Campylobacter jejuni is a prevalent cause of bacterial gastroenteritis in humans worldwide. The mechanism by which C. jejuni survives stomach acidity remains unknown. In this study, we have demonstrated that the ferric uptake regulator Fur plays an important role in Campylobacter acid survival. C. jejuni with a fur deletion was more sensitive to acid than the wild-type. Profiling the acid stimulon of the C. jejuni ∆fur mutant allowed us to uncover Fur-regulated genes under acidic conditions. The up-regulation of heat shock genes and the down-regulation of genes involved in flagellar and cell envelope biogenesis in the fur mutant highlight the importance of Fur in Campylobacter acid survival. Furthermore, prior exposure of C. jejuni to acid increased its capacity to survive other stresses, such as oxidative stress. This enhanced survival in the presence of oxidative stress was shown to be Fur-dependent through the regulation of catalase katA expression. Interestingly, Fur-mediated repression of katA was alleviated under low-pH conditions, allowing for higher catalase expression and defense against oxidative stress. Additionally, the transcriptome of C. jejuni under acidic conditions revealed that many genes involved in Campylobacter pathogenesis were differentially expressed. Prior exposure of C. jejuni to acid significantly increased its adherence to and invasion of human epithelial cells. Furthermore, in vivo experiments using Galleria mellonella larvae showed that acid exposure markedly enhanced Campylobacter virulence potential. In conclusion, this study demonstrates that the ferric uptake regulator Fur is a potential regulator of Campylobacter acid survival and cross-protection against other stresses. Moreover, our results suggest that the obligate passage of C. jejuni through the stomach acid barrier modulates the expression of its virulence factors and predisposes the bacterium for efficient gut colonization.

Escherichia coli has evolved in environments which may commonly be acidic and thus developed adaptive mechanisms to minimise acid-induced damage. It has previously been observed that adapted bacteria to moderately acidic conditions can grow in media considerably below their optimum growth pH. To explain this phenomenon, a hypothesis which suggested that diffusible molecules (alarmones) may serve as early warning systems of acidic conditions was proposed. Alarmones are thought to be produced upon exposure to mildly-acidic conditions. They then diffuse in the environment and elicit a protective response against acid in recipient cells. The protective activity of those putative alarmones against lethal acid was investigated. The main aim of this project is to determine the mode of action of those alarmones at the molecular level. Preliminary experiments confirmed acid resistance conferred by alarmones to populations of E. coli C600. The stability of those alarmones at different temperatures and following proteinase K treatment was investigated. Moreover, investigations into whether alarmones are autoinducer-2 (AI-2) molecules and whether alarmones increase the percentage of persisters in an E. coli population were undertaken. Subsequently, microarray analyses of both alarmone-induced and non-induced cultures were performed to reveal E. coli genes induced by alarmones. Moreover, proteomic studies using two-dimensional gel electrophoresis were conducted to reveal proteins induced by alarmones. Supernatants from alarmone-induced cultures conferred statistically significant protection (p<0.01) on recipient cultures against lethal acid (pH3). Alarmones were inactivated by heat (60oC) and by proteinase K. The autoinducer-2 (AI-2) assay revealed that alarmones are not AI-2 molecules. In addition, alarmones did not increase the percentage of persisters. In order to elucidate potential mechanisms for alarmone-mediated protection, the genomic expression and protein induction of alarmone-induced cells using microarray analysis and two-dimensional gel electrophoresis, respectively, were performed. Two-dimensional gel electrophoresis of transduced cultures indicated that around 13 proteins were induced in the alarmone-protected populations of E. coli C600. Mass-spectrometric analysis revealed that these alarmone-inducible proteins include the acid stress chaperone HdeB and the DNA-binding transcriptional dual regulator, H-NS which plays an important role in stress adaptation. Microarray analyses of transduced cultures indicated that 671 open reading frames (ORFs) were significantly differentially expressed between alarmone-protected and control populations (p<0.05). 508 ORFs were upregulated in the induced cells including 10 genes related to acid-resistance and 36 different genes related to multidrug efflux system proteins whereas 163 ORFs including the autoinducer-2 system were downregulated. E. coli releases diffusible signalling compounds which mediate adaptation to acid stress of recipient cells. Microarray and proteomic data show that two acid fitness island genes (hdeB and hdeD) and three genes that encode the antiporters of the three amino-acid-dependent acid resistance mechanisms (gadC, adiC and cadB) were among the upregulated genes. This work confirms that there is communication between bacterial cells in general and warning messages amongst E. coli C600 cells in particular in the presence of stress.

The bacterium Streptococcus sanguinis is a primary member of the human oral microflora and also has been recognized as a key player in the bacterial colonization of the mouth. It is considered the most common viridians streptococcal species implicated in infective endocarditis. In all kingdoms of life, ATP binding cassette (ABC) transporters are essential to many cellular functions. Sequencing of the SK36 genome provided the opportunity to study ABC transporter mutants and their relationship with acidity of the oral environment. Despite numerous studies that have focused on carbohydrate uptake systems in closely related streptococcal species such as S. mutans, S. pneumonia and S. pyogenes, the mechanism of the response of these ABC transporters to acidic conditions in S. sanguinis is still unknown. The capability of S. sanguinis to adapt in these harsh environments suggests this bacterium is capable of responding to various environmental stimuli. The purpose of this study was to examine ABC mutants to identify functions that contribute to acid tolerance in S. sanguinis. This study demonstrates that two acid-sensitive mutant genes, SSA_1507 and SSA_1508, identify genes involved in acid tolerance. The two mutants grew on different sugars and none of them showed a defect in sugar utilization at acid pH. We couldn’t recognize any significant differences in sugar uptake for the two acid sensitive mutants or in mutants of their neighboring genes. Thus, the observed acid sensitivity is not due to a failure to take up any of the common sugars tested. The cytoplasmic pH of S. sanguinis was studied with the fluorescent pH indicator (BCECF) and SK36 was observed to have a wider pH range than either of the two acid-sensitive mutants SSA_1507 or SSA_1508. In these two mutants, intracellular pH was not as well maintained. At all pH values tested, the mutants displayed a lower intracellular pH than the wild type. These observations indicate that the cell membrane of these two mutants is unable to protect the interior components from adverse effects of higher pH values and lower pH values, and prove that these two mutant genes SSA_1507 and SSA_1508 are unable to grow in lower pH values. These results support a role for these ABC transporters in proton pump or export and indicate that the mutants are less able to pump out protons.

Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.

Acid-stress adaptation in Listeria monocytogenes (Lm) serotype 4b and 1/2a occurred when cells were pre-exposed to pH 5.0 tryptic soy broth supplemented with yeast extract (TSB-YE) at 22°C or 37°C but not at 4°C. Prolonged time, varied sublethal acid pH, substitute of acidulants and addition of sodium chloride during 4°C mild acid pre-exposure still did not induce acid-stress adaptation in Lm. This finding was also validated using an acidic cheese, similar to what has seen for Gram-negative bacteria E. coli and Salmonella. Further investigation revealed that major cold shock protein in Lm CspL was not responsible for repressed acid-stress adaptation at 4°C. A bead beating treatment prior to mild acid pre-exposure at 4°C partially induced acid-stress adaptation after pre-exposure in 4°C to mild acid stress. Our data suggests that cold processing or cold storage temperature can lower the possibility of activating acid-stress adaptation in Lm.

Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. How Haa1 is activated by weak acids is not clear. This study proposes that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation. YRO2, one of the targets of Haa1, shows increase in expression during stationary phase. This increase is due to basal activity of Haa1 and another, unknown, transcription factor. This study proposes that Gsm1 is another transcription factor that regulates YRO2 expression in the stationary phase. Finally, the mechanism of regulation of YRO2 by Haa1 is largely unknown. This study identifies two possible Haa1-medated cis-acting elements in the YRO2 promoter.

<p>Understanding the diversity of life is one of the main aims of evolutionary biology, and requires knowledge of the occurrence and causes of adaptive genetic differentiation among geographically distinct populations. Environmental stress caused by acidity may cause strong directional selection in natural populations, but is little explored from an evolutionary perspective. In this thesis, a series of laboratory experiments and field data was used to study evolutionary and ecological responses of amphibians to environmental acidity. </p><p>Local adaptation to acid stress was studied in the moor frog (<i>Rana arvalis)</i>.The results show that acid origin populations have higher acid stress tolerance during the embryonic stages than neutral origin populations, and that acid and neutral origin populations have diverged in embryonic and larval life-histories. The mechanisms underlying adaptive differentiation are partially mediated by maternal effects related to extra-embryonic membranes and egg size. Acid origin females invest in larger eggs and have a stronger egg size-fecundity trade-off than females from neutral areas, likely reflecting adaptive differentiation in maternal investment patterns. </p><p>Potential carry-over effects of low pH, and the effects of UV-b/pH interaction were investigated in the common frog (<i>R. temporaria</i>). The results suggest that amphibian larvae are able to compensate for the negative effects of acidity experienced early in life, if conditions later turn beneficial. <i>R. temporaria</i> populations differed in their sensitivity to synergistic effects of low pH/UV-B, indicating variation in population responses to environmental stress.</p><p>In conclusion, these results suggest rapid evolution in response to human induced environmental change, much of which may be mediated via adaptive maternal effects. Acidification may be a powerful selective force shaping life-history evolution.</p>

Understanding the diversity of life is one of the main aims of evolutionary biology, and requires knowledge of the occurrence and causes of adaptive genetic differentiation among geographically distinct populations. Environmental stress caused by acidity may cause strong directional selection in natural populations, but is little explored from an evolutionary perspective. In this thesis, a series of laboratory experiments and field data was used to study evolutionary and ecological responses of amphibians to environmental acidity. Local adaptation to acid stress was studied in the moor frog (Rana arvalis).The results show that acid origin populations have higher acid stress tolerance during the embryonic stages than neutral origin populations, and that acid and neutral origin populations have diverged in embryonic and larval life-histories. The mechanisms underlying adaptive differentiation are partially mediated by maternal effects related to extra-embryonic membranes and egg size. Acid origin females invest in larger eggs and have a stronger egg size-fecundity trade-off than females from neutral areas, likely reflecting adaptive differentiation in maternal investment patterns. Potential carry-over effects of low pH, and the effects of UV-b/pH interaction were investigated in the common frog (R. temporaria). The results suggest that amphibian larvae are able to compensate for the negative effects of acidity experienced early in life, if conditions later turn beneficial. R. temporaria populations differed in their sensitivity to synergistic effects of low pH/UV-B, indicating variation in population responses to environmental stress. In conclusion, these results suggest rapid evolution in response to human induced environmental change, much of which may be mediated via adaptive maternal effects. Acidification may be a powerful selective force shaping life-history evolution.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.