Vitexin alters Staphylococcus aureus surface hydrophobicity to obstruct biofilm formation.

Cell Surface hydrophobicity is one of the determinant biophysical parameters of bacterial aggregation for being networked to form a biofilm. Phytoconstituent, like vitexin, has long been in use for their antibacterial effect. The present work demonstrates the role of vitexin in modulating Staphylococcus aureus surface hydrophobicity while aggregating to form biofilm and pathogenesis in a host. In planktonic form, vitexin shows minimum inhibitory concentration at 252 µg/ml against S. aureus. Sub-MIC doses of vitexin and antibiotics (26 µg/ml of vitexin, 55 µg/ml of azithromycin, and 2.5 µg/ml of gentamicin) were selected to treat S. aureus. Dead cell counts after treatment were studied through flow cytometry. As dead cell counts were minimal (<5 %), these doses were considered for all subsequent experiments. While studying aggregating cells, it was observed that vitexin reduces S. aureus surface hydrophobicity and membrane permeability at the sub-MIC dose of 26 µg/ml. The in silico binding analysis showed a higher binding affinity of vitexin with surface proteins (IcaA, DltA, and SasG) of S. aureus. Down-regulation of dltA and icaAB expression, along with the reduction in membrane potential with a sub-MIC dose of vitexin, explains reduced S. aureus surface hydrophobicity. Vitexin was found to interfere with S. aureus biofilm-associated protein biomass, EPS production, and swarming movement. Subsequently, the suppression of proteases production and down-regulation of icaAB and agrAC gene expression with a sub-MIC dose of vitexin explained the inhibition of S. aureus virulence in vitro. Besides, vitexin was also found to potentiate the antibiofilm activity of sub-MIC doses of gentamicin and azithromycin. Treatment with vitexin exhibits a protective response in S. aureus infected macrophages through modulation of expression of cytokines like IL-10 and IL-12p40 at protein and mRNA levels. Furthermore, CFU count and histological examination of infected mouse tissue (liver and spleen) justify the in vivo protective effect of vitexin from S. aureus biofilm-associated infection. From this study, it can be inferred that vitexin can reduce S. aureus surface hydrophobicity, leading to interference with aggregation at the time of biofilm formation and subsequent pathogenesis in a host.

The respiratory lipoquinone, menaquinone, functions as an inducer of genes regulated by the Mycobacterium smegmatis repressor MSMEG_2295.

A previous study reported that the Mycobacterium smegmatis (Msm) protein MSMEG_2295 is a repressor controlling the expression of several genes, including that for MSMEG_5125, a putative isoprenoid binding protein belonging to the YceI family, and DinB2, a DNA damage repair enzyme. This repressor is encoded by the first gene of the operon that also expresses the gene for DinB2. Targeted inhibition of MSMEG_5125 using CRISPRi technology resulted in a significant loss of Msm's respiratory activity and viability. Since this protein has been predicted to be an isoprenoid binding protein, we suspected a role of menaquinones, which are isoprenoid naphthoquinones, in the observed phenomenon. Accordingly, we tested whether MSMEG_5125's deficiency-induced lethality could be reversed by adding menaquinone. The result was positive, implying cooperation between MSMEG_5125 and menaquinone in bringing about respiration. Inhibition of MSMEG_5125 expression led to the induction of MSMEG_0089 and 2296, two hallmark genes of the MSMEG_2295 regulon. This result suggests that when MSMEG_5125 becomes limiting, a feedback-loop derepresses the MSMEG_2295 regulon genes, including its own. Interestingly, menaquinone functioned as an inducer of MSMEG_5125, indicating that it is likely to mediate the feedback mechanism. This result also strengthens our hypothesis that the functions of menaquinone and MSMEG_5125 are interrelated. Menaquinone also induced the MSMEG_2295-controlled operon MSMEG_2295-2294 (dinB2) not induced following the inactivation of MSMEG_5125. Therefore, the activation mechanism of MSMEG_2295-regulated genes may not be the same for all, although derepression is likely to be a common feature. In vitro, menaquinone abolished MSMEG_2295's DNA binding activity by interacting with it, confirming its role as an inducer. Therefore, a menaquinone-MSMEG_5125-regulated gene expression circuit controls Msm respiration and possibly oxidative stress-induced DNA damage repair.

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.

DNA binding and gene regulatory functions of MSMEG_2295, a repressor encoded by the dinB2 operon of Mycobacterium smegmatis.

MSMEG_2295 is a TetR family protein encoded by the first gene of a Mycobacterium smegmatis (Msm) operon that expresses the gene for DinB2 (MSMEG_2294), a translesion DNA repair enzyme. We have carried out investigations to understand its function by performing DNA binding studies and gene knockout experiments. We found that the protein binds to a conserved inverted repeat sequence located upstream of the dinB2 operon and several other genes. Using a knockout of MSMEG_2295, we show that MSMEG_2295 controls the expression of at least five genes, the products of which could potentially influence carbohydrate and fatty acid metabolism as well as antibiotic and oxidative stress resistance. We have demonstrated that MSMEG_2295 is a repressor by performing complementation analysis. Knocking out of MSMEG_2295 had a significant impact on pyruvate metabolism. Pyruvate dehydrogenase activity was virtually undetectable in ΔMSMEG_2295, although in the complemented strain, it was high. We also show that knocking out of MSMEG_2295 causes resistance to H2O2, reversed in the complemented strain. We have further found that the mycobacterial growth inhibitor plumbagin, a compound of plant origin, acts as an inducer of MSMEG_2295 regulated genes. We, therefore, establish that MSMEG_2295 functions by exerting its role as a repressor of multiple Msm genes and that by doing so, it plays a vital role in controlling pyruvate metabolism and response to oxidative stress.

Induced expression of the zwf gene in the presence of glucose contributes to lowering of glucose 6-phosphate level and consequently reduction of growth rate of Mycobacterium smegmatis.

In Mycobacterium smegmatis (renamed Mycolicibacterium smegmatis), glucose 6-phosphate (G6P) level is exceptionally high as compared to other bacteria, E. coli for example. Earlier investigations have indicated that G6P protects M. smegmatis (Msm) against oxidative stress-inducing agents. G6P is a glycolytic intermediate formed either directly through the phosphorylation of glucose or indirectly via the gluconeogenic pathway. Its consumption is catalysed by several enzymes, one of which being the NADPH dependent G6P dehydrogenase (G6PDH) encoded by zwf (msmeg_0314). While investigating the extent to which the carbon sources glucose and glycerol influence Msm growth, we observed that intracellular concentration of G6P was lower in the former's presence than the latter. We could correlate this difference with that in the growth rate, which was higher in glycerol than glucose. We also found that lowering of G6P content in glucose-grown cells was triggered by the induced expression of zwf and the resultant increase in G6PDH activity. When we silenced zwf using CRISPR-Cas9 technology, we observed a significant rise in the growth rate of Msm. Therefore, we have found that depletion of G6P in glucose-grown cells due to increased G6PDH activity is at least one reason why the growth rate of Msm in glucose is less than glycerol. However, we could not establish a similar link-up between slow growth in glucose and lowering of G6P level in the case of Mycobacterium tuberculosis (Mtb). Mycobacteria, therefore, may have evolved diverse mechanisms to ensure that they use glycerol preferentially over glucose for their growth.

Network approach to mutagenesis sheds insight on phage resistance in mycobacteria.

MOTIVATION: A rigorous yet general mathematical approach to mutagenesis, especially one capable of delivering systems-level perspectives would be invaluable. Such systems-level understanding of phage resistance is also highly desirable for phage-bacteria interactions and phage therapy research. Independently, the ability to distinguish between two graphs with a set of common or identical nodes and identify the implications thereof, is important in network science. RESULTS: Herein, we propose a measure called shortest path alteration fraction (SPAF) to compare any two networks by shortest paths, using sets. When SPAF is one, it can identify node pairs connected by at least one shortest path, which are present in either network but not both. Similarly, SPAF equalling zero identifies identical shortest paths, which are simultaneously present between a node pair in both networks. We study the utility of our measure theoretically in five diverse microbial species, to capture reported effects of well-studied mutations and predict new ones. We also scrutinize the effectiveness of our procedure through theoretical and experimental tests on Mycobacterium smegmatis mc2155 and by generating a mutant of mc2155, which is resistant to mycobacteriophage D29. This mutant of mc2155, which is resistant to D29 exhibits significant phenotypic alterations. Whole-genome sequencing identifies mutations, which cannot readily explain the observed phenotypes. Exhaustive analyses of protein-protein interaction network of the mutant and wild-type, using the machinery of topological metrics and differential networks does not yield a clear picture. However, SPAF coherently identifies pairs of proteins at the end of a subset of shortest paths, from amongst hundreds of thousands of viable shortest paths in the networks. The altered functions associated with the protein pairs are strongly correlated with the observed phenotypes. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mycobacterium tuberculosis thymidylate synthase (ThyX) is a target for plumbagin, a natural product with antimycobacterial activity.

Plumbagin derived from the plant Plumbago indica, known as Chitrak in India, is an example of a medicinal compound used traditionally to cure a variety of ailments. Previous reports have indicated that it can inhibit the growth of Mycobacterium tuberculosis (Mtb), the causative agent of the deadly disease TB. In this investigation, we provide an insight into its mode of action. We show here that a significant mycobacterial target that is inhibited by plumbagin is the enzyme ThyX, a form of thymidylate synthase, that is responsible for the synthesis of dTMP from dUMP in various bacterial pathogens, including Mtb. Using a purified preparation of the recombinant version of Mtb ThyX, we demonstrate that plumbagin, a 2,4 napthoquinone, but not lawsone, a structurally related medicinal compound, inhibits its activity in vitro. We also show that the intracellular [dTTP]/[dATP] ratio in Mycobacterium smegmatis (Msm) cells decrease upon treatment with plumbagin, and this, in turn, leads to cell death. Such a conclusion is supported by the observation that over-expression of thyx in the plumbagin treated Msm cells leads to the restoration of viability. The results of our investigation indicate that plumbagin kills mycobacterial cells primarily by targeting ThyX, a vital enzyme required for their survival.

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.

Mutual interaction enables the mycobacterial plasmid pAL5000 origin binding protein RepB to recruit RepA, the plasmid replicase, to the origin.

The Mycobacterium fortuitum plasmid, pAL5000, is the most-studied member of a family of plasmids that are found in Actinobacteria. Its replication is brought about by the combined action of two plasmid-encoded replication proteins, RepA and RepB. RepB has earlier been shown to be a sigma factor homologue that possesses origin-binding activity. The mechanism by which RepA functions, and its relationship with RepB, if any, has not been explored yet. In this study, we show that RepA shares a common catalytic domain, with proteins belonging to the primase-polymerase and DNA polymerase X families. We demonstrate that RepA is functionally a DNA polymerase and that mutations that alter two conserved aspartic acid residues present within the catalytic core lead to inactivation of plasmid replication. Replication of pAL5000 was shown not to depend on the host primase, and thus it is most likely that RepA is responsible for the priming act. We further demonstrate that RepA and RepB function as a pair and that the functional cooperation between the two requires physical contact. The C-terminal domain of RepA, which is structurally a helical bundle, is responsible for unwinding the origin in a site-specific manner and also for the establishment of contacts with RepB. The results presented show that RepB functions by recruiting RepA to the origin in much the same way as sigma factors recruit RNA polymerase core enzyme to promoters.

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.

Dynamics of Mycobacteriophage-Mycobacterial Host Interaction: Evidence for Secondary Mechanisms for Host Lethality.

Mycobacteriophages infect mycobacteria, resulting in their death. Therefore, the possibility of using them as therapeutic agents against the deadly mycobacterial disease tuberculosis (TB) is of great interest. To obtain better insight into the dynamics of mycobacterial inactivation by mycobacteriophages, this study was initiated using mycobacteriophage D29 and Mycobacterium smegmatis as the phage-host system. Here, we implemented a goal-oriented iterative cycle of experiments on one hand and mathematical modeling combined with Monte Carlo simulations on the other. This integrative approach lends valuable insight into the detailed kinetics of bacterium-phage interactions. We measured time-dependent changes in host viability during the growth of phage D29 in M. smegmatis at different multiplicities of infection (MOI). The predictions emerging out of theoretical analyses were further examined using biochemical and cell biological assays. In a phage-host interaction system where multiple rounds of infection are allowed to take place, cell counts drop more rapidly than expected if cell lysis is considered the only mechanism for cell death. The phenomenon could be explained by considering a secondary factor for cell death in addition to lysis. Further investigations reveal that phage infection leads to the increased production of superoxide radicals, which appears to be the secondary factor. Therefore, mycobacteriophage D29 can function as an effective antimycobacterial agent, the killing potential of which may be amplified through secondary mechanisms.

A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene.

UNLABELLED: Mycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc(2)155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ΔDRKIN, a strain derived from mc(2)155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ΔDRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2. IMPORTANCE: Mycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves the way for the development of drugs that can kill mycobacteria by inducing a dTTP-deficient state.

Biochemical Characterization of a Mycobacteriophage Derived DnaB Ortholog Reveals New Insight into the Evolutionary Origin of DnaB Helicases.

The bacterial replicative helicases known as DnaB are considered to be members of the RecA superfamily. All members of this superfamily, including DnaB, have a conserved C- terminal domain, known as the RecA core. We unearthed a series of mycobacteriophage encoded proteins in which the RecA core domain alone was present. These proteins were phylogenetically related to each other and formed a distinct clade within the RecA superfamily. A mycobacteriophage encoded protein, Wildcat Gp80 that roots deep in the DnaB family, was found to possess a core domain having significant sequence homology (Expect value < 10-5) with members of this novel cluster. This indicated that Wildcat Gp80, and by extrapolation, other members of the DnaB helicase family, may have evolved from a single domain RecA core polypeptide belonging to this novel group. Biochemical investigations confirmed that Wildcat Gp80 was a helicase. Surprisingly, our investigations also revealed that a thioredoxin tagged truncated version of the protein in which the N-terminal sequences were removed was fully capable of supporting helicase activity, although its ATP dependence properties were different. DnaB helicase activity is thus, primarily a function of the RecA core although additional N-terminal sequences may be necessary for fine tuning its activity and stability. Based on sequence comparison and biochemical studies we propose that DnaB helicases may have evolved from single domain RecA core proteins having helicase activities of their own, through the incorporation of additional N-terminal sequences.

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.

Mycobacteriophage L5Gp56, a novel member of the NrdH family of redoxins.

Mycobacteriophage L5 gene 56 encodes a putative thioredoxin family protein. Phylogenetic analysis revealed that Gp56 and related proteins are distantly related to NrdH - a glutaredoxin homolog which has thioredoxin-like properties. To understand its function, the recombinant version of the protein was biochemically characterized. For the sake of comparison, a mycobacterial thioredoxin, TrxB, was included in the study. Results show that Gp56 can be reduced by dithiothreitol, but only at a higher concentration as compared with TrxB, indicating that the standard redox potential of Gp56 is lower than that of TrxB. The reduced protein can subsequently act as a reductant of protein disulfide bonds. Gp56 can be reduced by NADPH with the help of thioredoxin reductase (TrxR) but less efficiently as compared with TrxB. The abilities of Gp56 and TrxB to reduce Gp50, the L5-encoded ribonucleotide reductase, was examined. While both are capable of executing this function, the former needs more reducing equivalents in the process as compared with the latter. This study shows that L5Gp56 represents a new class of NrdH-like proteins that function optimally in a reducing environment.

DnaK dependence of the mycobacterial stress-responsive regulator HspR is mediated through its hydrophobic C-terminal tail.

HspR is a repressor known to control expression of heat shock operons in a number of Eubacteria. In mycobacteria and in several other actinobacteria, this protein is synthesized from the dnaKJE-hspR operon. Previous investigations revealed that HspR binds to the operon promoter, thereby controlling its expression in an autoregulatory manner. DnaK, which is a product of the same operon, further aids this autoregulatory process by stimulating the operator binding activity of HspR. The molecular mechanism by which DnaK assists HspR in executing its function is not clearly understood. In this study, it has been shown that DnaK can augment DNA binding activity of HspR by two mechanisms: (i) DnaK can restore the activity of completely denatured HspR by forming a complex with it, and (ii) DnaK can prevent thermal instability of HspR renatured by other means. Unlike the first mechanism, the latter function does not involve complex formation. The C-terminal hydrophobic tail of HspR was found to play a significant role in determining its thermal stability and DnaK dependence properties. A deletion mutant in which this region is removed does not respond to thermal stress and functions independent of DnaK. The hydrophobic C-terminal tails of HspRs of Mycobacterium tuberculosis and related Actinomycetales therefore may have evolved to make these HspRs more sensitive to thermal stress and, at the same time, subject to regulation by DnaK.

Interactions of sulfur oxidation repressor with its promoters involve different binding geometries.

The divergently transcribed sulfur oxidation (sox) operon of a sulfur chemolithotrophs, Pseudaminobacter salicylatoxidans KCT001, comprising sox TRS-VW-XYZABCD, is regulated by a repressor (SoxR). SoxR binds to two disparate operators, sv (present in between soxS and soxV) and wx (present in between soxW and soxX). Here we report details of the interaction between SoxR and these two operator regions of the sox operon, using methylation interference and hydroxyl radical footprinting. We propose that the sv operator is symmetric and compact, while the wx operator is asymmetric and extended. We report an interesting difference between the SoxR-sv interaction and the SoxR-wx interaction through a competition assay involving groove-specific ligands. SoxR binds in the major groove of the sv operator, but binds in the minor groove of the wx operator. The structural flexibility of the SoxR helps it to act differentially in its interactions with these two operators. Mutational analysis shows that SoxR uses different amino acid residues when binding to the sv operator versus the wx operator. Taken together, the results indicate that interaction between SoxR and the two operator sites involves different binding geometries. This makes SoxR the only known example of a ArsR-family protein that binds differentially to different operators.

Evolutionary link between the mycobacterial plasmid pAL5000 replication protein RepB and the extracytoplasmic function family of σ factors.

Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in the Actinobacteria. It replicates using two plasmid-encoded proteins, RepA and RepB. While BLAST searches indicate that RepA is a replicase family protein, the evolutionary connection of RepB cannot be established, as no significant homologous partner (E < 10(-3)) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic, and mutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operon may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix domain characteristic of region 4 of extracytoplasmic function (ECF) σ factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of σ factors. Mutational analysis identified two arginines indispensable for RepB activity, one each located within the C- and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interaction modules.

Phages specific for mycobacterial lipoarabinomannan help serodiagnosis of tuberculosis.

This study evaluated the possibility to use six phages specific to the Mycobacterium tuberculosis lipoarabinomannan (LAM) as tools for tubercular serodiagnosis. We analysed sera samples from 30 subjects with active tuberculosis (TB+), 30 with latent tubercular infection (LTBI) and 60 healthy subjects as controls (K). Our data indicated a good antibody response of the TB+ and LTBI patients against the phage Ri(7)17; the optical density (OD) values obtained from sera patients was statistically significant when compared to the control samples. Our results confirm that phage display technology might be useful to develop new tools for diagnosis of tuberculosis.