Overall goals and aims are to:

a) map and understand the physiological state of mycobacteria grown under different conditions/ environments and to elucidate the underlying molecular mechanisms that dictate the morphological transitions in mycobacteria in response to growth conditions and environmental stress. We are particularly interested in identifying and examining the switches and factors that activate and/or repress alternative pathways leading to changes in cell morphology such as transitions between rod- and coccoid cell shapes, sporulation and biofilm formation in mycobacteria and genes involved in these processes from an evolutionary perspective. An important aim is to understand how and if cell shape plays a role during mycobacterial infections, expression of virulence and maintenance of dormancy.

b) increase our understanding of non-coding RNA (ncRNAs) and regulatory RNAs, their roles, processing and evolution in mycobacteria with emphasis on their role in regulating transitions between different cell forms and during infection.

c) understand the underlying mechanism behind RNA mediated catalysis and its impact on the overall physiology of E. coli with respect to the global levels of RNA and proteins.

Background

Our research focuses on three major areas in two systems

A) Mycobacterial cell morphology and physiology in adaptation to stress

B) RNA biology in Mycobacteria

C) RNA processing in Escherichia coli with an emphasis on RNA mediated catalysis

A) Mycobacterial cell morphology and physiology in adaptation to stress

Bacteria of the genus Mycobacterium are acid fast, robust and they can inhabit various environmental reservoirs, e.g. ground and tap water, soil, animals and humans. This genus includes non-pathogenic environmental bacteria, opportunistic and highly successful pathogens, e.g., M. tuberculosis that causes tuberculosis (TB; ≈9 million new cases per year). M. tuberculosis is human-specific while the closely related M. bovis can infect humans and animals and cause TB in both.

Mycobacteria's adaptability to different conditions of stress from nutritional and oxidative deprivation is well established. It has also been reported that they display widely diverse morphological variants at different stages of their life cycles. Recent discovery in our and other laboratories of mycobacteria's capacity for sporulation has opened up unexpected avenues of enquiry into the molecular basis of pleiomorphism in response to stress and variation in growth conditions. If these morphological changes influence pathogenicity and persistence, then studies of the causes for pleiomorphic variation and the mechanisms of transition between different forms might provide new insights into the mechanisms of transfer, spread, dormancy and treatment of mycobacterial infections.

The aim of our studies is to understand the molecular mechanisms underlying morphological changes seen in mycobacteria in response to different environmental stimuli and whether these changes play a role at and during infection. In our studies we focus on the fish pathogen Mycobacterium marinum, considered to be a model for M. tuberculosis. However, within the frame of our studies we also have taken a global approach where we study selected mycobacteria in order to map the physiological state in response to various growth conditions these mycobacteria are exposed to. These studies encompass sequencing the genomes of several mycobacterial species.

B) RNA biology in Mycobacteria

To date a large number of bacterial small (sRNA) and antisense (asRNA, cis- and trans-acting) RNAs as well as cis-regulatory RNA elements in the 5' UTR (untranslated region, e.g., attenuators and riboswitches) and in the 3' UTR have been identified and characterized. These RNAs are typically 50 to 500 nucleotides long. Understanding their functions have shed light on an underestimated layer of activity, regulation and diversity among a variety of bacteria, e.g., E. coli and other g-proteobacteria, Streptomyces spp. and low GC-content Gram-positive bacteria such as Bacillus subtilis. These RNAs have been shown to be involved in adaptation to various growth conditions including heat stress, biofilm formation, oxidative stress and expression of virulence genes. In addition, other RNAs like RNase P RNA, tmRNA, 4.5S RNA, 6S RNA and tRNA play important roles for cell survival and growth adaptation: RNase P with its catalytic RNA (RPR) is involved the processing of tRNA, small RNA and mRNA, tmRNA has a role in rescuing stalled ribosomes during translation, 4.5S RNA is part of the signal recognition particle involved in protein secretion, 6S RNA binds to the house keeping transcription sigma (sA) factor and represses the expression of sA driven promoters in stationary growth phase, and tRNA transfer amino acids to the ribosome as well as acting as regulators and as such tRNA is a key factor in gene expression.

Recent studies have indeed identified sRNA and regulatory RNAs in mycobacteria with a focus on M. tuberculosis. Examining 22 mycobacterial species others have predicted >2000 candidate RNAs including CRISPR genes; among these, the expression of ≈200 has been validated. We have identified sequence motifs, which can form catalytically active hammer-heads in certain mycobacteria and in our genome data we have detected variation in the number of Rfam classified RNA genes. However, information on and understanding of their function and regulation are limited.

On the basis of the above and our understanding of the role of various RNAs in other bacteria we are studying the role (and turnover/ stability) of sRNAs, regulatory RNAs and tRNA in mycobacteria in response to environmental changes and adaptation to various physiological conditions. In addition, we are interested in understanding their function (if any) with respect to changes in cell morphology, differentiation (biofilm and spore formation) and pathogenicity.

C RNA processing in Escherichia coli with an emphasis on RNA mediated catalysis

The tRNA genes are transcribed as precursors that need to be trimmed to generate matured and functional tRNAs. Almost all tRNAs carry a phosphate at their 5' ends due to the action of the endoribonuclease P, RNase P, which is responsible for the maturation of the 5'-termini of almost all known tRNAs in prokaryotes as well as in eukaryotes and as such it has a central role in precursor tRNA processing. In addition, it has become more evident that RNase P participates in the processing of certain mRNAs, and other ncRNAs including regulatory sRNAs.

RNase P is a ribonucleoprotein composed of one RNA subunit and depending on its origin, varying numbers of protein subunits (one in Bacteria, ≥ 4 in Archaea and 9-10 in Eukarya). However, irrespective of origin, the catalytic activity is associated with the RNA and RNase P RNA (RPR) can catalyze the trimming of the tRNA 5' end even without its protein component. Catalytic RNA molecules are referred to as ribozymes. Moreover, RNase P activity based solely on proteins, PRORP, has recently been demonstrated in plants and human mitochondria. This adds further intrigue to the variation in the composition of the enzymatic activity responsible for generating tRNAs with matured 5' ends.

Over the years we have focused our studies to understand the mechanism of RNA mediated cleavage of RNA using E. coli RPR as a model system. We study the interaction between the RPR and its substrate as well as interactions between RNA and small ligands, e.g., metal ions and antibiotics. Our findings have increased our knowledge of: (a) RNA mediated cleavage in general, (b) how in particular the naturally occurring ribozyme RNase P RNA interacts with its substrates and mediates cleavage and (c) factors that are important for RNA metal ion and antibiotic interactions. More recently we have included PRORP in our studies with the aim to understand differences and similarities in processing of various precursors. Currently we are also interested in understanding the impact of RNase P activity on the global RNA and protein expression.