Characterization of the Interaction Between the Small Regulatory Peptide SgrT and the EIICBGlc of the Glucose-Phosphotransferase System of E. coli K-12

In this paper, we present a non-fluidic microsystem for the simultaneous visualization and electrochemical evaluation of confined, growing bacteria on solid media. Using a completely automated platform, real-time monitoring of bacterial and image-based computer characterization of growth were performed. Electrochemical tests, using Escherichia coli K-12 as the model microorganism, revealed the development of a faradaic process at the bacteria–microelectrode interface inside the microsystem, as implied by cyclic voltammetry and electrochemical impedance spectrometry measurements. The electrochemical information was used to determine the moment in which bacteria colonized the electrode-enabled area of the microsystem. This microsystem shows potential advantages for long-term electrochemical monitoring of the extracellular environment of cell culture and has been designed using readily available technologies that can be easily integrated in routine protocols. Complementarily, these methods can help elucidate fundamental questions of the electron transfer of bacterial cultures and are potentially feasible to be integrated into current characterization techniques.

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Characterization of a Shiga Toxin-Encoding Temperate Bacteriophage of Shigella sonnei

Infection and Immunity ◽

10.1128/iai.69.12.7588-7595.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7588-7595 ◽

Cited By ~ 65

Author(s):

Eckhard Strauch ◽

Rudi Lurz ◽

Lothar Beutin

Keyword(s):

Shiga Toxin ◽

Regulatory Region ◽

Shigella Sonnei ◽

Shigella Dysenteriae ◽

Nucleotide Sequencing ◽

Temperate Bacteriophage ◽

Cross Hybridization ◽

E Coli ◽

K 12

ABSTRACT A Shiga toxin (Stx)-encoding temperate bacteriophage ofShigella sonnei strain CB7888 was investigated for its morphology, DNA similarity, host range, and lysogenization inShigella and Escherichia coli strains. Phage 7888 formed plaques on a broad spectrum of Shigella strains belonging to different species and serotypes, including Stx-producingShigella dysenteriae type 1. With E. coli, only strains with rough lipopolysaccharide were sensitive to this phage. The phage integrated into the genome of nontoxigenic S. sonneiand laboratory E. coli K-12 strains, which became Stx positive upon lysogenization. Moreover, phage 7888 is capable of transducing chromosomal genes in E. coli K-12. The relationships of phage 7888 with the E. coli Stx1-producing phage H-19B and the E. coli Stx2-producing phage 933W were investigated by DNA cross-hybridization of phage genomes and by nucleotide sequencing of an 8,053-bp DNA region of the phage 7888 genome flanking the stx genes. By these methods, a high similarity was found between phages 7888 and 933W. Much less similarity was found between phages H-19B and 7888. As in the other Stx phages, a regulatory region involved in Q-dependent expression is found upstream of stxA and stxB (stx gene) in phage 7888. The morphology of phage 7888 was similar to that of phage 933W, which shows a hexagonal head and a short tail. Our findings demonstrate that stx genes are naturally transferable and are expressed in strains of S. sonnei, which points to the continuous evolution of human-pathogenic Shigella by horizontal gene transfer.

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Characterization of the Plesiomonas shigelloides Genes Encoding the Heme Iron Utilization System

Journal of Bacteriology ◽

10.1128/jb.183.9.2715-2723.2001 ◽

2001 ◽

Vol 183 (9) ◽

pp. 2715-2723 ◽

Cited By ~ 38

Author(s):

D. P. Henderson ◽

E. E. Wyckoff ◽

C. E. Rashidi ◽

H. Verlei ◽

A. L. Oldham

Keyword(s):

Laboratory Strain ◽

Heme Iron ◽

Plesiomonas Shigelloides ◽

E Coli ◽

Iron Source ◽

Genes Encoding ◽

Tonb System ◽

K 12 ◽

Iron Utilization

ABSTRACT Plesiomonas shigelloides is a gram-negative pathogen which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a laboratory strain of Escherichia coli were isolated. In the present study, the cloned P. shigelloides sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes, hugA, hugZ, and tonB, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When theP. shigelloides genes were tested in E. coliK-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, orhugZ were required for heme iron utilization. When the genes were tested in a hemA entB mutant of E. coli, hugWXZ were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source. hugAcould replace the Vibrio cholerae hutA in a heme iron utilization assay, and V. cholerae hutA could complement aP. shigelloides heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonBmutants of both E. coli and V. cholerae and that it was similar to the V. cholerae TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium.

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Characterization of rapidly labelled ribonucleic acid in Escherichia coli by deoxyribonucleic acid–ribonucleic acid hybridization

Biochemical Journal ◽

10.1042/bj1100251 ◽

1968 ◽

Vol 110 (2) ◽

pp. 251-263 ◽

Cited By ~ 27

Author(s):

G. H. Pigott ◽

J. E. M. Midgley

Keyword(s):

Escherichia Coli ◽

Ribosomal Rna ◽

Ribonucleic Acid ◽

Messenger Rna ◽

Rna Binding ◽

Hybridization Technique ◽

E Coli ◽

K 12 ◽

Dna 2

1. Rapidly labelled RNA from Escherichia coli K 12 was characterized by hybridization to denatured E. coli DNA on cellulose nitrate membrane filters. The experiments were designed to show that, if sufficient denatured DNA is offered in a single challenge, practically all the rapidly labelled RNA will hybridize. With the technique employed, 75–80% hybridization efficiency could be obtained as a maximum. Even if an excess of DNA sites were offered, this value could not be improved upon in any single challenge of rapidly labelled RNA with denatured E. coli DNA. 2. It was confirmed that the hybridization technique can separate the rapidly labelled RNA into two fractions. One of these (30% of the total) was efficiently hybridized with the low DNA/RNA ratio (10:1, w/w) used in tests. The other fraction (70% of the total) was hybridized to DNA at low efficiencies with the DNA/RNA ratio 10:1, and was hybridized progressively more effectively as the amount of denatured DNA was increased. A practical maximum of 80% hybridization of all the rapidly labelled RNA was first achieved at a DNA/RNA ratio 210:1 (±10:1). This fraction was fully representative of the rapidly labelled RNA with regard to kind and relative amount of materials hybridized. 3. In competition experiments, where additions were made of unlabelled RNA prepared from E. coli DNA, DNA-dependent RNA polymerase (EC 2.7.7.6) and nucleoside 5′-triphosphates, the rapidly labelled RNA fraction hybridized at a low (10:1) DNA/RNA ratio was shown to be competitive with a product from genes other than those responsible for ribosomal RNA synthesis and thus was presumably messenger RNA. At higher DNA/rapidly labelled RNA ratios (200:1), competition with added unlabelled E. coli ribosomal RNA (without messenger RNA contaminants) lowered the hybridization of the rapidly labelled RNA from its 80% maximum to 23%. This proportion of rapidly labelled RNA was not competitive with E. coli ribosomal RNA even when the latter was in large excess. The ribosomal RNA would also not compete with the 23% rapidly labelled RNA bound to DNA at low DNA/RNA ratios. It was thus demonstrated that the major part of E. coli rapidly labelled RNA (70%) is ribosomal RNA, presumably a precursor to the RNA in mature ribosomes. 4. These studies have shown that, when earlier workers used low DNA/RNA ratios (about 10:1) in the assay of messenger RNA in bacterial rapidly labelled RNA, a reasonable estimate of this fraction was achieved. Criticisms that individual messenger RNA species may be synthesized from single DNA sites in E. coli at rates that lead to low efficiencies of messenger RNA binding at low DNA/RNA ratios are refuted. In accordance with earlier results, estimations of the messenger RNA content of E. coli in both rapidly labelled and randomly labelled RNA show that this fraction is 1·8–1·9% of the total RNA. This shows that, if any messenger RNA of relatively long life exists in E. coli, it does not contribute a measurable weight to that of rapidly labelled messenger RNA.

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Characterization of Glucose-Specific Catabolite Repression-Resistant Mutants of Bacillus subtilis: Identification of a Novel Hexose:H+ Symporter

Journal of Bacteriology ◽

10.1128/jb.180.3.498-504.1998 ◽

1998 ◽

Vol 180 (3) ◽

pp. 498-504 ◽

Cited By ~ 37

Author(s):

Ian T. Paulsen ◽

Sylvie Chauvaux ◽

Peter Choi ◽

Milton H. Saier

Keyword(s):

Bacillus Subtilis ◽

Catabolite Repression ◽

Genetic Background ◽

Insertional Mutagenesis ◽

Sequence Similarity ◽

Major Facilitator Superfamily ◽

Phosphotransferase System ◽

E Coli ◽

Major Facilitator

ABSTRACT Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H+ symporter ofEscherichia coli and the glucose/galactose:H+symporter of Brucella abortus. In a wild-type B. subtilis genetic background, theglcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [14C]glucose and [14C]methyl α-glucoside uptake. In a Δptsgenetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a Δptsmutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl α-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.

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Identification and Characterization of a New Lipoprotein, NlpI, in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.181.14.4318-4325.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4318-4325 ◽

Cited By ~ 42

Author(s):

Masaru Ohara ◽

Henry C. Wu ◽

Krishnan Sankaran ◽

Paul D. Rick

Keyword(s):

Escherichia Coli ◽

Direct Consequence ◽

Insertion Mutant ◽

Polynucleotide Phosphorylase ◽

Wild Type ◽

E Coli ◽

K 12 ◽

Identification And Characterization ◽

Lines Of Evidence

ABSTRACT We report here the identification of a new lipoprotein, NlpI, inEscherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp(polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.

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Characterization of the hca Cluster Encoding the Dioxygenolytic Pathway for Initial Catabolism of 3-Phenylpropionic Acid in Escherichia coliK-12

Journal of Bacteriology ◽

10.1128/jb.180.11.2915-2923.1998 ◽

1998 ◽

Vol 180 (11) ◽

pp. 2915-2923 ◽

Cited By ~ 51

Author(s):

Eduardo Díaz ◽

Abel Ferrández ◽

José L. García

Keyword(s):

Sequence Similarity ◽

Recombinant Bacteria ◽

Sequence Comparisons ◽

Phenylpropionic Acid ◽

E Coli ◽

Catabolic Genes ◽

Dihydrodiol Dehydrogenase ◽

Class Iib ◽

K 12

ABSTRACT We have identified, cloned, and sequenced the hcacluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid (PP) in Escherichia coli K-12. This cluster maps at min 57.5 of the chromosome and is composed of five catabolic genes arranged as a putative operon (hcaA1A2CBD) and two additional genes transcribed in the opposite direction that encode a potential permease (hcaT) and a regulator (hcaR). Sequence comparisons revealed that whilehcaA1A2CD genes encode the four subunits of the 3-phenylpropionate dioxygenase, the hcaB gene codes for the corresponding cis-dihydrodiol dehydrogenase. This type of catabolic module is homologous to those encoding class IIB dioxygenases and becomes the first example of such a catabolic cluster in E. coli. The inducible expression of the hca genes requires the presence of the hcaR gene product, which acts as a transcriptional activator and shows significant sequence similarity to members of the LysR family of regulators. Interestingly, the HcaA1A2CD and HcaB enzymes are able to oxidize not only PP to 3-(2,3-dihydroxyphenyl)propionate (DHPP) but also cinnamic acid (CI) to its corresponding 2,3-dihydroxy derivative. Further catabolism of DHPP requires the mhp-encoded meta fission pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) (A. Ferrández, J. L. Garcı́a, and E. Dı́az, J. Bacteriol. 179:2573–2581, 1997). Expression in Salmonella typhimurium of the mhp genes alone or in combination with the hca cluster allowed the growth of the recombinant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively. Thus, the convergent mhp- and hca-encoded pathways are also functional in S. typhimurium, and they are responsible for the catabolism of different phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely available in nature.

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Integrated Genomic Map from Uropathogenic Escherichia coli J96

Infection and Immunity ◽

10.1128/iai.68.10.5933-5942.2000 ◽

2000 ◽

Vol 68 (10) ◽

pp. 5933-5942 ◽

Cited By ~ 13

Author(s):

Lyla J. Melkerson-Watson ◽

Christopher K. Rode ◽

Lixin Zhang ◽

Betsy Foxman ◽

Craig A. Bloch

Keyword(s):

Escherichia Coli ◽

Virulence Genes ◽

Housekeeping Genes ◽

E Coli ◽

Restriction Sites ◽

Physical And Genetic Map ◽

K 12 ◽

Genomic Map ◽

Insertion Mutants

ABSTRACT Escherichia coli J96 is a uropathogen having both broad similarities to and striking differences from nonpathogenic, laboratoryE. coli K-12. Strain J96 contains three large (>100-kb) unique genomic segments integrated on the chromosome; two are recognized as pathogenicity islands containing urovirulence genes. Additionally, the strain possesses a fourth smaller accessory segment of 28 kb and two deletions relative to strain K-12. We report an integrated physical and genetic map of the 5,120-kb J96 genome. The chromosome contains 26 NotI, 13 BlnI, and 7 I-CeuI macrorestriction sites. Macrorestriction mapping was rapidly accomplished by a novel transposon-based procedure: analysis of modified minitransposon insertions served to align the overlapping macrorestriction fragments generated by three different enzymes (each sharing a common cleavage site within the insert), thus integrating the three different digestion patterns and ordering the fragments. The resulting map, generated from a total of 54 mini-Tn10insertions, was supplemented with auxanography and Southern analysis to indicate the positions of insertionally disrupted aminosynthetic genes and cloned virulence genes, respectively. Thus, it contains not only physical, macrorestriction landmarks but also the loci for eight housekeeping genes shared with strain K-12 and eight acknowledged urovirulence genes; the latter confirmed clustering of virulence genes at the large unique accessory chromosomal segments. The 115-kb J96 plasmid was resolved by pulsed-field gel electrophoresis inNotI digests. However, because the plasmid lacks restriction sites for the enzymes BlnI and I-CeuI, it was visualized in BlnI and I-CeuI digests only of derivatives carrying plasmid inserts artificially introducing these sites. Owing to an I-SceI site on the transposon, the plasmid could also be visualized and sized from plasmid insertion mutants after digestion with this enzyme. The insertional strains generated in construction of the integrated genomic map provide useful physical and genetic markers for further characterization of the J96 genome.

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Molecular Characterization of UpaB and UpaC, Two New Autotransporter Proteins of Uropathogenic Escherichia coli CFT073

Infection and Immunity ◽

10.1128/iai.05322-11 ◽

2011 ◽

Vol 80 (1) ◽

pp. 321-332 ◽

Cited By ~ 55

Author(s):

Luke P. Allsopp ◽

Christophe Beloin ◽

Glen C. Ulett ◽

Jaione Valle ◽

Makrina Totsika ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Characterization ◽

Host Responses ◽

Upec Strain ◽

Content Type ◽

Pcr Screening ◽

E Coli ◽

K 12 ◽

Encoding Genes

ABSTRACTUropathogenicEscherichia coli(UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genome-sequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. In this study, we have performed a detailed molecular characterization of two closely related AT adhesins from CFT073: UpaB (c0426) and UpaC (c0478). PCR screening revealed that theupaBandupaCAT-encoding genes are common inE. coli. TheupaBandupaCgenes were cloned and characterized in a recombinantE. coliK-12 strain background. This revealed that they encode proteins located at the cell surface but possess different functional properties: UpaB mediates adherence to several ECM proteins, while UpaC expression is associated with increased biofilm formation. In CFT073,upaBis expressed whileupaCis transcriptionally repressed by the global regulator H-NS. In competitive colonization experiments employing the mouse UTI model, CFT073 significantly outcompeted itsupaB(but notupaC) isogenic mutant strain in the bladder. This attenuated phenotype was also observed in single-challenge experiments, where deletion of theupaBgene in CFT073 significantly reduced early colonization of the bladder.

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Modulation of transcription and characterization of the promoter organization of the autotransporter adhesin heptosyltransferase and the autotransporter adhesin AIDA-I

Microbiology ◽

10.1099/mic.0.032292-0 ◽

2010 ◽

Vol 156 (4) ◽

pp. 1155-1166 ◽

Cited By ~ 7

Author(s):

Inga Benz ◽

Tessa van Alen ◽

Julia Bolte ◽

Mirka E. Wörmann ◽

M. Alexander Schmidt

Keyword(s):

Protein Translocation ◽

Growth Conditions ◽

Limited Attention ◽

Gram Negative Bacteria ◽

Regulation Of Expression ◽

Gene Encoding ◽

E Coli ◽

K 12 ◽

Transcriptional Fusions

In Gram-negative bacteria, autotransporter proteins constitute the largest family of secreted proteins, and exhibit many different functions. In recent years, research has largely focused on mechanisms of autotransporter protein translocation, where several alternative models are still being discussed. In contrast, the biogenesis of only a few autotransporters has been studied and, likewise, regulation of expression has received only very limited attention. The glycosylated autotransporter adhesin involved in diffuse adherence (AIDA)-I system consists of the aah gene, encoding a specific autotransporter adhesin heptosyltransferase (AAH), and the aidA gene, encoding the autotransporter protein (AIDA-I). In this study, we investigated the promoter organization and transcription of these two genes using reporter plasmids carrying lacZ transcriptional fusions. The two genes, aah and aidA, are transcribed as a bicistronic message. However, aidA is additionally transcribed from its own promoter. There are two distinct start sites for each of the two genes. Interestingly, transcription of both genes is enhanced in hns and rfaH mutant backgrounds. Furthermore, we addressed the influence of environmental factors and different genetic backgrounds of Escherichia coli K-12 strains on transcription activity. We found that transcription varied considerably in different E. coli K-12 laboratory strains and under different growth conditions.

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