RESUMO
Three Gram-positive-staining strains FJAT-49754T, FJAT-49682 and FJAT-49731 were isolated from the citrus rhizosphere soil sample. These strains showed the highest 16S rRNA gene sequence similarity with the type strain of Lederbergia panacisoli (97.8-97.9â%). The 16S rRNA gene sequence similarities between strains FJAT-49754T, FJAT-49682, and FJAT-49731 were 99.9â%. The average nucleotide identity (ANI) values between strains FJAT-49754T, FJAT-49682 and FJAT-49731 were above 96â%, while the ANI values with the members of the genus Lederbergia were below 95â%, which were below the cut-off level for prokaryotic species delineation. The above results suggest that strains FJAT-49754T, FJAT-49682 and FJAT-49731 belong to a novel species of the genus Lederbergia. Growth of strain FJAT-49754T was observed at 10-40 °C (optimum at 30 °C, pH 6.0-10.0 (optimum at pH 8.0), and NaCl tolerance up to 7â% (w/v) (optimum at 1â%). MK-7 was the only menaquinone detected in strain FJAT-49754T, and the main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids of strain FJAT-49754T were anteiso-C15â:â0, iso-C15â:â0, and C16â:â0. The genomic DNA G+C content of strain FJAT-49754T was 38.7â%. Based on the above results, strain FJAT-49754T represents a novel species of the genus Lederbergia, for which the name Lederbergia citrea sp. nov., is proposed. The type strain is FJAT-49754T (=CCTCC AB 2019211T=LMG 31589T).
Assuntos
Ácidos Graxos , Rizosfera , Ácidos Graxos/química , RNA Ribossômico 16S/genética , DNA Bacteriano/genética , Hibridização de Ácido Nucleico , Composição de Bases , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Ácido Diaminopimélico/química , Análise de Sequência de DNA , Parede Celular/química , Peptidoglicano/químicaRESUMO
Cellulosomes are multi-enzymatic nanomachines that have been fine-tuned through evolution to efficiently deconstruct plant biomass. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between the various enzyme-borne dockerin modules and the multiple copies of the cohesin modules located on the scaffoldin subunit. Recently, designer cellulosome technology was established to provide insights into the architectural role of catalytic (enzymatic) and structural (scaffoldin) cellulosomal constituents for the efficient degradation of plant cell wall polysaccharides. Owing to advances in genomics and proteomics, highly structured cellulosome complexes have recently been unraveled, and the information gained has inspired the development of designer-cellulosome technology to new levels of complex organization. These higher-order designer cellulosomes have in turn fostered our capacity to enhance the catalytic potential of artificial cellulolytic complexes. In this chapter, methods to produce and employ such intricate cellulosomal complexes are reported.
Assuntos
Celulose , Celulossomas , Celulose/metabolismo , Parede Celular/metabolismo , Membrana Celular/metabolismo , Genômica , Celulossomas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
Expansins are proteins that loosen plant cell walls but lack enzymatic activity. Here we describe two protocols tailored to measure the biomechanical activity of bacterial expansin. The first assay relies on the weakening of filter paper by expansin. The second assay is based on induction of creep (long-term, irreversible extension) of plant cell wall samples.
Assuntos
Bactérias , Parede Celular , Bactérias/metabolismo , Parede Celular/metabolismo , Membrana Celular/metabolismo , Proteínas de Plantas/metabolismoRESUMO
The interactions of proteins involved in plant cell wall hydrolysis, such as enzymes and CBMs, significantly determine their role and efficiency. In order to go beyond the characterization of interactions with simple ligands, bioinspired assemblies combined with the measurement of diffusion and interaction by FRAP offer a relevant alternative for highlighting the importance of different parameters related to the protein affinity and to the polymer type and organization in the assembly.
Assuntos
Parede Celular , Polímeros , Polímeros/metabolismo , Parede Celular/metabolismo , Membrana Celular , CarboidratosRESUMO
Fourier transform mid-infrared spectroscopy (FTIR) is a powerful tool for compositional analysis of plant cell walls. The infrared spectrum generates a fingerprint of a sample with absorption peaks corresponding to the frequency of vibrations between the bonds of the atoms making up the material. Here we describe a method focused on the use of FTIR in combination with principal component analysis (PCA) to characterize the composition of the plant cell wall. The FTIR method described here facilitates high-throughput identification of the major compositional differences across a large set of samples in a low-cost and non-destructive manner.
Assuntos
Carboidratos , Parede Celular , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Análise de Fourier , Carboidratos/análise , Parede Celular/química , Membrana CelularRESUMO
Immunocytochemistry is a widely used technique to localize antigen within intact tissues. Plant cell walls are complex matrixes of highly decorated polysaccharides and the large number of CBM families displaying specific substrate recognition reflects this complexity. The accessibility of large proteins, such as antibodies, to their cell wall epitopes may be sometimes difficult due to steric hindrance problems. Due to their smaller size, CBMs are interesting alternative probes. The aim of this chapter is to describe the use of CBM as probes to explore complex polysaccharide topochemistry in muro and to quantify enzymatic deconstruction.
Assuntos
Parede Celular , Polissacarídeos , Humanos , Imuno-Histoquímica , Parede Celular/metabolismo , Polissacarídeos/metabolismo , Membrana Celular/metabolismoRESUMO
Secondary cell wall (SCW) thickening has a significant effect on the growth and development of plants, as well as in the resistance to various biotic and abiotic stresses. Lignin accounts for the strength of SCW. It is synthesized through the phenylpropanoid pathway that also leads to flavonoid synthesis. The coupling strategies for lignin and flavonoid syntheses are diverse in plants. How their syntheses are balanced by transcriptional regulation in fleshy fruits is still unclear. The diploid strawberry (Fragaria vesca) is a model for fleshy fruits research due to its small genome and wide scope of genetic transformation. SCW thickening is regulated by a multilevel transcriptional regulatory network wherein vascular-related NAC domains (VNDs) act as key regulators. In this study, we systematically characterized VNDs in Fragaria vesca and explored their functions. The overexpression of FvVND4c in diploid strawberry fruits resulted in SCW thickening and fruit color changes accompanied with the accumulation of lignin and flavonoids. Genes related to these phenotypes were also induced upon FvVND4c overexpression. Among the induced genes, we found FvMYB46 to be a direct downstream regulator of FvVND4c. The overexpression of FvMYB46 resulted in similar phenotypes as FvVND4c, except for the color change. Transcriptomic analyses suggest that both FvVND4c and FvMYB46 act on phenylpropanoid and flavonoid biosynthesis pathways, and induce lignin synthesis for SCW. These results suggest that FvVND4c and FvMYB46 cooperatively regulate SCW thickening and flavonoid accumulation in Fragaria vesca.
Assuntos
Fragaria , Fragaria/metabolismo , Flavonoides/farmacologia , Expressão Ectópica do Gene , Lignina/metabolismo , Frutas/metabolismo , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismoRESUMO
Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE-MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE-MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE-MurF interacts with other Mur ligases via its central domains with KDs in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival.
Assuntos
Bactérias , Proteínas de Bactérias , Proteínas de Bactérias/metabolismo , Bactérias/metabolismo , Ligases/metabolismo , Parede Celular/metabolismo , Genômica , Peptidoglicano/metabolismo , Peptídeo Sintases/metabolismoRESUMO
The peptidoglycan (PG) cell wall produced by the bacterial division machinery is initially shared between the daughters and must be split to promote cell separation and complete division. In gram-negative bacteria, enzymes that cleave PG called amidases play major roles in the separation process. To prevent spurious cell wall cleavage that can lead to cell lysis, amidases like AmiB are autoinhibited by a regulatory helix. Autoinhibition is relieved at the division site by the activator EnvC, which is in turn regulated by the ATP-binding cassette (ABC) transporter-like complex called FtsEX. EnvC is also known to be autoinhibited by a regulatory helix (RH), but how its activity is modulated by FtsEX and the mechanism by which it activates the amidases have remained unclear. Here, we investigated this regulation by determining the structure of Pseudomonas aeruginosa FtsEX alone with or without bound ATP, in complex with EnvC, and in a FtsEX-EnvC-AmiB supercomplex. In combination with biochemical studies, the structures reveal that ATP binding is likely to activate FtsEX-EnvC and promote its association with AmiB. Furthermore, the AmiB activation mechanism is shown to involve a RH rearrangement. In the activated state of the complex, the inhibitory helix of EnvC is released, freeing it to associate with the RH of AmiB, which liberates its active site for PG cleavage. These regulatory helices are found in many EnvC proteins and amidases throughout gram-negative bacteria, suggesting that the activation mechanism is broadly conserved and a potential target for lysis-inducing antibiotics that misregulate the complex.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Hidrólise , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Amidoidrolases/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Parede Celular/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peptidoglicano/metabolismo , Endopeptidases/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
The integrity of the cell wall is important for plant cells. Mechanical or chemical distortions, tension, pH changes in the apoplast, disturbance of the ion homeostasis, leakage of cell compounds into the apoplastic space or breakdown of cell wall polysaccharides activate cellular responses which often occur via plasma membrane-localized receptors. Breakdown products of the cell wall polysaccharides function as damage-associated molecular patterns and derive from cellulose (cello-oligomers), hemicelluloses (mainly xyloglucans and mixed-linkage glucans as well as glucuronoarabinoglucans in Poaceae) and pectins (oligogalacturonides). In addition, several types of channels participate in mechanosensing and convert physical into chemical signals. To establish a proper response, the cell has to integrate information about apoplastic alterations and disturbance of its wall with cell-internal programs which require modifications in the wall architecture due to growth, differentiation or cell division. We summarize recent progress in pattern recognition receptors for plant-derived oligosaccharides, with a focus on malectin domain-containing receptor kinases and their crosstalk with other perception systems and intracellular signaling events.
Assuntos
Células Vegetais , Polissacarídeos , Células Vegetais/metabolismo , Polissacarídeos/metabolismo , Pectinas/metabolismo , Parede Celular/metabolismo , Membrana Celular/metabolismo , PercepçãoRESUMO
The cell envelope of Gram-negative bacteria contains two distinct membranes, an inner (IM) and an outer (OM) membrane, separated by the periplasm, a hydrophilic compartment that includes a thin layer of peptidoglycan [...].
Assuntos
Proteínas da Membrana Bacteriana Externa , Lipopolissacarídeos , Lipopolissacarídeos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Periplasma/metabolismo , Parede Celular/metabolismo , Peptidoglicano/metabolismoRESUMO
Brewer's spent yeast (BSY) mannoproteins have been reported to possess thickening and emulsifying properties. The commercial interest in yeast mannoproteins might be boosted considering the consolidation of their properties supported by structure/function relationships. This work aimed to attest the use of extracted BSY mannoproteins as a clean label and vegan source of ingredients for the replacement of food additives and protein from animal sources. To achieve this, structure/function relationships were performed by isolating polysaccharides with distinct structural features from BSY, either by using alkaline extraction (mild treatment) or subcritical water extraction (SWE) using microwave technology (hard treatment), and assessment of their emulsifying properties. Alkaline extractions solubilized mostly highly branched mannoproteins (N-linked type; 75%) and glycogen (25%), while SWE solubilized mannoproteins with short mannan chains (O-linked type; 55%) and (1â4)- and (ß1â3)-linked glucans, 33 and 12%, respectively. Extracts with high protein content yielded the most stable emulsions obtained by hand shaking, while the extracts composed of short chain mannans and ß-glucans yielded the best emulsions by using ultraturrax stirring. ß-Glucans and O-linked mannoproteins were found to contribute to emulsion stability by preventing Ostwald ripening. When applied in mayonnaise model emulsions, BSY extracts presented higher stability and yet similar texture properties as the reference emulsifiers. When used in a mayonnaise formulation, the BSY extracts were also able to replace egg yolk and modified starch (E1422) at 1/3 of their concentration. This shows that BSY alkali soluble mannoproteins and subcritical water extracted ß-glucans can be used as replacers of animal protein and additives in sauces.
Assuntos
Saccharomyces cerevisiae , beta-Glucanas , Animais , Humanos , Saccharomyces cerevisiae/metabolismo , Emulsões/metabolismo , Veganos , Polissacarídeos/química , Mananas/metabolismo , Água/análise , Parede Celular/química , beta-Glucanas/metabolismo , Extratos Vegetais/análiseRESUMO
The microalgae Haematococcus pluvialis is a commercial source of natural astaxanthin. However, mature cells develop rigid three-layer wall structures and a repulsive odor. This study applied a liquid static fermentation system to screen hydrolyzing microorganisms for cell wall hydrolysis. Baijiu jiuqu and Gutian hongqu were found to have promising potential for application. The fermentation using 2% baijiu jiuqu and 2% glucose for pre-activation achieved comparable recovery of carotenoids to homogenizer disruption methods and produced stable fragrance which may be attributed to ethyl octanoate, hexyl formate, and phenethyl butyrate, as revealed by gas chromatography-mass spectrometry analysis. The abundance of astaxanthin molecules was slightly affected by fermentation with fold change < 2, while molecules with higher fold change (>10) were mainly carbohydrates, lipids, and steroids proving the safety of the fermentation. This study provides a new scheme for the biorefining of Haematococcus. pluvialis, potentially contributing to the industrial production of natural astaxanthin.
Assuntos
Clorofíceas , Fermentação , Biomassa , Bebidas , Parede CelularRESUMO
Phytophthora capsici is a highly destructive phytopathogenic oomycete with a broad host range and is responsible for tremendous losses. Euphorbia factor L3 (EFL3) is a natural plant-derived compound that has been widely studied in medicine and cosmetic applications. In this study, the sensitivity of 105 P. capsici isolates to EFL3 was determined, and the biological activity and physiological effects of EFL3 against P. capsici were investigated. The median effective concentration (EC50) values for EFL3 inhibition mycelial growth and spore germination ranged from 0.66 to 8.94 µg/mL (mean, 2.96 ± 0.91 µg/mL) and 1.63 to 13.16 µg/mL (mean, 5.30 ± 1.64 µg/mL), respectively. EFL3 treatment resulted in cell wall and cell membrane damage of P. capsici, which was revealed by morphological and ultrastructural observations, propidium iodide (PI) and calcofluor white (CFW) staining, and measurements of relative conductivity as well as malondialdehyde (MDA) and glycerol contents. In addition, the contents of phospholipid and cellulose, which are the major components of cell membrane and cell wall, were significantly reduced following EFL3 treatment. Furthermore, EFL3 provided protective as well as curative efficacies against P. capsici on detached tomato leaves and pepper seedlings in vivo. These data show that EFL3 exhibits strong inhibitory activity against P. capsici, thereby suggesting that it could be an effective alternative for controlling P. capsici-induced diseases.
Assuntos
Euphorbia , Phytophthora , Membrana Celular , Parede Celular , Folhas de Planta , Doenças das Plantas/prevenção & controleRESUMO
The lack of effective therapeutics against emerging multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) prompts the identification of novel anti-tuberculosis targets. The essential nature of the peptidoglycan (PG) layer of the mycobacterial cell wall, which features several distinctive modifications, such as the N-glycolylation of muramic acid and the amidation of D-iso-glutamate, makes it a target of particular interest. To understand their role in susceptibility to beta-lactams and in the modulation of host-pathogen interactions, the genes encoding the enzymes responsible for these PG modifications (namH and murT/gatD, respectively) were silenced in the model organism Mycobacterium smegmatis using CRISPR interference (CRISPRi). Although beta-lactams are not included in TB-therapy, their combination with beta-lactamase inhibitors is a prospective strategy to treat MDR-TB. To uncover synergistic effects between the action of beta-lactams and the depletion of these PG modifications, knockdown mutants were also constructed in strains lacking the major beta-lactamase of M. smegmatis BlaS, PM965 (M. smegmatis ΔblaS1) and PM979 (M. smegmatis ΔblaS1 ΔnamH). The phenotyping assays affirmed the essentiality of the amidation of D-iso-glutamate to the survival of mycobacteria, as opposed to the N-glycolylation of muramic acid. The qRT-PCR assays confirmed the successful repression of the target genes, along with few polar effects and differential knockdown level depending on PAM strength and target site. Both PG modifications were found to contribute to beta-lactam resistance. While the amidation of D-iso-glutamate impacted cefotaxime and isoniazid resistance, the N-glycolylation of muramic acid substantially promoted resistance to the tested beta-lactams. Their simultaneous depletion provoked synergistic reductions in beta-lactam MICs. Moreover, the depletion of these PG modifications promoted a significantly faster bacilli killing by J774 macrophages. Whole-genome sequencing revealed that these PG modifications are highly conserved in a set of 172 clinical strains of Mtb, demonstrating their potential as therapeutic targets against TB. Our results support the development of new therapeutic agents targeting these distinctive mycobacterial PG modifications.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Peptidoglicano/genética , Ácidos Murâmicos/farmacologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Tuberculose/microbiologia , Resistência beta-Lactâmica , Parede Celular , beta-Lactamas/farmacologia , Glutamatos/genética , Glutamatos/farmacologia , Antibacterianos/farmacologiaRESUMO
Sudan grass is a high-quality forage of sorghum. The degree of lignification of Sudan grass is the main factor affecting its digestibility in ruminants such as cattle and sheep. Almost all lignocellulose in Sudan grass is stored in the secondary cell wall, but the mechanism and synthesis of the secondary cell wall in Sudan grass is still unclear. In order to study the mechanism of secondary cell wall synthesis in Sudan grass, we used an in vitro induction system of Sudan grass secondary cell wall. Through transcriptome sequencing, it was found that the NAC transcription factor CcNAC1 gene was related to the synthesis of the Sudan grass secondary cell wall. This study further generated CcNAC1 overexpression lines of Arabidopsis to study CcNAC1 gene function in secondary cell wall synthesis. It was shown that the overexpression of the CcNAC1 gene can significantly increase lignin content in Arabidopsis lines. Through subcellular localization analysis, CcNAC1 genes could be expressed in the nucleus of a plant. In addition, we used yeast two-hybrid screening to find 26 proteins interacting with CcNAC1. GO and KEGG analysis showed that CcNAC1 relates to the metabolic pathways and biosynthesis of secondary metabolites. In summary, the synthesis of secondary cell wall of Sudan grass can be regulated by CcNAC1.
Assuntos
Arabidopsis , Sorghum , Animais , Bovinos , Ovinos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Sorghum/genética , Lignina/metabolismo , Perfilação da Expressão Gênica , Parede Celular/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the D-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the D-arabinan or D-galactan components of arabinogalactan. Using one of these isolates with exo-D-galactofuranosidase activity, we generated enriched D-arabinan and used it to identify a strain of Dysgonomonas gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-D-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-D-arabinanases with different preferences for the D-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.
Assuntos
Mycobacterium tuberculosis , Polissacarídeos , Humanos , Polissacarídeos/metabolismo , Mycobacterium tuberculosis/metabolismo , Glicosídeo Hidrolases/metabolismo , Parede Celular/metabolismoRESUMO
In fungi, the cell wall plays a crucial role in morphogenesis and response to stress from the external environment. Chitin is one of the main cell wall components in many filamentous fungi. In Aspergillus nidulans, a class III chitin synthase ChsB plays a pivotal role in hyphal extension and morphogenesis. However, little is known about post-translational modifications of ChsB and their functional impacts. In this study, we showed that ChsB is phosphorylated in vivo. We characterized strains that produce ChsB using stepwise truncations of its N-terminal disordered region or deletions of some residues in that region and demonstrated its involvement in ChsB abundance on the hyphal apical surface and in hyphal tip localization. Furthermore, we showed that some deletions in this region affected the phosphorylation states of ChsB, raising the possibility that these states are important for the localization of ChsB to the hyphal surface and the growth of A. nidulans. Our findings indicate that ChsB transport is regulated by its N-terminal disordered region.
Assuntos
Aspergillus nidulans , Aspergillus nidulans/genética , Hifas , Parede Celular/metabolismo , Quitina Sintase/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
Control of cell size and morphology is of paramount importance for bacterial fitness. In the opportunistic pathogen Enterococcus faecalis, the formation of diplococci and short cell chains facilitates innate immune evasion and dissemination in the host. Minimisation of cell chain size relies on the activity of a peptidoglycan hydrolase called AtlA, dedicated to septum cleavage. To prevent autolysis, AtlA activity is tightly controlled, both temporally and spatially. Here, we show that the restricted localization of AtlA at the septum occurs via an unexpected mechanism. We demonstrate that the C-terminal LysM domain that allows the enzyme to bind peptidoglycan is essential to target this enzyme to the septum inside the cell before its translocation across the membrane. We identify a membrane-bound cytoplasmic protein partner (called AdmA) involved in the recruitment of AtlA via its LysM domains. This work reveals a moonlighting role for LysM domains, and a mechanism evolved to restrict the subcellular localization of a potentially lethal autolysin to its site of action.
Assuntos
Enterococcus faecalis , Peptidoglicano , Enterococcus faecalis/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Separação CelularRESUMO
The mycobacterial cell envelope consists of a typical plasma membrane, surrounded by a complex cell wall and a lipid-rich outer membrane. The biogenesis of this multilayer structure is a tightly regulated process requiring the coordinated synthesis and assembly of all its constituents. Mycobacteria grow by polar extension and recent studies showed that cell envelope incorporation of mycolic acids, the major constituent of the cell wall and outer membrane, is coordinated with peptidoglycan biosynthesis at the cell poles. However, there is no information regarding the dynamics of incorporation of other families of outer membrane lipids during cell elongation and division. Here, we establish that the translocation of non-essential trehalose polyphleates (TPP) occurs at different subcellular locations than that of the essential mycolic acids. Using fluorescence microscopy approaches, we investigated the subcellular localization of MmpL3 and MmpL10, respectively involved in the export of mycolic acids and TPP, in growing cells and their colocalization with Wag31, a protein playing a critical role in regulating peptidoglycan biosynthesis in mycobacteria. We found that MmpL3, like Wag31, displays polar localization and preferential accumulation at the old pole whereas MmpL10 is more homogenously distributed in the plasma membrane and slightly accumulates at the new pole. These results led us to propose a model in which insertion of TPP and mycolic acids into the mycomembrane is spatially uncoupled.
