Defining the nitrogen regulated transcriptome of Mycobacterium smegmatis using continuous culture.

BACKGROUND: Nitrogen is essential for microbial growth and its importance is demonstrated by the complex regulatory systems used to control the transport, assimilation and metabolism of nitrogen. Recent studies are beginning to shed light on how mycobacteria respond to nitrogen limitation and several regulators (e.g., GlnR, PII) have been characterized at a molecular level. However, despite this progress, our knowledge of the transcriptional response of mycobacteria to nitrogen limitation and its regulation is confined to batch culture. METHODS: To gain further insight into the response of mycobacteria to nitrogen limitation, we developed a nitrogen-limited chemostat. We compared the transcriptional response of nitrogen-limited cells to carbon-limited cells using RNA-seq analysis in a continuous culture model at a constant growth rate. CONCLUSIONS: Our findings revealed significant changes in the expression of 357 genes (208 upregulated, 149 downregulated; >2-fold change, false discovery rate <5 %) in response to nitrogen limitation in continuous culture. The vast majority of the GlnR regulon (68 %) was differentially expressed under nitrogen limitation in continuous culture and approximately 52 % of the 357 genes overlapped with a previously published study investigating the response of M. smegmatis to nitrogen limitation in batch culture, while expression of only 17 % of the genes identified in batch culture were affected in our chemostat model. Moreover, we identified a unique set of 45 genes involved in the uptake and metabolism of nitrogen that were exclusive to our chemostat model. We observed strong downregulation of pathways for amino acid catabolism (i.e., alanine, aspartate, valine, proline and lysine), suggesting preservation of these amino acids for critical cellular function. We found 16 novel transcriptional regulators that were directly or indirectly involved in the global transcriptomic response of M. smegmatis to nitrogen limitation and identified several non-coding RNAs that might be involved in the transcriptional or post-transcriptional regulation of nitrogen-regulated gene expression. RESULTS: Using nitrogen-limited continuous culture we identified the nitrogen-responsive transcriptome of M. smegmatis, including a number of small non-coding RNAs implicated in controlling nitrogen-regulated gene expression.

Flexible nitrogen utilisation by the metabolic generalist pathogen Mycobacterium tuberculosis.

Bacterial metabolism is fundamental to survival and pathogenesis. We explore how Mycobacterium tuberculosis utilises amino acids as nitrogen sources, using a combination of bacterial physiology and stable isotope tracing coupled to mass spectrometry metabolomics methods. Our results define core properties of the nitrogen metabolic network from M. tuberculosis, such as: (i) the lack of homeostatic control of certain amino acid pool sizes; (ii) similar rates of utilisation of different amino acids as sole nitrogen sources; (iii) improved nitrogen utilisation from amino acids compared to ammonium; and (iv) co-metabolism of nitrogen sources. Finally, we discover that alanine dehydrogenase is involved in ammonium assimilation in M. tuberculosis, in addition to its essential role in alanine utilisation as a nitrogen source. This study represents the first in-depth analysis of nitrogen source utilisation by M. tuberculosis and reveals a flexible metabolic network with characteristics that are likely a product of evolution in the human host.

Microarray dataset on the genome-wide expression profile of an M. smegmatis amtR mutant (JR258) compared to M. smegmatis mc2155.

The dataset presented here describes a microarray experiment to identify the AmtR regulon of Mycobacterium smegmatis comparing the transcription profile of a M. smegmatis amtR mutant to M. smegmatis wild-type. The raw and processed microarray data are available in the ArrayExpress database under Accession Number E-MTAB-4857 and interpretation of this data is found in the research article "Structure and function of AmtR in Mycobacterium smegmatis: implications for post-transcriptional regulation of urea metabolism through a small antisense RNA" (Petridis et al., in press) [1].

Structure and Function of AmtR in Mycobacterium smegmatis: Implications for Post-Transcriptional Regulation of Urea Metabolism through a Small Antisense RNA.

Soil-dwelling bacteria of the phylum actinomycetes generally harbor either GlnR or AmtR as a global regulator of nitrogen metabolism. Mycobacterium smegmatis harbors both of these canonical regulators; GlnR regulates the expression of key genes involved in nitrogen metabolism, while the function and signal transduction pathway of AmtR in M. smegmatis remains largely unknown. Here, we report the structure and function of the M. smegmatis AmtR and describe the role of AmtR in the regulation of nitrogen metabolism in response to nitrogen availability. To determine the function of AmtR in M. smegmatis, we performed genome-wide expression profiling comparing the wild-type versus an ∆amtR mutant and identified significant changes in the expression of 11 genes, including an operon involved in urea degradation. An AmtR consensus-binding motif (CTGTC-N4-GACAG) was identified in the promoter region of this operon, and ligand-independent, high-affinity AmtR binding was validated by both electrophoretic mobility shift assays and surface plasmon resonance measurements. We confirmed the transcription of a cis-encoded small RNA complementary to the gene encoding AmtR under nitrogen excess, and we propose a post-transcriptional regulatory mechanism for AmtR. The three-dimensional X-ray structure of AmtR at 2.0Å revealed an overall TetR-like dimeric structure, and the alignment of the M. smegmatis AmtR and Corynebacterium glutamicum AmtR regulatory domains showed poor structural conservation, providing a potential explanation for the lack of M. smegmatis AmtR interaction with the adenylylated PII protein. Taken together, our data suggest an AmtR (repressor)/GlnR (activator) competitive binding mechanism for transcriptional regulation of urea metabolism that is controlled by a cis-encoded small antisense RNA.

Do tissue levels of autoantigenic aminoacyl-tRNA synthetase predict clinical disease?

The etiologies of most autoimmune diseases are not completely understood. Aminoacyl-tRNA synthetases (AARS) are a family of heterogenous enzymes responsible for protein synthesis and whose secondary functions include a role in autoimmune myositis. A subset of patients with idiopathic inflammatory myopathies demonstrate autoantibody against specific cytoplasmic AARS and the human asparaginyl-tRNA synthetase (AsnRS) has been shown to be a potent chemokine that interacts with CCR3 chemokine receptors. One way in which a chemotactic cytoplasmic enzyme might contribute to tissue inflammation is if it were abundant in a specific injured tissue and thereby released to the microenvironment at times of cellular damage. To test this hypothesis, the relative levels of AsnRS mRNA were studied in six human tissues. A 1.6 kbF RNA probe identified highly variable levels of the corresponding mRNA in Northern blot analysis of human lung, brain, heart, skeletal muscle, pancreas and liver. The highest levels of signal were noted in muscle and pancreas. Polyclonal antibody raised against recombinant human AsnRS identified abundant antigenic material in the pancreas, in particular in islet cells. Thus, the local abundance of an endogenous pro-inflammatory autoantigen may provide one explanation for perpetuation or exacerbation of tissue specific immune-mediated pathologies.

Expression, localization and alternative function of cytoplasmic asparaginyl-tRNA synthetase in Brugia malayi.

Aminoacyl-tRNA synthetases (AARS) are a family of enzymes that exhibit primary and various secondary functions in different species. In Brugia malayi, the gene for asparaginyl-tRNA synthetase (AsnRS), a class II AARS, previously has been identified as a multicopy gene encoding an immunodominant antigen in the serum of humans with lymphatic filariasis. However, the relative level of expression and alternative functions of AARS in nematode parasites is not well understood. We searched the Filarial Genome Project database to identify the number and amino acid specificity of B. malayi AARS cDNAs to gain insight into the role of different AARS in filaria. These data showed that cytoplasmic AsnRS was present in five gene clusters, and is the most frequently represented member of the aminoacyl-tRNA synthetase family in adult B. malayi. The relative level of AsnRS transcribed in adult female B. malayi was compared to the levels of a low abundance and medium abundance AARS by quantitative real-time RT-PCR. By this method, AsnRS cDNA was 11 times greater than arginyl-tRNA synthetase and methionyl-tRNA synthetase cDNA. In situ hybridization using a B. malayi AsnRS-specific oligonucleotide probe identified abundant cytoplasmic mRNA, particularly in the hypodermis of adult B. malayi. In the absence of tRNA, AsnRS synthesizes diadenosine triphosphate, a potent regulator of cell growth in other eukaryotes. These data support the hypothesis that all AARS are not equally expressed in B. malayi and that these enzymes may demonstrate important alternative functions in filaria.

Characterization of an intergenic polymorphic site (pp-hC1A_5) in Wolbachia pipientis (wPip).

Wolbachia pipientis (wPip) is an intracellular bacterium causing cytoplasmic incompatibility in arthropods, including mosquitoes of the Culex pipiens complex. Here, we present a method useful for genotyping within the wPip group. Primers were designed using a Tandem Repeat Finder program to amplify an intergenic, polymorphic site (pp-hC1A_5) of wPip. The polymorphic site is located between genes that code for polynucleotide phosphorylase and a hypothetical protein (C1A_5). Comparison of these wPip genomic regions from C. pipiens mosquitoes sampled in different geographic regions revealed deletions of fragments that proved useful in phylogenetic analysis.

Can Anopheles gambiae be infected with Wolbachia pipientis? Insights from an in vitro system.

Wolbachia pipientis are maternally inherited endosymbionts associated with cytoplasmic incompatibility, a potential mechanism to drive transgenic traits into Anopheles populations for malaria control. W. pipientis infections are common in many mosquito genera but have never been observed in any Anopheles species, leading to the hypothesis that Anopheles mosquitoes are incapable of harboring infection. We used an in vitro system to evaluate the ability of Anopheles gambiae cells to harbor diverse W. pipientis infections. We successfully established W. pipientis infections (strains wRi and wAlbB) in the immunocompetent Anopheles gambiae cell line Sua5B. Infection was confirmed by PCR, antibiotic curing, DNA sequencing, and direct observation using fluorescence in situ hybridization. The infections were maintained at high passage rates for >30 passages. Our results indicate that there is no intrinsic genetic block to W. pipientis infection in A. gambiae cells, suggesting that establishment of in vivo W. pipientis infections in Anopheles mosquitoes may be feasible.