scholarly journals High Frequency of Virulence Factor Genestdh,trh, andtlhin Vibrio parahaemolyticus Strains Isolated from a Pristine Estuary

2013 ◽  
Vol 79 (7) ◽  
pp. 2247-2252 ◽  
Author(s):  
Casandra K. Gutierrez West ◽  
Savannah L. Klein ◽  
Charles R. Lovell
Keyword(s):  
Virulence Factor ◽  
Dna Sequences ◽  
Vibrio Parahaemolyticus ◽  
Pcr Primers ◽  
North Inlet ◽  
Estuarine Ecosystem ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding ◽  
Thermostable Direct Hemolysin

ABSTRACTVirulence factor genes encoding the thermostable direct hemolysin (tdh) and the thermostable direct hemolysin-related hemolysin (trh) are strongly correlated with virulence of the emergent human pathogenVibrio parahaemolyticus. The gene encoding the thermolabile hemolysin (tlh) is also considered a signature molecular marker for the species. These genes are typically reported in very low percentages (1 to 2%) of nonclinical strains.V. parahaemolyticusstrains were isolated from various niches within a pristine estuary (North Inlet, SC) and were screened for these genes using both newly designed PCR primers and more commonly used primers. DNA sequences oftdhandtrhwere recovered from 48% and 8.3%, respectively, of these North Inlet strains. The recovery of pathogenicV. parahaemolyticusstrains in such high proportions from an estuarine ecosystem that is virtually free of anthropogenic influences indicates the potential for additional, perhaps environmental roles of thetdhandtrhgenes.

scholarly journals Searching for Genes of Lactococcus lactis subsp. lactis Encoding the Bacteriocin Nisin using DNA/DNA Hybridisation

2009 ◽  
Vol 27 (Special Issue 1) ◽  
pp. S366-S368
Author(s):  
M. Dušková ◽  
A. Španová ◽  
V. Dráb ◽  
B. Rittich
Keyword(s):  
Lactococcus Lactis ◽  
Dna Sequences ◽  
Pcr Primers ◽  
Culture Collection ◽  
Dot Blot ◽  
Gene Encoding ◽  
Pcr Product ◽  
Dna Hybridisation ◽  
Genes Encoding ◽  
Blot Hybridisation

In this work, PCR primers P8 and P9 were used for amplification of a 320 bp long PCR product specific to the nisin gene. The PCR product was labelled with digoxigenine during amplification and used as a DNA probe for the screening of homologous DNA sequences in 7 <I>Lactococcus lactis</I> subsp.<I> lactis</I> strains from the Culture Collection of Dairy Microorganisms (CCDM). Dot blot hybridisation and hybridisation of colonies were used for DNA/DNA hybridisation. It was shown that 6 tested strains of <I>Lactococcus lactis </I>subsp. <I>lactis </I>have genes encoding nisin. One strain had probably a defective gene encoding nisin.

scholarly journals Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

2013 ◽  
Vol 79 (18) ◽  
pp. 5566-5575 ◽  
Author(s):  
Jens Buchholz ◽  
Andreas Schwentner ◽  
Britta Brunnenkan ◽  
Christina Gabris ◽  
Simon Grimm ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Fed Batch ◽  
Complex Activity ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding ◽  
Cell Densities ◽  
Dehydrogenase Complex

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

scholarly journals ISOLASI FRAGMEN GEN PENYANDI PUTRESIN N-METILTRANSFERASE DAN QUINOLINAT FOSFORIBOSILTRANSFERASE ASAL TEMBAKAU LOKAL TEMANGGUNG (Nicotiana tabacum)

2020 ◽  
Vol 17 (3) ◽  
pp. 109 ◽  
Author(s):  
SESANTI BASUKI ◽  
NURHAJATI AA MATTJIK ◽  
SUWARSO SUWARSO ◽  
DESTA WIRNAS ◽  
SUDARSONO SUDARSONO
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Data Bank ◽  
Degenerate Primer ◽  
Gene Encoding ◽  
Methyl Transferase ◽  
Dna Database ◽  
Genes Encoding ◽  
Tobacco Cultivar ◽  
Dna Template

<p>ABSTRAK</p><p>Upaya untuk menurunkan kandungan nikotin merupakan salah satuprioritas utama penelitian tembakau. Nikotin adalah senyawa alkaloidutama berpotensi dikonversi menjadi senyawa nor-nikotin yang bersifatkarsinogen. Gen PMT sebagai penyandi enzim putresin n-metiltransferase(PMT) dan gen QPT - penyandi enzim quinolinat fosforibosiltransferase(QPT) merupakan dua gen kunci yang berperan penting pada proses bio-sintesis nikotin. Penelitian ini bertujuan untuk mengisolasi potongan genPMT dan QPT asal tembakau lokal Indonesia, mengkarakterisasi danmenganalisis runutan DNA-nya. Tahapan penelitian dimulai dengan me-rancang primer degenerate berdasarkan informasi yang ada di pangkalandata Bank Gen NCBI (National Centre for Biotechnology Information),mengamplifikasi PCR menggunakan templat DNA genomik tembakaulokal cv. Sindoro1, mengklon potongan DNA hasil PCR dan menentukanrunutan DNA-nya. Hasil penelitian menunjukkan dari dua belas pasangprimer degenerate yang dirancang, hanya dua pasang primer yang meng-hasilkan potongan DNA hasil amplifikasi PCR, yaitu pasangan primerPMt-7 (F &amp; R) untuk gen PMT dan primer QPt-3 (F &amp; R) untuk gen QPT.Setelah dilakukan penentuan runutan DNA-nya, amplikon yang didapatdari hasil PCR dengan pasangan primer PMt-7 sebesar 1418 bp, sedangkanuntuk primer QPt-3 sebesar 205 bp. Runutan DNA gen PMT dan gen QPTasal tembakau lokal cv. Sindoro1 mempunyai tingkat kesamaan yang ting-gi dengan gen PMT dan gen QPT asal tembakau lainnya yang ada dipangkalan data Bank Gen NCBI.</p><p>Kata kunci : Gen PMT, gen QPT, lintasan biosintesis nikotin, perunutanDNA, amplifikasi PCR, primer degenerate</p><p>ABSTRACT</p><p>Isolation of Genes encoding Putrescine N-Methyl-transferase and Quinolinat Phosphoribosyl transferasederived from Temanggung Tobacco Cultivar (Nicotianatabacum)</p><p>Reduction of nicotine content is one of the major objective intobacco research. Nicotine is the main alcaloid compound that potentiallycould be converted into a carcinogenic compound (nor-nicotine). The PMTgene encoding putrescine N-methyl transferase (PMT) and the QPT gene -encoding quinolinate phosphoribosyl transferase (QPT) are the two keyenzymes involved in nicotine biosynthesis. The objectives of this researchwere to isolate PMT and QPT gene fragments originated from Indonesianlocal tobacco, to characterize, and to analyze their DNA sequences. Theresearch activities included: degenerate primer design based oninformation available in the GenBank DNA Database NCBI (NationalCentre for Biotechnology Information), PCR amplification usingdegenerate primer and genomic DNA template of a local tobacco cv.Sindoro1, clone the PCR amplified products, and determine their DNAnucleotide sequences. Results of the experiment indicated that from 12degenerate primer pairs synthesized, only two were able to yield positivePCR amplified products. These primer pairs were PMt-7 (F &amp; R primers)for PMT and QPt-3 (F &amp; R primers) for QPT. After DNA sequencing, theamplified DNA product amplified using PMt-7 degenerate primer pairswere 1418 bp, while that using QPt-3 primer pairs were only 205 bp.Nucleotide sequences of PMT or QPT gene fragments originated fromlocal tobacco cv. Sindoro1 showed a high nucleotide sequences identity ascompared to that of the respective genes from other tobacco species thatwere available in the GenBank DNA Database NCBI.</p><p>Key words: PMT gene, QPT gene, nicotine biosynthetic pathways, DNAsequencing, PCR amplification, degenerate primer</p>

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

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.

scholarly journals Coordinated Zinc Homeostasis Is Essential for the Wild-Type Virulence of Brucella abortus

2015 ◽  
Vol 197 (9) ◽  
pp. 1582-1591 ◽  
Author(s):  
Lauren M. Sheehan ◽  
James A. Budnick ◽  
R. Martin Roop ◽  
Clayton C. Caswell
Keyword(s):  
Mouse Model ◽  
Brucella Abortus ◽  
Zinc Uptake ◽  
Zinc Homeostasis ◽  
Zinc Toxicity ◽  
Uptake System ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding

ABSTRACTMetal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence ofBrucella abortus; however, the capacity ofBrucellastrains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region ofznuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion ofzurorzntRalone did not result in increased zinc toxicity in the corresponding mutants; however, deletion ofzntAled to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion ofzntRresulted in significant attenuation ofB. abortusin a mouse model of chronic infection, and subsequent experiments revealed that overexpression ofzntAin thezntRmutant is the molecular basis for its decreased virulence.IMPORTANCEThe importance of zinc uptake forBrucellapathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export inBrucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation ofBrucellavirulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria.

scholarly journals Genetic and Functional Investigation of Zn 2 Cys 6 Transcription Factors RSE2 and RSE3 in Podospora anserina

2013 ◽  
Vol 13 (1) ◽  
pp. 53-65 ◽  
Author(s):  
Elodie Bovier ◽  
Carole H. Sellem ◽  
Adeline Humbert ◽  
Annie Sainsard-Chanet
Keyword(s):  
Transcription Factors ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Constitutive Expression ◽  
Podospora Anserina ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Content Type ◽  
Zinc Cluster ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT In Podospora anserina , the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase ( aox ) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase ( pck ) and fructose-1,6-biphosphatase ( fbp ). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3 . Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3 . It showed that in addition to aox , fbp , and pck , RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn 2 Cys 6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.

scholarly journals Genetic Diversity of Clinical and Environmental Vibrio parahaemolyticus Strains from the Pacific Northwest

2012 ◽  
Vol 78 (24) ◽  
pp. 8631-8638 ◽  
Author(s):  
Rohinee Paranjpye ◽  
Owen S. Hamel ◽  
Asta Stojanovski ◽  
Martin Liermann
Keyword(s):  
Pacific Northwest ◽  
Vibrio Parahaemolyticus ◽  
The United States ◽  
Assessment Tools ◽  
Clinical Isolates ◽  
Environmental Isolates ◽  
Content Type ◽  
The Pacific ◽  
Geographical Regions ◽  
Thermostable Direct Hemolysin

ABSTRACTSince 1997, cases ofVibrio parahaemolyticus-related gastroenteritis from the consumption of raw oysters harvested in Washington State have been higher than historical levels. These cases have shown little or no correlation with concentrations of potentially pathogenicV. parahaemolyticus(positive for the thermostable direct hemolysin gene,tdh) in oysters, although significant concentrations oftdh+V. parahaemolyticusstrains were isolated from shellfish-growing areas in the Pacific Northwest (PNW). We compared clinical and environmental strains isolated from the PNW to those from other geographic regions within the United States and Asia for the presence of virulence-associated genes, including the thermostable direct hemolysin (tdh), the thermostable-related hemolysin (trh), urease (ureR), the pandemic group specific markersorf8andtoxRS, and genes encoding both type 3 secretion systems (T3SS1 and T3SS2). The majority of clinical strains from the PNW were positive fortdh,trh, andureRgenes, while a significant proportion of environmental isolates weretdh+buttrhnegative. Hierarchical clustering grouped the majority of these clinical isolates into a cluster distinct from that including the pandemic strain RIMD2210633, clinical isolates from other geographical regions, andtdh+,trh-negative environmental isolates from the PNW. We detected T3SS2-related genes (T3SS2β) in environmental strains that weretdhandtrhnegative. The presence of significant concentrations oftdh+,trh-negative environmental strains in the PNW that have not been responsible for illness and T3SS2β intdh- andtrh-negative strains emphasizes the diversity in this species and the need to identify additional virulence markers for this bacterium to improve risk assessment tools for the detection of this pathogen.

scholarly journals Chitin Heterodisaccharide, Released from Chitin by Chitinase and Chitin Oligosaccharide Deacetylase, Enhances the Chitin-Metabolizing Ability of Vibrio parahaemolyticus

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Takako Hirano ◽  
Manabu Okubo ◽  
Hironobu Tsuda ◽  
Masahiro Yokoyama ◽  
Wataru Hakamata ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Pcr Analysis ◽  
Content Type ◽  
Chitin Degradation ◽  
Expression Of Genes ◽  
Peptide Mass ◽  
Degrading Bacteria ◽  
Genes Encoding ◽  
Chitinase Production

ABSTRACT Vibrio parahaemolyticus RIMD2210633 secretes both chitinase and chitin oligosaccharide deacetylase and produces β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) from chitin. Previously, we reported that GlcNAc-GlcN induces chitinase production by several strains of Vibrio harboring chitin oligosaccharide deacetylase genes (T. Hirano, K. Kadokura, T. Ikegami, Y. Shigeta, et al., Glycobiology 19:1046–1053, 2009). The metabolism of chitin by Vibrio was speculated on the basis of the findings of previous studies, and the role of chitin oligosaccharide produced from chitin has been well studied. However, the role of GlcNAc-GlcN in the Vibrio chitin degradation system, with the exception of the above-mentioned function as an inducer of chitinase production, remains unclear. N,N′-Diacetylchitobiose, a homodisaccharide produced from chitin, is known to induce the expression of genes encoding several proteins involved in chitin metabolism in Vibrio strains (K. L. Meibom, X. B. Li, A. Nielsen, C. Wu, et al., Proc Natl Acad Sci U S A 101:2524–2529, 2004). We therefore hypothesized that GlcNAc-GlcN also affects the expression of enzymes involved in chitin metabolism in the same manner. In this study, we examined the induction of protein expression by several sugars released from chitin using peptide mass fingerprinting and confirmed the expression of genes encoding enzymes involved in chitin metabolism using real-time quantitative PCR analysis. We then confirmed that GlcNAc-GlcN induces the expression of genes encoding many soluble enzymes involved in chitin degradation in Vibrio parahaemolyticus. Here, we demonstrate that GlcNAc-GlcN enhances the chitin-metabolizing ability of V. parahaemolyticus. IMPORTANCE We demonstrate that β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) enhances the chitin-metabolizing ability of V. parahaemolyticus. Members of the genus Vibrio are chitin-degrading bacteria, and some species of this genus are associated with diseases affecting fish and animals, including humans (F. L. Thompson, T. Iida, and J. Swings, Microbiol Mol Biol Rev 68:403–431, 2004; M. Y. Ina-Salwany, N. Al-Saari, A. Mohamad, F.-A. Mursidi, et al., J Aquat Anim Health 31:3–22, 2019). Studies on Vibrio are considered important, as they may facilitate the development of solutions related to health, food, and aquaculture problems attributed to this genus. This report enhances the current understanding of chitin degradation by Vibrio bacteria.

scholarly journals Salivaricin D, a Novel Intrinsically Trypsin-Resistant Lantibiotic from Streptococcus salivarius 5M6c Isolated from a Healthy Infant

2011 ◽  
Vol 78 (2) ◽  
pp. 402-410 ◽  
Author(s):  
Dagim Jirata Birri ◽  
Dag Anders Brede ◽  
Ingolf F. Nes
Keyword(s):  
Abc Transporters ◽  
Response Regulator ◽  
Probiotic Bacterium ◽  
Streptococcus Salivarius ◽  
Human Pathogens ◽  
Gene Encoding ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Genes Encoding ◽  
Immunity Function

ABSTRACTIn this work, we purified and characterized a newly identified lantibiotic (salivaricin D) fromStreptococcus salivarius5M6c. Salivaricin D is a 34-amino-acid-residue peptide (3,467.55 Da); the locus of the gene encoding this peptide is a 16.5-kb DNA segment which contains genes encoding the precursor of two lantibiotics, two modification enzymes (dehydratase and cyclase), an ABC transporter, a serine-like protease, immunity proteins (lipoprotein and ABC transporters), a response regulator, and a sensor histidine kinase. The immunity gene (salI) was heterologously expressed in a sensitive indicator and provided significant protection against salivaricin D, confirming its immunity function. Salivaricin D is a naturally trypsin-resistant lantibiotic that is similar to nisin-like lantibiotics. It is a relatively broad-spectrum bacteriocin that inhibits members of many genera of Gram-positive bacteria, including the important human pathogensStreptococcus pyogenesandStreptococcus pneumoniae. Thus,Streptococcus salivarius5M6c may be a potential biological agent for the control of oronasopharynx-colonizing streptococcal pathogens or may be used as a probiotic bacterium.

scholarly journals Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

2014 ◽  
Vol 81 (1) ◽  
pp. 309-319 ◽  
Author(s):  
Kristina M. Mahan ◽  
Joseph T. Penrod ◽  
Kou-San Ju ◽  
Natascia Al Kass ◽  
Watumesa A. Tan ◽  
...  
Keyword(s):  
Active Site ◽  
Sole Source ◽  
Degradation Pathway ◽  
Short Term ◽  
Gene Encoding ◽  
Α Subunit ◽  
Content Type ◽  
Term Selection ◽  
Genes Encoding ◽  
Related Enzyme

ABSTRACTAcidovoraxsp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.

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