Preliminary Characterization of an Antibiotic Produced by Xanthomonas albilineans Which Inhibits DNA Synthesis in Escherichia coli

Birch, Robert G.; Patil, Suresh S.

doi:10.1099/00221287-131-5-1069

Cited by 56 publications

(90 citation statements)

References 10 publications

(8 reference statements)

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“…Escherichia coli DH5a, used as an albicidin biosensor and as the host strain for DNA cloning and expression of the GST-AlbA fusion protein, was grown at 37˚C in Luria-Bertani medium. X. albilineans XA13, used for albicidin production, was grown at 28˚C in SP medium (Birch & Patil, 1985b). Broth cultures were aerated by shaking at 200 r.p.m.…”

Section: Methodsmentioning

confidence: 99%

“…The protein concentration was determined by means of the DC protein assay (Bio-Rad) with BSA as the standard. Albicidins were produced in culture by X. albilineans as described previously (Birch & Patil, 1985b;Zhang et al, 1998a). The major albicidin peak after C18 HPLC was crystallized before use in kinetic and stoichiometry studies.…”

Section: Methodsmentioning

confidence: 99%

“…The exquisite sensitivity is due to illicit uptake of albicidins through a nucleoside uptake channel (Birch et al, 1990). Inhibition of DNA replication in bacteria and plastids is the primary mechanism of action (Birch & Patil, 1985b, 1987. Several albicidin-resistance mechanisms have been identified, including albicidin-binding proteins (Walker et al, 1988;Basnayake & Birch, 1995) and an esterase which hydrolyses and detoxifies albicidin (Zhang & Birch, 1997).…”

Section: Introductionmentioning

confidence: 99%

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Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

Weng

et al. 2003

Microbiology

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The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30 %, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K. oxytoca ATCC 13182 T to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His 78 , His 125 , His 141 and His 189 ) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95 %. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1: 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that His 125 might be the residue required for albicidin binding. Mutation of His 125 to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His 125 totally abolished binding activity. Mutation of His 125 to arginine and tyrosine had no effect. These results indicate that His 125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

Weng

et al. 2003

Microbiology

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“…Albicidins are bactericidal at nanomolar concentrations against a range of Gram-positive and Gramnegative bacteria (Birch & Patil, 1985b). Inhibition of prokaryote DNA replication is the primary mechanism of action (Birch & Patil, 1985a, 1987b.…”

Section: Introductionmentioning

confidence: 99%

High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca

Zhang

Birch

1998

Microbiology

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Albicidins are a family of phytotoxins and antibiotics which play an important role in the pathogenesis of sugarcane leaf scald disease. The albA gene from Klebsiella oxytoca encodes a protein which inactivates albicidin by heat-reversible binding. Albicidin ligand binding to a recombinant AlbA protein, purified by means of a glutathione S-transferase gene fusion system, is an almost instant and saturable reaction. Kinetic and stoichiometric analysis of the binding reaction indicated the presence of a single high affinity binding site with a dissociation constant of 6.4 x 10−8 M. The AlbA-albicidin complex is stable from 4 to 40 °, from pH 5 to 9 and in high salt solutions. Treatment with protein denaturants released all bound albicidin. These properties indicate that AlbA may be a useful affinity matrix for selective purification of albicidin antibiotics. AlbA does not bind to p-nitrophenyl butyrate or α-naphthyl butyrate, the substrates of the albicidin detoxification enzyme AlbD from Pantoea dispersa. The potential exists to pyramid genes for different mechanisms in transgenic plants to protect plastid DNA replication from inhibition by albicidins.

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“…Inhibition of prokaryote DNA replication was identified as the primary mode of action of albicidin, based on studies of incorporation of labelled precursors (Birch & Patil, 1985a). A combination of ultrastructural, genetic and biochemical evidence indicates that a member of the albicidin family of toxins produced by X. albilineans in invaded xylem causes chlorosis in sugarcane leaf scald disease by blocking plastid DNA replication, resulting in blocked chloroplast differentiation (Birch & Patil, 1983, 1987a. Albicidins are, therefore, of interest as phytotoxins, potential clinical antibiotics, and as tools to study prokaryotic DNA replication.…”

Section: Introductionmentioning

confidence: 99%

Stable albicidin resistance in Escherichia coli involves an altered outer-membrane nucleoside uptake system

Birch¹,

Pemberton²,

Basnayake³

1990

Journal of General Microbiology

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Albicidin blocked DNA synthesis in intact cells of a PolA-EndA-Escherichia cofi strain, and in permeabilized cells supplied with all necessary precursor nucleotides, indicating a direct effect on prokaryote DNA replication. Replication of phages T4 and T7 was also blocked by albicidin in albicidin-sensitive (Albs) but not in albicidinresistant (Alb') E. coli host-cells. All stable spontaneous Albr mutants of E. cofi simultaneously became resistant to phage T6. The locus determining albicidin sensitivity mapped at tsx, the structural gene for an outer-membrane protein used as a receptor by phage T6 and involved in transport through the outer membrane of nucleosides present at submicromolar extracellular concentrations. Albicidin does not closely resemble a nucleoside in structure. However, Albs E. cofi strains rapidly accumulated both nucleosides and albicidin from the surrounding medium whereas the Albr mutants were defective in uptake of nucleosides and albicidin at low extracellular concentrations. An insertion mutation blocking Tsx protein production also blocked albicidin uptake and conveyed albicidin resistance. Albicidin supplied at approximately 0-1 PM blocked DNA replication within seconds in intact Albs E. cofi cells, but a 100-fold higher albicidin concentration was necessary for a rapid inhibition of DNA replication in permeabilized cells. We conclude that albicidin is effective at very low concentrations against E. cofi because it is rapidly concentrated within cells by illicit transport through the tsx-encoded outer-membrane channel normally involved in nucleoside uptake. Albicidin resistance results from loss of the mechanism of albicidin transport through the outer membrane.

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Preliminary Characterization of an Antibiotic Produced by Xanthomonas albilineans Which Inhibits DNA Synthesis in Escherichia coli

Cited by 56 publications

References 10 publications

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca

Stable albicidin resistance in Escherichia coli involves an altered outer-membrane nucleoside uptake system

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