Fermentative degradation of acetone by an enrichment culture in membrane-separated culture devices and in cell suspensions

Abstract We have recently demonstrated that cultured human intestinal HT-29 and Caco-2 cell lines express receptors for the F1845 fimbrial adhesin harbored by the diarrheagenic C1845 Escherichia coli (Kernéis et al., Infect. Immun. 59 (1991) 4013–4018). This adhesinn belongs to a family of adhesins including the Dr hemagglutinin and the afimbrial adhesin AFA-1 harbored by uropathogenic E. coli . Here we investigated the cell association of laboratory E. coli strains expressing the Dr hemagglutinin and the afimbrial adhesin AFA-I with human cultured enterocyte-like or mucosecreting cells. We observed that the E. coli strains bearing these adhesins adhere both to human intestinal undifferentiated and differentiated fluid-transporting cells, and to mucus-secreting cells. This result strongly suggests a high capacity of intestinal colonization for the uropathogenic E. coli harboring adhesive factors belonging to the Dr adhesin family. These results further corroborate the intestinal colonization by uropathogenic E. coli of the Dr family related to the fecal-perineal-urethral hypothesis of urinary tract infection pathogenesis.     

Anaerobic degradation of α-resorcylate by Thauera aromatica strain AR-1 proceeds via oxidation and decarboxylation to hydroxyhydroquinone

Anaerobic degradation of α-resorcylate (3,5-dihydroxybenzoate) was studied with the denitrifying strain AR-1, which was assigned to the described species Thauera aromatica. α-Resorcylate degradation does not proceed via the benzoyl-CoA, the resorcinol, or the phloroglucinol pathway. Instead, α-resorcylate is converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by dehydrogenative oxidation and decarboxylation. Nitrate, K₃[Fe(CN)₆], dichlorophenol indophenol, and the NAD⁺ analogue 3-acetylpyridine adeninedinucleotide were suitable electron acceptors for the oxidation reaction; NAD⁺ did not function as an electron acceptor. The oxidation reaction was strongly accelerated by the additional presence of the redox carrier phenazine methosulfate, which could also be used as sole electron acceptor. Oxidation of α-resorcylate with molecular oxygen in cell suspensions or in cell-free extracts of α-resorcylate- and nitrate-grown cells was also detected although this bacterium did not grow with α-resorcylate under an air atmosphere. α-Resorcylate degradation to hydroxyhydroquinone proceeded in two steps. The α-resorcylate-oxidizing enzyme activity was membrane-associated and exhibited maximal activity at pH 8.0. The primary oxidation product was not hydroxyhydroquinone. Rather, formation of hydroxyhydroquinone by decarboxylation of the unknown intermediate in addition required the cytoplasmic fraction and needed lower pH values since hydroxyhydroquinone was not stable at alkaline pH. Received: 8 July 1997 / Accepted: 20 October 1997     

Heterologous expression and identification of the genes involved in anaerobic degradation of 1,3-dihydroxybenzene (resorcinol) in Azoarcus anaerobius

Azoarcus anaerobius, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and energy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this bacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the formation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To identify the A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related species Thauera aromatica, which cannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the ability to metabolize resorcinol. Its cosmid, designated R(+), carries a 29.88-kb chromosomal DNA fragment containing 22 putative genes. In cell extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R(+) carried all of the genes necessary for resorcinol degradation. On the basis of the physiological characterization of T. aromatica transconjugants carrying transposon insertions in different genes of cosmid R(+), eight open reading frames were found to be essential for resorcinol mineralization. Resorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have also been proposed. This work provides the first example of the identification of genes involved in the anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.     

Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3,4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-¹³C]naphthalene or deuterated D₈-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [¹³C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, ¹³C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.     

Hydroxyhydroquinone reductase, the initial enzyme involved in the degradation of hydroxyhydroquinone (1,2,4-trihydroxybenzene) by Desulfovibrio inopinatus

The recently isolated sulfate reducer Desulfovibrio inopinatus oxidizes hydroxyhydroquinone (1,2,4trihydroxybenzene; 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.     

Carbonylation as a Key Reaction in Anaerobic Acetone Activation by Desulfococcus biacutus

Acetone is activated by aerobic and nitrate-reducing bacteria via an ATP-dependent carboxylation reaction to form acetoacetate as the first reaction product. In the activation of acetone by sulfate-reducing bacteria, acetoacetate has not been found to be an intermediate. Here, we present evidence of a carbonylation reaction as the initial step in the activation of acetone by the strictly anaerobic sulfate reducer Desulfococcus biacutus. In cell suspension experiments, CO was found to be a far better cosubstrate for acetone activation than CO₂. The hypothetical reaction product, acetoacetaldehyde, is extremely reactive and could not be identified as a free intermediate. However, acetoacetaldehyde dinitrophenylhydrazone was detected by mass spectrometry in cell extract experiments as a reaction product of acetone, CO, and dinitrophenylhydrazine. In a similar assay, 2-amino-4-methylpyrimidine was formed as the product of a reaction between acetoacetaldehyde and guanidine. The reaction depended on ATP as a cosubstrate. Moreover, the specific activity of aldehyde dehydrogenase (coenzyme A [CoA] acylating) tested with the putative physiological substrate was found to be 153 ± 36 mU mg⁻¹ protein, and its activity was specifically induced in extracts of acetone-grown cells. Moreover, acetoacetyl-CoA was detected (by mass spectrometry) after the carbonylation reaction as the subsequent intermediate after acetoacetaldehyde was formed. These results together provide evidence that acetoacetaldehyde is an intermediate in the activation of acetone by sulfate-reducing bacteria.     

Acetone utilization by sulfate-reducing bacteria: draft genome sequence of Desulfococcus biacutus and a proteomic survey of acetone-inducible proteins

Background

The sulfate-reducing bacterium Desulfococcus biacutus is able to utilize acetone for growth by an inducible degradation pathway that involves a novel activation reaction for acetone with CO as a co-substrate. The mechanism, enzyme(s) and gene(s) involved in this acetone activation reaction are of great interest because they represent a novel and yet undefined type of activation reaction under strictly anoxic conditions.

Results

In this study, a draft genome sequence of D. biacutus was established. Sequencing, assembly and annotation resulted in 159 contigs with 5,242,029 base pairs and 4773 predicted genes; 4708 were predicted protein-encoding genes, and 3520 of these had a functional prediction. Proteins and genes were identified that are specifically induced during growth with acetone. A thiamine diphosphate-requiring enzyme appeared to be highly induced during growth with acetone and is probably involved in the activation reaction. Moreover, a coenzyme B₁₂- dependent enzyme and proteins that are involved in redox reactions were also induced during growth with acetone.

Conclusions

We present for the first time the genome of a sulfate reducer that is able to grow with acetone. The genome information of this organism represents an important tool for the elucidation of a novel reaction mechanism that is employed by a sulfate reducer in acetone activation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-584) contains supplementary material, which is available to authorized users.     

Anaerobic and aerobic oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria

Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30° C. Anaerobic nitrate-dependent ferrous iron oxidation was a biological process. One strain isolated from brackish-water sediment (strain HidR2, a motile, nonsporeforming, gram-negative rod) was chosen for further investigation of ferrous iron oxidation in the presence of acetate as cosubstrate. Strain HidR2 oxidized between 0.7 and 4.9 mM ferrous iron aerobically and anaerobically at pH 7.2 and 30° C in the presence of small amounts of acetate (between 0.2 and 1.1 mM). The strain gained energy for growth from anaerobic ferrous iron oxidation with nitrate, and the ratio of iron oxidized to acetate provided was constant at limiting acetate supply. The ability to oxidize ferrous iron anaerobically with nitrate at approximately pH 7 appears to be a widespread capacity among mesophilic denitrifying bacteria. Since nitrate-dependent iron oxidation closes the iron cycle within the anoxic zone of sediments and aerobic iron oxidation enhances the reoxidation of ferrous to ferric iron in the oxic zone, both processes increase the importance of iron as a transient electron carrier in the turnover of organic matter in natural sediments. Received: 24 April 1997 / Accepted: 22 September 1997     

Factors influencing the cultivability of lake water bacteria

Counting bacteria in natural water samples by cultivation yields only low recovery efficiencies (ca. 1%), compared to total counts obtained after 4,6-diamidino-2-phenylindol (DAPI) staining. In order to optimize the cultivation of heterotrophic planktonic bacteria from Lake Constance (Germany), selected parameters of the medium composition were modified. The most important factor was the concentration of organic substrate (nutrient broth plus yeast extract), which significantly influenced the "most probable number" obtained in liquid growth medium. Reduced oxygen concentrations (3-12%) lowered the "most probable number". Addition of N-acyl homoserine lactones to the medium increased the cultivability slightly. Low substrate concentrations [0.03-0.06% (w/v)], an incubation atmosphere of 21% oxygen at 16 degrees C for 4 weeks were optimal and increased the cultivability ("most probable number" related to total bacterial counts) to an average cultivability of 18+/-11%, (n=8). The results indicate that cultivabilities of heterotrophic bacteria from lakewater samples can be significantly increased by modifying the cultivation methods.