Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes

Kovach, Michael E.; Elzer, Philip H.; Hill, D. Steven; Robertson, Gregory T.; Farris, Michael A.; Roop, R. Martin; Peterson, Kenneth M.

doi:10.1016/0378-1119(95)00584-1

Cited by 3,043 publications

(1,638 citation statements)

References 6 publications

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“…The pXF20-PEMIK plasmid with an addiction system, described above, may be successful in allowing for complementation of mutants, which has been a major limiting factor in X. fastidiosa research. To our knowledge, only Chatterjee et al (2010) performed studies with complemented knockouts in both plants and insects using the broadhost-range plasmid pBRR-5 (Kovach et al 1995), which was previously used by Reddy et al (2007).…”

Section: Genetic Tractabilitymentioning

confidence: 99%

Genomic Insights into Xylella fastidiosa Interactions with Plant and Insect Hosts

Retchless

Labroussaa

Shapiro

et al. 2014

Genomics of Plant-Associated Bacteria

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Section: Genetic Tractabilitymentioning

confidence: 99%

Genomic Insights into Xylella fastidiosa Interactions with Plant and Insect Hosts

Retchless

Labroussaa

Shapiro

et al. 2014

Genomics of Plant-Associated Bacteria

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“…pCR2.1-TOPO vector (Invitrogen, CA) was used in the cloning of PCR amplification products. The expression vector pBBR1MCS-5 was used for expression of cloned genes in complementation studies as described below (23). Pseudomonas putida CA-3 was routinely grown on E2 minimal medium (24) containing 23 mM citrate as the sole carbon source.…”

Section: Methodsmentioning

confidence: 99%

GacS-Dependent Regulation of Polyhydroxyalkanoate Synthesis in Pseudomonas putida CA-3

Ryan

O’Leary

O’Mahony

et al. 2013

Appl Environ Microbiol

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To date, limited reports are available on the regulatory systems exerting control over bacterial synthesis of the biodegradable polyester group known as polyhydroxyalkanoates (PHAs). In this study, we performed random mini-Tn5 mutagenesis of the Pseudomonas putida CA-3 genome and screened transconjugants on nitrogen-limited medium for reduced PHA accumulation phenotypes. Disruption of a GacS sensor kinase in one such mutant was found to eliminate medium-chain-length PHA production in Pseudomonas putida CA-3. Recombinant expression of wild-type gacS from a pBBRgacS vector fully restored PHA accumulation capacity in the mutant strain. PCR-based screening of the P. putida CA-3 genome identified gene homologues of the GacS/GacA-rsm small RNA (sRNA) regulatory cascade with 96% similarity to published P. putida genomes. However, reverse transcription-PCR (RT-PCR) analyses revealed active transcription of the rsmY and rsmZ sRNAs in gacS-disrupted P. putida CA-3, which is atypical of the commonly reported Gac/Rsm regulatory cascade. Quantitative real-time RT-PCR analyses of the phaC1 synthase responsible for polymer formation in P. putida CA-3 indicated no statistically significant difference in transcript levels between the wild-type and gacS-disrupted strains. Subsequently, SDS-PAGE protein analyses of these strains identified posttranscriptional control of phaC1 synthase as a key aspect in the regulation of PHA synthesis by P. putida CA-3.

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“…Genes of huPON2 mutants D2 and E3 and aiiA were subcloned to a broad-host vector pBBR1MCS4 at XhoI and XbaI sites 47 …”

Section: P Aeruginosa Pao1 Swarming and Swimming Motility Testsmentioning

confidence: 99%

Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

Wang

Mulchandani

et al. 2016

ACS Chem. Biol.

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Many pathogenic bacteria utilize quorum sensing (QS) systems to regulate the expression of their virulence genes and promote the formation of biofilm, which renders pathogens with extreme resistance to conventional antibiotic treatments. As a novel approach for attenuating antibiotic resistance and in turn fighting chronic infections, enzymatic inactivation of QS signaling molecules, such as N-acyl homoserine lactones (AHLs), holds great promises. Instead of using bacterial lactonases that can evoke immune response when administered, we focus on the human paraoxonase 2 (huPON2). However, insolubility when heterologously overexpressed hinders its application as anti-infection therapeutics. In this study, huPON2 was engineered for soluble expression with minimal introduction of foreign sequences. On the basis of structure modeling, degenerate linkers were exploited for the removal of hydrophobic helices of huPON2 without disrupting its folding structure and thus retaining its enzymatic function. High soluble expression levels were achieved with a yield of 76 mg of fully human PON2 variants per liter of culture media. Particularly, two clones, D2 and E3, showed significant quorum quenching (QQ) bioactivities and effectively impeded Pseudomonas aeruginosa swimming and swarming motilities, signs of an early stage of biofilm formation. In addition, by correlating QQ with luminescence signal readouts, quantitative analysis of QQ toward natural or non-natural AHL-regulator combinations suggested that D2 and E3 exhibited strong lactone hydrolysis activities toward five AHLs of different side chain lengths and modifications widely utilized by a variety of biomedically important pathogens.

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Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes

Cited by 3,043 publications

References 6 publications

Genomic Insights into Xylella fastidiosa Interactions with Plant and Insect Hosts

Genomic Insights into Xylella fastidiosa Interactions with Plant and Insect Hosts

GacS-Dependent Regulation of Polyhydroxyalkanoate Synthesis in Pseudomonas putida CA-3

Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

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