Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli

Nazos, Penelope M.; Antonucci, Tammy K.; Landick, Robert; Oxender, Dale L.

doi:10.1128/jb.166.2.565-573.1986

Cited by 26 publications

(15 citation statements)

References 35 publications

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“…This revealed a clear difference in the rate of degradation of the transcripts ( mRNA degradation (1, 3, 4, 12, 13, 21). Differential expression is especially prevalent when one or more of the gene products is an integral membrane protein (2,8,10,20), as is the case with arsB (26). The molecular mechanism responsible for the decreased expression of these proteins is obscure.…”

Section: Methodsmentioning

confidence: 99%

Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon

Owolabi

Rosen

1990

J Bacteriol

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The arsenical resistance (ars) operon of the conjugative plasmid R773 encodes an ATP-driven anion extrusion pump, conferring bacterial resistance to arsenicals. The operon contains a regulatory gene, arsR, and three structural genes, arsA, arsB, and arsC. The hydrophilic ArsA and ArsC proteins are produced in large amounts, but the hydrophobic ArsB protein, an integral membrane polypeptide, is synthesized in limited quantities. Northern (RNA-DNA) hybridizations provide evidence that the inducible operon is regulated at the level of transcription. The genes were transcribed in the presence of an inducer (arsenite) as a single polycistronic mRNA with an approximate size of 4.4 kilobases (kb). This transcript was processed to generate relatively stable mRNA species: one of 2.7 kb, encoding the ArsR and ArsA proteins, and a second of 0.5 kb, encoding the ArsC protein. Segmental differences in stability within the polycistronic transcript are proposed to account for the differential expression of the ars genes. In addition, analysis of the mRNA structure at the 5' end of arsB suggests a potential translational block to the synthesis of this membrane protein.The arsenical resistance (ars) operon of resistance plasmid R773 confers resistance to arsenite, arsenate, and antimonite on Escherichia coli cells by the synthesis of an anion pump (23). This unique oxyanion-translocating ATPase, induced by the presence of its substrates, mediates their active extrusion from cells with energy derived from ATP (18,22,23,28). Thus, resistance results from a lowering of the intracellular concentration of the toxic oxyanion.A 4.3-kilobase (kb) HindIll fragment from R factor R773 was cloned into the vector pBR322 to produce a recombinant plasmid which produces constitutive resistance to arsenicals (17). Analysis of the nucleotide sequence of this fragment reveals three structural genes: arsA, arsB, and arsC (6). From the genetic evidence (7,22) and from the nucleotide sequence (6), the oxyanion pump was predicted to be composed of a complex of the 63-kilodalton ArsA and the 45.5-kilodalton ArsB proteins. The 16-kilodalton ArsC protein appears to act as a modifier subunit and is not necessary for arsenite resistance or transport (22). The ArsA protein was purified from the cytosol and shown to be an oxyanionstimulated ATPase (23). The hydrophobic ArsB protein has been identified as an inner membrane protein by creation of a gene fusion of the arsB gene with lacZ (26). It can be visualized as a [35S]methionine-labeled membrane protein when made in a T7 expression vector but is not present in amounts sufficient to be visible as a Coomassie blue-or silver-stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even though the operon is transcribed in high amounts by using the T7 expression system (31).Recently, the regulatory gene, arsR, has been identified on a 0.73-kb EcoRI-HindIII fragment contiguous with the 4. quence of the arsR gene has been determined, and its product, the ArsR protein, has been identified.Alt...

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Section: Methodsmentioning

confidence: 99%

Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon

Owolabi

Rosen

1990

J Bacteriol

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“…Previous studies involving purified BPs showed that LIV-BP, encoded by the livJ gene, binds isoleucine, leucine, and valine with K d values of 10 Ϫ6 to 10 Ϫ7 M and threonine, serine, and alanine with lower affinity and that LS-BP, encoded by the livK gene, binds leucine with a K d value of approximately 10 Ϫ6 M (24). To enable the ATP-hydrolysis-coupled transport of their substrates into the cytoplasm, LIV-BP and LS-BP interact with the common inner-membrane components LivHMGF, which constitute the LIV-I and LS systems, respectively (1,28,29,45,50). These six liv genes are clustered at 77 min on the chromosome (45) and divided into two transcription units, one for livJ and the other for livKHMGF (1,18,45).…”

Section: Vol 186 2004mentioning

confidence: 99%

Identification of the LIV-I/LS System as the Third Phenylalanine Transporter in Escherichia coli K-12

et al. 2004

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In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain (⌬aroP ⌬pheP ⌬mtr ⌬tna ⌬tyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The K m values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 M, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.It has been reported that Escherichia coli has five distinct transport systems (AroP, Mtr, PheP, TnaB, and TyrP) for the accumulation of aromatic amino acids (36). A general amino acid permease, encoded by the aroP gene, transports three aromatic amino acids with high affinity (8,11,21,36). The closely related PheP protein transports phenylalanine in preference to tyrosine but does not exhibit tryptophan uptake activity (10,(34)(35)(36). Mtr and TyrP are specific for tryptophan and tyrosine, respectively (19,36,42,51,52), and TnaB is a low-affinity, tryptophan-specific transporter encoded in the tryptophanase operon together with the tnaA gene (13,36,43).In a previous study, we cloned the tyrosine transporter tutB gene of Erwinia herbicola and used E. coli cells to determine the properties of its product (23). In the course of that study, we found that the aromatic amino acid transporter-deficient E. coli strain TK1135 (⌬aroP ⌬pheP mtr24 ⌬tna ⌬tyrP) (23) has the ability to accumulate phenylalanine in an energy-dependent manner, although the initial rate of uptake, as well as the steady-state level, was quite low. This finding prompted us to examine the basis for this activity and whether this transport activity is physiologically important in E. coli. Here, we present evidence indicating that a branched-chain amino acid transporter, the LIV-I/LS system (1-3, 18, 24, 28, 29, 32, 37, 38, 45, 50), acts as the third phenylalanine transporter, plays a significant role in the accumu...

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“…The brnQ gene product, BrnQ protein, is very hydrophobic, similar to that found in the LivH protein, which is a membrane-bound component of the LIV-I system in Escherichia coli (Nazos et al, 1986). The BrnQ protein contains several hydrophobic segments consisting of an average of 30 amino acid residues, which may span the cell membrane (Fig.…”

Section: Discussionmentioning

confidence: 80%

Cloning and nucleotide sequence of the brnQ gene, the structural gene for a membrane-associated component of the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium.

Ohnishi

Hasegawa

Matsubara

et al. 1988

The Japanese journal of genetics

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Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli

Cited by 26 publications

References 35 publications

Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon

Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon

Identification of the LIV-I/LS System as the Third Phenylalanine Transporter in Escherichia coli K-12

Cloning and nucleotide sequence of the brnQ gene, the structural gene for a membrane-associated component of the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium.

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