The Riboflavin Transporter RibU in Lactococcus lactis: Molecular Characterization of Gene Expression and the Transport Mechanism

ABSTRACT This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.

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Transcriptional control of the bovine leukemia virus genome: role and characterization of a non-immunoglobulin plasma protein from bovine leukemia virus-infected cattle.

Journal of Virology ◽

10.1128/jvi.50.1.267-270.1984 ◽

1984 ◽

Vol 50 (1) ◽

pp. 267-270 ◽

Cited By ~ 26

Author(s):

P Gupta ◽

S V Kashmiri ◽

J F Ferrer

Keyword(s):

Plasma Protein ◽

Bovine Leukemia Virus ◽

Transcriptional Control ◽

Leukemia Virus ◽

Virus Genome ◽

Infected Cattle

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Structural Characterization of HP1264 Reveals a Novel Fold for the Flavin Mononucleotide Binding Protein

Biochemistry ◽

10.1021/bi301714a ◽

2013 ◽

Vol 52 (9) ◽

pp. 1583-1593 ◽

Cited By ~ 3

Author(s):

Ki-Young Lee ◽

Ji-Hun Kim ◽

Kyu-Yeon Lee ◽

Jiyun Lee ◽

Ingyun Lee ◽

...

Keyword(s):

Structural Characterization ◽

Binding Protein ◽

Flavin Mononucleotide

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Molecular Characterization of TRI12 Which Encodes an Apparent Transport Protein Involved in Trichothecene Production by Fusarium sporotrjchioides.

Cereal Research Communications ◽

10.1007/bf03543723 ◽

1997 ◽

Vol 25 (3) ◽

pp. 347-348 ◽

Cited By ~ 2

Author(s):

N. J. Alexander ◽

T. M. Hohn ◽

S. P. McCormick

Keyword(s):

Molecular Characterization ◽

Transport Protein

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Mitochondrial phosphate transport. Large scale isolation and characterization of the phosphate transport protein from beef heart mitochondria.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)47273-5 ◽

1984 ◽

Vol 259 (14) ◽

pp. 9115-9120 ◽

Cited By ~ 11

Author(s):

H V Kolbe ◽

D Costello ◽

A Wong ◽

R C Lu ◽

H Wohlrab

Keyword(s):

Large Scale ◽

Phosphate Transport ◽

Transport Protein ◽

Heart Mitochondria ◽

Beef Heart ◽

Isolation And Characterization ◽

Beef Heart Mitochondria

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Location and characterization of aminopeptidase N in Lactococcus lactis subsp. cremoris HP

Applied Microbiology and Biotechnology ◽

10.1007/bf00174202 ◽

1992 ◽

Vol 37 (1) ◽

pp. 46-54 ◽

Cited By ~ 25

Author(s):

Fred A. Exterkate ◽

Marian de Jong ◽

Gerrie J. C. M. de Veer ◽

Ronald Baankreis

Keyword(s):

Lactococcus Lactis ◽

Aminopeptidase N ◽

Lactococcus Lactis Subsp

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Transcriptional Analysis of the Adeno-Associated Virus Integration Site

Journal of Virology ◽

10.1128/jvi.01754-09 ◽

2009 ◽

Vol 83 (23) ◽

pp. 12512-12525 ◽

Cited By ~ 5

Author(s):

Nathalie Dutheil ◽

Els Henckaerts ◽

Erik Kohlbrenner ◽

R. Michael Linden

Keyword(s):

Transcriptional Activity ◽

Integration Site ◽

Regulatory Elements ◽

Transcriptional Analysis ◽

Start Codon ◽

Adeno Associated Virus ◽

Site Specific ◽

Complex Interactions ◽

Site Specific Integration ◽

Virus Integration

ABSTRACT The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

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Gene Cloning and Molecular Characterization of a Two-Enzyme System Catalyzing the Oxidative Detoxification of β-Endosulfan

Applied and Environmental Microbiology ◽

10.1128/aem.68.12.6237-6245.2002 ◽

2002 ◽

Vol 68 (12) ◽

pp. 6237-6245 ◽

Cited By ~ 36

Author(s):

Tara D. Sutherland ◽

Irene Horne ◽

Robyn J. Russell ◽

John G. Oakeshott

Keyword(s):

Shuttle Vector ◽

Open Reading Frame ◽

Cosmid Library ◽

Flavin Mononucleotide ◽

Flavin Reductase ◽

Reading Frame ◽

Reductase Gene ◽

Degradation Activity ◽

Layer Chromatography

ABSTRACT The gram-positive bacterium Mycobacterium sp. strain ESD is able to use the cyclodiene insecticide endosulfan as a source of sulfur for growth. This activity is dependent on the absence of sulfite or sulfate in the growth medium. A cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia coli shuttle vector and screened for endosulfan-degrading activity in Mycobacterium smegmatis, a species that does not degrade endosulfan. Using this method, we identified a single cosmid that conferred sulfur-dependent endosulfan-degrading activity on the host strain. An open reading frame (esd) was identified within this cosmid that, when expressed behind a constitutive promoter in a mycobacterial expression vector, conferred sulfite- and sulfate-independent β-endosulfan degradation activity on the recombinant strain. The translation product of this gene (Esd) had up to 50% sequence identity with an unusual family of monooxygenase enzymes that use reduced flavins, provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial open reading frame was located upstream of the Esd gene that had sequence homology to the same monooxygenase family. A flavin reductase gene, identified in the M. smegmatis genome, was cloned, expressed, and used to provide reduced flavin mononucleotide for Esd in enzyme assays. Thin-layer chromatography and gas chromatography analyses of the enzyme assay mixtures revealed the disappearance of β-endosulfan and the appearance of the endosulfan metabolites, endosulfan monoaldehyde and endosulfan hydroxyether. This suggests that Esd catalyzes the oxygenation of β-endosulfan to endosulfan monoaldehyde and endosulfan hydroxyether. Esd did not degrade either α-endosulfan or the metabolite of endosulfan, endosulfan sulfate.

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Cloning and characterization of four SIR genes of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.688 ◽

1986 ◽

Vol 6 (2) ◽

pp. 688-702 ◽

Cited By ~ 144

Author(s):

J M Ivy ◽

A J Klar ◽

J B Hicks

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Blot Analysis ◽

Transcriptional Control ◽

Genomic Library ◽

Yeast Saccharomyces Cerevisiae ◽

Mating Type Genes ◽

Rna Blots

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.

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