Biomedical subjects

J M Tiedje

Publications and source records attributed to J M Tiedje.

At least 19 recordsLinked to original sources

PubMed2001

DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes.

DNA microarrays constructed with full length ORFs from Shewanella oneidensis, MR-1, were hybridized with genomic DNA from nine other Shewanella species and Escherichia coli K-12. This approach enabled visualization of relationships between organisms by comparing individual ORF hybridizations to 164 genes and is further amenable to high-density high-throughput analyses of complete microbial genomes. Conserved genes (arcA and ATP synthase) were identified among all species investigated. The mtr operon, which is involved in iron reduction, was poorly conserved among other known metal-reducing Shewanella species. Results were most informative for closely related organisms with small subunit rRNA sequence similarities greater than 93% and gyrB sequence similarities greater than 80%. At this level of relatedness, the similarity between hybridization profiles was strongly correlated with sequence divergence in the gyrB gene. Results revealed that two strains of S. oneidensis (MR-1 and DLM7) were nearly identical, with only 3% of the ORFs hybridizing poorly, in contrast to hybridizations with Shewanella putrefaciens, formerly considered to be the same species as MR-1, in which 63% of the ORFs hybridized poorly (log ratios below -0.75). Genomic hybridizations showed that genes in operons had consistent levels of hybridization across an operon in comparison to a randomly sampled data set, suggesting that similar applications will be informative for identification of horizontally acquired genes. The full value of microbial genomic hybridizations lies in providing the ability to understand and display specific differences between closely related organisms providing a window into understanding microheterogeneity, bacterial speciation, and taxonomic relationships.

Bacterial Proteins↗

PubMed2001

Development of a Rhodococcus recombinant strain for degradation of products from anaerobic dechlorination of PCBs.

The Gram-positive bacterium Rhodococcus sp. strain RHA1, naturally containing the biphenyl pathway, was electroporated with a broad host range plasmid containing the 4-chlorobenzoate (4-CBA) degradation operon (fcb) isolated from Arthrobacter globiformis strain KZT1. The recombinant strain grew in medium containing 4-CBA and 4-chlorobiphenyl (4-CB) as the only source of carbon, with stoichiometric release of chloride and a molar growth yield on 4-CB that suggested utilization of both biphenyl rings. In resting cell assays, similar rates of degradation were observed for wild-type and recombinant strains for the most common eight congeners from the anaerobic dechlorination of Aroclor 1242, but the recombinant strain accumulated lower amounts of chlorinated meta-cleavage products and no 4-CBA. Recombinant cells inoculated at 10(4) cells/g into nonsterile soil amended with 4-CB grew to 6-10(5) cells/g, a density consistent with the 4-CB consumed. 4-CB was removed only in the inoculated soil, and the recombinant strain did not grow in the same soil when it was not amended with 4-CB. The fcb operon remained stable in the recombinant strain reisolated from soil after 60 days. This work provides proof of concept that a Rhodococcus strain constructed to grow on a PCB would grow in nonsterile soil if the appropriate chlorobiphenyl is available.

Biodegradation, Environmental↗

PubMed2001

Factors controlling the rate of DDE dechlorination to DDMU in Palos Verdes margin sediments under anaerobic conditions.

Marine sediments off the coast of the Palos Verdes Peninsula in California have been designated a Superfund site primarily because of the presence of DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethene]. For decades, it was believed that DDE was not microbially transformed, but anaerobic bacteria in the Palos Verdes sediments reductively dechlorinate DDEto DDMU [1-chloro-2,2-bis(p-chlorophenyl)ethene], which is also found in the sediments. The effects of electron donor to sulfate ratio, available carbon, sampling sites, sediment depth, and temperature on the rate and extent of DDE dechlorination in anaerobic Palos Verdes sediment microcosms were investigated. Dechlorination rates varied, depending on the site and depth from which the sediments were collected, but DDE dechlorination occurred with sediments from all locations studied. Sulfate and low temperatures slowed dechlorination, but in the presence of sulfate and at in situ temperature, the dechlorination rates observed in the microcosms agree well with the observed rate of DDE disappearance from the Palos Verdes margin sediments.

California↗

PubMed2001

The RDP-II (Ribosomal Database Project).

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [Nucleic Acids Res. (2000), 28, 173-174], continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 8.0 (June 1, 2000) consisted of 16 277 aligned prokaryotic small subunit (SSU) rRNA sequences while the number of eukaryotic and mitochondrial SSU rRNA sequences in aligned form remained at 2055 and 1503, respectively. The number of prokaryotic SSU rRNA sequences more than doubled from the previous release 14 months earlier, and approximately 75% are longer than 899 bp. An RDP-II mirror site in Japan is now available (http://wdcm.nig.ac.jp/RDP/html/index.h tml). RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/). Analysis services include rRNA probe checking, approximate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment polymorphism experiments. The RDP-II email address for questions and comments has been changed from curator@cme.msu.edu to rdpstaff@msu.edu.

Databases, Factual↗

PubMed2001

The impact of fermentative organisms on carbon flow in methanogenic systems under constant low-substrate conditions.

We compared carbon flow under constant low-substrate conditions (below 20 microM glucose in situ) in laboratory-scale glucose-fed methanogenic bioreactors containing two very different microbial communities that removed chemical oxygen demand at similar rates. One community contained approximately equal proportions of spiral and cocci morphologies, while the other community was dominated by cocci. In the former bioreactor, over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Spirochaetaceae-related sequences, while in the latter bioreactor over 50% of the cloned SSU rRNA genes and the most common SSU rDNA terminal restriction fragment corresponded to Streptococcus-related sequences. Carbon flow was assessed by measuring 14C-labeled metabolites derived from a feeding of [U-14C]glucose that did not alter the concentration of glucose in the bioreactors. Acetate and ethanol were detected in the Spirochaetaceae-dominated reactor, whereas acetate and propionate were detected in the Streptococcus-dominated reactor. A spirochete isolated from a Spirochaetaceae-dominated reactor fermented glucose to acetate, ethanol, and small amounts of lactate. Maximum substrate utilization assays carried out on fluid from the same reactor indicated that acetate and ethanol were rapidly utilized by this community. These data indicate that an acetate- and ethanol-based food chain was present in the Spirochaetaceae-dominated bioreactor, while the typical acetate- and propionate-based food chain was prevalent in the Streptococcus-dominated bioreactor.

Acetates↗

PubMed2001

Desulfomonile limimaris sp. nov., an anaerobic dehalogenating bacterium from marine sediments.

Strains DCB-MT and DCB-F were isolated from anaerobic 3-chlorobenzoate (3CB)-mineralizing cultures enriched from marine sediments. The isolates are large, Gram-negative rods with a collar girdling each cell. The isolates are obligate anaerobes capable of reductive dechlorination of 3CB to benzoate. Growth by chlororespiration in strain DCB-MT yielded 1.7 g protein mol(-1) 3CB dechlorinated with lactate as the electron donor. Strain DCB-MT also used fumarate, sulfate, sulfite, thiosulfate and nitrate as physiological electron acceptors for growth, but grew poorly on sulfate and nitrate. Reductive dechlorination was inhibited completely by sulfite and thiosulfate but not by sulfate. Both strains were incapable of growth at NaCl concentrations below 0.32% (w/v). They grew well at sea-water salt concentrations; however, the optimum growth rate was achieved at a NaCl concentration half that of sea water. The 16S rDNA sequence analysis shows strains DCB-MT and DCB-F to be 99% similar to each other and 93% similar to their closest relative, Desulfomonile tiedjei strain DCB-1T. Strain DCB-MT can also be distinguished from strain DCB-1T by its inability to use acetate for growth on 3CB and by its requirement for NaCl. The morphology, physiology and 16S rDNA sequences of DCB-MT and DCB-F suggest that these strains represent a new, marine-adapted species of the genus Desulfomonile, designated Desulfomonile limimaris sp. nov. The type strain is strain DCB-MT (= ATCC 700979T).

Chlorobenzoates↗

PubMed2001

Simultaneous recovery of RNA and DNA from soils and sediments.

Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316-322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0 degrees C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 microg g of soil(-1) dry weight), whereas the DNA yield ranged from 23 to 435 microg g(-1). In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.

DNA, Bacterial↗

PubMed2001

Pseudomonas stutzeri nitrite reductase gene abundance in environmental samples measured by real-time PCR.

We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 10(6) gene copies (r2 = 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. Although P. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier.

DNA, Bacterial↗

PubMed2001

Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning.

To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.

Artifacts↗

PubMed2001

Community structure of denitrifiers, bacteria, and archaea along redox gradients in Pacific Northwest marine sediments by terminal restriction fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes.

Steep vertical gradients of oxidants (O(2) and NO(3)(-)) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096-2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.

Archaea↗

PubMed2001

Bacterial species determination from DNA-DNA hybridization by using genome fragments and DNA microarrays.

Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.

DNA, Bacterial↗

PubMed2000

Use of non-porous reversed-phase high-performance liquid chromatography for protein profiling and isolation of proteins induced by temperature variations for Siberian permafrost bacteria with identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry.

Non-porous reversed-phase high-performance liquid chromatography (NP-RP-HPLC) has been used to separate and isolate proteins from whole cell lysates of ED 7-3, a bacterium from the buried Siberian permafrost sediment. The proteins collected from the liquid eluent of this separation were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS). In order to study the differences in expression of cold-shock proteins (CSPs) at different growth temperatures, cultures of the ED 7-3 strain were prepared at 4 degrees C and 25 degrees C. The goals of this work were twofold: firstly, to identify the presence of CSPs and other proteins that are highly expressed at 4 degrees C but not at 25 degrees C; and secondly, to isolate these proteins for MALDI-TOF-MS and CE-ESI-MS identification. In this initial work, distinct protein profiles were observed for these cultures as a function of temperature. Fraction collection from the eluent of NP-RP-HPLC of some of the highly expressed proteins was performed and the proteins were mass analyzed for molecular mass. Peptide maps of the proteins were generated by tryptic digestion and were analyzed by CE-ESI-MS and MALDI-TOF-MS for database identification of the expressed proteins.

Amino Acid Sequence↗

PubMed2000

The RDP (Ribosomal Database Project) continues.

The Ribosomal Database Project (RDP-II), previously described by Maidak et al., continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 7.1 (September 17, 1999) included more than 10 700 small subunit rRNA sequences. More than 850 type strain sequences were identified and added to the prokaryotic alignment, bringing the total number of type sequences to 3324 representing 2460 different species. Availability of an RDP-II mirror site in Japan is also near completion. RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/ ). Analysis services include rRNA probe checking, approx-i-mate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment length polymorphism (T-RFLP) experiments.

Databases, Factual↗

PubMed2000

Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments.

Permafrost represents a unique ecosystem that has allowed the prolonged survival of certain bacterial lineages at subzero temperatures. To better understand the permafrost microbial community, it is important to identify isolation protocols that optimize the recovery of genetically diverse bacterial lineages. We have investigated the impact of different low-temperature isolation protocols on recovery of aerobic bacteria from northeast Siberian permafrost of variable geologic origin and frozen for 5000 to 3 million years. Low-nutrient media enhanced the quantitative recovery of bacteria, whereas the isolation of diverse morphotypes was maximized on rich media. Cold enrichments done directly in natural, undisturbed permafrost led not only to recovery of increased numbers of bacteria but also to isolation of genotypes not recovered by means of liquid low-temperature enrichments. On the other hand, direct plating and growth at 4 degrees C also led to recovery of diverse genotypes, some of which were not recovered following enrichment. Strains recovered from different permafrost samples were predominantly oligotrophic and non-spore-forming but were otherwise variable from each other in terms of a number of bacteriological characteristics. Our data suggest that a combination of isolation protocols from different permafrost samples should be used to establish a culture-based survey of the different bacterial lineages in permafrost.

Bacteria↗

PubMed2000

Leifsonia poae gen. nov., sp. nov., isolated from nematode galls on Poa annua, and reclassification of 'Corynebacterium aquaticum' Leifson 1962 as Leifsonia aquatica (ex Leifson 1962) gen. nov., nom. rev., comb. nov. and Clavibacter xyli Davis et al. 1984 with two subspecies as Leifsonia xyli (Davis et al. 1984) gen. nov., comb. nov.

The new genus Leifsonia gen. nov. with two new species, Leifsonia poae sp. nov. (type strain VKM Ac-1401T) and Leifsonia aquatica (ex Leifson 1962) nom. rev., comb. nov. (the type species, with VKM Ac-1400T = DSM 20146T = JCM 1368T as type strain), is proposed to accommodate bacteria found in Poa annua root gall, induced by the nematode Subanguina radicicola, and 'Corynebacterium aquaticum' Leifson 1962. Further, it is proposed to reclassify Clavibacter xyli Davis et al. 1984 with two subspecies in the new genus as Leifsonia xyli (Davis et al. 1984) comb. nov., Leifsonia xyli subsp. xyli (Davis et al. 1984) comb. nov. and Leifsonia xyli subsp. cynodontis (Davis et al. 1984) comb. nov. Members of the proposed genus are characterized by coryneform morphology, peptidoglycans based upon 2,4-diaminobutyric acid, the major menaquinone MK-11, phosphatidylglycerol and diphosphatidylglycerol as principal phospholipids, the high content of anteiso- and iso-branched saturated fatty acids, and a DNA G+C base composition of 66-73 mol%. They form a distinct phylogenetic branch attached to the line of descent of Agromyces spp. The new and reclassified species of the new genus clearly differ from each other phylogenetically and phenetically and can be recognized by their morphologies, the cell wall sugar composition, the requirement of complex media for growth, and numerous physiological characteristics, including the oxidase reaction.

Actinomycetales↗

PubMed2000

Nocardiopsis tropica sp. nov., Nocardiopsis trehalosi sp. nov., nom. rev. and Nocardiopsis dassonvillei subsp. albirubida subsp. nov., comb. nov.

Sixteen strains isolated from different soils and marine sediments and 'Nocardiopsis trehalosi' VKM Ac-942T were subjected to taxonomic analyses. The morphological and chemotaxonomic characteristics of the strains matched those described for members of the Nocardiopsis. Based on 16S rDNA sequence analysis, DNA-DNA hybridization values and phenotypic characteristics, including the composition of menaquinones and cell wall teichoic acids, two new species are proposed, Nocardiopsis tropica sp. nov. (type strain VKM Ac-1457T) and Nocardiopsis trehalosi sp. nov., nom. rev. (type strain VKM Ac-942T = JCM 3357T = NRRL 12026T). Nocardiopsis dassonvillei VKM Ac-1882T (formerly Nocardiopsis albirubida) differs from other strains of this species by the presence of MK-10 as the major menaquinone, cell wall teichoic acid structure, and numerical analysis based on 115 growth and physiological characteristics. The strain is reclassified as the subspecies Nocardiopsis dassonvillei subsp. albirubida, with VKM Ac-1882T (= DSM 43465T = ATCC 23612T = ISP 5465T) designated the type.

Actinomycetales↗

PubMed2000

Methylocella palustris gen. nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-pathway methanotrophs.

A new genus, Methylocella, and a new species, Methylocella palustris, are proposed for three strains of methane-oxidizing bacteria isolated from acidic Sphagnum peat bogs. These bacteria are aerobic, Gram-negative, colourless, non-motile, straight and curved rods that utilize the serine pathway for carbon assimilation, multiply by normal cell division and contain intracellular poly-beta-hydroxybutyrate granules (one at each pole). These strains use methane and methanol as sole sources of carbon and energy and are moderately acidophilic organisms with growth between pH 4.5 and pH 7.0, the optimum being at pH 5.0-5.5. The temperature range for growth is 10-28 degrees C with the optimum at 15-20 degrees C. The intracytoplasmic membrane system is different from those of type I and II methanotrophs. Cells contain an extensive periplasmic space and a vesicular membrane system connected to the cytoplasmic membrane. The strains grew only on media with a low salt content (0.2-0.5 g l(-1)). All three strains were found to possess soluble methane monooxygenase and are able to fix atmospheric nitrogen via an oxygen-sensitive nitrogenase. No products were observed in a PCR with particulate methane monooxygenase-targeted primers; hybridization with a pmoA probe was also negative. The major phospholipid fatty acids are 18:1 acids. The G+C content of the DNA is 61.2 mol%. The three strains share identical 16S rRNA gene sequences and represent a novel lineage of methane-oxidizing bacteria within the alpha-subclass of the class Proteobacteria and are only moderately related to type II methanotrophs of the Methylocystis-Methylosinus group. The three strains are most closely related to the acidophilic heterotrophic bacterium Beijerinckia indica subsp. indica (96.5% 16S rDNA sequence similarity). Collectively, these strains comprise a new species and genus Methylocella palustris gen. nov., sp. nov.; strain KT (= ATCC 700799T) is the type strain.

Base Composition↗

PubMed2000

Pacific Northwest marine sediments contain ammonia-oxidizing bacteria in the beta subdivision of the Proteobacteria.

The diversity of ammonia-oxidizing bacteria in aquatic sediments was studied by retrieving ammonia monooxygenase and methane monooxygenase gene sequences. Methanotrophs dominated freshwater sediments, while beta-proteobacterial ammonia oxidizers dominated marine sediments. These results suggest that gamma-proteobacteria such as Nitrosococcus oceani are minor members of marine sediment ammonia-oxidizing communities.

Ammonia↗