scholarly journals A Combination of Three Mutations, dcd,pyrH, and cdd, Establishes Thymidine (Deoxyuridine) Auxotrophy in thyA+ Strains of Salmonella typhimurium

1998 ◽  
Vol 180 (22) ◽  
pp. 5891-5895 ◽  
Author(s):  
Nevan J. Krogan ◽  
Michelle L. Zaharik ◽  
Jan Neuhard ◽  
Rod A. Kelln
Keyword(s):  
Salmonella Typhimurium ◽  
Genetic Background ◽  
Kinase Activity ◽  
Null Mutation ◽  
Wild Type ◽  
Gene Encoding ◽  
Deoxycytidine Deaminase ◽  
In The Wild ◽  
Ump Kinase ◽  
Thymidine Auxotrophy

ABSTRACT The dum gene of Salmonella typhimurium was originally identified as a gene involved in dUMP synthesis (C. F. Beck et al., J. Bacteriol. 129:305–316, 1977). In the genetic background used in their selection, the joint acquisition of adcd (dCTP deaminase) and a dum mutation established a condition of thymidine (deoxyuridine) auxotrophy. In this study, we show that dum is identical to pyrH, the gene encoding UMP kinase. The level of UMP kinase activity in thedum mutant was found to be only 30% of that observed for the dum + strain. Thymidine prototrophy was restored to the original dum dcd mutant (KP1361) either by transduction using a pyrH + donor or by complementation with either of twopyrH +-carrying plasmids. Thymidine auxotrophy could be reconstructed in the dum + derivative (KP1389) by the introduction of a mutant pyrH allele. To define the minimal mutational complement necessary to produce thymidine auxotrophy in thyA + strains, adcd::Km null mutation was constructed. In the wild-type background, dcd::Km alone or in combination with a pyrH (dum) mutation did not result in a thymidine requirement. A third mutation, cdd(cytidine-deoxycytidine deaminase), was required together with thedcd and pyrH mutations to impart thymidine auxotrophy.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

scholarly journals A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development inDictyostelium

2003 ◽  
Vol 14 (4) ◽  
pp. 1727-1743 ◽  
Author(s):  
Binggang Sun ◽  
Richard A. Firtel
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Kinase Activity ◽  
Site Directed Mutagenesis ◽  
Membrane Localization ◽  
Pleckstrin Homology Domain ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Gene Encoding

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ∼50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ∼40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ∼10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Gα2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.

scholarly journals Characterization and Analysis of Anthocyanin-Related Genes in Wild-Type Blueberry and the Pink-Fruited Mutant Cultivar ‘Pink Lemonade’: New Insights into Anthocyanin Biosynthesis

Agronomy ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1296
Author(s):  
Jose V. Die ◽  
Richard W. Jones ◽  
Elizabeth L. Ogden ◽  
Mark K. Ehlenfeldt ◽  
Lisa J. Rowland
Keyword(s):  
Fruits And Vegetables ◽  
Anthocyanin Biosynthesis ◽  
Mechanistic Explanation ◽  
Comparative Sequence Analysis ◽  
Wild Type ◽  
Flavonoid Pathway ◽  
Transient Expression Assay ◽  
Gene Encoding ◽  
In The Wild ◽  
Anthocyanin Production

Blueberries are one of the richest sources of antioxidants, such as anthocyanins, among fruits and vegetables. Anthocyanin mutants, like the pink-fruited cultivar ‘Pink Lemonade’, are valuable resources for investigating anthocyanin biosynthesis in blueberries. In this study, we examined expression of flavonoid pathway genes during fruit development in wild-type, blue-fruited blueberries using quantitative real-time PCR. Expression was also compared between wild-type and the pink-fruited ‘Pink Lemonade’. This revealed significantly lower expression in ‘Pink Lemonade’ than in wild-type of nearly all the structural genes examined suggesting that a transcriptional regulator of the pathway was affected. Hence, we compared expression of three known regulatory genes and found that the gene encoding the transcription factor MYB1 was expressed at a significantly lower level in ‘Pink Lemonade’ than in the wild-type. To validate the capacity of this MYB1 to regulate the transcription of anthocyanin genes in blueberries, a transient expression assay was conducted. Results indicated MYB1 overexpression enhanced anthocyanin production. Comparative sequence analysis between wild-type and mutant MYB1 variants found differences in highly conserved features suggesting a mechanistic explanation for the mutant phenotype. Collectively, the results presented here contribute to a better understanding of mechanisms regulating anthocyanin biosynthesis in Vaccinium.

scholarly journals Chlorobium tepidum Mutant Lacking Bacteriochlorophyll c Made by Inactivation of the bchK Gene, Encoding Bacteriochlorophyll c Synthase

2002 ◽  
Vol 184 (12) ◽  
pp. 3368-3376 ◽  
Author(s):  
Niels-Ulrik Frigaard ◽  
Ginny D. Voigt ◽  
Donald A. Bryant
Keyword(s):  
Photosynthetic Reaction Center ◽  
Chlorobium Tepidum ◽  
Wild Type ◽  
Bacteriochlorophyll C ◽  
Gene Encoding ◽  
Thin Sections ◽  
Insertional Inactivation ◽  
In The Wild ◽  
Green Sulfur ◽  
Isoprenoid Quinones

ABSTRACT The gene encoding bacteriochlorophyll (BChl) c synthase was identified by insertional inactivation in the photosynthetic green sulfur bacterium Chlorobium tepidum and was named bchK. The bchK mutant of C. tepidum was rusty-orange in color and completely lacked BChl c. Because of the absence of the BChl c antenna, the mutant grew about seven times slower than the wild type at light intensities that were limiting to the wild type (<90 μmol m−2 s−1). Various pheophorbides, which probably represent precursors of BChl c which had lost magnesium, accumulated in the mutant cells. A small fraction of these pheophorbides were apparently esterified by the remaining chlorophyll (Chl) a and BChl a synthases in cells. The amounts of BChl a, Chl a, isoprenoid quinones, carotenoids, Fenna-Matthews-Olson protein, and chlorosome envelope protein CsmA were not significantly altered on a cellular basis in the mutant compared to in the wild type. This suggests that the BChl a antennae, photosynthetic reaction centers, and remaining chlorosome components were essentially unaffected in the mutant. Electron microscopy of thin sections revealed that the mutant lacked normal chlorosomes. However, a fraction containing vestigial chlorosomes, denoted “carotenosomes,” was partly purified by density centrifugation; these structures contained carotenoids, isoprenoid quinones, and a 798-nm-absorbing BChl a species that is probably protein associated. Because of the absence of the strong BChl c absorption found in the wild type, the bchK mutant should prove valuable for future analyses of the photosynthetic reaction center and of the roles of BChl a in photosynthesis in green bacteria. An evolutionary implication of our findings is that the photosynthetic ancestor of green sulfur bacteria could have evolved without chlorosomes and BChl c and instead used only BChl a-containing proteins as the major light-harvesting antennae.

scholarly journals A Null Mutation in the Gene Encoding the Herpes Simplex Virus Type 1 UL37 Polypeptide Abrogates Virus Maturation

2001 ◽  
Vol 75 (21) ◽  
pp. 10259-10271 ◽  
Author(s):  
Prashant Desai ◽  
Gerry L. Sexton ◽  
J. Michael McCaffery ◽  
Stanley Person
Keyword(s):  
Herpes Simplex ◽  
Gene Product ◽  
Vero Cells ◽  
Mutant Virus ◽  
Null Mutation ◽  
Wild Type ◽  
Gene Encoding ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT The tegument is an integral and essential structural component of the herpes simplex virus type 1 (HSV-1) virion. The UL37 open reading frame of HSV-1 encodes a 120-kDa virion polypeptide which is a resident of the tegument. To analyze the function of the UL37-encoded polypeptide a null mutation was generated in the gene encoding this protein. In order to propagate this mutant virus, transformed cell lines that express the UL37 gene product in trans were produced. The null mutation was transferred into the virus genome using these complementing cell lines. A mutant virus designated KΔUL37 was isolated based on its ability to form plaques on the complementing cell line but not on nonpermissive (noncomplementing) Vero cells. This virus was unable to grow in Vero cells; therefore, UL37 encodes an essential function of the virus. The mutant virus KΔUL37 produced capsids containing DNA as judged by sedimentation analysis of extracts derived from infected Vero cells. Therefore, the UL37 gene product is not required for DNA cleavage or packaging. The UL37 mutant capsids were tagged with the smallest capsid protein, VP26, fused to green fluorescent protein. This fusion protein decorates the capsid shell and consequently the location of the capsid and the virus particle can be visualized in living cells. Late in infection, KΔUL37 capsids were observed to accumulate at the periphery of the nucleus as judged by the concentration of fluorescence around this organelle. Fluorescence was also observed in the cytoplasm in large puncta. Fluorescence at the plasma membrane, which indicated maturation and egress of virions, was observed in wild-type-infected cells but was absent in KΔUL37-infected cells. Ultrastructural analysis of thin sections of infected cells revealed clusters of DNA-containing capsids in the proximity of the inner nuclear membrane. Occasionally enveloped capsids were observed between the inner and outer nuclear membranes. Clusters of unenveloped capsids were also observed in the cytoplasm of KΔUL37-infected cells. Enveloped virions, which were observed in the cytoplasm of wild-type-infected cells, were never detected in the cytoplasm of KΔUL37-infected cells. Crude cell fractionation of infected cells using detergent lysis demonstrated that two-thirds of the UL37 mutant particles were associated with the nuclear fraction, unlike wild-type particles, which were predominantly in the cytoplasmic fraction. These data suggest that in the absence of UL37, the exit of capsids from the nucleus is slowed. UL37 mutant particles can participate in the initial envelopment at the nuclear membrane, although this process may be impaired in the absence of UL37. Furthermore, the naked capsids deposited in the cytoplasm are unable to progress further in the morphogenesis pathway, which suggests that UL37 is also required for egress and reenvelopment. Therefore, the UL37 gene product plays a key role in the early stages of the maturation pathway that give rise to an infectious virion.

scholarly journals Overproduction of l-Lysine from Methanol by Methylobacillus glycogenes Derivatives Carrying a Plasmid with a Mutated dapA Gene

2001 ◽  
Vol 67 (7) ◽  
pp. 3064-3070 ◽  
Author(s):  
Hiroaki Motoyama ◽  
Hiroshi Yano ◽  
Yoko Terasaki ◽  
Hideharu Anazawa
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Test Tube ◽  
Amino Acid Sequences ◽  
Nutritional Requirement ◽  
Wild Type ◽  
Gene Encoding ◽  
Lysine Production ◽  
Obligate Methylotroph ◽  
In The Wild

ABSTRACT The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by l-lysine, was cloned from an l-threonine- andl-lysine-coproducing mutant of the obligate methylotrophMethylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapAmutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an l-threonine producing mutant, elevated the specific activity of DDPS 20-fold andl-lysine production 2- to 3-fold with concomitant reduction of l-threonine in test tube cultures. AL119 containing thedapA gene produced 8 g of l-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M r of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition byl-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector,l-lysine.

scholarly journals Pleiotropic Effects of Inactivating a Carboxyl-Terminal Protease, CtpA, in Borrelia burgdorferi

2004 ◽  
Vol 186 (7) ◽  
pp. 2074-2084 ◽  
Author(s):  
Yngve Östberg ◽  
James A. Carroll ◽  
Marija Pinne ◽  
Jonathan G. Krum ◽  
Patricia Rosa ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Higher Plants ◽  
Bacterial Species ◽  
Immunoblot Analysis ◽  
Pleiotropic Effect ◽  
Wild Type ◽  
Gene Encoding ◽  
Two Dimensional Gel Electrophoresis ◽  
In The Wild ◽  
Carboxyl Terminal

ABSTRACT A gene encoding a putative carboxyl-terminal protease (CtpA), an unusual type of protease, is present in the Borrelia burgdorferi B31 genome. The B. burgdorferi CtpA amino acid sequence exhibits similarities to the sequences of the CtpA enzymes of the cyanobacterium Synechocystis sp. strain PCC 6803 and higher plants and also exhibits similarities to the sequences of putative CtpA proteins in other bacterial species. Here, we studied the effect of ctpA gene inactivation on the B. burgdorferi protein expression profile. Total B. burgdorferi proteins were separated by two-dimensional gel electrophoresis, and the results revealed that six proteins of the wild type were not detected in the ctpA mutant and that nine proteins observed in the ctpA mutant were undetectable in the wild type. Immunoblot analysis showed that the integral outer membrane protein P13 was larger and had a more acidic pI in the ctpA mutant, which is consistent with the theoretical change in pI for P13 not processed at the carboxyl terminus. Matrix-assisted laser desorption ionization—time of flight data indicated that in addition to P13, the BB0323 protein may serve as a substrate for carboxyl-terminal processing by CtpA. Complementation analysis of the ctpA mutant provided strong evidence that the observed effect on proteins depended on inactivation of the ctpA gene alone. We show that CtpA in B. burgdorferi is involved in the processing of proteins such as P13 and BB0323 and that inactivation of ctpA has a pleiotropic effect on borrelial protein synthesis. To our knowledge, this is the first analysis of both a CtpA protease and different substrate proteins in a pathogenic bacterium.

scholarly journals The recovery of tryptophan A auxotrophs at high frequency in a strain of Salmonella typhimurium

1967 ◽  
Vol 10 (2) ◽  
pp. 127-134 ◽  
Author(s):  
S. Riyasaty ◽  
G. W. P. Dawson
Keyword(s):  
Salmonella Typhimurium ◽  
Anthranilic Acid ◽  
High Frequency ◽  
Minimal Medium ◽  
Genetic Change ◽  
Wild Type ◽  
In The Wild ◽  
Slow Growing

1. Auxotrophs were sought in a slow-growing reversion of the tryA47 strain of S. typhimurium. This reversion differs from tryA47 by a genetic change that is inseparable from the 47 site and has been designated 47S. Out of thirty-nine auxotrophs that derived from independent mutations, twenty-two grew on minimal medium supplemented with anthranilic acid. Eight of these auxotrophs were examined and each was shown to have the 47S site unchanged and to carry a further mutation in the tryA gene. These further mutations were shown to be at different sites in different auxotrophs.2. Auxotrophs were sought in the wild-type LT-2 strain. None out of thirty-six were mutant in the tryA gene.3. The 47S site in tryA47S was replaced by 47+ transduced from wild-type LT-2. Auxotrophs were sought in this strain and only one out of nineteen was mutant in the tryA gene.4. Auxotrophs were sought in a wild-type reversion of tryA47. Out of twenty-six none were mutant in the tryA gene.5. The 47+ site in wild-type LT-2 was replaced by 47S transduced from tryA47S. Auxotrophs were sought in this strain and twenty-two out of fifty-five were mutant in the tryA gene.6. We conclude that tryA auxotrophs are only recovered at a high frequency when the 47S site is present in the tryA gene.7. In strains with the 47S site in the tryA gene the frequency of auxotrophs that will grow on minimal medium supplemented with indole but not on minimal medium supplemented with anthranilic acid is appreciably higher than in those strains without the 47S site in the tryA gene. These auxotrophs are mutant in genes that are in the same operon as tryA.

scholarly journals Galactose Metabolism by Streptococcus mutans

2004 ◽  
Vol 70 (10) ◽  
pp. 6047-6052 ◽  
Author(s):  
Jacqueline Abranches ◽  
Yi-Ywan M. Chen ◽  
Robert A. Burne
Keyword(s):  
Streptococcus Mutans ◽  
Wild Type Strain ◽  
Sugar Metabolism ◽  
Growth Defect ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Galactose Metabolism ◽  
Gene Encoding ◽  
Leloir Pathway ◽  
In The Wild

ABSTRACT The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.

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