Structural investigation of Mycobacterial protein complexes involved in the stationary phase stress response

Large protein complexes play key roles in mediating biological processes in the cell. Little structural information is known on the protein complex mediators governing persistence in the host for Mycobacterium tuberculosis (Mtb). We applied the ‘shotgun EM’ method for the structural characterisation of protein complexes produced after exposure to stationary phase stress for the model Mycobacterium, M smegmatis (Msm). We identified glutamine synthetase I, essential for Mtb virulence, in addition to bacterioferritin, critical for Mtb iron regulation, and encapsulin, which produces a cage-like structure to enclose target proteins. Further investigation found that encapsulin carries dye-decolourising peroxidase (DyP), a potent protein antioxidant, as the primary cargo during stationary phase stress. Our ‘proof-of-concept’ application of this method offers insight into identifying potential key-mediators in Mtb persistence.

Noncoding RNA of Glutamine Synthetase I Modulates Antibiotic Production in Streptomyces coelicolor A3(2)

ABSTRACT Overexpression of antisense chromosomal cis-encoded noncoding RNAss (ncRNAs) in glutamine synthetase I resulted in a decrease in growth, protein synthesis, and antibiotic production in Streptomyces coelicolor. In addition, we predicted 3,597 cis-encoded ncRNAs and validated 13 of them experimentally, including several ncRNAs that are differentially expressed in bacterial hormone-defective mutants.

The Rhizobium GstI Protein Reduces the NH4+ Assimilation Capacity of Rhizobium leguminosarum

We show that the protein encoded by the glutamine synthetase translational inhibitor (gstI) gene reduces the NH4+ assimilation capacity of Rhizobium leguminosarum. In this organism, gstI expression is regulated by the ntr system, including the PII protein, as a function of the nitrogen (N) status of the cells. The GstI protein, when expressed from an inducible promoter, inhibits glutamine synthetase II (glnII) expression under all N conditions tested. The induction of gstI affects the growth of a glutamine synthetase I (glnA-) strain and a single amino acid substitution (W48D) results in the complete loss of GstI function. During symbiosis, gstI is expressed in young differentiating symbiosomes (SBs) but not in differentiated N2-fixing SBs. In young SBs, the PII protein modulates the transcription of NtrC-regulated genes such as gstI and glnII. The evidence presented herein strengthens the idea that the endocytosis of bacteria inside the cytoplasm of the host cells is a key step in the regulation of NH4+ metabolism.

A maternal requirement for glutamine synthetase I for the mitotic cycles of syncytial Drosophila embryos

We describe the maternal effect phenotype of a hypomorphic mutation in the Drosophila gene for glutamine synthetase I (GSI). The extent of development of embryos derived from homozygous mutant females is variable, although most mutant embryos fail to survive past germband elongation and none develop into larvae. These embryos are characterised by an increase in the number of yolk-like nuclei following nuclear migration to the cortex. These nuclei appear to fall into the interior of the embryo from the cortex at blastoderm. As they do so, the majority continue to show association with PCNA in synchrony with nuclei at the cortex, suggesting some continuity of the synchrony of DNA replication. However, the occurrence of nuclei that have lost cell cycle synchrony with their neighbours is not uncommon. Immunostaining of mutant embryos revealed a range of mitotic defects, ultimately resulting in nuclear fusion events, division failure or other mitotic abnormalities. A high proportion of these mitotic figures show chromatin bridging at anaphase and telophase consistent with progression through mitosis in the presence of incompletely replicated DNA. GSI is responsible for the ATP-dependent amination of glutamate to produce glutamine, which is required in the formation of amino acids, purines and pyrimidines. We discuss how the loss of glutamine could depress both protein and DNA synthesis and lead to a variety of mitotic defects in this embryonic system that lacks certain checkpoint controls.