Nucleotide-induced ClpC oligomerization and its non-preferential association with ClpP isoforms of pathogenic Leptospira.

Bacterial caseinolytic protease-chaperone complexes participate in the elimination of misfolded and aggregated protein substrates. The spirochete Leptospira interrogans possess a set of Clp-chaperones (ClpX, ClpA, and ClpC), which may associate functionally with two different isoforms of LinClpP (ClpP1 and ClpP2). The L. interrogans ClpC (LinClpC) belongs to class-I chaperone with two active ATPase domains separated by a middle domain. Using the size exclusion chromatography, ANS dye binding, and dynamic light scattering analysis, the LinClpC is suggested to undergo nucleotide-induced oligomerization. LinClpC associates with either pure LinClpP1 or LinClpP2 isoforms non-preferentially and with equal affinity. Regardless, pure LinClpP isoforms cannot constitute an active protease complex with LinClpC. Interestingly, the heterocomplex LinClpP1P2 in association with LinClpC forms a functional proteolytic machinery and degrade ß-casein or FITC-casein in an energy-independent manner. Adding either ATP or ATPγS further fosters the LinClpCP1P2 complex protease activity by nurturing the functional oligomerization of LinClpC. The antibiotic, acyldepsipeptides (ADEP1) display a higher activatory role on LinClpP1P2 protease activity than LinClpC. Altogether, this work illustrates an in-depth study of hetero-tetradecamer LinClpP1P2 association with its cognate ATPase and unveils a new insight into the structural reorganization of LinClpP1P2 in the presence of chaperone, LinClpC to gain protease activity.

Lipopolysaccharide and Glycolipoprotein Coordinately Triggered Necroptosis Contributes to the Pathogenesis of Leptospira Infection in Mice.

Leptospirosis is a recurring but neglected zoonotic disease caused by pathogenic Leptospira. The explicit underlying mechanism of necroptosis and its role in Leptospira infection have not yet been elucidated. Here we reported that leptospiral pathogen-associated molecular patterns, lipopolysaccharide, and glycolipoprotein activate the necroptotic RIPK1-RIPK3-MLKL cascade through the TLR4 signaling pathway in mouse macrophages. Using the murine acute leptospirosis model, we reveal that abolition of necroptosis exhibited significantly improved outcomes in acute phases, with enhanced eradication of Leptospira from liver, mild clinical symptoms, and decreased cytokine production. RIPK3 was also found to exert a necroptosis-independent function in CXCL1 production and neutrophil recruitment, with the consequence of improved Leptospira control. These findings improve our understanding of the mechanism of Leptospira-macrophage interactions, indicating potential therapeutic values by targeting necroptosis signaling pathways.

Leptospirosis kidney disease: Evolution from acute to chronic kidney disease.

Leptospirosis is a neglected bacterial disease caused by leptospiral infection that carries a substantial mortality risk in severe cases. Research has shown that acute, chronic, and asymptomatic leptospiral infections are closely linked to acute and chronic kidney disease (CKD) and renal fibrosis. Leptospires affect renal function by infiltrating kidney cells via the renal tubules and interstitium and surviving in the kidney by circumventing the immune system. The most well-known pathogenic molecular mechanism of renal tubular damage caused by leptospiral infection is the direct binding of the bacterial outer membrane protein LipL32 to toll-like receptor-2 expressed in renal tubular epithelial cells (TECs) to induce intracellular inflammatory signaling pathways. These pathways include the production of tumor necrosis factor (TNF)-α and nuclear factor kappa activation, resulting in acute and chronic leptospirosis-related kidney injury. Few studies have investigated the relationship between acute and chronic renal diseases and leptospirosis and further evidence is necessary. In this review, we intend to discuss the roles of acute kidney injury (AKI) to/on CKD in leptospirosis. This study reviews the molecular pathways underlying the pathogenesis of leptospirosis kidney disease, which will assist in concentrating on potential future research directions.

Leptospira and Leptospirosis: New Systems Science Insights on Proteome, Posttranslational Modifications, and Pathogen-Host Interaction.

Leptospirosis is one of the most important zoonotic diseases for planetary health. It is caused by Leptospira spp., which poses a formidable challenge in both rural and urban geographies. Discovery of molecular targets is crucial for developing interventions, including vaccines, against leptospirosis. We report here novel systems science insights on Leptospira proteome, posttranslational modifications (PTMs), and pathogen-host interactions, with an eye to bacterial pathophysiology from a functional standpoint. A systematic reanalysis of unassigned spectra from our previous total proteome identification was used for a multi-PTM search. Notably, we identified 3693 unique high-confidence PTM sites corresponding to 1266 proteins (PTM-profiling probability cutoff value ≥75%). The majority of the phosphorylated peptides were found to be GroEL molecular chaperones. Notably, the molecular docking of PTM-GroEL with STAT3, an important signaling protein in cytokine production, resulted in the prediction of druggable "hotspots." These energetically significant smaller subsets of amino acids (hotspot residues) offer promise for practical applications in planetary health, rational drug design, and peptide engineering. Furthermore, the prediction strategies described here could serve as a starting point for narrowing down the more extensive interface in protein-protein interactions that currently exist. Going forward, systems science approaches and the new insights reported here offer veritable prospects for innovation in preventing and treating leptospirosis.

Norfloxacin suppresses Leptospira-induced inflammation through inhibiting p65 and ERK phosphorylation and NLRP3 inflammasome activation.

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira. Inflammatory storms induced by Leptospira are the reason to induce immunoparalysis and organ failures. Antibiotics are still the current mainstream treatment for leptospirosis. In addition to their antibacterial action, the immunomodulatory function of antibiotics has been paid more and more attention. In this study, the role of norfloxacin on Leptospira-induced inflammation was investigated. Treatment with norfloxacin down-regulated Leptospira-induced IL-1ß and TNF-α both in vivo and vitro models. Further study showed that norfloxacin inhibited Leptospira-induced phosphorylation of p65 and ERK. Norfloxacin also inhibited the Leptospira-induced NLRP3 inflammasome activation with the increased level of Na/K-ATPase Pump ß1 subunit and decreased level of Kcnk6. These results indicated that norfloxacin suppressed Leptospira-induced inflammation through inhibiting p65 and ERK phosphorylation and NLRP3 inflammasome activation. Norfloxacin may be a potential candidate for suppressing inflammatory storms caused by Leptospira.

Crosstalk between E-Cadherin/ß-Catenin and NF-κB Signaling Pathways: The Regulation of Host-Pathogen Interaction during Leptospirosis.

Approximately 1 million cases of leptospirosis, an emerging infectious zoonotic disease, are reported each year. Pathogenic Leptospira species express leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic Leptospira species. The LRR domain-containing protein family is vital for the virulence of pathogenic Leptospira species. In this study, the biological mechanisms of an essential LRR domain protein from pathogenic Leptospira were examined. The effects of Leptospira and recombinant LRR20 (rLRR20) on the expression levels of factors involved in signal transduction were examined using microarray, quantitative real-time polymerase chain reaction, and western blotting. The secreted biomarkers were measured using an enzyme-linked immunosorbent assay. rLRR20 colocalized with E-cadherin on the cell surface and activated the downstream transcription factor ß-catenin, which subsequently promoted the expression of MMP7, a kidney injury biomarker. Additionally, MMP7 inhibitors were used to demonstrate that the secreted MMP7 degrades surface E-cadherin. This feedback inhibition mechanism downregulated surface E-cadherin expression and inhibited the colonization of Leptospira. The degradation of surface E-cadherin activated the NF-κB signal transduction pathway. Leptospirosis-associated acute kidney injury is associated with the secretion of NGAL, a downstream upregulated biomarker of the NF-κB signal transduction pathway. A working model was proposed to illustrate the crosstalk between E-cadherin/ß-catenin and NF-κB signal transduction pathways during Leptospira infection. Thus, rLRR20 of Leptospira induces kidney injury in host cells and inhibits the adhesion and invasion of Leptospira through the upregulation of MMP7 and NGAL.

GroEL protein of the Leptospira spp. interacts with host proteins and induces cytokines secretion on macrophages.

BACKGROUND: Leptospirosis is a zoonotic disease caused by infection with spirochetes from Leptospira genus. It has been classified into at least 17 pathogenic species, with more than 250 serologic variants. This wide distribution may be a result of leptospiral ability to colonize the renal tubules of mammalian hosts, including humans, wildlife, and many domesticated animals. Previous studies showed that the expression of proteins belonging to the microbial heat shock protein (HSP) family is upregulated during infection and also during various stress stimuli. Several proteins of this family are known to have important roles in the infectious processes in other bacteria, but the role of HSPs in Leptospira spp. is poorly understood. In this study, we have evaluated the capacity of the protein GroEL, a member of HSP family, of interacting with host proteins and of stimulating the production of cytokines by macrophages. RESULTS: The binding experiments demonstrated that the recombinant GroEL protein showed interaction with several host components in a dose-dependent manner. It was also observed that GroEL is a surface protein, and it is secreted extracellularly. Moreover, two cytokines (tumor necrosis factor-α and interleukin-6) were produced when macrophages cells were stimulated with this protein. CONCLUSIONS: Our findings showed that GroEL protein may contribute to the adhesion of leptospires to host tissues and stimulate the production of proinflammatory cytokines during infection. These features might indicate an important role of GroEL in the pathogen-host interaction in the leptospirosis.

Deciphering the Role of Leptospira Surface Protein LigA in Modulating the Host Innate Immune Response.

Leptospira, a zoonotic pathogen, is known to infect various hosts and can establish persistent infection. This remarkable ability of bacteria is attributed to its potential to modulate (activate or evade) the host immune response by exploiting its surface proteins. We have identified and characterized the domain of the variable region of Leptospira immunoglobulin-like protein A (LAV) involved in immune modulation. The 11th domain (A11) of the variable region of LigA (LAV) induces a strong TLR4 dependent innate response leading to subsequent induction of humoral and cellular immune responses in mice. A11 is also involved in acquiring complement regulator FH and binds to host protease Plasminogen (PLG), there by mediating functional activity to escape from complement-mediated killing. The deletion of A11 domain significantly impaired TLR4 signaling and subsequent reduction in the innate and adaptive immune response. It also inhibited the binding of FH and PLG thereby mediating killing of bacteria. Our study discovered an unprecedented role of LAV as a nuclease capable of degrading Neutrophil Extracellular Traps (NETs). This nuclease activity was primarily mediated by A11. These results highlighted the moonlighting function of LigA and demonstrated that a single domain of a surface protein is involved in modulating the host innate immune defenses, which might allow the persistence of Leptospira in different hosts for a long term without clearance.

Enhanced early immune response of leptospiral outer membrane protein LipL32 stimulated by narrow band mid-infrared exposure.

Previous studies revealed significant impact on cancer cell by mid-infrared (MIR) radiation. However, the effects of narrow band MIR on immune reaction and infectious disease are still unknown. In this study, an enhanced innate immune response was observed through the interaction between Leptospiral outer membrane protein (LipL32) and toll-like receptor 2 (TLR2). Thereafter, human kidney proximal tubular cells (HK-2 cells) initiated a serial reaction of enhanced MCP-1 production. The 6 µm narrow bandwidth light source emitted by waveguide thermal emitter (WTE) was applied to induce carbonyl group (CO bond) stretching vibration during the stage of antigen-receptor complex formation. The amount of MCP-1 gene expression had 2.5 folds increase after narrow band MIR illumination comparing to non-MIR illumination at low dose LipL32 condition. Besides, both ELISA and confocal microscopy results also revealed that the chemokine concentration increased significantly after narrow band MIR illumination either at low or high concentration of LipL32. Furthermore, a specific phenomenon that narrow band MIR can amplify the signal of weak immune response by enhancing sensitivity of the interaction between antigen and receptor was observed. This study exhibits clear evidence that the narrow band MIR exposure can modulate the early immune response of infectious disease and play a potential role to develop host-directed therapy in the future.

Leptospiral phosphatidylinositol-specific phospholipase C: a novel conserved antigen for the definitive diagnosis of Leptospirosis.

Identification and characterisation of novel antigenic proteins of Leptospira are necessary to unravel the secrets of the Leptospiral disease biology. Besides, the current scenario of Leptospirosis demands the development of advanced diagnostics with the potential of an early and specific diagnosis. This work reports the diagnostic efficacy of a PI-PLC (LA_1375) of Leptospira interrogans serovar Lai strain 56601. Phospholipases are enzymes that catalyse the degradation of membrane phospholipids, with the after effects of pathophysiological consequences through the impairment of cellular signalling mechanisms. The immunoblotting analysis allows to identify the Leptospirosis positive serum samples demonstrated the expression and exposure of the protein to the immune system and confirmed the antigenic nature of the protein. Furthermore, evaluation of diagnostic efficacy of an ELISA to detect antibodies against recombinant PI-PLC has tested in indirect IgM ELISA using the purified 50 kDa recombinant protein as the coating antigen. A cut-off value of 0.218 at mean + 2SD and a sensitivity value of 100% and a specificity value of 95.65% explored for the rPI-PLC IgM ELISA. The PPV and NPV of ELISA for the Leptospirosis confirmed acute phase samples were 95.87% and 100% respectively. Comparison of the ELISA assay with MAT for the detection of Leptospiral antibodies showed a positive correlation (r value 0.345, p < 0.05), and signified the diagnostic efficacy of rPI-PLC for the definitive diagnosis of the Leptospirosis. In addition, the revealed conservation nature of PI-PLC among different Leptospira species highlights the diagnostic and vaccine candidate potential. Thus, the study emphasises the Leptospiral PI-PLC as a potential antigen still the importance as a novel biomarker candidate for the serodiagnosis and pathogenesis of Leptospirosis have to be investigated.

Leptospiral flagellar sheath protein FcpA interacts with FlaA2 and FlaB1 in Leptospira biflexa.

Leptospira spp. are spirochete bacteria that possess periplasmic flagella (PFs) underneath the outer membrane; each flagellum is attached to each end of the protoplasmic cylinder. PFs of Leptospira have a coiled shape that bends the end of the cell body. However, the molecular mechanism by which multiple flagellar proteins organize to form the distinctively curled PF of Leptospira remains unclear. Here we obtained a slow-motility mutant of L. biflexa MD4-3 by random insertion mutagenesis using a Himar1 transposon. In MD4-3, the gene encoding the flagellar sheath protein, flagellar-coiling protein A (FcpA), which was recently identified in L. interrogans, was inactivated. As with L. interrogans ΔfcpA strains, the L. biflexa ΔfcpA strain lacked a distinct curvature at both ends of the cell body, and its motility was significantly reduced as compared with that of the wild-type strain. PFs isolated from the ΔfcpA strain were straight and were thinner than those isolated from the wild-type strain. Western blot analysis revealed that flagellar proteins FlaA1, FlaA2, FlaB1, and FlaB2 were expressed in the ΔfcpA strain but the flagellar proteins, except for FlaB2 were not incorporated in its PFs. Immunoprecipitation assay using anti-FcpA antiserum demonstrated that FcpA associates with FlaA2 and FlaB1. The association between FcpA and FlaA2 was also verified using pull-down assay. The regions of FlaA2 and FlaB1 interacting with FcpA were determined using a bacterial two-hybrid assay. These results suggest that FcpA together with FlaA2, produces coiling of PF of the Leptospira, and the interaction between the sheath and core filament may be mediated by FcpA and FlaB1.

WC1 is a hybrid γδ TCR coreceptor and pattern recognition receptor for pathogenic bacteria.

WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. While present in variable, and sometimes high, numbers in the genomes of mammals and birds, in cattle there are 13 distinct genes (WC1-1 to WC1-13). All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation dependent manner, and some are required for the γδ T cell response to Leptospira. We hypothesized that individual WC1 receptors encode Ag specificity via coligation of bacteria with the γδ TCR. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species, L. borgpetersenii, and L. interrogans. Three to five of eleven WC1-3 SRCR domains, but none of the eleven WC1-4 SRCR domains, interacted with Leptospira spp. and Borrelia burgdorferi, but not with Escherichia coli or Staphylococcus aureus. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is composed of amino acids in three discontinuous regions. Recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. Our data suggest that WC1 gene arrays play a multifaceted role in the γδ T cell response to bacteria, including acting as hybrid pattern recognition receptors and TCR coreceptors, and they may function as antimicrobials.

Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway.

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif ((161)DDDDDGDD(168)). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp(132), Thr(133), Asp(164), Asp(165), and Tyr(178). The goals of this study were to determine possible roles of the Ca(2+)-binding cluster for the interaction of LipL32 and Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca(2+)-binding residues of LipL32 to Ala, and their effects subsequently were observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells.

Downregulation of the Na/K-ATPase pump by leptospiral glycolipoprotein activates the NLRP3 inflammasome.

Leptospira interrogans is responsible for a zoonotic disease known to induce severe kidney dysfunction and inflammation. In this work, we demonstrate that L. interrogans induces NLRP3 inflammasome-dependent secretion of IL-1ß through the alteration of potassium transport in bone marrow-derived macrophages. Lysosome destabilization also contributed to the IL-1ß production upon stimulation with live, but not dead, bacteria. Using bone marrow-derived macrophages from various TLRs and nucleotide-binding oligomerization domain-deficient mice, we further determined that IL-1ß production was dependent on TLR2 and TLR4, suggesting a participation of the leptospiral LPS to this process. Hypokaliemia in leptospirosis has been linked to the presence of glycolipoprotein, a cell wall component of L. interrogans that is known to inhibit the expression and functions of the Na/K-ATPase pump. We show in this study that glycolipoprotein activates the inflammasome and synergizes with leptospiral LPS to produce IL-1ß, mimicking the effect of whole bacteria. These results were confirmed in vivo, as wild-type mice expressed more IL-1ß in the kidney than TLR2/4-deficient mice 3 d postinfection with L. interrogans. Collectively, these findings provide the first characterization, to our knowledge, of bacteria-induced activation of the NLRP3 inflammasome through the downregulation of a specific host potassium transporter.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Leptospira santorosai Serovar Shermani detergent extract induces an increase in fibronectin production through a Toll-like receptor 2-mediated pathway.

Leptospirosis can activate inflammatory responses through Toll-like receptors (TLRs) and may cause renal tubulointerstitial fibrosis characterized by the accumulation of extracellular matrix (ECM). We have previously demonstrated that Leptospira santorosai serovar Shermani detergent extract stimulates ECM accumulation in vitro. The aim of this study was to examine the mechanistic basis of these previous observations and, in particular, to examine the potential involvement of TLRs. The addition of serovar Shermani detergent extract led to an increase in fibronectin gene expression and production. Inhibition of TLR2 but not TLR4 expression abrogated serovar Shermani detergent extract-mediated increases in fibronectin production. This response was also blocked by the knockdown of the gene expression of the TLR2 downstream transducers myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). Serovar Shermani detergent extract also activated nuclear factor-κB, and its inhibition by curcumin-attenuated serovar Shermani detergent extract induced increases in fibronectin production. These effects were also mimicked by the specific TLR2 agonist, Pam(3)CsK(4), a response that was also abrogated by the knockdown of MyD88 and TRAF6. Similarly, the administration of live leptospires to cells also induced fibronectin production that was blocked by inhibition of TLR2 and MyD88 expression. In conclusion, serovar Shermani detergent extract can induce fibronectin production through the TLR2-associated cascade, providing evidence of an association between TLRs and leptospirosis-mediated ECM deposition.

Protein identification false discovery rates for very large proteomics data sets generated by tandem mass spectrometry.

Comprehensive characterization of a proteome is a fundamental goal in proteomics. To achieve saturation coverage of a proteome or specific subproteome via tandem mass spectrometric identification of tryptic protein sample digests, proteomics data sets are growing dramatically in size and heterogeneity. The trend toward very large integrated data sets poses so far unsolved challenges to control the uncertainty of protein identifications going beyond well established confidence measures for peptide-spectrum matches. We present MAYU, a novel strategy that reliably estimates false discovery rates for protein identifications in large scale data sets. We validated and applied MAYU using various large proteomics data sets. The data show that the size of the data set has an important and previously underestimated impact on the reliability of protein identifications. We particularly found that protein false discovery rates are significantly elevated compared with those of peptide-spectrum matches. The function provided by MAYU is critical to control the quality of proteome data repositories and thereby to enhance any study relying on these data sources. The MAYU software is available as standalone software and also integrated into the Trans-Proteomic Pipeline.

[Expression and identification of gene Loa22 from the virulent strain of serovar Lai of L. interrogans and its cytotoxicity to macrophages].

OBJECTIVE: To study the function of Loa22 gene from virulent serovar Lai. L. interrogans by expressing its protein. METHODS: The recombinant plasmid with Loa22 was constructed and its expression was induced by IPTG. The expression product was identified by SDS-PAGE and Western Blotting and purified with GST-affinity chromatography. The purified protein was applied to mice macrophages ANA-1 cells purified by GST-affinity column and exerted to ANA-1 cells. The cytotoxicity of purified protein to ANA-1 was evaluated by detecting LDH activity in culture medium, XTT aborbtion and apoptosis ration. RESULTS: The recombinant plasmid with Loa22 mature peptide was constructed successfully and the protein was purified subsequently. Significant higher level of LDH activity and apoptosis ratio and lower XTT absorption were observed by comparison of expression protein treated cells and those untreated. CONCLUSION: Loa22 had a toxic effect on ANA-1 and the gene Loa22 might be a virulent-related gene of L. interrogans.

Thermodynamic and kinetic characterization using process dynamics: acidophilic ferrous iron oxidation by Leptospirillum ferrooxidans.

Kinetic and stoichiometric properties of acidophilic aerobic ferrous iron oxidation by growing and non-growing Leptospirillum ferrooxidans cultures were investigated. The use of a continuous stirred tank reactor operated at a variable dilution rate and equipped with on-line measurement of the electron donor, acceptor and anabolic substrate uptake rate enabled detailed kinetic characterization from a single experiment. It was demonstrated that substrate conversion and microbial growth are tightly coupled processes in L. ferrooxidans, and uncoupling occurs only due to the minor impact of substrate conversion for growth-independent maintenance purposes. The tight stoichiometric coupling implies bioenergetic uncoupling of the catabolism and anabolism because the Gibbs energy change for ferrous iron oxidation as a function of the actual growth rate of the culture ranges from -45 to -25 kJ mol-FeII(-1). Bioenergetic description of the process could only be achieved by introduction of a growth rate dependent Gibbs energy dissipation term. Removal of carbon dioxide from the influent gas stopped biomass growth, but the biomass specific respiration rate was unaffected or slightly stimulated. The uncoupling of the catabolism and anabolism is suggested to induce instantaneously an energy dissipation pathway. Also dosage of a low concentration propionic acid resulted in complete inhibition of the anabolism. Propionic acid served as an uncoupler of the membrane potential and all catabolic energy is required for the increased maintenance requirements. Recovery of the anabolism after reestablishment of the normal cultivation conditions was obtained only after 1-2 days. The results obtained provide additional constraints on cultivation of L. ferrooxidans for biotechnological application.