Stationary phase mutagenesis in Bacillus subtilis: The interaction between transcription and error-prone replication in conditions of stress

While under conditions of stress, non-dividing cells may acquire beneficial mutations. This is referred to as stationary phase mutagenesis, or adaptive mutagenesis. Previous research has shown that actively transcribed genes and those under selective pressure are prone to mutations that confer escape from non-dividing conditions. Accordingly, strains lacking transcription factors have shown a drastically lower number of mutations that confer escape while under amino acid starvation than those observed in the wildtype background. Also, error-prone DNA polymerases are known to be active in cells under stress and it has been shown that strains lacking an error-prone DNA polymerase display reduced levels of stationary phase mutagenesis. It is possible to speculate that when active transcription stalls, perhaps due to pre-mutagenic lesions in the template DNA strand, error-prone polymerases are recruited to the site of stalled transcription as part of DNA repair processes. This interaction between transcription and DNA repair is likely to bias the accumulation of mutations at highly transcribed loci. This model may be tested with strains carrying deficiencies in Mfd (transcription factor), YqjH (error-prone DNA polymerase), or both. We expect the double-knockout strain to show a similar level of mutagenesis to those observed in strains carrying only one deficiency, and lower levels compared to those in the wildtype. Alternatively, if these factors influence mutation separately, a double-knockout should show even lower accumulation of adaptive mutants than either the Mfd- or YqjH-. We are currently constructing the double-knockout strain in Bacillus subtilis

Stationary phase mutagenesis in Bacillus subtilisis independent of genome replication

Stationary phase mutagenesis is defined as cellular mechanisms that produce genetic diversity in cells experiencing conditions of stress. These processes are associated with many biological phenomena, including those that produce the formation of cancers in animal cells and other degenerative diseases. Also, these mechanisms are associated with the accumulation of beneficial mutations in bacteria, but follow stochastic processes and are controlled by genetic factors. The current models explaining the generation of stress-induced mutations are predicated on the formation of DNA replication intermediates that are formed during the repair of damaged DNA or during DNA replication and transcription encounters. Here we test the hypothesis that genome (DNA) replication is not required for the generation of stress-induced mutations. Our experiments compared the accumulation of mutations in cells differing in their ability to initiate and elongate genome replication at high temperatures (45° C) and showed that both types of cells accumulate mutations at very similar rates. These results then suggest that resting cells possess replication-independent mechanisms that generate mutations and therefore add novel aspects to our view of the evolutionary process

Abiotic, Biotic, and Bio-Enhanced Reduction of Hexavalent Chromium, Chloroform and Co-Contaminants Using Nano-Scale Zero Valent Iron in Highly Contaminated Groundwater

Investigations of groundwater in a former industrial perchlorate manufacturing site have shown high contamination with perchlorate, chlorate, nitrate, hexavalent chromium (Cr (VI)), and chloroform (CF) with levels greater than 3,000, 30,000, 300, 100, and 4 mg/L, respectively. Remediation efforts using biological reduction to desired contaminant levels at this site has been challenging due to high contaminant concentrations, and high total dissolved solids (TDS). Furthermore, removal of Cr(VI) and CF in the presence of nitrate, chlorate, and perchlorate has not been examined at the contaminated site. Nano-scale Zero-Valent-Iron (NZVI) has been effective at reducing groundwater contamination both with and without bacterial augmentation. The objective of this research was to investigate the removal of CF, Cr(VI) and co-contaminants in contaminated industrial groundwater using NZVI alone or in combination with biological reduction (bio-enhancement). The effectiveness of abiotic reduction using NZVI, biotic reduction using a 1ml bacterial sludge inoculum enriched with 20 ml/L of molasses and additional nutrients, and bio-enhanced reduction using both NZVI and bacteria was evaluated in this study. Bench-scale reactors were monitored for Cr(VI), CF, nitrate, chlorate, and perchlorate removal over 8 weeks. The use of NZVI resulted in 100% reduction of Cr(VI) in only 4 hours with doses of 5,000 mg Fe^0/L. As 100% reduction of Cr(VI) occurred at a much faster rate in abiotic treatments than biotic treatments, bio-enhancement for Cr(VI reduction relies more on NZVI reduction. For CF, removal showed 15%-40% greater results under bio-enhancement conditions than abiotic treatments. However, a bio-enhanced NZVI dose of at least 8,500 mg Fe^0/L is needed to achieve higher removal than biotic treatments alone. A bio-enhanced NZVI dose of 17,000 mg Fe^0/L resulted in 100% CF removal in 7 days. Bio-enhancement also achieved greater nitrate and chlorate removal, showing 100% removal at NZVI doses of 17,000 and 5,000 mg Fe^0/L, respectively. No abiotic perchlorate reduction was observed using NZVI. Perchlorate showed 25-50% removal only in biotic and bio-enhanced conditions. Bio-enhancement showed greater and more consistent removal for all the examined contaminants. This endorses bio-enhancement as the best treatment for groundwater from the examined site

Antibiotic resistance in Bacillus subtilis as affected by transcriptional derepression and the stringent response

Bacterial cells under conditions of starvation or prolonged non-lethal selective pressures accumulate mutations in highly transcribed genes. This process is part of cellular programs to increase genetic diversity in conditions of stress, also known as stationary phase or stress-induced mutagenesis. This experiment investigated mutation frequencies for antibiotic resistance as affected by the stringent response. The stringent response is a global cellular process that initiates at the cessation of growth and mediates changes in gene expression that repress synthesis of ribosome components. We used Bacillus subtilis strains that differ in RelA proficiency. The relA gene controls the synthesis of (p)ppGpp, the signaling molecule which mediates the stringent response. Since genes involved in protein synthesis are repressed during the stringent response, we hypothesize that relaxed mutants express a higher accumulation of mutations that confer resistance to tetracycline than cells that become stringent. Resistance to tetracycline may be acquired by altering components of the small subunit of bacterial ribosomes. Utilizing an overlay procedure and increasing times of incubation under nutritional stress, stationary cells were prompted for resistance to tetracycline. Our results showed that relA- cells expressed a higher accumulation of Tcr mutations than the one observed in wild type cells. These results provide evidence that transcriptional derepression in cells under non-lethal stress mediates mutagenic events. Implications in antibiotic resistance are further discussed

Examination of germination receptors of B. subtilis and B. megaterium

Many bacterial species including those in the Bacilli group form spores as a mechanism to survive harsh conditions such as extreme temperature, radiation, chemicals, and nutrient starvation. By forming spores, they can remain metabolically dormant for an extended period and revert to their vegetative form when environment becomes favorable. This resumption of metabolism and growth is marked by a process called germination that is triggered by exogenous nutrients such as amino acids, sugars, and nucleotides. The (Ger) germination receptors that are postulated to respond to these germinants, in the case of B. subtilis and B. megaterium, are a complex of at least three different proteins (the A-, B-, and C- subunits) transcribed from the same operon. While similar in gene arrangement and protein complex formation, these two Bacilli sp. respond to different germinants. This experiment investigates the GerA receptor of B. subtilis and the GerU receptor from B. megaterium. GerA of B. subtilis is activated with L-alanine, while GerU of B. megaterium is activated with L-proline. In order to determine the location of the binding site, different fragments of the GerAB gene and the GerUB genes encoding for protein A and B from each operon were amplified and fused together in frame to make a chimeric gene product. recombination. Spores from B. subtilis mutant strains expressing chimeric protein complexes will be tested for germination in the presence of L-proline and/or L-alanine. These studies will provide insights into how bacteria sense their environment and possible strategies to control and prevent growth

Synthesis of chimeric receptors essential for spore germination

Various species of bacteria have been reported to form an endospore, a metabolically dormant cell, during times of nutrient deficiencies and extreme stress. These said structures are outstandingly resistant to harsh chemicals, extreme temperatures, and can revert back to a metabolically active cell, through a process known as germination, when the necessary conditions are met. The rigid membrane of the endospore contains various germination (Ger) receptors which sense the external environment for necessary metabolites and germinants. Ger receptors are encoded by tricistronic operons that produce three distinct membrane proteins, the A, B, and C subunits. Although the function of the Ger receptor has been established by genetics, no information is currently available for germinant binding site. Bioinformatic and genetic approaches has predicted that the C-terminus of the B subunit is the most likely candidate to contain the germinant binding site. B. Subtilis and B. Megaterium, two species of the Bacilli genus, germinate in response to different germinants; B. Subtilis germinates in response to L-alanine by activation of the GerA receptor, while B. Megaterium germinates in response to L-leucine by the activation of its GerU receptor. The focus of this study is to construct chimeric genes in which fragments of B. Subtilis GerA receptors and B. Megaterium receptors are fused together. These B. Subtilis::B. Megaterium chimeric receptors will be introduced into the B. Subtilis genome and the mutant B. Subtilis spores will then be tested for the ability to germinate with leucine in order to establish the leucine binding site of GerUB. During the initial pilot studies, the regions coding for the Nterminus of the GerA receptor from B. Subtilis and the C-terminus of the GerU receptor from B. Megaterium were amplified using polymerase chain reaction with primer ends complementary to each other in order to further produce the desired hybrid genes without the use of restriction enzymes

DNA secondary structures and their contribution to mutagenesis in B. subtilis stationary phase cells

It is widely known and accepted that the cause of many mutations in cells are generated during the replication process of actively dividing cells, however more recent research has shown that mutations also arise in non growing conditions, a phenomenon known as stationary phase mutagenesis. Much of what is known come from studies in eukaryotic and bacterial models. It has been proposed that in non~growing cells, the process of transcription plays an important role in mutagenesis. We test the hypothesis that DNA secondary structures, formed during transcription, promote mutagenesis. The transcription-generated structures are speculated to be prone to mutations by exposing regions of single stranded DNA to lesions. We examined the Bacillus subtilis gene thiF, predicted by in silica analysis to be prone to mutations at particular locations during transcription. By altering the base sequence of this gene, the stability of its stem-loop structures is affected, thereby allowing us to test whether transcription of the altered sequence influences accumulation of mutations in thiF. Our assay for detection of mutations is based on reversion to thiamine prototrophy in cells under conditions of starvation. Ultimately, these experiments will increase our understanding of how mutations occur in cells of all domains of life

The Effect of CodY on stationary phase mutagenesis in Bacillus subtilis

We examine the notion that cells in conditions of stress accumulate mutation is in genes under selection via transcription processes. CodY is a global transcriptional regulator in many Gram positives, including soil and pathogenic microbes. In conditions of exponential growth and when branch chain amino acids and GTP are in abundance CodY acts as a transcriptional repressor of many metabolic operons. This transitional repression saves the cell energy and allows efficient use of resources. In conditions of starvation, CodY relieves repression of genes involved in acquisition of nutrients and degradation of carbon sources (genes under selection). Here, we compare the accumulation of mutations in genes under selection in wild type and CodY

Mfd Protects Against Oxidative Stress in Bacillus Subtilis Independently of its Canonical Function in DNA Repair

Background: Previous reports showed that mutagenesis in nutrient-limiting conditions is dependent on Mfd in Bacillus subtilis. Mfd initiates one type of transcription-coupled repair (TCR); this type of repair is known to target bulky lesions, like those associated with UV exposure. Interestingly, the roles of Mfd in repair of oxidative-promoted DNA damage and regulation of transcription differ. Here, we used a genetic approach to test whether Mfd protected B. subtilis from exposure to two different oxidants. Results: Wild-type cells survived tert-butyl hydroperoxide (t-BHP) exposure significantly better than Mfd-deficient cells. This protective effect was independent of UvrA, a component of the canonical TCR/nucleotide excision repair (NER) pathway. Further, our results suggest that Mfd and MutY, a DNA glycosylase that processes 8-oxoG DNA mismatches, work together to protect cells from lesions generated by oxidative damage. We also tested the role of Mfd in mutagenesis in starved cells exposed to t-BHP. In conditions of oxidative stress, Mfd and MutY may work together in the formation of mutations. Unexpectedly, Mfd increased survival when cells were exposed to the protein oxidant diamide. Under this type of oxidative stress, cells survival was not affected by MutY or UvrA. Conclusions: These results are significant because they show that Mfd mediates error-prone repair of DNA and protects cells against oxidation of proteins by affecting gene expression; Mfd deficiency resulted in increased gene expression of the OhrR repressor which controls the cellular response to organic peroxide exposure. These observations point to Mfd functioning beyond a DNA repair factor in cells experiencing oxidative stress

DNA secondary structures and their contribution to mutagenesis in B. subtilis stationary phase cells

It is widely known and accepted that the cause of many mutations in cells are generated during the replication process of actively dividing cells, however more recent research has shown that mutations also arise in non growing conditions, a phenomenon known stationary phase mutagenesis. Much of what is known come from studies in eukaryotic and bacterial models. It is proposed that in nongrowing cells, the process of transcription plays an important role in mutagenesis. I will test the hypothesis that secondary structures formed of DNA generated transcription promote mutagenesis. The sequences transcriptiongenerated structures are speculated to be prone to mutations by exposing regions of single stranded DNA to lesions. To test this hypothesis, I examined the Bacillus subtilis gene thiF, predicted by in silico analysis to be prone to mutations at particular locations during transcription. By altering the base sequence of this gene, the stability of its stem-loop structures is affected, thereby allowing us to test whether transcription of the altered sequence influences accumulation of in thiF. Our assay for detection of mutations is based on reversion to thiamine auxotrophy in cells under conditions of starvation. Ultimately, these experiments will increase our understanding of how mutations occur in cells of all domains of life