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Enterococcus faecalis is a robust bacterium, which is able to survive in and adapt to hostile environments such as the urinary tract and bladder. In this label-free quantitative proteomic study based on MaxQuant LFQ algorithms, we identified 127 proteins present in the secretome of the clinical vancomycin-resistant isolate E. faecalis V583 and we compared proteins secreted in the initial phase of cultivation in urine with the secretome during cultivation in standard laboratory medium, 2xYT. Of the 54 identified proteins predicted to be secreted, six were exclusively found after cultivation in urine including the virulence factor EfaA (\"endocarditis specific antigen\") and its homologue EF0577 (\"adhesion lipoprotein\"). These two proteins are both involved in manganese transport, known to be an important determinant of colonization and infection, and may additionally function as adhesins. Other detected urine-specific proteins are involved in peptide transport (EF0063 and EF3106) and protease inhibition (EF3054). In addition, we found an uncharacterized protein (EF0764), which had not previously been linked to the adaptation of V583 to a urine environment, and which is unique to E. faecalis. Proteins found in both environments included a histone-like protein, EF1550, that was up-regulated during cultivation in urine and that has a homologue in streptococci (HlpA) known to be involved in bacterial adhesion to host cells. Up-regulated secreted proteins included autolysins. These results from secretome analyses are largely compatible with previously published data from transcriptomics studies. All in all, the present data indicate that transport, in particular metal transport, adhesion, cell wall remodelling and the unknown function carried out by the unique EF0764 are important for enterococcal adaptation to the urine environment. These results provide a basis for a more targeted exploration of novel proteins involved in the adaptability and pathogenicity of E. faecalis.

Beneficial modulation of the gut microbiome has high-impact implications not only in humans, but also in livestock that sustain our current societal needs. In this context, we have tailored an acetylated galactoglucomannan (AcGGM) fibre to match unique enzymatic capabilities of Roseburia and Faecalibacterium species, both renowned butyrate-producing gut commensals. Here, we test the accuracy of AcGGM within the complex endogenous gut microbiome of pigs, wherein we resolve 355 metagenome-assembled genomes together with quantitative metaproteomes. In AcGGM-fed pigs, both target populations differentially express AcGGM-specific polysaccharide utilization loci, including novel, mannan-specific esterases that are critical to its deconstruction. However, AcGGM-inclusion also manifests a “butterfly effect”, whereby numerous metabolic changes and interdependent cross-feeding pathways occur in neighboring non-mannanolytic populations that produce short-chain fatty acids. Our findings show how intricate structural features and acetylation patterns of dietary fibre can be customized to specific bacterial populations, with potential to create greater modulatory effects at large. Here, the authors tailor an acetylated galactoglucomannan (AcGGM) fibre from spruce wood to specifically enrich Roseburia and Faecalibacterium - beneficial species which have the enzymatic machinery to breakdown the fibre and generate butyrate. They subsequently perform a piglet feeding trial, metagenomics and metaproteomics, together showing that AcGGM-fed pigs exhibit not only increased Roseburia and Faecalibacterium populations with AcGGM-specific mannan-specific esterases, but also secondary metabolic pathways.

Gammaproteobacteria from the family Endozoicomonadaceae have emerged as widespread associates of dense marine animal communities. Their abundance in coral reefs involves symbiotic relationships and possibly host nutrition. We explored functions encoded in the genome of an uncultured Endozoicomonadaceae ‘Candidatus Acestibacter aggregatus’ that lives inside gill cells of large Acesta excavata clams in deep-water coral reefs off mid-Norway. The dominance and deep branching lineage of this symbiont was confirmed using 16S rRNA gene sequencing and phylogenomic analysis from shotgun sequencing data. The 4.5 Mb genome binned in this study has a low GC content of 35% and is enriched in transposon and chaperone gene annotations indicating ongoing adaptation. Genes encoding functions potentially involved with the symbiosis include ankyrins, repeat in toxins, secretion and nutritional systems. Complete pathways were identified for the synthesis of eleven amino acids and six B-vitamins. A minimal chitinolytic machinery was indicated from a glycosyl hydrolase GH18 and a lytic polysaccharide monooxygenase LPMO10. Expression of the latter was confirmed using proteomics. Signal peptides for secretion were identified for six polysaccharide degrading enzymes, ten proteases and three lipases. Our results suggest a nutritional symbiosis fuelled by enzymatic products from extracellular degradation processes.

Background: Rhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids.Results: R. toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose.Conclusions: The results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through PPP although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.

Murein hydrolases (or peptidoglycan hydrolases) play diverse roles in bacteria, from controlled remodeling of the bacterial cell wall to lytic agents. In streptococci, a subset of these hydrolases is associated with competence-induced fratricide, a process where bacteria kill closely related cells to release DNA that can be taken up during natural transformation. Here, we characterize ScrM, a competence-induced murein hydrolase from Streptococcus dysgalactiae comprising a CHAP domain, an SH3b domain and an uncharacterized C-terminal domain (CCD). ScrM displayed lytic activity against pyogenic and salivarius group streptococci. Microscopy analysis of fluorescent fusions revealed that ScrM specifically localizes to the division zone of sensitive cells, with binding and localization mediated primarily by CCD. Upon competence induction, cells became immune to ScrM due to expression of ScrI, a Fem-transferase-like protein. We show by LC-MS/MS that ScrI incorporates Thr in place of Ala into the interpeptide bridges of peptidoglycan, which in turn prevents ScrM binding to the division zone, thereby protecting the cells from self-lysis during competence. ScrM and ScrI are conserved among pyogenic streptococcal pathogens and represent new players in the cell wall biogenesis of these bacteria that may form a platform for development of novel antimicrobial strategies.

Rhizobia living as microsymbionts inside nodules have stable access to carbon substrates, but also have to survive as free-living bacteria in soil where they are starved for carbon and energy most of the time. Many rhizobia can denitrify, thus switch to anaerobic respiration under low O2 tension using N-oxides as electron acceptors. The cellular machinery regulating this transition is relatively well-known from studies under optimal laboratory conditions, while little is known about this regulation in starved organisms. It is, for example, not known if the strong preference for N2O- over NO3--reduction in bradyrhizobia is retained under carbon limitation. Here we show that starved cultures of a Bradyrhizobium strain with respiration rates 1-18% of well-fed cultures, reduced all available N2O before touching provided NO3-. Proteomics showed similar abundance of Nap (periplasmic NO3- reductase) and NosZ (N2O reductase), suggesting that competition between electron pathways to Nap and NosZ favoured N2O reduction also in starved cells, similar to well-fed cultures. This contrasts the general notion that NosZ activity diminishes under carbon limitation. The results suggest that bradyrhizobia carrying NosZ can act as strong sinks for N2O under natural conditions and that this criterion should be considered in the development of biofertilizers.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Title and Importance section were revised