scholarly journals The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

2007 ◽  
Vol 20 (11) ◽  
pp. 1421-1430 ◽  
Author(s):  
Christian Sohlenkamp ◽  
Kanaan A. Galindo-Lagunas ◽  
Ziqiang Guan ◽  
Pablo Vinuesa ◽  
Sally Robinson ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Minimal Medium ◽  
Polymyxin B ◽  
Growth Conditions ◽  
Membrane Lipid ◽  
Low Ph ◽  
Wild Type ◽  
Rhizobium Tropici ◽  
Gram Negative ◽  
Inducible Genes

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed lpiA, have been identified in the gram-negative α-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

scholarly journals Phosphatidylethanolamine Is Not Essential for Growth of Sinorhizobium meliloti on Complex Culture Media

2004 ◽  
Vol 186 (6) ◽  
pp. 1667-1677 ◽  
Author(s):  
Christian Sohlenkamp ◽  
Karel E. E. de Rudder ◽  
Otto Geiger
Keyword(s):  
Sinorhizobium Meliloti ◽  
Minimal Medium ◽  
Membrane Lipids ◽  
Culture Media ◽  
Membrane Lipid ◽  
Deficient Mutant ◽  
Wild Type ◽  
Phosphatidylserine Synthase ◽  
Growth Phenotype ◽  
Complex Culture

ABSTRACT In addition to phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylethanolamine (PE), Sinorhizobium meliloti also possesses phosphatidylcholine (PC) as a major membrane lipid. The biosynthesis of PC in S. meliloti can occur via two different routes, either via the phospholipid N-methylation pathway, in which PE is methylated three times in order to obtain PC, or via the phosphatidylcholine synthase (Pcs) pathway, in which choline is condensed with CDP-diacylglycerol to obtain PC directly. Therefore, for S. meliloti, PC biosynthesis can occur via PE as an intermediate or via a pathway that is independent of PE, offering the opportunity to uncouple PC biosynthesis from PE biosynthesis. In this study, we investigated the first step of PE biosynthesis in S. meliloti catalyzed by phosphatidylserine synthase (PssA). A sinorhizobial mutant lacking PE was complemented with an S. meliloti gene bank, and the complementing DNA was sequenced. The gene coding for the sinorhizobial phosphatidylserine synthase was identified, and it belongs to the type II phosphatidylserine synthases. Inactivation of the sinorhizobial pssA gene leads to the inability to form PE, and such a mutant shows a greater requirement for bivalent cations than the wild type. A sinorhizobial PssA-deficient mutant possesses only PG, CL, and PC as major membrane lipids after growth on complex medium, but it grows nearly as well as the wild type under such conditions. On minimal medium, however, the PE-deficient mutant shows a drastic growth phenotype that can only partly be rescued by choline supplementation. Therefore, although choline permits Pcs-dependent PC formation in the mutant, it does not restore wild-type-like growth in minimal medium, suggesting that it is not only the lack of PC that leads to this drastic growth phenotype.

scholarly journals Phosphorus stress induces the synthesis of novel glycolipids in Pseudomonas aeruginosa that confer protection against a last-resort antibiotic

2021 ◽  
Author(s):  
Rebekah A. Jones ◽  
Holly Shropshire ◽  
Caimeng Zhao ◽  
Andrew Murphy ◽  
Ian Lidbury ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Sensitivity ◽  
Polymyxin B ◽  
Growth Conditions ◽  
Membrane Lipid ◽  
Comparative Genomic ◽  
P Limitation ◽  
Lung Microbiome ◽  
Glycolipid Synthesis

AbstractPseudomonas aeruginosa is a nosocomial pathogen with a prevalence in immunocompromised individuals and is particularly abundant in the lung microbiome of cystic fibrosis patients. A clinically important adaptation for bacterial pathogens during infection is their ability to survive and proliferate under phosphorus-limited growth conditions. Here, we demonstrate that P. aeruginosa adapts to P-limitation by substituting membrane glycerophospholipids with sugar-containing glycolipids through a lipid renovation pathway involving a phospholipase and two glycosyltransferases. Combining bacterial genetics and multi-omics (proteomics, lipidomics and metatranscriptomic analyses), we show that the surrogate glycolipids monoglucosyldiacylglycerol and glucuronic acid-diacylglycerol are synthesised through the action of a new phospholipase (PA3219) and two glycosyltransferases (PA3218 and PA0842). Comparative genomic analyses revealed that this pathway is strictly conserved in all P. aeruginosa strains isolated from a range of clinical and environmental settings and actively expressed in the metatranscriptome of cystic fibrosis patients. Importantly, this phospholipid-to-glycolipid transition comes with significant ecophysiological consequence in terms of antibiotic sensitivity. Mutants defective in glycolipid synthesis survive poorly when challenged with polymyxin B, a last-resort antibiotic for treating multi-drug resistant P. aeruginosa. Thus, we demonstrate an intriguing link between adaptation to environmental stress (nutrient availability) and antibiotic resistance, mediated through membrane lipid renovation that is an important new facet in our understanding of the ecophysiology of this bacterium in the lung microbiome of cystic fibrosis patients.

scholarly journals MtBZR1 Plays an Important Role in Nodule Development in Medicago truncatula

2019 ◽  
Vol 20 (12) ◽  
pp. 2941
Author(s):  
Can Cui ◽  
Hongfeng Wang ◽  
Limei Hong ◽  
Yiteng Xu ◽  
Yang Zhao ◽  
...  
Keyword(s):  
Medicago Truncatula ◽  
Sinorhizobium Meliloti ◽  
Normal Growth ◽  
Dry Mass ◽  
Growth Conditions ◽  
Nodule Development ◽  
Functional Study ◽  
Loss Of Function ◽  
Wild Type

Brassinosteroid (BR) is an essential hormone in plant growth and development. The BR signaling pathway was extensively studied, in which BRASSINAZOLE RESISTANT 1 (BZR1) functions as a key regulator. Here, we carried out a functional study of the homolog of BZR1 in Medicago truncatula R108, whose expression was induced in nodules upon Sinorhizobium meliloti 1021 inoculation. We identified a loss-of-function mutant mtbzr1-1 and generated 35S:MtBZR1 transgenic lines for further analysis at the genetic level. Both the mutant and the overexpression lines of MtBZR1 showed no obvious phenotypic changes under normal growth conditions. After S. meliloti 1021 inoculation, however, the shoot and root dry mass was reduced in mtbzr1-1 compared with the wild type, caused by partially impaired nodule development. The transcriptomic analysis identified 1319 differentially expressed genes in mtbzr1-1 compared with wild type, many of which are involved in nodule development and secondary metabolite biosynthesis. Our results demonstrate the role of MtBZR1 in nodule development in M. truncatula, shedding light on the potential role of BR in legume–rhizobium symbiosis.

A New Genetic Locus in Sinorhizobium meliloti Is Involved in Stachydrine Utilization

1998 ◽  
Vol 64 (10) ◽  
pp. 3954-3960 ◽  
Author(s):  
Donald A. Phillips ◽  
Eve S. Sande ◽  
J. A. C. Vriezen ◽  
Frans J. de Bruijn ◽  
Daniel Le Rudulier ◽  
...  
Keyword(s):  
Salt Stress ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Genetic Locus ◽  
Wild Type ◽  
Single Strain ◽  
Reading Frame ◽  
Carbon And Nitrogen ◽  
Promoter Probe ◽  
Inducible Genes

ABSTRACT Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in Sinorhizobium meliloti. Two stachydrine-inducible genes were found in S. meliloti1021 by mutation with a Tn5-luxAB promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [14C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [14C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (P ≤ 0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as putA, and the luxABfusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame, stcD. All data are consistent with the concept that stcD codes for an enzyme that produces proline by demethylation of N-methylproline, a degradation product of stachydrine.

scholarly journals Sinorhizobium meliloti Mutants Deficient in Phosphatidylserine Decarboxylase Accumulate Phosphatidylserine and Are Strongly Affected during Symbiosis with Alfalfa

2008 ◽  
Vol 190 (20) ◽  
pp. 6846-6856 ◽  
Author(s):  
Miguel Angel Vences-Guzmán ◽  
Otto Geiger ◽  
Christian Sohlenkamp
Keyword(s):  
Sinorhizobium Meliloti ◽  
Minimal Medium ◽  
Root Nodule ◽  
Membrane Lipids ◽  
Nodule Formation ◽  
Deficient Mutant ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Root Nodule Symbiosis ◽  
Nodule Symbiosis

ABSTRACT Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids. PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss) condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A sinorhizobial mutant deficient in psd is unable to form PE but accumulates the anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or Psd were compared with those of the S. meliloti wild type. Whereas both PE-deficient mutants grew in a wild-type-like manner on many complex media, they were unable to grow on minimal medium containing high phosphate concentrations. Surprisingly, the psd-deficient mutant could grow on minimal medium containing low concentrations of inorganic phosphate, while the pss-deficient mutant could not. Addition of choline to the minimal medium rescued growth of the pss-deficient mutant, CS111, to some extent but inhibited growth of the psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with their alfalfa host plant, they behaved strikingly differently. The Pss-deficient mutant, CS111, initiated nodule formation at about the same time point as the wild type but did form about 30% fewer nodules than the wild type. In contrast, the PS-accumulating mutant, MAV01, initiated nodule formation much later than the wild type and formed 90% fewer nodules than the wild type. The few nodules formed by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish, indicating that the plants were starved for nitrogen. Therefore, changes in lipid composition, including the accumulation of bacterial PS, prevent the establishment of a nitrogen-fixing root nodule symbiosis.

scholarly journals Phosphorus stress induces the synthesis of novel glycolipids in Pseudomonas aeruginosa that confer protection against a last-resort antibiotic

2021 ◽  
Author(s):  
Rebekah Jones ◽  
Holly Shropshire ◽  
Caimeng Zhao ◽  
Andrew Murphy ◽  
Ian Lidbury ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Sensitivity ◽  
Polymyxin B ◽  
Growth Conditions ◽  
Membrane Lipid ◽  
Comparative Genomic ◽  
P Limitation ◽  
Lung Microbiome ◽  
Glycolipid Synthesis

Pseudomonas aeruginosa is a nosocomial pathogen with a prevalence in immunocompromised individuals and is particularly abundant in the lung microbiome of cystic fibrosis patients. A clinically important adaptation for bacterial pathogens during infection is their ability to survive and proliferate under phosphorus limited growth conditions. Here, we demonstrate that P. aeruginosa adapts to P-limitation by substituting membrane glycerophospholipids with sugar-containing glycolipids through a lipid renovation pathway involving a phospholipase and two glycosyltransferases. Combining bacterial genetics and multi-omics (proteomics, lipidomics and metatranscriptomic analyses), we show that the surrogate glycolipids monoglucosyldiacylglycerol and glucuronic acid-diacylglycerol are synthesised through the action of a new phospholipase (PA3219) and two glycosyltransferases (PA3218 and PA0842). Comparative genomic analyses revealed that this pathway is strictly conserved in all P. aeruginosa strains isolated from a range of clinical and environmental settings and actively expressed in the metatranscriptome of cystic fibrosis patients. Importantly, this phospholipid-to-glycolipid transition comes with significant ecophysiological consequence in terms of antibiotic sensitivity. Mutants defective in glycolipid synthesis survive poorly when challenged with polymyxin B, a last-resort antibiotic for treating multi-drug resistant P. aeruginosa. Thus, we demonstrate an intriguing link between adaptation to environmental stress (nutrient availability) and antibiotic resistance, mediated through membrane lipid renovation that is an important new facet in our understanding of the ecophysiology of this bacterium in the lung microbiome of cystic fibrosis patients.

scholarly journals The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 3049-3059 ◽  
Author(s):  
Wayne G. Reeve ◽  
Lambert Bräu ◽  
Joanne Castelli ◽  
Giovanni Garau ◽  
Christian Sohlenkamp ◽  
...  
Keyword(s):  
Cell Growth ◽  
Sinorhizobium Meliloti ◽  
Calcium Concentration ◽  
Transmembrane Protein ◽  
Sequence Similarity ◽  
Membrane Lipid ◽  
Start Codon ◽  
Major Constituent ◽  
Rhizobium Tropici ◽  
Sinorhizobium Medicae

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor–regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.

scholarly journals PmrD Is Required for Modifications to Escherichia coli Endotoxin That Promote Antimicrobial Resistance

2015 ◽  
Vol 59 (4) ◽  
pp. 2051-2061 ◽  
Author(s):  
Erica J. Rubin ◽  
Carmen M. Herrera ◽  
Alexander A. Crofts ◽  
M. Stephen Trent
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Lipid A ◽  
Bacterial Resistance ◽  
Functional Divergence ◽  
Polymyxin B ◽  
Growth Conditions ◽  
Wild Type ◽  
Content Type ◽  
E Coli

ABSTRACTInSalmonella enterica, PmrD is a connector protein that links the two-component systems PhoP-PhoQ and PmrA-PmrB. WhileEscherichia coliencodes a PmrD homolog, it is thought to be incapable of connecting PhoPQ and PmrAB in this organism due to functional divergence from theS. entericaprotein. However, our laboratory previously observed that low concentrations of Mg2+, a PhoPQ-activating signal, leads to the induction of PmrAB-dependent lipid A modifications in wild-typeE. coli(C. M. Herrera, J. V. Hankins, and M. S. Trent, Mol Microbiol 76:1444–1460, 2010,http://dx.doi.org/10.1111/j.1365-2958.2010.07150.x). These modifications include phosphoethanolamine (pEtN) and 4-amino-4-deoxy-l-arabinose (l-Ara4N), which promote bacterial resistance to cationic antimicrobial peptides (CAMPs) when affixed to lipid A. Here, we demonstrate thatpmrDis required for modification of the lipid A domain ofE. colilipopolysaccharide (LPS) under low-Mg2+growth conditions. Further, RNA sequencing shows thatE. colipmrDinfluences the expression ofpmrAand its downstream targets, including genes coding for the modification enzymes that transfer pEtN andl-Ara4N to the lipid A molecule. In line with these findings, apmrDmutant is dramatically impaired in survival compared with the wild-type strain when exposed to the CAMP polymyxin B. Notably, we also reveal the presence of an unknown factor or system capable of activatingpmrDto promote lipid A modification in the absence of the PhoPQ system. These results illuminate a more complex network of protein interactions surrounding activation of PhoPQ and PmrAB inE. colithan previously understood.

scholarly journals Crz1p Regulates pH Homeostasis in Candida glabrata by Altering Membrane Lipid Composition

2016 ◽  
Vol 82 (23) ◽  
pp. 6920-6929 ◽  
Author(s):  
Dongni Yan ◽  
Xiaobao Lin ◽  
Yanli Qi ◽  
Hui Liu ◽  
Xiulai Chen ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Lipid Composition ◽  
Candida Glabrata ◽  
Membrane Integrity ◽  
Membrane Lipid ◽  
Low Ph ◽  
Wild Type ◽  
Content Type ◽  
Membrane Lipid Composition ◽  
Acidic Conditions

ABSTRACTThe asexual facultative aerobic haploid yeastCandida glabratais widely used in the industrial production of various organic acids. To elucidate the physiological function of theC. glabratatranscription factor Crz1p (CgCrz1p) and its role in tolerance to acid stress, we deleted or overexpressed the corresponding gene,CgCRZ1. Deletion ofCgCRZ1resulted in a 60% decrease in the dry weight of cells (DCW) and a 50% drop in cell viability compared with those of the wild type at pH 2.0. Expression of lipid metabolism-associated genes was also significantly downregulated. Consequently, the proportion of C18:1fatty acids, the ratio of unsaturated to saturated fatty acids, and the ergosterol content decreased by 30%, 46%, and 30%, respectively. Additionally, membrane integrity, fluidity, and H+-ATPase activity were reduced by 45%, 9%, and 50%, respectively. In contrast, overexpression of CgCrz1p increased C18:1and ergosterol contents by 16% and 40%, respectively. Overexpression also enhanced membrane integrity, fluidity, and H+-ATPase activity by 31%, 6%, and 20%, respectively. Moreover, in the absence of pH buffering, the DCW and pyruvate titers increased by 48% and 60%, respectively, compared to that of the wild type. Together, these results suggest that CgCrz1p regulates tolerance to acidic conditions by altering membrane lipid composition inC. glabrata.IMPORTANCEThis study provides insight into the metabolism ofCandida glabrataunder acidic conditions, such as those encountered during the industrial production of organic acids. We found that overexpression of the transcription factor CgCrz1p improved viability, biomass, and pyruvate yields at a low pH. Analysis of plasma membrane lipid composition indicated that CgCrz1p might play an important role in its integrity and fluidity and that it enhanced the pumping of protons in acidic environments. We propose that altering the structure of the cell membrane may provide a successful strategy for increasingC. glabrataproductivity at a low pH.

scholarly journals GuaB Activity Is Required in Rhizobium tropici During the Early Stages of Nodulation of Determinate Nodules but Is Dispensable for the Sinorhizobium meliloti-Alfalfa Symbiotic Interaction

2005 ◽  
Vol 18 (7) ◽  
pp. 742-750 ◽  
Author(s):  
Mónica Collavino ◽  
Pablo M. Riccillo ◽  
Daniel H. Grasso ◽  
Martín Crespi ◽  
O. Mario Aguilar
Keyword(s):  
Common Bean ◽  
Host Plants ◽  
Sinorhizobium Meliloti ◽  
Nodule Formation ◽  
Wild Type ◽  
Rhizobium Tropici ◽  
Symbiotic Interaction ◽  
Early Stages ◽  
Wild Type Phenotype ◽  
Macroptilium Atropurpureum

The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis with common bean, forming nodules that lack rhizobial content. In order to investigate thetiming of the guaB requirement during the nodule formation on the host common bean by the strain CIAT899-10.T, we constructed gene fusions in which the guaB gene is expressed under the control of the symbiotic promoters nodA, bacA, and nifH. Our data indicated that the guaB is required from the early stages of nodulation because full recovery of the wild-type phenotype was accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R. tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant also is defective in its symbiosis with Macroptilium atropurpureum and Vigna unguiculata but normal with Leucaena leucocephala. These results show that the requirement of the rhizobial guaB for symbiosis is found to be associated with host plants that form determinate type of nodules.

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