A bidirectional rho-independent transcription terminator between the E. coli tonB gene and an opposing gene

Postle, Kathleen; Good, Rhonda F.

doi:10.1016/s0092-8674(85)80030-1

Cited by 74 publications

(52 citation statements)

References 42 publications

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“…The putative terminator has a stable stem-loop structure that is preceded by four A's and followed by four U's. This arrangement is characteristic of bidirectional rho-independent terminators (32,41).…”

Section: Resultsmentioning

confidence: 98%

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Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum

et al. 1991

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Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.Bacteriocins are antimicrobial peptides or proteins formed by bacteria. The potential applications for bacteriocin-producing lactic acid bacteria in food preservation has stimulated interest in the characterization of these substances. However, maintaining activity during isolation and purification has proved difficult, and the full or partial amino acid sequences of only five such bacteriocins have been reported. The most extensively studied is nisin A (2, 3, 7, 34), a posttranslationally modified bacteriocin from Lactococcus lactis subsp. lactis. Nisin has been approved for use as a preservative in foods in over 45 countries (9). It is ribosomally synthesized (6, 10) and is one of a group of lantibiotics that possess lanthionine-or methyllanthionine-containing rings resulting from the attack of cysteine sulfhydryl groups on dehydroalanine or dehydrobutyrine residues (derived from serine or threonine). Partial characterization of lactacin 481 from L. lactis shows that it also contains lanthionine rings, but the full sequence has not yet been published (31). In contrast, lactocin S from Lactobacillus sake (27)

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Section: Resultsmentioning

confidence: 98%

“…6). This has the features associated with a bidirectional rho-independent termination site (32). The phy, dialysis, and high pH conditions were avoided because they resulted in large losses in activity.…”

Section: Resultsmentioning

confidence: 99%

Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum

et al. 1991

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“…Since tetA and the terminator were inserted in the direction opposite to secY, the stem-loop structure of the terminator introduced downstream of secY was expected to stabilize the secY transcript, as postulated by Ueguchi et al 9) A &-independent terminator has been reported to function in both directions. 24,25) The tetA gene encodes a membrane protein and might enhance the defective phenotype of the ÁrpmJ mutant by jamming the translocase. Hence we constructed another ÁrpmJ mutant, AY201, by replac- The rpmJ gene of the spc operon was replaced with tetA and cat to construct AY101 and AY201 respectively, as described in ''Materials and Methods''.…”

Section: Resultsmentioning

confidence: 99%

Disruption ofrpmJEncoding Ribosomal Protein L36 Decreases the Expression ofsecYUpstream of thespcOperon and Inhibits Protein Translocation inEscherichia coli

Ikegami

Nishiyama

Matsuyama

et al. 2005

Bioscience, Biotechnology, and Biochemistry

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The spc operon of Escherichia coli encodes 11 ribosomal proteins and SecY. The secY gene and downstream rpmJ encoding a ribosomal protein, L36, are located distal to the promoter of the spc operon. It has been suggested that the stability of SecY mRNA depends on rpmJ unless a &-independent terminator is inserted immediately downstream of secY. Moreover, it has been suggested that RpmJ is dispensable for E. coli. We constructed rpmJ null strains, AY101 (ÁrpmJ::tetA) and AY201 (ÁrpmJ::cat), by replacing rpmJ with tetA, which encodes a membrane protein responsible for tetracycline-resistance, and cat, which encodes a cytoplasmic chloramphenicol acetyltransferase, respectively. Depletion of RpmJ did not inhibit protein synthesis, whereas the growth of AY101 was defective at high temperatures. The level of SecY mRNA decreased significantly in both disruptants even though the &-independent terminator was inserted immediately downstream of secY. Some periplasmic proteins were missing in the disruptants with a concomitant increase in the amount of phage shock protein in the inner membrane. These phenotypes caused by the rpmJ null mutation were corrected by a plasmid carrying secY, but not by one carrying rpmJ.

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“…Downstream from the 3' ORF stop codon two possible stemloop structures were found covering nucleotides 6717 -6741 and nucleotides 6743 -6772. The first is preceded by a stretch of adenine residues (eight out of nine, nucleotides 671 1 -6719), suggesting that it may function as a rho-independent transcription terminator in the reverse orientation [26]. The 4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320 4330 4340 4350 4360 4370 4380 4390 4400 4410 4420 4430 5410 5420 5430 5440 5450 5460 5470 5480 5490 5500 5510 5520 TTTCCTTGATCGAGTGGCTGTCGGGGCGGGACCGGCCGTTTTTCCGCGGCTTGATGTTGTGCGTCTTCATGTACATAGCGTTTTTACTGGCGCGCCAGCTCGGCTTGGCGhCG~GC~.G 6490 6500 6510 6520 6530 6540 6550 6560 6570 6580 6590 6600 - d G values predicted for these stem-loop structures are -55.2 kJ/mol and -48.5 kJ/mol, respectively (see Fig.…”

Section: Resultsmentioning

confidence: 99%

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Borges

Hawkins

Packman

et al. 1990

European Journal of Biochemistry

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A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5' end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191,, was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, M , 46265) and 469 amino acids (E3, M , 49193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-0x0-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D ) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.The oxidative decarboxylation of 2-0x0-acids is accomplished in most organisms by the 2-0x0-acid dehydrogenase multienzyme complexes, producing the corresponding acyl-CoA and NADH. For the pyruvate dehydrogenase (PDH) complex, the constituent enzymes that function successively in the mechanism are pyruvate dehydrogenase (El), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3) (for recent reviews see [I, 21).The PDH complex from the thermophilic, Gram-positive bacterium Bacillus stearothermophilus is assembled round a core of the E2 component of 60 copies of the E2 polypeptide chain organized with icosahedral symmetry [3]. Multiple cop- ies of the El (comprising two polypeptide subunits, Elcl and Elp) and E3 components are attached to this core tightly but non-covalently. The M, of the individual components as well as the morphology of the E2 core [3, 41 closely resemble those of the PDH complexes of eukaryotes. Its thermostability makes the B. stearothermophilus PDH complex a good system for studying the structure of a 2-0x0-acid dehydrogenase complex based on icosahedral symmetry, which differs from the PDH and 2-oxoglutarate dehydrogenase complexes of Escherichia coli in that the latter have octahedral E2 cores [5, 61. The sequence of the first 21 1 amino acid residues from the N-terminus of the B. stearothermophilus E2 chain has been determined [7], showing that this ace...

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A bidirectional rho-independent transcription terminator between the E. coli tonB gene and an opposing gene

Cited by 74 publications

References 42 publications

Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum

Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum

Disruption ofrpmJEncoding Ribosomal Protein L36 Decreases the Expression ofsecYUpstream of thespcOperon and Inhibits Protein Translocation inEscherichia coli

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

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