Stenotrophomonas maltophilia is one of the most frequently isolated multidrug resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis patients and is frequently isolated from wounds, infected tissues or catheter surfaces. On these diverse surfaces S. maltophilia lives in single or multi-species biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4 and Sm2 - Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43 ± 1.36 % of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analysis represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings. IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. The nosocomial infections among others caused by S. maltophilia, particularly lung infection among CF patients, increased in prevalence in the last years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells, makes the treatment of S. maltophilia infection difficult. The significance of our research is based in understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among this species and in identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms.
Small ORF (sORF)‐encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding‐ and noncoding‐predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull‐down by affinity chromatography followed by MS analysis, reverse pull‐down, microscale thermophoresis, size‐exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1) with high affinity (app. KD = 0.76 µm± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1. Upon interaction with sP26, GlnA1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2‐oxoglutarate (2‐OG). Besides, strong interaction of sP26 with the PII‐like protein GlnK1 was demonstrated (app. KD = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2‐OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1.
Diorcinols and related prenylated diaryl ethers were reported to exhibit activity againstm ethicillin-resistant clinicali solates of Staphylococcus aureus (MRSA). Within these lines, we report the first total synthesis of diorcinol D, I, J, the proposed structure of verticilatin and recently isolated antibacterial diaryl ether by using an efficient and highly divergent synthetic strategy.T hese total syntheses furnish the diaryl ethers in only five to seven steps employingaPd-catalyzed diaryl ether coupling as the key step. The total synthesis led to the structural revision of the natural product verticilatin, which has been isolated from ap lant pathogenic fungus. Furthermore, these structures were tested in order to determine their antibacterial activities against different MRSA strains as well as furtherGram-positive and-negative bacteria.
Small ORFs (sORF) encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding and non-coding predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved within numerous Methanosarcina strains on the amino acid as well as on nucleotide level strongly arguing for a cellular function of the small protein. spRNA26 is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. His-tagged sP26 was heterologously expressed and purified by fractionated ammonium sulfate precipitation, affinity chromatography and size exclusion centrifugation. Using independent biochemical approaches (pull-down by affinity chromatography followed by MS analysis, revers pull-down, microscale thermophoresis and size exclusion chromatography) we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA 1 ) with high affinity (app. KD = 45 +/-14 µM). Upon interaction with sP26, GlnA 1 activity was significantly stimulated independently and in addition to the known activation by the metabolite 2-oxoglutarate. Besides strong interaction of sP26 with the PII-like protein GlnK 1 was demonstrated (KD= 1.4 µM +/-0.9 µM). On the basis of these findings, we hypothesize that in addition to 2-oxoglutarate, sP26activates GlnA 1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA 1 .
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