A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: Application to Mycobacterium tuberculosis

Abstract

We describe a postgenomic in silico approach for identifying genes that are likely to be essential and estimate their proportion in haploid genomes. With the knowledge of all sites eligible for mutagenesis and an experimentally determined partial list of nonessential genes from genome mutagenesis, a Bayesian statistical method provides reasonable predictions of essential genes with a subsaturation level of random mutagenesis. For mutagenesis, a transposon such as Himar1 is suitable as it inserts randomly into TA sites. All of the possible insertion sites may be determined a priori from the genome sequence and with this information, data on experimentally hit TA sites may be used to predict the proportion of genes that cannot be mutated. As a model, we used the Mycobacterium tuberculosis genome. Using the Himar1 transposon, we created a genetically defined collection of 1,425 insertion mutants. Based on our Bayesian statistical analysis using Markov chain Monte Carlo and the observed frequencies of transposon insertions in all of the genes, we estimated that the M. tuberculosis genome contains 35% (95% confidence interval, 28%-41%) essential genes. This analysis further revealed seven functional groups with high probabilities of being enriched in essential genes. The PE-PGRS (Pro-Glu polymorphic GC-rich repetitive sequence) family of genes, which are unique to mycobacteria, the polyketide͞ nonribosomal peptide synthase family, and mycolic and fatty acid biosynthesis gene families were disproportionately enriched in essential genes. At subsaturation levels of mutagenesis with a random transposon such as Himar1, this approach permits a statistical prediction of both the proportion and identities of essential genes of sequenced genomes.

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

“…2) and the estimates of their midpoint potential (E m ) values (supplemental Table 5) (53,58) allow us to propose that electron transfer from FAD to FMN takes place with two one-electron transfer processes that follow the same pathway. However, the energetics of transfer of the first (empty circle, continuous arrows, steps [2][3][4] and the second electron (full circle, dashed arrows, steps 5-7) differ due to the different E m values of oxidized/semiquinone and semiquinone/hydroquinone forms of both flavin cofactors. In these schemes, the two low potential [4Fe-4S] clusters of GltS (53) are assumed to be equipotential with E m values in the range of that of the NADP ϩ /NADPH couple (Ϫ340 mV).…”

“…Earlier SAXS results suggested a tetrameric (␣␤) 4 assembly of GltS in solution (54), assuming monodispersity of the GltS samples. Given the new evidence for the co-existence of different GltS oligomers, the previous data were reanalyzed, leading us to the conclusion that the samples studied at that time were actually mixtures of about 20% ␣␤ protomers and 80% (␣␤) 6 hexamers (supplemental Fig.…”

Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two‐component response regulator

“…Recent genetic studies catalogued M. tuberculosis genes essential for survival in broth, macrophages and animals (Sassetti et al, 2001;Lamichhane et al, 2003;Sassetti and Rubin, 2003;Rengarajan et al, 2005). For the present study, we focused on dnaA, ftsZ and devR genes.…”

Defining mycobacteria: Shared and specific genome features for different lifestyles

“…The systematic and impressive effort by Lamichhane et al [26], capitalizing on genome sequence availability of M. tuberculosis CDC1551 is noteworthy. By using the Himar1 phage to transpose randomly into the M. tuberculosis genome, picking individual surviving insertion clones, and identifying the point of insertion of the transposon in each by sequencing the fl anking genomic regions, they found that up to 65% of the predicted coding sequences (CDSs) could be interrupted, i.e., these CDSs were not 'lethal' (or 'non-essential') for growth on agar plates.…”

“…However, other biological activities have been discovered, such as a role in intracellular macrophage survival in M. marinum [46]. Only a few of the genes are 'essential' by the Lamichhane and Sassetti criteria [26,30].…”