Colicin-mediated killing is an example of allelopathy, which has been found among several bacteria. Screening of 42 strains of Shigella sonnei isolated from diarrheal patients revealed that 39 (93%) S. sonnei strains were positive for colicin production against Escherichia coli DH5␣. In the PCR-based detection of the colicin types, 36 (92.3%) were identified as E3, 2 (5.1%) as E3 and E8, and 1 (2.6%) as E3 and E2. Representative S. sonnei strains producing heterologous colicins exhibited antagonism against diarrheagenic Escherichia coli (DEC) groups. Although it is known that mutation in the colicin receptor renders the host resistant to colicin, there is a dearth of information on the genetic characterization of such mutants. In the fluctuation test, colicin-resistant E. coli mutants were found to occur spontaneously at the rates of 2.51 ؋ 10 ؊8 and 5.52 ؋ 10 ؊8 per generation when exposed to colicins E3 and E8 and colicins E3 and E2, respectively. Genotypic characterization of colicin-resistant E. coli (EC Cr ) and S. sonnei (SS Cr ) strains displayed mutations in the btuB gene, which encodes the receptor for vitamin B 12 uptake. This gene was interrupted by various insertion sequences, such as IS1, IS2, and IS911. Complementation of EC Cr and SS Cr with plasmid-borne btuB (pbtuB) accomplished restoration of the colicin-susceptible phenotype. The vitamin B 12 uptake assay gave an insight into the physiological relevance of the btuB mutation. Our studies provide insights into the latent influence of S. sonnei colicins in governing the existence of some of the shigellae and all of the DEC and the genetic mechanism underlying the emergence of resistance.A llelopathy refers to the production of toxic metabolites that suppress both the growth and survival of distinct competitors in a common niche (1, 2). Colicin is one such antimicrobial biomolecule produced by certain members of the family Enterobacteriaceae, which provides a competitive edge against microorganisms that are not immune or resistant (3-6). Colicin producers express the immunity protein constitutively, which forms a complex with the colicin protein, thereby preventing "cell suicide." Resistance to killing is attributable to two factors: (i) a mutation in the receptor, which serves as a portal for entry into the target cell (true resistance), and (ii) an alteration in some component, which constitutes a part of the translocation machinery (tolerance) (7,8).In the present study, we have focused primarily on the role of colicin produced by the Shigella sonnei strains isolated from acute diarrheal patients in preventing the growth of diarrheagenic Escherichia coli (DEC) groups and spontaneous emergence of resistance to colicin due to mutation in the btuB gene, which encodes the receptor for vitamin B 12 uptake. The BtuB protein also serves to localize type A/E colicins and T5-like phages (BF23 and EPS7) on the target cell surface (9, 10). The btuB gene consists of a single open reading frame, which is translated into a 614-amino-acid polypeptide. The fir...

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.

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Colicinogenic plasmids encode toxic proteins which have antagonistic activity against closely related bacteria. This study describes the molecular characterization of three colicinogenic plasmids designated as pSSE3, pSSE and pSSE2, each with a molecular size of ∼6 kb, identified in clinical isolates of Shigella sonnei. Sequence analysis revealed that pSSE and pSSE2 shared extensive sequence homology with each other and with Escherichia coli E-type colicinogenic plasmids. The plasmid pSSE3 lacked an additional gene imparting immunity to colicin E8, a unique feature not observed in any of the previously reported sequences of colicin E3 plasmids. Incomplete digestion of colicinogenic plasmids by restriction endonucleases, metachromatic staining with acridine orange and presence of single stranded initiation (ssi) region confirmed the coexistence of ssDNA along with dsDNA. Plasmid copy number as determined by real-time PCR was found to be about 20. Transmission electron microscopy revealed DNA impairment in test bacteria after colicin exposure. We hypothesize that S. sonnei has acquired E-group colicin plasmids from its close relative E. coli, with their sequences undergoing subtle changes depending on the cohabitation in the same milieu.

Mycobacteriophages are phages that infect mycobacteria resulting in their killing. Although lysis is the primary mechanism by which mycobacteriophages cause cell death, others such as abortive infection may also be involved. We took recourse to perform immunofluorescence and electron microscopic studies using mycobacteriophage D29 infected Mycobacterium smegmatis cells to investigate this issue. We could observe the intricate details of the infection process using these techniques such as adsorption, the phage tail penetrating the thick mycolic acid layer, formation of membrane pores, membrane blebbing, and phage release. We observed a significant increase in DNA fragmentation and membrane depolarization using cell-biological techniques symptomatic of programmed cell death (PCD). As Toxin-Antitoxin (TA) systems mediate bacterial PCD, we measured their expression profiles with and without phage infection. Of the three TAs examined, MazEF, VapBC, and phd/doc, we found that in the case of VapBC, a significant decrease in the antitoxin (VapB): toxin (VapC) ratio was observed following phage infection, implying that high VapC may have a role to play in the induction of mycobacterial apoptotic cell death following phage infection. This study indicates that D29 infection causes mycobacteria to undergo morphological and molecular changes that are hallmarks of apoptotic cell death.

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Mycobacteriophages are phages that interact with mycobacteria resulting in their killing. Although lysis is the major mechanism by which mycobacteriophages cause cell death, other mechanisms may also be involved. The present study was in i tiated with the objective of investigating the changes that take place at the cellular level following the infection of mycobacterial cells by phage D29.  To investigate th is issue, we took recourse to performing immunofluorescence and electron microscopic studies . Transmission electron microscopic examination reveal ed the adsorption of phages on to the surface of mycobacteria , f ollowing which penetration of the tail through the thick mycol o ic acid layer was seen . At later time points discrete populations of cells at different stages of lysis we re observed , which comprised of complete ly lys ed cells , in which the cells were fragmented and those at the early onset stage exhibited formation of membrane pores through which the phages and intracellular contents were released.   SEM results also indicate d that phages may come out through the entire surface of the cell, or alternatively through gaps in the surface. In some of the images we observed structures that apparently resembled membrane blebs which are normally encountered when cells undergo programmed cell death (PCD). In addition, we observed significant increase in DNA fragmentation as well as membrane depolarization, which are also indicative of occurrence of PCD. As several bacterial PCD pathways are mediated by the toxin-antitoxin (TA) modules, the expression profile of all the TA systems was examined before and after phage infection. Apart from specifically addressing the issue of PCD in mycobacteriophage infected cells, this investigation has led to the development of facile tools necessary for investigating mycobacteriophage-mycobacteria interactions by means of microscopic methods.