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Archives of Microbiology (v.179, #4)

Use of the mannitol pathway in fructose fermentation of Oenococcus oeni due to limiting redox regeneration capacity of the ethanol pathway by Hanno Richter; Inka Hamann; Gottfried Unden (pp. 227-233).

The heterolactic bacterium Oenococcus oeni ferments fructose by a mixed heterolactic/mannitol fermentation. For heterolactic fermentation of fructose, the phosphoketolase pathway is used. The excess NAD(P)H from the phosphoketolase pathway is reoxidized by fructose (yielding mannitol). It is shown here that, under conditions of C-limitation or decreased growth rates, fructose can be fermented by heterolactic fermentation yielding nearly stoichiometric amounts of lactate, ethanol and CO₂. Quantitative evaluation of NAD(P)H-producing (phosphoketolase pathway) and -reoxidizing (ethanol, mannitol and erythritol pathways) reactions demonstrated that at high growth rates or in batch cultures the ethanol pathway does not have sufficient capacity for NAD(P)H reoxidation, requiring additional use of the mannitol pathway to maintain the growth rate. In addition, insufficient capacities to reoxidize NAD(P)H causes inhibition of growth, whereas increased NAD(P)H reoxidation by electron acceptors such as pyruvate increases the growth rate.

Keywords: Oenococcus oeni Heterolactic fermentation Fructose Phosphoketolase pathway NAD(P)H reoxidation Mannitol

Biodegradation of bis(1-chloro-2-propyl) ether via initial ether scission and subsequent dehalogenation by Rhodococcus sp. strain DTB by Marcus Moreno Horn; Leif-Alexander Garbe; Roland Tressl; Lorenz Adrian; Helmut Görisch (pp. 234-241).

Rhodococcus sp. strain DTB (DSM 44534) grows on bis(1-chloro-2-propyl) ether (DDE) as sole source of carbon and energy. The non-chlorinated diisopropyl ether and bis(1-hydroxy-2-propyl) ether, however, did not serve as substrates. In ether degradation experiments with dense cell suspensions, 1-chloro-2-propanol and chloroacetone were formed, which indicated that scission of the ether bond is the first step while dehalogenation of the chlorinated C₃-compounds occurs at a later stage of the degradation pathway. Inhibition of ether scission by methimazole suggested that the first step in degradation is catalyzed by a flavin-dependent enzyme activity. The non-chlorinated compounds 1,2-propanediol, hydroxyacetone, lactate, pyruvate, 1-propanol, propanal, and propionate also supported growth, which suggested that the intermediates 1,2-propanediol and hydroxyacetone are converted to pyruvate or to propionate, which can be channeled into the citric acid cycle by a number of routes. Total release of chloride and growth-yield experiments with DDE and non-chlorinated C₃-compounds suggested complete biodegradation of the chlorinated ether.

Keywords: Rhodococcus Bis(1-chloro-2-propyl) ether Biodegradation Flavin-dependent monooxygenase Methimazole

Sporotomaculum syntrophicum sp. nov., a novel anaerobic, syntrophic benzoate-degrading bacterium isolated from methanogenic sludge treating wastewater from terephthalate manufacturing by Yan-Ling Qiu; Yuji Sekiguchi; Hiroyuki Imachi; Yoichi Kamagata; I-Cheng Tseng; Sheng-Shung Cheng; Akiyoshi Ohashi; Hideki Harada (pp. 242-249).

An anaerobic, mesophilic, syntrophic benzoate-degrading bacterium, designated strain FB^T, was isolated from methanogenic sludge which had been used to treat wastewater from the manufacture of terephthalic acid. Cells were non-motile gram-positive rods that formed spores. The optimum temperature for growth was 35–40 °C, and the optimum pH was 7.0–7.2. A co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei converted benzoate to acetate, carbon dioxide, and methane. Butyrate transiently accumulated at a high concentration of 2.5 mM during degradation. Besides benzoate, no other compound tested supported growth of the co-culture. Crotonate supported growth of strain FB^T in pure culture. Furthermore, the strain degraded benzoate in pure culture with crotonate as co-substrate to produce acetate and butyrate. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, or Fe(III) as electron acceptor. The G+C content of the DNA was 46.8 mol%. Strain FB^T contained MK-7 as the major quinone and C_16:1 as the major fatty acid. 16S rDNA sequence analysis revealed that the strain was a member of the genus Sporotomaculum, even though it exhibited significant differences, such as the capacity for syntrophic growth, to the known member of the genus. Hence, we propose the name Sporotomaculum syntrophicum sp. nov. for strain FB^T. The type strain is strain FB^T (DSM 14795, JCM 11475).

Keywords: Anaerobic benzoate oxidation Sporotomaculum syntrophicum sp. nov.

Characterization and phylogenetic analysis of a thermostable N-carbamoyl-l-amino acid amidohydrolase from Bacillus kaustophilus CCRC11223 by Hui-Yu Hu; Wen-Hwei Hsu; Hungchien Roger Chien (pp. 250-257).

A thermostable N-carbamoyl-l-amino acid amidohydrolase (l-N-carbamoylase) gene composed of an 1,230-bp ORF encoding a 44.3-kDa protein was cloned from the thermophile Bacillus kaustophilus CCRC11223. This l-N-carbamoylase contained six cysteine residues that form three disulfide bridges. The purified l-N-carbamoylase was stringently l-specific and exhibited high activity in the hydrolysis of N-carbamoyl-l-homophenylalanine. N-carbamoyl derivatives of β-alanine, β-aminoisobutyric acids, l-tryptophan, and d-specific amino acids were not recognized as substrates. The l-N-carbamoylase required the divalent metal ions Mn²⁺, Co²⁺, and Ni²⁺ for increasing activity. The pH and temperature optima of the enzyme were pH 7.4 and 70 °C, respectively. This enzyme was completely thermostable at 50 °C for 36 days in the presence of d- and/or l-specific substrates. Phylogenetic analysis of the available amino acid sequences of N-carbamoyl and N-acyl amino acid amidohydrolases from the three main kingdoms of life showed that they can be divided into four distinct families. The B. kaustophilus enzyme could be classified into the family of l-N-carbamoylases and some β-ureidopropionases, but did not hydrolyze β-ureidopropionates.

Keywords: N-carbamoyl-l-amino acid amidohydrolase Bacillus kaustophilus N-carbamoyl-l-homophenylalanine Thermostable enzyme Phylogeny

Characterization of a new keratinolytic bacterium that completely degrades native feather keratin by Alessandro Riffel; Françoise Lucas; Philipp Heeb; Adriano Brandelli (pp. 258-265).

A novel feather-degrading microorganism was isolated from poultry waste, producing a high keratinolytic activity when cultured on broth containing native feather. Complete feather degradation was achieved during cultivation. The bacterium presents potential use for biotechnological processes involving keratin hydrolysis. Chryseobacterium sp. strain kr6 was identified based on morphological and biochemical tests and 16S rRNA sequencing. The bacterium presented optimum growth at pH 8.0 and 30 °C; under these conditions, maximum feather-degrading activity was also achieved. Maximum keratinase production was reached at 25 °C, while concentration of soluble protein was similar at both 25 and 30 °C. Reduction of disulfide bridges was also observed, increasing with cultivation time. The keratinase of strain kr6 was active on azokeratin and azocasein as substrates, and presented optimum pH and temperature of 7.5 and 55 °C, respectively. The keratinase activity was inhibited by 1,10-phenanthroline, EDTA, Hg²⁺, and Cu²⁺ and stimulated by Ca²⁺.

Keywords: Keratin Proteolysis Poultry waste Chicken feather Bacteria

Cloning and characterization of a gene cluster involved in tetrahydrofuran degradation in Pseudonocardia sp. strain K1 by Barbara Thiemer; Jan R. Andreesen; Thomas Schräder (pp. 266-277).

A gene cluster involved in the utilization of tetrahydrofuran by Pseudonocardia sp. strain K1 was cloned and sequenced. Analysis of a 9.2-kb DNA fragment revealed eight ORFs. The genes designated as thmADBC encode the components of a putative monooxygenase exhibiting a high similarity to different binuclear-iron-containing multicomponent monooxygenases. thmA encodes the derived 545-amino-acid oxygenase α-subunit, thmD the 360-amino-acid reductase component, thmB the 346-amino-acid oxygenase β-subunit, and thmC the 117-amino-acid coupling protein. Upstream of the thm genes, an additional ORF (sad) was identified coding for a protein with high similarity to various aldehyde dehydrogenases. A succinate semialdehyde dehydrogenase activity was specifically expressed in tetrahydrofuran-grown cells. N-terminal sequence analysis of the purified protein revealed that it is encoded by sad. Northern blot analysis indicated that transcription of the thm genes and sad was specifically induced during growth on tetrahydrofuran. Mono-, di- and polycistronic transcripts of these genes were detected. Primer-extension analysis identified transcriptional start sites 37, 61, and 41 bp upstream of the translation start of sad, thmA, and thmB, respectively. Additional ORFs were identified upstream (orfY) and downstream (orfZ and aldH) of the thm genes. Furthermore, the data indicated that the analyzed gene cluster was present as a single copy and located on a plasmid.

Keywords: Tetrahydrofuran Monooxygenase Ether degradation Pseudonocardia 1,4-Butandiol

Genome-wide transcriptional changes during the lag phase of Saccharomyces cerevisiae by Jeanette Brejning; Lene Jespersen; Nils Arneborg (pp. 278-294).

The set of physiological and metabolic changes occurring immediately after inoculation and during the lag phase is thought to be of vital importance for optimal offset of fermentation. The transcriptional changes taking place during the lag phase after inoculation of a late-respiratory-phase yeast culture into fresh, minimal medium were investigated by use of Yeast GeneFilters. In response to the nutritional up-shift, 240 open reading frames were at least five-fold induced and 122 were at least five-fold repressed. These genes were hierarchically clustered according to their lag-phase expression patterns. The majority of the induced genes were most highly induced early in the lag phase, whereas strong repression generally occurred later. Clustering of the genes showed that many genes with similar roles had similar expression patterns. Repressed genes, however, did not cluster as tightly according to function as induced genes. Genes involved in RNA and protein synthesis and processing showed a peak in expression early in the lag phase, except most ribosomal protein genes, which were induced early and whose expression was sustained. Genes involved in chromatin/chromosome structure showed late induction. The correlation between function and expression pattern for these genes indicates regulation by similar mechanisms. Much of the transcriptional response observed appeared to be due to the presence of glucose in the new medium.

Keywords: Lag phase Gene expression Saccharomyces cerevisiae Temporal transcriptional profiling GeneFilters Microarrays

Disruption of mitochondrial function in Candida albicans leads to reduced cellular ergosterol levels and elevated growth in the presence of amphotericin B by Patrick Geraghty; Kevin Kavanagh (pp. 295-300).

A respiratory-deficient mutant of Candida albicans MEN was generated by culturing cells in medium supplemented with ethidium bromide at 37 °C for 5 days. The respiratory-deficient mutant (C. albicans MMU11) was incapable of growth on glycerol, had a reduced oxygen uptake rate and demonstrated an altered mitochondrial cytochrome profile. Respiratory-competent cybrids were formed by mitochondrial transfer following fusion of protoplasts with those of C. albicans ATCC 44990. Mutant MMU11 possessed lower levels of ergosterol than the parental isolates and the cybrids, and demonstrated a small but statistically significant increase in tolerance to amphotericin B. The results demonstrated that disruption of mitochondrial function in C. albicans increases the tolerance to amphotericin B, possibly mediated by a reduction in cellular ergosterol content.

Keywords: Amphotericin B Candida Ergosterol Mitochondrion

Application of crossover-PCR-mediated deletion-insertion mutagenesis to analysis of the bdhA-xdhA2-xdhB2 mixed-function operon of Sinorhizobium meliloti by Nicole Sukdeo; Trevor C. Charles (pp. 301-304).

The bdhA-xdhA2-xdhB2 mixed-function operon was used to demonstrate the application of crossover PCR to the construction of in-frame, non-polar deletion-insertion mutations in Sinorhizobium meliloti. Replacement of a 474-bp internal portion of the 774-bp coding sequence of bdhA with a 21-bp in-frame synthetic sequence resulted in loss of the bdhA-encoded d-3-hydroxybutyrate dehydrogenase activity. Such mutants retained the xanthine oxidase activity encoded by xdhA2-xdhB2, thus illustrating the non-polar nature of the mutation. This method of constructing unmarked, in-frame deletions should be generally applicable to functional genomics studies in S. meliloti and other α-Proteobacteria.

Keywords: PHB cycle Purine degradation Crossover PCR

Novel carotenoid glucoside esters from alkaliphilic heliobacteria by Shinichi Takaichi; Hirozo Oh-oka; Takashi Maoka; Deborah O. Jung; Michael T. Madigan (pp. 305-305).

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Archives of Microbiology (v.179, #4)