RESUMEN

Peptidoglycan hydrolases contribute to the generation of helical cell shape in Campylobacter and Helicobacter bacteria, while cytoskeletal or periskeletal proteins determine the curved, vibrioid cell shape of Caulobacter and Vibrio. Here, we identify a peptidoglycan hydrolase in the vibrioid-shaped predatory bacterium Bdellovibrio bacteriovorus which invades and replicates within the periplasm of Gram-negative prey bacteria. The protein, Bd1075, generates cell curvature in B. bacteriovorus by exerting LD-carboxypeptidase activity upon the predator cell wall as it grows inside spherical prey. Bd1075 localizes to the outer convex face of B. bacteriovorus; this asymmetric localization requires a nuclear transport factor 2-like (NTF2) domain at the protein C-terminus. We solve the crystal structure of Bd1075, which is monomeric with key differences to other LD-carboxypeptidases. Rod-shaped Δbd1075 mutants invade prey more slowly than curved wild-type predators and stretch invaded prey from within. We therefore propose that the vibrioid shape of B. bacteriovorus contributes to predatory fitness.

Asunto(s)

Bdellovibrio bacteriovorus , Bdellovibrio , Bdellovibrio/genética , Bdellovibrio bacteriovorus/genética , Bdellovibrio bacteriovorus/metabolismo , Pared Celular/metabolismo , Peptidoglicano/metabolismo , Periplasma/metabolismo

RESUMEN

The asymmetric Gram-negative outer membrane (OM) is the first line of defense for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here, we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active-site plug. Informed by our structure, we demonstrate that free movement of the active-site plug is essential for BepA function, suggesting that the protein is autoregulated by the active-site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the tetratricopeptide repeat (TPR) cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Last, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid.IMPORTANCE M48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.

Asunto(s)

Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Metaloproteasas/química , Metaloproteasas/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cristalografía por Rayos X , Proteínas de Escherichia coli/aislamiento & purificación , Metaloproteasas/aislamiento & purificación , Permeabilidad , Sensibilidad y Especificidad , Relación Estructura-Actividad

RESUMEN

The bifunctional alcohol/aldehyde dehydrogenase (AdhE) comprises both an N-terminal aldehyde dehydrogenase (AldDH) and a C-terminal alcohol dehydrogenase (ADH). In vivo, full-length AdhE oligomerizes into long oligomers known as spirosomes. However, structural analysis of AdhE is challenging owing to the heterogeneity of the spirosomes. Therefore, the domains of AdhE are best characterized separately. Here, the structure of ADH from the pathogenic Escherichia coli O157:H7 was determined to 1.65 Šresolution. The dimeric crystal structure was confirmed in solution by small-angle X-ray scattering.

Asunto(s)

Alcohol Deshidrogenasa/química , Aldehído Oxidorreductasas/química , Escherichia coli O157/enzimología , Proteínas de Escherichia coli/química , Hierro/química , NAD/química , Subunidades de Proteína/química , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cationes Bivalentes , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hierro/metabolismo , Modelos Moleculares , NAD/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Cyclic di-GMP (c-di-GMP) is a second messenger that modulates multiple responses to environmental and cellular signals in bacteria. Here we identify CdbA, a DNA-binding protein of the ribbon-helix-helix family that binds c-di-GMP in Myxococcus xanthus. CdbA is essential for viability, and its depletion causes defects in chromosome organization and segregation leading to a block in cell division. The protein binds to the M. xanthus genome at multiple sites, with moderate sequence specificity; however, its depletion causes only modest changes in transcription. The interactions of CdbA with c-di-GMP and DNA appear to be mutually exclusive and residue substitutions in CdbA regions important for c-di-GMP binding abolish binding to both c-di-GMP and DNA, rendering these protein variants non-functional in vivo. We propose that CdbA acts as a nucleoid-associated protein that contributes to chromosome organization and is modulated by c-di-GMP, thus revealing a link between c-di-GMP signaling and chromosome biology.

Asunto(s)

Proteínas Bacterianas/metabolismo , Núcleo Celular/metabolismo , Segregación Cromosómica , GMP Cíclico/análogos & derivados , Proteínas de Unión al ADN/metabolismo , Myxococcus xanthus/metabolismo , Proteínas Bacterianas/química , Secuencia de Bases , Cromosomas Bacterianos/metabolismo , GMP Cíclico/metabolismo , ADN Bacteriano/metabolismo , Sitios Genéticos , Modelos Moleculares , Multimerización de Proteína , Estructura Secundaria de Proteína , Transcripción Genética

RESUMEN

The bacterial second messenger cyclic-di-GMP is a widespread, prominent effector of lifestyle change. An example of this occurs in the predatory bacterium Bdellovibrio bacteriovorus, which cycles between free-living and intraperiplasmic phases after entering (and killing) another bacterium. The initiation of prey invasion is governed by DgcB (GGDEF enzyme) that produces cyclic-di-GMP in response to an unknown stimulus. Here, we report the structure of DgcB, and demonstrate that the GGDEF and sensory forkhead-associated (FHA) domains form an asymmetric dimer. Our structures indicate that the FHA domain is a consensus phosphopeptide sensor, and that the ligand for activation is surprisingly derived from the N-terminal region of DgcB itself. We confirm this hypothesis by determining the structure of a FHA:phosphopeptide complex, from which we design a constitutively-active mutant (confirmed via enzyme assays). Our results provide an understanding of the stimulus driving DgcB-mediated prey invasion and detail a unique mechanism of GGDEF enzyme regulation.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bdellovibrio/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Liasas de Fósforo-Oxígeno/química , Liasas de Fósforo-Oxígeno/metabolismo , Secuencia de Aminoácidos , Activación Enzimática , Ligandos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Dominios Proteicos , Multimerización de Proteína , Relación Estructura-Actividad

RESUMEN

Bacterial usage of the cyclic dinucleotide c-di-GMP is widespread, governing the transition between motile/sessile and unicellular/multicellular behaviors. There is limited information on c-di-GMP metabolism, particularly on regulatory mechanisms governing control of EAL c-di-GMP phosphodiesterases. Herein, we provide high-resolution structures for an EAL enzyme Bd1971, from the predatory bacterium Bdellovibrio bacteriovorus, which is controlled by a second signaling nucleotide, cAMP. The full-length cAMP-bound form reveals the sensory N-terminus to be a domain-swapped variant of the cNMP/CRP family, which in the cAMP-activated state holds the C-terminal EAL enzyme in a phosphodiesterase-active conformation. Using a truncation mutant, we trap both a half-occupied and inactive apo-form of the protein, demonstrating a series of conformational changes that alter juxtaposition of the sensory domains. We show that Bd1971 interacts with several GGDEF proteins (c-di-GMP producers), but mutants of Bd1971 do not share the discrete phenotypes of GGDEF mutants, instead having an elevated level of c-di-GMP, suggesting that the role of Bd1971 is to moderate these levels, allowing "action potentials" to be generated by each GGDEF protein to effect their specific functions.

Asunto(s)

Bdellovibrio bacteriovorus/metabolismo , AMP Cíclico/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bdellovibrio bacteriovorus/química , Bdellovibrio bacteriovorus/genética , Sitios de Unión , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Nucleótidos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Unión Proteica , Conformación Proteica , Transducción de Señal

RESUMEN

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting ß-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.

Asunto(s)

Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfolípidos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte Biológico , Cristalografía por Rayos X , Bacterias Gramnegativas/metabolismo , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Periplasma/metabolismo , Unión Proteica , Conformación Proteica en Lámina beta

RESUMEN

Growth and division by most bacteria requires remodelling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides, which are recycled in many model organisms. Bacteria and hosts can harness the unique nature of muropeptides as a signal for cell wall damage and infection, respectively. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core mycobacterial gene lpqI, encodes an authentic NagZ ß-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.

Asunto(s)

Acetilglucosaminidasa/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Peptidoglicano/metabolismo , Antibióticos Antituberculosos/farmacología , Pared Celular/química , Pared Celular/metabolismo , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad Microbiana , Ácidos Murámicos/metabolismo , Muramidasa/farmacología , Mycobacterium bovis/química , Mycobacterium tuberculosis/química , Peptidoglicano/química

RESUMEN

The sulfate reducing bacterium Desulfovibrio desulfuricans inhabits both the human gut and external environments. It can reduce nitrate and nitrite as alternative electron acceptors to sulfate to support growth. Like other sulphate reducing bacteria, it can also protect itself against nitrosative stress caused by NO generated when nitrite accumulates. By combining in vitro experiments with bioinformatic and RNA-seq data, metabolic responses to nitrate or NO and how nitrate and nitrite reduction are coordinated with the response to nitrosative stress were revealed. Although nitrate and nitrite reduction are tightly regulated in response to substrate availability, the global responses to nitrate or NO were largely regulated independently. Multiple NADH dehydrogenases, transcription factors of unknown function and genes for iron uptake were differentially expressed in response to electron acceptor availability or nitrosative stress. Amongst many fascinating problems for future research, the data revealed a YtfE orthologue, Ddes_1165, that is implicated in the repair of nitrosative damage. The combined data suggest that three transcription factors coordinate this regulation in which NrfS-NrfR coordinates nitrate and nitrite reduction to minimize toxicity due to nitrite accumulation, HcpR1 serves a global role in regulating the response to nitrate, and HcpR2 regulates the response to nitrosative stress.

Asunto(s)

Desulfovibrio desulfuricans/genética , Estrés Nitrosativo , Transcriptoma , Desulfovibrio desulfuricans/efectos de los fármacos , Desulfovibrio desulfuricans/metabolismo , Regulación Bacteriana de la Expresión Génica , Nitratos/farmacología , Óxido Nítrico/farmacología , Nitritos/farmacología

RESUMEN

In silico analyses identified a Crp/Fnr family transcription factor (HcpR) in sulfate-reducing bacteria that controls expression of the hcp gene, which encodes the hybrid cluster protein and contributes to nitrosative stress responses. There is only one hcpR gene in the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, but two copies in Desulfovibrio desulfuricans 27774, which can use nitrate as an alternative electron acceptor to sulfate. Structures of the D. desulfuricans hcpR1, hcpR2 and hcp operons are reported. We present evidence that hcp expression is regulated by HcpR2, not by HcpR1, and that these two regulators differ in both their DNA-binding site specificity and their sensory domains. HcpR1 is predicted to be a b-type cytochrome. HcpR1 binds upstream of the hcpR1 operon and its synthesis is regulated coordinately with hcp in response to NO. In contrast, hcpR2 expression was not induced by nitrate, nitrite or NO. HcpR2 is an iron-sulfur protein that reacts with NO and O2 . We propose that HcpR1 and HcpR2 use different sensory mechanisms to regulate subsets of genes required for defense against NO-induced nitrosative stress, and that diversification of signal perception and DNA recognition by these two proteins is a product of D. desulfuricans adaptation to its particular environmental niche.

Asunto(s)

Desulfovibrio desulfuricans/metabolismo , Nitratos/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Biología Computacional , Simulación por Computador , Desulfovibrio desulfuricans/genética , Proteínas Hierro-Azufre/metabolismo , Nitritos/metabolismo , Nitrosación/fisiología , Operón , Factores de Transcripción/genética

RESUMEN

Predatory Bdellovibrio bacteriovorus are natural antimicrobial organisms, killing other bacteria by whole-cell invasion. Self-protection against prey-metabolizing enzymes is important for the evolution of predation. Initial prey entry involves the predator's peptidoglycan DD-endopeptidases, which decrosslink cell walls and prevent wasteful entry by a second predator. Here we identify and characterize a self-protection protein from B. bacteriovorus, Bd3460, which displays an ankyrin-based fold common to intracellular pathogens of eukaryotes. Co-crystal structures reveal Bd3460 complexation of dual targets, binding a conserved epitope of each of the Bd3459 and Bd0816 endopeptidases. Complexation inhibits endopeptidase activity and cell wall decrosslinking in vitro. Self-protection is vital - ΔBd3460 Bdellovibrio deleteriously decrosslink self-peptidoglycan upon invasion, adopt a round morphology, and lose predatory capacity and cellular integrity. Our analysis provides the first mechanistic examination of self-protection in Bdellovibrio, documents protection-multiplicity for products of two different genomic loci, and reveals an important evolutionary adaptation to an invasive predatory bacterial lifestyle.

Asunto(s)

Ancirinas/metabolismo , Proteínas Bacterianas/metabolismo , Bdellovibrio/fisiología , Escherichia coli , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Bacterianas/genética , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Conformación Proteica

RESUMEN

The process of septation requires precise temporal and spatial organization of penicillin binding proteins (PBPs) and associated proteins for the deposition of new cell wall material. In most bacteria, the filamentous protein FtsZ organises PBPs into assemblies at the midcell which then constrict inwards as peptidoglycan is synthesised, eventually closing the septa. Tsui et al. (2014), through the use of fluorescent d-amino acids and high resolution microscopy, report that PBP2x of Streptococcus pneumoniae is directed to a discrete location within the septal aperture during the later stages of cell division. Once at this new site, PBP2x catalyses the de novo synthesis of peptidoglycan, which is imaged by the authors as a central 'spot', distinct from material made by other PBPs at the outer ring. This discovery, which represents a novel mode of cell wall assembly, was made in a directed capsular knockout of strain D39, thereby avoiding potential mechanistic complications in commonly used laboratory strain R6. These findings prompt not only a partial rethink of septum formation in S. pneumoniae, but consideration of the modes of PBP localization and the subtleties that can influence phenotypic study.

Asunto(s)

División Celular , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/biosíntesis , Streptococcus pneumoniae/citología

RESUMEN

In this issue of Structure, Hoyland and colleagues describe the structure of a peptidoglycan L,D-carboxypeptidase in both substrate-bound and apoenzyme forms. These studies reveal the basis for enzyme specificity and assist greatly in a field where form and function overlap.

Asunto(s)

Proteínas Bacterianas/química , Carboxipeptidasas/química , Peptidoglicano/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína

RESUMEN

The Escherichia coli CRP (cAMP receptor protein), is a global regulator of transcription that modulates gene expression by activation or repression at a range of promoters in E. coli. A major function is to regulate the selection of nutrients required for growth. The anaerobic sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC27774 is capable of utilizing sulfate, nitrite and nitrate as terminal electron acceptors. In the presence of both sulfate and nitrate, sulfate is reduced preferentially despite nitrate being the thermodynamically more favourable electron acceptor. Three inverted repeat sequences upstream of the D. desulfuricans ATCC27774 nap (nitrate reduction in the periplasm) operon have high levels of similarity to the consensus sequence for the E. coli CRP DNA-binding site. In other Desulfovibrio species a putative CRP homologue, HcpR [regulator of hcp (hybrid cluster protein) transcription], has a predicted regulon comprising genes involved in sulfate reduction and nitrosative stress. The presence of CRP consensus sites within the D. desulfuricans ATCC27774 nap promoter prompted a search for CRP homologues in the genomes of sulfate-reducing bacteria. This revealed the presence of a potential CRP homologue that we predict binds to CRP consensus sites such as those of the nap operon. Furthermore, we predict that much of the core HcpR regulon predicted in other Desulfovibrio species is conserved in D. desulfuricans.

Asunto(s)

Proteínas Bacterianas/metabolismo , Desulfovibrio desulfuricans/metabolismo , Nitratos/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Estrés Fisiológico , Proteínas Bacterianas/genética , Secuencia de Bases , Desulfovibrio desulfuricans/genética , Datos de Secuencia Molecular , Oxidación-Reducción , Regiones Promotoras Genéticas