Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics

The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosis genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of approximately 150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.

Comparative genomics of Esx genes from clinical isolates of Mycobacterium tuberculosis provides evidence for gene conversion and epitope variation

Stewart Cole, Jacques Rougemont, Swapna Uplekar

The 23-membered Esx protein family is involved in the host-pathogen interactions of Mycobacterium tuberculosis. These secreted proteins are among the most immunodominant antigens recognized by the human immune system and have thus been used to develop vaccines and immunodiagnostic tests for tuberculosis (TB). Gene pairs for 10 Esx proteins are contained in the ESX-1 to ESX-5 loci, encoding type VII secretion systems. A subset of Esx proteins can be further classified into the Mtb9.9, QILSS, and TB10.4 subfamilies. To survey genetic diversity in the Esx family and its potential for antigenic variation, we sequenced all esx genes from 108 clinical isolates of M. tuberculosis from different clades by using a targeted approach. A total of 109 unique single nucleotide polymorphisms (SNPs) were observed, and 59 of these were nonsynonymous. Some of the resultant amino acid substitutions affect known Esx epitopes, including two in the EsxB (CFP-10) and EsxH (TB10.4) antigens. Assessment of the SNP distribution across the Esx proteins revealed high genetic variability, especially in the Mtb9.9 and QILSS subfamilies, and more conservation in the ESX-1 to ESX-4 loci. Comparison of the DNA sequences of variable esx genes provided clear evidence for recombination events between different genes in the same strain, some of which are predicted to truncate the corresponding protein. Many of these polymorphisms escape detection by ultrahigh-throughput sequencing using short sequence reads, as such approaches cannot distinguish between closely related genes. The esx gene family is dynamic, and sequence changes likely lead to immune variation.

2011

The Phosphate Specific Transport (Pst) System in Fast- and Slow-Growing Mycobacteria

Cyntia De Piano

Although mycobacteria are known to accumulate polyphosphate (PolyP), a linear polymer of orthophosphate, when subjected to stresses, the exact roles of this molecule are not yet clearly understood. We found that deletion of the pstA gene encoding for a transmembrane domain of the high-affinity phosphate-specific ABC transporter system (Pst system) resulted in accumulation of high amounts of PolyP during growth in phosphate (Pi)-replete conditions in Mycobacterium tuberculosis and Mycobacterium smegmatis. These PolyP stores were gradually used during Pi starvation by the ApstA mutants in order to sustain growth and this allowed the mutant strains to reach a higher cell density than the wild-type (WT) parental strain. Neither the mRNA nor the protein levels of polyphosphate kinase 1 (PPK1) could explain the PolyP accumulation observed in the mutant, suggesting that another regulatory mechanism is involved. The ApstA mutant of M. smegmatis was hypersensitive to several in vitro stresses, including detergent, oxidative stress, and cell wall targeting antibiotics. Deletion of the main enzyme involved in PolyP synthesis, PPK1, in the ApstA mutant background of M. smegmatis completely reversed the PolyP accumulation and partially reversed the stress sensitivity as well as the derepression of PHO regulon genes observed in the ApstA mutant. The mechanism of PolyP accumulation in M. smegmatis was investigated using a genetic approach. We found that this mechanism differs from the (p)ppGpp-mediated PolyP accumulation observed in Escherichia coli, since deletion of the RelA enzyme in a ApstA mutant background did not prevent PolyP accumulation. The mechanism of PolyP degradation during Pi starvation in the M. smegmatis ApstA mutant was also analyzed. Deletion of both polyphosphatase enzymes (PPX1 and PPX2) as well as the polyphosphate kinase 2 (PPK2) enzyme did not have any impact on PolyP degradation. PPK1 is a bifunctional enzyme that can either synthesize or degrade PolyP. Our current hypothesis is that PPK1 might perform the reverse reaction (PolyP degradation) during Pi starvation. The aim of the second part of my project was to make a comparative study of the expression and regulation of the phosphate specific transporter (pst) operon in fast-growing [M. smegmatis] and slow-growing [M. tuberculosis] mycobacteria. To achieve this goal, pst reporter strains of M. smegmatis and M. tuberculosis were constructed by precise insertion of a modified dest_gfp gene (provided by Giulia Manina) encoding a destabilized green fluorescent protein (dest_GFP) into the corresponding pst locus as transcriptional ) fusions. The transcriptional reporter construct [p) was introduced in both the WT and the ApstA mutant backgrounds. The kinetics of induction of the pst-gfp reporters in WT and ApstA mutant strains in response to Pi starvation was analyzed. Population-averaged measurements were made using qRT-PCR and Western blotting. Single-cell measurements using flow cytometry and timelapse fluorescence microscopy were also performed. For the microscopy experiments, the bacteria were cultured in custom-made microfluidic devices that permit rapid switching of the culture medium, e.g., switching between Pi-replete and Pi-depleted media. Because these studies require precise environmental control and resolution of individual and temporal phenotypes, my principal experimental tool was the integrated microfluidics and timelapse fluorescence microscopy systems that have recently been developed in the laboratory. Although there have been many studies published on the population-averaged behavior of bacteria responding to external stresses such as Pi limitation, we are not aware of any studies addressing the individuality of such responses using a real-time single-cell approach. We observed that the kinetics of induction of the Pst operon upon Pi starvation scaled with the growth rate of the bacteria and peak induction occurred within about two generations for both smegmatis and tuberculosis. Interestingly, we found that the basal level of Pst expression and the steady state level reached upon induction by Pi starvation were similar in tuberculosis and M. smegmatis, although the kinetics of induction were much faster in M. smegmatis. These studies contributed to improve the understanding of the regulation and the kinetics of response of the PHO regulon in fast- and slow-growing mycobacteria. The finding that PolyP plays a role in this process could also apply to other organisms where the PHO regulon is regulated in a similar way.

EPFL2013

The cysteine desulfurase IscS of Mycobacterium tuberculosis is involved in iron sulfur cluster biogenesis and oxidative stress defence

Jeffrey Chen, Stewart Cole, Gaëlle Séraphine Kolly, Florence Pojer, Jan Lars Rybniker

The complex multiprotein systems for the assembly of protein-bound iron sulfur (Fe S) clusters are well defined in Gram-negative model organisms. However, little is known about Fe S cluster biogenesis in other bacterial species. The ISC (iron sulfur cluster) operon of Mycobacterium tuberculosis lacks several genes known to be essential for the function of this system in other organisms. However, the cysteine desulfurase IscSMtb (Rv number Rv3025c; Mtb denotes M. tuberculosis) is conserved in this important pathogen. The present study demonstrates that deleting iscSMtb renders the cells microaerophilic and hypersensitive to oxidative stress. Moreover, the Delta iscSMtb mutant shows impaired Fe S cluster-dependent enzyme activity, clearly indicating that IscSMtb is associated with Fe S cluster assembly. An extensive interaction network of IscSMtb with Fe S proteins was identified, suggesting a novel mechanism of sulfur transfer by direct interaction with apoproteins. Interestingly, the highly homologous IscS of Escherichia coli failed to complement the Delta iscSMtb mutant and showed a less diverse protein-interaction profile. To identify a structural basis for these observations we determined the crystal structure of IscSMtb, which mirrors adaptations made in response to an ISC operon devoid of IscU-like Fe S cluster scaffold proteins. We conclude that in M. tuberculosis IscS has been redesigned during evolution to compensate for the deletion of large parts of the ISC operon.

Portland Press2014

The Phosphate Specific Transport (Pst) System in Fast- and Slow-Growing Mycobacteria

Cyntia De Piano

EPFL2013