Construction and validation of a GFP-based vector for promoter expression analysis in the fish pathogen Flavobacterium psychrophilum

Gómez, Esther Romero; Pérez-Pascual, David; Fernández, Lucía; Méndez, Jessica; Reimundo, Pilar; Navais, Roberto; Guijarro, José A.

doi:10.1016/j.gene.2012.01.069

Cited by 11 publications

(18 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Experiments under starvation conditions are time consuming and contamination of the culture is a constant threat. Moreover, genetic systems for flavobacteria are very limited [78], [79]. Thus, the most precise experiment for an analysis of the global physiological role of PR, i.e.…”

Section: Discussionmentioning

confidence: 99%

Genomics and Physiology of a Marine Flavobacterium Encoding a Proteorhodopsin and a Xanthorhodopsin-Like Protein

et al. 2013

View full text Add to dashboard Cite

Proteorhodopsin (PR) photoheterotrophy in the marine flavobacterium Dokdonia sp. PRO95 has previously been investigated, showing no growth stimulation in the light at intermediate carbon concentrations. Here we report the genome sequence of strain PRO95 and compare it to two other PR encoding Dokdonia genomes: that of strain 4H-3-7-5 which shows the most similar genome, and that of strain MED134 which grows better in the light under oligotrophic conditions. Our genome analysis revealed that the PRO95 genome as well as the 4H-3-7-5 genome encode a protein related to xanthorhodopsins. The genomic environment and phylogenetic distribution of this gene suggest that it may have frequently been recruited by lateral gene transfer. Expression analyses by RT-PCR and direct mRNA-sequencing showed that both rhodopsins and the complete β-carotene pathway necessary for retinal production are transcribed in PRO95. Proton translocation measurements showed enhanced proton pump activity in response to light, supporting that one or both rhodopsins are functional. Genomic information and carbon source respiration data were used to develop a defined cultivation medium for PRO95, but reproducible growth always required small amounts of yeast extract. Although PRO95 contains and expresses two rhodopsin genes, light did not stimulate its growth as determined by cell numbers in a nutrient poor seawater medium that mimics its natural environment, confirming previous experiments at intermediate carbon concentrations. Starvation or stress conditions might be needed to observe the physiological effect of light induced energy acquisition.

show abstract

Section: Discussionmentioning

confidence: 99%

Genomics and Physiology of a Marine Flavobacterium Encoding a Proteorhodopsin and a Xanthorhodopsin-Like Protein

et al. 2013

View full text Add to dashboard Cite

show abstract

“… [ 39 ] pCP23 ColE1 ori, (pCP1 ori), Ap r (Tc r ), E. coli – F. psychrophilum shuttle plasmid [ 40 ] pCP23-fpgA FP1638 gene, derived from pCP23. This study pCP23-Gfpp2 pCP23-G carrying fpp2-fpp1 promoter [ 41 ] Bacterial strains F. psychrophilum THC02/90 Wild-type. [ 42 ] fpgA − FP1638 mutant created by Tn 4351 transposition.…”

Section: Methodsmentioning

confidence: 99%

“… This study fpgA+ Mutant ΔFP1638 carrying pCP23-FP1638 plasmid, complemented strain. This study THC02/90-G THC02/90 strain carrying pCP23-G plasmid [ 41 ] THC02/90 -fpp2 THC02/90 strain carrying pCP23-Gfpp2 plasmid [ 41 ] fpgA − -fpp2 ΔFP1638 strain carrying pCP23-Gfpp2 plasmid [ 41 ] E. coli S17-1 λpir λ pir hsdR pro thi ; RP4-2 Tc::Mu Km::Tn 7 [ 34 ] BW19851 RP4-2 tet ::Mu-1 kan ::Tn 7 integrant; _ uidA :: pir _ recA1 hsdR17 creB510 endA1 zbf-5 thi [ 35 ] Primers b 26-F 5′ACTG GGATCC AGTTTTAAGCCCGCAAA 3′ This study 26-R 5′ ACTG CTGCAG CAATGAACTTCGTCTTG 3′ This study TN-1 5′ GGACCTACCTCATAGACAA 3′ [ 19 ] IS4351-F 5′ TCAGAGTGAGAGAAAGGG 3′ [ 19 ] promfpp2-F 5′ ATCA GGATCC GAGCACTACACTTTCTAGA 3′ [ 41 ] promfpp2-R 5′ GATT GGATCC TGTTCGGTAGTGTAGCA 3′ [ 41 ] a Antibiotic resistance phenotypes: ampicillin, Ap r ; tetracycline, Tc r ; erythromycin, Emr b . Antibiotic resistance phenotypes and other features listed in parentheses are those expressed by F. psychrophilum but not by E. coli .…”

Section: Methodsmentioning

confidence: 99%

“…To complement the fpgA mutant, the DNA sequence corresponding to the encoding gene was amplified from the parental strain by PCR, using 26-F and 26-R primers and the Expand Long Template PCR System (Roche), obtaining a fragment of 1105 pb (Table 1 ). Bam HI and Pst I restriction sites were introduced into the sequences of 26-F and 26-R respectively, in order to clone the PCR product into the plasmid pCP23 that has promoter activity [ 41 ] (Table 1 ). The resulting plasmid was designated pCP23-fpgA (Table 1 ).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence

Pérez-Pascual

Gómez

Guijarro

2015

Vet Res

Self Cite

109

134

View full text Add to dashboard Cite

Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD50 experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine.

show abstract

“…Flavobacterium psychrophilum is considered a ‘fastidious’ bacterium because it is difficult to culture, isolate and manipulate, but, in spite of this, the need for an effective vaccine has motivated research into this organism. Thus, in the last few years, the development of genetic techniques (Alvarez et al ., ; Perez‐Pascual et al ., ; Gomez et al ., ) and culture media (Alvarez and Guijarro, ; Perez‐Pascual et al ., ) together with the availability of the complete genome sequence of the bacterium (Duchaud et al ., ) have facilitated its study and, in particular, that of its virulence determinants. In this respect, some factors associated with pathogenesis have been described.…”

Section: Introductionmentioning

confidence: 99%

Flavobacterium psychrophilum vaccine development: a difficult task

Gómez

Méndez

Cascales

et al. 2014

Microbial Biotechnology

Self Cite

View full text Add to dashboard Cite

Bacterial cold water disease (BCWD) is a globally distributed freshwater fish disease caused by the Gram-negative bacterium Flavobacterium psychrophilum. It is a particularly devastating infection in fry salmonids and may lead to high levels of mortality. In spite of its economic impact on fish farms, neither the biology of the bacterium nor the bacterium–host interactions are well understood. This review provides a synopsis of the major problems related to critical remaining questions about research into the use of vaccines against F. psychrophilum and the development of a commercial vaccine against this disease. Studies using sera from convalescent rainbow trout have shown the antigenic properties of different proteins such as OmpH, OmpA and FspA, as well as low and high molecular mass lipopolysaccharide of F. psychrophilum, which are potential candidates for subunit vaccines. Inactivated F. psychrophilum bacterins have been successfully tested as vaccines under laboratory conditions by both immersion and intraperitoneal routes. However, the efficacy and the practical usefulness of these preparations still have to be proved. The use of attenuated and wild-type strains to immunize fish showed that these systems offer high levels of protection. Nevertheless, their application clashes with the regulations for environmental protection in many countries. In conclusion, protective vaccines against BCWD are theoretically possible, but substantial efforts still have to be made in order to permit the development of a commercial vaccine.

show abstract

Construction and validation of a GFP-based vector for promoter expression analysis in the fish pathogen Flavobacterium psychrophilum

Cited by 11 publications

References 27 publications

Genomics and Physiology of a Marine Flavobacterium Encoding a Proteorhodopsin and a Xanthorhodopsin-Like Protein

Genomics and Physiology of a Marine Flavobacterium Encoding a Proteorhodopsin and a Xanthorhodopsin-Like Protein

Lack of a type-2 glycosyltransferase in the fish pathogen Flavobacterium psychrophilum determines pleiotropic changes and loss of virulence

Flavobacterium psychrophilum vaccine development: a difficult task

Contact Info

Product

Resources

About