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1.
Chemosphere ; 238: 124571, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31472351

RESUMO

Rhodococcus exhibits strong adaptability to environmental stressors and plays a crucial role in environmental bioremediation. However, seasonal changes in ambient temperature, especially rapid temperature drops exert an adverse effect on in situ bioremediation. In this paper, we studied the cell morphology and fatty acid composition of an aniline-degrading strain Rhodococcus sp. CNS16 at temperatures of 30 °C, 20 °C, and 10 °C. At suboptimal temperatures, cell morphology of CNS16 changed from short rod-shaped to long rod or irregular shaped, and the proportion of unsaturated fatty acids was upregulated. Transcriptomic technologies were then utilized to gain detailed insights into the adaptive mechanisms of CNS16 subjected to suboptimal temperatures. The results showed that the number of gene responses was significantly higher at 10 °C than that at 20 °C. The inhibition of peptidoglycan synthase expression and up-regulation of Filamentous Temperature Sensitive as well as unsaturated fatty acid synthesis genes at suboptimal temperatures might be closely related to corresponding changes in cell morphology and fatty acids composition. Strain CNS16 showed loss of catalase and superoxide dismutase activity, and utilized thioredoxin-dependent thiol peroxidase to resist oxidative stress. The up-regulation of carotenoid and Vitamin B2 synthesis at 10 °C might also be involved in the resistance to oxidative stress. Amino acid metabolism, coenzyme and vitamin metabolism, ABC transport, and energy metabolism are essential for peptidoglycan synthesis and regulation of cellular metabolism; therefore, synergistically resisting environmental stress. This study provides a mechanistic basis for the regulation of aniline degradation in Rhodococcus sp. CNS16 at low temperatures.


Assuntos
Aclimatação/fisiologia , Compostos de Anilina/metabolismo , Ácidos Graxos/metabolismo , Peptidoglicano/biossíntese , Rhodococcus/metabolismo , Biodegradação Ambiental , Catalase/metabolismo , Temperatura Baixa , Estresse Oxidativo/fisiologia , Proteínas de Ligação às Penicilinas/biossíntese , Peroxirredoxinas/metabolismo , Rhodococcus/genética , Estações do Ano , Superóxido Dismutase/metabolismo , Transcriptoma
2.
Gene ; 720: 144094, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476407

RESUMO

Fourteen different insertion sequences belonging to seven families were identified in the genome of Streptococcus agalactiae. Among them, IS1548, a mobile element of the ISAs1 family, was linked to clonal complex (CC) 19 strains associated with neonatal meningitis and endocarditis. IS1548 impacts S. agalactiae in two reported ways: i) inactivation of virulence genes by insertion in an open reading frame (e.g. hylB or cpsD), ii) positive modulation of the expression of a downstream gene by insertion in an intergenic region (e.g. lmb). We previously identified an unknown integration site of IS1548 in the intergenic region between the folK and the murB genes involved in folate and peptidoglycan biosynthesis, respectively. In this work, we analyzed the prevalence of IS1548 in a large collection of nine hundred and eleven S. agalactiae strains. IS1548 positive strains belong to twenty-nine different sequence types and to ten CCs. The majority of them were, however, clustered within sequence type 19 and sequence type 22, belonging to CC19 and CC22, respectively. In contrast, IS1548 targets the folK-murB intergenic region exclusively in CC19 strains. We evaluated the impact of the insertion of IS1548 on the expression of murB by locating transcriptional promoters influencing its expression in the presence or absence of IS1548 and by comparative ß-galactosidase transcriptional fusion assays. We found that in the absence of IS1548, genes involved in folate biosynthesis are co-transcribed with murB. As it was postulated that a folic acid mediated reaction may be involved in cell wall synthesis, this co-transcription could be necessary to synchronize these two processes. The insertion of IS1548 in the folK-murB intergenic region disrupt this co-transcription. Interestingly, we located a promoter at the right end of IS1548 that is able to initiate additional transcripts of murB. The insertion of IS1548 in this region has thus a dual and divergent impact on the expression of murB. By comparative ß-galactosidase transcriptional fusion assays, we showed that, consequently, the overall impact of the insertion of IS1548 results in a minor decrease of murB gene transcription. This study provides new insights into gene expression effects mediated by IS1548 in S. agalactiae.


Assuntos
Proteínas de Bactérias/genética , DNA Intergênico , Regulação Bacteriana da Expressão Gênica , Sequências Repetitivas Dispersas , Mutagênese Insercional , Peptidoglicano/biossíntese , Streptococcus agalactiae/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Regiões Promotoras Genéticas , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/metabolismo
3.
Int J Med Microbiol ; 309(6): 151335, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31378704

RESUMO

The type VI secretion system (T6SS) injects effector proteins into neighboring bacteria and host cells. Effector translocation is driven by contraction of a tubular sheath in the cytoplasm that expels an inner needle across the cell envelope. The AAA + ATPase ClpV disassembles and recycles the contracted sheath. While ClpV-1-GFP of the Burkholderia T6SS-1, which targets prokaryotic cells, assembles into randomly localized foci, ClpV-5-GFP of the virulence-associated T6SS-5 displays a polar distribution. The mechanisms underlying the localization of T6SSs to a particular site in the bacterial cell are currently unknown. We recently showed that ClpV-5-GFP retains its polar localization in the absence of all T6SS-5 components during infection of host cells. Herein, we set out to identify factors involved in the distribution of ClpV-5 and ClpV-1 in Burkholderia thailandensis. We show that focal assembly and polar localization of ClpV-5-GFP is not dependent on the intracellular host cell environment, known to contain the signal to induce T6SS-5 gene expression. In contrast to ClpV-5-GFP, localization of ClpV-1-GFP was dependent on the cognate T6SS. Foci formation of both ClpV5-GFP and ClpV-1-GFP was decreased by D cycloserine-mediated inhibition of peptidoglycan synthesis while treatment of B. thailandensis with A22 blocking the cytoskeletal protein MreB did not affect assembly of ClpV-5 and ClpV-1 into single discrete foci. Furthermore, we found that surface contact promotes but is not essential for localization of ClpV-5-GFP to the pole whereas expression of clpV-1-gfp appears to be induced by surface contact. In summary, the study provides novel insights into the localization of ClpV ATPases of T6SSs targeting prokaryotic and eukaryotic cells.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Burkholderia/fisiologia , Sistemas de Secreção Tipo VI/metabolismo , Fatores de Virulência/metabolismo , Aderência Bacteriana , Burkholderia/efeitos dos fármacos , Burkholderia/genética , Ciclosserina/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células HeLa , Humanos , Peptidoglicano/biossíntese , Peptidoglicano/efeitos dos fármacos , Transporte Proteico/fisiologia , Deleção de Sequência , Sistemas de Secreção Tipo VI/genética
4.
Int J Med Microbiol ; 309(6): 151332, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31350128

RESUMO

Peptidoglycan (PG) is a bacteria specific cell surface layer that ensures the bacterial shape and integrity. The two actinomycetes Amycolatopsis balhimycina and Microbispora sp. PTA-5024 are producers of PG targeting antibiotics. To prevent the binding of their secreted product to their own PG, they developed specific self-resistance mechanisms. Modifications of PG, which are applied by both strains, are the introduction of amide-residues at the PG precursors and the alternative crosslinks within the nascent PG. The PG modifications found in Microbispora sp. PTA-5024 seemed to be an intrinsic characteristic of the genus Microbispora, rather than a specific mechanism of NAI-107 resistance. In contrast, the modifications in A. balhimycina represent an alternative way to avoid suicide specific for glycopeptide producers. The different PG modifications reflect the fact that antibiotic producing organisms contain not only one but multiple mechanisms to ensure protection against biologically active molecules produced by themselves.


Assuntos
Actinobacteria/fisiologia , Antibacterianos/metabolismo , Farmacorresistência Bacteriana , Peptidoglicano/biossíntese , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glicopeptídeos/metabolismo , Peptidoglicano/química , Polimerização
5.
PLoS Genet ; 15(6): e1008195, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181062

RESUMO

To characterize the consequences of eliminating essential functions needed for peptidoglycan synthesis, we generated deletion mutations of Acinetobacter baylyi by natural transformation and visualized the resulting microcolonies of dead cells. We found that loss of genes required for peptidoglycan precursor synthesis or polymerization led to the formation of polymorphic giant cells with diameters that could exceed ten times normal. Treatment with antibiotics targeting early or late steps of peptidoglycan synthesis also produced giant cells. The giant cells eventually lysed, although they were partially stabilized by osmotic protection. Genome-scale transposon mutant screening (Tn-seq) identified mutations that blocked or accelerated giant cell formation. Among the mutations that blocked the process were those inactivating a function predicted to cleave murein glycan chains (the MltD murein lytic transglycosylase), suggesting that giant cell formation requires MltD hydrolysis of existing peptidoglycan. Among the mutations that accelerated giant cell formation after ß-lactam treatment were those inactivating an enzyme that produces unusual 3->3 peptide cross-links in peptidoglycan (the LdtG L,D-transpeptidase). The mutations may weaken the sacculus and make it more vulnerable to further disruption. Although the study focused on A. baylyi, we found that a pathogenic relative (A. baumannii) also produced giant cells with genetic dependencies overlapping those of A. baylyi. Overall, the analysis defines a genetic pathway for giant cell formation conserved in Acinetobacter species in which independent initiating branches converge to create the unusual cells.


Assuntos
Acinetobacter/genética , Genes Essenciais/genética , Glicosiltransferases/genética , Peptidoglicano/genética , Acinetobacter/enzimologia , Antibacterianos/biossíntese , Ciclo Celular/genética , Divisão Celular/genética , Parede Celular/enzimologia , Parede Celular/genética , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Deleção de Genes , Genoma Bacteriano/genética , Peptidoglicano/biossíntese , Peptidil Transferases/genética , Deleção de Sequência/genética
6.
PLoS Genet ; 15(2): e1007897, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30707707

RESUMO

Many bacteria have complex cell shapes, but the mechanisms producing their distinctive morphologies are still poorly understood. Caulobacter crescentus, for instance, exhibits a stalk-like extension that carries an adhesive holdfast mediating surface attachment. This structure forms through zonal peptidoglycan biosynthesis at the old cell pole and elongates extensively under phosphate-limiting conditions. We analyzed the composition of cell body and stalk peptidoglycan and identified significant differences in the nature and proportion of peptide crosslinks, indicating that the stalk represents a distinct subcellular domain with specific mechanical properties. To identify factors that participate in stalk formation, we systematically inactivated and localized predicted components of the cell wall biosynthetic machinery of C. crescentus. Our results show that the biosynthesis of stalk peptidoglycan involves a dedicated peptidoglycan biosynthetic complex that combines specific components of the divisome and elongasome, suggesting that the repurposing of preexisting machinery provides a straightforward means to evolve new morphological traits.


Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Parede Celular/metabolismo , Peptidoglicano/biossíntese , Fosfatos/metabolismo
7.
Nat Chem ; 11(4): 335-341, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804500

RESUMO

Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic D-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D- and L,D-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.


Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Peptidoglicano/biossíntese , Peptidil Transferases/metabolismo , Aminoácidos/química , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Parede Celular/metabolismo , Ensaios Enzimáticos/métodos , Cinética , Streptomyces/enzimologia , Streptomyces/metabolismo
8.
Proc Natl Acad Sci U S A ; 116(8): 3211-3220, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718427

RESUMO

Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Proteínas de Ligação às Penicilinas/genética , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Divisão Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/ultraestrutura , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , Humanos , Microscopia de Fluorescência , Peptidoglicano/biossíntese , Peptidoglicano/genética , Infecções Pneumocócicas/genética , Streptococcus pneumoniae/patogenicidade , Streptococcus pneumoniae/ultraestrutura
9.
Nat Commun ; 10(1): 261, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651563

RESUMO

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here, we describe critical structural features for the interaction of GpsB with peptidoglycan synthases from three bacterial species (Bacillus subtilis, Listeria monocytogenes and Streptococcus pneumoniae) and suggest their importance for cell wall growth and viability in L. monocytogenes and S. pneumoniae. We use these structural motifs to identify novel partners of GpsB in B. subtilis and extend the members of the GpsB interactome in all three bacterial species. Our results support that GpsB functions as an adaptor protein that mediates the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Parede Celular/metabolismo , Listeria monocytogenes/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Streptococcus pneumoniae/metabolismo , Fatores de Virulência/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Divisão Celular , Cristalografia por Raios X , Citosol/metabolismo , Proteínas de Membrana/metabolismo , Mutagênese , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/isolamento & purificação , Peptidoglicano/biossíntese , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/isolamento & purificação
10.
mBio ; 10(1)2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622193

RESUMO

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, PG synthesis localizes at midcell under the control of a multiprotein complex, the divisome, allowing the safe formation of two new cell poles and separation of daughter cells. Genetic studies in Escherichia coli pointed out that FtsBLQ and FtsN participate in the regulation of septal PG (sPG) synthesis; however, the underlying molecular mechanisms remained largely unknown. Here we show that FtsBLQ subcomplex directly interacts with the PG synthase PBP1b and with the subcomplex FtsW-PBP3, mainly via FtsW. Strikingly, we discovered that FtsBLQ inhibits the glycosyltransferase activity of PBP1b and that this inhibition was antagonized by the PBP1b activators FtsN and LpoB. The same results were obtained in the presence of FtsW-PBP3. Moreover, using a simple thioester substrate (S2d), we showed that FtsBLQ also inhibits the transpeptidase domain of PBP3 but not of PBP1b. As the glycosyltransferase and transpeptidase activities of PBP1b are coupled and PBP3 activity requires nascent PG substrate, the results suggest that PBP1b inhibition by FtsBLQ will block sPG synthesis by these enzymes, thus maintaining cell division as repressed until the maturation of the divisome is signaled by the accumulation of FtsN, which triggers sPG synthesis and the initiation of cell constriction. These results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which seems to counterbalance the inhibitory effect of FtsBLQ to restore PBP1b activity.IMPORTANCE Bacterial cell division is governed by a multiprotein complex called divisome, which facilitates a precise cell wall synthesis at midcell and daughter cell separation. Protein-protein interactions and activity studies using different combinations of the septum synthesis core of the divisome revealed that the glycosyltransferase activity of PBP1b is repressed by FtsBLQ and that the presence of FtsN or LpoB suppresses this inhibition. Moreover, FtsBLQ also inhibits the PBP3 activity on a thioester substrate. These results provide enzymatic evidence of the regulation of the peptidoglycan synthase PBP1b and PBP3 within the divisome. The results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which functions to relieve the repression on PBP1b by FtsBLQ and to initiate septal peptidoglycan synthesis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Divisão Celular , Parede Celular/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Peptidoglicano/biossíntese
11.
Appl Microbiol Biotechnol ; 103(2): 843-851, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30456576

RESUMO

Increasing drug resistance in pathogens including Mycobacterium tuberculosis (MTB) has been ascribed to mutations in the known target genes. However, many of these drugs have multiple targets; some of which have not been identified so far. Understanding the mechanism of action of these drugs holds a great promise in better management of disease especially by drug-resistant strains. In this study, we report glutamate racemase (MurI), a crucial enzyme of phase I peptidoglycan (PG) biosynthesis pathway of MTB, as an additional target of ethambutol (EMB). The effect on EMB on the MurI protein at structural and functional level was studied using different spectroscopic, biochemical, and insilico approaches. Spectroscopic analysis revealed that EMB-modified protein undergoes conformational alterations. Furthermore, in vitro racemization studies of the MurI protein suggest that EMB decreases its functional activity. Docking studies revealed that EMB interacts with most of the active residues at the binding site and blocks the binding pocket. Overall, data suggests that EMB, a primary drug used for the treatment of tuberculosis (TB), acts as a competitive inhibitor of substrate for binding to mycobacterial MurI protein. The study also points out to our lacunae in understanding the site and mechanism of action of existing drugs. Furthermore, glutamate racemase is a conserved protein of the bacterial kingdom; therefore, ethambutol could be a promising candidate as a broad-spectrum antibiotic for many other bacterial diseases.


Assuntos
Isomerases de Aminoácido/antagonistas & inibidores , Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Etambutol/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Peptidoglicano/biossíntese , Isomerases de Aminoácido/química , Isomerases de Aminoácido/metabolismo , Sítios de Ligação , Parede Celular/metabolismo , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Conformação Proteica , Análise Espectral
12.
Appl Microbiol Biotechnol ; 103(2): 793-806, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30417310

RESUMO

Most recombinant proteins in Escherichia coli are not efficiently secreted to the extracellular space. Structural stabilisation of the cell wall is essential for extracellular protein production in E. coli, for which D,D-carboxypeptidases are essential. Herein, we perturbed the peptidoglycan structure of the E. coli cell wall by overexpressing D,D-carboxypeptidase genes dacA or dacB, and investigated the effect on extracellular protein production. Overexpression of dacA or dacB promoted the accumulation of intracellular soluble peptidoglycan, altered cell morphology (shape and size) and led to the formation of transparent globular structures in E. coli cells. Compared with controls (CK), extracellular production of recombinant green fluorescent protein (GFP) was increased by 1.7- and 2.3-fold upon overexpression of dacA and dacB, respectively. Similarly, extracellular production of recombinant amylase and α-galactosidase was increased by 4.5- and 2.8-fold, respectively, upon overexpression of dacA, and by 11.9- and 2.5-fold, respectively, upon overexpression of dacB. Overexpression of dacA or dacB enhanced both the outer and inner membrane permeability of E. coli. This cell wall engineering strategy opens up a new direction for enhancing extracellular protein and chemical production in E. coli.


Assuntos
Carboxipeptidases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Peptidoglicano/biossíntese , Proteínas Recombinantes/metabolismo , Carboxipeptidases/genética , Membrana Celular/fisiologia , Parede Celular/química , Escherichia coli/genética , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Permeabilidade , Proteínas Recombinantes/genética
13.
Curr Genet ; 65(1): 99-101, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30056491

RESUMO

Bacterial cells need to divide. This process requires more than 30 different proteins, which gather at the division site. It is widely assumed that these proteins assemble into a macromolecular complex (the divisome), but capturing the molecular layout of this complex has proven elusive. Super-resolution microscopy can provide spatial information, down to a few tens of nanometers, about how the division proteins assemble into complexes and how their activities are co-ordinated. Herein we provide insight into recent work from our laboratories, where we used super-resolution gSTED nanoscopy to explore the molecular organization of FtsZ, FtsI and FtsN. The resulting images show that all three proteins form discrete densities organised in patchy pseudo-rings at the division site. Significantly, two-colour imaging highlighted a radial separation between FtsZ and FtsN, indicating that there is more than one type of macromolecular complex operating during division. These data provide a first glimpse into the spatial organisation of PG-synthesising enzymes during division in Gram-negative bacteria.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Complexos Multiproteicos/metabolismo , Peptidoglicano/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Complexos Multiproteicos/genética , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/genética , Peptidoglicano Glicosiltransferase/metabolismo
14.
Subcell Biochem ; 93: 273-289, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31939154

RESUMO

The bacterial cell wall is the validated target of mainstream antimicrobials such as penicillin and vancomycin. Penicillin and other ß-lactams act by targeting Penicillin-Binding Proteins (PBPs), enzymes that play key roles in the biosynthesis of the main component of the cell wall, the peptidoglycan. Despite the spread of resistance towards these drugs, the bacterial cell wall continues to be a major Achilles' heel for microbial survival, and the exploration of the cell wall formation machinery is a vast field of work that can lead to the development of novel exciting therapies. The sheer complexity of the cell wall formation process, however, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. New developments in genetic and biochemical screens, as well as different aspects of structural biology, have shed new light on the importance of complexes formed by PBPs, notably within the cell wall elongation machinery. This chapter summarizes structural and functional details of PBP complexes involved in the periplasmic and membrane steps of peptidoglycan biosynthesis with a focus on cell wall elongation. These assemblies could represent interesting new targets for the eventual development of original antibacterials.


Assuntos
Bactérias/citologia , Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Parede Celular/química , Peptidoglicano/biossíntese
15.
J Biosci ; 43(5): 1001-1013, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30541959

RESUMO

The ribosome-binding GTPase HflX is required for manganese homeostasis in E. coli. While under normal conditions ΔhflX cells behave like wild type E. coli with respect to growth pattern and morphology, deletion of hflX makes E. coli cells extremely sensitive to manganese, characterized by arrested cell growth and filamentation. Here we demonstrate that upon complementation by hflX, manganese stress is relieved. In phenotypic studies done in a manganese-rich environment, ΔhflX cells were highly sensitive to antibiotics that bind the penicillin binding protein 3 (PBP3), suggesting that the manganese stress led to impaired peptidoglycan biosynthesis. An irregular distribution of dark bands of constriction along filaments, delocalization of the dark bands from midcell towards poles and subpoles, lack of septum formation and arrested cell division were observed in ΔhflX cells under manganese stress. However, chromosome replication and segregation of nucleoids were unaffected under these conditions, as observed from confocal microscopy imaging and FACS studies. We conclude that absence of HflX leads to manganese accumulation in E. coli cells, affecting cell septum formation, probably by modulating the activity of the cell division protein PBP3 (FtsI), a major component of the divisome apparatus. We propose that HflX acts as a gatekeeper, regulating the influx of manganese into the cell.


Assuntos
Cloretos/farmacologia , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Proteínas de Ligação ao GTP/genética , Regulação Bacteriana da Expressão Gênica , Compostos de Manganês/farmacologia , Proteínas de Ligação às Penicilinas/genética , Estresse Fisiológico/genética , Antibacterianos/farmacologia , Divisão Celular/efeitos dos fármacos , Cloretos/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/ultraestrutura , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/deficiência , Deleção de Genes , Teste de Complementação Genética , Homeostase/genética , Compostos de Manganês/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/biossíntese , Estresse Fisiológico/efeitos dos fármacos
16.
Sci Rep ; 8(1): 17704, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30531805

RESUMO

Peptidoglycan (PG) biosynthesis and assembly are needed for bacterial cell wall formation. Lipid II is the precursor in the PG biosynthetic pathway and carries a nascent PG unit that is processed by glycosyltransferases. Despite its immense therapeutic value as a target of several classes of antibiotics, the conformational ensemble of lipid II in bacterial membranes and its interactions with membrane-anchored enzymes remain elusive. In this work, lipid II and its elongated forms (lipid VI and lipid XII) were modeled and simulated in bilayers of POPE (palmitoyl-oleoyl-phosphatidyl-ethanolamine) and POPG (palmitoyl-oleoyl-phosphatidyl-glycerol) that mimic the prototypical composition of Gram-negative cytoplasmic membranes. In addition, penicillin-binding protein 1b (PBP1b) from Escherichia coli was modeled and simulated in the presence of a nascent PG to investigate their interactions. Trajectory analysis reveals that as the glycan chain grows, the non-reducing end of the nascent PG displays much greater fluctuation along the membrane normal and minimally interacts with the membrane surface. In addition, dihedral angles within the pyrophosphate moiety are determined by the length of the PG moiety and its surrounding environment. When a nascent PG is bound to PBP1b, the stem peptide remains in close contact with PBP1b by structural rearrangement of the glycan chain. Most importantly, the number of nascent PG units required to reach the transpeptidase domain are determined to be 7 or 8. Our findings complement experimental results to further understand how the structure of nascent PG can dictate the assembly of the PG scaffold.


Assuntos
Membrana Celular/metabolismo , Peptidoglicano/biossíntese , Peptidoglicano/metabolismo , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Difosfatos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidil Transferases/metabolismo , Fosfatidiletanolaminas/farmacologia , Fosfatidilgliceróis/farmacologia , Polissacarídeos/metabolismo , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
17.
mBio ; 9(6)2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425148

RESUMO

Small regulatory RNAs play an important role in the adaptation to changing conditions. Here, we describe a differentially expressed small regulatory RNA (sRNA) that affects various cellular processes in the plant pathogen Agrobacterium tumefaciens Using a combination of bioinformatic predictions and comparative proteomics, we identified nine targets, most of which are positively regulated by the sRNA. According to these targets, we named the sRNA PmaR for peptidoglycan biosynthesis, motility, and ampicillin resistance regulator. Agrobacterium spp. are long known to be naturally resistant to high ampicillin concentrations, and we can now explain this phenotype by the positive PmaR-mediated regulation of the beta-lactamase gene ampC Structure probing revealed a spoon-like structure of the sRNA, with a single-stranded loop that is engaged in target interaction in vivo and in vitro Several riboregulators have been implicated in antibiotic resistance mechanisms, such as uptake and efflux transporters, but PmaR represents the first example of an sRNA that directly controls the expression of an antibiotic resistance gene.IMPORTANCE The alphaproteobacterium Agrobacterium tumefaciens is able to infect various eudicots causing crown gall tumor formation. Based on its unique ability of interkingdom gene transfer, Agrobacterium serves as a crucial biotechnological tool for genetic manipulation of plant cells. The presence of hundreds of putative sRNAs in this organism suggests a considerable impact of riboregulation on A. tumefaciens physiology. Here, we characterized the biological function of the sRNA PmaR that controls various processes crucial for growth, motility, and virulence. Among the genes directly targeted by PmaR is ampC coding for a beta-lactamase that confers ampicillin resistance, suggesting that the sRNA is crucial for fitness in the competitive microbial composition of the rhizosphere.


Assuntos
Agrobacterium/genética , Parede Celular/genética , Resistência Microbiana a Medicamentos/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Agrobacterium/efeitos dos fármacos , Agrobacterium/crescimento & desenvolvimento , Ampicilina/farmacologia , Proteínas de Bactérias/genética , Parede Celular/fisiologia , Biologia Computacional , Peptidoglicano/biossíntese , Peptidoglicano/genética , Plantas/microbiologia , Proteômica , Rizosfera , beta-Lactamases/genética
18.
Artigo em Inglês | MEDLINE | ID: mdl-30275084

RESUMO

The Gram-negative human pathogen Neisseria gonorrhoeae has progressively developed resistance to antibiotic monotherapies, and recent failures of dual-drug therapy have heightened concerns that strains resistant to all available antibiotics will begin circulating globally. Targeting bacterial cell wall assembly has historically been effective at treating infections with N. gonorrhoeae, but as the effectiveness of ß-lactams (including cephalosporins) is challenged by increasing resistance, research has expanded into compounds that target the numerous other enzymes with roles in peptidoglycan metabolism. One example is the dithiazoline compound JNJ-853346 (DTZ), which inhibits the activity of an Escherichia coli serine protease l,d-carboxypeptidase (LdcA). Recently, the characterization of an LdcA homolog in N. gonorrhoeae revealed localization and activity differences from the characterized E. coli LdcA, prompting us to explore the effectiveness of DTZ against N. gonorrhoeae We found that DTZ is effective at inhibiting N. gonorrhoeae in all growth phases, unlike the specific stationary-phase inhibition seen in E. coli Surprisingly, DTZ does not inhibit gonococcal LdcA enzyme activity, and DTZ sensitivity is not significantly decreased in ldcA mutants. While effective against numerous N. gonorrhoeae strains, including recent multidrug-resistant isolates, DTZ is much less effective at inhibiting growth of the commensal species Lactobacillus gasseri DTZ treatment during coinfections of epithelial cells resulted in significant lowering of gonococcal burden and interleukin-8 secretion without significantly impacting recovery of viable L. gasseri This selective toxicity presents a possible pathway for the use of DTZ as an effective antigonococcal agent at concentrations that do not impact vaginal commensals.


Assuntos
Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Lactobacillus gasseri/efeitos dos fármacos , Neisseria gonorrhoeae/efeitos dos fármacos , Tiazóis/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboxipeptidases A/genética , Carboxipeptidases A/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/metabolismo , Expressão Gênica , Células HCT116 , Humanos , Interleucina-8/genética , Interleucina-8/imunologia , Lactobacillus gasseri/crescimento & desenvolvimento , Lactobacillus gasseri/metabolismo , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mutação , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/crescimento & desenvolvimento , Neisseria gonorrhoeae/metabolismo , Peptidoglicano/biossíntese , Peptidoglicano/efeitos dos fármacos , Probióticos/química , Especificidade da Espécie
19.
Appl Environ Microbiol ; 84(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30341076

RESUMO

Corynebacterium glutamicum is frequently engineered to serve as a versatile platform and model microorganism. However, due to its complex cell wall structure, transformation of C. glutamicum with exogenous DNA is inefficient. Although efforts have been devoted to improve the transformation efficiency by using cell wall-weakening agents, direct genetic engineering of cell wall synthesis for enhancing cell competency has not been explored thus far. Herein, we reported that engineering of peptidoglycan synthesis could significantly increase the transformation efficiency of C. glutamicum Comparative analysis of C. glutamicum wild-type strain ATCC 13869 and a mutant with high electrotransformation efficiency revealed nine mutations in eight cell wall synthesis-related genes. Among them, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA dramatically increased the electrotransformation of strain ATCC 13869 by 19.25-fold in the absence of cell wall-weakening agents, with no inhibition on growth. The Y489C mutation had no effect on the membrane localization of PonA but affected the peptidoglycan structure. Deletion of the ponA gene led to more dramatic changes to the peptidoglycan structure but only increased the electrotransformation by 4.89-fold, suggesting that appropriate inhibition of cell wall synthesis benefited electrotransformation more. Finally, we demonstrated that the PonAY489C mutation did not cause constitutive or enhanced glutamate excretion, making its permanent existence in C. glutamicum ATCC 13869 acceptable. This study demonstrates that genetic engineering of genes involved in cell wall synthesis, especially peptidoglycan synthesis, is a promising strategy to improve the electrotransformation efficiency of C. glutamicum IMPORTANCE Metabolic engineering and synthetic biology are now the key enabling technologies for manipulating microorganisms to suit the practical outcomes desired by humankind. The introduction of exogenous DNA into cells is an indispensable step for this purpose. However, some microorganisms, including the important industrial workhorse Corynebacterium glutamicum, possess a complex cell wall structure to shield cells against exogenous DNA. Although genes responsible for cell wall synthesis in C. glutamicum are known, engineering of related genes to improve cell competency has not been explored yet. In this study, we demonstrate that mutations in cell wall synthesis genes can significantly improve the electrotransformation efficiency of C. glutamicum Notably, the Y489C mutation in bifunctional peptidoglycan glycosyltransferase/peptidoglycan dd-transpeptidase PonA increased electrotransformation efficiency by 19.25-fold by affecting peptidoglycan synthesis.


Assuntos
Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Mutação , Peptidoglicano/biossíntese , Peptidoglicano/genética , Aminoaciltransferases/genética , Proteínas de Transporte , Parede Celular/metabolismo , DNA Bacteriano/genética , Ácido Glutâmico/metabolismo , Engenharia Metabólica , Proteínas dos Microfilamentos , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano/química , Peptidoglicano Glicosiltransferase/genética
20.
Elife ; 72018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30198841

RESUMO

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.


Assuntos
Alanina/biossíntese , Proteínas de Bactérias/biossíntese , Parede Celular/química , Peptidoglicano/biossíntese , Alanina/química , Proteínas de Bactérias/química , Ciclo Celular/genética , Divisão Celular/genética , Parede Celular/genética , Dipeptídeos/química , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Penicilinas/química , Peptidoglicano/química
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