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1.
STAR Protoc ; 4(4): 102676, 2023 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-38048219

RESUMEN

Recent studies have revealed cellular heterogeneity of mesenchymal stromal cells and immune cells in adipose tissue and emphasized the need for quantitative analysis of small numbers of functionally distinct cells using state-of-the-art "omics" technologies. Here, we present an optimized protocol for precise protein quantification from minute amounts of samples. We describe steps for isolation of mouse adipose progenitor cells, proteomics sample preparation, mass spectrometry measurement, and computational analysis. This protocol can be adapted to other samples with limited amounts. For complete details on the use and execution of this protocol, please refer to Shan et al. (2022).1.


Asunto(s)
Células Madre Mesenquimatosas , Proteómica , Animales , Ratones , Tejido Adiposo , Espectrometría de Masas
2.
Cell Metab ; 34(5): 783-799.e7, 2022 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-35447091

RESUMEN

Single-cell RNA sequencing (scRNA-seq) has revealed that adult white adipose tissue (WAT) harbors functionally diverse subpopulations of mesenchymal stromal cells that differentially impact tissue plasticity. To date, the molecular basis of this cellular heterogeneity has not been fully defined. Here, we describe a multilayered omics approach to dissect adipose progenitor cell heterogeneity in three dimensions: progenitor subpopulation, sex, and anatomical localization. We applied state-of-the-art mass spectrometry methods to quantify 4,870 proteins in eight different stromal cell populations from perigonadal and inguinal WAT of male and female mice and acquired transcript expression levels of 15,477 genes using RNA-seq. Our data unveil molecular signatures defining sex differences in preadipocyte differentiation and identify regulatory pathways that functionally distinguish adipose progenitor subpopulations. This multilayered omics analysis, freely accessible at http://preadprofiler.net/, provides unprecedented insights into adipose stromal cell heterogeneity and highlights the benefit of complementary proteomics to support findings from scRNA-seq studies.


Asunto(s)
Adipocitos , Adipogénesis , Adipocitos/metabolismo , Tejido Adiposo , Tejido Adiposo Blanco/metabolismo , Animales , Diferenciación Celular , Femenino , Masculino , Ratones , Células Madre/metabolismo
3.
Microbiology (Reading) ; 164(5): 740-750, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29595414

RESUMEN

Campylobacter jejuni cells have bipolar flagella. Both flagella have similar lengths of about one helical turn, or 3.53±0.52 µm. The flagellar filament is composed of two homologous flagellins: FlaA and FlaB. Mutant strains that express either FlaA or FlaB alone produce filaments that are shorter than those of the wild-type. It is reported that the flaG gene could affect filament length in some species of bacteria, but its function remains unknown. We introduced a flaG-deletion mutation into the C. jejuni wild-type strain and flaA- or flaB-deletion mutant strains, and observed their flagella by microscopy. The ΔflaG mutant cells produced long filaments of two helical turns in the wild-type background. The ΔflaAG double mutant cells produced very short FlaB filaments. On the other hand, ΔflaBG double mutant cells produced long FlaA filaments and their morphology was not helical but straight. Furthermore, FlaG was secreted, and a pulldown assay showed that sigma factor 28 was co-precipitated with purified polyhistidine-tagged FlaG. We conclude that FlaG controls flagella length by negatively regulating FlaA filament assembly and discuss the role of FlaA and FlaB flagellins in C. jejuni flagella formation.


Asunto(s)
Proteínas Bacterianas/metabolismo , Campylobacter jejuni/fisiología , Flagelos/genética , Flagelos/metabolismo , Proteínas Bacterianas/genética , Campylobacter jejuni/citología , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Flagelos/ultraestructura , Flagelina/genética , Flagelina/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Locomoción , Microscopía Electrónica de Transmisión , Unión Proteica , Factor sigma/metabolismo
4.
BMC Biol ; 15(1): 97, 2017 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-29078764

RESUMEN

BACKGROUND: In a macro-molecular complex, any minor change may prove detrimental. For a supra-molecular nano-machine like the bacterial flagellum, which consists of several distinct parts with specific characteristics, stability is important. During the rotation of the bacterial flagellar motor, which is located in the membrane, the flagella rotate at speeds between 200 and 2000 rpm, depending on the bacterial species. The hook substructure of the bacterial flagellum acts as a universal joint connecting the motor to the flagellar filament. We investigated the formation of the bacterial flagellar hook and its overall stability between the FlgE subunits that make up the hook and attempted to understand how this stability differs between bacteria. RESULTS: An intrinsically disordered segment plays an important role for overall hook stability and for its structural cohesion during motor rotation. The length of this linker segment depends on the species of bacteria; for Salmonella enterica and Campylobacter jejuni it is approximately 37 and 54 residues, respectively. Few residues of the linker are conserved and mutating the conserved residues of the linker yields non-flagellated cells. In the case of Campylobacter, which rotates its flagella at a speed much higher than that of Salmonella, shortening the linker leads to a rupture of the hook at its base, decreasing cell motility. Our experiments show that this segment is required for polymerization and stability of the hook, demonstrating a surprising role for a disordered region in one of the most finely tuned and closely studied macromolecular machines. CONCLUSIONS: This study reveals a detailed functional characteristic of an intrinsically disordered segment in the hook protein. This segment evolved to fulfill a specific role in the formation of the hook, and it is at the core of the stability and flexibility of the hook. Its length is important in the case of bacteria with high-speed rotating flagella. Finding a way of disrupting this linker in Campylobacter might help in preventing infections.


Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Bacterias/genética , Proteínas Bacterianas/genética
5.
Nat Commun ; 7: 13425, 2016 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-27811912

RESUMEN

The bacterial flagellar hook is a tubular helical structure made by the polymerization of multiple copies of a protein, FlgE. Here we report the structure of the hook from Campylobacter jejuni by cryo-electron microscopy at a resolution of 3.5 Å. On the basis of this structure, we show that the hook is stabilized by intricate inter-molecular interactions between FlgE molecules. Extra domains in FlgE, found only in Campylobacter and in related bacteria, bring more stability and robustness to the hook. Functional experiments suggest that Campylobacter requires an unusually strong hook to swim without its flagella being torn off. This structure reveals details of the quaternary organization of the hook that consists of 11 protofilaments. Previous study of the flagellar filament of Campylobacter by electron microscopy showed its quaternary structure made of seven protofilaments. Therefore, this study puts in evidence the difference between the quaternary structures of a bacterial filament and its hook.


Asunto(s)
Proteínas Bacterianas/química , Campylobacter jejuni , Flagelos/ultraestructura , Estructura Cuaternaria de Proteína , Microscopía por Crioelectrón , Modelos Moleculares , Multimerización de Proteína
6.
Sci Rep ; 6: 35552, 2016 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-27759043

RESUMEN

Across bacteria, the protein that makes the flagellar hook, FlgE, has a high variability in amino acid residue composition and sequence length. We hereby present the structure of two fragments of FlgE protein from Campylobacter jejuni and from Caulobacter crescentus, which were obtained by X-ray crystallography, and a high-resolution model of the hook from Caulobacter. By comparing these new structures of FlgE proteins, we show that bacterial hook can be divided in two distinct parts. The first part comprises domains that are found in all FlgE proteins and that will make the basic structure of the hook that is common to all flagellated bacteria. The second part, hyper-variable both in size and structure, will be bacteria dependent. To have a better understanding of the C. jejuni hook, we show that a special strain of Salmonella enterica, which was designed to encode a gene of flgE that has the extra domains found in FlgE from C. jejuni, is fully motile. It seems that no matter the size of the hook protein, the hook will always have a structure made of 11 protofilaments.


Asunto(s)
Proteínas Bacterianas/metabolismo , Campylobacter jejuni/fisiología , Caulobacter crescentus/fisiología , Flagelos/fisiología , Salmonella enterica/fisiología , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Microorganismos Modificados Genéticamente , Modelos Moleculares , Polimorfismo Genético , Conformación Proteica , Especificidad de la Especie , Homología Estructural de Proteína , Relación Estructura-Actividad
7.
Mol Microbiol ; 100(2): 278-88, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26691662

RESUMEN

The Type III flagellar protein export apparatus of bacteria consists of five or six membrane proteins, notably FlhA, which controls the export of other proteins and is homologous to the large family of FHIPEP export proteins. FHIPEP proteins contain a highly-conserved cytoplasmic domain. We mutagenized the cloned Salmonella flhA gene for the 692 amino acid FlhA, changing a single, conserved amino acid in the 68-amino acid FHIPEP region. Fifty-two mutations at 30 positions mostly led to loss of motility and total disappearance of microscopically visible flagella, also Western blot protein/protein hybridization showed no detectable export of hook protein and flagellin. There were two exceptions: a D199A mutant strain, which produced short-stubby flagella; and a V151L mutant strain, which did not produce flagella and excreted mainly un-polymerized hook protein. The V151L mutant strain also exported a reduced amount of hook-cap protein FlgD, but when grown with exogenous FlgD it produced polyhooks and polyhook-filaments. A suppressor mutant in the cytoplasmic domain of the export apparatus membrane protein FlhB rescued export of hook-length control protein FliK and facilitated growth of full-length flagella. These results suggested that the FHIPEP region is part of the gate regulating substrate entry into the export apparatus pore.


Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Clonación Molecular , Citoplasma/metabolismo , Flagelina/metabolismo , Proteínas de la Membrana/genética , Mutación , Dominios Proteicos , Salmonella/genética , Salmonella/metabolismo , Especificidad por Sustrato
8.
J Bacteriol ; 196(23): 4001-11, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25201947

RESUMEN

The type III export apparatus of the Salmonella flagellum consists of six transmembrane proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR) and three soluble proteins (FliH, FliI, and FliJ). Deletion of the fliO gene creates a mutant strain that is poorly motile; however, suppressor mutations in the fliP gene can partially rescue motility. To further understand the mechanism of suppression of a fliO deletion mutation, we isolated new suppressor mutant strains with partially rescued motility. Whole-genome sequence analysis of these strains found a missense mutation that localized to the clpP gene [clpP(V20F)], which encodes the ClpP subunit of the ClpXP protease, and a synonymous mutation that localized to the fliA gene [fliA(+36T→C)], which encodes the flagellar sigma factor, σ(28). Combining these suppressor mutations with mutations in the fliP gene additively rescued motility and biosynthesis of the flagella in fliO deletion mutant strains. Motility was also rescued by an flgM deletion mutation or by plasmids carrying either the flhDC or fliA gene. The fliA(+36T→C) mutation increased mRNA translation of a fliA'-lacZ gene fusion, and immunoblot analysis revealed that the mutation increased levels of σ(28). Quantitative real-time reverse transcriptase PCR showed that either the clpP(V20F) or fliA(+36T→C) mutation rescued expression of class 3 flagellar and chemotaxis genes; still, the suppressor mutations in the fliP gene had a greater effect on bypassing the loss of fliO function. This suggests that the function of FliO is closely associated with regulation of FliP during assembly of the flagellum.


Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Flagelos/metabolismo , Proteínas de la Membrana/deficiencia , Complejos Multiproteicos/metabolismo , Salmonella typhimurium/metabolismo , Supresión Genética , Proteínas Bacterianas/genética , Análisis Mutacional de ADN , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Perfilación de la Expresión Génica , Genoma Bacteriano , Locomoción , Mutación Missense , Reacción en Cadena en Tiempo Real de la Polimerasa , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Factor sigma/genética , Factor sigma/metabolismo
9.
Microbiology (Reading) ; 160(Pt 6): 1075-1086, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24692644

RESUMEN

The primary mobile electron-carrier in the aerobic respiratory chain of Salmonella is ubiquinone. Demethylmenaquinone and menaquinone are alternative electron-carriers involved in anaerobic respiration. Ubiquinone biosynthesis was disrupted in strains bearing deletions of the ubiA or ubiE genes. In soft tryptone agar both mutant strains swam poorly. However, the ubiA deletion mutant strain produced suppressor mutant strains with somewhat rescued motility and growth. Six independent suppressor mutants were purified and comparative genome sequence analysis revealed that they each bore a single new missense mutation, which localized to genes for subunits of NADH : quinone oxidoreductase-1. Four mutants bore an identical nuoG(Q297K) mutation, one mutant bore a nuoM(A254S) mutation and one mutant bore a nuoN(A444E) mutation. The NuoG subunit is part of the hydrophilic domain of NADH : quinone oxidoreductase-1 and the NuoM and NuoN subunits are part of the hydrophobic membrane-embedded domain. Respiration was rescued and the suppressed mutant strains grew better in Luria-Bertani broth medium and could use l-malate as a sole carbon source. The quinone pool of the cytoplasmic membrane was characterized by reversed-phase HPLC. Wild-type cells made ubiquinone and menaquinone. Strains with a ubiA deletion mutation made demethylmenaquinone and menaquinone and the ubiE deletion mutant strain made demethylmenaquinone and 2-octaprenyl-6-methoxy-1,4-benzoquinone; the total quinone pool was reduced. Immunoblotting found increased NADH : quinone oxidoreductase-1 levels for ubiquinone-biosynthesis mutant strains and enzyme assays measured electron transfer from NADH to demethylmenaquinone or menaquinone. Under certain growth conditions the suppressor mutations improved electron flow activity of NADH : quinone oxidoreductase-1 for cells bearing a ubiA deletion mutation.


Asunto(s)
Locomoción , Redes y Vías Metabólicas/genética , Quinona Reductasas/metabolismo , Salmonella/enzimología , Salmonella/fisiología , Supresión Genética , Ubiquinona/análisis , Membrana Celular/química , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Genoma Bacteriano , Mutación Missense , Quinona Reductasas/genética , Salmonella/genética , Salmonella/crecimiento & desarrollo , Análisis de Secuencia de ADN
10.
PLoS One ; 8(7): e68384, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23874605

RESUMEN

The membrane protein FlhB is a highly conserved component of the flagellar secretion system, and it plays an active role in the regulation of protein export. In this study conserved properties of FlhB that are important for its function were investigated. Replacing the flhB gene (or part of the gene) in Salmonella typhimurium with the flhB gene of the distantly related bacterium Aquifex aeolicus greatly reduces motility. However, motility can be restored to some extent by spontaneous mutations in the part of flhB gene coding for the cytoplasmic domain of Aquifex FlhB. Structural analysis suggests that these mutations destabilize the structure. The secondary structure and stability of the mutated cytoplasmic fragments of FlhB have been studied by circular dichroism spectroscopy. The results suggest that conformational flexibility could be important for FlhB function. An extragenic suppressor mutation in the fliS gene, which decreases the affinity of FliS to FliC, partially restores motility of the FlhB substitution mutants.


Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Proteínas de la Membrana/metabolismo , Salmonella typhimurium/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Citoplasma/genética , Citoplasma/metabolismo , Flagelos/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Salmonella typhimurium/genética , Alineación de Secuencia , Supresión Genética/genética
11.
PLoS One ; 7(8): e44030, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22952860

RESUMEN

The bacterial type III export apparatus is found in the flagellum and in the needle complex of some pathogenic Gram-negative bacteria. In the needle complex its function is to secrete effector proteins for infection into Eukaryotic cells. In the bacterial flagellum it exports specific proteins for the building of the flagellum during its assembly. The export apparatus is composed of about five membrane proteins and three soluble proteins. The mechanism of the export apparatus is not fully understood. The five membrane proteins are well conserved and essential. Here a cross-complementation assay was performed: substituting in the flagellar system of Salmonella one of these membrane proteins, FlhB, by the FlhB ortholog from Aquifex aeolicus (an evolutionary distant hyperthermophilic bacteria) or a chimeric protein (AquSalFlhB) made by the combination of the trans-membrane domain of A. aeolicus FlhB with the cytoplasmic domain of Salmonella FlhB dramatically reduced numbers of flagella and motility. From cells expressing the chimeric AquSalFlhB protein, suppressor mutants with enhanced motility were isolated and the mutations were identified using whole genome sequencing. Gain-of-function mutations were found in the gene encoding FlhA, another membrane protein of the type III export apparatus. Also, mutations were identified in genes encoding 4-hydroxybenzoate octaprenyltransferase, ubiquinone/menaquinone biosynthesis methyltransferase, and 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, which are required for ubiquinone biosynthesis. The mutations were shown by reversed-phase high performance liquid chromatography to reduce the quinone pool of the cytoplasmic membrane. Ubiquinone biosynthesis could be restored for the strain bearing a mutated gene for 4-hydroxybenzoate octaprenyltransferase by the addition of excess exogenous 4-hydroxybenzoate. Restoring the level of ubiquinone reduced flagella biogenesis with the AquSalFlhB chimera demonstrating that the respiratory chain quinone pool is responsible for this phenomenon.


Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Prueba de Complementación Genética , Proteínas de la Membrana/metabolismo , Salmonella/genética , Bacterias/efectos de los fármacos , Western Blotting , Flagelos/efectos de los fármacos , Genes Bacterianos/genética , Movimiento/efectos de los fármacos , Parabenos/farmacología , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Salmonella/efectos de los fármacos , Supresión Genética/efectos de los fármacos , Ubiquinona/biosíntesis
12.
J Bacteriol ; 193(16): 4057-68, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21685294

RESUMEN

The Escherichia coli flagellar master regulator, FlhD(4)C(2), binds to the promoter regions of flagellar class II genes, yet, despite extensive analysis of the FlhD(4)C(2)-regulated promoter region, a detailed consensus sequence has not emerged. We used in vitro and in vivo experimental approaches to determine the nucleotides in the class II promoter, fliAp, required for the binding and function of FlhD(4)C(2). FlhD(4)C(2) protects 48 bp (positions -76 to -29 relative to the σ(70)-dependent transcriptional start site) in the fliA promoter. We divided the 48-bp footprint region into 5 sections to determine the requirement of each DNA segment for the binding and function of FlhD(4)C(2). Results from an in vitro binding competition assay between the wild-type FlhD(4)C(2)-protected fragment and DNA fragments possessing mutations in one section of the 48-bp protected region showed that only one-third of the 48 bp protected by FlhD(4)C(2) is required for FlhD(4)C(2) binding and fliA promoter activity. This in vitro binding result was also seen in vivo with fliA promoter-lacZ fusions carrying the same mutations. Only seven bases (A(12), A(15), T(34), A(36), T(37), A(44), and T(45)) are absolutely required for the promoter activity. Moreover, A(12), A(15), T(34), T(37), and T(45) within the 7 bases are highly specific to fliA promoter activity, and those bases form an asymmetric recognition site for FlhD(4)C(2). The implications of the asymmetry of the FlhD(4)C(2) binding site and its potential impact on FlhD(4)C(2) are discussed.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Flagelos/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Unión Proteica , Transactivadores/metabolismo , Secuencia de Bases , ADN Bacteriano/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas/fisiología , Transactivadores/genética
13.
PLoS Genet ; 6(9): e1001143, 2010 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-20941389

RESUMEN

The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the ΔfliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N-terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO43-125 or FliO1-95 was able to rescue motility of the ΔfliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO43-125, should contain beta-structure and alpha-helices. FliO43-125-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and its structure was a mixture of beta-sheet and random coil. Coexpression of full-length FliO with FliP increased expression levels of FliP, but coexpression with the cytoplasmic domain of FliO did not enhance FliP expression levels. Overexpression of the cytoplasmic domain of FliO further rescued motility of strains deleted for the fliO gene expressing bypass mutations in FliP. These results suggest FliO maintains FliP stability through transmembrane domain interaction. The results also demonstrate that the cytoplasmic domain of FliO has functionality, and it presumably becomes structured while interacting with its binding partners.


Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Proteínas de la Membrana/metabolismo , Salmonella enterica/metabolismo , Agar/farmacología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dicroismo Circular , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Análisis Mutacional de ADN , Flagelos/efectos de los fármacos , Eliminación de Gen , Prueba de Complementación Genética , Leucina/metabolismo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Movimiento/efectos de los fármacos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Periplasma/efectos de los fármacos , Periplasma/metabolismo , Estructura Terciaria de Proteína , Salmonella enterica/citología , Salmonella enterica/efectos de los fármacos , Relación Estructura-Actividad
14.
J Bacteriol ; 186(22): 7529-37, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15516564

RESUMEN

The flhD operon is the master operon of the flagellar regulon and a global regulator of metabolism. The genome sequence of the Escherichia coli K-12 strain MG1655 contained an IS1 insertion sequence element in the regulatory region of the flhD promoter. Another stock of MG1655 was obtained from the E. coli Genetic Stock Center. This stock contained isolates which were poorly motile and had no IS1 element upstream of the flhD promoter. From these isolates, motile subpopulations were identified after extended incubation in motility agar. Purified motile derivatives contained an IS5 element insertion upstream of the flhD promoter, and swarm rates were sevenfold higher than that of the original isolate. For a motile derivative, levels of flhD transcript had increased 2.7-fold, leading to a 32-fold increase in fliA transcript and a 65-fold increase in flhB::luxCDABE expression from a promoter probe vector. A collection of commonly used lab strains was screened for IS element insertion and motility. Five strains (RP437, YK410, MC1000, W3110, and W2637) contained IS5 elements upstream of the flhD promoter at either of two locations. This correlated with high swarm rates. Four other strains (W1485, FB8, MM294, and RB791) did not contain IS elements in the flhD regulatory region and were poorly motile. Primer extension determined that the transcriptional start site of flhD was unaltered by the IS element insertions. We suggest that IS element insertion may activate transcription of the flhD operon by reducing transcriptional repression.


Asunto(s)
Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Escherichia coli K12/fisiología , Regulación Bacteriana de la Expresión Génica , Operón , Transactivadores/genética , Secuencia de Bases , Medios de Cultivo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Movimiento , Transactivadores/química , Transactivadores/metabolismo , Transcripción Genética
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