Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

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.

Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

The 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.

Characteristics of two myoviruses induced from the coastal photoheterotrophic bacterium Porphyrobacter sp. YT40

ABSTRACT In this study, we characterized two induced myoviruses from one marine photoheterotrophic bacterium Porphyrobacter sp. YT40 belonging to the Sphingomonadales family in Alphaproteobacteria. The genome sequence of prophage A is ∼36.9 kb with an average GC content of 67.1%, and its core or functional genes are homologous to Mu or Mu-like phages. Furthermore, induced viral particles from prophage A show a knob-like neck structure, which is only found in bacteriophage Mu. The genome size of prophage B is ∼36.8 kb with an average GC content of 65.3%. Prophage B contains a conserved gene cluster Q-P-O-N-M-L, which is unique in P2 phages. Induced viral particles from prophage B display an icosahedral head with a diameter of ∼55 nm and a 130 ± 5 nm long contractile tail. To our knowledge, this is the first report that characterizes the induced P2-like phage in marine Alphaproteobacteria. Phylogeny analyses suggest that these two types of prophages are commonly found in sequenced bacteria of the Sphingomonadales family. This study sheds light on the ongoing interaction between marine bacteria and phages, and improves our understanding of bacterial genomic plasticity and evolution.

Observation of unexpected molecular binding activity for Mu phage tail fibre chaperones

In the history of viral research, one of the important biological features of bacteriophage Mu is the ability to expand its host range. For extending the host range, the Mu phage encodes two alternate tail fibre genes. Classical amber mutation experiments and genome sequence analysis of Mu phage suggested that gene products (gp) of geneS (gpS = gp49) and gene S’ (gpS’ = gp52) are tail fibres and that gene products of geneU (gpU = gp50) and geneU’ (gpU’ = gp51) work for tail fibre assembly or tail fibre chaperones. Depending on the gene orientation, a pair of genes 49-50 or 52-51 is expressed for producing different tail fibres that enable Mu phage to recognize different host cell surface. Since several fibrous proteins including some phage tail fibres employ their specific chaperone to facilitate folding and prevent aggregation, we expected that gp50 or gp51 would be a specific chaperone for gp49 and gp52, respectively. However, heterologous overexpression results for gp49 or gp52 (tail fibre subunit) together with gp51 and gp50, respectively, were also effective in producing soluble Mu tail fibres. Moreover, we successfully purified non-native gp49-gp51 and gp52-gp50 complexes. These facts showed that gp50 and gp51 were fungible and functional for both gp49 and gp52 each other.

Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy

There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of −0.022, and pSVβ-Gal −0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.

Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal.