scholarly journals Quantification of Expression of Staphylococcus epidermidis Housekeeping Genes with Taqman Quantitative PCR during In Vitro Growth and under Different Conditions

2001 ◽  
Vol 183 (24) ◽  
pp. 7094-7101 ◽  
Author(s):  
S. J. Vandecasteele ◽  
W. E. Peetermans ◽  
R. Merckx ◽  
J. Van Eldere
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Quantitative Pcr ◽  
Housekeeping Genes ◽  
Rrna Gene ◽  
In Vitro Growth ◽  
The 16S Rrna Gene

ABSTRACT The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene;hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10°C heat shock (37 to 47°C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings forEscherichia coli that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).

scholarly journals Yersinia nurmii sp. nov.

2011 ◽  
Vol 61 (10) ◽  
pp. 2368-2372 ◽  
Author(s):  
Anna Murros-Kontiainen ◽  
Maria Fredriksson-Ahomaa ◽  
Hannu Korkeala ◽  
Per Johansson ◽  
Riitta Rahkila ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Trees ◽  
Housekeeping Genes ◽  
Rrna Gene ◽  
Yersinia Ruckeri ◽  
Phenotypic Properties ◽  
Broiler Meat ◽  
Dna Reassociation ◽  
The 16S Rrna Gene

This study was set up to identify three Gram-negative, rod-shaped strains originating from broiler meat packaged under a modified atmosphere. A polyphasic taxonomic approach, including multilocus sequence analysis (MLSA) of five genes (16S rRNA, glnA, gyrB, recA and HSP60), DNA–DNA reassociation between the closest phylogenetic neighbours and determination of relevant phenotypic properties, was applied. Phylogenetic analysis of the 16S rRNA gene sequences grouped these strains together and within the genus Yersinia. MLSA of the 16S rRNA gene and four housekeeping genes showed that the strains formed a monophyletic group separate from other Yersinia species in all phylogenetic trees constructed. The strains had a phenotypic profile different from those of other representatives of the genus Yersinia, but most similar to that of Yersinia ruckeri. Typical virulence markers for pathogenic Yersinia were not detected. Based on phylogenetic, phenotypic and DNA–DNA reassociation data, a novel species, Yersinia nurmii sp. nov., is proposed for the isolated strains. The type strain is APN3a-cT ( = DSM 22296T  = LMG 25213T).

Genetic analysis of housekeeping genes of members of the genus Acholeplasma: Phylogeny and complementary molecular markers to the 16S rRNA gene

2007 ◽  
Vol 44 (2) ◽  
pp. 699-710 ◽  
Author(s):  
Dmitriy V. Volokhov ◽  
Alexander A. Neverov ◽  
Joseph George ◽  
Hyesuk Kong ◽  
Sue X. Liu ◽  
...  
Keyword(s):  
Molecular Markers ◽  
16S Rrna ◽  
Genetic Analysis ◽  
16S Rrna Gene ◽  
Housekeeping Genes ◽  
Rrna Gene ◽  
The 16S Rrna Gene

scholarly journals Proposal of Vespertiliibacter pulmonis gen. nov., sp. nov. and two genomospecies as new members of the family Pasteurellaceae isolated from European bats

2014 ◽  
Vol 64 (Pt_7) ◽  
pp. 2424-2430 ◽  
Author(s):  
Kristin Mühldorfer ◽  
Stephanie Speck ◽  
Gudrun Wibbelt
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Housekeeping Genes ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Content Type ◽  
Link Type ◽  
The Family ◽  
The 16S Rrna Gene

Five bacterial strains isolated from bats of the family Vespertilionidae were characterized by phenotypic tests and multilocus sequence analysis (MLSA) using the 16S rRNA gene and four housekeeping genes (rpoA, rpoB, infB, recN). Phylogenetic analyses of individual and combined datasets indicated that the five strains represent a monophyletic cluster within the family Pasteurellaceae . Comparison of 16S rRNA gene sequences demonstrated a high degree of similarity (98.3–99.9 %) among the group of bat-derived strains, while searches in nucleotide databases indicated less than 96 % sequence similarity to known members of the Pasteurellaceae . The housekeeping genes rpoA, rpoB, infB and recN provided higher resolution compared with the 16S rRNA gene and subdivided the group according to the bat species from which the strains were isolated. Three strains derived from noctule bats shared 98.6–100 % sequence similarity in all four genes investigated, whereas, based on rpoB, infB and recN gene sequences, 91.8–96 % similarity was observed with and between the remaining two strains isolated from a serotine bat and a pipistrelle bat, respectively. Genome relatedness as deduced from recN gene sequences correlated well with the results of MLSA and indicated that the five strains represent a new genus. Based on these results, it is proposed to classify the five strains derived from bats within Vespertiliibacter pulmonis gen. nov., sp. nov. (the type species), Vespertiliibacter genomospecies 1 and Vespertiliibacter genomospecies 2. The genus can be distinguished phenotypically from recognized genera of the Pasteurellaceae by at least three characteristics. All strains are nutritionally fastidious and require a chemically defined supplement with NAD for growth. The DNA G+C content of strain E127/08T is 38.2 mol%. The type strain of Vespertiliibacter pulmonis gen. nov., sp. nov. is E127/08T ( = CCUG 64585T = DSM 27238T). The reference strains of Vespertiliibacter genomospecies 1 and 2 are E145/08 and E157/08, respectively.

scholarly journals Usefulness of rpoB Gene Sequencing for Identification of Afipia and Bosea Species, Including a Strategy for Choosing Discriminative Partial Sequences

2003 ◽  
Vol 69 (11) ◽  
pp. 6740-6749 ◽  
Author(s):  
Atieh Khamis ◽  
Philippe Colson ◽  
Didier Raoult ◽  
Bernard La Scola
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Housekeeping Genes ◽  
Rpob Gene ◽  
Universal Primers ◽  
Strain Identification ◽  
Rrna Gene ◽  
Dna Relatedness ◽  
The 16S Rrna Gene

ABSTRACT Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability “hot spots” in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

scholarly journals Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae

2004 ◽  
Vol 54 (5) ◽  
pp. 1601-1609 ◽  
Author(s):  
Henrik Christensen ◽  
Peter Kuhnert ◽  
John Elmerdahl Olsen ◽  
Magne Bisgaard
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Sensu Stricto ◽  
Rrna Gene ◽  
Ratio Test ◽  
Sequence Comparisons ◽  
The Individual ◽  
The 16S Rrna Gene

Phylogenies of housekeeping gene and 16S rRNA gene sequences were compared to improve the classification of the bacterial family Pasteurellaceae and knowledge of the evolutionary relationships of its members. Deduced partial protein sequences of the housekeeping genes atpD, infB and rpoB were compared in 28, 36 and 28 representative taxa of the Pasteurellaceae, respectively. The monophyly of representatives of the genus Gallibacterium was recognized by analysis of all housekeeping genes, while members of Mannheimia, Actinobacillus sensu stricto and the core group of Pasteurella sensu stricto formed monophyletic groups with two out of three housekeeping genes. Representatives of Mannheimia, Actinobacillus sensu stricto, [Haemophilus] ducreyi and [Pasteurella] trehalosi formed a monophyletic unit by analysis of all three housekeeping genes, which was in contrast to the 16S rRNA gene-derived phylogeny, where these taxa occurred at separate positions in the phylogenetic tree. Representatives of the Rodent, Avian and Aphrophilus–Haemophilus 16S rRNA gene groups were weakly supported by phylogenetic analysis of housekeeping genes. Phylogenies derived by comparison of the housekeeping genes diverged significantly from the 16S rRNA gene-derived phylogeny as evaluated by the likelihood ratio test. A low degree of congruence was also observed between the individual housekeeping gene-derived phylogenies. Estimates on speciation derived from 16S rRNA and housekeeping gene sequence comparisons resulted in quite different evolutionary scenarios for members of the Pasteurellaceae. The phylogeny based on the housekeeping genes supported observed host associations between Mannheimia, Actinobacillus sensu stricto and [Pasteurella] trehalosi and animals with paired hooves.

scholarly journals Genetic diversity of European phytoplasmas of the 16SrV taxonomic group and proposal of ‘Candidatus Phytoplasma rubi’

2011 ◽  
Vol 61 (9) ◽  
pp. 2129-2134 ◽  
Author(s):  
Sylvie Malembic-Maher ◽  
Pascal Salar ◽  
Luisa Filippin ◽  
Patricia Carle ◽  
Elisa Angelini ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Housekeeping Genes ◽  
Taxonomic Group ◽  
Rrna Gene ◽  
Insect Vector ◽  
Flavescence Dorée ◽  
The 16S Rrna Gene ◽  
Specific Sequences

In addition to the grapevine flavescence dorée phytoplasmas, other members of taxonomic group 16SrV phytoplasmas infect grapevines, alders and species of the genera Clematis and Rubus in Europe. In order to investigate which phytoplasmas constitute discrete, species-level taxa, several strains were analysed by comparing their 16S rRNA gene sequences and a set of five housekeeping genes. Whereas 16S rRNA gene sequence similarity values were >97.5 %, the proposed threshold to distinguish two ‘Candidatus Phytoplasma’ taxa, phylogenetic analysis of the combined sequences of the tuf, rplV-rpsC, rplF-rplR, map and uvrB-degV genetic loci showed that two discrete phylogenetic clusters could be clearly distinguished. The first cluster grouped flavescence dorée (FD) phytoplasmas, alder yellows (AldY) phytoplasmas, Clematis (CL) phytoplasmas and the Palatinate grapevine yellows (PGY) phytoplasmas. The second cluster comprised Rubus stunt (RS) phytoplasmas. In addition to the specificity of the insect vector, the Rubus stunt phytoplasma contained specific sequences in the 16S rRNA gene. Hence, the Rubus stunt phytoplasma 16S rRNA gene was sufficiently differentiated to represent a novel putative taxon: ‘Candidatus Phytoplasma rubi’.

Identification of 16S rRNA mutations in Mycoplasma genitalium potentially associated with tetracycline resistance in vivo but not selected in vitro in M. genitalium and Chlamydia trachomatis

2021 ◽  
Author(s):  
Chloé Le Roy ◽  
Arabella Touati ◽  
Carla Balcon ◽  
Justine Garraud ◽  
Jean-Michel Molina ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Tetracycline Resistance ◽  
Mycoplasma Genitalium ◽  
Rrna Gene ◽  
Resistant Mutants ◽  
The 16S Rrna Gene

Abstract Objectives Tetracyclines are widely used for the treatment of bacterial sexually transmitted infections (STIs) and recently have been used successfully for post-exposure prophylaxis of STIs in MSM. We investigated the in vitro and in vivo development of tetracycline resistance in Chlamydia trachomatis and Mycoplasma genitalium and evaluated 16S rRNA mutations associated with acquired resistance in other bacteria. Methods In vitro selection of resistant mutants of reference strains of C. trachomatis and M. genitalium was undertaken by serial passage in medium containing subinhibitory concentrations of tetracycline or doxycycline, respectively. The 16S rRNA gene of the two microorganisms was amplified and sequenced at different passages, as were those of 43 C. trachomatis- and 106 M. genitalium-positive specimens collected in France from 2013 to 2019. Results No tetracycline- or doxycycline-resistant strains of C. trachomatis and M. genitalium, respectively, were obtained after 30 serial passages. The tetracycline and doxycycline MICs were unchanged and analysis of the 16S rRNA gene, the molecular target of tetracyclines, of C. trachomatis and M. genitalium revealed no mutation. No mutation in the 16S rRNA gene was detected in C. trachomatis-positive specimens. However, six M. genitalium-positive specimens harboured a mutation potentially associated with tetracycline resistance without known prior tetracycline treatment for patients. Conclusions Tetracyclines did not select in vitro-resistant mutants of C. trachomatis or M. genitalium. However, 16S rRNA mutations either responsible for or associated with tetracycline resistance in other bacteria, including mycoplasma species, were identified in several M. genitalium-positive specimens.

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