Biology subjects

Azam, M. S.

Publications and source records attributed to Azam, M. S..

8 recordsLinked to original sources

bioRxiv2026

Role of staphylococcal EzrA as a molecular organizer of cell division

Envelope biogenesis in Staphylococcus aureus is concentrated at the septum and includes peptidoglycan synthesis, lipo- and wall-teichoic acid production, and the targeted secretion of YSIRK/GXXS signal peptide-bearing proteins. How S. aureus confines these processes to the dividing crosswall remains unclear. EzrA, a scaffolding protein structurally related to eukaryotic spectrins, has been implicated in linking cell division to envelope synthesis, yet its precise role is poorly understood. Here, we re-examine the function of EzrA for its contribution to envelope biogenesis and homeostasis. We observe that ezrA null mutants synthesize excess peptidoglycan that is incorporated in a dispersed pattern, no longer strictly confined to the septum. A similar loss of spatial restriction was observed for protein A, a surface protein whose YSIRK/GXXS signal peptide directs septal secretion and anchoring. In wild-type cells, newly synthesized peptidoglycan co-localized with nascent protein A anchoring sites at the septum, whereas this spatial coupling was disrupted in the absence of EzrA. In addition, loss of EzrA resulted in impaired nucleoid occlusion with septal guillotining of the chromosome. Together, these findings support a model in which EzrA acts as a molecular organizer of cell division, coordinating septal biosynthesis and envelope assembly while ensuring proper nucleoid occlusion.

microbiology↗

bioRxiv2022

Small RNAs activate Salmonella pathogenicity island 1 by modulating mRNA stability through the hilD mRNA 3' UTR

Salmonella enterica serovar Typhimurium is an enteric pathogen associated with food-borne disease. Salmonella invades the intestinal epithelium using a type three secretion system encoded in Salmonella pathogenicity Island 1 (SPI-1). SPI-1 genes are tightly regulated by a complex feed-forward loop to ensure proper spatial and temporal expression. Most regulatory input is integrated at HilD, through control of hilD mRNA translation or HilD protein activity. The hilD mRNA possesses a 310-nucleotide 3' untranslated region (UTR) that influences HilD and SPI-1 expression, and this regulation is dependent on Hfq and RNase E, cofactors known to mediate small RNA (sRNA) activities. Thus, we hypothesized that the hilD mRNA 3' UTR is a target for sRNAs. Here we show that the sRNAs, SdsR and Spot 42 regulate SPI-1 by targeting different regions of the hilD mRNA 3' UTR. Regulatory activities of these sRNAs depend on Hfq and RNase E, in agreement with previous roles found for both at the hilD 3' UTR. We show that SdsR and RNase E are responsible for the accumulation of variable fragments of the hilD mRNA 3' UTR. Collectively, this work suggests that these sRNAs targeting the hilD mRNA 3' UTR regulate hilD mRNA levels by interfering with RNase E-dependent mRNA degradation. Our work provides novel insights into mechanisms of sRNA regulation at bacterial mRNA 3' UTRs and adds to our knowledge of post-transcriptional regulation of the SPI-1 complex feed-forward loop. ImportanceSalmonella are prominent food-borne pathogens, infecting millions of people a year. To express virulence genes at the correct time and place in the host, Salmonella uses a complex regulatory network that senses environmental conditions. Known for their role in allowing quick responses to stress and virulence conditions, we investigate the role of small RNAs in facilitating precise expression of these genes. We provide evidence that the 3' untranslated region of the hilD mRNA, encoding a key virulence regulator, is a target for small RNAs and the ribonuclease RNase E. The small RNAs play a role in stabilizing hilD mRNA to allow proper expression of Salmonella virulence genes in the host.

microbiology↗

bioRxiv2021

A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism in Bacteroides thetaiotaomicron

Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes. Here we report the identification of RBPs that are broadly distributed across the Bacteroidetes, with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron {Delta}rbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The {Delta}rbpB mutant had reduced expression of BT1871, encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes. ImportanceThe human colon houses hundreds of bacterial species, including many belonging to the genus Bacteroides, that aid in breaking down our food to keep us healthy. Bacteroides have many genes responsible for breaking down different dietary carbohydrates and complex regulatory mechanisms ensure that specific genes are only expressed when the right carbohydrates are available. In this study, we discovered that Bacteroides use a family of RNA-binding proteins as global regulators to coordinate expression of carbohydrate utilization genes. The ability to turn different carbohydrate utilization genes on and off in response to changing nutrient conditions is critical for Bacteroides to live successfully in the gut, and thus the new regulators we have identified may be important for life in the host.

microbiology↗

bioRxiv2020

Effects of individual base-pairs on in vivo target search and destruction kinetics of small RNA

Base-pairing interactions mediate intermolecular target recognition in many biological systems and applications, including DNA repair, CRISPR, microRNA, small RNA (sRNA) and antisense oligo therapies. Even a single base-pair mismatch can cause a substantial difference in biological activity but presently we do not yet know how the target search kinetics in vivo are influenced by single nucleotide level changes. Here, we used high-throughput sequencing to identify functionally relevant single point mutants of the bacterial sRNA, SgrS, and quantitative super-resolution microscopy to probe the mutational impact on the regulation of its primary target, ptsG mRNA. Our super-resolution imaging and analysis platform allowed us to further dissect mutational effects on SgrS lifetimes, and even subtle changes in the in vivo rates of target association, kon, and dissociation, koff. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decreased kon and increased koff, providing an in vivo demonstration that Hfq directly facilitates sRNA-mRNA annealing. Single base-pair mismatches in the annealing region reduced kon by 24-31% and increased koff by 14-25%, extending the time it takes to find and destroy the target mRNA by about a third, depending on whether an AU or GC base-pair is disrupted. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq also buffers base-pair disruptions.

molecular biology↗

bioRxiv2020

Stochastic Analysis Probes the Role of the Chaperone Hfq in E. coli Sugar Shock Response

Small RNAs (sRNAs) play a crucial role in the regulation of bacterial gene expression by silencing the translation of target mRNAs. SgrS is an sRNA that relieves glucose-phosphate stress, or "sugar shock" in E. coli. The power of single cell measurements is their ability to obtain population level statistics that illustrate cell-to-cell variation. Here, we utilize single molecule super-resolution microscopy in single E. coli cells coupled with stochastic modeling to analyze glucose-phosphate stress regulation by SgrS. We present a kinetic model that captures the combined effects of transcriptional regulation, gene replication and chaperone mediated RNA silencing in the SgrS regulatory network. This more complete kinetic description, simulated stochastically, recapitulates experimentally observed cellular heterogeneity and characterizes the binding of SgrS to the chaperone protein Hfq as a slow process that not only stabilizes SgrS but also may be critical in restructuring the sRNA to facilitate association with its target ptsG mRNA.

biophysics↗

bioRxiv2019

Translation inhibition from a distance: the small RNA SgrS interferes with a ribosomal protein S1-dependent enhancer

Many bacterial small RNAs (sRNAs) efficiently inhibit translation of target mRNAs by forming a duplex that sequesters the Shine-Dalgarno (SD) sequence or start codon and prevents formation of the translation initiation complex. There are a growing number of examples of sRNA-mRNA binding interactions distant from the SD region, but how these mediate translational regulation remains unclear. Our previous work in Escherichia coli and Salmonella identified a mechanism of translational repression of manY mRNA by the sRNA SgrS through a binding interaction upstream of the manY SD. Here, we report that SgrS forms a duplex with a uridine-rich translation-enhancing element in the manY 5 untranslated region. Notably, we show that the enhancer is ribosome-dependent and that the small ribosomal subunit protein S1 interacts with the enhancer to promote translation of manY. In collaboration with the chaperone protein Hfq, SgrS interferes with the interaction between the translation enhancer and ribosomal protein S1 to repress translation of manY mRNA. Since bacterial translation is often modulated by enhancer-like elements upstream of the SD, sRNA-mediated enhancer silencing could be a common mode of gene regulation.

microbiology↗

bioRxiv2017

Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS

The mechanisms by which small RNA (sRNA) regulators select and prioritize target mRNAs remain poorly understood, but serve to promote efficient responses to environmental cues and stresses. We sought to uncover mechanisms that establish regulatory hierarchy for a model sRNA, SgrS, found in enteric bacteria and produced under conditions of metabolic stress when sugar transport and metabolism are unbalanced. SgrS post-transcriptionally controls a nine-gene regulon to restore growth and homeostasis under stress conditions. An in vivo reporter system was used to quantify SgrS-dependent regulation of target genes and established that SgrS exhibits a clear preference for certain targets, and regulates those targets efficiently even at low SgrS levels. Higher SgrS concentrations are required to regulate other targets. The position of targets in the regulatory hierarchy is not well-correlated with the predicted thermodynamic stability of SgrS-mRNA interactions or the SgrS-mRNA binding affinity as measured in vitro. Detailed analyses of SgrS interaction with asd mRNA demonstrate that SgrS binds cooperatively to two sites and remodels asd mRNA secondary structure. SgrS binding at both sites increases the efficiency of asd mRNA regulation compared to mutants that have only a single SgrS binding site. Our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features. The sRNA-mRNA interaction, including the number and position of sRNA binding sites on the mRNA and cofactors like the RNA chaperone Hfq, seem to tune the efficiency of regulation of specific mRNA targets.\n\nIMPORTANCETo survive, bacteria must respond rapidly to stress and simultaneously maintain metabolic homeostasis. The small RNA (sRNA) SgrS mediates the response to stress arising from imbalanced sugar transport and metabolism. To coordinate the stress response, SgrS regulates genes involved in sugar uptake and metabolism. Intrinsic properties of sRNAs such as SgrS allow them to regulate extensive networks of genes. To date, sRNA regulation of targets has largely been studied in the context of \"one sRNA-one target\", and little is known about coordination of multi-gene regulons and sRNA regulatory network structure. Here, we explore the molecular basis for regulatory hierarchy in sRNA regulons. Our results reveal a complex interplay of factors that influence the outcome of sRNA regulation. The number and location of sRNA binding sites on mRNA targets and the participation of an RNA chaperone dictate prioritized regulation of targets to promote an efficient response to stress.

microbiology↗

bioRxiv2017

Translational regulation by bacterial small RNAs via an unusual Hfq-dependent mechanism

In bacteria, the canonical mechanism of translational repression by small RNAs (sRNAs) involves sRNA-mRNA base pairing that occludes the ribosome binding site (RBS), directly preventing translation. In this mechanism, the sRNA is the direct regulator, while the RNA chaperone Hfq plays a supporting role by stabilizing the sRNA. There are a few examples where the sRNA does not directly interfere with ribosome binding, yet translation of the target mRNA is still inhibited. Mechanistically, this non-canonical regulation by sRNAs is poorly understood. Our previous work demonstrated repression of the mannose transporter manX mRNA by the sRNA SgrS, but the regulatory mechanism was unknown. Here, we report that manX translation is controlled by a molecular role-reversal mechanism where Hfq, not the sRNA, is the direct repressor. Hfq binding adjacent to the manX RBS is required for sRNA-mediated translational repression. Translation of manX is also regulated by another sRNA, DicF, via the same non-canonical Hfq-dependent mechanism. Our results suggest that the sRNAs recruit Hfq to its binding site or stabilize the mRNA-Hfq complex. This work adds to the growing number of examples of diverse mechanisms of translational regulation by sRNAs in bacteria.

microbiology↗