Biology subjects

Zeinert, R.

Publications and source records attributed to Zeinert, R..

8 recordsLinked to original sources

bioRxiv2026

AltORF-encoded microprotein recruits primary-ORF-encoded dehydrogenase to cell poles upon heat shock

Alternative open reading frames (altORFs) initiating from in-frame or out-of-frame start sites within annotated protein-coding genes represent a largely unexplored source of protein-coding potential and remain poorly understood in terms of regulation, function, and impact on physiology. Here we investigate Escherichia coli GndA, encoded by an out-of-frame altORF within gnd, which encodes 6-phosphogluconate dehydrogenase. We observe that the GndA protein is induced by heat shock, consistent with the initial identification in a quantitative membrane proteomics screen, but is unstable, while the levels of the stable Gnd dehydrogenase are unchanged. Using translational fusion assays and in vivo RNA structure probing, we show that an internal complementary sequence upstream of gndA acts as a thermo-responsive secondary RNA structure controlling GndA translation. Mutagenesis of either gnd or gndA reduce heat shock survival of E. coli indicating both gene products are important for resistance. GndA fractionates with membranes consistent with in silico predictions that the protein forms an amphipathic -helix. In contrast, Gnd is predominantly cytoplasmic but shows GndA-dependent relocalization to cell poles upon heat shock. Together, our results demonstrate that an altORF-encoded microprotein is required for stress-dependent relocalization of its primary ORF partner, revealing a functional interplay between proteins encoded by overlapping ORFs in bacterial stress adaptation.

microbiology↗

bioRxiv2025

Stress testing reveals selective vulnerabilities in protein homeostasis

Protein quality control (PQC) systems are essential for cellular resilience to proteotoxic stress. Despite intensive study for decades, functional redundancies in the system obscure the contributions of the collectively important individual genes. Here, we leverage transposon sequencing across bacteria strains lacking key chaperones and proteases to reveal hidden determinants of stress response in protein homeostasis. By profiling fitness under multiple proteotoxic stresses, we uncover stress-specific vulnerabilities and reveal how major players of PQC mask correlations between transcriptomic responses and gene fitness. As an illustration of unexpected connections, we identify a heat-specific synthetic lethality between the disaggregase ClpB and DNA Polymerase I (PolA) mediated by persistent aggregation of the RecA recombinase and toxic persistence of the heat shock regulon. Our findings reveal that stress-induced aggregation is not broadly toxic. Rather, it becomes lethal in specific genetic or environmental contexts due to the depletion of components only needed in those specific circumstances. This work presents a framework to reveal normally hidden fragility in stress responses using gene fitness scores adaptable to a variety of systems.

molecular biology↗

bioRxiv2024

Magnesium Transporter MgtA revealed as a Dimeric P-type ATPase

Magnesium (Mg2+) uptake systems are present in all domains of life given the vital role of this ion. Bacteria acquire Mg2+ via conserved Mg2+ channels and transporters. The transporters are required for growth when Mg2+ is limiting or during bacterial pathogenesis, but, despite their significance, there are no known structures for these transporters. Here we report the first structure of the Mg2+ transporter MgtA solved by single particle cryo-electron microscopy (cryo-EM). Using mild membrane extraction, we obtained high resolution structures of both a homodimeric form (2.9 [A]), the first for a P-type ATPase, and a monomeric form (3.6 [A]). Each monomer unit of MgtA displays a structural architecture that is similar to other P-type ATPases with a transmembrane domain and two soluble domains. The dimer interface consists of contacts between residues in adjacent soluble nucleotide binding and phosphotransfer regions of the haloacid dehalogenase (HAD) domain. We suggest oligomerization is a conserved structural feature of the diverse family of P-type ATPase transporters. The ATP binding site and conformational dynamics upon nucleotide binding to MgtA were characterized using a combination of cryo-EM, molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and mutagenesis. Our structure also revealed a Mg2+ ion in the transmembrane segments, which, when combined with sequence conservation and mutagenesis studies, allowed us to propose a model for Mg2+ transport across the lipid bilayer. Finally, our work revealed the N-terminal domain structure and cytoplasmic Mg2+ binding sites, which have implications for related P-type ATPases defective in human disease.

biophysics↗

bioRxiv2022

EstG is a novel esterase required for cell envelope integrity

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the -proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell wall-targeting antibiotics, EstG does not demonstrate biochemical activity towards cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which is important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that, instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

microbiology↗

bioRxiv2021

Model-based identification of conditionally-essential genes from transposon-insertion sequencing data

The understanding of bacterial gene function has been greatly enhanced by recent advancements in the deep sequencing of microbial genomes. Transposon insertion sequencing methods combines next-generation sequencing techniques with transposon mutagenesis for the exploration of the essentiality of genes under different environmental conditions. We propose a model-based method that uses regularized negative binomial regression to estimate the change in transposon insertions attributable to gene-environment changes without transformations or uniform normalization. An empirical Bayes model for estimating the local false discovery rate combines unique and total count information to test for genes that show a statistically significant change in transposon counts. When applied to RB-TnSeq (randomized barcode transposon sequencing) and Tn-seq (transposon sequencing) libraries made in strains of Caulobacter crescentus using both total and unique count data the model was able to identify a set of conditionally essential genes for each target condition that shed light on their functions and roles during various stress conditions. Author summaryTransposon insertion sequencing allows the study of bacterial gene function by combining next-generation sequencing techniques with transposon mutagenesis under different genetic and environmental perturbations. Our proposed regularized negative binomial regression method improves the quality of analysis of this data.

bioinformatics↗

bioRxiv2020

Degradation of ATFS-1 by LONP-1 promotes deleterious mitochondrial genome heteroplasmy

The accumulation of deleterious mitochondrial genomes ({Delta}mtDNAs) underlies inherited mitochondrial diseases and contributes to the aging-associated decline in mitochondrial function. In heteroplasmic cells, oxidative phosphorylation (OXPHOS) function declines as the population of {Delta}mtDNAs increase relative to wildtype mtDNAs. In response to mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcription program to promote the recovery of mitochondrial function. Paradoxically, ATFS-1 is also required to maintain {Delta}mtDNAs in heteroplasmic worms. However, the mechanism(s) by which ATFS-1 promotes {Delta}mtDNA accumulation relative to wildtype mtDNAs is unclear. Here, we show that mitochondrial-localized ATFS-1 binds almost exclusively to {Delta}mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the preferential binding of the mtDNA replicative polymerase (POLG) to {Delta}mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wildtype mtDNAs. Furthermore, LONP-1 inhibition in C. elegans and human cybrid cells improved the heteroplasmy ratio and restored OXPHOS function. Our findings suggest that ATFS-1 promotes mtDNA replication by recruiting POLG to mtDNA in a manner that is antagonized by LONP-1. We speculate that this mechanism promotes the repair and expansion of the mitochondrial network by synchronizing mtDNA replication with UPRmt activation driven by nuclear ATFS-1 activity. However, this repair mechanism cannot resolve OXPHOS defects in mitochondria harboring {Delta}mtDNAs, resulting in an accumulation of ATFS-1 in dysfunctional mitochondria and constitutive replication of {Delta}mtDNAs.

cell biology↗

bioRxiv2019

The conserved transcriptional regulator CdnL is required for metabolic homeostasis and morphogenesis in Caulobacter

Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor {sigma}70. The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where {sigma}70 localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show that Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Specifically,{Delta} cdnL cells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes. Notably, we find that{Delta} cdnL cells are severely limited in glutamate synthesis, rendering them auxotrophic for that amino acid. Moreover,{Delta} cdnL cells produce low amounts of the cell wall precursor lipid II, and{Delta} cdnL is synthetic lethal with other genetic perturbations that limit lipid II production.{Delta} cdnL cells also have aberrant localization of MreB and CtpS, cytoskeletal proteins required for maintaining proper cell width and curvature. Interestingly, the localization of CtpS is dependent on availability of CTP, which is predicted to be low in{Delta} cdnL cells. Our findings implicate CdnL as a global regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and through metabolite-mediated changes in localization of cytoskeletal regulators of cell shape.

microbiology↗

bioRxiv2018

Activation of SEDS-PBP cell wall synthases by an essential regulator of bacterial division

Bacterial growth and division require insertion of new peptidoglycan (PG) into the existing cell wall by PG synthase enzymes. Emerging evidence suggests that many PG synthases require activation to function, however it is unclear how activation of division-specific PG synthases occurs. The FtsZ cytoskeleton has been implicated as a regulator of PG synthesis during division, but the mechanisms through which it acts are unknown. Here we show that FzlA, an essential regulator of constriction in Caulobacter crescentus, links FtsZ to PG synthesis to promote division. We find that hyperactive mutants of the PG synthases FtsW and FtsI specifically render fzlA, but not other division genes, non-essential. However, FzlA is still required to maintain proper constriction rate and efficiency in a hyperactive PG synthase background. Intriguingly, loss of fzlA in the presence of hyperactivated FtsWI causes cells to rotate about the division plane during constriction and sensitizes cells to cell wall-specific antibiotics. We demonstrate that FzlA-dependent signaling to division-specific PG synthesis is conserved in another -proteobacterium, Agrobacterium tumefaciens. These data establish that FzlA links FtsZ to cell wall remodeling, serving both to activate and spatially orient PG synthesis during division. Overall, our findings support the paradigm that activation of SEDS-PBP PG synthases is a broadly conserved requirement for bacterial morphogenesis.

microbiology↗