Multi-Omics Profiling Specifies Involvement of Alternative Ribosomal Proteins in Response to Zinc Limitation in Mycobacterium smegmatis.

Zinc ion (Zn2+) is an essential micronutrient and a potent antioxidant. However, Zn2+ is often limited in the environment. Upon Zn2+ limitation, Mycolicibacterium (basonym: Mycobacterium) smegmatis (Msm) undergoes a morphogenesis, which relies on alternative ribosomal proteins (AltRPs); i.e., Zn2+-independent paralogues of Zn2+-dependent ribosomal proteins. However, the underlying physiological changes triggered by Zn2+ limitation and how AltRPs contribute to these changes were not known. In this study, we expand the knowledge of mechanisms utilized by Msm to endure Zn2+ limitation, by comparing the transcriptomes and proteomes of Zn2+-limited and Zn2+-replete Msm. We further compare, corroborate and contrast our results to those reported for the pathogenic mycobacterium, M. tuberculosis, which highlighted conservation of the upregulated oxidative stress response when Zn2+ is limited in both mycobacteria. By comparing the multi-omics analysis of a knockout mutant lacking AltRPs (ΔaltRP) to the Msm wild type strain, we specify the involvement of AltRPs in the response to Zn2+ limitation. Our results show that AltRP expression in Msm does not affect the conserved oxidative stress response during Zn2+ limitation observed in mycobacteria, but AltRPs do significantly impact expression patterns of numerous genes that may be involved in morphogenesis or other adaptive responses. We conclude that AltRPs are not only important as functional replacements for their Zn2+-dependent paralogues; they are also involved in the transcriptomic response to the Zn2+-limited environment.

Zinc limitation triggers anticipatory adaptations in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ sequestration is part of "nutritional immunity", where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.

RNA-Seq analysis of the T3SA regulon in Shigella flexneri reveals two new chromosomal genes upregulated in the on-state.

Shigella spp. are enterobacteria that invade human colonic mucosal cells using their Type Three Secretion Apparatus (T3SA). Shigella spp. possess a large plasmid that encodes most of its virulence factors and has been the focus of seminal work that defined the T3SA regulon. Thus, a global assessment of the transcriptional response regulated by the T3SA has been lacking. Herein we used RNA-Seq to identify genes that are differentially expressed when the T3SA is active (on-state) versus inactive (off-state). The quality of the RNA-Seq dataset was validated by its correlation with a prior microarray study. Using novel insights about the expression of non-coding regions, bioinformatic tools and experimentations, we demonstrated the existence of six operons and evidence that ipaH2.5 is a pseudogene. In addition, 86 chromosomal genes were downregulated in the on-state including several non-coding transcripts corresponding to short antisense RNA embedded in the 16S and 23S RNA genes, and 40 coding transcripts, whose cognate proteins were highly connected at the genetic and biochemical levels. Finally, we identified two novel chromosomal genes dubbed gem1 and gem3, which were upregulated in the on-state similarly to genes belonging to the T3SA regulon. The latter findings were validated on biological triplicates by droplet digital PCR. To our knowledge gem1 and gem3 are the first chromosomal members of the T3SA regulon that have no homologs on the plasmid. Our approach provides a path to optimizing RNA-Seq studies in case of bacterial models that had previously been the subject of medium to large scale studies.