Proteomics of Syntrophomonas zehnderi and Methanobacterium formicicum growing on long-chain fatty acids

Background: Conversion of long-chain fatty acids (LCFA) in anaerobic digesters relies on syntrophic relationship between acetogenic bacteria and methanogenic archaea. Conversion of unsaturated- and saturated-LCFA has been previously shown by a coculture of Syntrophomonas zehnderi and Methanobacterium formicium. Degradation of unsaturated-LCFA is rare among Syntrophomonas species; the best studied fatty acid oxidizer, S. wolfei, can only grow on saturated-LCFA. Objectives: Major differences are expected in the pathways and enzymes involved in the degradation of unsaturated-LCFA. In this work we used proteogenomic approach to study these differences. Methods: A draft genome of S. zehnderi was obtained by Illumina HiSeq sequencing. Genomes of S. zehnderi and S. wolfei (available at NCBI) were compared. S. zehnderi and M. formicicum co-cultures grown on oleate (unsaturated LCFA, C18:1) and on stearate (saturated LCFA, C18:0) were further studied using a proteomics approach. Conclusions: Genomic comparison of S. zehnderi and S. wolfei revealed approximately 900 different proteins and 1200 common proteins. In the genome of S. zehnderi, two replicates of the unsaturated acyl-CoA dehydrogenase genes were identified, one of which differs considerably from the acyl-CoA gene found in S. wolfei. Proteomic analysis of S. zehnderi and M. formicium co-cultures revealed high expression levels of proteins related to the -oxidation of LCFA (up to 30% of total proteins identified). Different protein expression levels were observed during the degradation of oleate (44% unique proteins) and stearate (23% unique proteins). In addition, proteins involved in electron transfer were highly expressed, including electron transfer flavoproteins, ATP synthases and a number of hydrogenases and formate dehydrogenases

Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry

Background: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. Methods: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. Results: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. Conclusion: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis

Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 β-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem

While Extended-Spectrum β-Lactamases (ESBL) and AmpC β-lactamases barely degrade carbapenem antibiotics, they are able to bind carbapenems and prevent them from interacting with penicillin-binding proteins, thereby inhibiting their activity. Further, it has been shown that Enterobacterales can become resistant to carbapenems when high concentrations of ESBL and AmpC β-lactamases are present in the bacterial cell in combination with a decreased influx of antibiotics (due to a decrease in porins and outer-membrane permeability). In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the detection of the Escherichia coli porins OmpC and OmpF, its chromosomal AmpC β-lactamase, and the plasmid-mediated CMY-2 β-lactamase. Bla CMY-2-like positive E. coli isolates were cultured in the presence of increasing concentrations of meropenem, and resistant mutants were analyzed using the developed LC-MS/MS assay, Western blotting, and whole genome sequencing. In five strains that became meropenem resistant, a decrease in OmpC and/or OmpF (caused by premature stop codons or gene interruptions) was the first event toward meropenem resistance. In four of these strains, an additional increase in MICs was caused by an increase in CMY-2 production, and in one strain this was most likely caused by an increase in CTX-M-15 production. The LC-MS/MS assay developed proved to be suitable for the (semi-)quantitative analysis of CMY-2-like β-lactamases and porins within 4 h. Targeted LC-MS/MS could have additional clinical value in the early detection of non-carbapenemase-producing carbapenem-resistant E. coli

Actinomyces glycerinitolerans strain G10T was isolated from rumen fluid, can metabolize a range of various substrates including complex carbohydrates to organic acids. Here, we report a -Mbp draft genome of Actinomyces glycerinitolerans.

Actinomyces glycerinitolerans strain G10T was isolated from rumen fluid, can metabolize a range of various substrates including complex carbohydrates to organic acids. Here, we report a -Mbp draft genome of Actinomyces glycerinitolerans

Actinomyces glycerinitolerans strain G10T was isolated from rumen fluid, can metabolize a range of various substrates including complex carbohydrates to organic acids. Here, we report a -Mbp draft genome of Actinomyces glycerinitolerans.

Actinomyces glycerinitolerans strain G10T was isolated from rumen fluid, can metabolize a range of various substrates including complex carbohydrates to organic acids. Here, we report a -Mbp draft genome of Actinomyces glycerinitolerans

Actinomyces glycerinitolerans strain G10T was isolated from rumen fluid, can metabolize a range of various substrates including complex carbohydrates to organic acids. Here, we report a -Mbp draft genome of Actinomyces glycerinitolerans.

Actinomyces glycerinitolerans strain G10T was isolated from rumen fluid, can metabolize a range of various substrates including complex carbohydrates to organic acids. Here, we report a -Mbp draft genome of Actinomyces glycerinitolerans

Genome assembly of a novel psychrotolerance bacterium, Trichococcus ART1 .

A psychrotolerant anaerobe, strain ART1T, was isolated from a psychrophilic anaerobic digester treating . 16S rRNA gene sequence of strain ART1T was highly similar to those of other Trichococcus species (> 99%), but digital DNA-DNA hybridization (dDDH) values were lower than 70% indicating that strain ART1 is a new species of the genus Trichococcus. Cells of strain ART1T were immotile cocci and stained Gram-positive. Growth was optimal at pH 7.5 and cells could grow in a temperature range of 0 to 37°C (optimum 30°C). Strain ART1T could degrade several carbohydrates, and the main products from glucose fermentation are lactate, acetate, formate, and ethanol.