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The broad host range bacteriophage Mu employs a novel ‘methylcarbamoyl’ modification to protect its DNA from diverse restriction systems of its hosts. The DNA modification is catalyzed by a phage-encoded protein Mom, whose mechanism of action is a mystery. Here, we characterized the co-factor and metal-binding properties of Mom and provide a molecular mechanism to explain ‘methylcarbamoyl’ation of DNA by Mom. Computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom. We demonstrate that Mom binds to acetyl CoA and identify the active site. We discovered that Mom is an iron-binding protein, with loss of Fe2+/3+-binding associated with loss of DNA modification activity. The importance of Fe2+/3+ is highlighted by the colocalization of Fe2+/3+ with acetyl CoA within the Mom active site. Puzzlingly, acid-base mechanisms employed by >309,000 GNAT members identified so far, fail to support methylcarbamoylation of adenine using acetyl CoA. In contrast, free-radical chemistry catalyzed by transition metals like Fe2+/3+ can explain the seemingly challenging reaction, accomplished by collaboration between acetyl CoA and Fe2+/3+. Thus, binding to Fe2+/3+, a small but unprecedented step in the evolution of Mom, allows a giant chemical leap from ordinary acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.

Gre, one of the conserved transcription factors in bacteria, modulates RnA polymerase (RnAp) activity to ensure processivity and fidelity of RNA synthesis. Gre factors regulate transcription by inducing the intrinsic-endonucleolytic activity of RnAp, allowing the enzyme to resume transcription from the paused and arrested sites. While Escherichia coli and a number of eubacteria harbor GreA and GreB, genus mycobacteria has a single Gre (GreA). to address the importance of the GreA in growth, physiology and gene expression of Mycobacterium smegmatis, we have constructed a conditional knockdown strain of GreA. the GreA depleted strain exhibited slow growth, drastic changes in cell surface phenotype, cell death, and increased susceptibility to front-line anti-tubercular drugs. transcripts and 2D-gel electrophoresis (2D-PAGE) analysis of the GreA conditional knock-down strain showed altered expression of the genes involved in transcription regulation. Among the genes analysed, expression of RnAp subunits (β, β' and ω), carD, hupB, lsr2, and nusA were affected to a large extent. Severe reduction in the expression of genes of rRnA operon in the knock-down strain reveal a role for GreA in regulating the core components of the translation process. Transcription is the central process in the cell. It is regulated by a variety of proteins at different stages. Many of these regulators control transcription by modulating the RNAP activity. The movement of RNAP along the template often gets interrupted by pauses resulting in backtracked RNAP. During backtracking, RNAP slides backwards along the DNA and the 3′ end of the newly synthesized RNA is disengaged from the active centre 1,2. In order to continue RNA extension from the 3′ end, bacteria have evolved strategies that serve to assist the backtracked RNAP to reinitiate elongation. The newly synthesized 3′ end of RNA is subjected to intrinsic cleavage by RNAP itself 3-6. Gre factors bind to the RNAP and assist transcript-cleavage 1,7-12. The absence of Gre would prolong or even prevent rescuing of backtracked RNAP complexes leading to a pause or arrest of transcription. This would limit overall rate and processivity of transcription and hence cell survival. Although Gre factors or their homologues are conserved in all forms of life, present understanding of their in vivo role in bacteria other than in E. coli is limited. Even in E. coli, a complete understanding of the effect of Gre mutants is hindered because of the presence of two Gre (GreA and GreB) and also the partial redundancy in function by the other secondary channel binding proteins such as DksA, Rnk and TraR 13-15. While GreA cleaves 2-3 nucleotide from 3′ end of RNA, GreB cleaves up to 9 nucleotide fragments. However, how the other secondary channel binding proteins complement their function is not clearly understood. It has been shown that GreA, GreB and DksA are mutually competing and exhibit functional redundancy 15. In E. coli, a double knockout of greA and greB did not show lethality, although colonies were s...

Mycobacterium tuberculosis (Mtb) has evolved several mechanisms to counter host defence arsenal for its proliferation. We show that Mtb employs multi-pronged approach to modify host epigenetic machinery for its survival upon infection. It secretes methyltransferase (MTase) Rv2067c into macrophages, trimethylating K79 of histone H3 in non-nucleosomal context. Rv2067c downregulates host MTase DOT1L, decreasing its H3K79me3 mark added nucleosomally on pro-inflammatory response genes. Consequent inhibition of caspase8-dependent apoptosis and enhancement of RIPK3-mediated necrosis results in increased pathogenesis. In parallel, Rv2067c enhances the expression of SESTRIN3, NLRC3 and TMTC1 enabling the pathogen to overcome host inflammatory and oxidative response. We provide structural basis for differential methylation of H3 by Rv2067c and DOT1L. The structure of Rv2067c and DOT1L explain how their action on H3K79 is temporally and spatially separated enabling Rv2067c to effectively intercept the host epigenetic circuit and downstream signalling.

AbstractThe broad host range bacteriophage Mu employs a novel ‘methylcarbamoyl’ modification to protect its DNA from diverse host restriction systems. Biosynthesis of the unusual modification is a longstanding mystery. Moreover, isolation of Mom, the phage protein involved in the modification has remained elusive to date. Here, we characterized the co-factor and metal binding properties of Mom and provide a molecular mechanism to explain ‘methylcarbamoyl’ation by Mom. Our computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom, predicting acetyl CoA as its co-factor. We demonstrate that Mom binds to acetyl CoA and identify the active site. Puzzlingly, none of the > 309,000 GNAT members identified so far catalyze Mom-like modification of their respective substrates. Besides, conventional acid-base catalysis deployed by typical acetyltransferases cannot support methylcarbamoylation of adenine seen in Mu phage. In contrast, free radical-chemistry, catalyzed by Fe-S cluster or transition metal ions can explain the seemingly challenging reaction between acetyl CoA and DNA. We discovered that Mom is an iron-binding protein, with the Fe2+/3+ ion colocalized with acetyl CoA in the active site of Mom. Mutants defective for binding Fe2+/3+ or acetyl CoA demonstrated compromised activity, indicating their importance in the DNA modification reaction. Iron-binding in the GNAT active site is unprecedented and represents a small step in the evolution of Mom from the ancestral acetyltransferase fold. Yet, the tiny step allows a giant chemical leap from usual acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.SummaryStudying the arms race between bacteria and their viruses (bacteriophages or phages) is key to understanding microbial life and its complexity. An unprecedented DNA modification shields phage Mu from bacterial restriction endonucleases that destroy incoming phage DNA. Nothing is known of how the modification is brought about, except that a phage protein Mom is involved. Here, we discover acetyl CoA and iron as key requirements for the modification. We explain how by evolving the ability to bind iron - a transition metal capable of generating highly reactive free radicals, a well-studied scaffold like the acetyltransferase fold can gain novel catalytic prowess in Mom. These findings have broad implications for gene editing technologies and therapeutic application of phages.