scholarly journals The CbiB Protein of Salmonella enterica Is an Integral Membrane Protein Involved in the Last Step of the De Novo Corrin Ring Biosynthetic Pathway

2007 ◽  
Vol 189 (21) ◽  
pp. 7697-7708 ◽  
Author(s):  
Carmen L. Zayas ◽  
Kathy Claas ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Membrane Protein ◽  
Salmonella Enterica ◽  
De Novo ◽  
Integral Membrane Protein ◽  
In Vivo Studies ◽  
Single Amino Acid ◽  
Serovar Typhimurium ◽  
Ethanolamine Phosphate ◽  
Coenzyme B

ABSTRACT We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamide-phosphate, the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme B12) pathway of Salmonella enterica serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted that CbiB would be an integral membrane protein. We used a computer-generated topology model of the primary sequence of CbiB to guide the construction of CbiB-LacZ and CbiB-PhoA protein fusions, which were used to explore the general topology of CbiB in the cell membrane. A refined model of CbiB as an integral membrane protein is presented. In vivo analyses of the effect of single-amino-acid changes showed that periplasm- and cytosol-exposed residues are critical for CbiB function. Results of in vivo studies also show that ethanolamine-phosphate (EA-P) is a substrate of CbiB, but l-Thr-P is not, and that CbiB likely activates AdoCby by phosphorylation. The latter observation leads us to suggest that CbiB is a synthetase not a synthase enzyme. Results from mass spectrometry and bioassay experiments indicate that serovar Typhimurium synthesizes norcobalamin (cobalamin lacking the methyl group at C176) when EA-P is the substrate of CbiB.

scholarly journals Rcs and PhoPQ Regulatory Overlap in the Control of Salmonella enterica Virulence

2007 ◽  
Vol 189 (18) ◽  
pp. 6635-6644 ◽  
Author(s):  
Clara B. García-Calderón ◽  
Josep Casadesús ◽  
Francisco Ramos-Morales
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Salmonella Enterica ◽  
In Vivo Studies ◽  
Genetic Screens ◽  
Signal Transduction System ◽  
Constitutive Activation ◽  
Regulatory Systems ◽  
Serovar Typhimurium

ABSTRACT Genetic screens based on the use of MudJ-generated lac fusions permitted the identification of novel genes regulated by the Rcs signal transduction system in Salmonella enterica serovar Typhimurium. Besides genes that are also found in the Escherichia coli genome, our screens identified Salmonella-specific genes regulated by RcsB, including bapA, siiE, srfA, and srfB. Here we show that the srfABC operon is negatively regulated by RcsB and by PhoP. In vivo studies using mutants with constitutive activation of the Rcs and/or PhoPQ system suggested that there is an overlap between these regulatory systems in the control of Salmonella virulence.

scholarly journals Insights into the Relationship between Cobamide Synthase and the Cell Membrane

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Victoria L. Jeter ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Cell Membrane ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
De Novo ◽  
Integral Membrane Protein ◽  
Phospholipid Bilayer ◽  
Multienzyme Complex ◽  
Coenzyme B ◽  
Lower Ligand

ABSTRACT Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced de novo by some bacteria and archaea. The “late steps” of the adenosylcobamide biosynthetic pathway are responsible for the assembly of the nucleotide loop and are required during de novo synthesis and precursor salvaging. These steps are characterized by activation of the corrin ring and lower ligand base, condensation of the activated precursors to adenosylcobamide phosphate, and removal of the phosphate, yielding a complete adenosylcobamide molecule. The condensation of the activated corrin ring and lower ligand base is performed by an integral membrane protein, cobamide (5′ phosphate) synthase (CobS), and represents an important convergence of two pathways necessary for nucleotide loop assembly. Interestingly, membrane association of this penultimate step is conserved among all cobamide producers, yet the physiological relevance of this association is not known. Here, we present the purification and biochemical characterization of the CobS enzyme of the enterobacterium Salmonella enterica subsp. enterica serovar Typhimurium strain LT2, investigate its association with liposomes, and quantify the effect of the lipid bilayer on its enzymatic activity and substrate affinity. We report a purification scheme that yields pure CobS protein, allowing in vitro functional analysis. Additionally, we report a method for liposome reconstitution of CobS, allowing for physiologically relevant studies of this inner membrane protein in a phospholipid bilayer. In vitro and in vivo data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis. IMPORTANCE Salmonella is a human pathogen of worldwide importance, and coenzyme B12 is critical for the pathogenic lifestyle of this bacterium. The importance of the work reported here lies on the improvements to the methodology used to isolate cobamide synthase, a polytopic integral membrane protein that catalyzes the penultimate step of coenzyme B12 biosynthesis. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during de novo coenzyme B12 biosynthesis and for the assimilation of incomplete corrinoids from the environment. We proposed that cobamide synthase is likely localized to the cell membrane of every coenzyme B12-producing bacterium and archaeum sequenced to date. The new knowledge of cobamide synthase advances our understanding of the functionality of the enzyme in the context of the lipid bilayer and sets the foundation for the functional-structural analysis of the aforementioned multienzyme complex.

scholarly journals In Salmonella enterica, 2-Methylcitrate Blocks Gluconeogenesis

2009 ◽  
Vol 192 (3) ◽  
pp. 771-778 ◽  
Author(s):  
Christopher J. Rocco ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Salmonella Enterica ◽  
Citrate Synthase ◽  
Krebs Cycle ◽  
Single Amino Acid ◽  
Inborn Errors ◽  
Cycle Enzyme ◽  
Serovar Typhimurium ◽  
Key Enzyme

ABSTRACT Strains of Salmonella enterica serovar Typhimurium LT2 lacking a functional 2-methylcitric acid cycle (2-MCC) display increased sensitivity to propionate. Previous work from our group indicated that this sensitivity to propionate is in part due to the production of 2-methylcitrate (2-MC) by the Krebs cycle enzyme citrate synthase (GltA). Here we report in vivo and in vitro data which show that a target of the 2-MC isomer produced by GltA (2-MCGltA) is fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis. Lack of growth due to inhibition of FBPase by 2-MCGltA was overcome by increasing the level of FBPase or by micromolar amounts of glucose in the medium. We isolated an fbp allele encoding a single amino acid substitution in FBPase (S123F), which allowed a strain lacking a functional 2-MCC to grow in the presence of propionate. We show that the 2-MCGltA and the 2-MC isomer synthesized by the 2-MC synthase (PrpC; 2-MCPrpC) are not equally toxic to the cell, with 2-MCGltA being significantly more toxic than 2-MCPrpC. This difference in 2-MC toxicity is likely due to the fact that as a si-citrate synthase, GltA may produce multiple isomers of 2-MC, which we propose are not substrates for the 2-MC dehydratase (PrpD) enzyme, accumulate inside the cell, and have deleterious effects on FBPase activity. Our findings may help explain human inborn errors in propionate metabolism.

scholarly journals SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast

1993 ◽  
Vol 122 (1) ◽  
pp. 79-94 ◽  
Author(s):  
EA Whitters ◽  
AE Cleves ◽  
TP McGee ◽  
HB Skinner ◽  
VA Bankaitis
Keyword(s):  
Membrane Protein ◽  
Actin Cytoskeleton ◽  
Protein Function ◽  
De Novo ◽  
Integral Membrane Protein ◽  
Secretory Function ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Golgi Secretory Function

Mutations in the SAC1 gene exhibit allele-specific genetic interactions with yeast actin structural gene defects and effect a bypass of the cellular requirement for the yeast phosphatidylinositol/phosphatidylcholine transfer protein (SEC14p), a protein whose function is essential for sustained Golgi secretory function. We report that SAC1p is an integral membrane protein that localizes to the yeast Golgi complex and to the yeast ER, but does not exhibit a detectable association with the bulk of the yeast F-actin cytoskeleton. The data also indicate that the profound in vivo effects on Golgi secretory function and the organization of the actin cytoskeleton observed in sac1 mutants result from loss of SAC1p function. This cosuppression of actin and SEC14p defects is a unique feature of sac1 alleles as mutations in other SAC genes that result in a suppression of actin defects do not result in phenotypic suppression of SEC14p defects. Finally, we report that sac1 mutants also exhibit a specific inositol auxotrophy that is not exhibited by the other sac mutant strains. This sac1-associated inositol auxotrophy is not manifested by measurable defects in de novo inositol biosynthesis, nor is it the result of some obvious defect in the ability of sac1 mutants to utilize inositol for phosphatidylinositol biosynthesis. Thus, sac1 mutants represent a novel class of inositol auxotroph in that these mutants appear to require elevated levels of inositol for growth. On the basis of the collective data, we suggest that SAC1p dysfunction exerts its pleiotropic effects on yeast Golgi function, the organization of the actin cytoskeleton, and the cellular requirement for inositol, through altered metabolism of inositol glycerophospholipids.

scholarly journals Reassessment of the Late Steps of Coenzyme B12 Synthesis in Salmonella enterica: Evidence that Dephosphorylation of Adenosylcobalamin-5′-Phosphate by the CobC Phosphatase Is the Last Step of the Pathway

2007 ◽  
Vol 189 (6) ◽  
pp. 2210-2218 ◽  
Author(s):  
Carmen L. Zayas ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Salmonella Enterica ◽  
De Novo ◽  
Growth Conditions ◽  
Phosphoryl Group ◽  
Coenzyme B12 ◽  
Assembly Pathway ◽  
Serovar Typhimurium ◽  
Cobyric Acid

ABSTRACT We report that cobC strains of Salmonella enterica serovar Typhimurium are impaired in the ability to salvage cobyric acid (Cby), a de novo corrin ring biosynthetic intermediate, under aerobic growth conditions. In vivo and in vitro evidence support the conclusion that this new phenotype of cobC strains is due to the inability of serovar Typhimurium to dephosphorylate adenosylcobalamin-5′-phosphate (AdoCbl-5′-P), the product of the condensation of α-ribazole-5′-phosphate (α-RP) and adenosylcobinamide-GDP by the AdoCbl-5′-P synthase (CobS, EC 2.7.8.26) enzyme. Increased flux through the 5,6-dimethylbenzimidazole and cobinamide (Cbi) activation branches of the nucleotide loop assembly pathway in cobC strains restored AdoCbl-5′-P synthesis from Cby in a cobC strain. The rate of the CobS-catalyzed reaction was at least 2 orders of magnitude higher with α-RP than with α-ribazole as substrate. On the basis of the data reported herein, we conclude that removal of the phosphoryl group from AdoCbl-5′-P is the last step in AdoCbl biosynthesis in serovar Typhimurium and that the reaction is catalyzed by the AdoCbl-5′-P phosphatase (CobC) enzyme. Explanations for the correction of the Cby salvaging phenotype are discussed.

scholarly journals Mutational Analysis of the Residue at Position 48 in the Salmonella enterica Serovar Typhimurium PhoQ Sensor Kinase

2003 ◽  
Vol 185 (6) ◽  
pp. 1935-1941 ◽  
Author(s):  
Sarah Sanowar ◽  
Alexandre Martel ◽  
Hervé Le Moual
Keyword(s):  
Amino Acid ◽  
Phosphatase Activity ◽  
Salmonella Enterica ◽  
Mutational Analysis ◽  
Regulatory System ◽  
Single Amino Acid ◽  
Serovar Typhimurium ◽  
Constitutively Active

ABSTRACT The PhoP/PhoQ two-component regulatory system of Salmonella enterica serovar Typhimurium plays an essential role in controlling virulence by mediating the adaptation to Mg2+ depletion. The pho-24 allele of phoQ harbors a single amino acid substitution (T48I) in the periplasmic domain of the PhoQ histidine kinase sensor. This mutation has been shown to increase net phosphorylation of the PhoP response regulator. We analyzed the effect on signaling by PhoP/PhoQ of various amino acid substitutions at this position (PhoQ-T48X [X = A, S, V, I, or L]). Mutations T48V, T48I, and T48L were found to affect signaling by PhoP/PhoQ both in vivo and in vitro. Mutations PhoQ-T48V and PhoQ-T48I increased both the expression of the mgtA::lacZ transcriptional fusion and the net phosphorylation of PhoP, conferring to cells a PhoP constitutively active phenotype. In contrast, mutation PhoQ-T48L barely responded to changes in the concentration of external Mg2+, in vivo and in vitro, conferring to cells a PhoP constitutively inactive phenotype. By analyzing in vitro the individual catalytic activities of the PhoQ-T48X sensors, we found that the PhoP constitutively active phenotype observed for the PhoQ-T48V and PhoQ-T48I proteins is solely due to decreased phosphatase activity. In contrast, the PhoP constitutively inactive phenotype observed for the PhoQ-T48L mutant resulted from both decreased autokinase activity and increased phosphatase activity. Our data are consistent with a model in which the residue at position 48 of PhoQ contributes to a conformational switch between kinase- and phosphatase-dominant states.

scholarly journals An Unexpected Role for the Periplasmic Phosphatase PhoN in the Salvage of B6 Vitamers in Salmonella enterica

2020 ◽  
Author(s):  
Huong N. Vu ◽  
Diana M. Downs
Keyword(s):  
Salmonella Enterica ◽  
De Novo ◽  
Active Form ◽  
Biologically Active ◽  
General Role ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Domains Of Life

Pyridoxal 5′-phosphate (PLP) is the biologically active form of vitamin B6 essential for cellular function in all domains of life. In many organisms, such as Salmonella enterica serovar Typhimurium and Escherichia coli, this cofactor can be synthesized de novo, or salvaged from B6 vitamers in the environment. Unexpectedly, S. enterica strains blocked in PLP biosynthesis were able to use exogenous PLP and pyridoxine 5′-phosphate (PNP) as the source of this required cofactor, while E. coli strains of the same genotype could not. Transposon mutagenesis found that phoN was essential for the salvage of PLP and PNP under the conditions tested. phoN encodes a class A nonspecific acid phosphatase (EC 3.1.3.2) that is transcriptionally regulated by the PhoPQ two-component system. The periplasmic location of PhoN was essential for PLP and PNP salvage, and in vitro assays confirmed PhoN has phosphatase activity with PLP and PNP as substrates. The data suggest that PhoN dephosphorylates B6 vitamers, after which they enter the cytoplasm and are phosphorylated by kinases of the canonical PLP salvage pathway. The connection of phoN with PhoPQ, and the broad specificity of the gene product suggests S. enterica is exploiting a moonlighting activity of PhoN for PLP salvage. IMPORTANCE Nutrient salvage is a strategy used by species across domains of life to conserve energy. Many organisms are unable to synthesize all required metabolites de novo and must rely exclusively on salvage. Others supplement de novo synthesis with the ability to salvage. This study identified an unexpected mechanism present in S. enterica that allows salvage of phosphorylated B6 vitamers. In vivo and in vitro data herein determined that the periplasmic phosphatase PhoN can facilitate the salvage of PLP and PNP. We suggest a mechanistic working model of PhoN-dependent utilization of PLP and PNP and discuss the general role of promiscuous phosphatases and kinases in organismal fitness.

scholarly journals A Novel Golgi Membrane Protein Is a Partner of the ARF Exchange Factors Gea1p and Gea2p

2003 ◽  
Vol 14 (6) ◽  
pp. 2357-2371 ◽  
Author(s):  
Sophie Chantalat ◽  
Rëgis Courbeyrette ◽  
Francesca Senic-Matuglia ◽  
Catherine L. Jackson ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Trafficking ◽  
Transmembrane Domain ◽  
Integral Membrane Protein ◽  
Membrane Dynamics ◽  
General Interest ◽  
Single Mutation ◽  
Golgi Membrane ◽  
Nucleotide Exchange

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking. The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors. However, these interactions remain largely unknown. Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs. Gmh1p is an evolutionarily conserved protein. We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p. This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins. A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain. We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER. Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function. These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins.

scholarly journals Regulation of igaA and the Rcs System by the MviA Response Regulator in Salmonella enterica

2009 ◽  
Vol 191 (8) ◽  
pp. 2743-2752 ◽  
Author(s):  
Clara B. García-Calderón ◽  
Josep Casadesús ◽  
Francisco Ramos-Morales
Keyword(s):  
Membrane Protein ◽  
Salmonella Enterica ◽  
Response Regulator ◽  
Regulatory System ◽  
Enteric Bacteria ◽  
Lon Protease ◽  
Dependent Manner ◽  
Independent Manner ◽  
Serovar Typhimurium

ABSTRACT IgaA is a membrane protein that prevents overactivation of the Rcs regulatory system in enteric bacteria. Here we provide evidence that igaA is the first gene in a σ70-dependent operon of Salmonella enterica serovar Typhimurium that also includes yrfG, yrfH, and yrfI. We also show that the Lon protease and the MviA response regulator participate in regulation of the igaA operon. Our results indicate that MviA regulates igaA transcription in an RpoS-dependent manner, but the results also suggest that MviA may regulate RcsB activation in an RpoS- and IgaA-independent manner.

scholarly journals Role of sapA and yfgA in Susceptibility to Antibody-Mediated Complement-Dependent Killing and Virulence of Salmonella enterica Serovar Typhimurium

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Edna M. Ondari ◽  
Jennifer N. Heath ◽  
Elizabeth J. Klemm ◽  
Gemma Langridge ◽  
Lars Barquist ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Protein Translocation ◽  
Invasive Disease ◽  
Immune Serum ◽  
Insertion Mutant ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Serum Killing ◽  
Increased Sensitivity

ABSTRACT The ST313 pathovar of Salmonella enterica serovar Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of S. Typhimurium. Vaccination is an attractive disease-prevention strategy, and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of S. Typhimurium to immune serum identified a repertoire of S. Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation, and metabolism. We generated defined mutant derivatives using S. Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S. Typhimurium yfgA mutant lost the characteristic Salmonella rod-shaped appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes, and demonstrated decreased ability to infect mice. In contrast, the S. Typhimurium sapA mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile and an increased ability to infect enterocytes compared with the wild type, but it had no increased ability to cause in vivo infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence and suggest the presence of different mechanisms of antibody resistance.

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