Biology subjects

Sohlenkamp, C.

Publications and source records attributed to Sohlenkamp, C..

4 recordsLinked to original sources

bioRxiv2024

Vibrio cholerae O1 El Tor A1552 encodes two functional ornithine lipid synthases and induces ornithine lipid formation under low phosphate and under low salinity growth conditions.

Ornithine lipids (OLs) are phosphorus-free membrane lipids that can be formed by a wide range of bacteria. The presence of OLs is frequently related to the resistance to abiotic stress conditions, and its synthesis is often induced as part of various stress responses. Two different pathways for synthesizing OLs are currently known: the OlsBA pathway first described in Sinorhizobium meliloti, and the OlsF pathway first described in Serratia proteamaculans. We identified in the genome of Vibrio cholerae O1 El Tor A1552 two genes encoding OlsF homologs, VC0489 is located on chromosome 1, whereas VCA0646 is located on chromosome 2. Both synthases, when expressed in Escherichia coli, caused the synthesis of OLs. Single mutants deficient in each of the OL synthases, double mutants deficient in both OL synthases, and mutants deficient in the transcriptional regulator PhoB were constructed and characterized. We corroborated that VC0489 is solely responsible for the synthesis of OLs under phosphate-limitation. The deletion of VC0489 reduced the growth velocity compared to the wildtype under phosphate-limiting conditions but not under phosphate-replete conditions. The expression of VCA0646 is favored under low salt growth conditions, and its deletion abrogates OL synthesis at low salinities. The absence of VCA0646 and, therefore, the lack of OLs under low salt conditions makes the respective mutant more susceptible to polymyxin than OL-forming strains. None of the mutants was affected in biofilm formation, swimming, or virulence assays using Caenorhabditis elegans or Galleria mellonella. Here, we describe two functional OL synthases present in a single bacterium for the first time, and we show evidence that OLs have an important function during the V. cholerae lifecycle.

microbiology↗

bioRxiv2024

TamL is a key player of the outer membrane homeostasis in Bacteroidetes.

In Proteobacteria, the outer membrane protein TamA and the inner membrane-anchored protein TamB form the Translocation and Assembly Module (TAM) complex, which facilitates the transport of autotransporters, virulence factors, and likely lipids across the two membranes. In Bacteroidetes TamA is replaced by TamL, a TamA-like lipoprotein with a lipid modification at its N-terminus that likely anchors it to the outer membrane. This structural difference suggests that TamL may have a distinct function compared to TamA. However, the role of TAM in bacterial phyla other than Proteobacteria remains unexplored. Our study aimed to elucidate the functional importance of TamL in Flavobacterium johnsoniae, an environmental Bacteroidetes. Unlike its homologues in Proteobacteria, we found that TamL and TamB are essential in F. johnsoniae. Through genetic, phenotypic, proteomic, and lipidomic analyses, we discovered that TamL depletion severely compromises outer membrane integrity, as evidenced by reduced cell viability, altered cell shape, increased susceptibility to membrane-disrupting agents, and elevated levels of outer membrane lipoproteins. Notably, we did not observe any impact on outer membrane lipid composition. Via pull-down protein assays, we confirmed that TamL interacts with TamB in F. johnsoniae, likely forming the TAM complex. Furthermore, our in silico analysis revealed that the presence of TamL and TamB monocistronic genes is a shared genetic feature among Bacteroidetes members, including the human pathogen Capnocytophaga canimorsus where we also confirmed the essentiality of the TamL and TamB homologs. To our knowledge, this study is the first to provide functional insights into a TAM subunit beyond Proteobacteria. SignificanceIn Proteobacteria, the outer membrane (OM) protein TamA forms with the inner membrane (IM)-anchored protein TamB the Translocation and Assembly Module Complex (TAM). which contributes to efficient biogenesis of the OM. In Bacteroidetes TamA is replaced by TamL, a TamA-like lipoprotein of unknown role. In this work, we studied TamL in the Bacteroidetes Flavobacterium johnsoniae. We found that TamL and TamB are essential for cell viability, and that TamL depletion disrupts outer membrane stability, increases outer membrane vesicle size, and lead to higher sensitivity to OM stressors. These findings highlight TamL critical role in maintaining OM structure in Bacteroidetes. To our surprise, we also identified multiple TamL, TamB and TamA homologs in Bacteroidetes. Altogether, our findings extend the current knowledge on TAM and provide novel insights into a field of research barely investigated outside Proteobacteria.

microbiology↗

bioRxiv2023

Engineering of Robust Host Strains: Enhancing Escherichia coli Abiotic Stress Resistance through Ornithine Lipid Formation

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KeypointsO_LIThe E. coli membrane composition was engineered by producing ornithine lipids C_LIO_LIOrnithine lipid production increase biomass yield under phosphate limitation C_LIO_LIEngineered strains show enhanced production phenotype under low pH stress C_LIO_LITranscriptome analysis and CCCP experiments revealed reduced proton leakage C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/544863v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1e261ecorg.highwire.dtl.DTLVardef@18ae062org.highwire.dtl.DTLVardef@915b57org.highwire.dtl.DTLVardef@103b74d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Cardiolipin synthesis in Pseudomonas fluorescens UM270 plays a relevant role in stimulating plant growth under salt stress

Membrane cardiolipin (CL) phospholipids play a fundamental role in the adaptation of bacteria to various environmental conditions, including saline stress. Here, we constructed deletion mutants of two CL synthetase genes, clsA and clsB, in the rhizobacterium Pseudomonas fluorescens UM270, and evaluated their role in plant growth promotion under salt stress. P. fluorescens UM270 {Delta}clsA and {Delta}clsB mutants showed a significant reduction in CL synthesis compared to the UM270 wild-type strain (58% {Delta}clsA and 53% {Delta}clsB), and their growth rate was not affected, except when grown at 100 and 200 mM NaCl. Additionally, the root colonization capacity of both mutant strains was impaired compared with that of the wild type. Concomitant with the deletion of clsA and clsB, some physiological changes were observed in the UM270 {Delta}clsA and {Delta}clsB mutants, such as a reduction in indole acetic acid and biofilm production. By contrast, an increase in siderophore biosynthesis was observed. Further, inoculation of the UM270 wild-type strain in tomato plants (Lycopersicon esculentum Saladette) grown under salt stress conditions (100 and 200 mM NaCl) resulted in an increase in root and shoot length, chlorophyll content, and dry weight. On the contrary, when each of the mutants ({Delta}clsA and {Delta}clsB) were inoculated in tomato plants, a reduction in root length was observed when grown at 200 mM NaCl, but the shoot length, chlorophyll content, and total plant dry weight parameters were significantly reduced under normal or saline conditions (100 and 200 mM NaCl), compared to UM270 wild-type-inoculated plants. In conclusion, these results suggest that CL synthesis in P. fluorescens UM270 plays an important role in the promotion of tomato plant growth under normal conditions, but to a greater extent, under salt-stress conditions.

microbiology↗