Cloning and expression of the tabtoxin biosynthetic region from Pseudomonas syringae

Kinscherf, Thomas G.; Coleman, Roger; Barta, T. M.; Willis, David K.

doi:10.1128/jb.173.13.4124-4132.1991

Cited by 58 publications

(69 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2, both toxin production and toxin resistance were associated with the span of the region encompassing blocks II and III; this span also includes the prophage fragment that seems likely to be a vestigial remnant of a transfer event. Previous results had demonstrated that Tn5 insertions within the tabtoxin region resulted in the loss of toxin production without loss of host resistance to the antibiotic [5]. Sequence analysis confirms that the insertions in two of those mutants occurred in the tabC and tblS genes indicating that those two genes are required for antibiotic biosynthesis.…”

Section: Iv) Gene Cluster Blockmentioning

confidence: 56%

“…2A) or that vector with the BamHI fragment insert (Fig. 2B), were inoculated into an M9 agar overlay and then challenged with a paper disk containing semipurified tabtoxin [5].…”

Section: Iv) Gene Cluster Blockmentioning

confidence: 99%

“…While most b -lactam antibiotics exert their antimicrobial influence through the inhibition of bacterial cell wall synthesis, TBL appears to irreversibly inhibit the enzyme glutamine synthetase, causing cells to become intoxicated by high levels of their own unprocessed ammonia [3,4]. The genes encoding the pathway for both tabtoxin production and host resistance were originally cloned as a single cosmid insert from BR2, a Pseudomonas syringae strain causal to wildfire disease on bean [5]. Here we report the analysis of the DNA sequence of this cloned region.…”

mentioning

confidence: 99%

See 2 more Smart Citations

The Biosynthetic Gene Cluster for the β-Lactam Antibiotic Tabtoxin in Pseudomonas syringae

Kinscherf

Willis

2005

J Antibiot

View full text Add to dashboard Cite

DNA sequence analysis revealed that the biosynthetic genes of the unusual b -lactam antibiotic tabtoxin reside at the att site adjacent to the lysC tRNA gene in Pseudomonas syringae BR2. ORFs encoded within the region included ones with similarity to b -lactam synthase and clavaminic acid synthase, as well as amino acid synthesis enzymes. Novel ORFs were present in a portion of the biosynthetic region associated with a toxin hypersensitivity phenotype. Tabtoxin resistance was associated with a fragment containing a major facilitator superfamily (MFS) transporter gene. (Fig. 1). The antibiotic is usually associated with bacteria of the Pseudomonas syringae species group causal to several types of chlorotic plant disease. While most b -lactam antibiotics exert their antimicrobial influence through the inhibition of bacterial cell wall synthesis, TBL appears to irreversibly inhibit the enzyme glutamine synthetase, causing cells to become intoxicated by high levels of their own unprocessed ammonia [3,4]. The genes encoding the pathway for both tabtoxin production and host resistance were originally cloned as a single cosmid insert from BR2, a Pseudomonas syringae strain causal to wildfire disease on bean [5]. Here we report the analysis of the DNA sequence of this cloned region.Subcloned fragments of the cosmid pRTBL823 were sequenced using a combination of primer walking with synthetic primers derived from endogenous sequence within the insert DNA and transposon priming (GPS-1, New England Biolabs). Sequence of plasmid miniprep DNA (Qiagen) was generated using dye terminator kits (ABI) analyzed by capillary electrophoresis (GeneAnalyser 310, ABI). The sequence of overlaps between subclones was confirmed by sequencing with the original cosmid

show abstract

Section: Iv) Gene Cluster Blockmentioning

confidence: 56%

“…2A) or that vector with the BamHI fragment insert (Fig. 2B), were inoculated into an M9 agar overlay and then challenged with a paper disk containing semipurified tabtoxin [5].…”

Section: Iv) Gene Cluster Blockmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

The Biosynthetic Gene Cluster for the β-Lactam Antibiotic Tabtoxin in Pseudomonas syringae

Kinscherf

Willis

2005

J Antibiot

View full text Add to dashboard Cite

show abstract

“…syringae, coronatine (22,40) produced by P. syringae pv. tomato, and tabtoxin (15,38) produced by P. syringae pv. tabaci, have also been reported to be clustered.…”

Section: Discussionmentioning

confidence: 99%

Genetic organization of a cluster of genes involved in the production of phaseolotoxin, a toxin produced by Pseudomonas syringae pv. phaseolicola

1993

View full text Add to dashboard Cite

Phaseolotoxin [NVf(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl-homoarginineI produced by Pseudomonas syringae pv. phaseolicola, the bean halo blight pathogen, is a potent inhibitor of ornithine carbamoyltransferase (OCT). Inhibition of OCT in infected plants leads to chlorosis and growth inhibition. A genomic cosmid clone, pHK120, containing a 25-kb fragment of DNA from a wild-type strain of P. syringae pv. phaseolicola restores toxin production in Tox-mutants. TnS mutagenesis of pHK120 and marker exchange of pHK120::TnS plasmids in the wild-type strain resulted in the isolation of 39 chromosomal mutants that harbor TnS insertions at known positions. Toxin bioassays revealed that 28 of the mutants, with TnS insertions distributed throughout the insert of pHK120, were Tox-, indicating that a functional locus for toxin production in each mutant was inactivated. Complementation analysis was done by testing for toxin production strains that carried a genomic TnS at one location and a plasmid-borne TnS at another location (pair complementation). Pair complementation analysis of nine marker exchange mutants and a random genomic TnS mutant revealed that there are a minimum of eight toxin loci (phtA through phtH) in pHK120. Mutants carrying Tn5 insertions in the phtA, phtD, and phtF loci were complemented by deletion subclones containing fragments from pHK120; mutants carrying Tn5 insertions in the phtC locus were partially complemented by a subclone, and mutants carrying TnS insertions in the phtB, phtE, phtG, and phtH loci were not complemented by any of the available subclones. A comparison of the insert from pHK120 with that from pRCP17, a clone reported previously

show abstract

“…Unkefer et al (40) suggested that the two pathways might diverge after L-dihydrodipicolinate (DHDPA). Reduction to L-2,3,4,5-tetrahydrodipicolinate (THDPA) by DapB (5) would lead to DAP, while hydration would place a hydroxyl group on the carbon that becomes carbon 5 of T␤L.All of the genes required for T␤L biosynthesis and resistance are located in a region of about 32 kb of the BR2 chromosome (19). Approximately 3 kb of that region has been sequenced, and two genes (tabA and tblA), essential for T␤L production but with unknown functions, have been identified.…”

mentioning

confidence: 99%

Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis

Liu

Shaw

1997

J Bacteriol

View full text Add to dashboard Cite

The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis.Pseudomonas syringae pv. tabaci BR2.024, a plasmid-free derivative of strain BR2, is a tabtoxin-producing bacterium that causes wildfire disease on green beans (27, 31). Tabtoxin, a dipeptide, is not biologically active, but hydrolysis by peptidases produces tabtoxinine-␤-lactam (T␤L), the active toxin (22). Feeding studies with labeled precursors (26,34,40) indicated that carbons 1 to 6 of T␤L ( Fig. 1) are derived from aspartate and pyruvate, and labelling patterns led to speculation that the T␤L and lysine biosynthetic pathways may have common initial steps. Although lysine and diaminopimelic acid (DAP) are structurally related to T␤L (Fig. 1), label from these amino acids is not effectively incorporated into T␤L. The methyl group of methionine can provide the ␤-lactam carbonyl carbon. Unkefer et al. (40) suggested that the two pathways might diverge after L-dihydrodipicolinate (DHDPA). Reduction to L-2,3,4,5-tetrahydrodipicolinate (THDPA) by DapB (5) would lead to DAP, while hydration would place a hydroxyl group on the carbon that becomes carbon 5 of T␤L.All of the genes required for T␤L biosynthesis and resistance are located in a region of about 32 kb of the BR2 chromosome (19). Approximately 3 kb of that region has been sequenced, and two genes (tabA and tblA), essential for T␤L production but with unknown functions, have been identified. The deduced tabA product has an amino acid sequence homologous to meso-DAP decarboxylase (the lysA gene product) characterized in other bacteria (9, 32); however, tabA is unable to complement an Escherichia coli lysA mutant (9). The second gene, tblA, encodes a product with an amino acid sequence unrelated to those of known polypeptides (2). The transcription of tblA is positively regulated by ...

show abstract

Cloning and expression of the tabtoxin biosynthetic region from Pseudomonas syringae

Cited by 58 publications

References 41 publications

The Biosynthetic Gene Cluster for the β-Lactam Antibiotic Tabtoxin in Pseudomonas syringae

The Biosynthetic Gene Cluster for the β-Lactam Antibiotic Tabtoxin in Pseudomonas syringae

Genetic organization of a cluster of genes involved in the production of phaseolotoxin, a toxin produced by Pseudomonas syringae pv. phaseolicola

Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis

Contact Info

Product

Resources

About