Supplementary MaterialsFIG?S1. natural replicates is usually denoted by shading. As a control, the promoterless reporter plasmid pMS402 in the K56-2 WT strain is also shown. Download FIG?S3, TIF file, 1.6 MB. Copyright ? 2019 Oppy et al. This content is distributed Doxapram under the terms of the Creative Commons Attribution 4.0 International license. FIG?S4. Growth and Lux reporter curves of strains. (A and B) Twenty-four-hour Doxapram curves of strains made up of reporter in LB plus TMP (100 g/ml) demonstrate comparable growth kinetics across strains for both the CepR promoter (pPromCepR) and CepI promoter (pcp300). The standard derivation of biological replicates is usually denoted by shading. (C) CepR promoter reporter measurements at 12, 16, and 20 h in the OGC mutant demonstrate a decrease in the transcription of CepR upon entrance into stationary phase. (D) Twenty-four-hour curves of strains made up of reporter in LB plus TMP (100 g/ml) demonstrate comparable growth kinetics across strains. Standard derivation of biological replicates is usually denoted by shading. (E) CepI promoter reporter measurements at 12, 16, and 20 h in the OGC mutant demonstrate a decrease in the transcription of CepI upon entrance into stationary phase. (F) Twenty-four-hour curves of strains made up of the reporter in LB plus TMP (100 g/ml) demonstrate comparable growth kinetics across strains. Standard derivation of biological replicates is usually denoted by shading. Download FIG?S4, TIF file, 1.3 MB. Copyright ? 2019 Oppy et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S5. Assessment of biofilm formation and siderophore activity across strains. (A) Independently created mutants demonstrate identical loss of biofilm phenotype and can partially restored by chromosomal complementation. (B) Disruption of OGC results in a marked increase in biofilm formation. (C and D) Disruption of OGC results in a marked decrease in siderophore activity. Doxapram Download FIG?S5, TIF file, 1.1 MB. Copyright ? 2019 Oppy et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S6. Proteomics and functional analysis of strains. Comparison of Z-scored proteomic data reveals no difference between protein abundances of nonmevalonate (A) and undecaprenyl diphosphate biosynthesis (B) pathway proteins in glycosylation-null versus glycosylation-competent strains. (C) Protease probe analysis of WT, mutant, and mutant, and J2315. J2315 genes defined as virulence associated used for enrichment analysis are provided with PMID references denoting the source of the assignment as a virulence factor. Download Table?S1, XLSX file, 0.1 MB. Copyright ? 2019 Oppy et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. Data Availability StatementProteomic data units have been deposited into the ProteomeXchange Consortium via the PRIDE (91) partner repository with the data set identifiers Doxapram PXD014429, PXD014516, PXD014581, PXD014614, and PXD014700. TABLE?S1Compiled virulence-associated genes in J2315. J2315 genes defined as virulence associated used for enrichment analysis are provided with PMID recommendations denoting the source of the assignment as a virulence factor. Download Table?S1, XLSX file, 0.1 MB. Copyright ? 2019 Oppy et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. ABSTRACT genus. The addition of the trisaccharide -Gal-(1,3)–GalNAc-(1,3)–GalNAc to membrane exported proteins in is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence Rabbit Polyclonal to 41185 of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the grasp quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, the large quantity of most of the known glycosylated proteins did not significantly switch in the glycosylation-defective mutants, except for BCAL1086 and BCAL2974, which were found in reduced amounts, suggesting they could be Doxapram degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, biofilm formation, or siderophore activity. Together, our results show that loss of.
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