The Small RNA GcvB Regulates sstT mRNA Expression in Escherichia coli

ABSTRACT In Escherichia coli, the gcvB gene encodes a nontranslated RNA (referred to as GcvB) that regulates OppA and DppA, two periplasmic binding proteins for the oligopeptide and dipeptide transport systems. An additional regulatory target of GcvB, sstT, was found by microarray analysis of RNA isolated from a wild-type strain and a gcvB deletion strain grown to mid-log phase in Luria-Bertani broth. The SstT protein functions to transport l-serine and l-threonine by sodium transport into the cell. Reverse transcription-PCR and translational fusions confirmed that GcvB negatively regulates sstT mRNA levels in cells grown in Luria-Bertani broth. A series of transcriptional fusions identified a region of sstT mRNA upstream of the ribosome binding site needed for negative regulation by GcvB. Analysis of the GcvB RNA identified a sequence complementary to this region of the sstT mRNA. The region of GcvB complementary to sstT mRNA is the same region of GcvB identified to regulate the dppA and oppA mRNAs. Mutations predicted to disrupt base pairing between sstT mRNA and GcvB were made in gcvB, which resulted in the identification of a small region of GcvB necessary for negative regulation of sstT-lacZ. Additionally, the RNA chaperone protein Hfq was found to be necessary for GcvB to negatively regulate sstT-lacZ in Luria-Bertani broth and glucose minimal medium supplemented with glycine. The sstT mRNA is the first target found to be regulated by GcvB in glucose minimal medium supplemented with glycine.

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Role of the sRNA GcvB in regulation of cycA in Escherichia coli

Microbiology ◽

10.1099/mic.0.023598-0 ◽

2009 ◽

Vol 155 (1) ◽

pp. 106-114 ◽

Cited By ~ 48

Author(s):

Sarah C. Pulvermacher ◽

Lorraine T. Stauffer ◽

George V. Stauffer

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Minimal Medium ◽

Luria Bertani ◽

Cell Physiology ◽

Rt Pcr ◽

Luria Bertani Broth ◽

Increased Sensitivity ◽

Transcriptional Fusions

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria–Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria–Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

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Multiple Roles for the sRNA GcvB in the Regulation of Slp Levels in Escherichia coli

ISRN Bacteriology ◽

10.1155/2013/918106 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

Lorraine T. Stauffer ◽

George V. Stauffer

Keyword(s):

Escherichia Coli ◽

Luria Bertani ◽

Multiple Roles ◽

Activation Mechanism ◽

Mrna Levels ◽

Rt Pcr ◽

E Coli ◽

Luria Bertani Broth ◽

Outer Membrane Lipoprotein ◽

Membrane Lipoprotein

The Escherichia coli gcvB gene encodes a small RNA that regulates many genes involved in the transport of dipeptides, oligopeptides, and amino acids (oppA, dppA, cycA, and sstT). A microarray analysis of RNA isolated from an E. coli wild-type and a ΔgcvB strain grown to midlog phase in Luria-Bertani broth indicated that genes not involved in transport are also regulated by GcvB. One gene identified was slp that encodes an outer membrane lipoprotein of unknown function induced when cells enter stationary phase. The aim of this study was to verify that slp is a new target for GcvB-mediated regulation. In this study we used RT-PCR to show that GcvB regulates slp mRNA levels. GcvB negatively controls slp::lacZ in cells grown in Luria-Bertani broth by preventing an Hfq-mediated activation mechanism for slp::lacZ expression. In contrast, in glucose minimal medium supplemented with glycine, GcvB is required for inhibition of slp::lacZ expression, and Hfq prevents GcvB-mediated repression. Thus, GcvB regulates slp in both LB and in glucose minimal + glycine media and likely by mechanisms different than how it regulates sstT, dppA, cycA, and oppA. Repression of slp by GcvB results in an increase in resistance to chloramphenicol, and overexpression of slp in a ΔgcvB strain results in an increase in sensitivity to chloramphenicol.

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Role of the Escherichia coli Hfq protein in GcvB regulation of oppA and dppA mRNAs

Microbiology ◽

10.1099/mic.0.023432-0 ◽

2009 ◽

Vol 155 (1) ◽

pp. 115-123 ◽

Cited By ~ 31

Author(s):

Sarah C. Pulvermacher ◽

Lorraine T. Stauffer ◽

George V. Stauffer

Keyword(s):

Escherichia Coli ◽

Small Rnas ◽

Target Genes ◽

Luria Bertani ◽

Transport Systems ◽

Regulation Of Transcription ◽

Luria Bertani Broth ◽

The Stability ◽

Dipeptide Transport

The gcvB gene encodes a small non-translated RNA (referred to as GcvB) that regulates oppA and dppA, two genes that encode periplasmic binding proteins for the oligopeptide and dipeptide transport systems. Hfq, an RNA chaperone protein, binds many small RNAs and is required for the small RNAs to regulate expression of their respective target genes. We showed that repression by GcvB of dppA : : lacZ and oppA : : phoA translational fusions is dependent upon Hfq. Double mutations in gcvB and hfq yielded similar expression levels of dppA : : lacZ and oppA : : phoA compared with gcvB or hfq single mutations, suggesting that GcvB and Hfq repress by the same mechanism. The effect of Hfq is not through regulation of transcription of gcvB. Hfq is known to increase the stability of some small RNAs and to facilitate the interactions between small RNAs and specific mRNAs. In the absence of Hfq, there is a marked decrease in the half-life of GcvB in cells grown in both Luria–Bertani broth and glucose minimal medium with glycine, suggesting that part of the role of Hfq is to stabilize GcvB. Overproduction of GcvB in wild-type Escherichia coli results in superrepression of a dppA : : lacZ fusion, but overproduction of GcvB in an hfq mutant does not result in significant repression of the dppA : : lacZ fusion. These results suggest that Hfq also is likely required for GcvB–mRNA pairing.

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EVALUASI HEMATOTOKSIK SECARA IN VITRO NANOPARTIKEL ZnS HASIL REDUKSI BIOMATRIKS Eschericia coli

Majalah Farmasi dan Farmakologi ◽

10.20956/mff.v23i3.9398 ◽

2020 ◽

Vol 23 (3) ◽

pp. 82-84

Author(s):

Lisa Kurniati ◽

Andi Arjuna ◽

Sukamto S Mamada

Keyword(s):

Escherichia Coli ◽

Luria Bertani ◽

Luria Bertani Broth

Nanopartikel ZnS merupakan material semi konduktor yang memiliki sifat unik dan manfaat yang besar dibidang kesehatan, terutama sebagai antibakteri dan biomarker kanker. Walaupun demikian, informasi mengenai toksisitas dari nanopartikel ZnS masih sangat terbatas. Oleh karena itu, pada penelitian ini telah dilakukan evaluasi hematotoksisitas secara in vitro nanopartikel ZnS hasil reduksi biomatriks Escherichia coli. Penyiapan nanopartikel ZnS diawali dengan pencampuran dispersi ZnSO4 konsentrasi 200 bpj ke dalam medium Luria Bertani Broth (LBB) yang ditumbuhi E.Coli sebagai bioreduktor. Produk yang dihasilkan dikarakterisasi dengan uji photolimunisence (PL) dan spektrofotometri pada rentang panjang gelombang 250-700 nm. Hasilnya, nanopartikel ZnS berpendar biru dan diidentifikasi pada λmax 288 nm dengan absorbansi 0,905. Partikel yang dihasilkan kemudian didispersikan dengan variasi volume 30 µl, 40 µl, 50 µl pada larutan tyrod. Data persentase hemolisis secara berturut-turut adalah 32%, 39%, 22%, 0% (kontrol negatif) dan 100% (kontrol positif). Sehingga dapat disimpulkan bahwa nanopartikel ZnS hasil reduksi E.coli memberikan efek toksik terhadap sel darah merah

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Roles of the Extraintestinal Pathogenic Escherichia coli ZnuACB and ZupT Zinc Transporters during Urinary Tract Infection

Infection and Immunity ◽

10.1128/iai.01082-08 ◽

2008 ◽

Vol 77 (3) ◽

pp. 1155-1164 ◽

Cited By ~ 70

Author(s):

Mourad Sabri ◽

Sébastien Houle ◽

Charles M. Dozois

Keyword(s):

Escherichia Coli ◽

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Minimal Medium ◽

Cumulative Effect ◽

Transport Systems ◽

Single Strain ◽

Pathogenic Escherichia Coli ◽

K 12

ABSTRACT Roles of the ZnuACB and ZupT transporters were assessed in Escherichia coli K-12 and uropathogenic E. coli (UPEC) CFT073. K-12 and CFT073 Δznu ΔzupT mutants demonstrated decreased 65Zn2+ uptake and growth in minimal medium. CFT073Δznu demonstrated an intermediate decrease of 65Zn2+ uptake and growth in minimal medium, whereas the CFT073ΔzupT mutant grew as well as CFT073 and exhibited a less marked decrease in 65Zn2+ uptake. CFT073 mutants grew as well as the wild type in human urine. In competitive infections in CBA/J mice, the ΔzupT mutant demonstrated no disadvantage during urinary tract infection. In contrast, the UPEC Δznu and Δznu ΔzupT strains demonstrated significantly reduced numbers in the bladders (mean 4.4- and 30-fold reductions, respectively) and kidneys (mean 41- and 48-fold reductions, respectively). In addition, in single-strain infection experiments, the Δznu and Δznu ΔzupT mutants were reduced in the kidneys (P = 0.0012 and P < 0.0001, respectively). Complementation of the CFT073 Δznu ΔzupT mutant with the znuACB genes restored growth in Zn-deficient medium and bacterial numbers in the bladder and kidneys. The loss of the zinc transport systems decreased both motility and resistance to hydrogen peroxide, which could be restored by supplementation with zinc. Overall, the results indicate that Znu and ZupT are required for growth in zinc limited-conditions, that Znu is the predominant zinc transporter, and that the loss of Znu and ZupT has a cumulative effect on fitness during UTI, which may in part be due to reduced resistance to oxidative stress and motility.

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Influence of aerobic and anaerobic conditions on survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in Luria–Bertani broth, farm-yard manure and slurry

Journal of Environmental Management ◽

10.1016/j.jenvman.2010.10.031 ◽

2011 ◽

Vol 92 (3) ◽

pp. 780-787 ◽

Cited By ~ 42

Author(s):

Alexander V. Semenov ◽

Leo van Overbeek ◽

Aad J. Termorshuizen ◽

Ariena H.C. van Bruggen

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica Serovar Typhimurium ◽

Luria Bertani ◽

Escherichia Coli O157 ◽

Anaerobic Conditions ◽

Farm Yard Manure ◽

Luria Bertani Broth ◽

Serovar Typhimurium ◽

Aerobic And Anaerobic ◽

Aerobic And Anaerobic Conditions

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Nitazoxanide Inhibits Biofilm Production and Hemagglutination by Enteroaggregative Escherichia coli Strains by Blocking Assembly of AafA Fimbriae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01279-09 ◽

2010 ◽

Vol 54 (4) ◽

pp. 1526-1533 ◽

Cited By ~ 26

Author(s):

Eliah R. Shamir ◽

Michelle Warthan ◽

Sareena P. Brown ◽

James P. Nataro ◽

Richard L. Guerrant ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Agents ◽

Luria Bertani ◽

Type I ◽

Reverse Transcription Pcr ◽

Fimbrial Subunit ◽

Biofilm Production ◽

Type I Fimbriae ◽

Fimbrial Adhesins

ABSTRACT Enteroaggregative Escherichia coli (EAEC) strains have emerged as common causes of persistent diarrhea and malnutrition among children and HIV-infected persons. During infection, EAEC typically adheres to the intestinal mucosa via fimbrial adhesins, which results in a characteristic aggregative pattern. In the study described here we investigated whether the broad-spectrum antiparasitic and antidiarrheal drug nitazoxanide (NTZ) might be active against EAEC in vitro. While E. coli strains were resistant to NTZ in rich Luria-Bertani medium (MIC > 64 μg/ml), the drug was slightly inhibitory in a minimal medium supplemented with glucose (MinA-G medium; MIC, ∼32 μg/ml). NTZ also inhibited biofilm production by strain EAEC 042 in both Dulbecco's modified Eagle's medium and MinA-G medium with a 50% inhibitory concentration of ∼12 μg/ml. Immunofluorescence and immunoblot analyses with antibody against the major fimbrial subunit AafA of aggregative adherence fimbriae vaariant II (AAF/II) established that the numbers of AAF/II filaments on bacteria grown in the presence of NTZ were dramatically reduced. Comparative quantitative reverse transcription-PCR and reporter gene fusions (aafA::phoA) indicated that aafA expression was unaffected by NTZ, while aggR transcript levels and aggR::lacZ expression were increased ∼10- and 2.5-fold, respectively, compared with that for the untreated controls. More generally, NTZ inhibited hemagglutination (HA) of red blood cells by the non-biofilm-producing strain JM221 expressing either AAF/I or type I fimbriae. Our findings suggest that the inhibitory action of NTZ on biofilm formation and HA is likely due to inhibition of fimbrial assembly. Antimicrobial agents that inhibit the assembly or function of fimbrial filaments should be good candidates for the prevention of infection.

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trans-Acting Factors Affecting Carbon Catabolite Repression of the hut Operon inBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.181.9.2883-2888.1999 ◽

1999 ◽

Vol 181 (9) ◽

pp. 2883-2888 ◽

Cited By ~ 27

Author(s):

Jill M. Zalieckas ◽

Lewis V. Wray ◽

Susan H. Fisher

Keyword(s):

Catabolite Repression ◽

Double Mutant ◽

Minimal Medium ◽

Carbon Catabolite Repression ◽

Regulatory Protein ◽

Luria Bertani ◽

Factors Affecting ◽

Glucose Minimal Medium ◽

Rates Of Growth ◽

In Cells

ABSTRACT In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, theptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither theptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, theptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnAmutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.

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Comparative Analysis of Shigella boydii 18 Foodborne Outbreak Isolate and Related Enteric Bacteria: Role of rpoS and adiA in Acid Stress Response

Journal of Food Protection ◽

10.4315/0362-028x-68.3.521 ◽

2005 ◽

Vol 68 (3) ◽

pp. 521-527 ◽

Cited By ~ 10

Author(s):

YVONNE C. CHAN ◽

HANS P. BLASCHEK

Keyword(s):

Minimal Medium ◽

Enteric Bacteria ◽

Arginine Decarboxylase ◽

Luria Bertani ◽

Decarboxylase Activity ◽

E Coli ◽

Rpos Gene ◽

Shigella Boydii ◽

Luria Bertani Broth ◽

K 12

Shigella boydii CDPH (Chicago Department of Public Health) serotype 18 was implicated in an outbreak of foodborne illness in 1998. The suspected food vehicles were parsley and cilantro imported from Mexico used to prepare bean salad. Previous studies revealed that S. boydii CDPH serotype 18 can survive in bean salad, which contains organic acids and whose pH decreases over time. Acid challenge assays in acidified tryptic soy broth at pH 4.5, acidified Luria-Bertani broth at pH 4.5, and acidified M9 minimal salts medium at pH 2.5 containing amino acids, arginine, or glutamic acid were performed using S. boydii CDPH, S. boydii ATCC 35966, S. flexneri 3136, Escherichia coli O157:H7 dd8872, and E. coli O157:H7 dd642 to compare differences in acid tolerance. Differences in survival of exponential-phase cells were detected in acidified tryptic soy broth and Luria-Bertani broth at pH 4.5. In acidified minimal medium containing arginine, S. boydii strains were able to survive at pH 2.5. The arginine decarboxylase gene (adiA) present in S. boydii is involved in survival at extremely low pH. The discovery of adiA expression in S. boydii serotype 18 by use of an acidified minimal medium challenge and arginine decarboxylase biochemical assay is significant because arginine decarboxylase activity was thought to be unique to E. coli. Sequencing of the rpoS gene from the S. boydii outbreak strain indicates that it is 99% conserved compared with the E. coli K-12 rpoS gene and plays a vital role in survival under acidic conditions.

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