Cluster of genes controlling synthesis and activation of 2,3-dihydroxybenzoic acid in production of enterobactin in Escherichia coli

Nahlik, M S; Fleming, Timothy P.; McIntosh, Mark A.

doi:10.1128/jb.169.9.4163-4170.1987

Cited by 62 publications

(59 citation statements)

References 44 publications

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“…To meet the specific genetic requirements of our mutagenesis strategy, a derivative of pSUP202 (Table 1) (37), pLG51, was created by restricting pSUP202 with EcoRI, filling in the cut ends with Klenow fragment, and recircularizing the plasmid; this procedure inactivated the chloramphenicol resistance marker of the parent plasmid. A 6.3-kb HindIII fragment of the B. japonicum genome containing the N-terminal portion of sucA was then subcloned from pMDH9.0 (Table 1) into the HindIII site of pLG51 to form pLG11 and transformed into E. coli MC4160 (27). The plasmid was then mutagenized with the transposon Tn10-miniKan, delivered via a lambda phage derivative (43).…”

Section: Methodsmentioning

confidence: 99%

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate

Green

Emerich

1997

J Bacteriol

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The sucA gene, encoding the E1 component of ␣-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of ␣-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, ␤-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of ␣-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.Bradyrhizobium japonicum is a gram-negative soil bacterium that forms nitrogen-fixing symbioses with soybean. During the development of the symbiosis, the bacteria induce and invade specialized organs, called nodules, on the root of the host plant (5,26,44). The nodules house the bacteria and provide an environment conducive to nitrogen fixation, including a reduced oxygen tension to protect nitrogenase and a steady supply of nutrients to the endosymbiont. Concomitantly with its invasion of the nodule, B. japonicum differentiates from a free-living to a symbiotic form (bacteroid), a process involving extensive adjustments to its nitrogen and carbon metabolism (25,40).Bacteroid respiration during nitrogen fixation depends entirely on reduced carbon supplied by the host plant. The main carbon compounds used by fully differentiated bacteroids are four-carbon dicarboxylic acids, the metabolism of which has been assumed to occur via the tricarboxylic acid (TCA) cycle (25,40). However, some studies indicate that the TCA cycle may not be operating to full capacity during bacteroid respiration. For example, the O 2 limitation of bacteroid respiration inside the soybean nodule can lead to increased NADH/NAD ratios and thus to significant inhibition of ␣-ketoglutarate dehydrogenase (34). Such studies imply that the respiration rate of B. japonicum bacteroids may in part be limited by carbon flux through certain steps of the TCA cycle.To maintain carbon flux under oxygen-limiting conditions, bacteroids coul...

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Section: Methodsmentioning

confidence: 99%

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate

Green

Emerich

1997

J Bacteriol

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“…Walsh and coworkers (39,40), Earhart and coworkers (41,42), and McIntosh and coworkers (43,44) have shown that enterobactin is synthesized from a fork in the aromatic amino acid pathway in a two-step process (40,41,45). Chorismic acid, an aromatic amino acid precursor, is converted into isochorismate, then into 2,3-dihydro-2,3-dihydroxybenzoate, and finally into 2,3-dihydroxybenzoic acid (DHB).…”

Section: Biosynthesis Of Enterobactinmentioning

confidence: 99%

Enterobactin: An archetype for microbial iron transport

Raymond

Dertz

Kim

2003

Proc. Natl. Acad. Sci. U.S.A.

805

692

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Bacteria have aggressive acquisition processes for iron, an essential nutrient. Siderophores are small iron chelators that facilitate cellular iron transport. The siderophore enterobactin is a triscatechol derivative of a cyclic triserine lactone. Studies of the chemistry, regulation, synthesis, recognition, and transport of enterobactin make it perhaps the best understood of the siderophore-mediated iron uptake systems, displaying a lot of function packed into this small molecule. However, recent surprises include the isolation of corynebactin, a closely related trithreonine triscatechol derivative lactone first found in Gram-positive bacteria, and the crystal structure of a ferric enterobactin complex of a protein identified as an antibacterial component of the human innate immune system.

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“…(The AN462 genotype was reported to result from Mu-induced polarity, and it is now clear that the rightmost operon of the Ent gene cluster consists of the genes entCEB[G]A and is transcribed from left to right [14,20,27,30], but more recently Laird and Young [19] suggested that a Mu-induced deletion of entB [G]A was responsible for the AN462 characteristics. Some recent workers [27,28] have adopted the latter suggestion.) Weak genetic complementation of the AN462 EntG defect occurs with plasmids containing an approximately 700-bp fragment that includes portions of entB and entA (28).…”

mentioning

confidence: 98%

“…Some recent workers [27,28] have adopted the latter suggestion.) Weak genetic complementation of the AN462 EntG defect occurs with plasmids containing an approximately 700-bp fragment that includes portions of entB and entA (28). However, recent sequencing data show that no separate and distinct open reading frame that could correspond to entG exists between entB and entA (20,27 MATERIALS AND METHODS Bacteria, plasmids, and bacteriophages.…”

mentioning

confidence: 98%

EntG activity of Escherichia coli enterobactin synthetase

Staab

Earhart

1990

J Bacteriol

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The last steps in the biosynthesis of the Escherichia coli siderophore enterobactin (Ent) are carried out by Ent synthetase, a multienzyme complex believed to be composed of the entD, -E, -F, and -G products (EntD to -G). However, sequencing data showed that there is no separate eniG gene and, unlike EntD to -F, no distinct EntG polypeptide has been identified. In this study, genetic, biochemical, and immunological approaches were used to study the anomalies associated with EntG activity. Two plasmids, pJS43 and pJS100, were isolated that had mutations resulting in truncated EntB proteins; both had the phenotype EntB+ EntG-. PJS43 had a TnS inserted 198 bp from the entB termination codon, and pJSlOO had the last 25 codons of entB deleted. Plasmids isolated with TnS insertions in the 5' half of entB had the phenotype EntB-EntG+. These latter TnS mutations were EntB-EntG-when moved to the bacterial chromosome. Polyclonal antiserum was prepared and shown to react only with intact EntB in Western immunoblots. Addition of anti-EntB antiserum to Ent synthetase assays resulted in complete inhibition of enzyme activity, whereas preimmune serum had no effect. Lastly, AN462, the type strain for entG which was derived by Mu insertion and which has the phenotype EntB-G-A-, was characterized. Southern blot data showed a Mu insertion, presumably with polar effects, in the vicinity of the 5' end of entB. In summary, EntG activity was found to be encoded by the entB 3' terminus. The evidence, while not rigorously eliminating the possibility that a separate EntG polypeptide exists, strongly supports the idea that EntB is a bifunctional protein.

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Cluster of genes controlling synthesis and activation of 2,3-dihydroxybenzoic acid in production of enterobactin in Escherichia coli

Abstract: The Escherichia coli gene cluster encoding enzymatic activities responsible for the synthesis and activation of 2,3-dihydroxybenzoic acid in the formation of the catechol siderophore enterobactin was localized to a 4.

Cited by 62 publications

References 44 publications

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate

Enterobactin: An archetype for microbial iron transport

EntG activity of Escherichia coli enterobactin synthetase

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