Characterization of a Pseudomonas putida ABC transporter (AatJMQP) required for acidic amino acid uptake: biochemical properties and regulation by the Aau two-component system

Singh, Birendra; Röhm, Klaus‐Heinrich

doi:10.1099/mic.0.2007/013185-0

Cited by 27 publications

(36 citation statements)

References 35 publications

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“…Cellular lysis was performed by sonication and the supernatant was loaded on Ni‐NTA column (Histrap FF Crude, GE Healthcare Biosciences), followed by washing step and elution with His‐tag elution buffer 50 mM Tris‐HCl, pH 7.5, containing 500 mM NaCl and 200 mM imidazole. The periplasmic fraction was separated according to a protocol described by Singh and Rohm (2008).…”

Section: Methodsmentioning

confidence: 99%

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

Singh

Blom

Ünal

et al. 2010

Molecular Microbiology

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SummaryThe serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K D) for vitronectin binding to UspA2 was 2.3 ¥ 10 -8 M, and the N-terminal region encompassing residues UspA2 30-170 bound vitronectin with a KD of 7.9 ¥ 10 -8 M. Electron microscopy verified that the active binding domain (UspA2 30-177 ) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2 30-177 bound to the C-terminal region of vitronectin residues 312-396. Finally, when human serum was pre-incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N-terminal domain of UspA2 and the C-terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis-dependent serum resistance.

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Section: Methodsmentioning

confidence: 99%

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

Singh

Blom

Ünal

et al. 2010

Molecular Microbiology

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“…Very few such solute specific binding proteins of glutamate ABC- transporter system in different bacterial species have been experimentally characterized. Three such proteins from E. coli K 12, Pseudomonas putida KT2440 and Campylobacter jejuni NCTC 11168 are GltI, AatJ (PP1071) and PEB1a (CJ0921c) respectively [11– 13]. The aligned consensus of these three sequences revealed putative gene/ORF for periplasmic glutamate binding protein in C. perfringens Type-A strains Table 2 (see supplementary material).…”

Section: Resultsmentioning

confidence: 99%

“…Amino acid sequences of experimentally characterized solute specific binding protein of glutamate ABC-transporter system from three different bacterial species ( E. coli K 12, P. putida KT2440 and Campylobacter jejuni NCTC 11168) [9– 13] were taken from Uniprot Knowledge Base (ID: P37902, Q88NY2, Q0P9X8).…”

Section: Methodsmentioning

confidence: 99%

Identification of glutamate ABC-Transporter component in Clostridium perfringens as a putative drug target

et al. 2014

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Clostridium perfringens is an anaerobic pathogen known to cause vast number of diseases in mammals and birds. Various toxins and hydrolysing enzymes released by the organism are responsible for the necrosis of soft tissues. Due to serious safety issues associated with current vaccines against C. perfringens, there is a need for new drug or vaccine targets. C. perfringens is extremely dependent on its host for nutrition which can be targeted for vaccine development or drug design. Therefore, it is of interest to identify the unique transport systems used by C. perfringens involved in uptake of essential amino acids that are synthesized by the host, so that therapeutic agents can be designed to target the specific transport systems. Use of bioinformatics tools resulted in the identification of a protein component of the glutamate transport system that is not present in the host. Analysis of the conservation profile of the protein domain indicated it to be a glutamate binding protein which also stimulates the ATPase activity of ATP Binding Cassettes (ABC) transporters. Homology modelling of the protein showed two distinct lobes, which is a characteristic of substrate binding proteins. This suggests that the carboxylates of glutamate might be stabilized by electrostatic interactions with basic residues as is observed with other binding proteins. Hence, the homology model of this potential drug target can be employed for in silico docking studies by suitable inhibitors.

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“…Protein expression was induced by addition of 1 mM IPTG, and protein purification was performed as described elsewhere (Singh and Rohm, 2008). Some Hsf fragments produced inclusion bodies that were purified by using an inclusion body purification protocol (Singh and Rohm, 2008). The purified proteins were dialyzed in PBS and concentrated by using Centricon cartridge (Millipore, Bedford, MA).…”

Section: Methodsmentioning

confidence: 99%

“…For purification purposes, E. coli BL21(DE3) containing pET26b-hsf(s) was grown in LB medium with kanamycin at 37C until OD 600 nm reached to 0.8-1. Protein expression was induced by addition of 1 mM IPTG, and protein purification was performed as described elsewhere (Singh and Rohm, 2008). Some Hsf fragments produced inclusion bodies that were purified by using an inclusion body purification protocol (Singh and Rohm, 2008).…”

Section: Methodsmentioning

confidence: 99%

Haemophilus influenzae surface fibril (Hsf) is a unique twisted hairpin-like trimeric autotransporter

Singh

Jubair

Mörgelin

et al. 2015

International Journal of Medical Microbiology

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The Haemophilus surface fibril (Hsf) is an extraordinary large (2413 amino acids) trimeric autotransporter, present in all encapsulated Haemophilus influenzae. It contributes to virulence by directly functioning as an adhesin. Furthermore, Hsf recruits the host factor vitronectin thereby inhibiting the host innate immune response resulting in enhanced survival in serum. Here we observed by electron microscopy that Hsf appears as an 100 nm long fibril at the bacterial surface albeit the length is approximately 200 nm according to a bioinformatics based model. To unveil this discrepancy, we denaturated Hsf at the surface of Hib by using guanidine hydrochloride (GuHCl). Partial denaturation induced in the presence of GuHCl unfolded the Hsf molecules, and resulted in an increased length of fibres in comparison to the native trimeric form. Importantly, our findings were also verified by E. coli expressing Hsf at its surface. In addition, a set of Hsf-specific peptide antibodies also indicated that the N-terminal of Hsf is located near the C-terminal at the base of the fibril. Taken together, our results demonstrated that Hsf is not a straight molecule but is folded and doubled over. This is the first report that provides the unique structural features of the trimeric autotransporter Hsf. Highlights Trimeric autotransporters are important virulence factors of Gram negative bacteria, and have a "lollipop-shape" structure or straight trimeric strand.  Haemophilus surface fibril is involved in adherence to host epithelium and for recruitment of vitronectin.  Here we show for the first time that Haemophilus surface fibril has a twisted hairpin-like structure.

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Characterization of a Pseudomonas putida ABC transporter (AatJMQP) required for acidic amino acid uptake: biochemical properties and regulation by the Aau two-component system

Cited by 27 publications

References 35 publications

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

Identification of glutamate ABC-Transporter component in Clostridium perfringens as a putative drug target

Haemophilus influenzae surface fibril (Hsf) is a unique twisted hairpin-like trimeric autotransporter

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