DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus - PubMed (original) (raw)

DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus

Tiegang Xu et al. BMC Microbiol. 2009.

Abstract

Background: A novel DNA phosphorothioate modification (DNA sulfur modification), in which one of the non-bridging oxygen atoms in the phosphodiester bond linking DNA nucleotides is exchanged by sulphur, was found to be genetically determined by dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats. A detailed mutational analysis of the individual genes within the dnd locus in Streptomyces lividans responsible for DNA phosphorothioation was performed and is described here. It should be of great help for the mechanistic study of this intriguing system.

Results: A 6,665-bp DNA region carrying just five ORFs (dndA-E) was defined as the sole determinant for modification of the DNA backbone in S. lividans to form phosphorothioate. This provides a diagnostically reliable and easily assayable Dnd (DNA degradation) phenotype. While dndA is clearly transcribed independently, dndB-E constitute an operon, as revealed by RT-PCR analysis. An efficient mutation-integration-complementation system was developed to allow for detailed functional analysis of these dnd genes. The Dnd- phenotype caused by specific in-frame deletion of the dndA, C, D, and E genes or the enhanced Dnd phenotype resulting from in-frame deletion of dndB could be restored by expression vectors carrying the corresponding dnd genes. Interestingly, overdosage of DndC or DndD, but not other Dnd proteins, in vivo was found to be detrimental to cell viability.

Conclusion: DNA phosphorothioation is a multi-enzymatic and highly coordinated process controlled by five dnd genes. Overexpression of some proteins in vivo prevented growth of host strain, suggesting that expression of the gene cluster is strictly regulated in the native host.

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Figures

Figure 1

Figure 1

Localization of the boundaries for dnd gene cluster. pSET152-derivatives with the ability to confer Dnd (+ or -) phenotypes are indicated in line with their insert fragments. Five arrows from left to right represent five the ORFs of the dnd gene cluster (dndA-E). Directions of the arrows indicate the transcriptional directions of the genes.

Figure 2

Figure 2

RT-PCR analysis of the dnd genes transcripts. dnd gene transcripts were reverse transcribed and amplified. (A) Relative positions and directions of corresponding primers are marked with black arrows. (B) Amplification products with sense primer (SP), anti sense primer (AP) and their corresponding lengths. Intra-dnd gene amplification products are indicated as dnd gene names, while products of regions between dnd genes are named linking two corresponding genes such as AB. Amplification of 16S rRNA is used as an internal control marker (IM). (C) Electrophoresis of RT-PCR products. The amplification products are labeled as in Figure 2B. Reverse transcriptase inactivation (BC*) and without DNase treatment (AB') were carried out as negative and positive controls. DNA markers are labeled as "M".

Figure 3

Figure 3

dnd mutants. Black arrows represent dnd genes and their transcriptional directions. White blocks/arrows represent in-frame deletions in the corresponding genes.

Figure 4

Figure 4

Dnd phenotype of 1326 and related dnd mutants. (A) Dnd phenotype of chromosomal DNA for 1326 and related dnd mutants. (B) Dnd phenotype of plasmids pHZ209 isolated from 1326 and related dnd mutants. (C) Dnd phenotype of chromosomal DNA from complemented dnd mutants. DNA was first treated with TAE (top panel) or peracetic acid TAE (bottom panel) before fractionation by electrophoresis in TAE with added thiourea. M: DNA markers; CCC: covalently closed circular plasmid; OC: open circular plasmid. L: linear plasmid.

Figure 5

Figure 5

Western blotting for detecting expression of Dnd proteins in S. lividans 1326 and derivative strains. Rabbit polyclonal antibody to DndD reacted with the protein extracted from wild-type S. lividans 1326 or strain XTG4/pJTU64 (a pHZ1272-derived dndD expression vector).

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