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1.
PLoS Genet ; 19(7): e1010669, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37428814

RESUMO

Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5'-untranslated region (5'-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37°C. The trxA gene is transcribed into two mRNA isoforms, of which the most abundant short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5'-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region of katY we discovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37°C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with the upregulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37°C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature.


Assuntos
Yersinia pseudotuberculosis , Animais , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo , Temperatura , Espécies Reativas de Oxigênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/metabolismo , RNA/metabolismo , Estresse Oxidativo/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mamíferos/genética
2.
J Biol Chem ; 300(4): 107143, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38458396

RESUMO

A promising yet clinically unexploited antibiotic target in difficult-to-treat Gram-negative bacteria is LpxC, the key enzyme in the biosynthesis of lipopolysaccharides, which are the major constituents of the outer membrane. Despite the development of dozens of chemically diverse LpxC inhibitor molecules, it is essentially unknown how bacteria counteract LpxC inhibition. Our study provides comprehensive insights into the response against five different LpxC inhibitors. All compounds bound to purified LpxC from Escherichia coli. Treatment of E. coli with these compounds changed the cell shape and stabilized LpxC suggesting that FtsH-mediated proteolysis of the inactivated enzyme is impaired. LpxC inhibition sensitized E. coli to vancomycin and rifampin, which poorly cross the outer membrane of intact cells. Four of the five compounds led to an accumulation of lyso-phosphatidylethanolamine, a cleavage product of phosphatidylethanolamine, generated by the phospholipase PldA. The combined results suggested an imbalance in lipopolysaccharides and phospholipid biosynthesis, which was corroborated by the global proteome response to treatment with the LpxC inhibitors. Apart from LpxC itself, FabA and FabB responsible for the biosynthesis of unsaturated fatty acids were consistently induced. Upregulated compound-specific proteins are involved in various functional categories, such as stress reactions, nucleotide, or amino acid metabolism and quorum sensing. Our work shows that antibiotics targeting the same enzyme do not necessarily elicit identical cellular responses. Moreover, we find that the response of E. coli to LpxC inhibition is distinct from the previously reported response in Pseudomonas aeruginosa.


Assuntos
Amidoidrolases , Inibidores Enzimáticos , Escherichia coli , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Lipopolissacarídeos/biossíntese , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos
3.
Mol Microbiol ; 121(1): 26-39, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37985428

RESUMO

Agrobacterium tumefaciens is a plant pathogen, broadly known as the causal agent of the crown gall disease. The soil bacterium is naturally resistant to beta-lactam antibiotics by utilizing the inducible beta-lactamase AmpC. Our picture on the condition-dependent regulation of ampC expression is incomplete. A known regulator is AmpR controlling the transcription of ampC in response to unrecycled muropeptides as a signal for cell wall stress. In our study, we uncovered the global transcriptional regulator LsrB as a critical player acting upstream of AmpR. Deletion of lsrB led to severe ampicillin and penicillin sensitivity, which could be restored to wild-type levels by lsrB complementation. By transcriptome profiling via RNA-Seq and qRT-PCR and by electrophoretic mobility shift assays, we show that ampD coding for an anhydroamidase involved in peptidoglycan recycling is under direct negative control by LsrB. Controlling AmpD levels by the LysR-type regulator in turn impacts the cytoplasmic concentration of cell wall degradation products and thereby the AmpR-mediated regulation of ampC. Our results substantially expand the existing model of inducible beta-lactam resistance in A. tumefaciens by establishing LsrB as higher-level transcriptional regulator.


Assuntos
Agrobacterium tumefaciens , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , beta-Lactamases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica , Resistência beta-Lactâmica/genética
4.
Mol Microbiol ; 119(1): 29-43, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36464488

RESUMO

The outer membrane (OM) of Gram-negative bacteria functions as an essential barrier and is characterized by an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet. The enzyme LpxC catalyzes the first committed step in LPS biosynthesis. It plays a critical role in maintaining the balance between LPS and phospholipids (PL), which are both derived from the same biosynthetic precursor. The essential inner membrane proteins YejM (PbgA, LapC), LapB (YciM), and the protease FtsH are known to account for optimal LpxC levels, but the mechanistic details are poorly understood. LapB is thought to be a bi-functional protein serving as an adaptor for FtsH-mediated turnover of LpxC and acting as a scaffold in the coordination of LPS biosynthesis. Here, we provide experimental evidence for the physical interaction of LapB with proteins at the biosynthetic node from where the LPS and PL biosynthesis pathways diverge. By a total of four in vivo and in vitro assays, we demonstrate protein-protein interactions between LapB and the LPS biosynthesis enzymes LpxA, LpxC, and LpxD, between LapB and YejM, the anti-adaptor protein regulating LapB activity, and between LapB and FabZ, the first PL biosynthesis enzyme. Moreover, we uncovered a new adaptor function of LapB in destabilizing not only LpxC but also LpxD. Overall, our study shows that LapB is a multi-functional protein that serves as a protein-protein interaction hub for key enzymes in LPS and PL biogenesis presumably by virtue of multiple tetratricopeptide repeat (TPR) motifs in its cytoplasmic C-terminal region.


Assuntos
Proteínas de Escherichia coli , Lipopolissacarídeos , Lipopolissacarídeos/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Amidoidrolases/metabolismo , Proteínas de Escherichia coli/metabolismo
5.
Appl Environ Microbiol ; 90(9): e0059024, 2024 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-39120150

RESUMO

Phosphatidylcholine (PC) is critical for the nitrogen-fixing symbiosis between rhizobia and legumes. We characterized three PC biosynthesis pathways in Rhizobium leguminosarum and evaluated their impact on nitrogen fixation in clover nodules. In the presence of choline, a PC synthase catalyzes the condensation of cytidine diphosphate-diacylglycerol with choline to produce PC. In the presence of lyso-PC, acyltransferases acylate this mono-acylated phospholipid to PC. The third pathway relies on phospholipid N-methyltransferases (Pmts), which sequentially methylate phosphatidylethanolamine (PE) through three rounds of methylation, yielding PC via the intermediates monomethyl-PE and dimethyl-PE. In R. leguminosarum, at least three Pmts participate in this methylation cascade. To elucidate the functions of these enzymes, we recombinantly produced and biochemically characterized them. We moved on to determine the phospholipid profiles of R. leguminosarum mutant strains harboring single and combinatorial deletions of PC biosynthesis genes. The cumulative results show that PC production occurs through the combined action of multiple enzymes, each with distinct substrate and product specificities. The methylation pathway emerges as the dominant PC biosynthesis route, and we pinpoint PmtS2, which catalyzes all three methylation steps, as the enzyme responsible for providing adequate PC amounts for a functional nitrogen-fixing symbiosis with clover. IMPORTANCE: Understanding the molecular mechanisms of symbiotic nitrogen fixation has important implications for sustainable agriculture. The presence of the phospholipid phosphatidylcholine (PC) in the membrane of rhizobia is critical for the establishment of productive nitrogen-fixing root nodules on legume plants. The reasons for the PC requirement are unknown. Here, we employed Rhizobium leguminosarum and clover as model system for a beneficial plant-microbe interaction. We found that R. leguminosarum produces PC by three distinct pathways. The relative contribution of these pathways to PC formation was determined in an array of single, double, and triple mutant strains. Several of the PC biosynthesis enzymes were purified and biochemically characterized. Most importantly, we demonstrated the essential role of PC formation by R. leguminosarum in nitrogen fixation and pinpointed a specific enzyme indispensable for plant-microbe interaction. Our study offers profound insights into bacterial PC biosynthesis and its pivotal role in biological nitrogen fixation.


Assuntos
Proteínas de Bactérias , Fixação de Nitrogênio , Fosfatidilcolinas , Rhizobium leguminosarum , Simbiose , Rhizobium leguminosarum/metabolismo , Rhizobium leguminosarum/genética , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Nódulos Radiculares de Plantas/microbiologia , Medicago/microbiologia
6.
PLoS Pathog ; 17(11): e1009650, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34767606

RESUMO

Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotuberculosis, delivery of the secreted effector protein cocktail through the T3SS depends on YopN, a molecular gatekeeper that controls access to the secretion channel from the bacterial cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence conditions. A dominant cue is the host body temperature. A temperature of 37°C is known to induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of temperature control. We show that another RNAT silences translation of the yopN mRNA at low environmental temperatures. The long and short 5'-untranslated region of both cellular yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hairpin structure with an internal loop melts at 37°C and thereby permits formation of the translation initiation complex as shown by mutational analysis, in vitro structure probing and toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT in the faithful control of type III secretion by using a point-mutated thermostable RNAT variant with a trapped SD sequence. Abrogated YopN production in this strain led to unrestricted effector protein secretion into the medium, bacterial growth arrest and delayed translocation into eukaryotic host cells. Cumulatively, our results show that substrate delivery by the Yersinia T3SS is under hierarchical surveillance of two RNATs.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Virulência , Infecções por Yersinia pseudotuberculosis/microbiologia , Yersinia pseudotuberculosis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fagocitose , Transporte Proteico , RNA Bacteriano/genética , Infecções por Yersinia pseudotuberculosis/metabolismo
7.
PLoS Pathog ; 17(3): e1009345, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33651854

RESUMO

Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5' untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5' UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5' UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi's "stealthy" pathogenesis.


Assuntos
Regulação Bacteriana da Expressão Gênica/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Salmonella typhi/genética , Temperatura , Febre Tifoide/microbiologia , Proteínas de Bactérias/metabolismo , Humanos , Evasão da Resposta Imune/imunologia , Salmonella typhi/imunologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
8.
PLoS Comput Biol ; 18(7): e1010240, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35797361

RESUMO

It is well-established that neural networks can predict or identify structural motifs of non-coding RNAs (ncRNAs). Yet, the neural network based identification of RNA structural motifs is limited by the availability of training data that are often insufficient for learning features of specific ncRNA families or structural motifs. Aiming to reliably identify intrinsic transcription terminators in bacteria, we introduce a novel pre-training approach that uses inverse folding to generate training data for predicting or identifying a specific family or structural motif of ncRNA. We assess the ability of neural networks to identify secondary structure by systematic in silico mutagenesis experiments. In a study to identify intrinsic transcription terminators as functionally well-understood RNA structural motifs, our inverse folding based pre-training approach significantly boosts the performance of neural network topologies, which outperform previous approaches to identify intrinsic transcription terminators. Inverse-folding based pre-training provides a simple, yet highly effective way to integrate the well-established thermodynamic energy model into deep neural networks for identifying ncRNA families or motifs. The pre-training technique is broadly applicable to a range of network topologies as well as different types of ncRNA families and motifs.


Assuntos
Redes Neurais de Computação , RNA não Traduzido , Humanos , Motivos de Nucleotídeos , RNA não Traduzido/química , RNA não Traduzido/genética
9.
Mol Microbiol ; 116(1): 126-139, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33560537

RESUMO

Small RNAs (sRNAs) are universal posttranscriptional regulators of gene expression and hundreds of sRNAs are frequently found in each and every bacterium. In order to coordinate cellular processes in response to ambient conditions, many sRNAs are differentially expressed. Here, we asked how these small regulators are regulated using Agrobacterium tumefaciens as a model system. Among the best-studied sRNAs in this plant pathogen are AbcR1 regulating numerous ABC transporters and PmaR, a regulator of peptidoglycan biosynthesis, motility, and ampicillin resistance. We report that the LysR-type regulator VtlR (also known as LsrB) controls expression of AbcR1 and PmaR. A vtlR/lsrB deletion strain showed growth defects, was sensitive to antibiotics and severely compromised in plant tumor formation. Transcriptome profiling by RNA-sequencing revealed more than 1,200 genes with altered expression in the mutant. Consistent with the function of VtlR/LsrB as regulator of AbcR1, many ABC transporter genes were affected. Interestingly, the transcription factor did not only control the expression of AbcR1 and PmaR. In the mutant, 102 sRNA genes were significantly up- or downregulated. Thus, our study uncovered VtlR/LsrB as the master regulator of numerous sRNAs. Thereby, the transcriptional regulator harnesses the regulatory power of sRNAs to orchestrate the expression of distinct sub-regulons.


Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , RNA Bacteriano/biossíntese , Pequeno RNA não Traduzido/biossíntese , Fatores de Transcrição/genética , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Agrobacterium tumefaciens/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Deleção de Genes , Peptidoglicano/biossíntese , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genética
10.
Antimicrob Agents Chemother ; 66(1): e0087821, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34748386

RESUMO

Pseudomonas aeruginosa is among the highest priority pathogens for drug development because of its resistance to antibiotics, extraordinary adaptability, and persistence. Antipseudomonal research is strongly encouraged to address the acute scarcity of innovative antimicrobial lead structures. In an effort to understand the physiological response of P. aeruginosa to clinically relevant antibiotics, we investigated the proteome after exposure to ciprofloxacin, levofloxacin, rifampicin, gentamicin, tobramycin, azithromycin, tigecycline, polymyxin B, colistin, ceftazidime, meropenem, and piperacillin-tazobactam. We further investigated the response to CHIR-090, which represents a promising class of lipopolysaccharide biosynthesis inhibitors currently under evaluation. Radioactive pulse-labeling of newly synthesized proteins followed by two-dimensional polyacrylamide gel electrophoresis was used to monitor the acute response of P. aeruginosa to antibiotic treatment. The proteomic profiles provide insights into the cellular defense strategies for each antibiotic. A mathematical comparison of these response profiles based on upregulated marker proteins revealed similarities of responses to antibiotics acting on the same target area. This study provides insights into the effects of commonly used antibiotics on P. aeruginosa and lays the foundation for the comparative analysis of the impact of novel compounds with precedented and unprecedented modes of action.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Proteômica , Infecções por Pseudomonas/tratamento farmacológico
11.
PLoS Pathog ; 16(1): e1008184, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31951643

RESUMO

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.


Assuntos
Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Infecções por Yersinia pseudotuberculosis/microbiologia , Yersinia pseudotuberculosis/metabolismo , Regiões 5' não Traduzidas , Animais , Toxinas Bacterianas/química , Feminino , Humanos , Sequências Repetidas Invertidas , Camundongos , Camundongos Endogâmicos BALB C , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , Temperatura , Virulência , Yersinia pseudotuberculosis/química , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidade
12.
Nucleic Acids Res ; 48(12): e71, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32463449

RESUMO

The dynamic conformation of RNA molecules within living cells is key to their function. Recent advances in probing the RNA structurome in vivo, including the use of SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) or kethoxal reagents or DMS (dimethyl sulfate), provided unprecedented insights into the architecture of RNA molecules in the living cell. Here, we report the establishment of lead probing in a global RNA structuromics approach. In order to elucidate the transcriptome-wide RNA landscape in the enteric pathogen Yersinia pseudotuberculosis, we combined lead(II) acetate-mediated cleavage of single-stranded RNA regions with high-throughput sequencing. This new approach, termed 'Lead-seq', provides structural information independent of base identity. We show that the method recapitulates secondary structures of tRNAs, RNase P RNA, tmRNA, 16S rRNA and the rpsT 5'-untranslated region, and that it reveals global structural features of mRNAs. The application of Lead-seq to Y. pseudotuberculosis cells grown at two different temperatures unveiled the first temperature-responsive in vivo RNA structurome of a bacterial pathogen. The translation of candidate genes derived from this approach was confirmed to be temperature regulated. Overall, this study establishes Lead-seq as complementary approach to interrogate intracellular RNA structures on a global scale.


Assuntos
Análise de Sequência de RNA/métodos , Transcriptoma , Acetatos/química , Chumbo/química , Conformação de Ácido Nucleico , RNA Bacteriano/química , Yersinia pseudotuberculosis/genética
13.
Environ Microbiol ; 23(11): 6993-7008, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34528360

RESUMO

The bacterial membrane is constantly remodelled in response to environmental conditions and the external supply of precursor molecules. Some bacteria are able to acquire exogenous lyso-phospholipids and convert them to the corresponding phospholipids. Here, we report that some soil-dwelling bacteria have alternative options to metabolize lyso-phosphatidylglycerol (L-PG). We find that the plant-pathogen Agrobacterium tumefaciens takes up this mono-acylated phospholipid and converts it to two distinct isoforms of the non-canonical lipid bis(monoacylglycero)phosphate (BMP). Chromatographic separation and quadrupole-time-of-flight MS/MS analysis revealed the presence of two possible BMP stereo configurations acylated at either of the free hydroxyl groups of the glycerol head group. BMP accumulated in the inner membrane and did not visibly alter cell morphology and growth behaviour. The plant-associated bacterium Sinorhizobium meliloti was also able to convert externally provided L-PG to BMP. Other bacteria like Pseudomonas fluorescens and Escherichia coli metabolized L-PG after cell disruption, suggesting that BMP production in the natural habitat relies both on dedicated uptake systems and on head-group acylation enzymes. Overall, our study adds two previously overlooked phospholipids to the repertoire of bacterial membrane lipids and provides evidence for the remarkable condition-responsive adaptation of bacterial membranes.


Assuntos
Sinorhizobium meliloti , Espectrometria de Massas em Tandem , Lisofosfolipídeos , Monoglicerídeos/química , Sinorhizobium meliloti/metabolismo
14.
Appl Environ Microbiol ; 87(19): e0110521, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34288711

RESUMO

One of the most common pathways for the biosynthesis of the phospholipid phosphatidylcholine (PC) in bacteria is the successive 3-fold N-methylation of phosphatidylethanolamine (PE) catalyzed by phospholipid N-methyltransferases (Pmts). Pmts with different activities have been described in a number of mesophilic bacteria. In the present study, we identified and characterized the substrate and product spectra of four Pmts from thermophilic bacteria. Three of these enzymes were purified in an active form. The Pmts from Melghirimyces thermohalophilus, Thermostaphylospora chromogena, and Thermobifida fusca produce monomethyl-PE (MMPE) and dimethyl-PE (DMPE). T. fusca encodes two Pmt candidates, one of which is inactivated by mutation and the other is responsible for the accumulation of large amounts of MMPE. The Pmt enzyme from Rubellimicrobium thermophilum catalyzes all three methylation reactions to synthesize PC. Moreover, we show that PE, previously reported to be absent in R. thermophilum, is in fact produced and serves as a precursor for the methylation pathway. In an alternative route, the strain is able to produce PC by the PC synthase pathway when choline is available. The activity of all purified thermophilic Pmt enzymes was stimulated by anionic lipids, suggesting membrane recruitment of these cytoplasmic proteins via electrostatic interactions. Our study provides novel insights into the functional characteristics of phospholipid N-methyltransferases in a previously unexplored set of thermophilic environmental bacteria. IMPORTANCE In recent years, the presence of phosphatidylcholine (PC) in bacterial membranes has gained increasing attention, partly due to its critical role in the interaction with eukaryotic hosts. PC biosynthesis via a three-step methylation of phosphatidylethanolamine, catalyzed by phospholipid N-methyltransferases (Pmts), has been described in a range of mesophilic bacteria. Here, we expand our knowledge on bacterial PC formation by the identification, purification, and characterization of Pmts from phylogenetically diverse thermophilic bacteria and thereby provide insights into the functional characteristics of Pmt enzymes in thermophilic actinomycetes and proteobacteria.


Assuntos
Bactérias/enzimologia , Fosfatidil-N-Metiletanolamina N-Metiltransferase/metabolismo , Fosfatidiletanolaminas/metabolismo , Bactérias/genética , Metilação , Fosfatidil-N-Metiletanolamina N-Metiltransferase/genética
15.
Appl Microbiol Biotechnol ; 105(23): 8837-8851, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34709431

RESUMO

Escherichia coli is the daily workhorse in molecular biology research labs and an important platform microorganism in white biotechnology. Its cytoplasmic membrane is primarily composed of the phospholipids phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). As in most other bacteria, the typical eukaryotic phosphatidylcholine (PC) is not a regular component of the E. coli membrane. PC is known to act as a substrate in various metabolic or catabolic reactions, to affect protein folding and membrane insertion, and to activate proteins that originate from eukaryotic environments. Options to manipulate the E. coli membrane to include non-native lipids such as PC might make it an even more powerful and versatile tool for biotechnology and protein biochemistry. This article outlines different strategies how E. coli can be engineered to produce PC and other methylated PE derivatives. Several of these approaches rely on the ectopic expression of genes from natural PC-producing organisms. These include PC synthases, lysolipid acyltransferases, and several phospholipid N-methyltransferases with diverse substrate and product preferences. In addition, we show that E. coli has the capacity to produce PC by its own enzyme repertoire provided that appropriate precursors are supplied. Screening of the E. coli Keio knockout collection revealed the lysophospholipid transporter LplT to be responsible for the uptake of lyso-PC, which is then further acylated to PC by the acyltransferase-acyl carrier protein synthetase Aas. Overall, our study shows that the membrane composition of the most routinely used model bacterium can readily be tailored on demand.Key points• Escherichia coli can be engineered to produce non-native methylated PE derivatives.• These lipids can be produced by foreign and endogenous proteins.• Modification of E. coli membrane offers potential for biotechnology and research.


Assuntos
Escherichia coli , Fosfolipídeos , Transporte Biológico , Membrana Celular/metabolismo , Escherichia coli/genética , Lipídeos , Fosfolipídeos/metabolismo
16.
J Bacteriol ; 202(14)2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32366590

RESUMO

Correct targeting of membrane proteins is essential for membrane integrity, cell physiology, and viability. Cotranslational targeting depends on the universally conserved signal recognition particle (SRP), which is a ribonucleoprotein complex comprised of the protein component Ffh and the 4.5S RNA in Escherichia coli About 25 years ago it was reported that Ffh is an unstable protein, but the underlying mechanism has never been explored. Here, we show that Lon is the primary protease responsible for adjusting the cellular Ffh level. When overproduced, Ffh is particularly prone to degradation during transition from exponential to stationary growth and the cellular Ffh amount is lowest in stationary phase. The Ffh protein consists of two domains, the NG domain, responsible for GTP hydrolysis and docking to the membrane receptor FtsY, and the RNA-binding M domain. We find that the NG domain alone is stable, whereas the isolated M domain is degraded. Consistent with the importance of Lon in this process, the M domain confers synthetic lethality to the lon mutant. The Ffh homolog from the model plant Arabidopsis thaliana, which forms a protein-protein complex rather than a protein-RNA complex, is stable, suggesting that the RNA-binding ability residing in the M domain of E. coli Ffh is important for proteolysis. Our results support a model in which excess Ffh not bound to 4.5S RNA is subjected to proteolysis until an appropriate Ffh concentration is reached. The differential proteolysis adjusts Ffh levels to the cellular demand and maintains cotranslational protein transport and membrane integrity.IMPORTANCE Since one-third of all bacterial proteins reside outside the cytoplasm, protein targeting to the appropriate address is an essential process. Cotranslational targeting to the membrane relies on the signal recognition particle (SRP), which is a protein-RNA complex in bacteria. We report that the protein component Ffh is a substrate of the Lon protease. Regulated proteolysis of Ffh provides a simple mechanism to adjust the concentration of the essential protein to the cellular demand. This is important because elevated or depleted SRP levels negatively impact protein targeting and bacterial fitness.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Protease La/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Protease La/genética , Domínios Proteicos , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/genética
17.
J Bacteriol ; 202(22)2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093235

RESUMO

In any given organism, approximately one-third of all proteins have a yet-unknown function. A widely distributed domain of unknown function is DUF1127. Approximately 17,000 proteins with such an arginine-rich domain are found in 4,000 bacteria. Most of them are single-domain proteins, and a large fraction qualifies as small proteins with fewer than 50 amino acids. We systematically identified and characterized the seven DUF1127 members of the plant pathogen Agrobacterium tumefaciens They all give rise to authentic proteins and are differentially expressed as shown at the RNA and protein levels. The seven proteins fall into two subclasses on the basis of their length, sequence, and reciprocal regulation by the LysR-type transcription factor LsrB. The absence of all three short DUF1127 proteins caused a striking phenotype in later growth phases and increased cell aggregation and biofilm formation. Protein profiling and transcriptome sequencing (RNA-seq) analysis of the wild type and triple mutant revealed a large number of differentially regulated genes in late exponential and stationary growth. The most affected genes are involved in phosphate uptake, glycine/serine homeostasis, and nitrate respiration. The results suggest a redundant function of the small DUF1127 paralogs in nutrient acquisition and central carbon metabolism of A. tumefaciens They may be required for diauxic switching between carbon sources when sugar from the medium is depleted. We end by discussing how DUF1127 might confer such a global impact on cell physiology and gene expression.IMPORTANCE Despite being prevalent in numerous ecologically and clinically relevant bacterial species, the biological role of proteins with a domain of unknown function, DUF1127, is unclear. Experimental models are needed to approach their elusive function. We used the phytopathogen Agrobacterium tumefaciens, a natural genetic engineer that causes crown gall disease, and focused on its three small DUF1127 proteins. They have redundant and pervasive roles in nutrient acquisition, cellular metabolism, and biofilm formation. The study shows that small proteins have important previously missed biological functions. How small basic proteins can have such a broad impact is a fascinating prospect of future research.


Assuntos
Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Fosfatos/metabolismo , Agrobacterium tumefaciens/genética , Arginina/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Domínios Proteicos , RNA Bacteriano/genética , RNA-Seq , Fatores de Transcrição/metabolismo
18.
Infect Immun ; 88(3)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31792074

RESUMO

RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Regulação Bacteriana da Expressão Gênica , Shigella dysenteriae , Temperatura , Fatores de Virulência , Virulência/genética , RNA Bacteriano/metabolismo , Sequências Reguladoras de Ácido Nucleico/fisiologia , Shigella dysenteriae/patogenicidade , Shigella dysenteriae/fisiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
19.
Mol Microbiol ; 111(1): 17-30, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30325563

RESUMO

Biological nitrogen fixation, the reduction of chemically inert dinitrogen to bioavailable ammonia, is a central process in the global nitrogen cycle highly relevant for life on earth. N2 reduction to NH3 is catalyzed by nitrogenases exclusively synthesized by diazotrophic prokaryotes. All diazotrophs have a molybdenum nitrogenase containing the unique iron-molybdenum cofactor FeMoco. In addition, some diazotrophs encode one or two alternative Mo-free nitrogenases that are less efficient at reducing N2 than Mo-nitrogenase. To permit biogenesis of Mo-nitrogenase and other molybdoenzymes when Mo is scarce, bacteria synthesize the high-affinity molybdate transporter ModABC. Generally, Mo supports expression of Mo-nitrogenase genes, while it represses production of Mo-free nitrogenases and ModABC. Since all three nitrogenases and ModABC can reach very high levels at suitable Mo concentrations, tight Mo-mediated control saves considerable resources and energy. This review outlines the similarities and differences in Mo-responsive regulation of nitrogen fixation and molybdate transport in diverse diazotrophs.


Assuntos
Bactérias/metabolismo , Molibdênio/metabolismo , Fixação de Nitrogênio , Oligoelementos/metabolismo , Amônia/metabolismo , Bactérias/enzimologia , Transporte Biológico , Metaloproteínas/metabolismo , Molibdoferredoxina/metabolismo , Nitrogênio/metabolismo , Nitrogenase/metabolismo , Oxirredução
20.
Mol Microbiol ; 111(1): 269-286, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30353924

RESUMO

Agrobacterium tumefaciens transfers oncogenic T-DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl-phosphatidylglycerol (L-PG) hydrolase, which modulates L-PG homeostasis. Through functional characterization of recombinant AcvB variants, we showed that the C-terminal domain of AcvB (residues 232-456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10-fold increase in L-PG in Agrobacterium membranes and abolished T-DNA transfer and tumor formation. Overproduction of the L-PG synthase gene (lpiA) in wild-type A. tumefaciens resulted in a similar increase in the L-PG content (~7-fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L-PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L-PG content by complementation with different acvB variants revealed that cellular L-PG levels above 3% of total phospholipids interfere with T-DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane lipid homeostasis and T-DNA transfer.


Assuntos
Agrobacterium tumefaciens/patogenicidade , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Homeostase , Lisina/metabolismo , Fosfatidilgliceróis/metabolismo , Fatores de Virulência/metabolismo , Agrobacterium tumefaciens/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Domínio Catalítico , Análise Mutacional de DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Deleção de Genes , Teste de Complementação Genética , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Transformação Genética , Virulência , Fatores de Virulência/genética
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