Probable insensitivity of mollicutes to rifampin and characterization of spiroplasmal DNA-dependent RNA polymerase

Gadeau, Alain‐Pierre; Mouchès, C.; Bové, Joseph M.

doi:10.1128/jb.166.3.824-828.1986

Cited by 36 publications

(20 citation statements)

References 19 publications

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“…This rifampin sensitivity argues against contaminating Mycoplasma RNAP activity, as Mycoplasma spp. are unique among eubacteria in exhibiting 100-fold greater resistance to rifampin (17). We thus conclude that the transcriptional activity of our RNAP extract is derived from C. trachomatis.…”

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confidence: 69%

Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter

Tan

Engel

1996

J Bacteriol

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Chlamydia trachomatis RNA polymerase was partially purified by heparin-agarose chromatography and used in conjunction with a plasmid-borne G-less cassette template to characterize the C. trachomatis rRNA P1 promoter in vitro. Stepwise mutational analysis revealed that sequences in the ؊10, ؊25, and ؊35 regions are necessary for promoter activity, but no sequence upstream of position ؊40 is required. Partially purified C. trachomatis RNA polymerase and purified Escherichia coli holoenzyme exhibited some differences in promoter specificity.Chlamydia trachomatis is a gram-negative obligate intracellular pathogen with a complex life cycle characterized by two serial morphologic forms (reviewed in references 26 and 34). Stage-specific expression has been demonstrated in early (38) and late (1-3, 8, 14, 19, 28) periods of the developmental cycle, but the molecular basis of this gene regulation is not understood. Direct genetic studies of promoter structures have not been feasible; transient transformation of chlamydiae has been described (35), but a practical DNA-mediated transformation system does not exist.In the absence of a genetic system, several approaches have been taken to identify and characterize chlamydial promoters. The transcription initiation sites of several abundant transcripts have been identified, and their upstream regions have been examined for putative promoter sequences. By inspection, appropriately spaced canonical Escherichia coli 70 -type promoter sequences are present in a few of these upstream regions. These include the C. trachomatis plasmid countertranscript (PCT) (15), envA P1 (13, 22), and the dnaK operon of the C. trachomatis mouse pneumonitis biovar (11). Others, including the hctB, ltuA, and ltuB genes, have 70 promoterlike sequences at the putative Ϫ10 region only. Interestingly, these three genes are all transcribed late in the developmental cycle (14). However, many chlamydial genes are not preceded by 70 -like promoter sequences (i.e., there is no more than a 50% match), and no consensus chlamydial promoter sequence is apparent (8). Explanations for this sequence diversity include the possibilities that (i) some of the presumptive chlamydial transcription initiation sites are instead processing sites, (ii) the putative promoters belong to more than one class of promoters, and (iii) there is latitude in the promoter specificity of C. trachomatis A RNA polymerase (RNAP). The subunits of C. trachomatis RNAP have been cloned and sequenced (9,10,18,21).A , a C. trachomatis 70 homolog (9, 21), is the only sigma factor identified in chlamydiae to date.A and 70 share striking amino acid sequence conservation in subregions 2.4 and 4.2 (9, 21) of 70 , including the specific amino acid residues which have been shown in E. coli to be involved in promoter recognition at the Ϫ10 and Ϫ35 positions (reviewed in reference 5). This conservation of amino acid residues involved in promoter recognition contrasts with the observed variability in the DNA sequences of the putative C. trachomatis promote...

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confidence: 69%

Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter

Tan

Engel

1996

J Bacteriol

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“…4) were identified upstream of ORFs I, II, IV, and V. The promoter of the spiralin gene was shown to be functional in an E. coli promoter selection vector. The spiroplasmal core RNA polymerase has the eubacterial subunit structure (a23PP') (11), which recognizes spiroplasmal promoters with the consensus sequence TATAAT at -10 and TTGACA at -35 (6).…”

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confidence: 99%

Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein

Chevalier¹,

Saillard²,

Bové³

1990

J Bacteriol

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The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf-X-spiralin gene-pfl-pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction.Spiroplasmas are mollicutes (mycoplasmas) (class Mollicutes) with motility and helical morphology (33). Analysis of the Spiroplasma genome by classical genetic techniques has been difficult. The dependence of these organisms on complex growth media and their poorly defined metabolic pathways explain the paucity of auxotrophic mutants and hence of genetic markers in these procaryotes. In the absence of such markers, characterization of cloned DNA fragments provides the means for analyzing the structural organization of Spiroplasma genes.The gene for spiralin seemed to be of particular interest. Spiralin (35) is the major membrane protein of Spiroplasma citri, an important plant pathogen (28). It is apparently a transmembrane amphiphilic protein (34), and it is acylated (37). Acylation seems to be a characteristic of several membrane proteins of mollicutes. The role of spiralin is not known. It must, however, be a key protein of the spiroplasmal membrane, since spiralinlike proteins occur in Spiroplasma species other than S. citri (37) and probably in all spiroplasmas. Elucidation of the amino acid sequence of spiralin as deduced from the nucleotide sequence of its gene would undoubtly contribute to a better understanding of its structure and function, as well as the organization of its gene. Sequence determination of the spiralin gene was made possible by the fact that we have previously cloned the gene.

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“…These authors found that Clostridium innocuum, Clostridium ramosum, and six species of mollicutes (belonging to four genera) were all resistant to high levels of rifampin. Generally, eubacteria are susceptible to rifampin, an inhibitor of ribonucleic acid polymerase; some mollicutes, however, are resistant (10).…”

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Pyrophosphate-Dependent Enzymes in Walled Bacteria Phylogenetically Related to the Wall-Less Bacteria of the Class Mollicutes

Petzel

Hartman

Allison

1989

International Journal of Systematic Bacteriology

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Some of the wall-less bacteria of the class Mollicutes (mycoplasmas) have pyrophosphate (PP,)-dependent enzymic activities, including PP,-dependent phosphofructokinase (PP,-PFK) , PP,-dependent nucleoside kinase, and pyruvate,orthophosphate dikinase (PPDK) activities. In most other bacteria, adenosine 5'-triphosphate (ATP), not PP,, is the cofactor of analogous enzymic reactions. Because PP,-dependent enzymes are more common among mollicutes than other bacteria, we describe here an examination of the six walled bacteria that have been reported to be phylogenetically related to the mollicutes (Clostridium innocuum, Clostridium ramosum, Erysipelothrix rhusiopathiae, Lactobacillus catenaformis, Lactobacillus vitulinus, and Streptococcus pleomorphus) for PP,-PFK, ATP-dependent PFK, phosphoenolpyruvate carboxytransphosphorylase, PPDK, and PP,-and ATP-dependent acetate kinases. Two anaerobic mollicutes, Anaeroplasma intermedium and Asteroleplasma anuerobium, were also tested. C. innocuum, E. rhusiopathiue, S . pleomorphus, and Anueroplasma intermedium had PPi-PFK activities, whereas C . ramosum, the two lactobacilli, and Asteroleplasma anaerobium had only ATP-dependent PFK activities. Asteroleplasma anaerobium and all of the walled bacteria except E. rhusiopathiue had PPDK activities. All of the species except Asteroleplasma anaerobium and E . rhusiopathiae also had pyruvate kinase activities; the effects of allosteric activators were tested. Phosphoenolpyruvate carboxytransphosphorylase was detected by using two methods in C . innocuum, C . ramosum, and S . pleomorphus. All of the species tested had ATP-dependent acetate kinase activities, but none had detectable PP,-dependent acetate kinase activity. The occurrence of one or more PPi-dependent enzymes in the mollicutes and their walled relatives is a phenotypic indicator of the phylogenetic relatedness of these organisms. The distribution of these enzymes among members of this group substantiates the subgroups proposed by other workers who used 16s ribosomal ribonucleic acid analysis.The wall-less eubacteria belonging to the genera Mycoplasma, Ureaplasma , Spiroplasma, Acholeplasma, Anaeroplasma, and Asteroleplasma are grouped together in the class Mollicutes. Phylogenetic analyses of 5s and 16s ribosomal ribonucleic acid (rRNA) sequences have revealed that the mollicutes cluster with the low-guanine-plus-cytosine content subdivision of gram-positive eubacteria that includes the bacillus-lactobacillus-streptococcus branch (31, 40, 41). Among the members of the low-guanine-plus-cytosine content gram-positive eubacteria, the mollicutes are most closely related to Clostridium innocuum, Clostridium ramosum, Erysipelothrix rhusiopathiae, Lactobacillus catenaformis (Lactobacillus catenaforme), Lactobacillus vitulinus, and Streptococcus pleomorphus (16,31,[40][41][42] The mollicutes and their walled phylogenetic relatives have few phenotypic similarities. The only comparative study of the phenotypes of the mollicutes and their walled phylogenetic relatives was conducted by...

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Probable insensitivity of mollicutes to rifampin and characterization of spiroplasmal DNA-dependent RNA polymerase

Cited by 36 publications

References 19 publications

Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter

Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter

Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein

Pyrophosphate-Dependent Enzymes in Walled Bacteria Phylogenetically Related to the Wall-Less Bacteria of the Class Mollicutes

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