scholarly journals Identification of Listeria monocytogenes Genes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening

2006 ◽  
Vol 188 (2) ◽  
pp. 556-568 ◽  
Author(s):  
Biju Joseph ◽  
Karin Przybilla ◽  
Claudia Stühler ◽  
Kristina Schauer ◽  
Jörg Slaghuis ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Proteins ◽  
Carbon Sources ◽  
Brain Heart Infusion ◽  
Nitrogen Sources ◽  
Pentose Phosphate ◽  
Intracellular Growth ◽  
Cell Infection ◽  
Phosphotransferase System ◽  
Isoleucine Leucine

ABSTRACT A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.

scholarly journals Transport and Catabolism of Pentitols by Listeria monocytogenes

2016 ◽  
Vol 26 (6) ◽  
pp. 369-380 ◽  
Author(s):  
Takfarinas Kentache ◽  
Eliane Milohanic ◽  
Thanh Nguyen Cao ◽  
Abdelhamid Mokhtari ◽  
Francine Moussan Aké ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Pentose Phosphate ◽  
Phosphotransferase System ◽  
Virulence Gene Expression ◽  
Transposon Insertion ◽  
Encoding Gene ◽  
High Concentrations ◽  
Transcription Activators

Transposon insertion into <i>Listeria monocytogenes lmo2665</i>, which encodes an EIIC of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), was found to prevent <smlcap>D</smlcap>-arabitol utilization. We confirm this result with a deletion mutant and show that Lmo2665 is also required for <smlcap>D</smlcap>-xylitol utilization. We therefore called this protein EIIC<sup>Axl</sup>. Both pentitols are probably catabolized via the pentose phosphate pathway (PPP) because <i>lmo2665</i> belongs to an operon, which encodes the three PTS<sup>Axl</sup> components, two sugar-P dehydrogenases, and most PPP enzymes. The two dehydrogenases oxidize the pentitol-phosphates produced during PTS-catalyzed transport to the PPP intermediate xylulose-5-P. <i>L. monocytogenes</i> contains another PTS, which exhibits significant sequence identity to PTS<sup>Axl</sup>. Its genes are also part of an operon encoding PPP enzymes. Deletion of the EIIC-encoding gene <i>(lmo0508)</i> affected neither <smlcap>D</smlcap>-arabitol nor <smlcap>D</smlcap>-xylitol utilization, although <smlcap>D</smlcap>-arabitol induces the expression of this operon. Both operons are controlled by MtlR/LicR-type transcription activators (Lmo2668 and Lmo0501, respectively). Phosphorylation of Lmo0501 by the soluble PTS<sup>Axl</sup> components probably explains why <smlcap>D</smlcap>-arabitol also induces the second pentitol operon. Listerial virulence genes are submitted to strong repression by PTS sugars, such as glucose. However, <smlcap>D</smlcap>-arabitol inhibited virulence gene expression only at high concentrations, probably owing to its less efficient utilization compared to glucose.

Global Gene Expression of Listeria monocytogenes to Salt Stress

2012 ◽  
Vol 75 (5) ◽  
pp. 906-912 ◽  
Author(s):  
DONGRYEOUL BAE ◽  
CONNIE LIU ◽  
TING ZHANG ◽  
MARCUS JONES ◽  
SCOTT N. PETERSON ◽  
...  
Keyword(s):  
Gene Expression ◽  
Salt Stress ◽  
Listeria Monocytogenes ◽  
Cell Growth ◽  
Expression Profiles ◽  
Brain Heart Infusion ◽  
Global Gene Expression ◽  
Phosphotransferase System ◽  
Transcript Levels ◽  
Qrt Pcr

Outbreaks of listeriosis caused by the ingestion of Listeria-contaminated ready-to-eat foods have been reported worldwide. Many ready-to-eat foods, such as deli meat products, contain high amounts of salt, which can disrupt the maintenance of osmotic balance within bacterial cells. To understand how Listeria monocytogenes adapts to salt stress, we examined the growth and global gene expression profiles of L. monocytogenes strain F2365 under salt stress using oligonucleotide probe-based DNA array and quantitative real-time PCR (qRT-PCR) analyses. The growth of L. monocytogenes in brain heart infusion (BHI) medium with various concentrations of NaCl (2.5, 5, and 10%) was significantly inhibited (P &lt; 0.01) when compared with growth in BHI with no NaCl supplementation. Microarray data indicated that growth in BHI medium with 1.2% NaCl upregulated 4 genes and down-regulated 24 genes in L. monocytogenes, which was confirmed by qRT-PCR. The transcript levels of genes involved in the uptake of glycine betaine/l-proline were increased, whereas genes associated with a putative phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS), metabolic enzymes, and virulence factor were down-regulated. Specifically, the expression levels of PTS transport genes were shown to be dependent on NaCl concentration. To further examine whether the down-regulation of PTS genes is related to decreased cell growth, the transcript levels of genes encoding components of enzyme II, involved in the uptake of various sugars used as the primary carbon source in bacteria, were also measured using qRT-PCR. Our results suggest that the decreased transcript levels of PTS genes may be caused by salt stress or reduced cell growth through salt stress. Here, we report global transcriptional profiles of L. monocytogenes in response to salt stress, contributing to an improved understanding of osmotolerance in this bacterium.

scholarly journals Phosphotransferase System-Dependent Extracellular Growth of Listeria monocytogenes Is Regulated by Alternative Sigma Factors σLand σH

2014 ◽  
Vol 80 (24) ◽  
pp. 7673-7682 ◽  
Author(s):  
Siyun Wang ◽  
Renato H. Orsi ◽  
Silin Tang ◽  
Wei Zhang ◽  
Martin Wiedmann ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Carbon Source ◽  
Parent Strain ◽  
Carbon Sources ◽  
Maximum Growth ◽  
Fine Tuning ◽  
Lag Phase ◽  
Phosphotransferase System ◽  
Phase Duration ◽  
Content Type

ABSTRACTAlternative sigma (σ) factors and phosphotransferase systems (PTSs) play pivotal roles in the environmental adaptation and virulence ofListeria monocytogenes. The growth of theL. monocytogenesparent strain 10403S and 15 isogenic alternative σ factor mutants was assessed in defined minimal medium (DM) with PTS-dependent or non-PTS-dependent carbon sources at 25°C or 37°C. Overall, our results suggested that the regulatory effect of alternative σ factors on the growth ofL. monocytogenesis dependent on the temperature and the carbon source. One-way analysis of variance (one-way ANOVA) showed that the factor “strain” had a significant effect on the maximum growth rate (μmax), lag phase duration (λ), and maximum optical density (ODmax) in PTS-dependent carbon sources (P< 0.05) but not in a non-PTS-dependent carbon source. Also, the ODmaxwas not affected by strain for any of the three PTS-dependent carbon sources at 25°C but was affected by strain at 37°C. Monitoring by quantitative real-time PCR (qRT-PCR) showed that transcript levels forlmo0027, a glucose-glucoside PTS permease (PTSGlc-1)-encoding gene, were higher in the absence of σL, and lower in the absence of σH, than in the parent strain. Our data thus indicate that σLnegatively regulateslmo0027and that the increased μmaxobserved for the ΔsigLstrain in DM with glucose may be associated with increased expression of PTSGlc-1 encoded bylmo0027. Our findings suggest that σHand σLmediate the PTS-dependent growth ofL. monocytogenesthrough complex transcriptional regulations and fine-tuning of the expression of specificptsgenes, includinglmo0027. Our findings also reveal a more important and complex role of alternative σ factors in the regulation of growth in different sugar sources than previously assumed.

scholarly journals Salt Stress Proteins Induced in Listeria monocytogenes

2002 ◽  
Vol 68 (4) ◽  
pp. 1491-1498 ◽  
Author(s):  
Ophélie Duché ◽  
Frédéric Trémoulet ◽  
Philippe Glaser ◽  
Jean Labadie
Keyword(s):  
Salt Stress ◽  
Listeria Monocytogenes ◽  
Food Processing ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Food Preservation ◽  
Terminal Sequence ◽  
Phosphotransferase System ◽  
General Metabolism ◽  
Survival Mechanisms

ABSTRACT The ability of Listeria monocytogenes to tolerate salt stress is of particular importance, as this pathogen is often exposed to such environments during both food processing and food preservation. In order to understand the survival mechanisms of L. monocytogenes, an initial approach using two-dimensional polyacrylamide gel electrophoresis was performed to analyze the pattern of protein synthesis in response to salt stress. Of 400 to 500 visible proteins, the synthesis of 40 proteins (P < 0.05) was repressed or induced at a higher rate during salt stress. Some of the proteins were identified on the basis of mass spectrometry or N-terminal sequence analysis and database searching. Twelve proteins showing high induction after salt stress were similar to general stress proteins (Ctc and DnaK), transporters (GbuA and mannose-specific phosphotransferase system enzyme IIAB), and general metabolism proteins (alanine dehydrogenase, CcpA, CysK, EF-Tu, Gap, GuaB, PdhA, and PdhD).

Evaluation of the growth and sporulation of different entomopathogenic fungi in different liquid and solid media at varied concentrations

2017 ◽  
Vol 6 (8) ◽  
pp. 5459
Author(s):  
Chandra Teja K. ◽  
Rahman S. J.
Keyword(s):  
Entomopathogenic Fungi ◽  
Large Scale ◽  
Insect Pests ◽  
Culture Media ◽  
Virulent Strain ◽  
Carbon Sources ◽  
Nitrogen Sources ◽  
Maximum Growth ◽  
Nutritional Requirements ◽  
Virulent Strains

Entomopathogenic fungi like Beauveria bassiana, Metarhizium anisopliae and Lecanicillium lecanii are used in biological control of agricultural insect pests. Their specific mode of action makes them an effective alternative to the chemical Insecticides. Virulent strains of Entomopathogenic fungi are effectively formulated and used as bio-insecticides world-wide. Amenable and economical multiplication of a virulent strain in a large scale is important for them to be useful in the field. Culture media plays a major role in the large-scale multiplication of virulent strains of Entomopathogens. Different substrates and media components are being used for this purpose. Yet, each strain differs in its nutritional requirements for the maximum growth and hence it is necessary to standardize the right components and their optimum concentrations in the culture media for a given strain of Entomopathogen. In the current study, three different nitrogen sources and two different carbon sources were tried to standardize the mass multiplication media for seven test isolates of Entomopathogenic fungi. A study was also conducted to determine the ideal grain media for the optimum conidial yields of the test isolates. Yeast extract was found to be the best Nitrogen source for the isolates. The isolates tested, differed in their nutritional requirements and showed variation in the best nitrogen and carbon sources necessary for their growth. Variation was also found in the optimum concentration of both the ingredients for the growth and sporulation of the isolates. In the solid-state fermentation study, rice was found to be the best grain for the growth of most of the fungi followed by barley. The significance of such a study in the development of an effective Myco-insecticide is vital and can be successfully employed in agriculture is discussed.

scholarly journals PSIX-18 Laurate and its glycerol monoester, monolaurin, as potential additives to control Listeria monocytogenes and tetracycline resistant Enterococcus faecalis in air-exposed silage

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 414-415
Author(s):  
Yamicela Castillo-Castillo ◽  
Marina Ontiveros ◽  
Eric J Scholljegerdes ◽  
Robin Anderson ◽  
Claudio Arzola-Alvarez ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Enterococcus Faecalis ◽  
Brain Heart Infusion ◽  
Bactericidal Effect ◽  
Complete Elimination ◽  
Gram Positive Bacteria ◽  
Viable Cells ◽  
Colony Forming Units ◽  
Feed Value ◽  
Risk Infection

Abstract Silages can harbor pathogenic and antimicrobial resistant microbes which risk infection of food-producing animals. Livestock producers need effective yet environmentally friendly interventions to preserve the feed value of these fermented materials. Medium chain fatty acids such as laurate and its glycerol monoester, monolaurin, are potent inhibitors of many Gram-positive bacteria and when tested at 5 mg/mL in anaerobic cultures (n = 3/treatment) inoculated with 105 colony forming units (CFU) of Listeria monocytogenes and grown at 37oC in ½ strength Brain Heart infusion broth achieved near complete elimination of viable cells after 6 h compared to a 2.2 ± 0.1 log10 CFU/mL increase observed in controls. Culture of a tetracycline-resistant Enterococcus faecalis with 5 mg laurate/mL likewise achieved near complete elimination of viable cells (5 log10 CFU/mL) by 6 h incubation. The bactericidal effect of 5 mg monolaurin was less against E. faecalis, achieving a decrease of 1.8 ± 0.2 log10 CFU/mL and not decreased further after 24 h. When tested against air-exposed silage, pH 7.53 (4 g), mixed with 4 mL water, 5 mg laurate or monolaurin decreased viability of experimentally-inoculated L. monocytogenes (105 CFU/g silage) more (P &lt; 0.05) than untreated controls after 24 h aerobic incubation (22oC), with viable counts being decreased 6.3 ± 0.1, 5.9 ± 0.8 and 4.5 ± 0.1 log10 CFU/g, respectively. In contrast, viable recovery of the experimentally-inoculated (105 CFU/g) tetracycline-resistant E. faecalis was reduced more (P &lt; 0.05) than controls (decreased 0.7 ± 0.1 log10 CFU/g) after 6 h incubation when similarly tested with laurate and monolaurin (1.7 ± 0.5 and 3.0 ± 0.9 log10 CFU/g, respectively) but counts after 24 h were similar, decreasing on average 2.0 ± 0.5 log10 CFU/g). Results indicate laurate and monolaurin may be useful in killing L. monocytogenes and tetracycline-resistant E. faecalis during silage feed-out.

scholarly journals Studies on the Role of the are a Gene in the Regulation of Nitrogen Catabolism in Aspergillus nidulans

1975 ◽  
Vol 28 (3) ◽  
pp. 301 ◽  
Author(s):  
MJ Hynes
Keyword(s):  
Nitrogen Source ◽  
Aspergillus Nidulans ◽  
Carbon Sources ◽  
Nitrogen Sources ◽  
Selection Methods ◽  
Growth Responses ◽  
Dominance Relationships ◽  
Regulatory Circuits ◽  
Specific Activities

Mutants of Apergillus nidulanswith lesions in a gene, areA (formerly called amdT), have been isolated by a variety of different selection methods. The areA mutants show a range of pleiotropic growth responses to a number of compounds as sole nitrogen sources, but are normal in utilization of carbon sources. The levels of two amidase enzymes as well as urease have been investigated in the mutants and have been shown to be affected by this gene. Most of the areA mutants have much lower amidase-specific activities when grown in ammonium-containing medium, compared with mycelium incubated in medium la9king a nitrogen source. Some of the areA. mutants do not show derepression of urease upon relief of ammonium repression. The dominance relationships of areA alleles have been investigated in� heterozygous diploids, and these studies lend support to the proposal that areA codes for a positively acting regulatory product. One of the new areA alleles is partially dominant to areA + and areA102. This may be a result of negative complementation or indicate that areA has an additional negative reiuIatory function. Investigation.of various amdR; areA double mutants has led to the conclusion that amdR and areA participate in independent regulatory circuits in the control of acetamide utilizatiol1. Studies on an amdRc; areA.double mutant indicate that areA is involved in derepression of acetamidase upon relief of ammo.nium repression.

scholarly journals Factorial Design to Optimize Biosurfactant Production byYarrowia lipolytica

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Gizele Cardoso Fontes ◽  
Priscilla Filomena Fonseca Amaral ◽  
Marcio Nele ◽  
Maria Alice Zarur Coelho
Keyword(s):  
Surface Tension ◽  
Factorial Design ◽  
Ammonium Sulfate ◽  
Yeast Extract ◽  
Carbon Sources ◽  
Nitrogen Sources ◽  
Biosurfactant Production ◽  
Standard Process ◽  
Emulsification Index ◽  
Full Factorial

In order to improve biosurfactant production byYarrowia lipolyticaIMUFRJ 50682, a factorial design was carried out. A24full factorial design was used to investigate the effects of nitrogen sources (urea, ammonium sulfate, yeast extract, and peptone) on maximum variation of surface tension (ΔST) and emulsification index (EI). The best results (67.7% of EI and 20.9 mNm−1ofΔST) were obtained in a medium composed of 10 g 1−1of ammonium sulfate and 0.5 g 1−1of yeast extract. Then, the effects of carbon sources (glycerol, hexadecane, olive oil, and glucose) were evaluated. The most favorable medium for biosurfactant production was composed of both glucose (4% w/v) and glycerol (2% w/v), which provided an EI of 81.3% and aΔST of 19.5 mN m−1. The experimental design optimization enhancedΔEI by 110.7% andΔST by 108.1% in relation to the standard process.

scholarly journals A Mannose Family Phosphotransferase System Permease and Associated Enzymes Are Required for Utilization of Fructoselysine and Glucoselysine in Salmonella enterica Serovar Typhimurium

2015 ◽  
Vol 197 (17) ◽  
pp. 2831-2839 ◽  
Author(s):  
Katherine A. Miller ◽  
Robert S. Phillips ◽  
Paul B. Kilgore ◽  
Grady L. Smith ◽  
Timothy R. Hoover
Keyword(s):  
Nitrogen Source ◽  
Nitrogen Sources ◽  
Maillard Reaction Products ◽  
Reaction Products ◽  
Phosphotransferase System ◽  
Carbon And Nitrogen Sources ◽  
Content Type ◽  
Carbon And Nitrogen ◽  
Serovar Typhimurium ◽  
Food Borne Illness

ABSTRACTSalmonella entericserovar Typhimurium, a major cause of food-borne illness, is capable of using a variety of carbon and nitrogen sources. Fructoselysine and glucoselysine are Maillard reaction products formed by the reaction of glucose or fructose, respectively, with the ε-amine group of lysine. We report here thatS. Typhimurium utilizes fructoselysine and glucoselysine as carbon and nitrogen sources via a mannose family phosphotransferase (PTS) encoded bygfrABCD(glucoselysine/fructoselysine PTS components EIIA, EIIB, EIIC, and EIID; locus numbers STM14_5449 to STM14_5454 inS. Typhimurium 14028s). Genes coding for two predicted deglycases within thegfroperon,gfrEandgfrF, were required for growth with glucoselysine and fructoselysine, respectively. GfrF demonstrated fructoselysine-6-phosphate deglycase activity in a coupled enzyme assay. The biochemical and genetic analyses were consistent with a pathway in which fructoselysine and glucoselysine are phosphorylated at the C-6 position of the sugar by the GfrABCD PTS as they are transported across the membrane. The resulting fructoselysine-6-phosphate and glucoselysine-6-phosphate subsequently are cleaved by GfrF and GfrE to form lysine and glucose-6-phosphate or fructose-6-phosphate. Interestingly, althoughS. Typhimurium can use lysine derived from fructoselysine or glucoselysine as a sole nitrogen source, it cannot use exogenous lysine as a nitrogen source to support growth. Expression ofgfrABCDEFwas dependent on the alternative sigma factor RpoN (σ54) and an RpoN-dependent LevR-like activator, which we designated GfrR.IMPORTANCESalmonellaphysiology has been studied intensively, but there is much we do not know regarding the repertoire of nutrients these bacteria are able to use for growth. This study shows that a previously uncharacterized PTS and associated enzymes function together to transport and catabolize fructoselysine and glucoselysine. Knowledge of the range of nutrients thatSalmonellautilizes is important, as it could lead to the development of new strategies for reducing the load ofSalmonellain food animals, thereby mitigating its entry into the human food supply.

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