scholarly journals Precise Excision of IS5 from the Intergenic Region between the fucPIK and the fucAO Operons and Mutational Control of fucPIK Operon Expression in Escherichia coli

2010 ◽  
Vol 192 (7) ◽  
pp. 2013-2019 ◽  
Author(s):  
Zhongge Zhang ◽  
Ming Ren Yen ◽  
Milton H. Saier
Keyword(s):  
Escherichia Coli ◽  
Insertion Site ◽  
Point Mutations ◽  
Wild Type ◽  
Sequence Element ◽  
Low Frequencies ◽  
Precise Excision ◽  
Wild Type Phenotype ◽  
Unique System ◽  
Operon Expression

ABSTRACT Excision of transposable genetic elements from host DNA occurs at low frequencies and is usually imprecise. A common insertion sequence element in Escherichia coli, IS5, has been shown to provide various benefits to its host by inserting into specific sites. Precise excision of this element had not previously been demonstrated. Using a unique system, the fucose (fuc) regulon, in which IS5 insertion and excision result in two distinct selectable phenotypes, we have demonstrated that IS5 can precisely excise from its insertion site, restoring the wild-type phenotype. In addition to precise excision, several “suppressor” insertion, deletion, and point mutations restore the wild-type Fuc+ phenotype to various degrees without IS5 excision. The possible bases for these observations are discussed.

scholarly journals Excision of IS492 Requires Flanking Target Sequences and Results in Circle Formation in Pseudoalteromonas atlantica

1999 ◽  
Vol 181 (16) ◽  
pp. 4937-4948 ◽  
Author(s):  
Donna Perkins-Balding ◽  
Guy Duval-Valentin ◽  
Anna C. Glasgow
Keyword(s):  
Insertion Site ◽  
Extracellular Polysaccharide ◽  
Inverse Pcr ◽  
Target Sequence ◽  
Sequence Element ◽  
E Coli ◽  
Precise Excision ◽  
Circular Form ◽  
Pseudoalteromonas Atlantica ◽  
Circle Formation

ABSTRACT The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced intoEscherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 inE. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.

scholarly journals Characterization, Expression, and Mutation of the Lactococcus lactis galPMKTE Genes, Involved in Galactose Utilization via the Leloir Pathway

2003 ◽  
Vol 185 (3) ◽  
pp. 870-878 ◽  
Author(s):  
Benoît P. Grossiord ◽  
Evert J. Luesink ◽  
Elaine E. Vaughan ◽  
Alain Arnaud ◽  
Willem M. de Vos
Keyword(s):  
Escherichia Coli ◽  
Lactococcus Lactis ◽  
Cell Separation ◽  
Sole Carbon Source ◽  
Wild Type ◽  
Leloir Pathway ◽  
Wild Type Phenotype ◽  
Mutant Strains ◽  
Galactose Utilization ◽  
Long Chain

ABSTRACT A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose 1-epimerase (GalM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.

scholarly journals Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005 ◽  
Vol 187 (9) ◽  
pp. 2974-2982 ◽  
Author(s):  
Laura I. Álvarez-Añorve ◽  
Mario L. Calcagno ◽  
Jacqueline Plumbridge
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Sugar Transport ◽  
Point Mutations ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

scholarly journals Effect of point mutations in the lac promoter on transient and severe catabolite repression of the lac operon of Escherichia coli

1971 ◽  
Vol 123 (4) ◽  
pp. 579-584 ◽  
Author(s):  
M. D. Yudkin
Keyword(s):  
Escherichia Coli ◽  
Catabolite Repression ◽  
Point Mutations ◽  
Severe Form ◽  
Lac Operon ◽  
Wild Type ◽  
Diploid Strain ◽  
Transient Repression ◽  
Lac Promoter

1. Experiments were devised to show whether the point mutations L8 and L29 in the lac promoter alleviate transient repression. 2. Several recombinants were picked from matings between a single F−p+strain and Hfr strains carrying mutations L8 and L29. All of the 19 p−recombinants tested proved to suffer no transient repression, whereas all of the eight p+recombinants tested suffered prolonged transient repression. 3. A diploid strain was constructed in which more than 90% of the thiogalactoside transacetylase is synthesized from the episome with a wild-type lac promoter, whereas 100% of the β-galactosidase is synthesized from the chromosome with a promoter carrying mutation L8. In this diploid the synthesis of thiogalactoside transacetylase suffered transient repression but the synthesis of β-galactosidase did not. 4. Exactly similar results were obtained with a diploid strain in which the chromosomal promoter carried mutation L29. 5. The same diploid strains were used in experiments to show whether mutations L8 and L29 alleviate the severe catabolite repression caused by growth in glucose plus gluconate. In both strains glucose+gluconate repressed the synthesis of β-galactosidase much less than the synthesis of thiogalactoside transacetylase. 6. These and previously reported results can be explained by assuming (a) that both mutations L8 and L29 render the lac promoter partially, but not completely, insensitive to catabolite repression, and (b) that transient repression is an exceptionally severe form of catabolite repression.

scholarly journals Chlamydomonas reinhardtii CNX1E Reconstitutes Molybdenum Cofactor Biosynthesis in Escherichia coli Mutants

2007 ◽  
Vol 6 (6) ◽  
pp. 1063-1067 ◽  
Author(s):  
Ángel Llamas ◽  
Manuel Tejada-Jimenez ◽  
David González-Ballester ◽  
José Javier Higuera ◽  
Guenter Schwarz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chlamydomonas Reinhardtii ◽  
Double Mutant ◽  
Molybdenum Cofactor ◽  
Wild Type ◽  
Eukaryotic Protein ◽  
E Coli ◽  
Wild Type Phenotype ◽  
Cofactor Biosynthesis ◽  
Molybdenum Cofactor Biosynthesis

ABSTRACT We have isolated and characterized the Chlamydomonas reinhardtii genes for molybdenum cofactor biosynthesis, namely, CNX1G and CNX1E, and expressed them and their chimeric fusions in Chlamydomonas and Escherichia coli. In all cases, the wild-type phenotype was restored in individual mutants as well as in a CNX1G CNX1E double mutant. Therefore, CrCNX1E is the first eukaryotic protein able to complement an E. coli moeA mutant.

Precise excision of bacteriophage Mu DNA

2001 ◽  
Vol 47 (8) ◽  
pp. 722-726 ◽  
Author(s):  
Chiraz Abbes ◽  
Guennadi Sezonov ◽  
Danièle Joseleau-Petit ◽  
Richard D'Ari ◽  
Jean-Claude Liébart
Keyword(s):  
Escherichia Coli ◽  
Transposable Element ◽  
Bacteriophage Mu ◽  
Temperate Bacteriophage ◽  
Wild Type ◽  
Prophage Induction ◽  
Bacterial Dna ◽  
Precise Excision ◽  
Type Sequence

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.Key words: transposable element, Escherichia coli, mini-Muduction.

scholarly journals Phenotypic Stability of Staphylococcus aureus Small Colony Variants (SCV) Isolates from Cystic Fibrosis (CF) Patients

2019 ◽  
Vol 16 (11) ◽  
pp. 1940 ◽  
Author(s):  
Clemens Kittinger ◽  
Daniela Toplitsch ◽  
Bettina Folli ◽  
Lilian Masoud Landgraf ◽  
Gernot Zarfel
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Point Mutations ◽  
High Variability ◽  
Wild Type ◽  
Phenotypic Stability ◽  
Small Colony Variant ◽  
Gene Coding ◽  
Wild Type Phenotype ◽  
Small Colony

One of the most interesting features of Staphylococcus aureus is its ability to switch to a small colony variant (SCV). This switch allows the pathogen to survive periods of antibiotic treatment or pressure from the immune system of the host and further enables it to start the infection once again after the environmental stress declines. However, so far only little is known about this reversion back to the more virulent wild type phenotype. Therefore, this study aimed to analyze the frequency of reversion to the wild type phenotype of thymidine auxotroph S. aureus SCV isolates (TD-SCVs) obtained from patients with cystic fibrosis (CF). With the use of single cell starting cultures, the occurrence of the thymidine prototroph revertants was monitored. The underlying mutational cause of the SCVs and subsequent revertants were analyzed by sequencing the gene coding for thymidylate synthase (ThyA), whose mutations are known to produce thymidine auxotroph S. aureus SCV. In our study, the underlying mutational cause for the switch to the TD-SCV phenotype was primarily point mutations. Out of twelve isolates, seven isolates showed an occurrence of revertants with a frequency ranging from 90.06% to 0.16%. This high variability in the frequency of reversion to the wild type was not expected. However, this variability in the frequency of reversion may also be the key to successful re-infection of the host. Sometimes quick reversion to the wild type proves necessary for survival, whereas other times, staying hidden for a bit longer leads to success in re-colonization of the host.

scholarly journals The Molybdate-Responsive Escherichia coli ModE Transcriptional Regulator Coordinates Periplasmic Nitrate Reductase (napFDAGHBC) Operon Expression with Nitrate and Molybdate Availability

2002 ◽  
Vol 184 (12) ◽  
pp. 3253-3259 ◽  
Author(s):  
Paul M. McNicholas ◽  
Robert P. Gunsalus
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Transcription Factor ◽  
Point Mutations ◽  
Dnase I Footprinting ◽  
Mode Recognition ◽  
Operon Expression ◽  
Essential Enzyme

ABSTRACT Expression of the Escherichia coli napFDAGHBC operon (also known as aeg46.5), which encodes the periplasmic molybdoenzyme for nitrate reduction, is increased in response to anaerobiosis and further stimulated by the addition of nitrate or to a lesser extent by nitrite to the cell culture medium. These changes are mediated by the transcription factors Fnr and NarP, respectively. Utilizing a napF-lacZ operon fusion, we demonstrate that napF gene expression is impaired in strain defective for the molybdate-responsive ModE transcription factor. This control abrogates nitrate- or nitrite-dependent induction during anaerobiosis. Gel shift and DNase I footprinting analyses establish that ModE binds to the napF promoter with an apparent Kd of about 35 nM at a position centered at −133.5 relative to the start of napF transcription. Although the ModE binding site sequence is similar to other E. coli ModE binding sites, the location is atypical, because it is not centered near the start of transcription. Introduction of point mutations in the ModE recognition site severely reduced or abolished ModE binding in vitro and conferred a modE phenotype (i.e., loss of molybdate-responsive gene expression) in vivo. In contrast, deletion of the upstream ModE region site rendered napF expression independent of modE. These findings indicate the involvement of an additional transcription factor to help coordinate nitrate- and molybdate-dependent napF expression by the Fnr, NarP, NarL, and ModE proteins. The upstream ModE regulatory site functions to override nitrate control of napF gene expression when the essential enzyme component, molybdate, is limiting in the cell environment.

scholarly journals NEGATIVE DOMINANT MUTATIONS OF THE uidR GENE IN ESCHERICHIA COLI: GENETIC PROOF FOR A COOPERATIVE REGULATION OF uidA EXPRESSION

Genetics ◽  
1986 ◽  
Vol 112 (2) ◽  
pp. 173-182
Author(s):  
Carlos Blanco ◽  
Paul Ritzenthaler ◽  
Mireille Mata-Gilsinger
Keyword(s):  
Escherichia Coli ◽  
Gene Dosage ◽  
Regulatory Gene ◽  
Active Form ◽  
Point Mutations ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Cooperative Process ◽  
Mutant Phenotypes ◽  
K 12

ABSTRACT The uidA gene is the first gene involved in the hexuronide-hexuronate pathway in Escherichia coli K-12 and is under the dual control of the uidR and uxuR encoded repressors. Point mutations affecting the uidR regulatory gene were sought to investigate the regulation of uidA. When the uidR mutant allele was on a multicopy plasmid and the wild-type allele was on the chromosome, some of the mutant phenotypes were dominant to the wild-type phenotype, indicating that the active form of the UidR repressor is multimeric. We have demonstrated that expression of the mutant phenotype is dependent on gene dosage. The dominance of the uidR allele was also sensitive to the presence of the wild-type uxuR allele in the cell. This behavior probably results from UidR-UxuR repressor interactions. A mechanism is proposed: we suggest that the UidR and UxuR repressors interact after their binding to the operator site of uidA; the binding of one regulatory molecule may facilitate the binding of the other one in a cooperative process.

scholarly journals Utilization of gluconate by Escherichia coli. A role of adenosine 3′:5′-cyclic monophosphate in the induction of gluconate catabolism

1975 ◽  
Vol 150 (1) ◽  
pp. 123-128 ◽  
Author(s):  
B Bächi ◽  
H L Kornberg
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Uptake System ◽  
Wild Type ◽  
Methyl Glyoxal ◽  
Cyclic Monophosphate ◽  
Inhibition Of Growth ◽  
Wild Type Phenotype ◽  
Specific Activities

1. Cultures of Escherichia coli growing on gluconate use both gluconate and glucose when glucose is added. 2. Glycerol-grown cells adapt to gluconate utilization even in media containing glucose as well as gluconate. 3. The rates of gluconate utilization by cells growing on a mixture of glucose and gluconate, and the specific activities of the gluconate uptake system and of gluconate kinase, are greater if adenosine 3′:5′-cyclic monophosphate (cyclic AMP) is present in the medium than in its absence. 4. Growth on media containing gluconate and cyclic AMP is accompanied by the formation of methyl glyoxal and pyruvate, and progressive inhibition of growth. 5. A mutant devoid of adenylate cyclase activity (cya) grew well on glucose in the absence of exogenous cyclic AMP but grew only poorly on gluconate; neither the gluconate uptake system nor gluconate kinase was adequately induced. The addition of cyclic AMP promoted growth on gluconate and facilitated the induction of proteins required for gluconate catabolism. 6. Phage Pl-mediated transduction of cya+ into the cya-mutant also restored the wild-type phenotype in its ability to adapt to gluconate utilization.

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