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ACS Chemical Biology Aug 2021Natural products are an essential source of bioactive compounds. Isotopic labeling is an effective way to identify natural products that incorporate a specific...
Natural products are an essential source of bioactive compounds. Isotopic labeling is an effective way to identify natural products that incorporate a specific precursor; however, this approach is limited by the availability of isotopically enriched precursors. We used an inverse stable isotopic labeling approach to identify natural products by growing bacteria on a C-carbon source and then identifying C-precursor incorporation by mass spectrometry. We applied this approach to methylotrophs, ecologically important bacteria predicted to have significant yet underexplored biosynthetic potential. We demonstrate that this method identifies acyl homoserine lactone quorum sensing signals produced by diverse methylotrophs grown on three different one-carbon compounds. We then apply this approach to simultaneously detect five previously unidentified signals produced by a methylotroph and link these compounds to their synthases. We envision that this method can be used to identify other natural product classes synthesized by methylotrophs and other organisms that grow on relatively inexpensive C-carbon sources.
Topics: Acyl-Butyrolactones; Carbon; Carbon Isotopes; Isotope Labeling; Methylobacteriaceae; Methylococcaceae; Proof of Concept Study; Quorum Sensing
PubMed: 34328722
DOI: 10.1021/acschembio.1c00329 -
PloS One 2013Chloromethane (CH3Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for...
The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12- and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study.
Chloromethane (CH3Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu) was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate-bound one-carbon units, and central metabolism. The mosaic organization of plasmid pCMU01 and the clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of gene acquisition related to chloromethane utilization.
Topics: Computational Biology; Genes, Bacterial; Metabolic Networks and Pathways; Methyl Chloride; Methylobacterium extorquens; Multigene Family; Plasmids; Proteomics; Stress, Physiological; Tetrahydrofolates; Vitamin B 12
PubMed: 23593113
DOI: 10.1371/journal.pone.0056598 -
MBio Mar 2020Population-level analyses are rapidly becoming inadequate to answer many of biomedical science and microbial ecology's most pressing questions. The role of microbial...
Population-level analyses are rapidly becoming inadequate to answer many of biomedical science and microbial ecology's most pressing questions. The role of microbial populations within ecosystems and the evolutionary selective pressure on individuals depend fundamentally on the metabolic activity of single cells. Yet, many existing single-cell technologies provide only indirect evidence of metabolic specialization because they rely on correlations between transcription and phenotype established at the level of the population to infer activity. In this study, we take a top-down approach using isotope labels and secondary ion mass spectrometry to track the uptake of carbon and nitrogen atoms from different sources into biomass and directly observe dynamic changes in anabolic specialization at the level of single cells. We investigate the classic microbiological phenomenon of diauxic growth at the single-cell level in the model methylotroph In nature, this organism inhabits the phyllosphere, where it experiences diurnal changes in the available carbon substrates, necessitating an overhaul of central carbon metabolism. We show that the population exhibits a unimodal response to the changing availability of viable substrates, a conclusion that supports the canonical model but has thus far been supported by only indirect evidence. We anticipate that the ability to monitor the dynamics of anabolism in individual cells directly will have important applications across the fields of ecology, medicine, and biogeochemistry, especially where regulation downstream of transcription has the potential to manifest as heterogeneity that would be undetectable with other existing single-cell approaches. Understanding how genetic information is realized as the behavior of individual cells is a long-term goal of biology but represents a significant technological challenge. In clonal microbial populations, variation in gene regulation is often interpreted as metabolic heterogeneity. This follows the central dogma of biology, in which information flows from DNA to RNA to protein and ultimately manifests as activity. At present, DNA and RNA can be characterized in single cells, but the abundance and activity of proteins cannot. Inferences about metabolic activity usually therefore rely on the assumption that transcription reflects activity. By tracking the atoms from which they build their biomass, we make direct observations of growth rate and substrate specialization in individual cells throughout a period of growth in a changing environment. This approach allows the flow of information from DNA to be constrained from the distal end of the regulatory cascade and will become an essential tool in the rapidly advancing field of single-cell metabolism.
Topics: Biomass; Carbon; Isotope Labeling; Methylobacterium extorquens; Nitrogen; Phenotype; Single-Cell Analysis; Spectrometry, Mass, Secondary Ion
PubMed: 32127448
DOI: 10.1128/mBio.01519-19 -
Journal of Bacteriology May 2003
Review
Topics: Citric Acid Cycle; Formates; Genes, Bacterial; Genome, Bacterial; Glyoxylates; Hydroxybutyrates; Methanol; Methylamines; Methylobacterium extorquens; Oxidation-Reduction; Polyesters; Serine
PubMed: 12730156
DOI: 10.1128/JB.185.10.2980-2987.2003 -
Journal of Pediatric Hematology/oncology Jul 2020Roseomonas gilardii is a Gram-negative coccobacillus identified in immunocompromised pediatric patients. A 5-year-old male with a history of HbSβ thalassemia status... (Review)
Review
Roseomonas gilardii is a Gram-negative coccobacillus identified in immunocompromised pediatric patients. A 5-year-old male with a history of HbSβ thalassemia status postsurgical splenectomy presented to the emergency department with fever. Blood cultures grew R. gilardii at 63 hours, but the patient had been discharged home at 48 hours. The patient was readmitted for repeat cultures and initiated on meropenem for 10 days as Roseomonas spp. are often resistant to third generation cephalosporins. R. gilardii is a rare cause of bacteremia in immunocompromised patients. Clinicians should consider Roseomonas in slow growing Gram-negative rod bacteremias, and consider meropenem as empiric coverage.
Topics: Bacteremia; Child, Preschool; Gram-Negative Bacterial Infections; Humans; Male; Methylobacteriaceae; Prognosis; Thalassemia
PubMed: 30951022
DOI: 10.1097/MPH.0000000000001476 -
Applied and Environmental Microbiology Jan 2016The genus Methylobacterium tolerates hygiene agents like benzalkonium chloride (BAC), and infection with this organism is an important public health issue. Here, we...
The genus Methylobacterium tolerates hygiene agents like benzalkonium chloride (BAC), and infection with this organism is an important public health issue. Here, we found that the combination of BAC with particular alcohols at nonlethal concentrations in terms of their solitary uses significantly reduced bacterial viability after only 5 min of exposure. Among the alcohols, Raman spectroscopic analyses showed that pentanol (pentyl alcohol [PeA]) and benzyl alcohol (BzA) accelerated the cellular accumulation of BAC. Fluorescence spectroscopic assays and morphological assays with giant vesicles indicated that PeA rarely attacked membrane structures, while BzA increased the membrane fluidity and destabilized the structures. Other fluorescent spectroscopic assays indicated that PeA and BzA inactivate bacterial membrane proteins, including an efflux pump for BAC transportation. These findings suggested that the inactivation of membrane proteins by PeA and BzA led to the cellular accumulation but that only BzA also enhanced BAC penetration by membrane fluidization at nonlethal concentrations.
Topics: Anti-Infective Agents, Local; Benzalkonium Compounds; Benzyl Alcohol; Drug Combinations; Drug Synergism; Humans; Membrane Fluidity; Membrane Proteins; Methylobacterium; Microbial Viability; Pentanols; Spectrometry, Fluorescence
PubMed: 26519389
DOI: 10.1128/AEM.02515-15 -
BMC Infectious Diseases Dec 2023Roseomonas mucosa (R. mucosa) is a pink-pigmented, Gram-negative short rod bacterium. It is isolated from moist environments and skin, resistant to multiple drugs,...
BACKGROUND
Roseomonas mucosa (R. mucosa) is a pink-pigmented, Gram-negative short rod bacterium. It is isolated from moist environments and skin, resistant to multiple drugs, including broad-spectrum cephalosporins, and a rare cause of infection with limited reports. R. mucosa mostly causes catheter-related bloodstream infections, with even fewer reports of skin and soft tissue infections.
CASE PRESENTATION
A 10-year-old boy received topical steroid treatment for sebum-deficient eczema. A few days before the visit, he was bitten by an insect on the front of his right lower leg and scratched it due to itching. The day before the visit, redness, swelling, and mild pain in the same area were observed. Based on his symptoms, he was diagnosed with cellulitis. He was treated with sulfamethoxazole/trimethoprim, and his symptoms improved. Pus culture revealed R. mucosa.
CONCLUSIONS
We report a rare case of cellulitis caused by R. mucosa. Infections caused by rare organisms that cause opportunistic infections, such as R. mucosa, should be considered in patients with compromised skin barrier function and regular topical steroid use. Gram stain detection of organisms other than Gram-positive cocci should be considered.
Topics: Male; Child; Humans; Cellulitis; Soft Tissue Infections; Methylobacteriaceae; Steroids
PubMed: 38082373
DOI: 10.1186/s12879-023-08875-9 -
PloS One 2012Understanding microbial populations in hospital environments is crucial for improving human health. Hospital-acquired infections are an increasing problem in intensive...
Understanding microbial populations in hospital environments is crucial for improving human health. Hospital-acquired infections are an increasing problem in intensive care units (ICU). In this work we present an exploration of bacterial diversity at inanimate surfaces of the ICU wards of the University Hospital A Coruña (Spain), as an example of confined hospital environment subjected to selective pressure, taking the entrance hall of the hospital, an open and crowded environment, as reference. Surface swab samples were collected from both locations and recovered DNA used as template to amplify a hypervariable region of the bacterial 16S rRNA gene. Sequencing of the amplicons was performed at the Roche 454 Sequencing Center using GS-FLX Titanium procedures. Reads were pre-processed and clustered into OTUs (operational taxonomic units), which were further classified. A total of 16 canonical bacterial phyla were detected in both locations. Members of the phyla Firmicutes (mainly Staphylococcus and Streptococcus) and Actinobacteria (mainly Micrococcaceae, Corynebacteriaceae and Brevibacteriaceae) were over-represented in the ICU with respect to the Hall. The phyllum Proteobacteria was also well represented in the ICU, mainly by members of the families Enterobacteriaceae, Methylobacteriaceae and Sphingomonadaceae. In the Hall sample, the phyla Proteobacteria, Bacteroidetes, Deinococcus-Thermus and Cyanobacteria were over-represented with respect to the ICU. Over-representation of Proteobacteria was mainly due to the high abundance of Enterobacteriaceae members. The presented results demonstrate that bacterial diversity differs at the ICU and entrance hall locations. Reduced diversity detected at ICU, relative to the entrance hall, can be explained by its confined character and by the existence of antimicrobial selective pressure. This is the first study using deep sequencing techniques made in hospital wards showing substantial hospital microbial diversity.
Topics: Bacteria; Databases, Nucleic Acid; Environmental Microbiology; Genetic Variation; Hospitalization; Hospitals; Humans; Intensive Care Units; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Spain; Temperature; Titanium
PubMed: 22952889
DOI: 10.1371/journal.pone.0044105 -
Microbes and Environments 2020The Vietnamese Mekong delta is one of the largest rice-producing areas globally. Methylobacterium spp. are persistent colonizers of the rice plant and exert beneficial...
The Vietnamese Mekong delta is one of the largest rice-producing areas globally. Methylobacterium spp. are persistent colonizers of the rice plant and exert beneficial effects on plant growth and health. Sixty-one Methylobacterium strains belonging to seven species were predominantly isolated from the phyllosphere of rice cultivated in six Mekong delta provinces. Inoculation tests revealed that some strains exhibited plant growth-promoting activity. Moreover, three strains possessed the novel characteristics of inducing leaf bleaching and killing rice seedlings. These results revealed the complex diversity of Methylobacterium in Mekong delta rice and that healthy and productive rice cultivation requires a proper balance of Methylobacterium.
Topics: Genetic Variation; Lactuca; Methylobacterium; Oryza; Plant Components, Aerial; Plant Growth Regulators; Vietnam
PubMed: 31969531
DOI: 10.1264/jsme2.ME19111 -
Journal of Bacteriology Apr 2016Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH...
UNLABELLED
Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH requires pyrroloquinoline quinone (PQQ) and Ca in its active site, while XoxF-MeDH requires PQQ and lanthanides, such as Ce and La. Using MeDH mutant strains to conduct growth analysis and MeDH activity assays, we demonstrate that M. extorquens AM1 has at least one additional lanthanide-dependent methanol oxidation system contributing to methanol growth. Additionally, the abilities of different lanthanides to support growth were tested and strongly suggest that both XoxF and the unknown methanol oxidation system are able to use La, Ce, Pr, Nd, and, to some extent, Sm. Further, growth analysis using increasing La concentrations showed that maximum growth rate and yield were achieved at and above 1 μM La, while concentrations as low as 2.5 nM allowed growth at a reduced rate. Contrary to published data, we show that addition of exogenous lanthanides results in differential expression from the xox1 and mxa promoters, upregulating genes in the xox1 operon and repressing genes in the mxa operon. Using transcriptional reporter fusions, intermediate expression from both the mxa and xox1 promoters was detected when 50 to 100 nM La was added to the growth medium, suggesting that a condition may exist under which M. extorquens AM1 is able to utilize both enzymes simultaneously. Together, these results suggest that M. extorquens AM1 actively senses and responds to lanthanide availability, preferentially utilizing the lanthanide-dependent MeDHs when possible.
IMPORTANCE
The biological role of lanthanides is a nascent field of study with tremendous potential to impact many areas in biology. Our studies demonstrate that there is at least one additional lanthanide-dependent methanol oxidation system, distinct from the MxaFI and XoxF MeDHs, that may aid in classifying additional environmental organisms as methylotrophs. Further, our data suggest that M. extorquens AM1 has a mechanism to regulate which MeDH is transcribed, depending on the presence or absence of lanthanides. While the mechanism controlling differential regulation is not yet understood, further research into how methylotrophs obtain and use lanthanides will facilitate their cultivation in the laboratory and their use as a biomining and biorecycling strategy for recovery of these commercially valuable rare-earth elements.
Topics: Alcohol Oxidoreductases; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Lanthanoid Series Elements; Methanol; Methylobacterium extorquens; Oxidation-Reduction; Promoter Regions, Genetic
PubMed: 26833413
DOI: 10.1128/JB.00937-15