EntG activity of Escherichia coli enterobactin synthetase

Staab, Janet F.; Earhart, Charles F.

doi:10.1128/jb.172.11.6403-6410.1990

Cited by 28 publications

(32 citation statements)

References 47 publications

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“…DPH-2 was a spontaneous heme utilization mutant isolated from P. shigelloides 9 after nalidixic acid enrichment as previously described (20). DHE-1, a hemA entB mutant of E. coli, was created by P1 transduction of a lysate from the entB mutant E. coli AB1515.24 (48) into the hemA mutant RK1065L.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Plesiomonas shigelloides Genes Encoding the Heme Iron Utilization System

et al. 2001

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Plesiomonas shigelloides is a gram-negative pathogen which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a laboratory strain of Escherichia coli were isolated. In the present study, the cloned P. shigelloides sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes, hugA, hugZ, and tonB, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the P. shigelloides genes were tested in E. coli K-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, or hugZ were required for heme iron utilization. When the genes were tested in a hemA entB mutant of E. coli, hugWXZ were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source. hugA could replace the Vibrio cholerae hutA in a heme iron utilization assay, and V. cholerae hutA could complement a P. shigelloides heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonB mutants of both E. coli and V. cholerae and that it was similar to the V. cholerae TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium.Plesiomonas shigelloides is a gram-negative bacterium associated with diarrheal disease in humans (4). The organism has been reported to cause several types of gastroenteritis, including acute secretory gastroenteritis (33), an invasive shigellosislike disease (35), and a cholera-like illness (55). Extraintestinal infections, such as meningitis, bacteremia (2), and pseudoappendicitis (13), are also associated with P. shigelloides infection.Many bacterial pathogens have iron transport systems that play a critical role in allowing the organism to establish an infection (for reviews, see references 31 and 41). Several bacterial pathogens, including P. shigelloides (9), obtain iron from heme or heme-containing compounds. Heme iron utilization systems have been examined in many gram-negative pathogens, including Vibrio cholerae (20,21,38), Vibrio vulnificus (30), Shigella dysenteriae (37, 60), Escherichia coli O157:H7 (54), Serratia marcescens (16, 26, 27), yersiniae (22, 50, 51, 53), Haemophilus (8,11,24,32), neisseriae (7,29,52,64), Pseudomonas (23,28,39), and Porphyromonas gingivalis (47). Many heme iron utilization systems studied to date require an outer membrane receptor which binds heme from the environment (for a review, see reference 58). TonB, with the aid of ExbBD, is thought to interact with the receptor to allow movement of heme into the periplasm. A periplasmic binding protein then moves the heme across th...

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Section: Methodsmentioning

confidence: 99%

Characterization of the Plesiomonas shigelloides Genes Encoding the Heme Iron Utilization System

et al. 2001

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“…Pre-stained protein molecular mass standards (high-range; Gibco-BRL), were used and where appropriate gels were stained with Coomassie Brilliant Blue G. Western blot analyses were performed as described by Staab & Earhart (1990). Antisera against EntB, E and F were prepared as described by Ammerlaan (1994) and Staab & Earhart (1990), anti-PhoA antiserum was purchased from 5 Prime + 3 Prime Inc. ; anti-T7gpl0 antiserum was from Novagen, and anti-acyl carrier protein antiserum was provided by C. Rock, St Jude Children's Research Hospital, respectively. Horseradish-peroxidase-conjugated goat antirabbit IgG (Bio-Rad) served as secondary antibody.…”

Section: Electrophoresis Of Proteins and Western Blot Analysismentioning

confidence: 99%

“…EntB/G and EntD are essential but their roles have not been determined. Ent B/G (32.5 kDa) seems to be a bifunctional protein (Staab & Earhart, 1990) required both for an early step in Ent biosynthesis (EntB activity ;Rusnak et al, 1990) and an unidentified later step (EntG activity) (Greenwood & Luke, 1976, 1980Woodrow et al, 1975); the early and late synthetic activities of Ent B/G are separable by mutation and appear to reside in the N-and C-termini of the protein, respectively. That EntB and EntG activities are on the same polypeptide is supported by the detection (unpublished results) of homology between the C-terminus of EntB/G and the N-terminus of iron-regulated HMWP2 of Yersinia enterocolitica (Guilvout et al, 1993) ; the homologous regions include a consensus substrate amino acid binding site for enzymes engaged in non-ribosomal peptide synthesis (Schlumbohm et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

Enterobactin Synthase Polypeptides of Escherichia Coli are Present in an Osmotic-Shock-Sensitive Cytoplasmic Locality

1997

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The terminal reactions in the synthesis of the siderophore enterobactin (Ent) by Escherichia coli require the EntD, E, F and B/G polypeptides. The idea that these molecules form a complex (Ent synthase) that is membrane-associated was re-evaluated. In vitro results provided no evidence in support of the proposal : (i) Ent synthase activity occurred normally under conditions where membrane was either absent or disrupted by high concentrations of neutral detergents, and (ii) immunoprecipitation experiments conducted on extracts engaged in E n t synthesis failed to detect any association among the Ent polypeptides. However, Western blot analyses showed that EntE, F and B/G were released from cells by osmotic shock and freeze/thaw treatment but not by conversion of cells to spheroplasts. These results demonstrated that EntE, F and B/G belong to the Beacham group D class of proteins. The shockability of a given group D Ent protein was unaffected by the absence of either EntB/G or EntD and, for EntB/G, the N-terminus was sufficient for release by osmotic shock. The behaviour of group D proteins is generally attributed to their association (partial, loose or transient) with cytoplasmic membrane; therefore, the results are indirect evidence that Ent synthase interacts with membrane in vivo. At the very least, the data indicate that EntE, F and B/G are compartmentalized in E. coli and, because other biosynthetic enzymes for siderophores and surfactants are related to these Ent proteins, suggest that this entire protein class may be sequestered in vivo.

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“…Enterobactin was first purified and characterized from Salmonella enterica serovar Typhimurium and E. coli culture supernatants (34,77,84,90,108). Figure 6A shows a schematic map of the enterobactin gene cluster, including the transcrip- (8,34,52,77).…”

Section: Introductionmentioning

confidence: 99%

Genetics and Assembly Line Enzymology of Siderophore Biosynthesis in Bacteria

Crosa

Walsh

2002

Microbiol Mol Biol Rev

727

697

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The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium

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EntG activity of Escherichia coli enterobactin synthetase

Cited by 28 publications

References 47 publications

Characterization of the Plesiomonas shigelloides Genes Encoding the Heme Iron Utilization System

Characterization of the Plesiomonas shigelloides Genes Encoding the Heme Iron Utilization System

Enterobactin Synthase Polypeptides of Escherichia Coli are Present in an Osmotic-Shock-Sensitive Cytoplasmic Locality

Genetics and Assembly Line Enzymology of Siderophore Biosynthesis in Bacteria

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