Promoter activity of ORF-less gene cassettes isolated from the oral metagenome

AbstractIntegrons are genetic elements consisting of a functional platform for recombination and expression of gene cassettes (GCs). GCs usually carry promoter-less open reading frames (ORFs), encoding proteins with various functions including antibiotic resistance. The transcription of GCs relies mainly on a cassette promoter (PC), located upstream of an array of GCs. Some integron GCs, called ORF-less GCs, contain no identifiable ORF with a small number shown to be involved in antisense mRNA mediated gene regulation.In this study, promoter sequences were identified, usingin silicoanalysis, within GCs PCR amplified from the oral metagenome. The promoter activity of ORF-less GCs was verified by cloning them upstream of agusAreporter, proving they can function as a promoter, presumably allowing bacteria to adapt to multiple stresses within the complex physico-chemical environment of the human oral cavity. A bi-directional promoter detection system was also developed allowing direct identification of clones with promoter-containing GCs on agar plates. Novel promoter-containing GCs were identified from the human oral metagenomic DNA using this construct, called pBiDiPD.This is the first demonstration and detection of promoter activity of ORF-less GCs and the development of an agar plate-based detection system will enable similar studies in other environments.

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Development of a 4-valent genotyping assay for direct identification of the most frequent Pseudomonas aeruginosa serotypes from respiratory specimens of pneumonia patients

Journal of Medical Microbiology ◽

10.1099/jmm.0.066043-0 ◽

2014 ◽

Vol 63 (4) ◽

pp. 508-517 ◽

Cited By ~ 1

Author(s):

Holger Koch ◽

Thomas Emrich ◽

Sandra Jampen ◽

Marianne Wyss ◽

Verena Gafner ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Limit Of Detection ◽

High Sensitivity ◽

Cross Reactivity ◽

Open Reading Frames ◽

Direct Identification ◽

Genotyping Assay ◽

Reading Frames ◽

Respiratory Specimens ◽

Single Tube Reaction

Pseudomonas aeruginosa is a common cause of nosocomial infections and is associated with high rates of mortality. In order to facilitate rapid and sensitive identification of the most prevalent serotypes of P. aeruginosa, we have developed a 4-valent real-time PCR-based assay using oligonucleotides specific for open-reading frames constituting the O-antigen-specific lipopolysaccharide loci of P. aeruginosa. The assay simultaneously detects and differentiates between each of the four serotypes IATS-O1, -O6, -O11 and serogroup 2 (IATS-O2, -O5, and -O16) with high sensitivity and specificity in a single-tube reaction. No cross-reactivity was observed with other serotypes of P. aeruginosa or other microbial species, and reproducibility was demonstrated regardless of template, i.e. purified DNA, bacterial culture and clinical specimens (broncho-alveolar lavage). The limit of detection of the assay was approximately 100 copies per reaction for IATS-O1, -O2 and -O11, and 50 copies per reaction for IATS-O6. Comparison of the assay specificity with a commercially available slide agglutination kit showed consistent results; however, the number of non-typable isolates was reduced by 15 % using the genotyping assay. Use of the 4-valent genotyping assay in the context of a clinical trial resulted in identification of pneumonia patients positive for the IATS-O11 serotype, and hence eligible for therapy with panobacumab (an investigational monoclonal antibody against the O-polysaccharide of serotype IATS-O11).

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ColE1-Like Plasmid pIP843 of Klebsiella pneumoniae Encoding Extended-Spectrum β-Lactamase CTX-M-17

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.5.1212-1217.2002 ◽

2002 ◽

Vol 46 (5) ◽

pp. 1212-1217 ◽

Cited By ~ 71

Author(s):

Van Cao ◽

Thierry Lambert ◽

Patrice Courvalin

Keyword(s):

Klebsiella Pneumoniae ◽

Transcriptional Start Site ◽

Open Reading Frames ◽

Single Amino Acid ◽

Consensus Sequences ◽

Promoter Sequences ◽

Initiation Of Replication ◽

The Stability ◽

Rna I ◽

Reading Frames

ABSTRACT The resistance of Klebsiella pneumoniae BM4493, isolated in Ho Chi Minh City, Vietnam, to cefotaxime and aztreonam was due to production of a novel β-lactamase, CTX-M-17. The bla CTX-M-17 gene was borne by 7,086-bp plasmid pIP843, which was entirely sequenced and which was found to belong to the ColE1 family. The 876-bp bla CTX-M-17 gene differed from bla CTX-M-14 by 2 nucleotides, which led to the single amino acid substitution Glu289→Lys. bla CTX-M-17 was flanked upstream by an ISEcp1-like element and downstream by an insertion sequence (IS) IS903 variant designated IS903-C. The transcriptional start site of bla CTX-M-17 was located 109 nucleotides upstream from the initiation codon in the ISEcp1-like element, which also provided the promoter sequences. Plasmid pIP843, which was non-self-transferable and nonmobilizable, contained five open reading frames transcribed in the same orientation. Regions homologous to sequences coding for putative RNA II and RNA I transcripts, a rom gene, which is involved in initiation of replication, and a cer-like gene, which is responsible for the stability of ColE1-like plasmids, were identified. Consensus sequences for putative replication (oriV) and transfer (oriT) origins were present. Results of primer extension experiments indicated that ISEcp1 provides the promoter for expression of bla CTX-M-17 and may contribute to dissemination of this gene.

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Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA

Applied and Environmental Microbiology ◽

10.1128/aem.67.11.5240-5246.2001 ◽

2001 ◽

Vol 67 (11) ◽

pp. 5240-5246 ◽

Cited By ~ 127

Author(s):

H. W. Stokes ◽

Andrew J. Holmes ◽

Blair S. Nield ◽

Marita P. Holley ◽

K. M. Helena Nevalainen ◽

...

Keyword(s):

Genetic Diversity ◽

Sequence Data ◽

Environmental Dna ◽

Gene Families ◽

Soil Samples ◽

Open Reading Frames ◽

Sequence Information ◽

Gene Cassettes ◽

Conserved Sequence ◽

Reading Frames

ABSTRACT The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.

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Independent regulation of H-NS-mediated silencing of the bgl operon at two levels: upstream by BglJ and LeuO and downstream by DnaKJ

Microbiology ◽

10.1099/mic.0.28080-0 ◽

2005 ◽

Vol 151 (10) ◽

pp. 3349-3359 ◽

Cited By ~ 24

Author(s):

S. Madhusudan ◽

Andreas Paukner ◽

Yvonne Klingen ◽

Karin Schnetz

Keyword(s):

Escherichia Coli ◽

Transcription Initiation ◽

Promoter Activity ◽

Transposon Mutagenesis ◽

Open Reading Frames ◽

Wild Type ◽

Coding Region ◽

First Time ◽

Insertion Mutations ◽

Reading Frames

Silencing of the Escherichia coli bgl operon by the histone-like nucleoid-structuring protein H-NS occurs at two levels. Binding of H-NS upstream of the promoter represses transcription initiation, whilst binding within the coding region is also proposed to repress transcription elongation. The latter, downstream level of repression is counteracted by the protease Lon and, thus, silencing of the bgl operon is more effective in lon mutants. Transposon-mutagenesis screens for suppression of this lon phenotype on bgl were performed and insertion mutations disrupting rpoS and crl were obtained, as well as mutations mapping upstream of the open reading frames of bglJ, leuO and dnaK. In rpoS and crl mutants, bgl promoter activity is known to be higher. Likewise, as shown here, bgl promoter activity is increased in the bglJ and leuO mutants, which express BglJ and LeuO constitutively. However, BglJ and LeuO have no impact on downstream repression. A dnaKJ mutant was isolated for the first time in the context of the bgl operon. The mutant expresses lower levels of DnaK than the wild-type. Interestingly, in this dnaKJ : : miniTn10 mutant, downstream repression of bgl by H-NS is less effective, whilst upstream repression by H-NS remains unaffected. Together, the data show that the two levels of bgl silencing by H-NS are regulated independently.

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Characterization of SepL of EnterohemorrhagicEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.182.22.6490-6498.2000 ◽

2000 ◽

Vol 182 (22) ◽

pp. 6490-6498 ◽

Cited By ~ 50

Author(s):

Andreas U. Kresse ◽

Fabrizio Beltrametti ◽

Astrid Müller ◽

Frank Ebel ◽

Carlos A. Guzmán

Keyword(s):

Protein Localization ◽

Open Reading Frames ◽

Start Codon ◽

Exponential Growth Phase ◽

Promoter Sequences ◽

Enterocyte Effacement ◽

Extension Analysis ◽

Reading Frames

ABSTRACT The sepL gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, thesepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from theesp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of thesepL start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions −5 and −36. Translational fusions using lacZ as a reporter gene demonstrated that sepL is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37°C, a nutrient-rich environment, high osmolarity, and the presence of Mn2+. Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. ThissepL mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sepL gene intrans. The EDL933ΔsepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sepL in trans. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.

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