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8.  U(VI) Reduction by Diverse Outer Surface c-Type Cytochromes of Geobacter sulfurreducens 
Applied and Environmental Microbiology  2013;79(20):6369-6374.
Early studies with Geobacter sulfurreducens suggested that outer-surface c-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of G. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface c-type cytochromes of G. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ± 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ± 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that G. sulfurreducens requires outer-surface c-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.
PMCID: PMC3811187  PMID: 23934497
9.  Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Brettanomyces bruxellensis 
Applied and Environmental Microbiology  2013;79(20):6264-6270.
The yeasts Zygosaccharomyces bailii, Dekkera bruxellensis (anamorph, Brettanomyces bruxellensis), and Saccharomyces cerevisiae are the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeasts Z. bailii, B. bruxellensis, and S. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% for Z. bailii, 92.3% for B. bruxellensis, and 98.6% for S. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.
PMCID: PMC3811183  PMID: 23913433
10.  The MAP Kinase Slt2 Is Involved in Vacuolar Function and Actin Remodeling in Saccharomyces cerevisiae Mutants Affected by Endogenous Oxidative Stress 
Applied and Environmental Microbiology  2013;79(20):6459-6471.
Oxidative stress causes transient actin cytoskeleton depolarization and also provokes vacuole fragmentation in wild-type cells. Under conditions of oxidative stress induced by hydrogen peroxide, the Slt2 protein is required to repolarize the actin cytoskeleton and to promote vacuole fusion. In this study, we show that grx3 grx4 and grx5 mutants are cellular models of endogenous oxidative stress. This stress is the result of alterations in iron homeostasis that lead to impairment of vacuolar function and also to disorganization of the actin cytoskeleton. Slt2 overexpression suppresses defects in vacuolar function and actin cytoskeleton organization in the grx3 grx4 mutant. Slt2 exerts this effect independently of the intracellular levels of reactive oxygen species (ROS) and of iron homeostasis. The deletion of SLT2 in the grx3 grx4 mutant results in synthetic lethality related to vacuolar function with substantial vacuole fragmentation. The observation that both Vps4 and Vps73 (two proteins related to vacuole sorting) suppress vacuole fragmentation and actin depolarization in the grx3 grx4 slt2 triple mutant strengthens the hypothesis that Slt2 plays a role in vacuole homeostasis related to actin dynamics. Here we show that in sod1, grx5, and grx3 grx4 slt2 mutants, all of which are affected by chronic oxidative stress, the overexpression of Slt2 favors vacuole fusion through a mechanism dependent on an active actin cytoskeleton.
PMCID: PMC3811184  PMID: 23956390
11.  Monitoring the Metabolic State of Fungal Hyphae and the Presence of Melanin by Nonlinear Spectral Imaging 
Applied and Environmental Microbiology  2013;79(20):6345-6350.
Label-free nonlinear spectral imaging microscopy (NLSM) records two-photon-excited fluorescence emission spectra of endogenous fluorophores within the specimen. Here, NLSM is introduced as a novel, minimally invasive method to analyze the metabolic state of fungal hyphae by monitoring the autofluorescence of NAD(P)H and flavin adenine dinucleotide (FAD). Moreover, the presence of melanin was analyzed by NLSM. NAD(P)H, FAD, and melanin were used as biomarkers for freshness of mushrooms of Agaricus bisporus (white button mushroom) that had been stored at 4°C for 0 to 17 days. During this period, the mushrooms did not show changes in morphology or color detectable by eye. In contrast, FAD/NAD(P)H and melanin/NAD(P)H ratios increased over time. For instance, these ratios increased from 0.92 to 2.02 and from 0.76 to 1.53, respectively, at the surface of mushroom caps that had been harvested by cutting the stem. These ratios were lower under the skin than at the surface of fresh mushrooms (0.78 versus 0.92 and 0.41 versus 0.76, respectively), indicative of higher metabolism and lower pigment formation within the fruiting body. Signals were different not only between tissues of the mushroom but also between neighboring hyphae. These data show that NLSM can be used to determine the freshness of mushrooms and to monitor the postharvest browning process at an early stage. Moreover, these data demonstrate the potential of NLSM to address a broad range of fundamental and applied microbiological processes.
PMCID: PMC3811185  PMID: 23934488
12.  Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study 
Applied and Environmental Microbiology  2013;79(20):6429-6438.
In this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) from Alkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. The kcat/Km was increased from 1.0 × 105 liters · mol−1 · s−1 for AmyK to 30.6 × and 23.2 × 105 liters · mol−1 · s−1 for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.
PMCID: PMC3811186  PMID: 23956385
13.  Exploring the Biosynthesis of Unsaturated Fatty Acids in Bacillus cereus ATCC 14579 and Functional Characterization of Novel Acyl-Lipid Desaturases 
Applied and Environmental Microbiology  2013;79(20):6271-6279.
At low temperatures, Bacillus cereus synthesizes large amounts of unsaturated fatty acids (UFAs) with double bonds in positions Δ5 and Δ10, as well as Δ5,10 diunsaturated fatty acids. Through sequence homology searches, we identified two open reading frames (ORFs) encoding a putative Δ5 desaturase and a fatty acid acyl-lipid desaturase in the B. cereus ATCC 14579 genome, and these were named BC2983 and BC0400, respectively. Functional characterization of ORFs BC2983 and BC0400 by means of heterologous expression in Bacillus subtilis confirmed that they both encode acyl-lipid desaturases that use phospholipids as the substrates and have Δ5 and Δ10 desaturase activities. Thus, these ORFs were correspondingly named desA (Δ5 desaturase) and desB (Δ10 desaturase). We established that DesA utilizes ferredoxin and flavodoxins (Flds) as electron donors for the desaturation reaction, while DesB preferably employs Flds. In addition, increased amounts of UFAs were found when B. subtilis expressing B. cereus desaturases was subjected to a cold shock treatment, indicating that the activity or the expression of these enzymes is upregulated in response to a decrease in growth temperature. This represents the first work reporting the functional characterization of fatty acid desaturases from B. cereus.
PMCID: PMC3811188  PMID: 23913431
14.  Metabolism of Four α-Glycosidic Linkage-Containing Oligosaccharides by Bifidobacterium breve UCC2003 
Applied and Environmental Microbiology  2013;79(20):6280-6292.
Members of the genus Bifidobacterium are common inhabitants of the gastrointestinal tracts of humans and other mammals, where they ferment many diet-derived carbohydrates that cannot be digested by their hosts. To extend our understanding of bifidobacterial carbohydrate utilization, we investigated the molecular mechanisms by which 11 strains of Bifidobacterium breve metabolize four distinct α-glucose- and/or α-galactose-containing oligosaccharides, namely, raffinose, stachyose, melibiose, and melezitose. Here we demonstrate that all B. breve strains examined possess the ability to utilize raffinose, stachyose, and melibiose. However, the ability to metabolize melezitose was not common to all B. breve strains tested. Transcriptomic and functional genomic approaches identified a gene cluster dedicated to the metabolism of α-galactose-containing carbohydrates, while an adjacent gene cluster, dedicated to the metabolism of α-glucose-containing melezitose, was identified in strains that are able to use this carbohydrate.
PMCID: PMC3811189  PMID: 23913435
15.  Comparative Genomic and Transcriptomic Analyses Reveal Habitat Differentiation and Different Transcriptional Responses during Pectin Metabolism in Alishewanella Species 
Applied and Environmental Microbiology  2013;79(20):6351-6361.
Alishewanella species are expected to have high adaptability to diverse environments because they are isolated from different natural habitats. To investigate how the evolutionary history of Alishewanella species is reflected in their genomes, we performed comparative genomic and transcriptomic analyses of A. jeotgali, A. aestuarii, and A. agri, which were isolated from fermented seafood, tidal flat sediment, and soil, respectively. Genomic islands with variable GC contents indicated that invasion of prophage and transposition events occurred in A. jeotgali and A. agri but not in A. aestuarii. Habitat differentiation of A. agri from a marine environment to a terrestrial environment was proposed because the species-specific genes of A. agri were similar to those of soil bacteria, whereas those of A. jeotgali and A. aestuarii were more closely related to marine bacteria. Comparative transcriptomic analysis with pectin as a sole carbon source revealed different transcriptional responses in Alishewanella species, especially in oxidative stress-, methylglyoxal detoxification-, membrane maintenance-, and protease/chaperone activity-related genes. Transcriptomic and experimental data demonstrated that A. agri had a higher pectin degradation rate and more resistance to oxidative stress under pectin-amended conditions than the other 2 Alishewanella species. However, expression patterns of genes in the pectin metabolic pathway and of glyoxylate bypass genes were similar among all 3 Alishewanella species. Our comparative genomic and transcriptomic data revealed that Alishewanella species have evolved through horizontal gene transfer and habitat differentiation and that pectin degradation pathways in Alishewanella species are highly conserved, although stress responses of each Alishewanella species differed under pectin culture conditions.
PMCID: PMC3811190  PMID: 23934491
16.  Development of a Tunable Wide-Range Gene Induction System Useful for the Study of Streptococcal Toxin-Antitoxin Systems 
Applied and Environmental Microbiology  2013;79(20):6375-6384.
Despite the plethora of genetic tools that have been developed for use in Streptococcus mutans, the S. mutans genetic system still lacks an effective gene induction system exhibiting low basal expression and strong inducibility. Consequently, we created two hybrid gene induction cassettes referred to as Xyl-S1 and Xyl-S2. Both Xyl-S cassettes are xylose inducible and controlled by the Bacillus megaterium xylose repressor. The Xyl-S cassettes each demonstrated >600-fold-increased reporter activity in the presence of 1.2% (wt/vol) xylose. However, the Xyl-S1 cassette yielded a much higher maximum level of gene expression, whereas the Xyl-S2 cassette exhibited much lower uninduced basal expression. The cassettes also performed similarly in Streptococcus sanguinis and Streptococcus gordonii, which suggests that they are likely to be useful in a variety of streptococci. We demonstrate how both Xyl-S cassettes are particularly useful for the study of toxin-antitoxin (TA) modules using both the previously characterized S. mutans mazEF TA module and a previously uncharacterized HicAB TA module in S. mutans. HicAB TA modules are widely distributed among bacteria and archaea, but little is known about their function. We show that HicA serves as the toxin component of the module, while HicB serves as the antitoxin. Our results suggest that, in contrast to that of typical TA modules, HicA toxicity in S. mutans is modest at best. The implications of these results for HicAB function are discussed.
PMCID: PMC3811191  PMID: 23934493
17.  Comparison of PCR versus Culture for Detection of Mycobacterium bovis after Experimental Inoculation of Various Matrices Held under Environmental Conditions for Extended Periods 
Applied and Environmental Microbiology  2013;79(20):6501-6506.
The purpose of this study was to compare the performance of a molecular detection technique (nested PCR) with that of mycobacterial culture in the detection of Mycobacterium bovis DNA in a set of 687 samples of experimentally inoculated environmental substrates (hay, soil, corn, water) exposed to natural weather conditions in Michigan. Four replicates of each substrate were used; half were autoclaved for sterilization, all were inoculated with 50,000 CFU of M. bovis isolated from Michigan livestock, and all were placed in outdoor enclosures, with half under shade and the other half exposed to direct sunlight. Samples were tested for the presence of M. bovis during one 12-month period, with monthly sample testing and during three 12-week periods (winter, spring, summer) with weekly sample testing. Samples were subjected to mycobacterial culture for isolation of M. bovis and a nested PCR with two primer sets targeting IS6110 to detect M. bovis DNA. In 128 samples tested during the 12-month period, M. bovis was not detectable by culture after 2 months but M. bovis DNA was detectable by PCR for at least 7 months. Of the 559 samples tested during the 12-week periods, PCR detected M. bovis DNA for up to 88 days in all of the sample types. There were no significant differences in the detection of M. bovis between shade and sun samples or between sterile and unsterilized samples, regardless of the detection method (PCR or culture). For use in epidemiologic investigations, the PCR assay was more rapid than mycobacterial culture, was not hindered by contaminating organisms, and detected M. bovis DNA in environment samples much longer after initial contamination than mycobacterial culture did.
PMCID: PMC3811193  PMID: 23956383
18.  Functional Identification of Rubber Oxygenase (RoxA) in Soil and Marine Myxobacteria 
Applied and Environmental Microbiology  2013;79(20):6391-6399.
The rubber oxygenase (RoxA) of Xanthomonas sp. strain 35Y (RoxAXsp) is so far the only known extracellular c-type diheme cytochrome that is able to cleave poly(cis-1,4-isoprene). All other rubber-degrading bacteria described are Gram positive and employ a nonheme protein (latex-clearing protein [Lcp]) for the postulated primary attack of polyisoprene. Here, we identified RoxA orthologs in the genomes of Haliangium ochraceum, Myxococcus fulvus, Corallococcus coralloides, and Chondromyces apiculatus. The roxA orthologs of H. ochraceum (RoxAHoc), C. coralloides BO35 (RoxACco), and M. fulvus (RoxAMfu) were functionally expressed in a ΔroxA Xanthomonas sp. 35Y background. All RoxA orthologs oxidatively cleaved polyisoprene, as revealed by restoration of clearing-zone formation and detection of 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) as a cleavage product. RoxAXsp, RoxAMfu, and RoxACco were purified and biochemically characterized. The optimal temperature of RoxACco and RoxAMfu was between 22 and 30°C. All RoxA orthologs as isolated showed an oxidized UV-visible spectrum. Chemical reduction of RoxACco and RoxAMfu indicated the presence of two slightly different heme centers with absorption maxima between 549 and 553 nm, similar to RoxAXsp. Sequence analysis and modeling of the three-dimensional structures of the RoxA orthologs revealed a high degree of similarity to the recently solved RoxAXsp structure and included several conserved residues, notably, W302, F317, and a MauG motif at about H517. Lcp-like sequences were not detected in the genomes of the Xanthomonas sp. 35Y, H. ochraceum, M. fulvus, and C. coralloides. No RoxA orthologs were found in Gram-positive bacteria, and this first description of functional RoxA in Gram-negative bacteria other than Xanthomonas proves that RoxA is more common among rubber degraders than was previously assumed.
PMCID: PMC3811194  PMID: 23934498
19.  Metagenome Survey of a Multispecies and Alga-Associated Biofilm Revealed Key Elements of Bacterial-Algal Interactions in Photobioreactors 
Applied and Environmental Microbiology  2013;79(20):6196-6206.
Photobioreactors (PBRs) are very attractive for sunlight-driven production of biofuels and capturing of anthropogenic CO2. One major problem associated with PBRs however, is that the bacteria usually associated with microalgae in nonaxenic cultures can lead to biofouling and thereby affect algal productivity. Here, we report on a phylogenetic, metagenome, and functional analysis of a mixed-species bacterial biofilm associated with the microalgae Chlorella vulgaris and Scenedesmus obliquus in a PBR. The biofilm diversity and population dynamics were examined through 16S rRNA phylogeny. Overall, the diversity was rather limited, with approximately 30 bacterial species associated with the algae. The majority of the observed microorganisms were affiliated with Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes. A combined approach of sequencing via GS FLX Titanium from Roche and HiSeq 2000 from Illumina resulted in the overall production of 350 Mbp of sequenced DNA, 165 Mbp of which was assembled in larger contigs with a maximum size of 0.2 Mbp. A KEGG pathway analysis suggested high metabolic diversity with respect to the use of polymers and aromatic and nonaromatic compounds. Genes associated with the biosynthesis of essential B vitamins were highly redundant and functional. Moreover, a relatively high number of predicted and functional lipase and esterase genes indicated that the alga-associated bacteria are possibly a major sink for lipids and fatty acids produced by the microalgae. This is the first metagenome study of microalga- and PBR-associated biofilm bacteria, and it gives new clues for improved biofuel production in PBRs.
PMCID: PMC3811195  PMID: 23913425
20.  The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System 
Applied and Environmental Microbiology  2013;79(20):6312-6324.
The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate bacterial effector proteins into eukaryotic host cells. The membrane-spanning secretion apparatus consists of 11 core components and several associated proteins with yet unknown functions. In this study, we analyzed the role of HrpB1, which was previously shown to be essential for T3S and the formation of the extracellular T3S pilus. We provide experimental evidence that HrpB1 localizes to the bacterial periplasm and binds to peptidoglycan, which is in agreement with its predicted structural similarity to the putative peptidoglycan-binding domain of the lytic transglycosylase Slt70 from Escherichia coli. Interaction studies revealed that HrpB1 forms protein complexes and binds to T3S system components, including the inner membrane protein HrcD, the secretin HrcC, the pilus protein HrpE, and the putative inner rod protein HrpB2. The analysis of deletion and point mutant derivatives of HrpB1 led to the identification of amino acid residues that contribute to the interaction of HrpB1 with itself and HrcD and/or to protein function. The finding that HrpB1 and HrpB2 colocalize to the periplasm and both interact with HrcD suggests that they are part of a periplasmic substructure of the T3S system.
PMCID: PMC3811196  PMID: 23934485
21.  Functional and Expression Analysis of the Metal-Inducible dmeRF System from Rhizobium leguminosarum bv. viciae 
Applied and Environmental Microbiology  2013;79(20):6414-6422.
A gene encoding a homolog to the cation diffusion facilitator protein DmeF from Cupriavidus metallidurans has been identified in the genome of Rhizobium leguminosarum UPM791. The R. leguminosarum dmeF gene is located downstream of an open reading frame (designated dmeR) encoding a protein homologous to the nickel- and cobalt-responsive transcriptional regulator RcnR from Escherichia coli. Analysis of gene expression showed that the R. leguminosarum dmeRF genes are organized as a transcriptional unit whose expression is strongly induced by nickel and cobalt ions, likely by alleviating the repressor activity of DmeR on dmeRF transcription. An R. leguminosarum dmeRF mutant strain displayed increased sensitivity to Co(II) and Ni(II), whereas no alterations of its resistance to Cd(II), Cu(II), or Zn(II) were observed. A decrease of symbiotic performance was observed when pea plants inoculated with an R. leguminosarum dmeRF deletion mutant strain were grown in the presence of high concentrations of nickel and cobalt. The same mutant induced significantly lower activity levels of NiFe hydrogenase in microaerobic cultures. These results indicate that the R. leguminosarum DmeRF system is a metal-responsive efflux mechanism acting as a key element for metal homeostasis in R. leguminosarum under free-living and symbiotic conditions. The presence of similar dmeRF gene clusters in other Rhizobiaceae suggests that the dmeRF system is a conserved mechanism for metal tolerance in legume endosymbiotic bacteria.
PMCID: PMC3811197  PMID: 23934501
22.  Effects of Inoculum Additions in the Presence of a Preestablished Arbuscular Mycorrhizal Fungal Community 
Applied and Environmental Microbiology  2013;79(20):6507-6515.
Communities of arbuscular mycorrhizal fungi (AMF) are crucial for promoting plant productivity in most terrestrial systems, including anthropogenically managed ecosystems. Application of AMF inocula has therefore become a widespread practice. It is, however, pertinent to understand the mechanisms that govern AMF community composition and their performance in order to design successful manipulations. Here we assess whether the composition and plant growth-promotional effects of a synthetic AMF community can be altered by inoculum additions of the isolates forming the community. This was determined by following the effects of three AMF isolates, each inoculated in two propagule densities into a preestablished AMF community. Fungal abundance in roots and plant growth were evaluated in three sequential harvests. We found a transient positive response in AMF abundance to the intraspecific inoculation only in the competitively weakest isolate. The other two isolates responded negatively to intra- and interspecific inoculations, and in some cases plant growth was also reduced. Our results suggest that increasing the AMF density may lead to increased competition among fungi and a trade-off with their ability to promote plant productivity. This is a key ecological aspect to consider when introducing AMF into soils.
PMCID: PMC3811198  PMID: 23956395
23.  Assessment of Survival and Body Size Variation of Culicoides imicola (Diptera: Ceratopogonidae) as Functions of “Candidatus Cardinium” (Bacteroidetes) Infection Status 
Applied and Environmental Microbiology  2013;79(20):6260-6263.
“Candidatus Cardinium hertigii” (Bacteroidetes) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of “Candidatus Cardinium” infection was documented as moderate, with no detectable sex bias. We therefore investigated whether “Candidatus Cardinium” affects important fitness parameters, such as survival and body size, in Culicoides imicola, a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account “Candidatus Cardinium” infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with “Candidatus Cardinium” infection data. The findings revealed no difference in survival of Culicoides infected with “Candidatus Cardinium” and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for “Candidatus Cardinium”-infected versus uninfected midges. In aggregate, these findings support our conclusion that “Candidatus Cardinium” does not have an overt effect on the survival and size of adult C. imicola midges. “Candidatus Cardinium” may affect immature stages or may alter adult reproductive performance.
PMCID: PMC3811199  PMID: 23913434
24.  Microgradients of pH Do Not Occur around Lactococcus Colonies in a Model Cheese 
Applied and Environmental Microbiology  2013;79(20):6516-6518.
Lactococci inoculated into cheese grow as colonies producing lactic acid. The pH microgradients were investigated around colonies in a complex food such as cheese. The results, obtained using a nondestructive technique, demonstrated that pH microgradients did not occur regardless of the acidification kinetics and the size of the colony.
PMCID: PMC3811200  PMID: 23934499
25.  Carbonylation as a Key Reaction in Anaerobic Acetone Activation by Desulfococcus biacutus 
Applied and Environmental Microbiology  2013;79(20):6228-6235.
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.
PMCID: PMC3811201  PMID: 23913429

Results 1-25 (28925)