scholarly journals Bifidobacterium lactis DSM 10140: Identification of the atp (atpBEFHAGDC) Operon and Analysis of Its Genetic Structure, Characteristics, and Phylogeny

2004 ◽  
Vol 70 (5) ◽  
pp. 3110-3121 ◽  
Author(s):  
Marco Ventura ◽  
Carlos Canchaya ◽  
Douwe van Sinderen ◽  
Gerald F. Fitzgerald ◽  
Ralf Zink
Keyword(s):  
Transcription Initiation ◽  
Bacterial Species ◽  
Blot Hybridization ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Rrna Gene ◽  
Specific Sequence ◽  
Bifidobacterium Lactis ◽  
Genes Encoding ◽  
Pcr Products

ABSTRACT The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F1F0-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.

scholarly journals A PCR Detection Method for Rapid Identification ofMelissococcus pluton in Honeybee Larvae

1998 ◽  
Vol 64 (5) ◽  
pp. 1983-1985 ◽  
Author(s):  
V. A. Govan ◽  
V. Br�zel ◽  
M. H. Allsopp ◽  
S. Davison
Keyword(s):  
Sequence Data ◽  
Bacterial Species ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Rrna Gene ◽  
16S Rrna Sequence ◽  
Growth Requirements ◽  
Detection And Identification ◽  
Pcr Products ◽  
Pure Cultures

ABSTRACT Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. plutonby sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.

scholarly journals Rapid Identification, Differentiation, and Proposed New Taxonomic Classification of Bifidobacterium lactis

2002 ◽  
Vol 68 (12) ◽  
pp. 6429-6434 ◽  
Author(s):  
Marco Ventura ◽  
Ralf Zink
Keyword(s):  
Pcr Primers ◽  
Rapid Identification ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Specific Detection ◽  
Bifidobacterium Lactis ◽  
Bifidobacterium Animalis ◽  
Subspecies Level ◽  
Internally Transcribed Spacer

ABSTRACT Identification of Bifidobacterium lactis and Bifidobacterium animalis is problematic because of phenotypic and genetic homogeneities and has raised the question of whether they belong to one unique taxon. Analysis of the 16S-23S internally transcribed spacer region of B. lactis DSM10140T, B. animalis ATCC 25527T, and six potential B. lactis strains suggested two distinct clusters. Two specific 16S-23S spacer rRNA gene-targeted primers have been developed for specific detection of B. animalis. All of the molecular techniques used (B. lactis or B. animalis PCR primers, enterobacterial repetitive intergenic consensus PCR) demonstrated that B. lactis and B. animalis form two main groups and suggest a revision of the strains assigned to B. animalis. We propose that B. lactis should be separated from B. animalis at the subspecies level.

scholarly journals PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria

1999 ◽  
Vol 65 (4) ◽  
pp. 1652-1657 ◽  
Author(s):  
Sara Hallin ◽  
Per-Eric Lindgren
Keyword(s):  
Wastewater Treatment ◽  
Activated Sludge ◽  
Nitrite Reductase ◽  
Wastewater Treatment Plants ◽  
Paracoccus Denitrificans ◽  
Pcr Primers ◽  
Genes Encoding ◽  
Pcr Products ◽  
Key Enzyme ◽  
Sequence Relationships

ABSTRACT Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplifycd 1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships ofnir genes were also established. Thecd 1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.

scholarly journals Specific PCR detection of Peptostreptococcus magnus

2003 ◽  
Vol 52 (4) ◽  
pp. 309-313 ◽  
Author(s):  
M.P. Riggio ◽  
A. Lennon
Keyword(s):  
Pcr Primers ◽  
Oral Bacteria ◽  
Rrna Gene ◽  
Subgingival Plaque ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Specific Pcr ◽  
Wide Range ◽  
Pcr Products ◽  
Peptostreptococcus Magnus

Peptostreptococcus magnus is the most pathogenic and one of the most common Gram-positive anaerobic cocci found in human clinical specimens. The organism has been isolated in pure culture from a range of serious infections, including meningitis and endocarditis. However, isolation of Peptostreptococcus magnus from the oral cavity has rarely been attempted. Identification of Peptostreptococcus magnus in clinical specimens is reliant upon microbiological culture and biochemical methods, which often give ambiguous results. The aim of this study was to develop a PCR assay for the specific detection of Peptostreptococcus magnus in oral clinical specimens. PCR primers specific for Peptostreptococcus magnus DNA were derived by comparison of 16S rRNA gene sequences and selection of primers that demonstrated specificity at their 3′ ends for Peptostreptococcus magnus. PCR positivity for Peptostreptococcus magnus DNA was indicated by the amplification of a 553 bp product. The PCR assay was then used to attempt detection of Peptostreptococcus magnus DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. The PCR assay was demonstrated to be highly specific for Peptostreptococcus magnus DNA, since no PCR products were obtained when genomic DNA from a wide range of other oral bacteria, including closely related Peptostreptococcus species, was used in the PCR assay. Confirmation of specific amplification of Peptostreptococcus magnus DNA was obtained by digestion of PCR products with the restriction endonuclease RsaI, which gives a unique restriction profile for Peptostreptococcus magnus. Of the 33 subgingival plaque samples analysed, 2 (6 %) were positive for Peptostreptococcus magnus DNA. None of the 60 pus aspirates analysed was positive for Peptostreptococcus magnus DNA. It is concluded that Peptostreptococcus magnus is not a major pathogen in adult periodontitis or dento-alveolar abscesses. The PCR assay provides a more rapid, specific and sensitive alternative to conventional methods for identification of Peptostreptococcus magnus in clinical specimens.

Differentiation of Schistosoma mansoni from S. rodhaini using cloned DNA probes

Parasitology ◽  
1989 ◽  
Vol 98 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Tina K. Walker ◽  
A. J. G. Simpson ◽  
D. Rollinson
Keyword(s):  
Schistosoma Mansoni ◽  
Blot Hybridization ◽  
Repeated Sequence ◽  
Transcription Unit ◽  
Hybridization Analysis ◽  
Rrna Gene ◽  
Dot Blot ◽  
Restriction Sites ◽  
Genes Encoding ◽  
Female Specific

SummaryThe ribosomal RNA (rRNA) gene units of Schistosoma mansoni and S. rodhaini, of the lateral spined egg group, have been studied. The schistosome rRNA gene unit consists of a regular interspersion of the two genes encoding the large and small rRNA units with two spacers. The large spacer is not transcribed while the small spacer is part of the transcription unit. Variation in the rRNA gene unit between the two species is demonstrated to take the form of loss or gain of restriction sites within the non-transcribed and transcribed spacers. This variation has been demonstrated to enable the differentiation of S. mansoni from S. rodhaini by Southern hybridization analysis. In addition, a DNA clone representing a female specific, tandemly repeated sequence, is demonstrated to enable differentiation of S. mansoni and S. rodhaini using dot blot hybridization analysis.

scholarly journals Molecular Characterization of Legionella Populations Present within Slow Sand Filters Used for Fungal Plant Pathogen Suppression in Horticultural Crops

2003 ◽  
Vol 69 (1) ◽  
pp. 533-541 ◽  
Author(s):  
Leo A. Calvo-Bado ◽  
J. Alun W. Morgan ◽  
Martin Sergeant ◽  
Tim R. Pettitt ◽  
John M. Whipps
Keyword(s):  
Bacterial Community ◽  
Quantitative Pcr ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Primers ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Sand Column ◽  
Dominant Band ◽  
Pcr Products

ABSTRACT The total bacterial community of an experimental slow sand filter (SSF) was analyzed by denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA gene PCR products. One dominant band had sequence homology to Legionella species, indicating that these bacteria were a large component of the SSF bacterial community. Populations within experimental and commercial SSF units were studied by using Legionella-specific PCR primers, and products were studied by DGGE and quantitative PCR analyses. In the experimental SSF unit, the DGGE profiles for sand column, reservoir, storage tank, and headwater tank samples each contained at least one intense band, indicating that a single Legionella strain was predominant in each sample. Greater numbers of DGGE bands of equal intensity were detected in the outflow water sample. Sequence analysis of these PCR products showed that several Legionella species were present and that the organisms exhibited similarity to strains isolated from environmental and clinical samples. Quantitative PCR analysis of the SSF samples showed that from the headwater sample through the sand column, the number of Legionella cells decreased, resulting in a lower number of cells in the outflow water. In the commercial SSF, legionellae were also detected in the sand column samples. Storing prefilter water or locating SSF units within greenhouses, which are often maintained at temperatures that are higher than the ambient temperature, increases the risk of growth of Legionella and should be avoided. Care should also be taken when used filter sand is handled or replaced, and regular monitoring of outflow water would be useful, especially if the water is used for misting or overhead irrigation.

scholarly journals Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

1998 ◽  
Vol 64 (2) ◽  
pp. 795-799 ◽  
Author(s):  
Julian R. Marchesi ◽  
Takuichi Sato ◽  
Andrew J. Weightman ◽  
Tracey A. Martin ◽  
John C. Fry ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Amplification ◽  
Environmental Samples ◽  
Bacterial Species ◽  
Pcr Primers ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Specific Pcr

ABSTRACT We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.

scholarly journals Identification by 16S rRNA Gene Analyses of a Potential Novel Mycobacterial Species as an Etiological Agent of Canine Leproid Granuloma Syndrome

2000 ◽  
Vol 38 (3) ◽  
pp. 953-959 ◽  
Author(s):  
M. S. Hughes ◽  
G. James ◽  
N. Ball ◽  
M. Scally ◽  
R. Malik ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Blot Hybridization ◽  
Etiological Agent ◽  
Rrna Gene ◽  
Fresh Tissue ◽  
Variable Regions ◽  
Pcr Products ◽  
Tissue Specimens ◽  
The 16S Rrna Gene

PCR amplifications of the 16S rRNA gene were performed on 46 specimens obtained from 43 dogs with canine leproid granuloma syndrome to help determine its etiology. Sequence capture PCR was applied to 37 paraffin-embedded specimens from 37 dogs, and nested PCR was attempted on DNA from 9 fresh tissue specimens derived from 3 of the 37 aforementioned dogs and from an additional 6 dogs. Molecular analyses of the paraffin-embedded tissues and fresh tissue specimen analyses were performed at separate institutions. PCR products with identical sequences over a 350-bp region encompassing variable regions 2 and 3 of the 16S rRNA gene were obtained from 4 of 37 paraffin-embedded specimens and from all 9 specimens of fresh tissue originating from 12 of the 43 dogs. Identical sequences were determined from amplicons obtained from paraffin-embedded and fresh specimens from one dog. The consensus DNA sequence, amplified from paraffin-embedded tissue and represented by GenBank accession no. AF144747, shared highest nucleotide identity (99.4% over 519 bp) with mycobacterial strain IWGMT 90413 but did not correspond exactly to any EMBL or GenBank database sequence. With a probe derived from the V2 region of the novel canine sequence, reverse cross blot hybridization identified an additional four paraffin-embedded specimens containing the same novel sequence. In total, molecular methodologies identified the proposed novel mycobacterial sequence in 16 of 43 dogs with canine leproid granuloma syndrome, indicating that the species represented by this sequence may be the principal etiological agent of canine leproid granuloma syndrome.

In Situ, Real-Time Catabolic Gene Expression: Extraction and Characterization of Naphthalene Dioxygenase mRNA Transcripts from Groundwater

1999 ◽  
Vol 65 (1) ◽  
pp. 80-87 ◽  
Author(s):  
Mark S. Wilson ◽  
Corien Bakermans ◽  
Eugene L. Madsen
Keyword(s):  
Coal Tar ◽  
Sodium Dodecyl ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Contaminated Site ◽  
Sequence Comparisons ◽  
Degrading Bacteria ◽  
Genes Encoding ◽  
Pcr Products

ABSTRACT We developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-μm-pore-size filters, which were then frozen in dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify nahAc homologs. Several primer pairs were found to amplify nahAc homologs representing the entire diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of nahAchomologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes ndoB anddntAc, previously cloned from Pseudomonas putida NCIB 9816-4 and Burkholderia sp. strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To our knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches.

scholarly journals Emergence of CTX-M-3, TEM-1 and a new plasmid-mediated MOX-4 AmpC in a multiresistant Aeromonas caviae isolate from a patient with pneumonia

2010 ◽  
Vol 59 (7) ◽  
pp. 843-847 ◽  
Author(s):  
Ying Ye ◽  
Xi-Hai Xu ◽  
Jia-Bin Li
Keyword(s):  
Pulmonary Infection ◽  
Blot Hybridization ◽  
Severe Pneumonia ◽  
Southern Blot Hybridization ◽  
Amino Acid Sequences ◽  
Aeromonas Caviae ◽  
Genes Encoding ◽  
Southern Blot Hybridization Analysis ◽  
First Time ◽  
Encoding Genes

Aeromonas species rarely cause pulmonary infection. We report, for what is believed to be the first time, a case of severe pneumonia in a cancer patient caused by Aeromonas caviae. Detailed microbiological investigation revealed that this isolate carried three β-lactamase-encoding genes (encoding MOX-4, CTX-M-3 and TEM-1) conferring resistance to all β-lactams but imipenem. The β-lactamase with a pI of 9.0 was transferred by conjugation and associated with a 7.3 kb plasmid, as demonstrated by Southern blot hybridization. Analysis of the nucleotide and amino acid sequences showed a new ampC gene that was closely related to those encoding the MOX-1, MOX-2 and MOX-3 β-lactamases. This new plasmid-mediated AmpC β-lactamase from China was named MOX-4. This is believed to be the first report of MOX-4, CTX-M-3 and TEM-1 β-lactamases in a multiresistant A. caviae.

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