Biomedical subjects

Allan Konopka

Publications and source records attributed to Allan Konopka.

11 recordsLinked to original sources

PubMed2006

Optimization of MALDI-TOF MS for strain level differentiation of Arthrobacter isolates.

Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to be a rapid and sensitive method for characterization of bacteria, but it has not yet become a routine microbiological procedure. Currently there are no standardized protocols that would allow development of large libraries of reproducible protein profiles from a broad range of microorganisms to use for identification purposes. Important variables that may affect spectrum quality are MALDI matrices, solvents, cell growth condition, and culture age. In the present study our aim was to: (1) to determine optimal sample preparation and MALDI conditions for discrimination at the strain level; (2) to determine if changes in growth cycle correlated with MALDI spectrum changes; and (3) to compare level of isolate discrimination based on their MALDI spectra versus their 16S rRNA gene sequence. Using 16 strains of the Gram positive bacterium Arthrobacter, optimal spectra were obtained using two-layer sample application of intact cells grown on solid surface overlaid with a matrix consisting of sinapinic acid (SA) or alpha-cyano-hydroxy-cinnaminic acid (CHCA) in 50:50 acetonitrile:water solvent with 2% trifluoroacetic acid. Spectrum changes paralleled the coccus-rod-coccus growth cycle indicative of Arthrobacter. Strain differences based on their MALDI profiles (using Pearson coefficient and UPGMA) corresponded with their 16S rRNA gene phylogeny but it had greater discrimination.

Arthrobacter↗

PubMed2006

Bacterial activity, community structure, and centimeter-scale spatial heterogeneity in contaminated soil.

In an anthropogenically disturbed soil (88% sand, 8% silt, 4% clay), 150-mg samples were studied to examine the fine-scale relationship of bacterial activity and community structure to heavy metal contaminants. The soils had been contaminated for over 40 years with aromatic solvents, Pb, and Cr. Samples from distances of <1, 5, 15, and 50 cm over a depth range of 40-90 cm underwent a sequential analysis to determine metabolic potential (from 14C glucose mineralization), bacterial community structure [using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)], and total extractable Pb and Cr levels. Metabolic potential varied by as much as 10,000-fold in samples <1 cm apart; log-log plots of metal concentration and microbial metabolic potential showed no correlation with each other. Overall, metal concentrations ranged from 9 to 29,000 mg kg(-1) for Pb and from 3 to 8500 mg kg(-1) for Cr with small zones of high contamination present. All regions exhibited variable metal concentrations, with some soil samples having 30-fold differences in metal concentration in sites <1 cm apart. Geostatistical analysis revealed a strong spatial dependence for all three parameters tested (metabolic activity, Pb, and Cr levels) with a range up to 30 cm. Kriging maps showed that in zones of high metal, the corresponding metabolic activity was low suggesting that metals negatively impacted the microbial community. PCR-DGGE analysis revealed that diverse communities were present in the soils with a random distribution of phylotypes throughout the sampling zones. These results suggest the presence of spatially isolated microbial communities within the soil profile.

Bacteria↗

PubMed2006

Microbial community analysis of soils contaminated with lead, chromium and petroleum hydrocarbons.

The impact on the microbial community of long-term environmental exposure to metal and organic contamination was investigated. Twenty-four soil samples were collected along a transect dug in soils contaminated with road paint and paint solvents, mainly toluene. Chemical analysis along the transect revealed a range from high to low concentrations of metals (lead and chromium) and organic solvent compounds. Principal components analysis of microbial community structure based on denaturing gradient gel electrophoresis of the V3 region of the 16S rRNA gene and fatty acid methyl esters derived from phospholipids (phospholipid fatty acid analysis) showing samples with similar fingerprints also had similar contaminant concentrations. There was also a weak positive correlation between microbial biomass and the organic carbon concentration. Results indicated that microbial populations are present despite some extreme contaminant levels in this mixed-waste contaminated site. Nucleotide sequence determination of the 16S rRNA gene indicated the presence of phylogenetically diverse bacteria belonging to the alpha-, beta-, gamma-, and delta-Proteobacteria, the high and low G + C Gram-positive bacteria, green nonsulfur, OP8, and others that did not group within a described division. This indicates that soils contaminated with both heavy metals and hydrocarbons for several decades have undergone changes in community composition, but still contain a phylogenetically diverse group of bacteria (including novel phylotypes) that warrant further investigation.

Bacteria↗

PubMed2006

Microbial community dynamics in nutrient-pulsed chemostats.

In nature, microbes are subject to nutrient fluxes. As the periodicity of nutrient flux lengthens, different physiological traits may be selected. The competitive exclusion principle stipulates that one organism will dominate these systems; however, interspecies interactions may produce a dynamic microbial community. These issues were investigated in chemostats pulsed with gelatin. Chemostats were run over 30 days with substrate addition continuously or at intervals of 0.5, 1 or 3 days. Growth rates were similar between pulse intervals. Ectoaminopeptidase activity levels remained relatively constant within a pulse interval. Bacterial community structure was monitored using denaturing gradient gel electrophoresis of PCR products of the 16S rRNA gene. There were dynamic changes at all periodicities; however, the pace of these changes decreased over time. Final communities were not identical between different treatments. The structure of persistent vs. active microbial populations was compared by denaturing gradient gel electrophoresis of the PCR and reverse transcriptase-PCR amplicons of 16S rDNA and rRNA templates, respectively. For all the chemostats, the rRNA profiles were not identical to the rDNA profiles for a sample. These experiments demonstrate that complex community dynamics can occur under environmental heterogeneities that are modest relative to those found in natural aquatic habitats. Furthermore, the physiological functionality of these dynamic communities was stable.

Aminopeptidases↗

PubMed2006

Responses of the anaerobic bacterial community to addition of organic C in chromium(VI)- and iron(III)-amended microcosms.

Chromium (VI) is toxic to microorganisms and can inhibit the biodegradation of organic pollutants in contaminated soils. We used microcosms amended with either glucose or protein (to drive bacterial community change) and Fe(III) (to stimulate iron-reducing bacteria) to study the effect of various concentrations of Cr(VI) on anaerobic bacterial communities. Microcosms were destructively sampled based on microbial activity (measured as evolution of CO2) and analyzed for the following: (i) dominant bacterial community by PCR-denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene; (ii) culturable Cr-resistant bacteria; and (iii) enrichment of iron-reducing bacteria of the Geobacteraceae family by real-time PCR. The addition of organic C stimulated the activities of anaerobic communities. Cr(VI) amendment resulted in lower rates of CO2 production in glucose microcosms and a slow mineralization phase in protein-amended microcosms. Glucose and protein amendments selected for different bacterial communities. This selection was modified by the addition of Cr(VI), since some DGGE bands were intensified and new bands appeared in Cr(VI)-amended microcosms. A second dose of Cr(VI), added after the onset of activity, had a strong inhibitory effect when higher levels of Cr were added, indicating that the developing Cr-resistant communities had a relatively low tolerance threshold. Most of the isolated Cr-resistant bacteria were closely related to previously studied Cr-resistant anaerobes, such as Pantoea, Pseudomonas, and Enterobacter species. Geobacteraceae were not enriched during the incubation. The studied Cr(VI)-contaminated soil contained a viable anaerobic bacterial community; however, Cr(VI) altered its composition, which could affect the soil biodegradation potential.

Carbon↗

PubMed2006

Effect of nutrient periodicity on microbial community dynamics.

When microbes are subjected to temporal changes in nutrient availability, growth rate and substrate affinity can contribute to competitive fitness and thereby affect microbial community structure. This hypothesis was tested using planktonic bacterial communities exposed to nutrient additions at 1-, 3-, 7-, or 14-day intervals. Growth rates after nutrient addition were inversely proportional to the pulse interval and declined from 0.5 h(-1) to 0.15 h(-1) as the pulse interval increased from 1 to 14 days. The dynamics of community structure were monitored by 16S rRNA gene PCR-denaturing gradient gel electrophoresis. At pulse intervals of more than 1 day, the community composition continued to change over 130 days. Although replicate systems exposed to the same pulse interval were physiologically similar, their community compositions could exhibit as much dissimilarity (Dice similarity coefficients of <0.5) as did systems operated at different intervals. Bacteria were cultivated from the systems to determine if the physiological characteristics of individual members were consistent with the measured performance of the systems. The isolates fell into three bacterial divisions, Bacteroidetes, Proteobacteria, and Actinobacteria. In agreement with community results, bacteria isolated from systems pulsed every day with nutrients had higher growth rates and ectoaminopeptidase specific activities than isolates from systems pulsed every 14 days. However, the latter isolates did not survive starvation longer than those provided with nutrients every day. The present study demonstrates the dynamic nature of microbial communities exposed to even simple and regular environmental discontinuities when a substantial pool of species that can catabolize the limiting substrate is present.

Actinobacteria↗

PubMed2005

Microbial catabolic diversity in soils contaminated with hydrocarbons and heavy metals.

Understanding indigenous microbial function in contaminated soil is crucial to the successful development and use of bioremediation technologies. We measured the catabolic diversity of indigenous microbial communities in soils with a 30-yr history of Pb, Cr, and hydrocarbon (HC) contamination using a modified substrate-induced respiration method. There were characteristic differences of microbial respirations in the response of highly versus less contaminated soils to the range of organic substrates used. The catabolic response to glucose as compared to succinic acid was approximately 1:5 in less contaminated soils, but 1:25 in highly contaminated soils. In contrast, the response ratio to glucose versus aromatics was about 1:0.4 in less contaminated soils and 1:1 in highly contaminated soils. Principal components analysis (PCA) of the responses confirmed that catabolic diversity differed between highly and less contaminated soils. Univariate analysis also indicated that catabolic diversity was reduced in highly contaminated soils. This catabolic difference was strongly associated with the alteration of microbial community composition. Statistical analyses suggested that the variation in microbial community catabolic diversity was attributed to HCs more than to Pb and Cr.

Bacteria↗

PubMed2005

Soil microbial community responses to additions of organic carbon substrates and heavy metals (Pb and Cr).

Microcosm experiments were conducted with soils contaminated with heavy metals (Pb and Cr) and aromatic hydrocarbons to determine the effects of each upon microbial community structure and function. Organic substrates were added as a driving force for change in the microbial community. Glucose represented an energy source used by a broad variety of bacteria, whereas fewer soil species were expected to use xylene. The metal amendments were chosen to inhibit the acute rate of organic mineralization by either 50% or 90%, and lower mineralization rates persisted over the entire 31-day incubation period. Significant biomass increases were abolished when metals were added in addition to organic carbon. The addition of organic carbon alone had the most significant impact on community composition and led to the proliferation of a few dominant phylotypes, as detected by PCR-denaturing gradient gel electrophoresis of bacterial 16S rRNA genes. However, the community-wide effects of heavy metal addition differed between the two carbon sources. For glucose, either Pb or Cr produced large changes and replacement with new phylotypes. In contrast, many phylotypes selected by xylene treatment were retained when either metal was added. Members of the Actinomycetales were very prevalent in microcosms with xylene and Cr(VI); gene copy numbers of biphenyl dioxygenase and phenol hydroxylase (but not other oxygenases) were elevated in these microcosms, as determined by real-time PCR. Much lower metal concentrations were needed to inhibit the catabolism of xylene than of glucose. Cr(VI) appeared to be reduced during the 31-day incubations, but in the case of glucose there was substantial microbial activity when much of the Cr(VI) remained. In the case of xylene, this was less clear.

Actinomycetales↗

PubMed2002

Aciduric Proteobacteria isolated from pH 2.9 soil.

Acidic (pH 2.9) soil was used as an inoculum to culture heterotrophic bacteria at pH values of 3-4. Four isolates were obtained; on the basis of 16S rDNA sequence, they were shown to be members of the beta- and gamma-Proteobacteria. The three isolates that were most closely related to Burkholderia spp. had simple nutritional requirements and could grow in glucose-mineral salts media; two of these used a broad array of organic substrates. The 16S rDNA sequence of the fourth isolate was most similar (96%) to Frateuria aurantia. The isolates were aciduric rather than acidophilic; their pH ranges for growth were approximately 3.5-8. Unlike many bacteria whose acid tolerance represents the capacity to survive acid exposure, these microorganisms carried out exponential growth at pH<4 and their growth rates at pH 3.9 ranged from 60 to 98% of those found at pH 7. The cell yields on glucose of two strains were identical at pH 4 and pH 7. The acidic soils appeared to contain a very diverse bacterial community as assessed by denaturing gradient gel electrophoresis fingerprinting of PCR amplicons of a portion of the 16S rDNA gene. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-002-0427-1.

Culture Media↗

PubMed2002

Evaluation of methods to solubilize and analyze cell-associated ectoenzymes.

To quantify the functional diversity of microbes that use hydrolytic ectoenzymes, the feasibility of separating cell-associated proteins on polyacrylamide gels and detecting enzyme activity via fluorescent substrate analogs for aminopeptidases, glucosidases, and esterases was determined. More than 87% of aminopeptidase activity was associated with particulate cell material in all of the 10 Gram-negative bacterial strains that were investigated. Although 7-amino-4-methylcoumarin-leucine (AMC-leucine) provided high activities after incubation with eight strains from the Cytophaga-Flexibacter-Bacteroides group, very poor responses were noted with two gamma Proteobacteria that grew well on protein. Therefore, this molecule was not a universal substrate for aminopeptidases. Methods of increasing stringency were evaluated to release enzyme activity from particulate material. Some methods (treatment with 0.1% Triton X-100) gave good results with some but not all strains. Cell lysis by shearing produced the most consistent results. Ectoenzyme activities could be localized on polyacrylamide gels using fluorescent substrate analogs. However, some activities were located in high molecular weight complexes, and methods that disrupted these complexes (such as treatment with sodium dodecyl sulfate at elevated temperature) destroyed enzyme activity. In addition, the enzymes from different strains showed the same electrophoretic mobility. Therefore, the analysis of functional diversity by this approach is limited by the difficulty in solubilizing particulate enzymes under conditions where they retain activity.

Aminopeptidases↗

PubMed2002

Analyses of microbial activity in biomass-recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products.

The relationship between mixed microbial community structure and physiology when grown under substrate-limited conditions was investigated using continuous-flow bioreactors with 100% biomass recycle. Community structure was analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR and RT-PCR amplified V3 region of 16S rDNA and 16S rRNA templates, respectively. Comparisons were made of communities exposed to different types of transient conditions (e.g., long- and short-term starvation, increasing nutrients). With progressively more stringent substrate limitation over time, the specific content of community RNA declined by more than 10-fold and closely followed the decline in specific growth rate. In contrast, the DNA content was variable (up to 3-fold differences) and did not follow the same trend. Cluster analysis of the presence or absence of individual bands indicated that the fingerprints generated by the two templates were different, and community response was first observed in the rRNA fraction. However, both the rDNA and rRNA fingerprints provided a picture of temporal population dynamics. Dice similarity coefficients gave a quantitative measure of the differences and changes between the communities. In comparison, standard cultivation techniques yielded only a quarter of the phylotypes detected by DGGE, but included the most dominant population based on rRNA. Nucleotide-sequence analyses of the almost complete 16S rRNA genes of these isolates place them in the same group of organisms that is typically cultivated from environmental samples: alpha, beta, and gamma Proteobacteria and the high GC and the low GC Gram-positive divisions.

Bacteria↗