, also grew (doubling time = 8 h) while reducing arsenate (3.1 mM) and sulfate (8.3 mM) concomitantly. No evidence was found, however, that this organism is able to grow using arsenate as the terminal electron acceptor. Instead, it appears that arsenate reduction by the Desulfovibrio strain Ben-RA is catalyzed by an arsenate reductase that is encoded by a chromosomally-borne gene shown to be homologous to the arsC gene of the Escherichia coli plasmid, R773 ars system.

Keywords: Key words Arsenate reduction; Sulfate reduction; Desulfovibrio sp.; Desulfomicrobium sp.; Arsenate ¶reductase; Cytochrome c; Arsenate resistance

Anaerobic oxidation of alkanes by newly isolated denitrifying bacteria by P. Ehrenreich; A. Behrends; J. Harder; F. Widdel (pp. 58-64).

The capacity of denitrifying bacteria for anaerobic utilization of saturated hydrocarbons (alkanes) was investigated with n-alkanes of various chain lengths and with crude oil in enrichment cultures containing nitrate as electron acceptor. Three distinct types of denitrifying bacteria were isolated in pure culture. A strain (HxN1) with oval-shaped, nonmotile cells originated from a denitrifying enrichment culture with crude oil and was isolated with n-hexane (C₆H₁₄). Another strain (OcN1) with slender, rod-shaped, motile cells was isolated from an enrichment culture with n-octane (C₈H₁₈). A third strain (HdN1) with oval, somewhat pleomorphic, partly motile cells originated from an enrichment culture with aliphatic mineral oil and was isolated with n-hexadecane (C₁₆H₃₄). Cells of hexane-utilizing strain HxN1 grew homogeneously in the growth medium and did not adhere to the alkane phase, in contrast to the two other strains. Quantification of substrate consumption and cell growth revealed the capacity for complete oxidation of alkanes under strictly anoxic conditions, with nitrate being reduced to dinitrogen.

Keywords: Key wordsn-Alkanes; Anaerobic hydrocarbon ¶oxidation; Denitrifying bacteria; Isolation; Degradation ¶balance

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001 by F. Miake; T. Satho; H. Takesue; F. Yanagida; N. Kashige; K. Watanabe (pp. 65-70).

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far.

Keywords: Key wordsα-l-Rhamnosidase; l-rhamnose; Pseudomonas paucimobilis

Anaerobic degradation of flavonoids by Eubacterium ramulus by H. Schneider; M. Blaut (pp. 71-75).

Eubacterium ramulus, a quercetin-3-glucoside-degrading anaerobic microorganism that occurs at numbers of approximately 10⁸/g dry feces in humans, was tested for its ability to transform other flavonoids. The organism degraded luteolin-7-glucoside, rutin, quercetin, kaempferol, luteolin, eriodictyol, naringenin, taxifolin, and phloretin to phenolic acids. It hydrolyzed kaempferol-3-sorphoroside-7-glucoside to kaempferol-3-sorphoroside and transformed 3,4-dihydroxyphenylacetic acid, a product of anaerobic quercetin degradation, very slowly to non-aromatic fermentation products. Luteolin-5-glucoside, diosmetin-7-rutinoside, naringenin-7-neohesperidoside, (+)-catechin, and (–)-epicatechin were not degraded. Cell extracts of E. ramulus contained α- and β-d-glucosidase activities, but were devoid of α-l-rhamnosidase activity. Based on the degradation patterns of these substrates, a pathway for the degradation of flavonoids by E. ramulus is proposed.

Keywords: Key wordsEubacterium ramulus; Degradation; Quercetin; Kaempferol; Luteolin; Eriodictyol; Naringenin; Taxifolin; Catechin; Glycosides

Salmonella typhimurium forms adenylcobamide and 2-methyladenylcobamide, but no detectable cobalamin during strictly anaerobic growth by B. Keck; P. Renz (pp. 76-77).

Under microaerophilic conditions Salmonella typhimurium LT2 synthesizes cobalamin, during which 5,6-dimethylbenzimidazole is formed from riboflavin. We report here that in an anoxic environment S. typhimurium did not form cobalamin, but rather adenylcobamide, 2-methyladenylcobamide, and cobyric acid. This indicated that S. typhimurium, like other microorganisms that synthesize 5,6-dimethylbenzimidazole from riboflavin, requires oxygen for the formation of the cobalamin base.

Keywords: Key words 5,6-Dimethylbenzimidazole; Factor A; Pseudovitamin B12; Riboflavin; Salmonella ¶typhimurium; Vitamin B12

Indole-inducible proteins in bacteria suggest membrane and oxidant toxicity by T. R. Garbe; M. Kobayashi; H. Yukawa (pp. 78-82).

Oxidant toxicity of indole was demonstrated by the induction of alkylhydroperoxide reductase subunit C (AhpC) in Escherichia coli K12 and by the constitutive overproduction of AhpC in a variant of E. coli JM109 with enhanced resistance to indole. Oxidant toxicity was also indicated in an indole-adapted variant of Brevibacterium flavum by the indole-inducible overproduction of a novel 36-kDa protein with N-terminal sequence similarity to proteins involved in superoxide and singlet oxygen resistance. It is proposed that indole dissolved in membrane lipids, which caused membrane derangement and enabled direct interaction of redox-cycling isoprenoid quinones and dioxygen, resulting in the generation of superoxide. ¶A direct indication of membrane derangement in E. coli may be the indole-inducible overproduction of spheroplast protein y (Spy).

Keywords: Key words Tetralin; Cumene hydroperoxide; Cpx ¶regulon; Membrane adhesion sites

Erratum: Arch Microbiol (1999) 172: 167–174 (pp. 83-83).

Erratum: Arch Microbiol (1999) 172: 204–212 (pp. 84-84).

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Archives of Microbiology (v.173, #1)