Replacing the general energy-coupling proteins of the phospho-enol-pyruvate:sugar phosphotransferase system ofSalmonella typhimuriumwith fructose-inducible counterparts results in the inability to utilize nonphosphotransferase system sugars

2007 ◽  
Vol 53 (5) ◽  
pp. 586-598 ◽  
Author(s):  
Sarah L. Sutrina ◽  
Patricia I. Inniss ◽  
Leslie-Ann Lazarus ◽  
Lizette Inglis ◽  
Jacqueline Maximilien
Keyword(s):  
Cyclic Amp ◽  
Rapid Growth ◽  
Regulatory Protein ◽  
Energy Coupling ◽  
Threshold Level ◽  
Luria Bertani ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Inducer Exclusion ◽  
Luria Bertani Broth

A Salmonella typhimurium mutant lacking Enzyme I and HPr, general proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but producing homologues EIFructoseand FPr constitutively, did not grow in minimal medium supplemented with non-PTS sugars (melibiose, glycerol, and maltose) in the absence of any trace of Luria–Bertani broth; adding cyclic AMP allowed growth. On melibiose, rapid growth began only when melibiose permease activity had reached a threshold level. Wild-type cultures reached this level within about 2 h, but the mutant only after a 12–14 h lag period, and then only when cyclic AMP had been added to the medium. On a mixture of melibiose and a PTS sugar, permease was undetectable in either the wild type or mutant until the PTS sugar had been exhausted. Permease then appeared, increasing with time, but in the mutant it never reached the threshold allowing rapid growth on melibiose unless cyclic AMP had been added. On rich medium supplemented with melibiose or glycerol, the mutant produced lower (30%) levels of melibiose permease or glycerol kinase compared with the wild type. We propose that poor phosphorylation of the regulatory protein Enzyme IIAGlucose, leading to constitutive inducer exclusion and catabolite repression in this strain, accounts for these results.

scholarly journals Regulatory Functions of Serine-46-Phosphorylated HPr in Lactococcus lactis

2001 ◽  
Vol 183 (11) ◽  
pp. 3391-3398 ◽  
Author(s):  
Vicente Monedero ◽  
Oscar P. Kuipers ◽  
Emmanuel Jamet ◽  
Josef Deutscher
Keyword(s):  
Lactococcus Lactis ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Inducer Exclusion ◽  
Gram Positive Bacteria ◽  
In Vivo Experiments ◽  
Regulatory Functions

ABSTRACT In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a β-glucoside-specific EII and a 6-P-β-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methylβ-d-thiogalactoside (TMG) and 2-deoxy-d-glucose (2-DG). In vivo experiments with theptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

Mutant ras gene induces elevated levels of inositol tris- and hexakisphosphates in Dictyostelium

1988 ◽  
Vol 89 (1) ◽  
pp. 13-20
Author(s):  
G.N. Europe-Finner ◽  
M.E. Luderus ◽  
N.V. Small ◽  
R. Van Driel ◽  
C.D. Reymond ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cyclic Amp ◽  
Gene Product ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Regulatory Protein ◽  
Signal Transduction Pathway ◽  
Mutant Form ◽  
Wild Type ◽  
Ras Gene

Previous studies of Europe-Finner & Newell indicated that in amoebae of Dictyostelium discoideum, signal transduction used for chemotaxis to cyclic AMP involved transient formation of inositol tris- and polyphosphates. Evidence was also presented for the involvement of a GTP-binding G-protein. Here we report evidence for the involvement of a ras gene product in the D. discoideum inositol phosphate pathway. Use was made of strains of Dictyostelium transformed with a wild-type D. discoideum ras gene (ras-Gly12) or a mutant form of the gene (ras-Thr12). Experiments using separation of soluble inositol phosphates by Dowex anion-exchange resin chromatography indicated that cells transformed with the wild-type ras-Gly12 gene were unaffected in their basal levels of inositol polyphosphates and in the inositol phosphates formed in response to stimulation with the chemotactic agent cyclic AMP. In contrast, cells transformed with the mutant ras-Thr12 gene showed a basal level of inositol polyphosphate that was several-fold elevated over the controls and stimulation of these cells with cyclic AMP produced only a small further elevation. When the inositol phosphates were analysed by h.p.l.c. it was found that the basal level of inositol 1,4,5-trisphosphate was raised three- to fivefold in the ras-Thr12 strain compared to the strain transformed with ras-Gly12, and that inositol hexakisphosphate (which was found to be present in large amounts relative to other inositol phosphates in D. discoideum cells) was also raised to a similar extent in the ras-Thr12-transformed cells. We propose that the Dictyostelium ras gene product codes for a regulatory protein involved in the inositol phosphate chemotactic signal-transduction pathway.

scholarly journals Salmonella-Induced Cell Death Is Not Required for Enteritis in Calves

2001 ◽  
Vol 69 (7) ◽  
pp. 4610-4617 ◽  
Author(s):  
Renato L. Santos ◽  
Renée M. Tsolis ◽  
Shuping Zhang ◽  
Thomas A. Ficht ◽  
Andreas J. Bäumler ◽  
...  
Keyword(s):  
Cell Death ◽  
Inflammatory Response ◽  
Fluid Secretion ◽  
Luria Bertani ◽  
Wild Type ◽  
Fluid Accumulation ◽  
Luria Bertani Broth ◽  
Serovar Typhimurium ◽  
In The Wild

ABSTRACT Salmonella enterica serovar Typhimurium causes cell death in bovine monocyte-derived and murine macrophages in vitro by asipB-dependent mechanism. During this process, SipB binds and activates caspase-1, which in turn activates the proinflammatory cytokine interleukin-1β through cleavage. We used bovine ileal ligated loops to address the role of serovar Typhimurium-induced cell death in induction of fluid accumulation and inflammation in this diarrhea model. Twelve perinatal calves had 6- to 9-cm loops prepared in the terminal ileum. They were divided into three groups: one group received an intralumen injection of Luria-Bertani broth as a control in 12 loops. The other two groups (four calves each) were inoculated with 0.75 × 109 CFU of either wild-type serovar Typhimurium (strain IR715) or a sopB mutant per loop in 12 loops. Hematoxylin and eosin-stained sections were scored for inflammation, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were detected in situ. Fluid accumulation began at 3 h postinfection (PI). Inflammation was detected in all infected loops at 1 h PI. The area of TUNEL-labeled cells in the wild-type infected loops was significantly higher than that of the controls at 12 h PI, when a severe inflammatory response and tissue damage had already developed. ThesopB mutant induced the same amount of TUNEL-positive cells as the wild type, but it was attenuated for induction of fluid secretion and inflammation. Our results indicate that serovar Typhimurium-induced cell death is not required to trigger an early inflammatory response and fluid accumulation in the ileum.

scholarly journals Characterization and nucleotide sequence of the cryptic cel operon of Escherichia coli K12.

Genetics ◽  
1990 ◽  
Vol 124 (3) ◽  
pp. 455-471 ◽  
Author(s):  
L L Parker ◽  
B G Hall
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Regulatory Protein ◽  
Wild Type ◽  
Phosphotransferase System ◽  
E Coli ◽  
Significant Homology ◽  
Chromosomal Genes ◽  
Alpha Galactosidase ◽  
Beta Glucosidase

Abstract Wild-type Escherichia coli are not able to utilize beta-glucoside sugars because the genes for utilization of these sugars are cryptic. Spontaneous mutations in the cel operon allow its expression and enable the organism to ferment cellobiose, arbutin and salicin. In this report we describe the structure and nucleotide sequence of the cel operon. The cel operon consists of five genes: celA, whose function is unknown; celB and celC which encode phosphoenolpyruvate-dependent phosphotransferase system enzyme IIcel and enzyme IIIcel, respectively, for the transport and phosphorylation of beta-glucoside sugars; celD, which encodes a negative regulatory protein; and celF, which encodes a phospho-beta-glucosidase that acts on phosphorylated cellobiose, arbutin and salicin. The mutationally activated cel operon is induced in the presence of its substrates, and is repressed in their absence. A comparison of proteins encoded by the cel operon with functionally equivalent proteins of the bgl operon, another cryptic E. coli gene system responsible for the catabolism of beta-glucoside sugars, revealed no significant homology between these two systems despite common functional characteristics. The celD and celF encoded repressor and phospho-beta-glucosidase proteins are homologous to the melibiose regulatory protein and to the melA encoded alpha-galactosidase of E. coli, respectively. Furthermore, the celC encoded PEP-dependent phosphotransferase system enzyme IIIcel is strikingly homologous to an enzyme IIIlac of the Gram-positive organism Staphylococcus aureus. We conclude that the genes for these two enzyme IIIs diverged much more recently than did their hosts, indicating that E. coli and S. aureus have undergone relatively recent exchange of chromosomal genes.

scholarly journals Evolutionary dynamics of asexual hypermutators adapting to a novel environment

2021 ◽  
Author(s):  
Wei-Chin Ho ◽  
Megan G. Behringer ◽  
Samuel F. Miller ◽  
Jadon Gonzales ◽  
Amber Nguyen ◽  
...  
Keyword(s):  
Adaptive Evolution ◽  
Evolutionary Biology ◽  
Evolutionary Dynamics ◽  
Fold Increase ◽  
Luria Bertani ◽  
Mutation Rates ◽  
Wild Type ◽  
Novel Environment ◽  
Luria Bertani Broth ◽  
Nucleotide Mutation

How microbes adapt to a novel environment is a central question in evolutionary biology. While adaptive evolution must be fueled by beneficial mutations, whether higher mutation rates facilitate the rate of adaptive evolution remains unclear. To address this question, we cultured Escherichia coli hypermutating populations, in which a defective methyl-directed mismatch repair pathway causes a 140-fold increase in single-nucleotide mutation rates. In parallel with wild-type E. coli, populations were cultured in tubes containing Luria-Bertani broth, a complex medium known to promote the evolution of subpopulation structure. After 900 days of evolution, in three transfer schemes with different population-size bottlenecks, hypermutators always exhibited similar levels of improved fitness as controls. Fluctuation tests revealed that the mutation rates of hypermutator lines converged evolutionarily on those of wild-type populations, which may have contributed to the absence of fitness differences. Further genome-sequence analysis revealed that, although hypermutator populations have higher rates of genomic evolution, this largely reflects the effects of genetic draft under strong linkage. Despite these linkage effects, the evolved populations exhibit parallelism in fixed mutations, including those potentially related to biofilm formation, transcription regulation, and mutation-rate evolution. Together, these results generally negate the presumed relationship between high mutation rates and high adaptive speed of evolution, providing insight into how clonal adaptation occurs in novel environments.

scholarly journals Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

2000 ◽  
Vol 182 (9) ◽  
pp. 2582-2590 ◽  
Author(s):  
Valérie Dossonnet ◽  
Vicente Monedero ◽  
Monique Zagorec ◽  
Anne Galinier ◽  
Gaspar Pérez-Martínez ◽  
...  
Keyword(s):  
Catabolite Repression ◽  
Type Strain ◽  
Lactobacillus Casei ◽  
Wild Type Strain ◽  
Carbon Sources ◽  
Lag Phase ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Inducer Exclusion ◽  
In The Wild

ABSTRACT We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion ofmethyl β-d-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative inptsH mutants altered at Ser-46 of HPr and thehprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.

scholarly journals Antagonistic Roles for GcvA and GcvB in hdeAB Expression in Escherichia coli

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Lorraine T. Stauffer ◽  
George V. Stauffer
Keyword(s):  
Luria Bertani ◽  
Low Ph ◽  
Wild Type ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Luria Bertani Broth ◽  
Periplasmic Proteins ◽  
Acidic Conditions ◽  
Survival And Growth ◽  
Minimal Media

In E. coli, the periplasmic proteins HdeA and HdeB have chaperone-like functions, suppressing aggregation of periplasmic proteins under acidic conditions. A microarray analysis of RNA isolated from an E. coli wild type and a ΔgcvB strain grown to mid-log phase in Luria-Bertani broth indicated the hdeAB operon, encoding the HdeA and HdeB proteins, is regulated by the sRNA GcvB. We wanted to verify that GcvB and its coregulator Hfq play a role in regulation of the hdeAB operon. In this study, we show that GcvB positively regulates hdeA::lacZ and hdeB::lacZ translational fusions in cells grown in Luria-Bertani broth and in glucose minimal media + glycine. Activation also requires the Hfq protein. Although many sRNAs dependent on Hfq regulate by an antisense mechanism, GcvB regulates hdeAB either directly or indirectly at the level of transcription. GcvA, the activator of gcvB, negatively regulates hdeAB at the level of transcription. Although expression of gcvB is dependent on GcvA, activation of hdeAB by GcvB occurs independently of GcvA’s ability to repress the operon. Cell survival and growth at low pH are consistent with GcvA negatively regulating and GcvB positively regulating the hdeAB operon.

scholarly journals CRYPTIC OPERON FOR β-GLUCOSIDE METABOLISM IN ESCHERICHIA COLI K12: GENETIC EVIDENCE FOR A REGULATORY PROTEIN

Genetics ◽  
1981 ◽  
Vol 97 (1) ◽  
pp. 11-25
Author(s):  
Roberto Defez ◽  
Maurilio De Felice
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Single Gene ◽  
Regulatory Protein ◽  
Regulatory Site ◽  
Structural Genes ◽  
Wild Type ◽  
Cis Acting ◽  
E Coli ◽  
Escherichia Coli K12

ABSTRACT Escherichia coli K12 does not metabolize β-glucosides such as arbutin and salicin because of lack of expression of the bglBSRC operon, which contains structural genes for transport (bglC) and hydrolysis (bglB) of phospho-β-glucosides. Mutants carrying lesions in the cis-acting regulatory site bglR metabolize β-glucosides as a consequence of expression of this cryptic operon (Prasad and Schaefler 1974). We isolated mutations promoting β-glucoside metabolism that were unlinked to bglR; some of these mutations were shown to be amber. All of them were mapped at 27 min on the E. coli K12 linkage map and appeared to define a single gene, for which we propose the designation bglY. Utilization of β-glucosides in bglY mutants appeared to be a consequence of expression of the bglBSRC operon, since bglB bglR and bglB bglY double mutants had the same phenotype. All bglY mutations analyzed were recessive to the wild-type bglY  + allele. Phospho-β-glucosidase B and β-glucoside transport activities are inducible in bglY mutants, as they are in bglR mutants. Metabolism of β-glucosides in both bglR and bglY mutants required cyclic AMP. We propose that bglY encodes a protein acting as a repressor of the bglBSRC operon, active in both the presence and absence of β-glucosides, whose recognition site would be within the bglR locus.

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