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1.
Mol Ecol ; 31(16): 4319-4331, 2022 08.
Article in English | MEDLINE | ID: mdl-35762848

ABSTRACT

After gastrulation, oviductal hypoxia maintains turtle embryos in an arrested state prior to oviposition. Subsequent exposure to atmospheric oxygen upon oviposition initiates recommencement of embryonic development. Arrest can be artificially extended for several days after oviposition by incubation of the egg under hypoxic conditions, with development recommencing in an apparently normal fashion after subsequent exposure to normoxia. To examine the transcriptomic events associated with embryonic arrest in green sea turtles (Chelonia mydas), RNA-sequencing analysis was performed on embryos from freshly laid eggs and eggs incubated in either normoxia (oxygen tension ~159 mmHg) or hypoxia (<8 mmHg) for 36 h after oviposition (n = 5 per group). The patterns of gene expression differed markedly among the three experimental groups. Normal embryonic development in normoxia was associated with upregulation of genes involved in DNA replication, the cell cycle, and mitosis, but these genes were commonly downregulated after incubation in hypoxia. Many target genes of hypoxia inducible factors, including the gene encoding insulin-like growth factor binding protein 1 (igfbp1), were downregulated by normoxic incubation but upregulated by incubation in hypoxia. Notably, some of the transcriptomic effects of hypoxia in green turtle embryos resembled those reported to be associated with hypoxia-induced embryonic arrest in diverse taxa, including the nematode Caenorhabditis elegans and zebrafish (Danio rerio). Hypoxia-induced preovipositional embryonic arrest appears to be a unique adaptation of turtles. However, our findings accord with the proposition that the mechanisms underlying hypoxia-induced embryonic arrest per se are highly conserved across diverse taxa.


Subject(s)
Turtles , Animals , Female , Hypoxia , Oxygen/metabolism , Transcriptome/genetics , Turtles/genetics , Zebrafish
2.
J Bacteriol ; 204(4): e0059221, 2022 04 19.
Article in English | MEDLINE | ID: mdl-35323048

ABSTRACT

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.


Subject(s)
Escherichia coli Proteins , Pasteurella multocida , RNA, Small Untranslated , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Host Factor 1 Protein/genetics , Host Factor 1 Protein/metabolism , Pasteurella multocida/genetics , Pasteurella multocida/metabolism , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Small Untranslated/genetics , RNA-Binding Proteins/metabolism
3.
Article in English | MEDLINE | ID: mdl-30745381

ABSTRACT

Hypermutable Pseudomonas aeruginosa isolates (hypermutators) have been identified in patients with cystic fibrosis (CF) and are associated with reduced lung function. Hypermutators display a greatly increased mutation rate and an enhanced ability to become resistant to antibiotics during treatment. Their prevalence has been established among patients with CF, but it has not been determined for patients with CF in Australia. This study aimed to determine the prevalence of hypermutable P. aeruginosa isolates from adult patients with CF from a health care institution in Australia and to characterize the genetic diversity and antibiotic susceptibility of these isolates. A total of 59 P. aeruginosa clinical isolates from patients with CF were characterized. For all isolates, rifampin (RIF) mutation frequencies and susceptibility to a range of antibiotics were determined. Of the 59 isolates, 13 (22%) were hypermutable. Whole-genome sequences were determined for all hypermutable isolates. Core genome polymorphisms were used to assess genetic relatedness of the isolates, both to each other and to a sample of previously characterized P. aeruginosa strains. Phylogenetic analyses showed that the hypermutators were from divergent lineages and that hypermutator phenotype was mostly the result of mutations in mutL or, less commonly, in mutS Hypermutable isolates also contained a range of mutations that are likely associated with adaptation of P. aeruginosa to the CF lung environment. Multidrug resistance was more prevalent in hypermutable than nonhypermutable isolates (38% versus 22%). This study revealed that hypermutable P. aeruginosa strains are common among isolates from patients with CF in Australia and are implicated in the emergence of antibiotic resistance.


Subject(s)
Cystic Fibrosis/microbiology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/isolation & purification , Adult , Anti-Bacterial Agents/therapeutic use , Australia , Bacterial Proteins/genetics , Cystic Fibrosis/drug therapy , Drug Resistance, Microbial/drug effects , Drug Resistance, Microbial/genetics , Humans , Mutation/genetics , Phylogeny , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Rifampin/therapeutic use
4.
Vet Microbiol ; 169(3-4): 163-70, 2014 Mar 14.
Article in English | MEDLINE | ID: mdl-24467929

ABSTRACT

Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjobovis) is the main causative agent of bovine leptospirosis in Australia, New Zealand, North America and elsewhere. Bovine leptospirosis can result in spontaneous abortion, stillbirth and reduced milk output. The organism is shed in the urine of infected animals and contact with contaminated materials can result in zoonotic infections in humans. Protective immunity in cattle against Hardjobovis involves stimulation of a Th1 cell mediated immune response, which can be characterized by the production of IFN-γ when blood from vaccinated animals is exposed to Hardjobovis antigens. However, the leptospiral components involved in stimulating this response have yet to be identified. In this study, 238 recombinant leptospiral proteins were evaluated for their ability to stimulate IFN-γ production in blood of cattle vaccinated with a commercial monovalent Hardjobovis vaccine. The conserved lipoprotein LipL32 is the major outer membrane protein of pathogenic Leptospira spp. A pool of soluble recombinant proteins which included LipL32, as well as LipL32 alone, stimulated significant IFN-γ production in blood of vaccinated cattle. A number of recombinant LipL32 fragments was generated, which identified the amino acids between 20 and 200 as containing the bovine T-cell reactive regions of LipL32. However, whether LipL32 plays a role in stimulating protective immunity in mammals has yet to be conclusively determined.


Subject(s)
Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/immunology , Cattle Diseases/immunology , Interferon-gamma/metabolism , Leptospira/immunology , Leptospirosis/veterinary , Lipoproteins/immunology , Recombinant Proteins/immunology , Amino Acids/immunology , Animals , Bacterial Outer Membrane Proteins/genetics , Cattle , Female , Immunity, Cellular/immunology , Interferon-gamma/blood , Leptospirosis/immunology , Lipoproteins/genetics , Recombinant Proteins/genetics
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