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1.
J Bacteriol ; 183(22): 6565-72, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11673426

RESUMO

The rpmA gene, which encodes 50S ribosomal subunit protein L27, was cloned from the extreme thermophile Aquifex aeolicus, and the protein was overexpressed and purified. Comparison of the A. aeolicus protein with its homologue from Escherichia coli by circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas the E. coli protein is unstructured under the same conditions. A mutant of E. coli that lacks L27 was found earlier to be impaired in the assembly and function of the 50S subunit; both defects could be corrected by expression of E. coli L27 from an extrachromosomal copy of the rpmA gene. When A. aeolicus L27 was expressed in the same mutant, an increase in the growth rate occurred and the "foreign" L27 protein was incorporated into E. coli ribosomes. However, the presence of A. aeolicus L27 did not promote 50S subunit assembly. Thus, while the A. aeolicus protein can apparently replace its E. coli homologue functionally in completed ribosomes, it does not assist in the assembly of E. coli ribosomes that otherwise lack L27. Possible explanations for this paradoxical behavior are discussed.


Assuntos
Proteínas de Bactérias , Escherichia coli/genética , Bacilos e Cocos Aeróbios Gram-Negativos/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/química , Alinhamento de Sequência , Transformação Bacteriana
3.
Biochim Biophys Acta ; 1353(2): 137-47, 1997 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-9294008

RESUMO

The rpmB,G operon of Escherichia coli codes for proteins L28 and L33 of the larger (50S) ribosomal subunit. Strains with mutations in this operon can help define the roles of these proteins in ribosome synthesis and function. One such strain, BM108, makes neither protein and is unable to synthesize completed ribosomes; instead ribonucleoproteins accumulate, in the form of '30S material' and '47S particles'. However, when protein L28 is supplied from a plasmid, the growth rate, the kinetics of ribosome synthesis and the coordination of ribosomal protein synthesis are no different from that in wild-type organisms even though protein L33 is missing. This suggests that the latter protein can be redundant for ribosome synthesis and function. Another mutant strain, BM81, has a frameshift mutation that gives rise to an oversized protein L28. This mutant accumulates 30S material and 47S particles during slow exponential growth. The composition of the 47S particles from strains BM81, BM108 and a third mutant strain, TP28, suggests that their defining feature is the absence of L28; this is further evidence for an important role for this protein in ribosome assembly. Accumulation of ribonucleoproteins in strains BM81 and BM108 leads to some loss of the ordinarily precise coordination of synthesis of ribosomal proteins. We describe and discuss the characteristic features of this unbalanced synthesis.


Assuntos
Compostos de Anilina , Proteínas de Bactérias , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Ribossômicas/biossíntese , Ribossomos/genética , Fracionamento Celular , Escherichia coli/fisiologia , Genes Bacterianos , Mutação , Óperon , Plasmídeos , Proteínas Ribossômicas/fisiologia , Ribossomos/fisiologia
4.
J Bacteriol ; 179(8): 2486-93, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9098043

RESUMO

The rpmBG operon of Escherichia coli codes for ribosomal proteins L28 and L33. Two strains with mutations in the operon are AM81, whose ribosomes lack protein L28, and AM90, whose ribosomes are without protein L33. Neither strain showed major defects in ribosome assembly. However, when the mutations were transferred to other strains of E. coli, ribosome synthesis was greatly perturbed and precursor ribonucleoproteins accumulated. In the new backgrounds, the mutation in rpmB was complemented by synthesis of protein L28 from a plasmid; the rpmG mutation was not complemented by protein L33 because synthesis of protein L28 from the upstream rpmB gene was also greatly reduced. The results suggest that protein L33, in contrast to protein L28, has at best a minor role in ribosome assembly and function.


Assuntos
Compostos de Anilina , Proteínas de Bactérias , Proteínas de Escherichia coli , Escherichia coli/genética , Mutação/genética , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Mutação da Fase de Leitura , Teste de Complementação Genética , Dados de Sequência Molecular , Óperon/genética , Fenótipo , Proteínas Ribossômicas/biossíntese , Análise de Sequência , Especificidade da Espécie , Supressão Genética , Transdução Genética
5.
Mol Microbiol ; 23(2): 237-45, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9044258

RESUMO

Strain BM108 of Escherichia coli has a chromosomal mutation in the rpmB,G operon that prevents synthesis of ribosomal proteins L28 and L33. The mutation was lethal unless synthesis of protein L28 was induced from a plasmid. Without protein L28, RNA and protein synthesis were linear rather than exponential. No 70S ribosomes were made. Instead, RNA accumulated in '30S material' and '47S particles'; the latter were distinct from 50S ribosomal subunits, lacked proteins L28 and L33 and had substoicheometric amounts of three other proteins. When L28 synthesis was induced (but protein L33 was still absent), the strain grew as well as, and assembled 70S ribosomes with similar kinetics to, a wild-type control. Thus, protein L28 is required for ribosome assembly in strain BM108 while protein L33 has no significant effect on ribosome synthesis or function.


Assuntos
Compostos de Anilina , Proteínas de Bactérias/fisiologia , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Ribossômicas/fisiologia , Ribossomos/fisiologia , Proteínas de Bactérias/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Vetores Genéticos , Mutação , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Transfecção
6.
Mol Microbiol ; 26(5): 1083-96, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9426144

RESUMO

In contrast to enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants. Although a chemotaxis operon has been identified containing homologues of the enteric cheA, cheW, cheR genes and two homologues of the cheY gene, deletion of the entire chemotaxis operon had only minor effects on chemotactic behaviour under the conditions tested. Responses to sugars were enhanced in tethered cells but in all other chemotaxis assays behaviour of the operon deletion mutant was wild type. The mutant also showed wild-type responses to weak organic acids such as acetate and propionate, the dominant chemoattractants for this organism, under all conditions. This is in direct contrast to the enterics in which CheA, CheW and CheY are absolutely essential for taxis to PTS sugars, oxygen and MCP-dependent chemoeffectors. The operon deletion mutant was subjected to Tn5 transposon mutagenesis and new mutants selected using a chemotaxis and phototaxis screen. One mutant, JPA203, was non-chemotactic on swarm plates and showed inverted responses when tethered or subjected to changes in light intensity. Characterization of the Tn5 insertion in JPA203 identified a second chemotaxis operon in R. sphaeroides that contains homologues of cheY, cheA and cheR, the first homologue of cheB and two homologues of cheW. The new genes were labelled orf10, cheY(III), cheA(II) cheW(II), cheW(III), cheR(II), cheB and tlpC. When introduced into a wild-type background, deletion of cheA(II) produced a chemotaxis minus phenotype in R. sphaeroides, suggesting that cheA(II) forms part of a dominant chemotactic pathway, whereas the earlier identified operon plays only a minor role under laboratory conditions. The data presented here support the existence of two chemosensory pathways in R. sphaeroides, a feature that so far is unique in bacterial chemotaxis.


Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia , Proteínas de Membrana/metabolismo , Proteínas Quinases/metabolismo , Rhodobacter sphaeroides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Elementos de DNA Transponíveis , DNA Bacteriano , Deleção de Genes , Genótipo , Proteínas de Membrana/genética , Proteínas Quimiotáticas Aceptoras de Metil , Metiltransferases/genética , Metiltransferases/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Óperon , Fenótipo , Proteínas Quinases/genética , Rhodobacter sphaeroides/genética , Homologia de Sequência de Aminoácidos
8.
J Gen Microbiol ; 139(4): 707-16, 1993 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7685806

RESUMO

The rpmB,G operon of Escherichia coli codes for the synthesis of ribosomal proteins L28 and L33. In one mutant strain (TP28), these two proteins are made at about half their normal rates, ribosome assembly is greatly perturbed and precursor particles accumulate. The mutation in strain TP28 is in a Shine-Dalgarno sequence in the leader region of the rpmB,G messenger RNA. Another mutant, strain AM108, makes neither protein because it has the mobile element IS1 inserted into the rpmB coding sequence. Surprisingly, ribosome assembly in this strain is virtually normal with respect to growth rate. Strain AM90, which fails to make protein L33, has the element IS3 inserted into rpmG and also shows no major defects in ribosome assembly.


Assuntos
Compostos de Anilina , Proteínas de Bactérias , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Sequência de Bases , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Escherichia coli/metabolismo , Genes Bacterianos , Dados de Sequência Molecular , Mutação , Óperon , RNA Bacteriano/metabolismo , Proteínas Ribossômicas/biossíntese , Ribossomos/metabolismo , Especificidade da Espécie
9.
J Gen Microbiol ; 135(3): 575-81, 1989 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2482857

RESUMO

Strains of Escherichia coli can be isolated that require erythromycin for growth. With one strain, AM, a range of antibiotics, including chloramphenicol, tetracycline, spectinomycin, kasugamycin and rifampicin, will substitute for erythromycin on solid and in liquid media; nalidixic acid supports growth in liquid but not on solid media. With a second strain, 103, chloramphenicol, tetracycline and spectinomycin support growth in liquid media but on solid medium only chloramphenicol substitutes for erythromycin. In media of higher than normal ionic strength, strain AM, but not strain 103, can grow in the absence of antibiotics. Possible reasons for these complex phenotypes are discussed.


Assuntos
Aminoglicosídeos , Eritromicina/metabolismo , Escherichia coli/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/biossíntese , Cloranfenicol/metabolismo , Meios de Cultura , DNA Bacteriano/biossíntese , Fenótipo , RNA Bacteriano/biossíntese , Rifampina/metabolismo , Espectinomicina/metabolismo , Tetraciclina/metabolismo
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