scholarly journals YjhS (NanS) Is Required for Escherichia coli To Grow on 9-O-Acetylated N-Acetylneuraminic Acid

2009 ◽  
Vol 191 (22) ◽  
pp. 7134-7139 ◽  
Author(s):  
Susan M. Steenbergen ◽  
Jamie L. Jirik ◽  
Eric R. Vimr
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Mammalian Host ◽  
E Coli ◽  
Acetylneuraminic Acid

ABSTRACT The nanATEK-yhcH, yjhATS, and yjhBC operons in Escherichia coli are coregulated by environmental N-acetylneuraminic acid, the most prevalent sialic acid in nature. Here we show that YjhS (NanS) is a probable 9-O-acetyl N-acetylneuraminic acid esterase required for E. coli to grow on this alternative sialic acid, which is commonly found in mammalian host mucosal sites.

Utilization of chymotrypsin as a sole carbon and (or) nitrogen source by Escherichia coli

1992 ◽  
Vol 38 (4) ◽  
pp. 290-295 ◽  
Author(s):  
Arthur S. Brecher ◽  
Timothy A. Moehlman ◽  
William D. Hann
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Nitrogen Source ◽  
Degradation Products ◽  
Defined Medium ◽  
Host Protein ◽  
Mammalian Host ◽  
Sole Nitrogen Source ◽  
E Coli ◽  
Nitrogen Nutrients

α-Chymotrypsin serves as a sole carbon source, sole nitrogen source, and as sole carbon plus nitrogen source for wild-type Escherichia coli in a totally defined medium. Hence, a mammalian host for E. coli may supply the necessary carbon and nitrogen nutrients for the microorganism. Growth is most rapid when chymotrypsin is a sole nitrogen source,and least rapid with chymotrypsin as a carbon source. The approximate doubling times for E. coli utilizing chymotrypsin as a nitrogen source, carbon plus nitrogen source, and carbon source are 1.6, 4.6, and 11.3 h, respectively. The activity of the residual enzyme in the culture supernates falls off asymptotically over the course of time, as followed by cleavage of glutaryl-L-phenylalanine-p-nitroanilide. Chymotrypsin hydrolyzes succinyl-L-ala-L-ala-L-ala-p-nitroanilide, the elastase substrate, to some extent. Peptidases do not appear to be secreted that hydrolyze such model substrates as benzoyl-DL-arginine-p-nitroanilide, the tryptic and cathepsin B substrate, L-leucine-p-nitroanilide, the leucine aminopeptidase substrate, or L-lysine-p-nitroanilide, the aminopeptidase B substrate. Growth of E. coli is generally directly related to the loss of chymotryptic activity in the medium. Hence, autolysis of chymotrypsin, i.e., self-degradation, is an important factor for the availability of degradation products of the enzyme to the bacterium for growth purposes. Accordingly, the degradation of a host protein by autolysis presents an opportunity for E. coli to survive during periods of host nutritional crisis by utilization of the degradation peptides that are produced during autolysis. Key words: chymotrypsin, Escherichia coli, growth, nutrition, peptide source.

scholarly journals Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria

2020 ◽  
Vol 295 (40) ◽  
pp. 13724-13736 ◽  
Author(s):  
Andrew Bell ◽  
Emmanuele Severi ◽  
Micah Lee ◽  
Serena Monaco ◽  
Dimitrios Latousakis ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
2D Nmr ◽  
Site Directed Mutagenesis ◽  
Bioinformatics Analyses ◽  
E Coli ◽  
Acetylneuraminic Acid ◽  
Protein Dimer

The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-N-acetylneuraminic acid intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.

K-1 Antigen Content and the Presence of an Additional Sialic Acid-Containing Antigen Among Bacteremic K-1 Escherichia coli : Correlation with Susceptibility to Opsonophagocytosis

1980 ◽  
Vol 29 (3) ◽  
pp. 1055-1061 ◽  
Author(s):  
Paul Stevens ◽  
Chung L. Chu ◽  
Lowell S. Young
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Blood Culture ◽  
Reference Group ◽  
Dry Weight ◽  
E Coli ◽  
Crossed Immunoelectrophoresis ◽  
Fresh Serum ◽  
Group B

Eighty percent of blood culture isolates of Escherichia coli K-1 are resistant to in vitro opsonophagocytosis by normal human granulocytes and fresh serum. To determine the basis for susceptibility to phagocytosis in 20% of bacteremic K-1 E. coli , we investigated possible quantitative and qualitative immunochemical differences in the K-1 antigen content among resistant and sensitive isolates. We prepared extracts of blood culture K-1 E. coli by sonication and determined the K-1 polysaccharide content per dry weight of bacteria by rocket immunoelectrophoresis using cross-reactive equine anti-group B meningococcal sera. We assessed qualitative differences in the antigen content by crossed immunoelectrophoresis, using an immune globulin fraction and isolated immunoglobulin G (IgG) and IgM from the group B antisera. Three different resistant K-1 isolates contained a mean K-1 content of 48.5 ± 7.6 μg/mg ± standard deviation of dry bacteria, and three sensitive isolates contained 23.2 ± 5.6 μg/mg ( P < 0.005). Crossed immunoelectrophoresis of extracts from both sensitive and resistant strains revealed a secondary sialic acid-containing antigen that was electrophoretically different from both the major K-1 antigen and a reference group B meningococcal antigen. This negatively charged secondary antigen was susceptible to Clostridium perfringens neuraminidase degradation and reacted only with IgG whereas the major K-1 antigen reacted only with IgM. This antigen was detected in the extracts of resistant isolates only at 10 10 but not at 10 9 colony-forming units per milliliter. This study demonstrates that (i) the degree of phagocytosis of bacteremic E. coli K-1 isolates is inversely associated with K-1 content, and (ii) more easily phagocytosed (sensitive) K-1 isolates have greater amounts of an additional sialic acid-containing antigen that appears to be unrelated to the previously described O acetyl K-1 antigen.

scholarly journals Unexpected Diversity of Escherichia coli SialateO-Acetyl Esterase NanS

2016 ◽  
Vol 198 (20) ◽  
pp. 2803-2809 ◽  
Author(s):  
Ariel Rangel ◽  
Susan M. Steenbergen ◽  
Eric R. Vimr
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Bacterial Species ◽  
Sialic Acids ◽  
Host Bacterium ◽  
Acetyl Esterase ◽  
Content Type ◽  
E Coli ◽  
Host Microbe Interactions ◽  
K 12

ABSTRACTThe sialic acids (N-acylneuraminates) are a group of nine-carbon keto-sugars existing mainly as terminal residues on animal glycoprotein and glycolipid carbohydrate chains. Bacterial commensals and pathogens exploit host sialic acids for nutrition, adhesion, or antirecognition, whereN-acetyl- orN-glycolylneuraminic acids are the two predominant chemical forms of sialic acids. Each form may be modified by acetyl esters at carbon position 4, 7, 8, or 9 and by a variety of less-common modifications. Modified sialic acids produce challenges for colonizing bacteria, because the chemical alterations toN-acetylneuraminic acid (Neu5Ac) confer increased resistance to sialidase and aldolase activities essential for the catabolism of host sialic acids. Bacteria withO-acetyl sialate esterase(s) utilize acetylated sialic acids for growth, thereby gaining a presumed metabolic advantage over competitors lacking this activity. Here, we demonstrate the esterase activity ofEscherichia coliNanS after purifying it as a C-terminal HaloTag fusion. Using a similar approach, we show thatE. colistrain O157:H7 Stx prophage or prophage remnants invariably include paralogs ofnanSoften located downstream of the Shiga-like toxin genes. These paralogs may include sequences encoding N- or C-terminal domains of unknown function where the NanS domains can act as sialateO-acetyl esterases, as shown by complementation of anE. colistrain K-12nanSmutant and the unimpaired growth of anE. coliO157nanSmutant onO-acetylated sialic acid. We further demonstrate thatnanShomologs inStreptococcusspp. also encode active esterase, demonstrating an unexpected diversity of bacterial sialateO-acetyl esterase.IMPORTANCEThe sialic acids are a family of over 40 naturally occurring 9-carbon keto-sugars that function in a variety of host-bacterium interactions. These sugars occur primarily as terminal carbohydrate residues on host glycoproteins and glycolipids. Available evidence indicates that diverse bacterial species use host sialic acids for adhesion or as sources of carbon and nitrogen. Our results show that the catabolism of the diacetylated form of host sialic acid requires a specialized esterase, NanS. Our results further show thatnanShomologs exist in bacteria other thanEscherichia coli, as well as part of toxigenicE. coliprophage. The unexpected diversity of these enzymes suggests new avenues for investigating host-bacterium interactions. Therefore, these original results extend our previous studies ofnanSto include mucosal pathogens, prophage, and prophage remnants. This expansion of thenanSsuperfamily suggests important, although as-yet-unknown, functions in host-microbe interactions.

scholarly journals Identification and Biochemical Characterization of the Novel α2,3-Sialyltransferase WbwA from Pathogenic Escherichia coli Serotype O104

2015 ◽  
Vol 197 (24) ◽  
pp. 3760-3768 ◽  
Author(s):  
Diana Czuchry ◽  
Paul Desormeaux ◽  
Melissa Stuart ◽  
Donald L. Jarvis ◽  
Khushi L. Matta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Biochemical Characterization ◽  
Enzyme Reaction ◽  
T Antigen ◽  
Glycan Structure ◽  
Content Type ◽  
E Coli ◽  
Pathogenic Escherichia Coli

ABSTRACTThe sialyl-T antigen sialylα2-3Galβ1-3GalNAc is a common O-glycan structure in human glycoproteins and is synthesized by sialyltransferase ST3Gal1. The enterohemorrhagicEscherichia coliserotype O104 has the rare ability to synthesize a sialyl-T antigen mimic. We showed here that thewbwAgene of theE. coliO104 antigen synthesis gene cluster encodes an α2,3-sialyltransferase WbwA that transfers sialic acid from CMP-sialic acid to Galβ1-3GalNAcα-diphosphate-lipid acceptor. Nuclear magnetic resonance (NMR) analysis of purified WbwA enzyme reaction product indicated that the sialyl-T antigen sialylα2-3Galβ1-3GalNAcα-diphosphate-lipid was synthesized. We showed that the conserved His-Pro (HP) motif and Glu/Asp residues of two EDG motifs in WbwA are important for the activity. The characterization studies showed that WbwA fromE. coliO104 is a monofunctional α2,3-sialyltransferase and is distinct from human ST3Gal1 as well as all other known sialyltransferases due to its unique acceptor specificity. This work contributes to knowledge of the biosynthesis of bacterial virulence factors.IMPORTANCEThis is the first characterization of a sialyltransferase involved in the synthesis of an O antigen inE. coli. The enzyme contributes to the mimicry of human sialyl-T antigen and has unique substrate specificity but very little sequence identity to other sialyltransferases. Thus, the bacterial sialyltransferase is related to the human counterpart only by the similarity of biochemical activity.

scholarly journals Uptake of N-acetylneuraminic acid by Escherichia coli K-235. Biochemical characterization of the transport system

1987 ◽  
Vol 246 (2) ◽  
pp. 287-294 ◽  
Author(s):  
L B Rodríguez-Aparicio ◽  
A Reglero ◽  
J M Luengo
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Transport System ◽  
Biochemical Characterization ◽  
Osmotic Shock ◽  
Metabolic Inhibitors ◽  
Acid Uptake ◽  
Uptake System ◽  
Acid Transport ◽  
Acetylneuraminic Acid

Kinetic measurement of the uptake of N-acetyl[4,5,6,7,8,9-14C]neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0. Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM. The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation [2,4-dinitrophenol (100%); NaN3 (66%]) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%). The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar. N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect. Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake. The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E. coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system. Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation. Protein synthesis is not needed for sialic acid transport.

scholarly journals Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase

1999 ◽  
Vol 343 (2) ◽  
pp. 397-402 ◽  
Author(s):  
Daniel M. STOUGHTON ◽  
Gerardo ZAPATA ◽  
Robert PICONE ◽  
Willie F. VANN
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Active Site ◽  
Positive Charge ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Escherichia Coli K1

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 → Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in kcat, Km and Ks for CTP, which implicates Arg-12 in catalysis and substrate binding.

scholarly journals Function and Expression of an N-Acetylneuraminic Acid-Inducible Outer Membrane Channel in Escherichia coli

2005 ◽  
Vol 187 (6) ◽  
pp. 1959-1965 ◽  
Author(s):  
Guy Condemine ◽  
Catherine Berrier ◽  
Jacqueline Plumbridge ◽  
Alexandre Ghazi
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Structural Genes ◽  
Membrane Channel ◽  
Positive Potential ◽  
E Coli ◽  
Acetylneuraminic Acid ◽  
Catabolite Activator Protein ◽  
Intergenic Regions ◽  
Outer Membrane Channel

ABSTRACT The Escherichia coli yjhA (renamed nanC) gene encodes a protein of the KdgM family of outer membrane-specific channels. It is transcribed divergently from fimB, a gene involved in the site-specific inversion of the region controlling transcription of the fimbrial structural genes but is separated from it by one of the largest intergenic regions in E. coli. We show that nanC expression is induced by N-acetylneuraminic acid and modulated by N-acetylglucosamine. This regulation occurs via the NanR and NagC regulators, which also control fimB expression. nanC expression is also activated by the regulators cyclic AMP-catabolite activator protein, OmpR, and CpxR. When the NanC protein was reconstituted into liposomes, it formed channels with a conductance of 450 pS at positive potential and 300 to 400 pS at negative potential in 800 mM KCl. The channels had a weak anionic selectivity. In an ompR background, where the general porins OmpF and OmpC are absent, NanC is required for growth of E. coli on N-acetylneuraminic acid as the sole carbon source. All these results suggest that NanC is an N-acetylneuraminic acid outer membrane channel protein.

scholarly journals EXPERIMENTAL GENETIC RECOMBINATION IN VIVO BETWEEN ESCHERICHIA COLI AND SALMONELLA TYPHIMURIUM

1961 ◽  
Vol 114 (1) ◽  
pp. 141-148 ◽  
Author(s):  
H. Schneider ◽  
Samuel B. Formal ◽  
L. S. Baron
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Intestinal Tract ◽  
Genetic Recombination ◽  
Mammalian Host ◽  
Experimental Conditions ◽  
E Coli

Antibiotic-pretreated mice were fed orally an Hfr culture of streptomycin-resistant E. coli and 1 day later, a streptomycin-resistant F- S. typhimurium culture. Hybrids were recovered in relatively small numbers from the feces of these mice within 24 hours demonstrating that genetic recombination can occur within the intestinal tract of a mammalian host under experimental conditions. These hybrids multiplied rapidly and persisted throughout the course of the experiment. In addition, hybrids were recovered which had not been observed in single matings performed in vitro.

scholarly journals The gusBC Genes of Escherichia coli Encode a Glucuronide Transport System

2005 ◽  
Vol 187 (7) ◽  
pp. 2377-2385 ◽  
Author(s):  
Wei-Jun Liang ◽  
Kate J. Wilson ◽  
Hao Xie ◽  
Jan Knol ◽  
Shun'ichi Suzuki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Expression Vector ◽  
Proton Motive Force ◽  
Mammalian Host ◽  
Transport Activity ◽  
Secondary Active Transport ◽  
E Coli ◽  
Fecal Isolate ◽  
Vector Transport

ABSTRACT Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of β-glucuronides with synthetic [14C]phenyl-1-thio-β-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of β-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.

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