The genes encoding coenzyme B 12 -dependent glycerol dehydratase of Citrobacter freundii were cloned and overexpressed in Escherichia coli. The B 12 -free enzyme was purified to homogeneity. It consists of three types of subunits whose N-terminal sequences are in accordance with those deduced from the open reading frames dhaB, dhaC, and dhaE, coding for subunits of 60,433 ( Glycerol dehydratase (EC 4.2.1.30) catalyzes the coenzyme B 12 -dependent conversion of glycerol, 1,2-propanediol, and 1,2-ethanediol to 3-hydroxypropionaldehyde, propionaldehyde, and acetaldehyde, respectively (14, 23). The enzyme is involved in anaerobic glycerol utilization by Citrobacter freundii. In the absence of an external oxidant, glycerol is fermented by a dismutation process using two pathways. Through one pathway glycerol is dehydrogenated by an NAD ϩ -linked glycerol dehydrogenase to dihydroxyacetone, which is then phosphorylated and funneled to glycolysis by dihydroxyacetone kinase (12). Through the other pathway glycerol is dehydrated by glycerol dehydratase to form 3-hydroxypropionaldehyde, which is reduced to the major fermentation product 1,3-propanediol by the NADH-linked 1,3-propanediol dehydrogenase, thereby regenerating NAD ϩ (10). The four key enzymes of this pathway are encoded by the dha regulon, the expression of which is induced when dihydroxyacetone or glycerol is present (10, 16). Recently we have cloned and expressed the dha regulon of C. freundii in Escherichia coli (6). The genes coding for 1,3-propanediol dehydrogenase, glycerol dehydrogenase, and dihydroxyacetone kinase have been identified and characterized (5, 7). Here we report on the coenzyme B 12 -dependent enzyme glycerol dehydratase.Cloning of the genes encoding glycerol dehydratase. The recombinant cosmid pRD1, which contains a 32-kb insert of C. freundii genomic DNA, harbors the entire dha regulon of C. freundii, as described previously (6). After digestion of pRD1 with HindIII, ligation into pBluescript SKϩ, and transformation into E. coli DH5␣, one subclone with glycerol dehydratase activity (0.5 U/mg) in cell extracts was obtained. Glycerol dehydratase was assayed according to the method of Toraya and Fukui (27). Protein concentrations were determined as described by Bradford (3). The recombinant plasmid recovered from this strain was designated pRD12 and contained a 10.9-kb HindIII insert of C. freundii genomic DNA (Fig. 1). The origin of the cloned DNA was established by Southern blot analysis (data not shown).Digestion of pRD12 with BstEII and XbaI, followed by treatment with Klenow fragment and self-ligation, yielded plasmid pMS2, containing a 5,468-bp HindIII-BstEII fragment from pRD12. Digestion of pRD12 with SmaI, followed by self-ligation, yielded plasmid pMS3, containing a 4,910-bp HindIII-SmaI fragment from pRD12 (Fig. 1). Both recombinant E. coli strains exhibited glycerol dehydratase activity (0.5 U/mg).However, recombinant E. coli strains harboring pRD11, which contained a 3,246-bp HindIII-PstI fragment derived from pRD1 (Fig. 1), sh...

The study of complex microbial communities typically entails high-throughput sequencing and downstream bioinformatics analyses. Here we expand and accelerate microbiota analysis by enabling cell type diversity quantification from multidimensional flow cytometry data using a supervised machine learning algorithm of standard cell type recognition (CellCognize). As a proof-of-concept, we trained neural networks with 32 microbial cell and bead standards. The resulting classifiers were extensively validated in silico on known microbiota, showing on average 80% prediction accuracy. Furthermore, the classifiers could detect shifts in microbial communities of unknown composition upon chemical amendment, comparable to results from 16S-rRNA-amplicon analysis. CellCognize was also able to quantify population growth and estimate total community biomass productivity, providing estimates similar to those from 14 C-substrate incorporation. CellCognize complements current sequencing-based methods by enabling rapid routine cell diversity analysis. The pipeline is suitable to optimize cell recognition for recurring microbiota types, such as in human health or engineered systems.

We previously showed that the relative response factors of volatile compounds were predictable from either combustion enthalpies or their molecular formulae only 1. We now extend this prediction to silylated derivatives by adding an increment in the ab initio calculation of combustion enthalpies. The accuracy of the experimental relative response factors database was also improved and its population increased to 490 values. In particular, more brominated compounds were measured, and their prediction accuracy was improved by adding a correction factor in the algorithm. The correlation coefficient between predicted and measured values increased from 0.936 to 0.972, leading to a mean prediction accuracy of ± 6%. Thus, 93% of the relative response factors values were predicted with an accuracy of better than ± 10%. The capabilities of the extended algorithm are exemplified by (i) the quick and accurate quantification of hydroxylated metabolites resulting from a biodegradation test after silylation and prediction of their relative response factors, without having the reference substances available; and (ii) the rapid purity determinations of volatile compounds. This study confirms that Gas chromatography with a flame ionization detector and using predicted relative response factors is one of the few techniques that enables quantification of volatile compounds without calibrating the instrument with the pure reference substance.

When grown on glycerol as sole carbon and energy source, cell extracts of Clostridium pasteurianum exhibited activities of glycerol dehydrogenase, dihydroxyacetone kinase, glycerol dehydratase and 1,3-propanediol dehydrogenase. The genes encoding the latter two enzymes were cloned by colony hybridization using the dhaT gene of Citrobacter freundii as a heterologous DNA probe and expressed in Escherichia coli. The native molecular mass of 1,3-propanediol dehydrogenase (DhaT) is 440,000 Da. The dhaT gene of C. pasteurianum was subcloned and its nucleotide sequence (1158 bp) was determined. The deduced gene product (41,776 Da) revealed high similarity to DhaT of C. freundii (80.5% identity; 89.8% similarity).

The degradation of Romandolide ([1-(3',3'-dimethyl-1'-cyclohexyl)ethoxycarbonyl] methyl propanoate), a synthetic alicyclic musk, by activated sludge inocula was investigated using both the manometric respirometry test OECD 301F and the CO₂ evolution test. In addition to measuring its biodegradability, key steps of the upper part of the metabolic pathway responsible for Romandolide degradation were identified using extracts at different time points of incubation. Early metabolism of Romandolide yielded ester hydrolysis products, including Cyclademol (1-(3,3-dimethylcyclohexyl)ethanol). The principal metabolites after 31 days were identified as 3,3-dimethyl cyclohexanone and 3,3-dimethyl cyclohexyl acetate. Formation of 3,3-dimethyl cyclohexanone from Cyclademol by sludge was confirmed in subsequent experiments using Cyclademol as a substrate, indicating the involvement of an oxygen insertion reminiscent of a Baeyer-Villiger oxidation. Further mineralization of 3,3-dimethyl cyclohexanone was also confirmed in subsequent studies. Three steps were thus required for complete biodegradation of the alicyclic musk: (1) successive ester hydrolyses leading to the formation of Cyclademol with concomitant degradation of the resulting acids, (2) conversion of Cyclademol into 3,3-dimethyl cyclohexanone, and (3) further mineralization via ring cleavage.