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published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

Ph.D. / Biochemistry and Molecular Biology / The Bacillus subtilis spx gene encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It resides in the yjbC-spx operon and is transcribed from at least four promoters, three (P[subscript]1, P[subscript]2 and P[subscript]B) residing upstream of yjbC and one (P[subscript]M) located in the intergenic region between yjbC and spx. We uncovered a second intergenic promoter, P[subscript]3, from which transcription is elevated in cells treated with the thiol-specific oxidant diamide, by primer extension analysis. P[subscript]3 is recognized by the σ[superscript]A form of RNA polymerase (RNAP) in vitro without the involvement of a transcriptional activator. Deletion analysis together with point mutation analysis uncovered two negative cis-acting control elements within the P[subscript]3 promoter. Previously published studies and transcription factor/transformation array technology uncovered two transcriptional repressors, PerR and YodB that were potential candidates for the missing trans-acting factors affecting P[subscript]3 promoter utilization. PerR was previously characterized as the regulator of the inducible peroxide stress response in B. subtilis, while YodB is a novel DUF24/MarR type repressor that controls genes that are induced in response to phenolic compounds and oxidative stress. The derepression of spx was detected in both perR and yodB mutants by examining the level of spx expression using the spx-bgaB fusion construct. The additive effect was observed in the perR yodB double mutant. The regions of spx P[subscript]3 DNA required for transcriptional repression by YodB and PerR were confirmed by DNase I footprinting analysis. PerR protects an area from approximately position -3 to +35. YodB binds a region from approximately positions -3 to -32. The binding of YodB and PerR proteins to spx P[subscript]3 promoter DNA was impaired by addition of diamide and H[subscript]2O[subscript]2 in vitro as determined by DNase I footprinting analysis. Besides spx, YodB also controls the divergently transcribed yodC gene which encodes a putative nitroreductase that is induced by disulfide stress. Microarray and proteome analyses were performed to identify other genes controlled by YodB. yocJ (azoR1), encoding the putative FMN-dependent NADH-azoreductase, was the most strongly derepressed by yodB null mutation and was induced in response to diamide, catechol, MHQ and nitrofurantoin stress. bsrB encoding a small 6S RNA located downstream of azoR1, is co-transcribed with azoR1 and increased in concentration in response to thiol-reactive compounds. The yodB mutant confers a catechol and MHQ resistance phenotype due to AzoR1 overproduction. In addition, the yodBmhqR double mutant, bearing the deletion of the mhqR gene encoding a MarR-like repressor, that overproduces AzoR1 and MhqR-regulated paralog AzoR2, exhibits hyper-resistance to thiol-reactive compounds. Thus, the detoxification of thiol-reactive substances in YodB and MhqR regulons show overlapping functions. DNase I footprinting analysis, together with promoter sequence alignments, uncovered YodB boxes which contain a common 15 bp consensus sequence for YodB-DNA interaction. The YodB protein contains three cysteine residues Cys6, Cys101 and Cys108. The conserved Cys6 contributes to the repression of spx and azoR1 transcription by YodB. Moreover, mass spectrometry revealed YodB Cys modifications by catechol and MHQ.

Genetic engineering tools are under continuous development. However, hesitation by consumers and governments regarding consumption of genetically modified organism (GMO) affects taking advantage of developments in biotechnology. While being a complicated issue to address, this challenge inspired us to investigate whether it is possible to engineer organisms without altering their wild-type genomes, but with the same customizability level offered by genetic engineering; that is, having the capacity of expressing foreign proteins not codified by the wild-type genome. I used B. subtilis spores as a model organism for this purpose. I took advantage of the sporulation process during which two compartments with differential expression, or different gene expression patterns co-exist, the mother cell and the forespore, and I programmed a single designer plasmid to behave differently in each compartment: the plasmid in the mother cell modifies the spore phenotype, while the plasmid in the forespore undergoes self-digestion. At the end of sporulation, the mother cell lyses and releases the final product — a plasmid-free engineered spore. Following this, I incorporated the forespore-specific "self-digestion" gene circuit into a variety of plasmids with different purposes, including the generation of spores expressing GFP on their protective coats and the artificial induction of sporulation, both of them as a proof-of-concept of genetically intact bioengineered organisms. Production of the different types of genetically intact bioengineered spores resulted in an average of nearly 90% of them free of detectible plasmid or genome alterations. Spores of B. subtilis and other species overall continue to gain attention in the biotechnology sector, with potential applications ranging from biopesticides, probiotics, and vaccines to energy-converting materials, self-healing concrete, and whole-cell biocatalysts. While spores represent a special case of multiple-compartment organisms among bacteria, most eukaryotic organisms possess multiple compartments, structures, or tissues with differential expression, including plants and animals. Therefore, our results in this study could serve as a starting point for new ideas and methods for the genetic modification-free engineering of complex organisms or parts of them.

The physiological role of the teichoic acid polymers found in Gram-positive bacterial cell walls is not known. Studies of Bacillus subtilis hybrid strains implicate a defined chromosomal region, which includes the tag-1 locus, as necessary for teichoic acid biosynthesis. A set of ten mutants carrying lesions in this region was identified from among forty-four temperature-sensitive (ts) mutants generated by nitrosoguanidine mutagenesis and bacteriophage 029 selection. This protocol gave a population enriched for ts, versus auxotrophic, mutants. For each of the ten mutants, the frequency of genetic reconstruction, or correction, of the ts phenotype indicated that it was due to change(s) in a single gene. Results of two-factor transformation crosses sorted the mutants into three complementation groups; all ten could complement tag-1. Mutants in two complementation groups were transformed to ts⁺ with cloned rodC DNA. The map order of the newly isolated ts markers was determined from the results of two factor crosses. Orientation with respect to the hisA marker was inferred from transduction experiments. The newly isolated strains were shown to be conditional rod⁻ mutants. Growth at 48°C resulted in reduced growth rates and spherically shaped cells. Additional phenotypes seen for some mutants, namely 029 phage resistance and ts spore outgrowth, appeared closely associated with the ts rod⁻ mutation. Wall phosphate content for two of the mutants, following growth at 48°C, was found to be reduced in comparison to the wild-type control. Taken together these results lend support to the argument that the tag-1 region of the chromosome, which most likely directs teichoic acid biosynthesis, is important for establishment and maintenance of the normal bacillary morphology seen for B. subtilis. The importance of other gene products to the organization of newly synthesized wall was examined using B. subtilis macrofibers. Left- and right-handed macrofibers were converted to spheroplasts and the multi-celled structures regenerated under the two sets of conditions conducive for production of the original, and inverse hand. The helix hands observed for the regenerated structures always corresponded to those expected on the basis of the parental genotype.

Multi-drug resistance among bacterial pathogens can be mediated by a number of mechanisms, including multidrug efflux pumps. One such pump in Bacillus spp. is ykkCD, a heterodimer of the SMR family consisting of C and D subunits. Previous studies suggest that the expression of ykkCD is controlled by a putative riboswitch and that the antibiotic tetracycline binds to the riboswitch in vitro. Additional studies have shown that two derivatives of tetracycline also bind to the putative riboswitch. These findings now need to be validated by an in vivo study. In this study, the effects that tetracycline and its commercially available derivatives—doxycycline, minocycline, anhydrotetracycline, and oxytetracycline—have on the expression levels of the ykkCD gene in Bacillus subtilis were explored. The level of ykkCD expression was quantified using two different methods: (1) ykkCD protein levels was determined using a ykkCD RNA--galactosidase reporter gene construct and (2) ykkCD mRNA levels was quantified by quantitative RT-PCR. Although the findings from method (1) were inconclusive, upregulation was observed for tetracycline and minocycline, in agreement with the results of the previous binding studies. / Department of Chemistry

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