Importance of Uracil DNA Glycosylase in Pseudomonas aeruginosa and Mycobacterium smegmatis, G+C-rich Bacteria, in Mutation Prevention, Tolerance to Acidified Nitrite, and Endurance in Mouse Macrophages

Jeganathan Venkatesh,Pradeep Kumar,Pulukuri Sai Murali Krishna,Ramanathapuram Manjunath,Umesh Varshney

doi:10.1074/jbc.m302121200

Jeganathan Venkatesh, Pradeep Kumar + Show 3 more

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https://doi.org/10.1074/jbc.m302121200

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Abstract

Uracil DNA glycosylase (Ung (or UDG)) initiates the excision repair of an unusual base, uracil, in DNA. Ung is a highly conserved protein found in all organisms. Paradoxically, loss of this evolutionarily conserved enzyme has not been seen to result in severe growth phenotypes in the cellular life forms. In this study, we chose G+C-rich genome containing bacteria (Pseudomonas aeruginosa and Mycobacterium smegmatis) as model organisms to investigate the biological significance of ung. Ung deficiency was created either by expression of a highly specific inhibitor protein, Ugi, and/or by targeted disruption of the ung gene. We show that abrogation of Ung activity in P. aeruginosa and M. smegmatis confers upon them an increased mutator phenotype and sensitivity to reactive nitrogen intermediates generated by acidified nitrite. Also, in a mouse macrophage infection model, P. aeruginosa (Ung-) shows a significant decrease in its survival. Infections of the macrophages with M. smegmatis show an initial increase in the bacterial counts that remain for up to 48 h before a decline. Interestingly, abrogation of Ung activity in M. smegmatis results in nearly a total abolition of their multiplication and a much-decreased residency in macrophages stimulated with interferon gamma. These observations suggest Ung as a useful target to control growth of G+C-rich bacteria.

Highlights

Among all the bases in DNA, cytosine is highly susceptible to deamination of its exocyclic amino group in response to normal physiological reactions or environmental pollutants, resulting in generation of promutagenic GϫU mismatches in the genome
We show that abrogation of uracil-DNA glycosylase (Ung) activity in P. aeruginosa and M. smegmatis confers upon them an increased mutator phenotype and sensitivity to reactive nitrogen intermediates generated by acidified nitrite
Ugi cloned under M. tuberculosis intiator tRNA gene promoter at Dral site Contains a temperature-sensitive origin of replication and a sacB marker for negative selection in mycobacteria pPR27 harboring disrupted M. smegmatis ung gene for allelic exchange of the chromosomal gene Same as pBBR1MCS (CmR)

Summary

Introduction

Among all the bases in DNA, cytosine is highly susceptible to deamination of its exocyclic amino group in response to normal physiological reactions or environmental pollutants, resulting in generation of promutagenic GϫU mismatches in the genome If these were to be left unrepaired, an exorbitant increase in accrual of G-C to A-T mutations would occur unabated after each replication cycle and pose a serious threat to the genomic integrity and the very survival of the organism. In a mouse macrophage infection model, P. aeruginosa (UngϪ) shows a significant decrease in survival under the conditions of increased RNI production In this assay loss of Ung in M. smegmatis results in near abolition of with 0.2% Tween 80; ORF, open reading frame; FCS, fetal calf serum; IFN, interferon; Eco, E. coli; kb, kilobase(s). A P. aeruginosa strain A high efficiency transformation strain of M. smegmatis Contains pDK20 (vector control) integrated at the L5 att site in genome of M

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