Pathway In Gram-negative Bacteria Research Articles

Assembly of the β-Barrel Outer Membrane Proteins in Gram-Negative Bacteria, Mitochondria, and Chloroplasts.

In the last decade, there has been an explosion of publications on the assembly of β-barrel outer membrane proteins (OMPs), which carry out diverse cellular functions, including solute transport, protein secretion, and assembly of protein and lipid components of the outer membrane. Of the three outer membrane model systems—Gram-negative bacteria, mitochondria and chloroplasts—research on bacterial and mitochondrial systems has so far led the way in dissecting the β-barrel OMP assembly pathways. Many exciting discoveries have been made, including the identification of β-barrel OMP assembly machineries in bacteria and mitochondria, and potentially the core assembly component in chloroplasts. The atomic structures of all five components of the bacterial β-barrel assembly machinery (BAM) complex, except the β-barrel domain of the core BamA protein, have been solved. Structures reveal that these proteins contain domains/motifs known to facilitate protein-protein interactions, which are at the heart of the assembly pathways. While structural information has been valuable, most of our current understanding of the β-barrel OMP assembly pathways has come from genetic, molecular biology, and biochemical analyses. This paper provides a comparative account of the β-barrel OMP assembly pathways in Gram-negative bacteria, mitochondria, and chloroplasts.

Pathoadaptive Conditional Regulation of the Type VI Secretion System in Vibrio cholerae O1 Strains

The most recently discovered secretion pathway in gram-negative bacteria, the type VI secretion system (T6SS), is present in many species and is considered important for the survival of non-O1 non-O139 Vibrio cholerae in aquatic environments. Until now, it was not known whether there is a functionally active T6SS in wild-type V. cholerae O1 strains, the cause of cholera disease in humans. Here, we demonstrate the presence of a functionally active T6SS in wild-type V. cholerae O1 strains, as evidenced by the secretion of the T6SS substrate Hcp, which required several gene products encoded within the putative vas gene cluster. Our analyses showed that the T6SS of wild-type V. cholerae O1 strain A1552 was functionally activated when the bacteria were grown under high-osmolarity conditions. The T6SS was also active when the bacteria were grown under low temperature (23°C), suggesting that the system may be important for the survival of the bacterium in the environment. A test of the interbacterial virulence of V. cholerae strain A1552 against an Escherichia coli K-12 strain showed that it was strongly enhanced under high osmolarity and that it depended on the hcp genes. Interestingly, we found that the newly recognized osmoregulatory protein OscR plays a role in the regulation of T6SS gene expression and secretion of Hcp from V. cholerae O1 strains.

Species-Specific and Inhibitor-Dependent Conformations of LpxC: Implications for Antibiotic Design

LpxC is an essential enzyme in the lipid A biosynthetic pathway in gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against gram-negative infections.

Assignment of 1H, 13C and 15N backbone resonances of Escherichia coli LpxC bound to L-161,240.

The UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase LpxC catalyzes the committed reaction of lipid A biosynthesis, an essential pathway in Gram-negative bacteria. We report the backbone resonance assignments of the 34 kDa LpxC from Escherichia coli in complex with the antibiotic L-161,240 using multidimensional, multinuclear NMR experiments. The (1)H chemical shifts of complexed L-161,240 are also determined.

Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 2. Substrate Modeling and Active Site Mutations

The N-acyl-l-homoserine lactone hydrolases (AHL lactonases) have attracted considerable attention because of their ability to quench AHL-mediated quorum-sensing pathways in Gram-negative bacteria and because of their relation to other enzymes in the metallo-β-lactamase superfamily. To elucidate the detailed catalytic mechanism of AHL lactonase, mutations are made on residues that presumably contribute to substrate binding and catalysis. Steady-state kinetic studies are carried out on both the wild-type and mutant enzymes using a spectrum of substrates. Two mutations, Y194F and D108N, present significant effects on the overall catalysis. On the basis of a high-resolution structural model of the enzyme−product complex, a hybrid quantum mechanical/molecular mechanical method is used to model the substrate binding orientation and to probe the effect of the Y194F mutation. Combining all experimental and computational results, we propose a detailed mechanism for the ring-opening hydrolysis of AHL substrates as catalyzed by the AHL lactonase from Bacillus thuringiensis. Several features of the mechanism that are also found in related enzymes are discussed and may help to define an evolutionary thread that connects the hydrolytic enzymes of this mechanistically diverse superfamily.

The global regulator H‐NS binds to two distinct classes of sites within the Tn10 transpososome to promote transposition

The histone-like nucleoid structuring protein (H-NS) is a global transcriptional regulator that influences stress response and virulence pathways in Gram-negative bacteria. H-NS also promotes Tn10 transposition by binding directly to the transpososome and inducing a conformational change in the transpososome that favours intermolecular transposition events. H-NS binds preferentially to curved DNA and can bend non-curved DNA, it self-oligomerizes and can interact with other proteins. To determine what functions of H-NS are important in promoting Tn10 transposition, we have examined the ability of two mutant forms of H-NS, P116S and 1-64, to act in Tn10 transposition. We provide evidence that the initial interaction of H-NS with the transpososome is dependent on H-NS binding to a specific structure in DNA flanking the transposon end. Additional molecules of H-NS then bind within the transposon end. This latter event appears to be directed by H-NS binding to the Tn10 transposase protein, and is important in maintaining the transpososome in a conformation that promotes intermolecular transposition. The binding of H-NS to a transposase protein is a novel function for this important regulatory molecule.

Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans

DNA adenine methyltransferase (DAM) plays critical roles in diverse biological pathways in gram-negative bacteria, and specifically in regulating the expression of virulence genes in several organisms. Actinobacillus actinomycetemcomitans plays an important role in the pathogenesis of juvenile and adult periodontal disease, yet little is known about its mechanisms of gene regulation. DAM is shown here to directly or indirectly affect well-known A. actinomycetemcomitans virulence factors. A mutant A. actinomycetemcomitans strain lacking the dam gene was created by homologous recombination and shows normal growth phenotypes when grown exponentially. This mutant strain has four sixfold increased levels of extracellular leukotoxin, altered cellular levels of leukotoxin, and significant changes in bacterial invasion of KB oral epithelial cells. These results provide a basis for further characterization of regulatory mechanisms that control A. actinomycetemcomitans virulence.

Translocator Proteins in the Two-partner Secretion Family Have Multiple Domains

The two-partner secretion pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane translocator protein. Previous work has demonstrated that the TpsB protein forms a beta-barrel structure with pore forming activity and facilitates translocation of the TpsA protein across the outer membrane. In this study, we characterized the functional domains of the Haemophilus influenzae HMW1B protein, a TpsB protein that interacts with the H. influenzae HMW1 adhesin. Using c-Myc epitope tag insertions and cysteine substitution mutagenesis, we discovered that HMW1B contains an N-terminal surface-localized domain, an internal periplasmic domain, and a C-terminal membrane anchor. Functional and biochemical analysis of the c-Myc epitope tag insertions and a series of HMW1B deletion constructs demonstrated that the periplasmic domain is required for secretion of HMW1 and that the C-terminal membrane anchor (HMW1B-(234-545)) is capable of oligomerization and pore formation. Similar to our observations with HMW1B, examination of a Bordetella pertussis TpsB protein called FhaC revealed that the C terminus of FhaC (FhaC-(232-585)) is capable of pore formation. We speculate that all TpsB proteins have a modular structure, with a periplasmic domain that interacts with the cognate TpsA protein and with pore forming activity contained within the C terminus.

Structure of a membrane-based steric chaperone in complex with its lipase substrate

Secretion via the type II secretion pathway in Gram-negative bacteria often relies crucially on steric chaperones in the periplasm. Here, we report the crystal structure of the soluble form of a lipase-specific foldase (Lif) from Burkholderia glumae in complex with its cognate lipase. The structure reveals how Lif uses a novel alpha-helical scaffold to embrace lipase, thereby creating an unusually extensive folding platform.

Channel Properties of TpsB Transporter FhaC Point to Two Functional Domains with a C-terminal Protein-conducting Pore

Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a beta-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition.

Mechanisms of Protein Export across the Bacterial Outer Membrane

Gram-negative bacteria secrete a wide range of proteins whose functions include biogenesis of organelles, such as pili and flagella; nutrient acquisition; virulence; and efflux of drugs and other toxins. Export of these proteins to the bacterial surface involves transport across the inner membrane (IM), periplasm, and outer membrane (OM) of the cell envelope. Several pathways have evolved to fulfill the task of secretion (Table ​(Table1).1). These pathways can be divided into two main groups: (i) Sec dependent and (ii) Sec independent (Fig. ​(Fig.1)1) (64). FIG. 1. Schematic presentation of Sec-dependent and Sec-independent secretion pathways in gram-negative bacteria. The type I pathway is exemplified by the hemolysin A (HlyA) secretion in pathogenic E. coli, the type II pathway by YopE secretion in Yersinia pestis ... TABLE 1. Representative members of the eight secretion systems Proteins secreted via the Sec-dependent pathways utilize a common machinery, the Sec translocase, for transport across the IM and are mainly differentiated based on their mechanisms of secretion across the OM (64). Sec-dependent pathways include the type II secretion (T2S); the autotransporter (AT), or type V secretion; the two-partner secretion (TPS); and the chaperone/usher (CU) secretion systems (34, 46, 58, 70, 73). The type IV secretion (T4S), or adapted-conjugation, pathway can be Sec dependent but is mostly considered Sec independent (21). Sec-independent pathways tend to allow direct export from the cytoplasm to the extracellular environment in one step and do not involve periplasmic intermediates. These pathways also include the type I secretion (T1S), or ABC (ATP-binding cassette), exporters (5) and the type III secretion (T3S) systems (31). An alternative Sec-independent pathway known as twin-arginine translocation (Tat) is employed for transport of already-folded proteins across the IM. Generally, Tat substrates are targeted to the periplasm of the cell (4) but can also be transported across the OM via the type II pathway, such as the phospholipases of Pseudomonas aeruginosa (77). The Sec and Tat machineries for transport across the IM will not be discussed further. In this review, we summarize the current knowledge of OM protein secretion in terms of the structure of the exporters and the mechanism of secretion and examine similarities and differences among the different export pathways (Table ​(Table2).2). Since many virulence factors are secreted proteins, a more complete understanding of the mechanisms underlying bacterial protein secretion is crucial to the development of new vaccines and treatments for a wide range of human pathogens. TABLE 2. Structural features of the OM components of protein secretion systems

A putative three-dimensional targeting motif of polygalacturonase (PehA), a protein secreted through the type II (GSP) pathway in Erwinia carotovora.

Intramolecular information specifying protein secretion through the type II (GSP) pathway of Gram-negative bacteria was investigated. Two regions of the polygalacturonase (PehA) of Erwinia carotovora containing residues proposed to be included in a targeting motif were located, one close to the C-terminus between residues 342 and 369 and another between residues 84 and 135 in the large central loops. The regions were required together to promote secretion. Further residues in the middle of the protein were required for proper positioning of the regions, suggesting that they were both involved in interaction with the GSP. To our knowledge, this is the first time that a possible three-dimensional targeting motif has been defined. At least one of the motifs comprises a cluster on the surface of the protein. The two motifs are structurally dissimilar, suggesting that there are two distinct recognition regions in the GSP apparatus. Finally, we propose that the targeting motifs are of a complex conformational nature with some variability accommodated, as illustrated by the observation that many mutations exhibited no clear phenotype individually but, in combination, severely compromised secretion.

Colicin A hybrids: a genetic tool for selection of type II secretion-proficient Pseudomonas strains.

The gram-negative bacterium Pseudomonas aeruginosa secretes the majority of its extracellular proteins by the type II secretion mechanism, a two-step process initiated by translocation of signal peptide-bearing exoproteins across the inner membrane. The periplasmic forms are transferred across the outer membrane by a machinery consisting of 12 xcp gene products. Although the type II secretion machinery is conserved among gram-negative bacteria, interactions between the secreted proteins and the machinery are specific. The lack of a selectable phenotype has hampered the development of genetic strategies for studying type II secretion. We report a novel strategy to identify rare events, such as those that allow heterologous secretion or identification of extragenic suppressors correcting xcp defects. This is based on creating a host-vector system where the non-secretory phenotype is lethal. The original tool we designed is a hybrid protein containing elastase and the pore-forming domain of colicin A.

Pilus formation and protein secretion by the same machinery in Escherichia coli.

The secreton (type II secretion) and type IV pilus biogenesis branches of the general secretory pathway in Gram-negative bacteria share many features that suggest a common evolutionary origin. Five components of the secreton, the pseudopilins, are similar to subunits of type IV pili. Here, we report that when the 15 genes encoding the pullulanase secreton of Klebsiella oxytoca were expressed on a high copy number plasmid in Escherichia coli, one pseudopilin, PulG, was assembled into pilus-like bundles. Assembly of the 'secreton pilus' required most but not all of the secreton components that are essential for pullulanase secretion, including some with no known homologues in type IV piliation machineries. Two other pseudopilins, pullulanase and two outer membrane-associated secreton components were not associated with pili. Thus, PulG is probably the major component of the pilus. Expression of a type IV pilin gene, the E.coli K-12 gene ppdD, led to secreton-dependent incorporation of PpdD pilin into pili without diminishing pullulanase secretion. This is the first demonstration that pseudopilins can be assembled into pilus-like structures.

Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae.

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.

6] Erwinia metalloprotease permease: Aspects of secretion pathway and secretion functions

This chapter discusses the secretion pathway and secretion functions of Erwinia chrysanthemi , which is a phytopathogenic gram-negative enterobacterium that causes soft rot disease among various plants. It secretes several hydrolytic enzymes, among which pectinases and cellulases are the main constituents of its pathogenicity. E . chrysanthemi also secretes several metalloproteases. The chapter reviews the E . chrysanthemi metalloprotease secretion pathway and describes different techniques used to study in this pathway. Protein secretion by the adenosine triphosphate-binding cassette (ABC) pathway in gram-negative bacteria is widespread in many species, allowing the secretion of several classes of proteins and presents several distinctive features. The secretion apparatus is made up of three proteins, the ABC protein itself being made of the ABC domain fused to a hydrophobic membrane domain and functioning as a dimer localized in the cytoplasmic membrane.

Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria – a review

The main terminal branch (MTB) of the general secretory pathway is used by a wide variety of Gram − bacteria to transport exoproteins from the periplasm to the outside milieu. Recent work has led to the identification of the function of two of its 14 (or more) components: an enzyme with type-IV prepilin peptidase activity and a chaperone-like protein required for the insertion of another of the MTB components into the outer membrane. Despite these important discoveries, little tangible progress has been made towards identifying MTB components that determine secretion specificity (presumably by binding to cognate exoproteins) or which form the putative channel through which exoproteins are transported across the outer membrane. However, the idea that the single integral outer membrane component of the MTB could line the wall of this channel, and the intriguing possibility that other components of the MTB form a rudimentary type-IV pilus-like structure that might span the periplasm both deserve more careful examination. Although Escherichia coli K-12 does not normally secrete exoproteins, its chromosome contains an apparently complete set of genes coding for MTB components. At least two of these genes code for functional proteins, but the operon in which twelve of the genes are located does not appear to be expressed. We are currently searching for conditions which allow these genes to be expressed with the eventual aim of identifying the protein(s) that E. coli K-12 can secrete.

Analysis of three 2,3-dihydroxybiphenyl 1,2-dioxygenases found in Rhodococcus globerulus P6. Identification of a new family of extradiol dioxygenases.

The polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6 contains three bphC genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenases. One of them, bphC1, is clustered with the bphB gene which encodes 2,3-dihydroxy-4-phenylhexa-4,6-diene dehydrogenase and constitutes part of the bph operon specifying the degradation of biphenyl. The nucleotide sequence of bphB and the three bphC genes has been determined. The protein products of the bphBC1 gene cluster were found to exhibit significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative bacteria; the highest homology was in those of the toluene degradation pathway of Pseudomonas putida strain F1. No homology was found between bphC2 and bphC3 and any other sequence in the database. At least two of the three meta cleavage enzymes are inducible by biphenyl. 2,3-Dihydroxybiphenyl 1,2-dioxygenase II, encoded by the bphC2 gene, was purified to apparent homogeneity from a recombinant Escherichia coli strain. The enzyme differed from other extradiol dioxygenases in having a subunit molecular mass of 21 kDa and a hexameric structure. The enzyme contains one tightly bound iron per subunit. These characteristics demonstrate that the 2,3-dihydroxybiphenyl 1,2-dioxygenases encoded by bphC2 and bphC3 belong to a new class of extradiol dioxygenases.

A superfamily of proteins involved in different secretion pathways in gram-negative bacteria: modular structure and specificity of the N-terminal domain.

The family of PulD proteins, which has been characterized in a wide variety of microorganisms, comprises several membrane-associated proteins essential for the transport of macromolecules across bacterial membranes. These proteins are involved in the transport of complex structures (such as phage particles, DNA) or various proteins (such as extracellular enzymes and pathogenicity determinants). Amino acid sequence analysis revealed a possible modular organisation of proteins of this superfamily, with highly conserved C-terminal domains and dissimilar N-terminal domains. In the C-terminal domain, four highly conserved regions have been found, one of them containing a remarkable common motif: (V, I)PXL(S, G)XIPXXGXLF. Structural comparisons between the N-terminal domains indicate that proteins of this superfamily can be divided into at least two subgroups, probably reflecting the existence of distinct secretion mechanisms. This implies that members of the superfamily of PulD-related proteins are independently involved in (1) the general secretory pathway, (2) a new signal-peptide-independent secretion pathway found in several bacterial pathogens, and possibly in (3) the translocation of bacteriophage particles through the bacterial cell envelope.

The complete general secretory pathway in gram-negative bacteria

The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins.