Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1723-1734 ◽  
Author(s):  
Ramakrishnan Srikumar ◽  
Leonie G. Mikael ◽  
Peter D. Pawelek ◽  
Ali Khamessan ◽  
Bernard F. Gibbs ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Pasteurella Multocida ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Actinobacillus Pleuropneumoniae ◽  
Sole Source ◽  
Genomic Libraries ◽  
Iron Deficient ◽  
Serotype 1 ◽  
Internal Peptide

From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of ∼105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for −35 and −10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.

Fhua and HgbA, outer membrane proteins ofActinobacilluspleuropneumoniae: their role as virulence determinants

2006 ◽  
Vol 52 (4) ◽  
pp. 391-396 ◽  
Author(s):  
Lara Shakarji ◽  
Leonie G Mikael ◽  
Ramakrishnan Srikumar ◽  
Marylène Kobisch ◽  
James W Coulton ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Growth Promotion ◽  
Outer Membrane Proteins ◽  
Actinobacillus Pleuropneumoniae ◽  
Membrane Receptors ◽  
Virulence Determinants ◽  
Serotype 1 ◽  
Specific Pathogen ◽  
Polymerase Chain ◽  
Different Sources

For the recently described serotype 15 of biotype I and serotypes 13 and 14 of biotype II of Actinobacillus pleuropneumoniae, fhuA and hgbA were detected by polymerase chain reaction and DNA sequencing. To determine the substrate specificity of the iron receptors FhuA and HgbA and to study their role in the virulence of A. pleuropneumoniae, we used two isogenic A. pleuropneumoniae serotype 1 deletion mutants of fhuA and hgbA. Different sources of iron and siderophores were tested in growth promotion assays. FhuA and HgbA are specific for their ligands ferrichrome and hemoglobin, respectively. The virulence of the two deletion mutant strains was evaluated in experimental infections using specific pathogen-free piglets. While the fhuA mutant (DG02) was as highly virulent as the parental strain S4074, the virulence of the hgbA mutant (ΔhgbA) was reduced. Our data indicate that both FhuA and HgbA are conserved among all serotypes and biotypes of A. pleuropneumoniae and that HgbA, the receptor for porcine hemoglobin, may play a role in virulence.Key words: Actinobacillus pleuropneumoniae, iron uptake, outer membrane receptors, virulence.

scholarly journals RGEN-seq For Highly Sensitive Amplification-free Screen Of Off-target Sites Of Gene Editors

2021 ◽  
Author(s):  
Alexander Kuzin ◽  
Brendan Redler ◽  
Jaya Onuska ◽  
Alexei Slesarev
Keyword(s):  
Target Detection ◽  
Dna Cleavage ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Detailed Comparison ◽  
Detection Methods ◽  
Guide Rnas ◽  
Coverage Bias ◽  
Biochemical Assays ◽  
Target Sites

Abstract Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, significant technical and methodological issues still remain.. Of note, existing methods rely on PCR amplification, tagging, and affinity purification which can introduce bias, contaminants, sample loss through handling, etc. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. Through use of novel sequencing adapters, the RGEN-Seq method saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs.

scholarly journals Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Janine T. Bossé ◽  
Yanwen Li ◽  
Leon G. Leanse ◽  
Liqing Zhou ◽  
Roy R. Chaudhuri ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Pasteurella Multocida ◽  
Insertion Site ◽  
Genomic Analysis ◽  
Actinobacillus Pleuropneumoniae ◽  
Functional Genomic ◽  
Transposase Gene ◽  
Functional Genomic Analysis ◽  
Veterinary Importance

AbstractComprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.

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