Detection of DNA replication errors and 8-oxo-dGTP-mediated mutations in E. coli by Duplex DNA Sequencing.

Mutation is a phenomenon inescapable for all life-forms, including bacteria. While bacterial mutation rates are generally low due to the operation of error-avoidance systems, sometimes they are elevated by many orders of magnitude. Such a state, known as a hypermutable state, can result from exposure to stress or to harmful environments. Studies of bacterial mutation frequencies and analysis of the precise types of mutations can provide insights into the mechanisms by which mutations occur and the possible involvement of error-avoidance pathways. Several approaches have been used for this, like reporter assays involving non-essential genes or mutation accumulation over multiple generations. However, these approaches give an indirect estimation, and a more direct approach for determining mutations is desirable. With the recent development of a DNA sequencing technique known as Duplex Sequencing, it is possible to detect rare variants in a population at a frequency of 1 in 107 base pairs or less. Here, we have applied Duplex Sequencing to study spontaneous mutations in E. coli. We also investigated the production of replication errors by using a mismatch-repair defective (mutL) strain as well as oxidative-stress associated mutations using a mutT-defective strain. For DNA from a wild-type strain we obtained mutant frequencies in the range of 10-7 to 10-8 depending on the specific base-pair substitution, but we argue that these mutants merely represent a background of the system, rather than mutations that occurred in vivo. In contrast, bona-fide in vivo mutations were identified for DNA from both the mutL and mutT strains, as indicated by specific increases in base substitutions that are fully consistent with their established in vivo roles. Notably, the data reproduce the specific context effects of in vivo mutations as well as the leading vs. lagging strand bias among DNA replication errors.

Mycobacteriophage D29 induced association of Mycobacterial RNA polymerase with ancillary factors leads to increased transcriptional activity.

Mycobacteriophage D29 infects species belonging to the genus Mycobacterium including the deadly pathogen Mycobacterium tuberculosis. D29 is a lytic phage, although, related to the lysogenic mycobacteriophage L5. This phage is unable to lysogenize in mycobacteria as it lacks the gene encoding the phage repressor. Infection by many mycobacteriophages cause various changes in the host that ultimately leads to inactivation of the latter. One of the host targets often modified in the process is RNA polymerase. During our investigations with phage D29 infected Mycobacterium smegmatis (Msm) we observed that the promoters from both phage, and to a lesser extent those of the host were found to be more active in cells that were exposed to D29, as compared to the unexposed. Further experiments indicate that the RNA polymerase purified from phage infected cells possessed higher affinity for promoters particularly those that were phage derived. Comparison of the purified RNA polymerase preparations from infected and uninfected cells showed that several ancillary transcription factors, Sigma factor F, Sigma factor H, CarD and RbpA are prominently associated with the RNA polymerase from infected cells. Based on our observations we conclude that the higher activity of RNA polymerase observed in D29 infected cells is due to its increased association with ancillary transcription factors.

A CRISPRi-based investigation reveals that multiple promoter elements drive gene expression from the genome of mycobacteriophage D29.

A unique feature found in the genomes of mycobacteriophages such as L5 belonging to the A cluster is the presence of multiple dispersed repeated elements known as stoperators. The phage repressor binds these repeat elements, shutting off transcription globally and thereby promoting lysogeny. Interestingly, the sequence of these stoperators closely matches that of the consensus -35 region of prokaryotic promoters, leading us to propose that they may have a role to play in the initiation of transcription by serving as RNA polymerase binding sites. Mycobacteriophage D29 is closely related to phage L5, and their genome organizations are very similar. As in L5, there are multiple stoperators in the genome of D29. The positions occupied by the stoperators in the two genomes are almost identical. The significant difference between the two phages is that D29 lacks the gene encoding the equivalent of the L5 repressor. Since phage D29 does not produce a repressor, we considered it to be a suitable model for testing our hypothesis that the stoperators function as promoters in the absence of the repressor. To prove our point, we targeted CRISPR guide RNAs against six stoperators. In the case of five out of the six, we found a significant reduction in downstream gene expression and phage growth. Based on this observation and primer extension assays, we conclude that promoting gene expression is likely to be the primary function of stoperators.

Complete Genome Sequence of Escherichia coli BL21-AI.

Escherichia coli BL21-AI is a commercially available strain possessing a phage T7-based protein-expression system. A combination of tight regulation and high yield makes it widely used for high-level expression of toxic recombinant proteins. Here, we present the complete genome sequence of BL21-AI and provide insights on its genome.

A transcriptomic analysis of the mycobacteriophage D29 genome reveals the presence of novel stoperator-associated promoters in its right arm.

Mycobacteriophage D29 is a lytic phage that infects various species of Mycobacterium including M. tuberculosis. Its genome has 77 genes distributed almost evenly between two converging operons designated as left and right. Transcription of the phage genome is negatively regulated by multiple copies of an operator-like element known as stoperator that acts by binding the phage repressor Gp71. The function of the D29 genes and their expression status are poorly understood and therefore we undertook a transcriptome analysis approach to address these issues. The results indicate that the average transcript intensity of the right arm genes was higher than of those on the left, at the early stage of infection. Moreover, the fold increase from early to the late stage was found to be less for the right arm genes than for the left. Both observations support the prediction that the right arm genes are expressed early whereas the left arm ones are expressed late. The analysis further revealed a break in the continuity of the right arm operon between 89, the first gene in it, and 88, the next. Gene 88 was found to be expressed from a newly identified promoter located between 88 and 89. Another new promoter was found upstream of 89. Thus, the promoter Pleft, identified earlier, is not the only one that drives expression of the right arm genes. All these promoters overlap with stoperators, with which they share a conserved sequence motif, TTGACA, commonly known as the -35 promoter element. We demonstrate mutually exclusive binding of RNA polymerase and Gp71 to the stoperator-promoters and conclude that stoperators can function as -35 promoter elements and that they can control gene expression not only negatively as was believed earlier but in many cases positively as well.

Vitamin C induced DevR-dependent synchronization of Mycobacterium smegmatis growth and its effect on the proliferation of mycobacteriophage D29.

Vitamin C is known to inhibit mycobacterial growth by acting as a hypoxia inducing agent. While investigating how mycobacteriophage growth is influenced by hypoxic conditions induced by vitamin C, using Mycobacterium smegmatis- mycobacteriophage D29 as a model system, it was observed that prior exposure of the host to such conditions resulted in increased burst size of the phage. Vitamin C pre-exposure was also found to induce synchronous growth of the host. A mutant defective in DevR, the response regulator that controls hypoxic responses in mycobacteria, neither supported higher phage bursts nor was it able to undergo synchronized growth following vitamin C pre-exposure, indicating thereby that the two phenomena are interrelated. Further evidence supporting such an interrelationship was obtained from the observation that phage burst sizes varied depending on the stage of synchronous growth that the host cells were in, at the time of infection-higher bursts were observed in the resting/synthetic phases and lower in the dividing ones. The effects were specific in nature as synchronization by an unrelated method, known as 'crowding', did not lead to the same consequence. The results indicate that growth synchronization induced by vitamin C treatment is a DevR-dependent phenomenon which is exploited by mycobacteriophage D29 to grow in larger numbers.

Gp66, a calcineurin family phosphatase encoded by mycobacteriophage D29, is a 2', 3' cyclic nucleotide phosphodiesterase that negatively regulates phage growth.

Mycobacteriophage D29 encodes a protein Gp66 which has been predicted to be a calcineurin family phosphoesterase. Phylogenetically Gp66 and related proteins mostly derived from mycobacteriophages form a distinct clade within this family. Interestingly, the presence of gene 66 orthologs can be traced to bacteria of diverse phylogenetic lineages such as Aquifex aeolicus, a deep branching eubacteria and Methanococcus jannaschii, an archaebacteria. The promiscuous nature of gene 66 suggests that it may have been transferred across genus barriers by horizontal gene transfer mechanisms. The biological function of members of this novel clade comprising mostly the mycobacteriophage phosphoesterases have not been elucidated so far. In this investigation, it has been demonstrated for the first time that Gp66, a member of this novel family, is a 2', 3' cyclic phosphodiesterase. The gene is expressed during phage infection and the net result is negative regulation of bacteriophage as well as bacterial growth.