Hongtai Zhang

Genome sequencing of 161 Mycobacterium tuberculosis isolates from China identifies genes and intergenic regions associated with drug resistance.

The worldwide emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis threatens to make this disease incurable. Drug resistance mechanisms are only partially understood, and whether the current understanding of the genetic basis of drug resistance in M. tuberculosis is sufficiently comprehensive remains unclear. Here we sequenced and analyzed 161 isolates with a range of drug resistance profiles, discovering 72 new genes, 28 intergenic regions (IGRs), 11 nonsynonymous SNPs and 10 IGR SNPs with strong, consistent associations with drug resistance. On the basis of our examination of the dN/dS ratios of nonsynonymous to synonymous SNPs among the isolates, we suggest that the drug resistance–associated genes identified here likely contain essentially all the nonsynonymous SNPs that have arisen as a result of drug pressure in these isolates and should thus represent a near-complete set of drug resistance–associated genes for these isolates and antibiotics. Our work indicates that the genetic basis of drug resistance is more complex than previously anticipated and provides a strong foundation for elucidating unknown drug resistance mechanisms.

Identification by cDNA cloning of abundant sRNAs in a human- avirulent Yersinia pestis strain grown under five different growth conditions

AIMS sRNA regulation is supposedly involved in the stress response of a pathogen during infection. Yersinia pestis, the etiologic agent of plague, must encounter temperature and microenvironment changes, given its lifestyle. Here, we used the cDNA cloning approach to discover full-length sRNA candidates that are highly expressed in Y. pestis under five different growth conditions. MATERIALS & METHODS The cDNA cloning approach was improved by combining the traditional cDNA library construction with the prevalent rapid amplification of cDNA ends and RNA size selection techniques. RESULTS In total, 43 RNA species, including six previously annotated sRNAs, were identified. Of these, 25 sRNAs were encoded on the antisense strand of the annotated genes. Interestingly, two of these sRNAs were found on the complementary strand of noncoding RNAs. In addition, eight novel sRNAs encoded in the intergenic regions were also revealed. Ten sRNA candidates chosen for the northern blot analysis were successfully detected. Analysis of the expression patterns of 29 candidate sRNAs showed that 24 sRNAs are highly abundant in Y. pestis upon entry into the stationary growth phase. CONCLUSION Our preliminary attempt at screening the novel sRNA candidates will lay the foundation for understanding the roles of sRNAs in Y. pestis physiology and pathogenesis.

Mutations Found in the pncA Gene of Mycobacterium tuberculosis in Clinical Pyrazinamide-resistant Isolates from a Local Region of China

This study was designed to investigate the presence of mutations in the pncA gene, minimum inhibitory concentrations and pyrazinamidase activity of pyrazinamide-resistant Mycobacterium tuberculosis. In total, 47 M. tuberculosis clinical isolates from a local region of China were assayed. Pyrazinamidase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and a 721 bp region, including the entire pncA open-reading frame, 104 bp of the upstream sequence and 59 bp of the downstream sequence, was determined by DNA sequencing of purified polymerase chain reaction products. Of the 47 isolates resistant to pyrazinamide, 44 lost pyrazinamidase activity and had pncA mutations that occurred mainly near pyrazinamidases active or metal ion binding sites; nine of them have not been reported previously. Three pyrazinamide-resistant isolates carried the wild-type pncA sequence and retained pyrazinamidase activity. These results show the molecular mechanism of pyrazinamide resistance in China and may also contribute towards the prevention of tuberculosis in China.

The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity

DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an αβ2ε polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its α subunit, Mtb DNA pol III has two potential proofreading subunits; the α and ε subunits. During DNA replication, the presence of the β2 clamp strongly promotes the polymerization of the αβ2ε replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb.

MSMEG_2731, an uncharacterized nucleic acid binding protein from Mycobacterium smegmatis, physically interacts with RPS1.

While the M. smegmatis genome has been sequenced, only a small portion of the genes have been characterized experimentally. Here, we purify and characterize MSMEG_2731, a conserved hypothetical alanine and arginine rich M. smegmatis protein. Using ultracentrifugation, we show that MSMEG_2731 is a monomer in vitro. MSMEG_2731 exists at a steady level throughout the M. smegmatis life-cycle. Combining results from pull-down techniques and LS-MS/MS, we show that MSMEG_2731 interacts with ribosomal protein S1. The existence of this interaction was confirmed by co-immunoprecipitation. We also show that MSMEG_2731 can bind ssDNA, dsDNA and RNA in vitro. Based on the interactions of MSMEG_2731 with RPS1 and RNA, we propose that MSMEG_2731 is involved in the transcription-translation process in vivo.

The dimer state of GyrB is an active form: implications for the initial complex assembly and processive strand passage

In a previous study, we presented the dimer structure of DNA gyrase B′ domain (GyrB C-terminal domain) from Mycobacterium tuberculosis and proposed a ‘sluice-like’ model for T-segment transport. However, the role of the dimer structure is still not well understood. Cross-linking and analytical ultracentrifugation experiments showed that the dimer structure exists both in the B′ protein and in the full-length GyrB in solution. The cross-linked dimer of GyrB bound GyrA very weakly, but bound dsDNA with a much higher affinity than that of the monomer state. Using cross-linking and far-western analyses, the dimer state of GyrB was found to be involved in the ternary GyrA–GyrB–DNA complex. The results of mutational studies reveal that the dimer structure represents a state before DNA cleavage. Additionally, these results suggest that the dimer might also be present between the cleavage and reunion steps during processive transport.

Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis, the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III‐A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA‐seq approaches, we show it is active in invader defense and has features atypical of type III‐A systems: mature CRISPR RNA (crRNA) in its crRNA‐CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3′‐end processing after Cas6 cleavage of repeat RNA 8 nt from its 3′ end. crRNAs generated resemble mature crRNA in type I systems, having both 5′ (8 nt) and 3′ (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3′ hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis, providing a foundation for investigating the potential of a type III‐A–based genome editing system.—Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Y., Zhou, Y., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.‐E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III‐A CRISPR/Cas system crRNA and its maturation have atypical features. FASEB J. 33, 1496–1509 (2019). www.fasebj.org

Magnetic reconnection and plasmoid events in the magnetopause boundary layer

Abstract An important role of velocity shear in producing transient magnetic reconnection in the magneto- pause boundary layer region is supposed. Two kinds of velocity shears may be there in the magnetopause boundary layer: the velocity shear of field-aligned flow occuring at the high latitude boundary layer region in the dayside magnetopause outside the subsolar point region or cusp region; and the velocity shear of transverse flow near the subpolar point region. The influence of velocity shear on magnetic reconnection processes has been investigated systematically in the magnetopause boundary layer, including: vortex induced reconnection(VIR), single X line bursty reconnection(SBXR), transverse shear flow rushed reconnection, stochastic reconnection, and local transient reconnection in the high latitude boundary layer under different IMF conditions. Finally, the plamoid events in the magnetospheric boundary layer are discussed.