The Bacillus subtilis tyrZ Gene Encodes a Highly Selective Tyrosyl-tRNA Synthetase and Is Regulated by a MarR Regulator and T Box Riboswitch

ABSTRACTMisincorporation ofd-tyrosine (d-Tyr) into cellular proteins due to mischarging of tRNATyrwithd-Tyr by tyrosyl-tRNA synthetase inhibits growth and biofilm formation ofBacillus subtilis. Furthermore, manyB. subtilisstrains lack a functional gene encodingd-aminoacyl-tRNA deacylase, which prevents misincorporation ofd-Tyr in most organisms.B. subtilishas two genes that encode tyrosyl-tRNA synthetase:tyrSis expressed under normal growth conditions, andtyrZis known to be expressed only whentyrSis inactivated by mutation. We hypothesized thattyrZencodes an alternate tyrosyl-tRNA synthetase, expression of which allows the cell to grow whend-Tyr is present. We show that TyrZ is more selective forl-Tyr overd-Tyr than is TyrS; however, TyrZ is less efficient overall. We also show that expression oftyrZis required for growth and biofilm formation in the presence ofd-Tyr. BothtyrSandtyrZare preceded by a T box riboswitch, buttyrZis found in an operon withywaE, which is predicted to encode a MarR family transcriptional regulator. Expression oftyrZis repressed by YwaE and also is regulated at the level of transcription attenuation by the T box riboswitch. We conclude that expression oftyrZmay allow growth when excessd-Tyr is present.IMPORTANCEAccurate protein synthesis requires correct aminoacylation of each tRNA with the cognate amino acid and discrimination against related compounds.Bacillus subtilisproducesd-Tyr, an analog ofl-Tyr that is toxic when incorporated into protein, during stationary phase. Most organisms utilize ad-aminoacyl-tRNA deacylase to prevent misincorporation ofd-Tyr. This work demonstrates that the increased selectivity of the TyrZ form of tyrosyl-tRNA synthetase may provide a mechanism by whichB. subtilisprevents misincorporation ofd-Tyr in the absence of a functionald-aminoacyl-tRNA deacylase gene.

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Aminoacyl-tRNA synthetase genes of Bacillus subtilis: organization and regulation

Biochemistry and Cell Biology ◽

10.1139/o99-040 ◽

1999 ◽

Vol 77 (4) ◽

pp. 343-347 ◽

Cited By ~ 10

Author(s):

Martin Pelchat ◽

Jacques Lapointe

Keyword(s):

Bacillus Subtilis ◽

Initiation Site ◽

Trna Synthetase ◽

Translation Initiation Site ◽

Aminoacyl Trna Synthetase ◽

Gene Encoding ◽

T Box ◽

Trna Synthetases ◽

Genes Encoding ◽

Mature Mrnas

In Bacillus subtilis, 14 of the 24 genes encoding aminoacyl-tRNA synthetases (aaRS) are regulated by tRNA-mediated antitermination in response to starvation for their cognate aminoacid. Their transcripts have an untranslated leader mRNA of about 300 nucleotides, including alternative and mutually exclusive terminator-antiterminator structures, just upstream from the translation initiation site. Following antitermination, some of these transcripts are cleaved leaving at the 5prime-end of the mature mRNAs, stable secondary structures that can protect them against degradation. Although most B. subtilis aaRS genes are expressed as monocistronic mRNAs, the gltX gene encoding the glutamyl-tRNA synthetase is cotranscribed with cysE and cysS encoding serine acetyl-transferase and cysteinyl-tRNA synthetase, respectively. Transcription of gltX is not controlled by a tRNA, but tRNACys-mediated antitermination regulates the elongation of transcription into cysE and cysS. The full-length gltX-cysE-cysS transcript is then cleaved into a monocistronic gltX mRNA and a cysE-cysS mRNA.Key words: regulation, aminoacyl-tRNA synthetase, T-Box, processing.

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A gene encoding a tyrosine tRNA synthetase is located near Sacs in Bacillus subtilis

DNA Sequence ◽

10.3109/10425179109020780 ◽

1991 ◽

Vol 1 (4) ◽

pp. 251-261 ◽

Cited By ~ 23

Author(s):

P. Glaser ◽

F. Kunst ◽

M. Débarbouillé ◽

A. Vertès ◽

A. Danchin ◽

...

Keyword(s):

Bacillus Subtilis ◽

Trna Synthetase ◽

Gene Encoding

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Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12

Microbiology ◽

10.1099/mic.0.000947 ◽

2020 ◽

Vol 166 (9) ◽

pp. 880-890 ◽

Cited By ~ 1

Author(s):

Hiroshi Ogasawara ◽

Toshiyuki Ishizuka ◽

Shuhei Hotta ◽

Michiko Aoki ◽

Tomohiro Shimada ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Cell Aggregation ◽

Master Regulator ◽

Gene Encoding ◽

Content Type ◽

Promoter Probe ◽

K 12 ◽

Specific Environmental Condition

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study ‘promoter-specific transcription-factor’ (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

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Mapping of the Denitrification Pathway in Burkholderia thailandensis by Genome-Wide Mutant Profiling

Journal of Bacteriology ◽

10.1128/jb.00304-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Alessandra Vitale ◽

Sarah Paszti ◽

Kohei Takahashi ◽

Masanori Toyofuku ◽

Gabriella Pessi ◽

...

Keyword(s):

Biofilm Formation ◽

Burkholderia Pseudomallei ◽

Anaerobic Growth ◽

Gene Encoding ◽

Terminal Electron Acceptor ◽

Burkholderia Thailandensis ◽

Content Type ◽

Sufficient Energy ◽

Genes Encoding ◽

Human Infections

ABSTRACT Burkholderia thailandensis is a soil saprophyte that is closely related to the pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans. The environmental niches and infection sites occupied by these bacteria are thought to contain only limited concentrations of oxygen, where they can generate energy via denitrification. However, knowledge of the underlying molecular basis of the denitrification pathway in these bacteria is scarce. In this study, we employed a transposon sequencing (Tn-Seq) approach to identify genes conferring a fitness benefit for anaerobic growth of B. thailandensis. Of the 180 determinants identified, several genes were shown to be required for growth under denitrifying conditions: the nitrate reductase operon narIJHGK2K1, the aniA gene encoding a previously unknown nitrite reductase, and the petABC genes encoding a cytochrome bc1, as well as three novel regulators that control denitrification. Our Tn-Seq data allowed us to reconstruct the entire denitrification pathway of B. thailandensis and shed light on its regulation. Analyses of growth behaviors combined with measurements of denitrification metabolites of various mutants revealed that nitrate reduction provides sufficient energy for anaerobic growth, an important finding in light of the fact that some pathogenic Burkholderia species can use nitrate as a terminal electron acceptor but are unable to complete denitrification. Finally, we demonstrated that a nitrous oxide reductase mutant is not affected for anaerobic growth but is defective in biofilm formation and accumulates N2O, which may play a role in the dispersal of B. thailandensis biofilms. IMPORTANCE Burkholderia thailandensis is a soil-dwelling saprophyte that is often used as surrogate of the closely related pathogen Burkholderia pseudomallei, the causative agent of melioidosis and a classified biowarfare agent. Both organisms are adapted to grow under oxygen-limited conditions in rice fields by generating energy through denitrification. Microoxic growth of B. pseudomallei is also considered essential for human infections. Here, we have used a Tn-Seq approach to identify the genes encoding the enzymes and regulators required for growth under denitrifying conditions. We show that a mutant that is defective in the conversion of N2O to N2, the last step in the denitrification process, is unaffected in microoxic growth but is severely impaired in biofilm formation, suggesting that N2O may play a role in biofilm dispersal. Our study identified novel targets for the development of therapeutic agents to treat meliodiosis.

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Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

mBio ◽

10.1128/mbio.01464-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 6

Author(s):

Jan Kampf ◽

Jan Gerwig ◽

Kerstin Kruse ◽

Robert Cleverley ◽

Miriam Dormeyer ◽

...

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Selective Pressure ◽

Wild Type ◽

Master Regulator ◽

Form Complex ◽

Content Type

ABSTRACT Biofilm formation by Bacillus subtilis requires the expression of genes encoding enzymes for extracellular polysaccharide synthesis and for an amyloid-like protein. The master regulator SinR represses all the corresponding genes, and repression of these key biofilm genes is lifted when SinR interacts with its cognate antagonist proteins. The YmdB phosphodiesterase is a recently discovered factor that is involved in the control of SinR activity: cells lacking YmdB exhibit hyperactive SinR and are unable to relieve the repression of the biofilm genes. In this study, we have examined the dynamics of gene expression patterns in wild-type and ymdB mutant cells by microfluidic analysis coupled to time-lapse microscopy. Our results confirm the bistable expression pattern for motility and biofilm genes in the wild-type strain and the loss of biofilm gene expression in the mutant. Moreover, we demonstrated dynamic behavior in subpopulations of the wild-type strain that is characterized by switches in sets of the expressed genes. In order to gain further insights into the role of YmdB, we isolated a set of spontaneous suppressor mutants derived from ymdB mutants that had regained the ability to form complex colonies and biofilms. Interestingly, all of the mutations affected SinR. In some mutants, large genomic regions encompassing sinR were deleted, whereas others had alleles encoding SinR variants. Functional and biochemical studies with these SinR variants revealed how these proteins allowed biofilm gene expression in the ymdB mutant strains. IMPORTANCE Many bacteria are able to choose between two mutually exclusive lifestyles: biofilm formation and motility. In the model bacterium Bacillus subtilis, this choice is made by each individual cell rather than at the population level. The transcriptional repressor SinR is the master regulator in this decision-making process. The regulation of SinR activity involves complex control of its own expression and of its interaction with antagonist proteins. We show that the YmdB phosphodiesterase is required to allow the expression of SinR-repressed genes in a subpopulation of cells and that such subpopulations can switch between different SinR activity states. Suppressor analyses revealed that ymdB mutants readily acquire mutations affecting SinR, thus restoring biofilm formation. These findings suggest that B. subtilis cells experience selective pressure to form the extracellular matrix that is characteristic of biofilms and that YmdB is required for the homeostasis of SinR and/or its antagonists.

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Stress-Induced MazF-Mediated Proteins in Escherichia coli

mBio ◽

10.1128/mbio.00340-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 8

Author(s):

Akanksha Nigam ◽

Tamar Ziv ◽

Adi Oron-Gottesman ◽

Hanna Engelberg-Kulka

Keyword(s):

Escherichia Coli ◽

Proteomic Analysis ◽

Normal Growth ◽

Growth Conditions ◽

Translation Machinery ◽

Content Type ◽

E Coli ◽

Adverse Conditions ◽

Almost All ◽

Induced Proteins

ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. By that means, under stress, the induced MazF generates a stress-induced translation machinery (STM) composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated through the chromosomally borne mazF gene. We show that the mRNAs of almost all of them are characterized by the presence of an ACA site up to 100 nucleotides upstream of the AUG initiator. Therefore, under stressful conditions, induced MazF processes mRNAs that are translated by STM. Furthermore, the presence of the ACA sites far upstream (up to 100 nucleotides) of the AUG initiator may still permit translation by the canonical translation machinery. Thus, such dual-translation mechanisms enable the bacterium under stress also to prepare proteins for immediate functions while coming back to normal growth conditions. IMPORTANCE The stress response, the strategy that bacteria have developed in order to cope up with all kinds of adverse conditions, is so far understood at the level of transcription. Our previous findings of a uniquely modified stress-induced translation machinery (STM) generated in E. coli under stress by the endoribonucleolytic activity of the toxin MazF opens a new chapter in understanding microbial physiology under stress at the translational level. Here, we performed a proteomic analysis of all the E. coli stress-induced proteins that are mediated by chromosomally borne MazF through STM.

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Galactose Metabolism Plays a Crucial Role in Biofilm Formation by Bacillus subtilis

mBio ◽

10.1128/mbio.00184-12 ◽

2012 ◽

Vol 3 (4) ◽

Cited By ~ 83

Author(s):

Yunrong Chai ◽

Pascale B. Beauregard ◽

Hera Vlamakis ◽

Richard Losick ◽

Roberto Kolter

Keyword(s):

Bacillus Subtilis ◽

Biofilm Formation ◽

Carbon Sources ◽

Galactose Metabolism ◽

Content Type ◽

Leloir Pathway ◽

The Matrix ◽

Living Organisms ◽

Galactose Metabolites ◽

Biofilm Matrix

ABSTRACTGalactose is a common monosaccharide that can be utilized by all living organisms via the activities of three main enzymes that make up the Leloir pathway: GalK, GalT, and GalE. InBacillus subtilis, the absence of GalE causes sensitivity to exogenous galactose, leading to rapid cell lysis. This effect can be attributed to the accumulation of toxic galactose metabolites, since thegalEmutant is blocked in the final step of galactose catabolism. In a screen for suppressor mutants restoring viability to agalEnull mutant in the presence of galactose, we identified mutations insinR, which is the major biofilm repressor gene. These mutations caused an increase in the production of the exopolysaccharide (EPS) component of the biofilm matrix. We propose that UDP-galactose is the toxic galactose metabolite and that it is used in the synthesis of EPS. Thus, EPS production can function as a shunt mechanism for this toxic molecule. Additionally, we demonstrated that galactose metabolism genes play an essential role inB. subtilisbiofilm formation and that the expressions of both thegalandepsgenes are interrelated. Finally, we propose thatB. subtilisand other members of theBacillusgenus may have evolved to utilize naturally occurring polymers of galactose, such as galactan, as carbon sources.IMPORTANCEBacteria switch from unicellular to multicellular states by producing extracellular matrices that contain exopolysaccharides. In such aggregates, known as biofilms, bacteria are more resistant to antibiotics. This makes biofilms a serious problem in clinical settings. The resilience of biofilms makes them very useful in industrial settings. Thus, understanding the production of biofilm matrices is an important problem in microbiology. In studying the synthesis of the biofilm matrix ofBacillus subtilis, we provide further understanding of a long-standing microbiological observation that certain mutants defective in the utilization of galactose became sensitive to it. In this work, we show that the toxicity observed before was because cells were grown under conditions that were not propitious to produce the exopolysaccharide component of the matrix. When cells are grown under conditions that favor matrix production, the toxicity of galactose is relieved. This allowed us to demonstrate that galactose metabolism is essential for the synthesis of the extracellular matrix.

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Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

Applied and Environmental Microbiology ◽

10.1128/aem.01638-14 ◽

2014 ◽

Vol 80 (19) ◽

pp. 6167-6174 ◽

Cited By ~ 20

Author(s):

Xiaohui Gao ◽

Xiao Dong ◽

Sundharraman Subramanian ◽

Paige M. Matthews ◽

Caleb A. Cooper ◽

...

Keyword(s):

Bacillus Subtilis ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Threshold Level ◽

Metabolic Enzymes ◽

Guanosine Monophosphate ◽

Content Type ◽

Diguanylate Cyclases ◽

Rapid Characterization ◽

Green Fluorescent

ABSTRACTMicrobial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite ofBacillus subtilisstrains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs inClostridium difficileusing parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putativeC. difficile630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility inBacillus subtilis.

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Identification and Characterization of a NaCl-Responsive Genetic Locus Involved in Survival during Desiccation in Sinorhizobium meliloti

Applied and Environmental Microbiology ◽

10.1128/aem.01037-13 ◽

2013 ◽

Vol 79 (18) ◽

pp. 5693-5700 ◽

Cited By ~ 5

Author(s):

Jan A. C. Vriezen ◽

Frans J. de Bruijn ◽

Klaus Nüsslein

Keyword(s):

Sinorhizobium Meliloti ◽

Agricultural Soils ◽

Sequence Similarity ◽

Genetic Locus ◽

Model Organism ◽

Normal Growth ◽

Growth Conditions ◽

Amino Acid Sequence Similarity ◽

Content Type ◽

Genetic Mechanisms

ABSTRACTTheRhizobiaceaeare a bacterial family of enormous agricultural importance due to the ability of its members to fix atmospheric nitrogen in an intimate relationship with plants. Their survival as naturally occurring soil bacteria in agricultural soils as well as popular seed inocula is affected directly by drought and salinity. Survival after desiccation in the presence of NaCl is enabled by underlying genetic mechanisms in the model organismSinorhizobium meliloti1021. Since salt stress parallels a loss in water activity, the identification of NaCl-responsive loci may identify loci involved in survival during desiccation. This approach enabled identification of the lociasnOandnggby their reduced ability to grow on increased NaCl concentrations, likely due to their inability to produce the osmoprotectant N-acetylglutaminylglutamine (NAGGN). In addition, the mutant harboringngg::Tn5luxABwas affected in its ability to survive desiccation and responded to osmotic stress. The desiccation sensitivity may have been due to secondary functions of Ngg (N-acetylglutaminylglutamine synthetase)-like cell wall metabolism as suggested by the presence of ad-alanine-d-alanine ligase (dAla-dAla) domain and by sensitivity of the mutant to β-lactam antibiotics.asnO::Tn5luxABis expressed during the stationary phase under normal growth conditions. Amino acid sequence similarity to enzymes producing β-lactam inhibitors and increased resistance to β-lactam antibiotics may indicate thatasnOis involved in the production of a β-lactam inhibitor.

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