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1.
Nat Commun ; 11(1): 4157, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814767

RESUMO

Swarming is a form of collective bacterial motion enabled by flagella on the surface of semi-solid media. Swarming populations exhibit non-genetic or adaptive resistance to antibiotics, despite sustaining considerable cell death. Here, we show that antibiotic-induced death of a sub-population benefits the swarm by enhancing adaptive resistance in the surviving cells. Killed cells release a resistance-enhancing factor that we identify as AcrA, a periplasmic component of RND efflux pumps. The released AcrA interacts on the surface of live cells with an outer membrane component of the efflux pump, TolC, stimulating drug efflux and inducing expression of other efflux pumps. This phenomenon, which we call 'necrosignaling', exists in other Gram-negative and Gram-positive bacteria and displays species-specificity. Given that adaptive resistance is a known incubator for evolving genetic resistance, our findings might be clinically relevant to the rise of multidrug resistance.


Assuntos
Antibacterianos/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Resistência Microbiana a Medicamentos/fisiologia , Transdução de Sinais/fisiologia , Adaptação Fisiológica/fisiologia , Antibacterianos/farmacologia , Bactérias/classificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Periplasma/metabolismo , Especificidade da Espécie
2.
PLoS Comput Biol ; 16(7): e1008024, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609716

RESUMO

Vitamin B12 (or cobalamin) is an enzymatic cofactor essential both for mammals and bacteria. However, cobalamin can be synthesized only by few microorganisms so most bacteria need to take it up from the environment through the TonB-dependent transport system. The first stage of cobalamin import to E. coli cells occurs through the outer-membrane receptor called BtuB. Vitamin B12 binds with high affinity to the extracellular side of the BtuB protein. BtuB forms a ß-barrel with inner luminal domain and extracellular loops. To mechanically allow for cobalamin passage, the luminal domain needs to partially unfold with the help of the inner-membrane TonB protein. However, the mechanism of cobalamin permeation is unknown. Using all-atom molecular dynamics, we simulated the transport of cobalamin through the BtuB receptor embedded in an asymmetric and heterogeneous E. coli outer-membrane. To enhance conformational sampling of the BtuB loops, we developed the Gaussian force-simulated annealing method (GF-SA) and coupled it with umbrella sampling. We found that cobalamin needs to rotate in order to permeate through BtuB. We showed that the mobility of BtuB extracellular loops is crucial for cobalamin binding and transport and resembles an induced-fit mechanism. Loop mobility depends not only on the position of cobalamin but also on the extension of luminal domain. We provided atomistic details of cobalamin transport through the BtuB receptor showing the essential role of the mobility of BtuB extracellular loops. A similar TonB-dependent transport system is used also by many other compounds, such as haem and siderophores, and importantly, can be hijacked by natural antibiotics. Our work could have implications for future delivery of antibiotics to bacteria using this transport system.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Vitamina B 12/metabolismo , Algoritmos , Antibacterianos/química , Sítios de Ligação , Biologia Computacional , Cristalografia por Raios X , Heme/química , Íons , Bicamadas Lipídicas/química , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Distribuição Normal , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Sacarose/química , Água/química
3.
Proc Natl Acad Sci U S A ; 117(31): 18737-18743, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675245

RESUMO

The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral ß-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the ß-barrel assembly machine (BAM), which is composed of one OMP-BamA-and four lipoproteins-BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation, bamA E470K , that bypasses the requirement for BamD. Although bamD::kan bamA E470K cells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.


Assuntos
Proteínas da Membrana Bacteriana Externa , Membrana Externa Bacteriana , Proteínas de Escherichia coli , Mutação com Ganho de Função/genética , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
4.
Nature ; 583(7816): 473-478, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32528179

RESUMO

Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a ß-barrel structure1,2. ß-Barrels are sheets of ß-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3-5. One subunit of the machines is itself a ß-barrel protein that has a central role in folding other ß-barrels. In Gram-negative bacteria, the ß-barrel assembly machine (BAM) consists of the ß-barrel protein BamA, and four lipoproteins5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid ß-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10-12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/química , Cloroplastos/química , Proteínas de Escherichia coli/química , Bactérias Gram-Negativas/química , Ligação de Hidrogênio , Mitocôndrias/química , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
5.
Eur J Clin Microbiol Infect Dis ; 39(10): 1821-1830, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32557327

RESUMO

Helicobacter pylori (H. pylori) infection is associated with some gastric diseases, such as gastritis, peptic ulcer, and gastric cancer. CagA and VacA are known virulence factors of H. pylori, which play a vital role in severe clinical outcomes. Additionally, the expression of outer membrane proteins (OMPs) helps H. pylori attach to gastric epithelial cells at the primary stage and increases the virulence of H. pylori. In this review, we have summarized the paralogs of H. pylori OMPs, their genomic loci, and the different receptors of OMPs identified so far. We focused on five OMPs, BabA (HopS), SabA (HopP), OipA (HopH), HopQ, and HopZ, and one family of OMPs: Hom. We highlight the coexpression of OMPs with other virulence factors and their relationship with clinical outcomes. In conclusion, OMPs are closely related to the pathogenic processes of adhesion, colonization, persistent infection, and severe clinical consequences. They are potential targets for the prevention and treatment of H. pylori-related diseases.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Fatores de Virulência/metabolismo , Células Epiteliais/microbiologia , Expressão Gênica , Humanos , Fatores de Virulência/genética
6.
PLoS One ; 15(6): e0234306, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555615

RESUMO

Moraxella catarrhalis is a human-adapted, opportunistic bacterial pathogen of the respiratory mucosa. Although asymptomatic colonization of the nasopharynx is common, M. catarrhalis can ascend into the middle ear, where it is a prevalent causative agent of otitis media in children, or enter the lower respiratory tract, where it is associated with acute exacerbations of chronic obstructive pulmonary disease in adults. Phase variation is the high frequency, random, reversible switching of gene expression that allows bacteria to adapt to different host microenvironments and evade host defences, and is most commonly mediated by simple DNA sequence repeats. Bioinformatic analysis of five closed M. catarrhalis genomes identified 17 unique simple DNA sequence repeat tracts that were variable between strains, indicating the potential to mediate phase variable expression of the associated genes. Assays designed to assess simple sequence repeat variation under conditions mimicking host infection demonstrated that phase variation of uspA1 (ubiquitous surface protein A1) from high to low expression occurs over 72 hours of biofilm passage, while phase variation of uspA2 (ubiquitous surface protein A2) to high expression variants occurs during repeated exposure to human serum, as measured by mRNA levels. We also identify and confirm the variable expression of two novel phase variable genes encoding a Type III DNA methyltransferase (modO), and a conserved hypothetical permease (MC25239_RS00020). These data reveal the repertoire of phase variable genes mediated by simple sequence repeats in M. catarrhalis and demonstrate that modulation of expression under conditions mimicking human infection is attributed to changes in simple sequence repeat length.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Moraxella catarrhalis/genética , Aderência Bacteriana/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Expressão Gênica/genética , Humanos , Repetições de Microssatélites/genética , Moraxella catarrhalis/patogenicidade , Infecções por Moraxellaceae , Otite Média/microbiologia , Sequências Repetitivas de Ácido Nucleico/genética
7.
PLoS One ; 15(6): e0235139, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574205

RESUMO

Viral infections complicated by a bacterial infection are typically referred to as coinfections or superinfections. Streptococcus pyogenes, the group A streptococcus (GAS), is not the most common bacteria associated with influenza A virus (IAV) superinfections but did cause significant mortality during the 2009 influenza pandemic even though all isolates are susceptible to penicillin. One approach to improve the outcome of these infections is to use passive immunization targeting GAS. To test this idea, we assessed the efficacy of passive immunotherapy using antisera against either the streptococcal M protein or streptolysin O (SLO) in a murine model of IAV-GAS superinfection. Prophylactic treatment of mice with antiserum to either SLO or the M protein decreased morbidity compared to mice treated with non-immune sera; however, neither significantly decreased mortality. Therapeutic use of antisera to SLO decreased morbidity compared to mice treated with non-immune sera but neither antisera significantly reduced mortality. Overall, the results suggest that further development of antibodies targeting the M protein or SLO may be a useful adjunct in the treatment of invasive GAS diseases, including IAV-GAS superinfections, which may be particularly important during influenza pandemics.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Transporte/imunologia , Imunoterapia/métodos , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Estreptolisinas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Coinfecção/microbiologia , Coinfecção/terapia , Coinfecção/virologia , Feminino , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Soros Imunes/imunologia , Soros Imunes/farmacologia , Vírus da Influenza A/fisiologia , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/terapia , Infecções por Orthomyxoviridae/virologia , Coelhos , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/terapia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/fisiologia , Estreptolisinas/antagonistas & inibidores , Estreptolisinas/metabolismo , Superinfecção/microbiologia , Superinfecção/terapia , Superinfecção/virologia
8.
Nat Commun ; 11(1): 2155, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358557

RESUMO

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Proteínas de Transporte/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Espectrometria de Massas , Chaperonas Moleculares/genética , Peptidilprolil Isomerase/genética , Ligação Proteica
9.
Proc Natl Acad Sci U S A ; 117(17): 9302-9310, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32245809

RESUMO

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic-catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.


Assuntos
Lignina/metabolismo , Pseudomonas putida/enzimologia , Vesículas Secretórias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Pseudomonas putida/metabolismo
10.
Nat Commun ; 11(1): 1789, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286264

RESUMO

The mechanical properties of the cell envelope in Gram-negative bacteria are controlled by the peptidoglycan, the outer membrane, and the proteins interacting with both layers. In Escherichia coli, the lipoprotein Lpp provides the only covalent crosslink between the outer membrane and the peptidoglycan. Here, we use single-cell atomic force microscopy and genetically engineered strains to study the contribution of Lpp to cell envelope mechanics. We show that Lpp contributes to cell envelope stiffness in two ways: by covalently connecting the outer membrane to the peptidoglycan, and by controlling the width of the periplasmic space. Furthermore, mutations affecting Lpp function substantially increase bacterial susceptibility to the antibiotic vancomycin, indicating that Lpp-dependent effects can affect antibacterial drug efficacy.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Antibacterianos/farmacologia , Bacteriologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Microscopia de Força Atômica , Peptidoglicano/metabolismo , Vancomicina/farmacologia
11.
PLoS Pathog ; 16(4): e1008469, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324807

RESUMO

The molecular mechanisms that allow pathogenic bacteria to infect animals have been intensively studied. On the other hand, the molecular mechanisms by which bacteria acquire virulence functions are not fully understood. In the present study, we experimentally evaluated the evolution of a non-pathogenic strain of Escherichia coli in a silkworm infection model and obtained pathogenic mutant strains. As one cause of the high virulence properties of E. coli mutants, we identified amino acid substitutions in LptD (G580S) and LptE (T95I) constituting the lipopolysaccharide (LPS) transporter, which translocates LPS from the inner to the outer membrane and is essential for E. coli growth. The growth of the LptD and LptE mutants obtained in this study was indistinguishable from that of the parent strain. The LptD and LptE mutants exhibited increased secretion of outer membrane vesicles containing LPS and resistance against various antibiotics, antimicrobial peptides, and host complement. In vivo cross-linking studies revealed that the conformation of the LptD-LptE complex was altered in the LptD and LptE mutants. Furthermore, several clinical isolates of E. coli carried amino acid substitutions of LptD and LptE that conferred resistance against antimicrobial substances. This study demonstrated an experimental evolution of bacterial virulence properties in an animal infection model and identified functional alterations of the growth-essential LPS transporter that led to high bacterial virulence by conferring resistance against antimicrobial substances. These findings suggest that non-pathogenic bacteria can gain virulence traits by changing the functions of essential genes, and provide new insight to bacterial evolution in a host environment.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/patogenicidade , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Bombyx/microbiologia , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Modelos Moleculares , Ligação Proteica , Virulência/fisiologia
12.
Proc Natl Acad Sci U S A ; 117(16): 8941-8947, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32241888

RESUMO

The bacterial flagellum is an amazing nanomachine. Understanding how such complex structures arose is crucial to our understanding of cellular evolution. We and others recently reported that in several Gammaproteobacterial species, a relic subcomplex comprising the decorated P and L rings persists in the outer membrane after flagellum disassembly. Imaging nine additional species with cryo-electron tomography, here, we show that this subcomplex persists after flagellum disassembly in other phyla as well. Bioinformatic analyses fail to show evidence of any recent horizontal transfers of the P- and L-ring genes, suggesting that this subcomplex and its persistence is an ancient and conserved feature of the flagellar motor. We hypothesize that one function of the P and L rings is to seal the outer membrane after motor disassembly.


Assuntos
Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Flagelos/genética , Especiação Genética , Bactérias/citologia , Bactérias/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/ultraestrutura , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Biologia Computacional , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Flagelos/metabolismo , Genes Bacterianos , Filogenia
13.
Nat Commun ; 11(1): 1305, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161270

RESUMO

Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. This coordination requires the recruitment of the peptidoglycan-binding outer-membrane lipoprotein Pal at division sites by the Tol system. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Divisão Celular , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Lipoproteínas/metabolismo , Peptidoglicano/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana , Proteínas Periplásmicas/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(12): 6777-6783, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152098

RESUMO

Tol-Pal is a multiprotein system present in the envelope of Gram-negative bacteria. Inactivation of this widely conserved machinery compromises the outer membrane (OM) layer of these organisms, resulting in hypersensitivity to many antibiotics. Mutants in the tol-pal locus fail to complete division and form cell chains. This phenotype along with the localization of Tol-Pal components to the cytokinetic ring in Escherichia coli has led to the proposal that the primary function of the system is to promote OM constriction during division. Accordingly, a poorly constricted OM is believed to link the cell chains formed upon Tol-Pal inactivation. However, we show here that cell chains of E. coli tol-pal mutants are connected by an incompletely processed peptidoglycan (PG) layer. Genetic suppressors of this defect were isolated and found to overproduce OM lipoproteins capable of cleaving the glycan strands of PG. Among the factors promoting cell separation in mutant cells was a protein of previously unknown function (YddW), which we have identified as a divisome-localized glycosyl hydrolase that cleaves peptide-free PG glycans. Overall, our results indicate that the cell chaining defect of Tol-Pal mutants cannot simply be interpreted as a defect in OM constriction. Rather, the complex also appears to be required for the activity of several OM-localized enzymes with cell wall remodeling activity. Thus, the Tol-Pal system may play a more general role in coordinating OM invagination with PG remodeling at the division site than previously appreciated.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Divisão Celular , Parede Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Peptidoglicano/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Lipoproteínas/genética , Ligação Proteica
15.
J Biotechnol ; 310: 40-48, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32001255

RESUMO

The relevant microorganims driving efficiency changes in anaerobic digestion of phenol remains uncertain. In this study correlations were established between microbial population and the process performance in an anaerobic sequencing batch reactor (ASBR) treating increasing concentrations of phenol (from 120 to 1200 mg L-1). Sludge samples were taken at different operational stages and microbial community dynamics was analyzed by 16S rRNA sequencing. In addition, bamA gene was quantified in order to evaluate the dynamics of anaerobic aromatic degraders. The microbial community was dominated by Anaerolineae, Bacteroidia, Clostridia, and Methanobacteria classes. Correlation analysis between bamA gene copy number and phenol concentration were highly significant, suggesting that the increase of aromatic degraders targeted by bamA assay was due to an increase in the amount of phenol degraded over time. The incremental phenol concentration affected hydrogenotrophic archaea triggering a linear decrease of Methanobacterium and the growth of Methanobrevibacter. The best performance in the reactor was at 800 mg L-1 of phenol. At this stage, the highest relative abundances of Syntrophorhabdus, Chloroflexus, Smithella, Methanolinea and Methanosaeta were observed and correlated positively with initial degradation rate, suggesting that these microorganisms are relevant players to maintain a good performance in the ASBR.


Assuntos
Bactérias , Proteínas da Membrana Bacteriana Externa , Reatores Biológicos , Fenol , Anaerobiose/efeitos dos fármacos , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Relação Dose-Resposta a Droga , Fenol/metabolismo , Fenol/farmacologia
16.
J Am Soc Mass Spectrom ; 31(2): 190-195, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32031408

RESUMO

Molecular interactions between two different classes of ß-lactamase enzymes and outer membrane protein A (OmpA) were studied by in vivo chemical cross-linking of a multi-drug-resistant strain of Acinetobacter baumannii AB5075. Class A ß-lactamase blaGES-11 and Class D ß-lactamase Oxa23, responsible for hydrolysis of different types of ß-lactam antibiotics, were found to be cross-linked to similar lysine sites of the periplasmic domain of outer membrane protein OmpA, despite low sequence homology between the two enzymes. The findings from in vivo XL-MS suggest that the interacting surfaces between both ß-lactamase enzymes and OmpA are conserved during molecular evolution, and the OmpA C-terminus domain serves an important function of anchoring different types of ß-lactamase enzymes in the periplasmic space.


Assuntos
Acinetobacter baumannii/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , beta-Lactamases/metabolismo , Acinetobacter baumannii/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Sequência Conservada , Reagentes para Ligações Cruzadas/química , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , beta-Lactamases/química
17.
Nat Commun ; 11(1): 769, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034139

RESUMO

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH.


Assuntos
Histidina Quinase/química , Histidina Quinase/metabolismo , Thermotoga maritima/enzimologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Histidina/metabolismo , Histidina Quinase/genética , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação , Fosforilação , Conformação Proteica , Thermotoga maritima/genética , Transativadores/química , Transativadores/metabolismo
18.
World J Microbiol Biotechnol ; 36(3): 36, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32088790

RESUMO

As an interspecies and interkingdom signaling molecule, indole has recently received attention for its diverse effects on the physiology of both bacteria and hosts. In this study, indole increased the tetracycline resistance of Vibrio splendidus. The minimal inhibitory concentration of tetracycline was 10 µg/mL, and the OD600 of V. splendidus decreased by 94.5% in the presence of 20 µg/mL tetracycline; however, the OD600 of V. splendidus with a mixture of 20 µg/mL tetracycline and 125 µM indole was 10- or 4.5-fold higher than that with only 20 µg/mL tetracycline at different time points. The percentage of cells resistant to 10 µg/mL tetracycline was 600-fold higher in the culture with an OD600 of approximately 2.0 (higher level of indole) than that in the culture with an OD600 of 0.5, which also meant that the level of indole was correlated to the tetracycline resistance of V. splendidus. Furthermore, one differentially expressed protein, which was identified as the outer membrane porin OmpN using SDS-PAGE combined with MALDI-TOF/TOF MS, was upregulated. Consequently, the expression of the ompN gene in the presence of either tetracycline or indole and simultaneously in the presence of indole and tetracycline was upregulated by 1.8-, 2.54-, and 6.01-fold, respectively, compared to the control samples. The combined results demonstrated that indole enhanced the tetracycline resistance of V. splendidus, and this resistance was probably due to upregulation of the outer membrane porin OmpN.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Indóis/farmacologia , Resistência a Tetraciclina , Vibrio/crescimento & desenvolvimento , Proteínas da Membrana Bacteriana Externa/metabolismo , Meios de Cultura/química , Eletroforese em Gel de Poliacrilamida , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tetraciclina/farmacologia , Regulação para Cima , Vibrio/efeitos dos fármacos , Vibrio/metabolismo
19.
Vet Res ; 51(1): 5, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31973724

RESUMO

Avian pathogenic Escherichia coli (APEC), a pathotype of extraintestinal pathogenic E. coli, causes one of the most serious infectious diseases of poultry and shares some common virulence genes with neonatal meningitis-associated E. coli. TonB-dependent receptors (TBDRs) are ubiquitous outer membrane ß-barrel proteins; they play an important role in the recognition of siderophores during iron uptake. Here, in the APEC strain DE205B, we investigated the role of four putative TBDRs-ireA, 0007, 0008, and 2235-in iron uptake. Glutathione-S-transferase pulldown assays indicated that the proteins encoded by these genes directly interact with TonB. Moreover, the expression levels of all four genes were significantly upregulated under iron-depleted conditions compared with iron-rich conditions. The expression levels of several iron uptake-related genes were significantly increased in the ireA, 0007, 0008, and 2235 deletion strains, with the upregulation being the most prominent in the ireA deletion mutant. Furthermore, iron uptake by the ireA deletion strain was significantly increased compared to that by the wild-type strain. Moreover, a tonB mutant strain was constructed to study the effect of tonB deletion on the TBDRs. We found that regardless of the presence of tonB, the expression levels of the genes encoding the four TBDRs were regulated by fur. In conclusion, our findings indicated that ireA, 0007, 0008, and 2235 indeed encode TBDRs, with ireA having the most important role in iron uptake. These results should help future studies explore the mechanisms underlying the TonB-dependent iron uptake pathway.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Doenças das Aves Domésticas/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Galinhas , Escherichia coli/metabolismo , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Doenças das Aves Domésticas/microbiologia
20.
J Mol Biol ; 432(4): 1251-1264, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31931013

RESUMO

Outer membrane vesicles (OMVs) are spherical liposomes that are secreted by almost all forms of Gram-negative bacteria. The nanospheres contribute to bacterial pathogenesis by trafficking molecular cargo from bacterial membranes to target cells at the host-pathogen interface. We have simulated the interaction of OMVs with host cell membranes to understand why OMV uptake depends on the length of constituent lipopolysaccharide macromolecules. Using coarse-grained molecular dynamics simulations, we show that lipopolysaccharide lipid length affects OMV shape at the host-pathogen interface: OMVs with long (smooth-type) lipopolysaccharide lipids retain their spherical shape when they interact with host cell membranes, whereas OMVs with shorter (rough-type) lipopolysaccharide lipids distort and spread over the host membrane surface. In addition, we show that OMVs preferentially coordinate domain-favoring ganglioside lipids within host membranes to enhance curvature and affect the local lipid composition. We predict that these differences in shape preservation affect OMV internalization on long timescales: spherical nanoparticles tend to be completely enveloped by host membranes, whereas low sphericity nanoparticles tend to remain on the surface of cells.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Membrana Externa Bacteriana/metabolismo , Gangliosídeos/metabolismo , Interações Hospedeiro-Patógeno , Simulação de Dinâmica Molecular , Fatores de Virulência/metabolismo
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