The CsgD protein is the master regulator of a pathway leading to the formation of curli, in essence regulating the switch between a motile and a sessile lifestyle for bacteria. The 5’-UTR region of the csgD mRNA is a hotspot for multiple regulatory small RNAs (sRNA) involved in a complex regulatory network. Even though it is previously known how the interaction takes place it is unknown how sRNA binding affects the translational activity. In order to suggest a mode of regulation a mutational assay was performed by making changes in the csgD 5’-UTR and investigate what the translational effects were. Mutations in different regions are shown to affect the translation levels in various ways.

Antibiotikaresistenz/-toleranz und Evasion des menschlichen Immunsystem sind wesentliche Probleme persistierender chronischer Infektionen, die im Zusammenhang mit Biofilmen stehen. Eine Notwendigkeit alternativer Behandlungen liegt daher nahe. Für diese Studie wurden zehn ayurvedische Pflanzen ausgewählt, die die Produktion von Curli-Fasern und/oder pEtN-Cellulose in E. coli K-12 Makrokolonie-Biofilmen eindeutig hemmten. Eine Reihe molekularer Reporter wurde verwendet, um die molekularen Ziele im Modellorganismus E. coli zu identifizieren. Eine Kombination von mikrobiologischen, molekularbiologischen und enzymatischen Methoden und Experimenten wurde dann verwendet, um die Aktivitäten der Pflanzenextrakte weiter zu charakterisieren. Um ihre Wirkung auf Biofilme eines breiteren Spektrums von Bakterien zu testen, wurden einige relevante gramnegative Pathogene (EAEC, UPEC, P. aeruginosa) und grampositive Bakterien (B. subtilis, S. aureus) als Makrokolonie-Biofilme sowie als submerse Biofilme in Gegenwart der Pflanzenextrakte inkubiert. Die wichtigsten Ergebnisse dieser Studie sind, dass es kein „Allheilmittel“ gibt, das effektiv gegen verschiedene Biofilmstrukturen wirken kann. Es konnte gezeigt werden, dass fast alle Pflanzenextrakte die CsgA Amyloidogenese hemmen. Drei der zehn Pflanzenextrakte beeinflussten die Curli- und pEtN-Cellulose-Gene signifikant, indem sie csgB und dgcC über den Regulator CsgD herunterregulierten. Darüber hinaus wurde festgestellt, dass ein Extrakt die Expression flagellarer Gene in E. coli hochreguliert - eine neue Anti-Biofilm Strategie. Überraschenderweise wurde auch festgestellt, dass ein Pflanzenextrakt, das die Biofilmbildung des Kommensalen E. coli K-12 hemmt, während es die Biofilmbildung von UPEC fördert. Daher können Anti-Biofilm-Effekte stammspezifisch sein. Eine Strategie, bei der verschiedene Pflanzenextrakte kombiniert werden, könnte gegen Biofilme wirken, die aus mehreren Arten bestehen, erfordert jedoch weitere Forschung. / Antibiotic resistance/tolerance and evasion from the human immune system are major causes of concern associated with biofilm-related persistent chronic infections. So, the need for an alternative source of treatment is obvious. In this study, 10 Ayurvedic plants were selected as they clearly inhibited the production of curli fiber and pEtN-cellulose or of curli fibers only in E. coli K-12 macrocolony biofilms. A series of molecular reporters were used to determine the molecular targets using E. coli as model bacteria. A combination of microbiological, molecular biological, and enzymatic assays and experiments were then used to further characterize the activities of the plant extracts. To test anti-biofilm effects on a wider range of bacteria, some relevant Gram-negative pathogens (EAEC, UPEC, P. aeruginosa) and Gram-positive bacteria (B. subtilis, S. aureus) were grown in macrocolony biofilms and submerged biofilms in the presence of active plant extracts. The major findings of this study are that there is not one single “magic bullet” that can effectively work against the diverse biofilm compositions and structures. Nearly all plant extracts were found to inhibit CsgA amyloidogenesis. Three of the ten plant extracts affected the curli and pEtN-cellulose genes significantly by downregulating csgB and dgcC via the CsgD regulator. In addition, one extract was found to upregulate flagellar gene expression in E. coli - this is a new anti-biofilm strategy that had not considered before. Surprising, it was also noticed that one plant extract, which inhibits biofilm formation by commensal E. coli K-12, promotes biofilm formation by UPEC. Thus, anti-biofilm effects can be strain-specific because of the diversity of composition of the matrix within the same bacterial species. A strategy of combining different plant extracts may work to deal with biofilms involving multiple species, but requires more research and understanding.

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.