Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase

Arnau, José; Jørgensen, Flemming; Madsen, Søren; Vrang, Astrid; Israelsen, Hans

doi:10.1128/jb.179.18.5884-5891.1997

Cited by 49 publications

(55 citation statements)

References 39 publications

(30 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The assay nevertheless made possible an estimation of the impact of oxygen on the levels of ADH and PFL, which has not been possible before because of the extreme difficulty in measuring this activity (Table 3). While the oxygen-related trend observed for PFL was expected (2,19), no actual correlation between controlled microaerobic conditions and intracellular PFL protein levels had been established. Furthermore, to our knowledge, no reports are available on the influence of small amounts of oxygen on in vitro protein levels of ADH.…”

Section: Fig 3 Synthesis Of Nadmentioning

confidence: 99%

“…Interest in the microaerobic physiology of LAB is furthermore spurred by reports on the extreme sensitivity of pyruvate formate-lyase (PFL) to oxygen (1, 25) and the negative effect of oxygen on pfl gene expression (2,19). Similarly, it has recently been reported that vigorous aeration reduces the transcription of the adhE gene, which encodes alcohol dehydrogenase (ADH) in Lactococcus lactis (3).…”

Section: ؊1mentioning

confidence: 99%

“…In this light, it is surprising that, to our knowledge, no investigations have been carried out on the microaerobic physiology of starter cultures. A number of studies on the effect of oxygen in fully aerated cultures have been published, but little light has been shed on the window ranging from anaerobic to fully aerobic conditions of growth (4,7,8,20).Interest in the microaerobic physiology of LAB is furthermore spurred by reports on the extreme sensitivity of pyruvate formate-lyase (PFL) to oxygen (1, 25) and the negative effect of oxygen on pfl gene expression (2,19). Similarly, it has recently been reported that vigorous aeration reduces the transcription of the adhE gene, which encodes alcohol dehydrogenase (ADH) in Lactococcus lactis (3).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Metabolic Behavior of Lactococcus lactis MG1363 in Microaerobic Continuous Cultivation at a Low Dilution Rate

Jensen

Melchiorsen

Jokumsen

et al. 2001

Appl Environ Microbiol

View full text Add to dashboard Cite

Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h ؊1. More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, ␣-acetolactate synthase, and the NADH oxidases. Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration.Homofermentative lactic acid bacteria (LAB) are used primarily in the dairy industry but may also prove to be advantageous hosts for production of lactate as a bulk chemical or of food additives such as bacteriocins or amino acids using recombinant DNA technology (10, 15). The metabolism of these bacteria is constrained by the requirement for a balance between NADH-producing and -consuming reactions. In the absence of external electron acceptors (e.g., oxygen), the carbon fluxes of catabolism are tightly coupled. In anaerobic culture, glucose may be redox-neutrally converted to lactate or, alternatively, to the mixed acid products formate, acetate, and ethanol in a molar ratio of 2:1:1 (12, 26). However, when oxygen is present in the growth medium, the catabolic carbon fluxes are uncoupled from the redox metabolism due to the NAD ϩ -regenerating activity of NADH oxidases (NOX). This oxygenrelated potential for redirecting fluxes is used industrially in connection with the formation of other metabolites such as diacetyl in fermented milk products. Even submillimolar concentrations of diacetyl have a strong impact on the taste of buttermilk and cheese (22). Hence, minute amounts of oxygen may influence an industrial process greatly. In this light, it is surprising that, to our knowledge, no investigations have been carried out on the microaerobic physiology of starter cultures. A number of studies on the effect of oxygen in fully aerated cultures have been published, but little light has been shed on the window ranging from anaerobic to fully aerobic conditions of growth (4,7,8,20).Interest in the microaerobic physiology of LAB is furthermore spurred by reports on the extreme sensitivity of pyruvate formate-lyase (PFL) to oxygen (1, 25) and the negative effect of oxygen on pfl gene expression (2,19). Similarly, it has recently been reported that vigorous aeration reduces the...

show abstract

Section: Fig 3 Synthesis Of Nadmentioning

confidence: 99%

Section: ؊1mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Metabolic Behavior of Lactococcus lactis MG1363 in Microaerobic Continuous Cultivation at a Low Dilution Rate

Jensen

Melchiorsen

Jokumsen

et al. 2001

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…While acetate formation generates an additional molecule of ATP, ethanol production from acetylCoA regenerates two molecules of NAD ϩ (34,40). Unlike in other lactic acid bacteria (2,56), in S. pneumoniae there are no genes for PDHC nor any pyruvate dehydrogenase activity in cell extracts (19,49,52), therefore, mixed-acid fermentation is expected to occur mainly by the action of PFL. The regulation of PFL activity in other bacteria has been reported to arise at three levels (27,46).…”

mentioning

confidence: 99%

Pyruvate Formate Lyase Is Required for Pneumococcal Fermentative Metabolism and Virulence

et al. 2009

View full text Add to dashboard Cite

Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.Streptococcus pneumoniae is the leading cause of pneumonia in children and adults, and it is a major cause of otitis media, meningitis, and septicemia (22). Despite considerable progress in pneumococcal vaccine development, the serotype specificity and the genomic plasticity of the pneumococcus will hamper its success (36). In addition, the increasing frequency of antibiotic resistance (14) makes it important to understand the mechanisms of pathogenesis of pneumococcal disease.Probably one of the most understudied fields of pneumococcal biology is that of how the pathogen generates its metabolic energy. The pneumococcus is strictly fermentative, and sugars are the major sources of energy for biosynthesis and growth (19,52). Therefore, in vivo fitness is determined to a large extent by the processes governed by sugar metabolism. Accumulating evidence suggests that there is a direct link between pneumococcal virulence and sugar metabolism (6,16,20,21,50). This is not limited to proteins involved in polysaccharide degradation, transport, and regulation (16, 31) but also includes those involved in redox metabolism, such as NADH oxidase (Nox) (6), and in pyruvate metabolism (42,50). For example, mutation of nox caused diminished virulence in a systemic infec...

show abstract

“…The EhADH2 enzyme is part of a newly described family of multifunctional enzymes found in Gram-negative and Grampositive bacteria and the intestinal protozoan parasite Giardia lamblia (2,(7)(8)(9)(10)(11)(12). EhADH2 and other members of the family appear to be composed of separate C-terminal ADH and Nterminal ALDH domains linked together to create a fusion enzyme (8).…”

mentioning

confidence: 99%

The Bifunctional Entamoeba histolytica Alcohol Dehydrogenase 2 (EhADH2) Protein Is Necessary for Amebic Growth and Survival and Requires an Intact C-terminal Domain for Both Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase Activity

Espinosa

Yan²,

Zhang³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required for E. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N-and C-terminal halves of the molecule.The anaerobic intestinal protozoan parasite Entamoeba histolytica converts pyruvate to ethanol in its fermentation pathway (1). The last two steps of this pathway are the conversion of acetyl-CoA to acetaldehyde followed by the reduction of acetaldehyde to ethanol (1). E. histolytica possesses at least three enzymes with alcohol dehydrogenase (ADH) 1 activity: a NADP-dependent ADH (EhADH1); a 97-kDa NAD(ϩ)-dependent and Fe 2ϩ -dependent bifunctional enzyme with both ADH and acetaldehyde dehydrogenase (ALDH) activities (EhADH2, also known as EhADHE); and a 43-kDa NADP-dependent ADH with some sequence homology to class III microbial alcohol dehydrogenases (EhADH3) (2-5). There are at least two enzymes with ALDH activity, the EhADH2 enzyme and a NADPdependent ALDH, EhALDH1 (2, 5, 6). Given the presence of multiple ADH and ALDH enzymes in E. histolytica, an important question is whether any of these enzymes are essential for E. histolytica growth and survival and thus potential targets for anti-amebic therapy.The EhADH2 enzyme is part of a newly described family of multifunctional enzymes found in Gram-negative and Grampositive bacteria and the intestinal protozoan parasite Giardia lamblia (2, 7-12). EhADH2 and other members of the family appear to be composed of separate C-terminal ADH and Nterminal ALDH domains linked together to create a fusion enzyme (8). The EhADH2 enzyme utilizes NAD and Fe 2ϩ as co-factors and does not demonstrate homology with the zincdependent ADH enzymes (13). Regions of EhADH2 that could be involved in iron binding and NAD binding have been identified, but a requirement for specific residues in enzymatic activity has not been demo...

show abstract

Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase

Cited by 49 publications

References 39 publications

Metabolic Behavior of Lactococcus lactis MG1363 in Microaerobic Continuous Cultivation at a Low Dilution Rate

Metabolic Behavior of Lactococcus lactis MG1363 in Microaerobic Continuous Cultivation at a Low Dilution Rate

Pyruvate Formate Lyase Is Required for Pneumococcal Fermentative Metabolism and Virulence

The Bifunctional Entamoeba histolytica Alcohol Dehydrogenase 2 (EhADH2) Protein Is Necessary for Amebic Growth and Survival and Requires an Intact C-terminal Domain for Both Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase Activity

Contact Info

Product

Resources

About