scholarly journals Molecular Characterization of the Lactococcus lactis ptsHI Operon and Analysis of the Regulatory Role of HPr

1999 ◽  
Vol 181 (3) ◽  
pp. 764-771 ◽  
Author(s):  
Evert J. Luesink ◽  
Christel M. A. Beumer ◽  
Oscar P. Kuipers ◽  
Willem M. De Vos
Keyword(s):  
Lactococcus Lactis ◽  
Mutational Analysis ◽  
Dominant Role ◽  
Promoter Sequence ◽  
Regulatory Role ◽  
Phosphotransferase System ◽  
Galactose Metabolism ◽  
Wild Type Phenotype ◽  
Genes Encoding

ABSTRACT The Lactococcus lactis ptsH and ptsI genes, encoding the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system, HPr and enzyme I, respectively, were cloned, and the regulatory role of HPr was studied by mutational analysis of its gene. A promoter sequence was identified upstream of theptsHI operon, and the transcription start site was mapped by primer extension. The results of Northern analyses showed the presence of two glucose-inducible transcripts, one of 0.3 kb containingptsH and a second of 2.0 kb containing bothptsH and ptsI. Disruption of theptsH and ptsI genes in strain NZ9800 resulted in a reduced growth rate at the expense of glucose, but no growth at the expense of sucrose and fructose, confirming the dominant role of the phosphotransferase system in the uptake of these sugars in L. lactis. Complementation of the ptsH andptsI mutants with the intact genes under the control of a regulated promoter resulted in the restoration of the wild-type phenotype. The role of HPr(Ser-P) in the recently established CcpA-mediated control of galactose metabolism as well as glycolysis was analyzed by producing an HPr mutant carrying an aspartic acid on residue 46 which mimicks a phosphorylated serine. The results of these experiments demonstrated the role of HPr(Ser-P) as corepressor in the catabolite repression of the gal operon. Furthermore, we show for the first time that HPr(Ser-P) functions as a coactivator in the CcpA-mediated catabolite activation of the pyruvate kinase andl-lactate dehydrogenase genes.

scholarly journals Mutational Analysis of the Role of HPr inListeria monocytogenes

1999 ◽  
Vol 65 (5) ◽  
pp. 2112-2115 ◽  
Author(s):  
Douglas P. Christensen ◽  
Andrew K. Benson ◽  
Robert W. Hutkins
Keyword(s):  
Listeria Monocytogenes ◽  
Phosphatase Activity ◽  
Mutational Analysis ◽  
Active Form ◽  
Regulatory Role ◽  
Dependent Manner ◽  
Phosphotransferase System ◽  
Gram Positive ◽  
A Charge

ABSTRACT The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in Listeria monocytogenes. By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.

scholarly journals Towards Enhanced Galactose Utilization by Lactococcus lactis

2010 ◽  
Vol 76 (21) ◽  
pp. 7048-7060 ◽  
Author(s):  
Ana R. Neves ◽  
Wietske A. Pool ◽  
Ana Solopova ◽  
Jan Kok ◽  
Helena Santos ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Poor Quality ◽  
Genetically Engineered ◽  
Phosphotransferase System ◽  
Galactose Metabolism ◽  
Gene Encoding ◽  
Leloir Pathway ◽  
Lactose Fermentation ◽  
Consumption Rates ◽  
Galactose Utilization

ABSTRACT Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.

scholarly journals Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides

2013 ◽  
Vol 289 (3) ◽  
pp. 1243-1256 ◽  
Author(s):  
Mark J. Lee ◽  
Fabrice N. Gravelat ◽  
Robert P. Cerone ◽  
Stefanie D. Baptista ◽  
Paolo V. Campoli ◽  
...  
Keyword(s):  
Cell Wall ◽  
Aspergillus Fumigatus ◽  
Normal Growth ◽  
Biosynthetic Pathways ◽  
Cell Wall Synthesis ◽  
Cell Wall Polysaccharides ◽  
Galactose Metabolism ◽  
Double Deletion ◽  
Genes Encoding

The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.

Expression of mptC of Listeria monocytogenes induces sensitivity to class IIa bacteriocins in Lactococcus lactis

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2663-2668 ◽  
Author(s):  
Manilduth Ramnath ◽  
Safia Arous ◽  
Anne Gravesen ◽  
John W. Hastings ◽  
Yann Héchard
Keyword(s):  
Lactic Acid ◽  
Listeria Monocytogenes ◽  
Lactic Acid Bacteria ◽  
Lactococcus Lactis ◽  
Inhibitory Activity ◽  
Bacteriocin Activity ◽  
Phosphotransferase System ◽  
Enterocin A ◽  
Leucocin A

Sensitivity to class IIa bacteriocins from lactic acid bacteria was recently associated with the mannose phosphotransferase system (PTS) permease, , in Listeria monocytogenes. To assess the involvement of this protein complex in class IIa bacteriocin activity, the mptACD operon, encoding , was heterologously expressed in an insensitive species, namely Lactococcus lactis, using the NICE double plasmid system. Upon induction of the cloned operon, the recombinant Lc. lactis became sensitive to leucocin A. Pediocin PA-1 and enterocin A also showed inhibitory activity against Lc. lactis cultures expressing mptACD. Furthermore, the role of the three genes of the mptACD operon was investigated. Derivative plasmids containing various combinations of these three genes were made from the parental mptACD plasmid by divergent PCR. The results showed that expression of mptC alone is sufficient to confer sensitivity to class IIa bacteriocins in Lc. lactis.

scholarly journals Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis

2021 ◽  
Vol 12 ◽  
Author(s):  
Guillermo Ortiz Charneco ◽  
Philip Kelleher ◽  
Andrius Buivydas ◽  
Hugo Streekstra ◽  
Emiel Ver Loren van Themaat ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
High Frequency ◽  
Mutational Analysis ◽  
Genetic Dissection ◽  
R Gene ◽  
Conjugation System ◽  
Transfer Frequency ◽  
Preliminary Model ◽  
Conjugation Process

Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the traR gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery.

scholarly journals Autolysis of Lactococcus lactis Is Increased upon d-Alanine Depletion of Peptidoglycan and Lipoteichoic Acids

2005 ◽  
Vol 187 (1) ◽  
pp. 114-124 ◽  
Author(s):  
Anton Steen ◽  
Emmanuelle Palumbo ◽  
Marie Deghorain ◽  
Pier Sandro Cocconcelli ◽  
Jean Delcour ◽  
...  
Keyword(s):  
Cell Wall ◽  
Lactococcus Lactis ◽  
Double Mutant ◽  
Wild Type Strain ◽  
Bacterial Species ◽  
Genes Encoding ◽  
In The Wild ◽  
Zymographic Analysis ◽  
Lipoteichoic Acids

ABSTRACT Mutations in the genes encoding enzymes responsible for the incorporation of d-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external supply of d-Ala to be able to synthesize peptidoglycan and to incorporate d-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when d-Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of d-Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the d-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that d-alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking d-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of dltD in L. lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, d-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis. Second, reduced amounts of d-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.

scholarly journals A Novel Role for Enzyme I of the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System in Regulation of Growth in a Biofilm

2007 ◽  
Vol 190 (1) ◽  
pp. 311-320 ◽  
Author(s):  
Laetitia Houot ◽  
Paula I. Watnick
Keyword(s):  
Vibrio Cholerae ◽  
Sugar Transport ◽  
Phosphotransferase System ◽  
Potent Inducer ◽  
Inducer Exclusion ◽  
Genes Encoding ◽  
Cell Components ◽  
Specific Regulation ◽  
Enzyme I

ABSTRACT Glucose is a universal energy source and a potent inducer of surface colonization for many microbial species. Highly efficient sugar assimilation pathways ensure successful competition for this preferred carbon source. One such pathway is the phosphoenolpyruvate phosphotransferase system (PTS), a multicomponent sugar transport system that phosphorylates the sugar as it enters the cell. Components required for transport of glucose through the PTS include enzyme I, histidine protein, enzyme IIAGlc, and enzyme IIBCGlc. In Escherichia coli, components of the PTS fulfill many regulatory roles, including regulation of nutrient scavenging and catabolism, chemotaxis, glycogen utilization, catabolite repression, and inducer exclusion. We previously observed that genes encoding the components of the Vibrio cholerae PTS were coregulated with the vps genes, which are required for synthesis of the biofilm matrix exopolysaccharide. In this work, we identify the PTS components required for transport of glucose and investigate the role of each of these components in regulation of biofilm formation. Our results establish a novel role for the phosphorylated form of enzyme I in specific regulation of biofilm-associated growth. As the PTS is highly conserved among bacteria, the enzyme I regulatory pathway may be relevant to a number of biofilm-based infections.

scholarly journals Role of ptsO in Carbon-Mediated Inhibition of the Pu Promoter Belonging to the pWW0Pseudomonas putida Plasmid

2001 ◽  
Vol 183 (17) ◽  
pp. 5128-5133 ◽  
Author(s):  
Ildefonso Cases ◽  
Francisco Velázquez ◽  
Vı́ctor de Lorenzo
Keyword(s):  
Gene Dosage ◽  
Sugar Transport ◽  
Regulatory Elements ◽  
Open Reading Frames ◽  
Type I ◽  
Phosphotransferase System ◽  
Wild Type Phenotype ◽  
Genomic Search ◽  
Regulatory Functions

ABSTRACT An investigation was made into the role of the ptsOgene in carbon source inhibition of the Pu promoter belonging to the Pseudomonas putida upper TOL (toluene degradation) operon. ptsO is coexpressed withptsN, the loss of which is known to renderPu unresponsive to glucose. Both ptsN andptsO, coding for the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family proteins IIANtr and NPr, respectively, have been mapped adjacent to the rpoN gene of P. putida. The roles of these two genes in the responses of Pu to glucose were monitored by lacZ reporter technology with a P. putida strain engineered with all regulatory elements in monocopy gene dosage. In cells lacking ptsO,Pu activity seemed to be inhibited even in the absence of glucose. A functional relationship with ptsNwas revealed by the phenotype of a double ptsN ptsOmutant that was equivalent to the phenotype of a mutant with a singleptsN disruption. Moreover, phosphorylation of the product of ptsO seemed to be required for C inhibition of Pu, since an H15A change in the NPr sequence that prevents phosphorylation of this conserved amino acid residue did not restore the wild-type phenotype. A genomic search for proteins able to phosphorylate ptsO revealed the presence of two open reading frames, designated ptsP and mtp, with the potential to encode PTS type I enzymes in P. putida. However, neither an insertion in ptsPnor an insertion in mtp resulted in a detectable change in inhibition of Pu by glucose. These results indicate that some PTS proteins have regulatory functions in P. putida that are independent of their recognized role in sugar transport in other bacteria.

Role of phosphoenolpyruvate-phosphotransferase in glucose utilization by bacilli

1972 ◽  
Vol 182 (1067) ◽  
pp. 171-181 ◽  
Keyword(s):  
Bacillus Licheniformis ◽  
Glucose Utilization ◽  
Glycolytic Enzymes ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Wild Type Phenotype

The growth of mutant Z4 of Bacillus licheniformis on glucose and on a number of other carbohydrates is impaired, but growth on fructose, glycerol and on glucuronate is not. There are no significant differences between the mutant and its parent in the levels of glycolytic enzymes and in the ability of the organisms to take up labelled fructose; in contrast, the mutant takes up and phosphorylates labelled glucose, and its analogues methyl α -glucoside and 2-deoxyglucose, to a much smaller extent than does the wild-type. Extracts of the mutant are virtually devoid of the inducible phosphoenolpyruvate-dependent glucose phosphotransferase present in the parent, though fructose phosphotransferase activity is present in both organisms. Revertants of Z4 , selected for growth on glucose, fully regain the wild-type phenotype. These results show that the phosphotransferase system plays a necessary role in the utilization of glucose by bacilli.

A General Method for Selection of α-Acetolactate Decarboxylase-Deficient Lactococcus lactis Mutants To Improve Diacetyl Formation

1999 ◽  
Vol 65 (3) ◽  
pp. 1202-1206 ◽  
Author(s):  
Mirjana Curic ◽  
Birgitte Stuer-Lauridsen ◽  
Pierre Renault ◽  
Dan Nilsson
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Genetically Modified Organisms ◽  
Defined Medium ◽  
Isoleucine Leucine ◽  
Genes Encoding ◽  
Resistant Mutants ◽  
Branched Chain ◽  
Selection Of

ABSTRACT The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the α-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp.lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains.

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