Valakunja Nagaraja

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

SUMMARY Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.

DNA Gyrase Genes in Mycobacterium tuberculosis: a Single Operon Driven by Multiple Promoters

The two genes encoding DNA gyrase in Mycobacterium tuberculosis are present next to each other in the genome, with gyrB upstream of gyrA. We show that the primary transcript is dicistronic. However, in addition to the principal promoter, there are multiple weaker promoters that appear to fine-tune transcription. With these and other mycobacterial promoters, we propose consensus promoter sequences for two distinct sigma factors. In addition to this, the gyr genes in M. tuberculosis, as in other species, are subject to autoregulation, albeit with slower kinetics, probably reflecting the slower metabolism of the organism.

DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes

Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non‐specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzymes ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi‐directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process.

WebGeSTer DB--a transcription terminator database.

We present WebGeSTer DB, the largest database of intrinsic transcription terminators (http://pallab.serc.iisc.ernet.in/gester). The database comprises of a million terminators identified in 1060 bacterial genome sequences and 798 plasmids. Users can obtain both graphic and tabular results on putative terminators based on default or user-defined parameters. The results are arranged in different tiers to facilitate retrieval, as per the specific requirements. An interactive map has been incorporated to visualize the distribution of terminators across the whole genome. Analysis of the results, both at the whole-genome level and with respect to terminators downstream of specific genes, offers insight into the prevalence of canonical and non-canonical terminators across different phyla. The data in the database reinforce the paradigm that intrinsic termination is a conserved and efficient regulatory mechanism in bacteria. Our database is freely accessible.

DNA Topoisomerase I from Mycobacterium smegmatis AN ENZYME WITH DISTINCT FEATURES

A type I topoisomerase has been purified to homogeneity from Mycobacterium smegmatis. It is the largest single subunit enzyme of this class having molecular mass of 110 kDa. The enzyme is Mg2+ dependent and can relax negatively supercoiled DNA, catenate, and knot single-stranded DNA, thus having typical properties of type I topoisomerases. Furthermore, the enzyme makes single-stranded nicks and the 5′-phosphoryl end of the nicked DNA gets covalently linked with a tyrosine residue of the enzyme. However,M. smegmatis enzyme shows some distinctive features from the prototype Escherichia coli topoisomerase I. The enzyme is relatively stable at higher temperatures and not inhibited by spermidine. It apparently does not contain any bound Zn2+and on modification of cysteine residues retains the activity, suggesting the absence of the zinc-finger motif in DNA binding. Partially purified Mycobacterium tuberculosis topoisomerase I exhibits very similar properties with respect to size, stability, and reaction characteristics. Sequence comparison of topoisomerase I fromE. coli and M. tuberculosis shows the absence of zinc-finger motifs in mycobacterial enzyme. Using a two-substrate assay system, we demonstrate that the enzyme acts processively at low ionic strength and switches over to distributive mode at high Mg2+ concentration. Significantly, the enzyme activity is stimulated by single strand DNA-binding protein. There is a potential to exploit the characteristics of the enzyme to develop it as a molecular target against mycobacterial infections.

Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.

Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors

The nucleoid-associated protein HU plays an important role in maintenance of chromosomal architecture and in global regulation of DNA transactions in bacteria. Although HU is essential for growth in Mycobacterium tuberculosis (Mtb), there have been no reported attempts to perturb HU function with small molecules. Here we report the crystal structure of the N-terminal domain of HU from Mtb. We identify a core region within the HU-DNA interface that can be targeted using stilbene derivatives. These small molecules specifically inhibit HU-DNA binding, disrupt nucleoid architecture and reduce Mtb growth. The stilbene inhibitors induce gene expression changes in Mtb that resemble those induced by HU deficiency. Our results indicate that HU is a potential target for the development of therapies against tuberculosis.

Synthesis and antimycobacterial activities of novel 6-nitroquinolone-3-carboxylic acids.

Various 1-(substituted)-1,4-dihydro-6-nitro-4-oxo-7-(sub-secondary amino)-quinoline-3-carboxylic acids were synthesized from 2,4-dichlorobenzoic acid by six step synthesis. The compounds were evaluated for antimycobacterial in vitro and in vivo against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB) and Mycobacterium smegmatis (MC(2)) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the 48 synthesized compounds, 7-(4-((benzo[d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)-1-cyclopropyl-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid (8c) was found to be the most active compound in vitro with MIC of 0.08 and 0.16 microM against MTB and MDR-TB, respectively. In the in vivo animal model 8c decreased the bacterial load in lung and spleen tissues with 2.78 and 4.15-log10 protections, respectively, at the dose of 50 mg/kg body weight.

GyrI: a counter‐defensive strategy against proteinaceous inhibitors of DNA gyrase

DNA gyrase is the target of two plasmid‐encoded toxins CcdB and microcin B17, which ensure plasmid maintenance. These proteins stabilize gyrase–DNA covalent complexes leading to double‐strand breaks in the genome. In contrast, the physiological role of chromosomally encoded inhibitor of DNA gyrase (GyrI) in Escherichia coli is unclear and its mechanism of inhibition has not been established. We demonstrate that the mode of inhibition of GyrI is distinct from all other gyrase inhibitors. It inhibits DNA gyrase prior to, or at the step of, binding of DNA by the enzyme. GyrI reduces intrinsic as well as toxin‐stabilized gyrase–DNA covalent complexes. Furthermore, GyrI reduces microcin B17‐mediated double‐strand breaks in vivo, imparting protection to the cells against the toxin, substantiating the in vitro results. Thus, GyrI is an antidote to DNA gyrase‐specific proteinaceous poisons encoded by plasmid addiction systems.

The Additional 165 Amino Acids in the B Protein of Escherichia coli DNA Gyrase Have an Important Role in DNA Binding

DNA gyrase is the only enzyme known to negatively supercoil DNA. The enzyme is a heterotetramer of A2B2 subunit composition. Alignment of the primary sequence of gyrase B (GyrB) from various species shows that they can be grouped into two classes. The GyrB of Gram-negative eubacteria has a stretch of about 165 amino acids in the C-terminal half, which is lacking in other GyrB subunits and type II topoisomerases. In Escherichia coli, no function has so far been attributed to this stretch. In this study, we have tried to assess the function of this region both in vivo and in vitro. A deletant (GyrBΔ160) lacking this region is non-functional in vivo. The holoenzyme reconstituted from gyrase A (GyrA) and GyrBΔ160 shows reduced but detectable supercoiling and quinolone-induced cleavage activity in vitro. GyrBΔ160 retains its ability to bind to GyrA and novobiocin. However, when reconstituted with GyrA, the deletant shows greatly impaired DNA binding. The intrinsic ATPase activity of the GyrBΔ160 is comparable to that of wild type GyrB, but this activity is not stimulated by DNA. These studies indicate that the additional stretch present in GyrB is essential for the DNA binding ability ofE. coli gyrase.