scholarly journals Identification of themcpAandmcpMGenes, Encoding Methyl-Accepting Proteins Involved in Amino Acid and l-Malate Chemotaxis, and Involvement of McpM-Mediated Chemotaxis in Plant Infection by Ralstonia pseudosolanacearum (Formerly Ralstonia solanacearum Phylotypes I and III)

2015 ◽  
Vol 81 (21) ◽  
pp. 7420-7430 ◽  
Author(s):  
Akiko Hida ◽  
Shota Oku ◽  
Takeru Kawasaki ◽  
Yutaka Nakashimada ◽  
Takahisa Tajima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Gene Deletion ◽  
Pcr Analysis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Complete Collection ◽  
Content Type ◽  
Plant Infection ◽  
Tomato Roots ◽  
Ralstonia Pseudosolanacearum

ABSTRACTSequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in theRalstonia pseudosolanacearumGMI1000 genome. PCR analysis and DNA sequencing showed that the highly motileR. pseudosolanacearumstrain Ps29 possesses homologs of all 22R. pseudosolanacearumGMI1000mcpgenes. We constructed a complete collection of singlemcpgene deletion mutants ofR. pseudosolanacearumPs29 by unmarked gene deletion. Screening of the mutant collection revealed thatR. pseudosolanacearumPs29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis tol-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designatedmcpMandmcpA. While wild-typeR. pseudosolanacearumstrain Ps29 displayed attraction to 16 amino acids, themcpAmutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructedmcpAandmcpMdeletion mutants of highly virulentR. pseudosolanacearumstrain MAFF106611 to investigate the contribution of chemotaxis tol-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, themcpMmutant (but not themcpAmutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis tol-malate, facilitatesR. pseudosolanacearummotility to tomato roots in sand.

scholarly journals Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

2012 ◽  
Vol 79 (5) ◽  
pp. 1500-1507 ◽  
Author(s):  
Suk-Jin Ha ◽  
Heejin Kim ◽  
Yuping Lin ◽  
Myoung-Uoon Jang ◽  
Jonathan M. Galazka ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Single Amino Acid ◽  
Kinetic Properties ◽  
Wild Type ◽  
Scheffersomyces Stipitis ◽  
Content Type ◽  
Charged Amino Acids ◽  
Cellodextrin Transporter ◽  
Engineered Strain

ABSTRACTSaccharomyces cerevisiaecannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes fromNeurospora crassa. Here, we report that an engineeredS. cerevisiaestrain expressing the putative hexose transporter geneHXT2.4fromScheffersomyces stipitisandgh1-1can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter whenHXT2.4is overexpressed inS. cerevisiae. However, cellobiose fermentation by the engineered strain expressingHXT2.4andgh1-1was much slower and less efficient than that by an engineered strain that initially expressedcdt-1andgh1-1. The rate of cellobiose fermentation by theHXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolvedS. cerevisiaestrain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higherKmand 4-fold higherVmaxvalues than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed inS. cerevisiaeare suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineeredS. cerevisiaestrains.

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

scholarly journals Glutathione Synthesis Contributes to Virulence of Streptococcus agalactiae in a Murine Model of Sepsis

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Elizabeth A. Walker ◽  
Gary C. Port ◽  
Michael G. Caparon ◽  
Blythe E. Janowiak
Keyword(s):  
Streptococcus Agalactiae ◽  
De Novo ◽  
Single Gene ◽  
Neonatal Meningitis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Glutathione Synthesis ◽  
Content Type ◽  
Resistance Pathway

ABSTRACT Streptococcus agalactiae, a leading cause of sepsis and meningitis in neonates, utilizes multiple virulence factors to survive and thrive within the human host during an infection. Unique among the pathogenic streptococci, S. agalactiae uses a bifunctional enzyme encoded by a single gene (gshAB) to synthesize glutathione (GSH), a major antioxidant in most aerobic organisms. Since S. agalactiae can also import GSH, similar to all other pathogenic streptococcal species, the contribution of GSH synthesis to the pathogenesis of S. agalactiae disease is not known. In the present study, gshAB deletion mutants were generated in strains representing three of the most prevalent clinical serotypes of S. agalactiae and were compared against isogenic wild-type and gshAB knock-in strains. When cultured in vitro in a chemically defined medium under nonstress conditions, each mutant and its corresponding wild type had comparable growth rates, generation times, and growth yields. However, gshAB deletion mutants were found to be more sensitive than wild-type or gshAB knock-in strains to killing and growth inhibition by several different reactive oxygen species. Furthermore, deletion of gshAB in S. agalactiae strain COH1 significantly attenuated virulence compared to the wild-type or gshAB knock-in strains in a mouse model of sepsis. Taken together, these data establish that GSH is a virulence factor important for resistance to oxidative stress and that de novo GSH synthesis plays a crucial role in S. agalactiae pathogenesis and further suggest that the inhibition of GSH synthesis may provide an opportunity for the development of novel therapies targeting S. agalactiae disease. IMPORTANCE Approximately 10 to 30% of women are naturally and asymptomatically colonized by Streptococcus agalactiae. However, transmission of S. agalactiae from mother to newborn during vaginal birth is a leading cause of neonatal meningitis. Although colonized mothers who are at risk for transmission to the newborn are treated with antibiotics prior to delivery, S. agalactiae is becoming increasingly resistant to current antibiotic therapies, and new treatments are needed. This research reveals a critical stress resistance pathway, glutathione synthesis, that is utilized by S. agalactiae and contributes to its pathogenesis. Understanding the role of this unique bifunctional glutathione synthesis enzyme in S. agalactiae during sepsis may help elucidate why S. agalactiae produces such an abundance of glutathione compared to other bacteria.

scholarly journals Siderophores Are Not Involved in Fe(III) Solubilization during Anaerobic Fe(III) Respiration by Shewanella oneidensis MR-1

2010 ◽  
Vol 76 (8) ◽  
pp. 2425-2432 ◽  
Author(s):  
Christine M. Fennessey ◽  
Morris E. Jones ◽  
Martial Taillefert ◽  
Thomas J. DiChristina
Keyword(s):  
Electron Acceptor ◽  
Gene Deletion ◽  
Shewanella Oneidensis ◽  
Nitrilotriacetic Acid ◽  
Organic Ligands ◽  
Aerobic Respiration ◽  
Deletion Mutants ◽  
Wild Type ◽  
Voltammetric Techniques ◽  
Wide Range

ABSTRACT Shewanella oneidensis MR-1 respires a wide range of anaerobic electron acceptors, including sparingly soluble Fe(III) oxides. In the present study, S. oneidensis was found to produce Fe(III)-solubilizing organic ligands during anaerobic Fe(III) oxide respiration, a respiratory strategy postulated to destabilize Fe(III) and produce more readily reducible soluble organic Fe(III). In-frame gene deletion mutagenesis, siderophore detection assays, and voltammetric techniques were combined to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration were synthesized via siderophore biosynthesis systems and (ii) if the Fe(III)-siderophore reductase was required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. Genes predicted to encode the siderophore (hydroxamate) biosynthesis system (SO3030 to SO3032), the Fe(III)-hydroxamate receptor (SO3033), and the Fe(III)-hydroxamate reductase (SO3034) were identified in the S. oneidensis genome, and corresponding in-frame gene deletion mutants were constructed. ΔSO3031 was unable to synthesize siderophores or produce soluble organic Fe(III) during aerobic respiration yet retained the ability to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. ΔSO3034 retained the ability to synthesize siderophores during aerobic respiration and to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. These findings indicate that the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are not synthesized via the hydroxamate biosynthesis system and that the Fe(III)-hydroxamate reductase is not essential for respiration of Fe(III)-citrate or Fe(III)-nitrilotriacetic acid (NTA) as an anaerobic electron acceptor.

scholarly journals Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus

2014 ◽  
Vol 80 (7) ◽  
pp. 2299-2306 ◽  
Author(s):  
Axel Thiel ◽  
Grégoire Michoud ◽  
Yann Moalic ◽  
Didier Flament ◽  
Mohamed Jebbar
Keyword(s):  
Gene Disruption ◽  
Negative Selection ◽  
Deletion Mutants ◽  
Wild Type ◽  
Genetic Studies ◽  
Content Type ◽  
Genetic Manipulations ◽  
Starting Point ◽  
Flanking Regions ◽  
Markerless Deletion

ABSTRACTIn this study, we developed a gene disruption system forThermococcus barophilususing simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-typeT. barophilusMP cells. Initially, apyrFdeletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of thehisBgene was also constructed in the UBOCC-3256 (ΔpyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functionalpyrForhisBallele fromT. barophiluswas inserted into the chromosome of UBOCC-3256 (ΔpyrF) or UBOCC-3260 (ΔpyrFΔhisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.

scholarly journals A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Qusai Al Abdallah ◽  
Wenbo Ge ◽  
Jarrod R. Fortwendel
Keyword(s):  
Drug Resistance ◽  
Gene Deletion ◽  
Gene Replacement ◽  
Antifungal Drug ◽  
Wild Type ◽  
Gene Manipulation ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
Efficient Gene

ABSTRACT Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, we developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A. fumigatus. The system employs in vitro assembly of dual Cas9 ribonucleoproteins (RNPs) for targeted gene deletion. Additionally, our CRISPR-Cas9 system utilizes 35 to 50 bp of flanking regions for mediating homologous recombination at Cas9 double-strand breaks (DSBs). As a proof of concept, we first tested our system in the ΔakuB (ΔakuB ku80 ) laboratory strain and generated high rates (97%) of gene deletion using 2 µg of the repair template flanked by homology regions as short as 35 bp. Next, we inspected the portability of our system across other genetic backgrounds of A. fumigatus, namely, the wild-type strain Af293 and a clinical isolate, A. fumigatus DI15-102. In the Af293 strain, 2 µg of the repair template flanked by 35 and 50 bp of homology resulted in highly efficient gene deletion (46% and 74%, respectively) in comparison to classical gene replacement systems. Similar deletion efficiencies were also obtained in the clinical isolate DI15-102. Taken together, our data show that in vitro-assembled Cas9 RNPs coupled with microhomology repair templates are an efficient and universal system for gene manipulation in A. fumigatus. IMPORTANCE Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus.

scholarly journals A Novelmeso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d-Amino Acid Synthesis

2012 ◽  
Vol 78 (24) ◽  
pp. 8595-8600 ◽  
Author(s):  
Xiuzhen Gao ◽  
Xi Chen ◽  
Weidong Liu ◽  
Jinhui Feng ◽  
Qiaqing Wu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Enantiomeric Excess ◽  
Acid Synthesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Gene Encoding ◽  
Amino Acid Synthesis ◽  
Content Type

ABSTRACTmeso-Diaminopimelate dehydrogenase (meso-DAPDH) is an NADP+-dependent enzyme which catalyzes the reversible oxidative deamination on thed-configuration ofmeso-2,6-diaminopimelate to producel-2-amino-6-oxopimelate. In this study, the gene encoding ameso-diaminopimelate dehydrogenase fromSymbiobacterium thermophilumwas cloned and expressed inEscherichia coli. In addition to the native substratemeso-2,6-diaminopimelate, the purified enzyme also showed activity towardd-alanine,d-valine, andd-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generated-amino acids in up to 99% conversion and 99% enantiomeric excess. Sincemeso-diaminopimelate dehydrogenases are known to be specific tomeso-2,6-diaminopimelate, this is a unique wild-typemeso-diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential ford-amino acid synthesis. The enzyme is the most stablemeso-diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites ofS. thermophilum meso-DAPDH different from the sequences of other knownmeso-DAPDHs were replaced with the conserved amino acids in othermeso-DAPDHs, and assay of wild-type and mutant enzyme activities revealed that F146 and M152 are not critical in determining the enzyme's substrate specificity. The high thermostability and relaxed substrate profile ofS. thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effectived-amino acid dehydrogenases by protein engineering.

scholarly journals The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium psychrophilum

2020 ◽  
Vol 86 (16) ◽  
Author(s):  
Paul Barbier ◽  
Tatiana Rochat ◽  
Haitham H. Mohammed ◽  
Gregory D. Wiens ◽  
Jean-François Bernardet ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Gene Deletion ◽  
Cold Water ◽  
Secretion System ◽  
Gliding Motility ◽  
Control Measures ◽  
Fish Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Bacterial Cold Water Disease

ABSTRACT Flavobacterium psychrophilum causes bacterial cold-water disease in wild and aquaculture-reared fish and is a major problem for salmonid aquaculture. The mechanisms responsible for cold-water disease are not known. It was recently demonstrated that the related fish pathogen, Flavobacterium columnare, requires a functional type IX protein secretion system (T9SS) to cause disease. T9SSs secrete cell surface adhesins, gliding motility proteins, peptidases, and other enzymes, any of which may be virulence factors. The F. psychrophilum genome has genes predicted to encode components of a T9SS. Here, we used a SacB-mediated gene deletion technique recently adapted for use in the Bacteroidetes to delete a core F. psychrophilum T9SS gene, gldN. The ΔgldN mutant cells were deficient for secretion of many proteins in comparison to wild-type cells. Complementation of the mutant with wild-type gldN on a plasmid restored secretion. Compared to wild-type and complemented strains, the ΔgldN mutant was deficient in adhesion, gliding motility, and extracellular proteolytic and hemolytic activities. The ΔgldN mutant exhibited reduced virulence in rainbow trout and complementation restored virulence, suggesting that the T9SS plays an important role in the disease. IMPORTANCE Bacterial cold-water disease, caused by F. psychrophilum, is a major problem for salmonid aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. A targeted gene deletion method was adapted to F. psychrophilum and used to demonstrate the importance of the T9SS in virulence. Proteins secreted by this system are likely virulence factors and targets for the development of control measures.

scholarly journals Gene Deletion Strategy To Examine the Involvement of the Two Chondroitin Lyases in Flavobacterium columnare Virulence

2015 ◽  
Vol 81 (21) ◽  
pp. 7394-7402 ◽  
Author(s):  
Nan Li ◽  
Ting Qin ◽  
Xiao Lin Zhang ◽  
Bei Huang ◽  
Zhi Xin Liu ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Bacterial Pathogen ◽  
Infected Fish ◽  
Economic Losses ◽  
Flavobacterium Columnare ◽  
Wild Type ◽  
Single Strain ◽  
Content Type

ABSTRACTFlavobacterium columnareis an important bacterial pathogen of freshwater fish that causes high mortality of infected fish and heavy economic losses in aquaculture. The pathogenesis of this bacterium is poorly understood, in part due to the lack of efficient methods for genetic manipulation. In this study, a gene deletion strategy was developed and used to determine the relationship between the production of chondroitin lyases and virulence. TheF. johnsoniaeompApromoter (PompA) was fused tosacBto construct a counterselectable marker forF. columnare.F. columnarecarrying PompA-sacBfailed to grow on media containing 10% sucrose. A suicide vector carrying PompA-sacBwas constructed, and a gene deletion strategy was developed. Using this approach, the chondroitin lyase-encoding genes,cslAandcslB, were deleted. The ΔcslAand ΔcslBmutants were both partially deficient in digestion of chondroitin sulfate A, whereas a double mutant (ΔcslAΔcslB) was completely deficient in chondroitin lyase activity. Cells ofF. columnarewild-type strain G4and of the chondroitin lyase-deficient ΔcslAΔcslBmutant exhibited similar levels of virulence toward grass carp in single-strain infections. Coinfections, however, revealed a competitive advantage for the wild type over the chondroitin lyase mutant. The results indicate that chondroitin lyases are not essential virulence factors ofF. columnarebut may contribute to the ability of the pathogen to compete and cause disease in natural infections. The gene deletion method developed in this study may be employed to investigate the virulence factors of this bacterium and may have wide application in many other members of the phylumBacteroidetes.

scholarly journals Vibrio fischeriDarR Directs Responses to D-Aspartate and Represents a Group of Similar LysR-Type Transcriptional Regulators

2018 ◽  
Vol 200 (15) ◽  
Author(s):  
Richard M. Jones ◽  
David L. Popham ◽  
Alicia L. Schmidt ◽  
Ellen L. Neidle ◽  
Eric V. Stabb
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Control ◽  
Vibrio Fischeri ◽  
Transcriptional Response ◽  
Control Mechanisms ◽  
Wild Type ◽  
Acinetobacter Baylyi ◽  
Transcriptional Responses ◽  
Content Type

ABSTRACTMounting evidence suggests thatd-amino acids play previously underappreciated roles in diverse organisms. In bacteria, evend-amino acids that are absent from canonical peptidoglycan (PG) may act as growth substrates, as signals, or in other functions. Given these proposed roles and the ubiquity ofd-amino acids, the paucity of knownd-amino-acid-responsive transcriptional control mechanisms in bacteria suggests that such regulation awaits discovery. We found that DarR, a LysR-type transcriptional regulator (LTTR), activates transcription in response tod-Asp. Thed-Glu auxotrophy of aVibrio fischerimurI::Tn mutant was suppressed, with the wild-type PG structure maintained, by a point mutation indarR. ThisdarRmutation resulted in the overexpression of an adjacent operon encoding a putative aspartate racemase, RacD, which compensated for the loss of the glutamate racemase encoded bymurI. Using transcriptional reporters, we found that wild-type DarR activatedracDtranscription in response to exogenousd-Asp but not upon the addition ofl-Asp,l-Glu, ord-Glu. A DNA sequence typical of LTTR-binding sites was identified betweendarRand the divergently orientedracDoperon, and scrambling this sequence eliminated activation of the reporter in response tod-Asp. In several proteobacteria, genes encoding LTTRs similar to DarR are linked to genes with predicted roles ind- and/orl-Asp metabolism. To test the functional similarities in another bacterium,darRandracDmutants were also generated inAcinetobacter baylyi. InV. fischeriandA. baylyi, growth ond-Asp required the presence of bothdarRandracD. Our results suggest that multiple bacteria have the ability to sense and respond tod-Asp.IMPORTANCEd-Amino acids are prevalent in the environment and are generated by organisms from all domains of life. Although some biological roles ford-amino acids are understood, in other cases, their functions remain uncertain. Given the ubiquity ofd-amino acids, it seems likely that bacteria will initiate transcriptional responses to them. Elucidatingd-amino acid-responsive regulators along with the genes they control will help uncover bacterial uses ofd-amino acids. Here, we report the discovery of DarR, a novel LTTR inV. fischerithat mediates a transcriptional response to environmentald-Asp and underpins the catabolism ofd-Asp. DarR represents the founding member of a group of bacterial homologs that we hypothesize control aspects of aspartate metabolism in response tod-Asp and/or tod-Asp-containing peptides.

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