Bacteria are ubiquitous in all ecosystems and are often challenged by multiple stresses such as extreme temperatures, high salt concentrations, nutrient limitation, pH variations, radiation, predation and the presence of antibiotics/toxins. The most challenging among them is predation pressure which is one of the major causes of their mortality in different niches. Bacteria have evolved different adaptive measures to counter predation. Some of them include change in shape, size, motility, and unpalatable aggregate formation. Aromatic β-glucosides such as salicin, produced by plants as secondary metabolites, play a significant role in protecting them from herbivores. Members of the family Enterobaceriaceae primarily present in soil, e.g. Erwinia chrysanthemi (a phytopathogen) and Klebsiella aerogenes, can utilize the aromatic β-glucosides salicin and arbutin (likely to be present in soil derived from decomposing plant materials) as a carbon source unlike their fellow members such as Escherichia coli, Shigella sonnei, and Salmonella present in the gut environment. Bacteria can obtain energy by metabolizing β-glucosides in the form of glucose. Whether they can also use these molecules as defense tools in a manner similar to plants is an intriguing possibility. In such an event, Bgl+ bacteria could derive a dual advantage in terms of energy generation and protection from predation. The current study was initiated to investigate a possible link between β-glucoside metabolism and self-defense in Enterobacteriaceae. Different members of Enterobacteriaceae comprising of both laboratory strains and natural isolates were considered as prey. Predators included were laboratory strains and soil isolates of bacteriovorous nematodes of the Rhabditidae family, the amoeba Dictyostelium discoidium and a bacteriovorous Streptomyces sp. The predator-prey interaction was analyzed by performing viability and behavioral assays in the context of β-glucoside metabolism Results presented in Chapter 2 show that active catabolism of aromatic β¬glucosides like salicin, arbutin and esculin by Bgl+ bacteria decreases the viability of their predators. The aglycone products released during β-glucosides metabolism, e.g. saligenin in the case of salicin, are the causative agents of the mortality of the predators. The lethality is reversible up to a specific threshold of exposure. Saligenin acts as a chemo-attractant that lures and kills Caenorhabditis elegans N2. In the case of nematodes that succumb, bacteria can derive nutrition from the dead predators indicating a conversion of prey to predator. Experiments with mutant strains of Caenorhabditis elegans suggest that the dopaminergic receptor dop-1 is involved in mediating saligenin toxicity. Studies mentioned in Chapter 3 revolve around the relevance of the predator-prey interaction discussed in Chapter 2 in the natural environment. Members of Enterobacteriaceae and their predator amoebae (cellular slime molds) and nematodes were isolated from soil. They show coexistence in most of the soil samples analyzed. All the predators isolated from soil and other natural isolates of Caenorhabditis succumb to saligenin as their laboratory counterparts with higher sensitivity in some of the strains. Soil nematodes belonging to genera Oscheius and Mesorhabditis avoid saligenin unlike the members of Caenorhabditis genus which are attracted towards saligenin. This indicates that the soil nematodes are often exposed to saligenin or saligenin-like compounds, resulting in the evolution of a genetic machinery to avoid these toxic compounds. Studies with quasi-natural environments like soil and fruit indicate that β-glucoside metabolism have similar effects on predator prey interaction in these environments, reinforcing the relevance of these observations to the natural ecology of the organisms. The studies reported in Chapter 2 and 3 shed light on a novel defense strategy of otherwise non-pathogenic members of Enterobacteriaceae which comes with a dual advantage. These results have also brought into focus issues such as the benefit derived by bacterial populations that are genetically heterogeneous, consisting of both Bgl+ and Bgl-strains. The broad implications and future directions of the work are discussed in Chapter 4. Work presented in Appendix deals with the investigation of the pattern of cellobiose utilization in Shigella sonnei. As mentioned in Chapter 1, it is known that members of Enterobacteriaceae exhibit diversity in their pattern of β-glucoside utilization. Wild type strains of both E. coli and Shigella sonnei are unable to utilize Arbutin, Salicin and Cellobiose. While E. coli can acquire cellobiose utilizing ability directly from the wild type state (Arb-Sal-Cel-), Shigella sonnei strains, though closely related to E. coli, have to undergo a series of mutations in a specific sequence to become capable of utilizing these sugars. Characterization of a few Shigella sonnei Cel+ mutants showed a different mode of activation of the chb operon (known to be involved in cellobiose utilization in E. coli). Considering the ecological significance of the ability to hydrolyze aromatic β-glucosides, a detailed understanding of the metabolic capability of different strains and the molecular mechanism involved becomes significant.

The genetic systems involved in the utilisation of aromatic β-glucosides in E. coli consist of the bgl, asc, and chb operons and the locus bglA encoding phospho-β-glucosidase A. The bgl and asc operons are known as cryptic or silent systems since their expression is not sufficient for utilisation of these sugars in wild type strains of E. coli. Their transcriptional activation by different classes of mutations confers a Bgl+ phenotype to the mutant. The maintenance of cryptic genes without accumulating deleterious mutation in spite of being silent is an evolutionary puzzle. Several observations have suggested the possibility that these genes may be expressed under specific physiological conditions conferring a fitness advantage to the organism. The main aim of this study was to investigate the possible role of aromatic β-glucoside catabolic systems of E. coli in combating nutrient stress and microaerobic growth conditions. The results presented in Chapter 2 address the evolution of aromatic β-glucoside catabolic systems when exposed to a novel β-glucoside as the sole substrate. The results indicate that the bgl opeon, the primary system involved in the utilisation of the aromatic β-glucosides arbutin and salicin, is also involved in esculin utilisation. In the absence of bglB encoding the enzyme phospho-β-glucosidase B, activation of the silent asc operon enables esculin utilisation. The bglA gene encoding phospho-β-glucosidase A specific for arbutin, can undergo successive mutations to evolve the ability to hydrolyse esculin and salicin sequentially when bglB and ascB are absent. The Esc+ and Sal+ mutants retain their arbutin+ phenotype, indicating that the mutations enhance the promiscuity of the enzyme. Sequencing data indicate that the first step Esc+ mutant carries a four base insertion within the promoter of the bglA gene that results in enhanced transcription of bglA. RT-PCR studies confirm that both the steady-state levels as well as the half-life of the bglA mRNA are enhanced in the mutant. This is further corroborated by the observation that overexpression of wild type bglA in the parent strain using a multicopy plasmid confers an Esc+ phenotype. The second step Sal+ mutant carries a point mutation within bglA ORF, a thymine to guanine transversion at position 583 (T583G) of the bglA gene, resulting in an amino acid change from cysteine to glycine at position 195 (C195G) of the BglA ORF close to the active site. Presence of a plasmid carrying the T583G mutation, introduced by site-directed mutagenesis, results in a Sal+ phenotype, confirming the role of the transversion in conferring the Sal+ phenotype. Based on docking studies, the positioning of salicin into the substrate binding site of the mutant BglA enzyme is different compared to wild type BglA due to the loss of stearic hindrance for the binding of salicin when C195 is replaced by the smaller amino acid glycine in the mutant protein. These observations indicate that under conditions of nutrient deprivation, exposure to novel substrates can result in the evolution of new metabolic capabilities by the sequential modification of a pre-existing genetic system. In the case of one novel substrate, the mutation results in the overexpression of the hydrolytic enzyme, while in the case of the second substrate, a mutation close to its active site increases its substrate specificity. Results presented in Chapter 3 specifically deal with the involvement of the bgl operon under low levels of oxygen. Earlier observations have shown that there is a 22 fold enhancement in the expression of the bgl operon under anaerobic condition. The present results provide evidence that bgl expression has a physiological role under low levels of oxygen and in addition suggest a possible mechanism for the overexpression of the bgl operon that involves the ArcAB two component system known to mediate regulation under microaerobic and static conditions. Transcription studies using a lacZ reporter fused to the wild type bgl promoter show that there is enhanced transcription from the bgl promoter under microaerobic and static conditions in the presence of arcA encoding the response regulator compared to that in its absence. The positive effect of arcA on the expression of the bgl operon is dispensable in the absence of H-NS since presence or absence of arcA does not change the expression of the bgl operon in an hns-null background, implying that the involvement of ArcA is via antagonizing H-NS. Competition experiments indicate that there is growth advantage associated with the activated allele of the bgl operon under low levels of oxygen since Bgl+ strains carrying the activated allele of the bgl operon as well as strains expressing BglG constitutively can out-compete wild-type strains. Presence of the wild type arcA allele results in a strong growth advantage compared to its absence under static conditions but not aerobic condition. The bgl operon seems to be one of the possible downstream targets of ArcA under static condition since absence of the bgl operon results in a modest reduction of the growth advantage (GASP) phenotype conferred by arcA. The up-regulation of the bgl operon is likely to enable the cells to scavenge available nutrients from their niche more efficiently. These experiments also show that the GASP phenotype associated with BglG constitutive strains under static conditions involves downstream genes that are different from oppA known to be one of the downstream targets during aerobic growth. It is possible that under low level of oxygen, the bgl operon is regulating a different set of downstream genes involving a different mechanism. In summary, the results of this investigation show that the aromatic β-glucoside catabolic systems in E. coli play a role in the generation of new metabolic capabilities via mutations in pre-existing genetic systems as well as through changes in gene expression patterns. The mechanisms outlined in this study are likely to be of broader significance applicable to microbial evolution under stress in general.