scholarly journals Complex Oxidation of Apocytochromes c during Bacterial Cytochrome c Maturation

2019 ◽  
Vol 85 (24) ◽  
Author(s):  
Kailun Guo ◽  
Wei Wang ◽  
Haixia Wang ◽  
Zhenmei Lu ◽  
Haichun Gao
Keyword(s):  
Disulfide Bonds ◽  
Shewanella Oneidensis ◽  
Critical Factor ◽  
Binding Motif ◽  
Primary Role ◽  
Research Model ◽  
Gram Negative ◽  
Content Type ◽  
Cytochrome C Maturation ◽  
Cysteine Thiols

ABSTRACT c-Type cytochromes (cyts c) are proteins that contain covalently bound heme and that thus require posttranslational modification for activity, a process carried out by the cytochrome c (cyt c) maturation system (referred to as the Ccm system) in many Gram-negative bacteria. It has been established that during cyt c maturation (CCM), two cysteine thiols of the heme binding motif (CXXCH) within apocytochromes c (apocyts c) are first oxidized largely by DsbA to form a disulfide bond, which is later reduced through a thio-reductive pathway involving DsbD. However, the physiological impacts of DsbA proteins on CCM in fact vary significantly among bacteria. In this work, we used the cyt c-rich Gram-negative bacterium Shewanella oneidensis as the research model to clarify the roles of DsbA proteins in CCM. We show that in terms of the oxidation of apocyts c, DsbA proteins are an important but not critical factor, and, strikingly, oxygen is not either. By exploiting the DsbD-independent pathway, we identify DsbA1, DsbA2, and DsbA3 as oxidants contributing to the oxidation of apocyts c and reductants, such as cysteine, to be an effective antagonist against DsbA-independent oxidation. We further show that DsbB proteins are partially responsible for the reoxidization of reduced DsbA proteins. Overall, our results indicate that the DsbA-DsbB redox pair has a limited role in CCM, challenging the established notion that it is the main oxidant for apocyts c. IMPORTANCE DsbA is a powerful oxidase that functions in the bacterial periplasm to introduce disulfide bonds in many proteins, including apocytochromes c. It has been well established that although DsbA is not essential, it plays a primary role in cytochrome c maturation, based on studies in bacteria hosting several cyts c. Here, with cyt c-rich S. oneidensis as a research model, we show that this is not always the case. Moreover, we demonstrate that DsbB is also not essential for cytochrome c maturation. These results underscore the need to identify oxidants other than DsbA/DsbB that are crucial in the oxidation of apocyts c in bacteria.

scholarly journals Protein Machineries Involved in the Attachment of Heme to Cytochrome c: Protein Structures and Molecular Mechanisms

Scientifica ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-17 ◽  
Author(s):  
Carlo Travaglini-Allocatelli
Keyword(s):  
Molecular Mechanisms ◽  
Protein Structures ◽  
Eukaryotic Cells ◽  
Binding Motif ◽  
Gram Negative ◽  
Open Questions ◽  
Structural And Functional Properties ◽  
Cyt C ◽  
Electron Transfer Processes ◽  
Cysteine Thiols

Cytochromes c (Cyt c) are ubiquitous heme-containing proteins, mainly involved in electron transfer processes, whose structure and functions have been and still are intensely studied. Surprisingly, our understanding of the molecular mechanism whereby the heme group is covalently attached to the apoprotein (apoCyt) in the cell is still largely unknown. This posttranslational process, known as Cyt c biogenesis or Cyt c maturation, ensures the stereospecific formation of the thioether bonds between the heme vinyl groups and the cysteine thiols of the apoCyt heme binding motif. To accomplish this task, prokaryotic and eukaryotic cells have evolved distinctive protein machineries composed of different proteins. In this review, the structural and functional properties of the main maturation apparatuses found in gram-negative and gram-positive bacteria and in the mitochondria of eukaryotic cells will be presented, dissecting the Cyt c maturation process into three functional steps: (i) heme translocation and delivery, (ii) apoCyt thioreductive pathway, and (iii) apoCyt chaperoning and heme ligation. Moreover, current hypotheses and open questions about the molecular mechanisms of each of the three steps will be discussed, with special attention to System I, the maturation apparatus found in gram-negative bacteria.

scholarly journals PBP1a/LpoA but Not PBP1b/LpoB Are Involved in Regulation of the Major β-Lactamase GeneblaAin Shewanella oneidensis

2015 ◽  
Vol 59 (6) ◽  
pp. 3357-3364 ◽  
Author(s):  
Jianhua Yin ◽  
Yiyang Sun ◽  
Yinting Mao ◽  
Miao Jin ◽  
Haichun Gao
Keyword(s):  
Antibiotic Resistance ◽  
Shewanella Oneidensis ◽  
Natural Resistance ◽  
Aquatic Environments ◽  
Gram Negative Bacteria ◽  
Penicillin Binding Protein ◽  
Regulatory Pathway ◽  
Gram Negative ◽  
Content Type ◽  
Class D

ABSTRACTβ-Lactamase production is one of the most important strategies for Gram-negative bacteria to combat β-lactam antibiotics. Studies of the regulation of β-lactamase expression have largely been focused on the class C β-lactamase AmpC, whose induction by β-lactams requires LysR-type regulator AmpR and permease AmpG-dependent peptidoglycan recycling intermediates. InShewanella, which is ubiquitous in aquatic environments and is a reservoir for antibiotic resistance, production of the class D β-lactamase BlaA confers bacteria with natural resistance to many β-lactams. Expression of theblaAgene in the genus representativeShewanella oneidensisis distinct from the AmpC paradigm because of the lack of an AmpR homologue and the presence of an additional AmpG-independent regulatory pathway. In this study, using transposon mutagenesis, we identify proteins that are involved inblaAregulation. Inactivation ofmrcAandlpoA, which encode penicillin binding protein 1a (PBP1a) and its lipoprotein cofactor, LpoA, respectively, drastically enhancesblaAexpression in the absence of β-lactams. Although PBP1b and its cognate, LpoB, also exist inS. oneidensis, their roles inblaAinduction are dispensable. We further show that themrcA-mediatedblaAexpression is independent of AmpG.

scholarly journals Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

2016 ◽  
Vol 198 (16) ◽  
pp. 2192-2203 ◽  
Author(s):  
Alessandra M. Martorana ◽  
Mattia Benedet ◽  
Elisa A. Maccagni ◽  
Paola Sperandeo ◽  
Riccardo Villa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Critical Factor ◽  
Permeability Barrier ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
Essential Proteins ◽  
Outer Leaflet ◽  
Abc Protein

ABSTRACTThe assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made inEscherichia coliof seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level oflptBis a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carriesPa-LptC, the highly divergent LptC orthologue fromPseudomonas aeruginosa. We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carryingPa-LptC which is otherwise defective in interactions with theE. coliLptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery.IMPORTANCEThe asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. InEscherichia colithe LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a transenvelope complex. Here, we show that increased expression of the membrane-associated ABC protein LptB can suppress defects of LptC, which participates in the formation of the periplasmic bridge. This reveals functional interactions between these two components and supports a role of LptB in the assembly of the Lpt machine.

What is the substrate specificity of the System I cytochrome c biogenesis apparatus?

2006 ◽  
Vol 34 (1) ◽  
pp. 150-151 ◽  
Author(s):  
J.W.A. Allen ◽  
S.J. Ferguson
Keyword(s):  
Cytochrome C ◽  
Substrate Specificity ◽  
Nitrogen Cycle ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Cytochrome C Maturation ◽  
Cytochrome C Biogenesis

c-Type cytochromes are characterized by covalent attachment of haem to protein through thioether bonds between the vinyl groups of the haem and the thiols of a CXXCH motif. Proteins of this type play crucial roles in the biochemistry of the nitrogen cycle. Many Gram-negative bacteria use the Ccm (cytochrome c maturation) proteins for the post-translational haem attachment to their c-type cytochromes; in the present paper, we discuss the substrate specificity of the Ccm apparatus. The main conclusion is that the feature recognized and required in the apocytochrome is simply the two cysteines and the histidine of the haem-binding motif.

scholarly journals Distinct Roles of Shewanella oneidensis Thioredoxin in Regulation of Cellular Responses to Hydrogen and Organic Peroxides

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
Xue Feng ◽  
Weining Sun ◽  
Linggen Kong ◽  
Haichun Gao
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Cellular Response ◽  
Critical Role ◽  
Shewanella Oneidensis ◽  
Redox Status ◽  
Antioxidant Systems ◽  
Organic Peroxides ◽  
Research Model ◽  
Content Type

ABSTRACT The thioredoxin (Trx) and glutaredoxin (Grx) antioxidant systems are deeply involved in bacterial response to oxidative stress, but to date, we know surprisingly little about the roles of these systems in response to reactive oxygen species (ROS) other than hydrogen peroxide (H2O2). In this study, we used Shewanella oneidensis, an environmental bacterium, as a research model to investigate the roles of Trx and Grx in oxidative stress response because it has functionally intertwined ROS responsive regulators OxyR and OhrR. We found that Trx1 is the major thiol/disulfide redox system and that in its absence a Grx system becomes essential under normal conditions. Although overshadowed by Trx1 in the wild type, Trx2 can fully replace Trx1 in physiology when overproduced. Trx1 is required for OxyR to function as a repressor but, more importantly, plays a critical role in the cellular response to organic peroxide (OP) by mediating the redox status of OhrR but not OP scavenger OhrA. While none of the trx and grx genes are OxyR dependent, trxA and trxC are affected by OhrR indirectly. Additional data suggest that depletion of glutathione is likely the cue to trigger induced expression of trxA and trxC. These findings underscore the particular importance of Trx in the bacterial OP stress response. IMPORTANCE The Trx and Grx systems are deeply involved in bacterial responses to H2O2-induced oxidative stress. However, little is known about their roles in response to other ROS, such as organic peroxides (OPs). In this study, we used S. oneidensis as a research model to investigate the interplay between Trx/Grx and OxyR/OhrR. We show that Trxs mediate the redox status of transcriptional OP-responding regulator OhrR. Although none of the trx or grx genes are directly controlled by OxyR or OhrR, expression of trxA and trxC is induced by tert-butyl hydroperoxide (t-BHP). We further show that the trxA and trxC genes respond to effects of glutathione (GSH) depletion rather than oxidation. These findings underscore the particular importance of Trx in the bacterial OP stress response.

scholarly journals Draft Genome Sequence of Urease-Producing Pseudorhodobacter sp. Strain E13, Isolated from the Yellow Sea in Gunsan, South Korea

2019 ◽  
Vol 8 (23) ◽  
Author(s):  
Si Chul Kim ◽  
Hyo Jung Lee
Keyword(s):  
South Korea ◽  
Genome Sequence ◽  
Yellow Sea ◽  
Draft Genome ◽  
The Yellow Sea ◽  
Draft Genome Sequence ◽  
Protein Coding ◽  
Coding Sequences ◽  
Gram Negative ◽  
Content Type

Here, we report the draft genome sequence of Pseudorhodobacter sp. strain E13, a Gram-negative, aerobic, nonflagellated, and rod-shaped bacterium which was isolated from the Yellow Sea in South Korea. The assembled genome sequence is 3,878,578 bp long with 3,646 protein-coding sequences in 159 contigs.

scholarly journals Draft Genome Sequences of Three Flavobacterium psychrophilum Strains Isolated from Diseased Ayu (Plecoglossus altivelis altivelis) Caught at Three Sites in the Kagami River in Kochi, Japan

2019 ◽  
Vol 8 (34) ◽  
Author(s):  
Hazuki Yamashita ◽  
Takayuki Wada ◽  
Yusuke Kato ◽  
Takuji Ikeda ◽  
Masayuki Imajoh
Keyword(s):  
Draft Genome ◽  
Psychrophilic Bacterium ◽  
Genome Sequences ◽  
Plecoglossus Altivelis ◽  
Gram Negative ◽  
Content Type ◽  
Flavobacterium Psychrophilum ◽  
Skin Ulcers ◽  
The Family

Flavobacterium psychrophilum is a Gram-negative, psychrophilic bacterium within the family Flavobacteriaceae. Here, we report the draft genome sequences of three F. psychrophilum strains isolated from skin ulcers of diseased ayu caught by tomozuri angling at three sites in the Kagami River in Japan.

scholarly journals Retrospective Molecular Analysis of DIM-1 Metallo-β-Lactamase Discovered in Pseudomonas stutzeri from India in 2000

2013 ◽  
Vol 58 (1) ◽  
pp. 596-598 ◽  
Author(s):  
Lalitagauri M. Deshpande ◽  
Ronald N. Jones ◽  
Leah N. Woosley ◽  
Mariana Castanheira
Keyword(s):  
Molecular Analysis ◽  
Pseudomonas Stutzeri ◽  
Clinical Isolates ◽  
Gram Negative ◽  
Content Type ◽  
Environmental Source ◽  
Lactamase Gene ◽  
Encoding Genes

ABSTRACTAmong 220 clinical isolates of Gram-negative bacilli collected in India during 2000, 22 strains showing elevated imipenem MICs were evaluated for carbapenemase production. One DIM-1-producingPseudomonas stutzeriisolate was detected, and no other carbapenemase-encoding genes were identified. This detection of a DIM-1-producingP. stutzeriisolate from India predating the finding of this gene in the index Dutch strain and the very recent detection of DIM-1 in Africa suggest an unidentified environmental source of this metallo-β-lactamase gene.

scholarly journals Dynamics ofMycobacterium tuberculosisAg85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Joel D. Ernst ◽  
Amber Cornelius ◽  
Miriam Bolz
Keyword(s):  
Protein Secretion ◽  
Immunosorbent Assay ◽  
Bacterial Population ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Protein ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Bacterial Proteins

ABSTRACTSecretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteinsin vivoare poorly understood. We generated new monoclonal antibodies that recognize theMycobacteriumtuberculosissecreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generatedin vitroandin vivo. We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in theM. tuberculosiscomplex: compared with the commonly used H37Rv strain (lineage 4),Mycobacteriumafricanum(lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates ofM. tuberculosisH37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretionin vivois dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCEBacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted byM. tuberculosisand used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.

scholarly journals Structure and Molecular Recognition Mechanism of IMP-13 Metallo-β-Lactamase

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Charlotte A. Softley ◽  
Krzysztof M. Zak ◽  
Mark J. Bostock ◽  
Roberto Fino ◽  
Richard Xu Zhou ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Rational Drug Design ◽  
Tryptophan Residue ◽  
Resonance Spectroscopy ◽  
Global Public Health ◽  
Recognition Mechanism ◽  
Gram Negative ◽  
Content Type ◽  
Locking Mechanism

ABSTRACT Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-β-lactamases (MBLs) target the most widely used antibiotic class, the β-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-β-lactamase inhibitors, essential in the fight against antibiotic resistance.

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