Biomedical subjects

Mary E Lidstrom

Publications and source records attributed to Mary E Lidstrom.

At least 19 recordsLinked to original sources

PubMed2006

Physiological analysis of Methylobacterium extorquens AM1 grown in continuous and batch cultures.

Chemostat cultures of Methylobacterium extorquens AM1 grown on methanol or succinate at a range of dilution rates were compared to batch cultures in terms of enzyme levels, poly-beta-hydroxybutyrate content, and intracellular concentrations of adenine and pyridine nucleotides. In both chemostat and batch cultures, enzymes specific to C1 metabolism were up-regulated during growth on methanol and down-regulated during growth on succinate, polyhydroxybutyrate levels were higher on succinate, intracellular ATP levels and the energy charge were higher during growth on methanol, while the pools of reducing equivalents were higher during growth on succinate. For most of the tested parameters, little alteration occurred in response to growth rate. Overall, we conclude that the chemostat cultivation conditions developed in this study roughly mimic the growth in batch cultures, but provide a better control over the culturing conditions and a better data reproducibility, which are important for integrative functional studies. This study provides baseline data for future work using chemostat cultures, defining key similarities and differences in the physiology compared to existing batch culture data.

Adenine Nucleotides↗

PubMed2006

Development of a defined medium supporting rapid growth for Deinococcus radiodurans and analysis of metabolic capacities.

A morpholinepropanesulfonic acid (MOPS)-buffered rich defined medium (RDM) was optimized to support a reproducible 2.6-h doubling time at 35 degrees C for Deinococcus radiodurans R1 and used to gain insight into vitamin and carbon metabolism. D. radiodurans was shown to require biotin and niacin for growth in this medium. A glutamine-serine simple defined medium (SDM) was developed that supported a 4-h doubling time, and this medium was used to probe sulfur and methionine metabolism. Vitamin B(12) was shown to alleviate methionine auxotrophy, and under these conditions, sulfate was used as the sole sulfur source. Phenotypic characterization of a methionine synthase deletion mutant demonstrated that the B(12) alleviation of methionine auxotrophy was due to the necessity of the B(12)-dependent methionine synthase in methionine biosynthesis. Growth on ammonium as the sole nitrogen source in the presence of vitamin B(12) was demonstrated, but it was not possible to achieve reproducibly good growth in the absence of at least one amino acid as a nitrogen source. Growth on sulfate, cysteine, and methionine as sulfur sources demonstrated the function of a complete sulfur recycling pathway in this strain. These studies have demonstrated that rapid growth of D. radiodurans R1 can be achieved in a MOPS-based medium solely containing a carbon source, salts, four vitamins, and two amino acids.

Carbon↗

PubMed2006

Methylotenera mobilis gen. nov., sp. nov., an obligately methylamine-utilizing bacterium within the family Methylophilaceae.

A novel obligate methylamine utilizer (strain JLW8(T)), isolated from Lake Washington sediment, was characterized taxonomically. The isolate was an aerobic, Gram-negative bacterium. Cells were rod-shaped and motile by means of a single flagellum. Reproduction was by binary fission and no resting bodies were formed. Growth was observed within a pH range of 5-8.5, with optimum growth at pH 7.5. It utilized methylamine as a single source of energy, carbon and nitrogen. Methylamine was oxidized via methylamine dehydrogenase and formaldehyde was assimilated via the ribulose monophosphate cycle. The cellular fatty acid profile was dominated by C(16 : 0)omega7c and C(16 : 0) and the major phospholipid was phosphatidylethanolamine. The DNA G+C content was 54 mol%. 16S rRNA gene sequence analysis indicated that the new isolate was closely related (97-98 % similarity) to a broad group of sequences from uncultured or uncharacterized Betaproteobacteria, but only distantly related (93-96 % similarity) to known methylotrophs of the family Methylophilaceae. Strain JLW8(T) (=ATCC BAA-1282(T)=DSM 17540(T)) is proposed as the type strain of a novel species in a new genus within the family Methylophilaceae, Methylotenera mobilis gen. nov., sp. nov.

Bacterial Typing Techniques↗

PubMed2006

Methyloversatilis universalis gen. nov., sp. nov., a novel taxon within the Betaproteobacteria represented by three methylotrophic isolates.

The taxonomic positions and phylogenetic relationships of two new methylotrophic isolates from Lake Washington (USA) sediment, FAM5T and 500, and the previously described methylotrophic strain EHg5 isolated from contaminated soil in Estarreja (Portugal) were investigated. All three strains were facultative methylotrophs capable of growth on a variety of C1 and multicarbon compounds. Optimal growth occurred at pH 7.5-8 and 30-37 degrees C. The major fatty acids were C16:1omega7c and C16:0. The major quinone was ubiquinone Q8. Neither methanol dehydrogenase nor methanol oxidase activities were detectable in cells grown on methanol, suggesting an alternative, as-yet unknown, mechanism for methanol oxidation. The isolates assimilated C1 units at the level of formaldehyde, via the serine cycle. The DNA G+C content of the strains ranged between 64 and 65 mol%. 16S rRNA gene sequence similarity between the three new isolates was 99.85-100%, but was below 94% with other members of the Betaproteobacteria, indicating that the isolates represent a novel taxon. Based on physiological, phenotypic and genomic characteristics of the three isolates, a new genus, Methyloversatilis gen. nov., is proposed within the family Rhodocyclaceae. The type strain of Methyloversatilis universalis gen. nov., sp. nov. is FAM5T (=CCUG 52030T=JCM 13912T).

Alcohol Oxidoreductases↗

PubMed2006

Involvement of the S-layer proteins Hpi and SlpA in the maintenance of cell envelope integrity in Deinococcus radiodurans R1.

The potential functions have been investigated of two proteins in Deinococcus radiodurans R1 predicted to be involved in the maintenance and integrity of the S layer: the hexagonally packed intermediate (Hpi) protein, and SlpA (DR2577), a homologue of an S-layer SlpA protein in Thermus thermophilus. Deletion of the hpi gene had little effect on the structure of the cell envelope or on shear- or solvent-induced stress responses. However, deletion of the slpA gene caused substantial alterations in cell envelope structure, and a significant defect in resistance to solvent and shear stresses compared to the wild-type. Ultrastructural analysis of slpA mutant cells indicated loss of much of the outer Hpi protein carbohydrate coat, the 'pink envelope', and the membrane-like backing layer. Together these results suggest that the SlpA protein may be involved in attachment of the Hpi surface layer to the inner cell envelope, and that SlpA may play an important role in the maintenance of cell envelope integrity in D. radiodurans.

Bacterial Outer Membrane Proteins↗

PubMed2006

Fluorescence in situ hybridization-flow cytometry-cell sorting-based method for separation and enrichment of type I and type II methanotroph populations.

A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA, pmoA (encoding a subunit of particulate methane monooxygenase), and fae (encoding formaldehyde-activating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISH/FC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct whole-genome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 10(4) or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.

DNA Primers↗

PubMed2006

Measurement of respiration rates of Methylobacterium extorquens AM1 cultures by use of a phosphorescence-based sensor.

Respiration rates of bacterial cultures can be a powerful tool in gauging the effects of genetic manipulation and environmental changes affecting overall metabolism. We present an optical method for measuring respiration rates using a robust phosphorescence lifetime-based sensor and off-the-shelf technology. This method was tested with the facultative methylotroph Methylobacterium extorquens AM1 to demonstrate subtle mutant phenotypes.

Electrodes↗

PubMed2005

Methanotrophic populations in estuarine sediment from Newport Bay, California.

Methanotrophic populations have been studied in Newport Bay estuary, Southern California. Environmental clone banks were generated for 16S rRNA genes specific to methanotrophs and for a diagnostic functional gene, pmoA, encoding a conserved subunit of the particulate methane monooxygenase. These clone banks contained sequences specific to types I and II methanotrophs typically found in aquatic environments including freshwater lake and soda lake sediments, aquifers and rice paddies. However, a group of clones that were divergent (93% identity) from known methanotrophic 16S rRNA genes but represented in 16S rRNA gene libraries from other aquatic environments were detected. A group of pmoA sequences divergent (83% identity) from extant methanotrophs and not previously represented in any environmental clone libraries, were also detected. It is concluded that this environment contains significant methanotroph diversity and that some of these may represent novel groups of methanotrophic bacteria.

Bacteria↗

PubMed2005

A genomic view of methane oxidation by aerobic bacteria and anaerobic archaea.

Recent sequencing of the genome and proteomic analysis of a model aerobic methanotrophic bacterium, Methylococcus capsulatus (Bath) has revealed a highly versatile metabolic potential. In parallel, environmental genomics has provided glimpses into anaerobic methane oxidation by certain archaea, further supporting the hypothesis of reverse methanogenesis.

Anaerobiosis↗

PubMed2005

Flux analysis uncovers key role of functional redundancy in formaldehyde metabolism.

Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems. Here we describe direct flux measurements to determine the role of redundancy in three modules involved in formaldehyde assimilation and dissimilation in a bacterium growing on methanol. A combination of deuterium and (14)C labeling was used to measure the flux through each of the branches of metabolism for growth on methanol during transitions into and out of methylotrophy. The cells were found to differentially partition formaldehyde among the three modules depending on the flux of methanol into the cell. A dynamic mathematical model demonstrated that the kinetic constants of the enzymes involved are sufficient to account for this phenomenon. We demonstrate the role of redundancy in formaldehyde metabolism and have uncovered a new paradigm for coping with toxic, high-flux metabolic intermediates: a dynamic, interconnected metabolic loop.

Deuterium↗

PubMed2005

Methylosarcina lacus sp. nov., a methanotroph from Lake Washington, Seattle, USA, and emended description of the genus Methylosarcina.

An obligately methanotrophic bacterial strain, LW14T, isolated from the sediment of Lake Washington, Seattle, USA, is described taxonomically. The isolate is an aerobic, Gram-negative, non-motile bacterium capable of growth on methane, and possesses type I intracytoplasmic membranes (i.e. it is a type I methanotroph). The strain possesses particulate methane monooxygenase (MMO) and has no soluble MMO. Formaldehyde is assimilated via the ribulose monophosphate cycle. The isolate grows within a pH range of 4-8, with the optimum between pH 5.5 and 6.5. The cellular fatty acid profile is dominated by C(16 : )omega18c, C(16 : 1)omega7c and C(16 : 1)omega5t fatty acids. The DNA G+C content is 53.3+/-0.4 mol%. On the basis of sequence analysis of the 16S rRNA gene, isolate LW14T is related most closely to representatives of the genus Methylosarcina. However, DNA-DNA hybridization analysis reveals only a distant relationship between isolate LW14T and the previously described Methylosarcina species. On the basis of its phenotypic and genotypic characteristics, LW14T represents a novel species of the genus Methylosarcina, for which the name Methylosarcina lacus sp. nov. is proposed, with LW14T (=ATCC BAA-1047T=JCM 13284T) as the type strain.

Base Composition↗

PubMed2005

Labrys methylaminiphilus sp. nov., a novel facultatively methylotrophic bacterium from a freshwater lake sediment.

A new bacterial isolate from a methylamine enrichment culture is described, representing a novel species of facultatively methylotrophic bacteria. The non-motile bacterium is Gram-negative, replicates by budding and does not form endospores. The isolate utilizes methylated amines, as well as a variety of monosaccharides, disaccharides, amino acids, organic acids, aromatic compounds and alcohols as substrates, but does not utilize methanol. Growth factors are not required, although yeast extract stimulates growth. The major components of the fatty acid profile are C(18 : 1)omega7c, C(19 : 0) cyclo and C(16 : 0). The dominant cellular phospholipids are phosphatidyl acid, phosphatidylcholine and phosphatidylethanolamine. The G + C content of the DNA is 65.7 +/- 0.3 mol%. 16S rRNA gene-based phylogenetic analysis revealed that the novel isolate belongs to the alpha-Proteobacteria and is closely related to the only representative of the genus Labrys, Labrys monachus (97.4 % sequence similarity). However, the level of DNA-DNA relatedness with L. monachus is less than 3 %, justifying the placement of this isolate into a novel species of the genus Labrys. The name Labrys methylaminiphilus sp. nov. is proposed (type strain JLW10(T) = ATCC BAA-1080(T) = DSM 16812(T)).

Alcohols↗

PubMed2005

Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1.

A multiple A-tract sequence has been identified in the promoter regions for the mxaF, pqqA, mxaW, mxbD and mxcQ genes involved in methanol oxidation in Methylobacterium extorquens AM1, a facultative methylotroph. Site-directed mutagenesis was exploited to delete or change this conserved sequence. Promoter-xylE transcriptional fusions were used to assess promoter activity in these mutants. A fiftyfold drop in the XylE activity was observed for the mxaF and pqqA promoters without this sequence, and a five- to sixfold drop in the XylE activity was observed for the mxbD and mxcQ promoters without this sequence. Mutants were generated in the chromosomal copies in which this sequence was either deleted or altered, and these mutants were unable to grow on methanol. When one of these sequences was added to Plac of Escherichia coli, which is a weak constitutive promoter in M. extorquens AM1, the activity increased two- to threefold. These results suggest that this sequence is essential for normal expression of these genes in M. extorquens AM1, and may serve as a general enhancer element for genetic constructs in this bacterium.

Bacterial Proteins↗

PubMed2005

HspR is a global negative regulator of heat shock gene expression in Deinococcus radiodurans.

The HspR protein functions as a negative regulator of chaperone and protease gene expression in a diversity of bacteria. Here we have identified, cloned and deleted the Deinococcus radiodurans HspR homologue, DR0934. Delta hspR mutants exhibit moderate growth defects when shifted to mild heat shock temperatures, but are severely impaired for survival at 48 degrees C. Using quantitative reverse transcription polymerase chain reaction and global transcriptional analysis, we have identified 14 genes that are derepressed in the absence of stress in the delta hspR background, 11 of which encode predicted chaperones and proteases, including dnaKJgrpE, ftsH, lonB, hsp20 and clpB. Promoter mapping indicated that the transcription of these genes initiates from a promoter bearing a sigma70-type consensus, and that putative HspR binding sites (HAIR) were present in the 5'-untranslated regions. Electrophoretic mobility shift assays indicated that HspR binds to these promoters at the HAIR site in vitro. These results strongly suggest that DR0934 encodes the HspR-like global negative regulator of D. radiodurans that directly represses chaperone and protease gene expression by binding to the HAIR site in close proximity to promoter regions.

5' Untranslated Regions↗

PubMed2005

Development and application of polymerase chain reaction primers based on fhcD for environmental detection of methanopterin-linked C1-metabolism in bacteria.

In this work we describe development and testing of a novel pair of environmental primers targeting fhcD, a conserved gene in the H4MTP-linked C1-transfer pathway, and demonstrate that these primers enable confident detection of a broad variety of fhcD genes originating from phylogenetically diverse bacteria. The new primer pair was employed to analyse fhcD diversity in Lake Washington sediment, uncovering the presence of 40 fhcD phylotypes. Based on phylogenetic analyses, the phylotypes identified were affiliated with alpha-, beta- and gamma-proteobacteria, and Planctomycetes, while a number of sequences formed deep branches suggesting the presence of unknown groups of microorganisms. To assess the physiological potential and the possible substrate repertoire of the fhcD-containing species in Lake Washington, we conducted enrichments of natural populations on a variety of C1 substrates, and observed specific shifts in community structure in response to different C1 substrates. A specific shift in community structure was also observed in the presence of humic acids suggesting that C1 transfer metabolism linked to H4MPT may be part of the degradation pathway for this natural polymer, possibly involving formaldehyde production. Overall, our data suggest that C1 oxidation reactions linked to H4MPT are much more widespread in natural environments than previously thought.

Bacteria↗

PubMed2005

Bacterial populations active in metabolism of C1 compounds in the sediment of Lake Washington, a freshwater lake.

Active members of the bacterial community in the sediment of Lake Washington, with special emphasis on C1 utilizers, were identified by employing two complementary culture-independent approaches: reverse transcription of environmental mRNA and 16S rRNA combined with PCR (RT-PCR) and stable-isotope probing (SIP) of DNA with the 13C-labeled C1 substrates methanol, methylamine, formaldehyde, and formate. Analysis of RT-PCR-amplified fragments of 16S rRNA-encoding genes revealed that gammaproteobacterial methanotrophs belonging to Methylobacter and Methylomonas dominate the active methylotroph population, while only one other known methylotrophic lineage, Methylophilaceae, was detected via this approach. Analysis of RT-PCR-amplified functional genes, pmoA and fae, allowed detection of alphaproteobacterial (Methylosinus) and gammaproteobacterial (Methylobacter, Methylomonas, and Methylomicrobium) methanotrophs, methylotrophs of the genus Methylobacterium, and yet-unidentified proteobacteria. SIP experiments allowed detection of a broad variety of groups actively metabolizing C1 compounds. Comparisons between 16S rRNA gene pools amplified from [13C]DNA and from [12C]DNA revealed that the proportion of Methylophilus-related sequences increased in the presence of [13C]methanol, [13C]methylamine, and [13C]formaldehyde; Novosphingobium-related sequences were enriched in the presence of [13C]methanol; Gemmatimonadaceae-related sequences were enriched in the presence of [13C]formaldehyde and [13C]formate; and Xanthomonadaceae-related sequences were enriched in the presence of [13C]formate. Analysis of fae genes amplified from [13C]DNAs isolated from different microcosms revealed specific shifts in populations in response to a specific C1 compound: Methylosinus sequences dominated the [13C]methanol microcosm pool, and beta- and gammaproteobacterial sequences dominated the [13C]methylamine microcosm pool. The [13C]formaldehyde microcosm was dominated by betaproteobacterial sequences and by sequences of a nonaffiliated group, while the [13C]formate microcosm was dominated by alpha- and betaproteobacterial sequences. Overall, these data point toward the presence of a diverse population of active methylotrophs in Lake Washington sediments and toward the existence of yet-uncultivated organisms.

Bacterial Proteins↗

PubMed2005

Highly divergent genes for methanopterin-linked C1 transfer reactions in Lake Washington, assessed via metagenomic analysis and mRNA detection.

The origins and the evolutionary history of tetrahydromethanopterin-linked C1 transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated Proteobacteria, Planctomycetes, or Archaea (M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova, Microb. Ecol. 48:463-472, 2004; M. G. Kalyuzhnaya, O. Nercessian, M. E. Lidstrom, and L. Chistoserdova, Environ. Microbiol. 7:1269-1274, 2005). Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known Bacteria and Archaea, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.

Archaea↗

PubMed2005

Analysis of fae and fhcD genes in Mono Lake, California.

Genes for two enzymes of the tetrahydromethanopterin-linked C(1) transfer pathway (fae and fhcD) were detected in hypersaline, hyperalkaline Mono Lake (California), via PCR amplification and analysis. Low diversity for fae and fhcD was noted, in contrast to the diversity previously detected in a freshwater lake, Lake Washington (Washington).

Bacterial Proteins↗