Selective translation by alternative bacterial ribosomes

Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes fromMycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain ofM. smegmatisin which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.

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Selective translation by alternative bacterial ribosomes

10.1101/605931 ◽

2019 ◽

Cited By ~ 3

Author(s):

Yu-Xiang Chen ◽

Zhi-yu Xu ◽

Xueliang Ge ◽

Suparna Sanyal ◽

Zhi John Lu ◽

...

Keyword(s):

Mycobacterium Smegmatis ◽

Bacterial Species ◽

Growth Conditions ◽

Ribosome Profiling ◽

Protein Subunits ◽

Relative Contribution ◽

Selective Translation ◽

Cellular Context ◽

Different Strains ◽

Hostile Environments

AbstractAlternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in gene translation, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Our work convincingly confirms the distinct and non-redundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.Significance StatementMany organisms, including most bacteria code for multiple paralogues of some ribosomal protein subunits. The relative contribution of these alternative subunits to ribosome function and gene translation is unknown and controversial. Furthermore, many studies on alternative ribosomes have been confounded by isolation of alternative and canonical ribosomes from different strains and/ or different growth conditions, potentially confounding results. Here, we show unequivocally that one form of alternative ribosome from Mycobacterium smegmatis actively engages in gene translation, but its translational profile from an identical cellular context is subtly different from canonical ribosomes. Given the prevalence of alternative ribosomal genes in diverse organisms, our study suggests that alternative ribosomes may represent a further layer of regulation of gene translation.

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The Regulator PerR Is Involved in Oxidative Stress Response and Iron Homeostasis and Is Necessary for Full Virulence of Streptococcus pyogenes

Infection and Immunity ◽

10.1128/iai.70.9.4968-4976.2002 ◽

2002 ◽

Vol 70 (9) ◽

pp. 4968-4976 ◽

Cited By ~ 65

Author(s):

Susanna Ricci ◽

Robert Janulczyk ◽

Lars Björck

Keyword(s):

Oxidative Stress ◽

Streptococcus Pyogenes ◽

Stress Responses ◽

Metal Binding ◽

Iron Homeostasis ◽

Bacterial Species ◽

Transcriptional Analysis ◽

Wild Type ◽

Allelic Replacement

ABSTRACT Ferric uptake regulator (Fur) and Fur-like proteins form an important family of transcriptional regulators in many bacterial species. In this work we have characterized a Fur-like protein, the peroxide regulator PerR, in an M1 serotype of Streptococcus pyogenes. To determine the role of PerR in S. pyogenes, we inactivated the gene by allelic replacement. PerR-deficient bacteria showed 48% reduction of 55Fe incorporation from the culture medium. Transcriptional analysis revealed that mtsA, encoding a metal-binding protein of an ABC transporter in S. pyogenes, was transcribed at lower levels than were wild-type cells. Although total iron accumulation was reduced, the growth of the mutant strain was not significantly hampered. The mutant showed hyperresistance to hydrogen peroxide, and this response was induced in wild-type cells by growth in aerobiosis, suggesting that PerR acts as an oxidative stress-responsive repressor. PerR may also participate in the response to superoxide stress, as the perR mutant was more sensitive to the superoxide anion and had a reduced transcription of sodA, which encodes the sole superoxide dismutase of S. pyogenes. Complementation of the mutation with a functional perR gene restored 55Fe incorporation, response to peroxide stress, and transcription of both mtsA and sodA to levels comparable to those of wild-type bacteria. Finally, the perR mutant was attenuated in virulence in a murine air sac model of infection (P < 0.05). These results demonstrate that PerR is involved in the regulation of iron homeostasis and oxidative stress responses and that it contributes to the virulence of S. pyogenes.

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An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

10.1101/758037 ◽

2019 ◽

Author(s):

Yves-Marie Boudehen ◽

Maximillian Wallat ◽

Philippe Rousseau ◽

Olivier Neyrolles ◽

Claude Gutierrez

Keyword(s):

Antibiotic Resistance ◽

High Frequency ◽

Mycobacterium Smegmatis ◽

Bacterial Species ◽

Wild Type ◽

Model Species ◽

Mycobacterial Genome ◽

Bacterial Chromosomes ◽

Genetic Constructs ◽

Zeocin Resistance

SummaryXer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin-resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.Method summarydif-ZeoR-dif cassettes are used to replace non-essential genes in mycobacterial genome through recombineering. Spontaneous excision of the cassette is carried out under the action of the recombinase XerCD, resulting in unmarked deletions. Subsequent rounds of mutagenesis using cassettes flanked by a range of dif site variants allow construction of multiple mutants in which the different dif sites cannot recombine which each other, yielding stable genetic constructs.

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Transcriptional Response of Leptospira interrogans to Iron Limitation and Characterization of a PerR Homolog

Infection and Immunity ◽

10.1128/iai.00435-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4850-4859 ◽

Cited By ~ 54

Author(s):

Miranda Lo ◽

Gerald L. Murray ◽

Chen Ai Khoo ◽

David A. Haake ◽

Richard L. Zuerner ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Response ◽

Iron Homeostasis ◽

Storage Proteins ◽

Iron Acquisition ◽

Bacterial Species ◽

Transcriptional Response ◽

Oxidative Stress Response ◽

Wild Type ◽

In The Wild

ABSTRACT Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

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An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

BioTechniques ◽

10.2144/btn-2019-0119 ◽

2020 ◽

Vol 68 (2) ◽

pp. 106-110 ◽

Cited By ~ 1

Author(s):

Yves-Marie Boudehen ◽

Maximilian Wallat ◽

Philippe Rousseau ◽

Olivier Neyrolles ◽

Claude Gutierrez

Keyword(s):

Antibiotic Resistance ◽

Mycobacterium Tuberculosis ◽

Central Region ◽

High Frequency ◽

Mycobacterium Smegmatis ◽

Bacterial Species ◽

Wild Type ◽

Model Species ◽

Bacterial Chromosomes ◽

Zeocin Resistance

Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild-type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.

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The Role of Alcohol, LPS Toxicity, and ALDH2 in Dental Bony Defects

Biomolecules ◽

10.3390/biom11050651 ◽

2021 ◽

Vol 11 (5) ◽

pp. 651

Author(s):

Hsiao-Cheng Tsai ◽

Che-Hong Chen ◽

Daria Mochly-Rosen ◽

Yi-Chen Ethan Li ◽

Min-Huey Chen

Keyword(s):

Bone Loss ◽

Bacterial Infection ◽

Bone Regeneration ◽

Alcohol Drinking ◽

Bacterial Species ◽

Dominant Negative ◽

Enzyme Inactivation ◽

Wild Type ◽

Micro Computed Tomography ◽

Dominant Negative Mutation

It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.

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Identification ofvib-1, a Locus Involved in Vegetative Incompatibility Mediated byhet-cinNeurospora crassa

Genetics ◽

10.1093/genetics/162.1.89 ◽

2002 ◽

Vol 162 (1) ◽

pp. 89-101 ◽

Cited By ~ 6

Author(s):

Qijun Xiang ◽

N Louise Glass

Keyword(s):

Acid Phosphatase ◽

Recognition System ◽

Deletion Mutants ◽

Wild Type ◽

Vegetative Incompatibility ◽

Death Rates ◽

Self Recognition ◽

Allelic Differences ◽

Native Gel ◽

Type Strains

AbstractA non-self-recognition system called vegetative incompatibility is ubiquitous in filamentous fungi and is genetically regulated by het loci. Different fungal individuals are unable to form viable heterokaryons if they differ in allelic specificity at a het locus. To identify components of vegetative incompatibility mediated by allelic differences at the het-c locus of Neurospora crassa, we isolated mutants that suppressed phenotypic aspects of het-c vegetative incompatibility. Three deletion mutants were identified; the deletions overlapped each other in an ORF named vib-1 (vegetative incompatibility blocked). Mutations in vib-1 fully relieved growth inhibition and repression of conidiation conferred by het-c vegetative incompatibility and significantly reduced hyphal compartmentation and death rates. The vib-1 mutants displayed a profuse conidiation pattern, suggesting that VIB-1 is a regulator of conidiation. VIB-1 shares a region of similarity to PHOG, a possible phosphate nonrepressible acid phosphatase in Aspergillus nidulans. Native gel analysis of wild-type strains and vib-1 mutants indicated that vib-1 is not the structural gene for nonrepressible acid phosphatase, but rather may regulate nonrepressible acid phosphatase activity.

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HET-E and HET-D Belong to a New Subfamily of WD40 Proteins Involved in Vegetative Incompatibility Specificity in the Fungus Podospora anserina

Genetics ◽

10.1093/genetics/161.1.71 ◽

2002 ◽

Vol 161 (1) ◽

pp. 71-81

Author(s):

Eric Espagne ◽

Pascale Balhadère ◽

Marie-Louise Penin ◽

Christian Barreau ◽

Béatrice Turcq

Keyword(s):

Genetic Difference ◽

Podospora Anserina ◽

Wild Type ◽

Incompatible Interaction ◽

Vegetative Incompatibility ◽

Repeat Domain ◽

Upper Face ◽

E1a Gene ◽

Type Strains ◽

Wd40 Repeats

Abstract Vegetative incompatibility, which is very common in filamentous fungi, prevents a viable heterokaryotic cell from being formed by the fusion of filaments from two different wild-type strains. Such incompatibility is always the consequence of at least one genetic difference in specific genes (het genes). In Podospora anserina, alleles of the het-e and het-d loci control heterokaryon viability through genetic interactions with alleles of the unlinked het-c locus. The het-d2Y gene was isolated and shown to have strong similarity with the previously described het-e1A gene. Like the HET-E protein, the HET-D putative protein displayed a GTP-binding domain and seemed to require a minimal number of 11 WD40 repeats to be active in incompatibility. Apart from incompatibility specificity, no other function could be identified by disrupting the het-d gene. Sequence comparison of different het-e alleles suggested that het-e specificity is determined by the sequence of the WD40 repeat domain. In particular, the amino acids present on the upper face of the predicted β-propeller structure defined by this domain may confer the incompatible interaction specificity.

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Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer’s disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis

BMC Genomics ◽

10.1186/s12864-021-07509-1 ◽

2021 ◽

Vol 22 (1) ◽

Cited By ~ 1

Author(s):

Yang Dong ◽

Morgan Newman ◽

Stephen M. Pederson ◽

Karissa Barthelson ◽

Nhi Hin ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Oxidative Phosphorylation ◽

Transcriptome Analysis ◽

Iron Homeostasis ◽

Late Onset ◽

Wild Type ◽

Familial Alzheimer’S Disease ◽

Zebrafish Larvae ◽

Familial Alzheimer's Disease

Abstract Background Early-onset familial Alzheimer’s disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer’s disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1Q96_K97del, in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs. Results We generated clutches of wild type and heterozygous psen1Q96_K97del 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions. Conclusions Our analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1Q96_K97del brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3′ untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1Q96_K97del EOfAD-like mutant brains.

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