scholarly journals Host-Derived Metabolites Modulate Transcription ofSalmonellaGenes Involved in L-Lactate Utilization during Gut Colonization

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Caroline C. Gillis ◽  
Maria G. Winter ◽  
Rachael B. Chanin ◽  
Wenhan Zhu ◽  
Luisella Spiga ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Regulatory Protein ◽  
Transcriptional Level ◽  
Lactate Oxidation ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Gut Colonization ◽  
Serovar Typhimurium ◽  
Lactate Utilization

ABSTRACTDuringSalmonella entericaserovar Typhimurium infection, host inflammation alters the metabolic environment of the gut lumen to favor the outgrowth of the pathogen at the expense of the microbiota. Inflammation-driven changes in host cell metabolism lead to the release ofl-lactate and molecular oxygen from the tissue into the gut lumen.Salmonellautilizes lactate as an electron donor in conjunction with oxygen as the terminal electron acceptor to support gut colonization. Here, we investigated transcriptional regulation of the respiratoryl-lactate dehydrogenase LldDin vitroand in mouse models ofSalmonellainfection. The two-component system ArcAB repressed transcription ofl-lactate utilization genes under anaerobic conditionsin vitro. The ArcAB-mediated repression oflldDtranscription was relieved under microaerobic conditions. Transcription oflldDwas induced byl-lactate but notd-lactate. A mutant lacking the regulatory protein LldR failed to inducelldDtranscription in response tol-lactate. Furthermore, thelldRmutant exhibited reduced transcription ofl-lactate utilization genes and impaired fitness in murine models of infection. These data provide evidence that the host-derived metabolites oxygen andl-lactate serve as cues forSalmonellato regulate lactate oxidation metabolism on a transcriptional level.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge

2016 ◽  
Vol 60 (4) ◽  
pp. 2052-2062 ◽  
Author(s):  
Ky V. Hoang ◽  
Heather Curry ◽  
Michael A. Collier ◽  
Hassan Borteh ◽  
Eric M. Bachelder ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Lethal Challenge ◽  
Content Type ◽  
Human Macrophages ◽  
Gentamicin Treatment ◽  
Significant Toxicity ◽  
Serovar Typhimurium ◽  
Spectrum Activity

ABSTRACTFrancisella tularensiscauses tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activityin vitroagainstSalmonella entericaserovar Typhimurium andF. tularensis. We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin vivo. No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

scholarly journals Helicobacter pylori FlhA Binds the Sensor Kinase and Flagellar Gene Regulatory Protein FlgS with High Affinity

2015 ◽  
Vol 197 (11) ◽  
pp. 1886-1892 ◽  
Author(s):  
Jennifer Tsang ◽  
Takanori Hirano ◽  
Timothy R. Hoover ◽  
Jonathan L. McMurry
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Kinase Domain ◽  
Optical Biosensing ◽  
Content Type ◽  
High Affinity ◽  
Flagellar Assembly ◽  
Flagellar Biogenesis

ABSTRACTFlagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. InHelicobacter pylori, transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoN-dependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Deletion of its N-terminal cytoplasmic sequence dramatically decreased expression of two RpoN-dependent genes,flaBandflgE. Optical biosensing demonstrated a high-affinity interaction between FlgS and a peptide consisting of residues 1 to 25 of FlhA (FlhANT). TheKD(equilibrium dissociation constant) was 21 nM and was characterized by fast-on (kon= 2.9 × 104M−1s−1) and slow-off (koff= 6.2 × 10−4s−1) kinetics. FlgS did not bind peptides consisting of smaller fragments of the FlhANTsequence. Analysis of binding to purified fragments of FlgS demonstrated that the C-terminal portion of the protein containing the kinase domain binds FlhANT. FlhANTbinding did not stimulate FlgS autophosphorylationin vitro, suggesting that FlhA facilitates interactions between FlgS and other structures required to stimulate autophosphorylation.IMPORTANCEThe high-affinity binding of FlgS to FlhA characterized in this study points to an additional role for FlhA in flagellar assembly. Beyond its necessity for type III secretion, the N-terminal cytoplasmic sequence of FlhA is required for RpoN-dependent gene expression via interaction with the C-terminal kinase domain of FlgS.

scholarly journals Identification and Regulation of a Novel Citrobacter rodentium Gut Colonization Fimbria (Gcf)

2015 ◽  
Vol 197 (8) ◽  
pp. 1478-1491 ◽  
Author(s):  
Gustavo G. Caballero-Flores ◽  
Matthew A. Croxen ◽  
Verónica I. Martínez-Santos ◽  
B. Brett Finlay ◽  
José L. Puente
Keyword(s):  
Escherichia Coli ◽  
Intestinal Epithelium ◽  
Pathogenic Bacteria ◽  
Critical Role ◽  
Growth Conditions ◽  
Citrobacter Rodentium ◽  
Content Type ◽  
Gut Colonization ◽  
Successful Infection

ABSTRACTThe Gram-negative enteric bacteriumCitrobacter rodentiumis a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagicEscherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. ForC. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novelC. rodentiumcolonization factor, calledgutcolonizationfimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. AgcfAmutant shows a severe colonization defect within the first 10 days of infection. Thegcfpromoter is not active inC. rodentiumunder severalin vitrogrowth conditions; however, it is readily expressed in aC. rodentiumΔhns1mutant lacking the closest ortholog of theEscherichia colihistone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region ofgcf, further supporting its direct role as a repressor of thegcfpromoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. Thegcfoperon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures.IMPORTANCEFimbriae are surface bacterial structures implicated in a variety of biological processes. Some have been shown to play a critical role during host colonization and thus in disease. Pathogenic bacteria possess the genetic information for an assortment of fimbriae, but their function and regulation and the interplay between them have not been studied in detail. This work provides new insights into the function and regulation of a novel fimbria called Gcf that is important for early establishment of a successful infection byC. rodentiumin mice, despite being poorly expressed underin vitrogrowth conditions. This discovery offers an opportunity to better understand the individual role and the regulatory mechanisms controlling the expression of specific fimbrial operons that are critical during infection.

scholarly journals Mechanistic Basis of pH-Dependent 5-Flucytosine Resistance inAspergillus fumigatus

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Fabio Gsaller ◽  
Takanori Furukawa ◽  
Paul D. Carr ◽  
Bharat Rash ◽  
Christoph Jöchl ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Fungal Pathogens ◽  
Regulatory Protein ◽  
Transcriptional Regulators ◽  
Primary Factor ◽  
Ph Sensing ◽  
Content Type ◽  
Ph Dependent ◽  
Mechanistic Basis

ABSTRACTThe antifungal drug 5-flucytosine (5FC), a derivative of the nucleobase cytosine, is licensed for the treatment of fungal diseases; however, it is rarely used as a monotherapeutic to treatAspergillusinfection. Despite being potent against other fungal pathogens, 5FC has limited activity againstAspergillus fumigatuswhen standardin vitroassays are used to determine susceptibility. However, in modifiedin vitroassays where the pH is set to pH 5, the activity of 5FC increases significantly. Here we provide evidence thatfcyB, a gene that encodes a purine-cytosine permease orthologous to known 5FC importers, is downregulated at pH 7 and is the primary factor responsible for the low efficacy of 5FC at pH 7. We also uncover two transcriptional regulators that are responsible for the repression offcyBand, consequently, mediators of 5FC resistance, the CCAAT binding complex (CBC) and the pH regulatory protein PacC. We propose that the activity of 5FC might be enhanced by the perturbation of factors that repressfcyBexpression, such as PacC or other components of the pH-sensing machinery.

scholarly journals Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines againstStreptococcus pneumoniaeandSalmonella entericaSerovar Typhi

2018 ◽  
Vol 86 (9) ◽  
Author(s):  
Vivek Belde ◽  
Matthew P. Cravens ◽  
Dania Gulandijany ◽  
Justin A. Walker ◽  
Isabel Palomo-Caturla ◽  
...  
Keyword(s):  
Antibody Response ◽  
B Cell ◽  
Salmonella Enterica ◽  
Passive Immunization ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Human Infants ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTB cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) ofSalmonella entericaserovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/−and TdT−/−mice generated comparable antibody responses to Pneumovax23 and survivedStreptococcus pneumoniaechallenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/−or TdT−/−mice conferred protection. TdT+/−and TdT−/−mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity againstS. Typhiin vitro. To test the protective immunity conferred by ViPS immunizationin vivo, TdT+/−and TdT−/−mice were challenged with a chimericSalmonella entericaserovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts forS. Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/−and TdT−/−mice challenged with ViPS-expressingS. Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

scholarly journals Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

2014 ◽  
Vol 80 (18) ◽  
pp. 5854-5865 ◽  
Author(s):  
Maria H. Daleke-Schermerhorn ◽  
Tristan Felix ◽  
Zora Soprova ◽  
Corinne M. ten Hagen-Jongman ◽  
David Vikström ◽  
...  
Keyword(s):  
Immune Responses ◽  
Outer Membrane ◽  
Vaccine Development ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Heterologous Proteins ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Heterologous Antigens

ABSTRACTOuter membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of theMycobacterium tuberculosisantigens ESAT6, Ag85B, and Rv2660c were targeted to the surface ofEscherichia coliOMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolRΔtolAderivative of attenuatedSalmonella entericaserovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by theM. tuberculosisantigens and epitopes fromChlamydia trachomatismajor outer membrane protein (MOMP). Also, we showed that delivery ofSalmonellaOMVs displaying Ag85B to antigen-presenting cellsin vitroresults in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

scholarly journals Protective Role of Akt2 in Salmonella enterica Serovar Typhimurium-Induced Gastroenterocolitis

2011 ◽  
Vol 79 (7) ◽  
pp. 2554-2566 ◽  
Author(s):  
Winnie W. S. Kum ◽  
Bernard C. Lo ◽  
Hong B. Yu ◽  
B. Brett Finlay
Keyword(s):  
Salmonella Enterica ◽  
Annexin V ◽  
Immunohistochemical Analysis ◽  
Neutrophil Infiltration ◽  
Protective Role ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTTheSalmonellaeffector protein SopB has previously been shown to induce activation of Akt and protect epithelial cells from apoptosisin vitro. To characterize the role of Akt2 in host defense againstSalmonella entericaserovar Typhimurium infection, wild-type (WT) mice and mice lacking Akt2 (Akt2 knockout [KO] mice) were infected using aSalmonellaacute gastroenteritis model. Infected Akt2 KO mice showed a more pronounced morbidity and mortality associated with higher bacterial loads in the intestines and elevated levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and MCP-1, in the colons at 1 day postinfection compared to those shown in WT mice. Histopathological assessment and immunohistochemical analysis of cecal sections at 1 day postinfection revealed more severe inflammation and higher levels of neutrophil infiltration in the ceca of Akt2 KO mice. Flow cytometry analysis further confirmed an increase in the recruitment of Gr-1+CD11b+neutrophils and F4/80+CD11b+macrophages in the intestines of infected Akt2 KO mice. Additionally, enhanced levels of annexin V+and terminal transferase dUTP nick end labeling-positive (TUNEL+) apoptotic cells in the intestines of infected Akt2 KO mice were also observed, indicating that Akt2 plays an essential role in protection against apoptosis. Finally, the differences in bacterial loads and cecal inflammation in WT and Akt2 KO mice infected with WTSalmonellawere abolished when these mice were infected with thesopBdeletion mutant, indicating that SopB may play a role in protecting the mice fromSalmonellainfection through the activation of Akt2. These data demonstrate a definitive phenotypic abnormality in the innate response in mice lacking Akt2, underscoring the important protective role of Akt2 inSalmonellainfection.

scholarly journals Salmonella enterica Infection of Murine and Human Enteroid-Derived Monolayers Elicits Differential Activation of Epithelium-Intrinsic Inflammasomes

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Mayumi K. Holly ◽  
Xiao Han ◽  
Edward J. Zhao ◽  
Shauna M. Crowley ◽  
Joannie M. Allaire ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Bacterial Infections ◽  
Intestinal Epithelial ◽  
Major Pathway ◽  
Content Type ◽  
Caspase 1 ◽  
Serovar Typhimurium ◽  
Multiple Species ◽  
Inflammatory Caspases

ABSTRACT Recent studies have determined that inflammasome signaling plays an important role in driving intestinal epithelial cell (IEC) responses to bacterial infections, such as Salmonella enterica serovar Typhimurium. There are two primary inflammasome pathways, canonical (involving caspase-1) and noncanonical (involving caspase-4 and -5 in humans and caspase-11 in mice). Prior studies identified the canonical inflammasome as the major pathway leading to interleukin-18 (IL-18) release and restriction of S. Typhimurium replication in the mouse cecum. In contrast, the human C2Bbe1 colorectal carcinoma cell line expresses little caspase-1 but instead utilizes caspase-4 to respond to S. Typhimurium infection. Intestinal enteroid culture has enabled long-term propagation of untransformed IECs from multiple species, including mouse and human. Capitalizing on this technology, we used a genetic approach to directly compare the relative importance of different inflammatory caspases in untransformed mouse and human IECs and transformed human IECs upon S. Typhimurium infection in vitro. We show that caspase-1 is important for restricting intracellular S. Typhimurium replication and initiating IL-18 secretion in mouse IECs but is dispensable in human IECs. In contrast, restriction of intracellular S. Typhimurium and production of IL-18 are dependent on caspase-4 in both transformed and untransformed human IECs. Notably, cytosolic replication in untransformed cells from both species was less pronounced than in transformed human cells, suggesting that transformation may impact additional pathways that restrict S. Typhimurium replication. Taken together, these data highlight the differences between mouse and human IECs and the utility of studying transformed and untransformed cells in parallel.

scholarly journals NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

2015 ◽  
Vol 197 (13) ◽  
pp. 2239-2247 ◽  
Author(s):  
Chao Gao ◽  
Yujiao Wang ◽  
Yingxin Zhang ◽  
Min Lv ◽  
Peipei Dou ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Gene Cluster ◽  
Pseudomonas Stutzeri ◽  
Lactate Metabolism ◽  
Lactate Oxidation ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Lactate Utilization

ABSTRACTNAD-independentl-lactate dehydrogenases (l-iLDHs) play important roles inl-lactate utilization of different organisms. All of the previously reportedl-iLDHs were flavoproteins that catalyze the oxidation ofl-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA,lldB, andlldC) encoding a novel type ofl-iLDH was identified inPseudomonas stutzeriA1501. When the gene cluster was expressed inEscherichia coli, distinctivel-iLDH activity was detected. The expressedl-iLDH was purified by ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography. SDS-PAGE and successive matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis of the purifiedl-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded bylldA,lldB, andlldC, respectively). Purifiedl-iLDH (LldABC) is a dimer of three subunits (LldA, LldB, and LldC), and the ratio between LldA, LldB, and LldC is 1:1:1. Different from the FMN-containingl-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for thel-lactate oxidation. LldABC has narrow substrate specificity, and onlyl-lactate anddl-2-hydrobutyrate were rapidly oxidized. Mg2+could activatel-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for thel-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for thel-lactate metabolism ofP. stutzeriA1501. LldABC is the first purified and characterizedl-iLDH with different subunits that uses the iron-sulfur cluster as the cofactor.IMPORTANCEProviding new insights into the diversity of microbial lactate utilization could assist in the production of valuable chemicals and understanding microbial pathogenesis. An NAD-independentl-lactate dehydrogenase (l-iLDH) encoded by the gene clusterlldABCis indispensable for thel-lactate metabolism inPseudomonas stutzeriA1501. This novel type of enzyme was purified and characterized in this study. Different from the well-characterized FMN-containingl-iLDH in other microbes, LldABC inP. stutzeriA1501 is a dimer of three subunits (LldA, LldB, and LldC) and uses the iron-sulfur cluster as a cofactor.

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