Development of a Novel Genetic System To Create Markerless Deletion Mutants of
            <i>Bdellovibrio bacteriovorus</i>

Steyert, Susan R.; Piñeiro, Silvia A.

doi:10.1128/aem.00640-07

Cited by 49 publications

(42 citation statements)

References 31 publications

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“…2), as these chromosomal regions contain only small insertions or deletions of a few nucleotides in the hit gene. However, we were not able to identify mutants in which an allelic replacement of the wildtype hit gene by an altered hit gene had occurred due to a double crossover, even when we used the counterselection protocol with the addition of 5% sucrose to the medium (37).…”

Section: Resultsmentioning

confidence: 97%

“…The knockout of the Bd0108 gene was done using the plasmid pK18mobsacB (29), a suicide plasmid in B. bacteriovorus carrying the sacB gene for counterselection (37).…”

Section: Resultsmentioning

confidence: 99%

“…The ColE1 origin of replication of this plasmid is not functional in B. bacteriovorus, and, therefore, selection of chloramphenicol-resistant bdellovibrios is indicative for the insertion of the knockout construct into the Bdellovibrio genome. Furthermore, pK18mobsacB carries the sacB gene (33), which confers sucrose sensitivity and can be used to detect double-crossover events (37).…”

Section: Vol 193 2011 Prey-independent Growth Of B Bacteriovorus 1747mentioning

confidence: 99%

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Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100

et al. 2011

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Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that attacks and invades Gram-negative bacteria. The predator requires living bacteria to survive as growth and replication take place inside the bacterial prey. It is possible to isolate mutants that grow and replicate outside prey bacteria. Such mutants are designated host or prey independent, and their nutritional requirements vary. Some mutants are saprophytic and require prey extracts for extracellular growth, whereas other mutants grow axenically, which denotes the formation of colonies on complete medium in the absence of any prey components. The initial events leading to prey-independent growth are still under debate, and several genes may be involved. We selected new mutants by three different methods: spontaneous mutation, transposon mutagenesis, and targeted gene knockout. By all approaches we isolated mutants of the hit (host interaction) locus. As the relevance of this locus for the development of prey independence has been questioned, we performed whole-genome sequencing of five prey-independent mutants. Three mutants were saprophytic, and two mutants could grow axenically. Wholegenome analysis revealed that the mutation of a small open reading frame of the hit locus is sufficient for the conversion from predatory to saprophytic growth. Complementation experiments were performed by introduction of a plasmid carrying the wild-type hit gene into saprophytic mutants, and predatory growth could be restored. Whole-genome sequencing of two axenic mutants demonstrated that in addition to the hit mutation the colony formation on complete medium was shown to be influenced by the mutations of two genes involved in RNA processing. Complementation experiments with a wild-type gene encoding an RNA helicase, RhlB, abolished the ability to form colonies on complete medium, indicating that stability of RNA influences axenic growth.

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Section: Resultsmentioning

confidence: 97%

“…The knockout of the Bd0108 gene was done using the plasmid pK18mobsacB (29), a suicide plasmid in B. bacteriovorus carrying the sacB gene for counterselection (37).…”

Section: Resultsmentioning

confidence: 99%

Section: Vol 193 2011 Prey-independent Growth Of B Bacteriovorus 1747mentioning

confidence: 99%

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Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100

et al. 2011

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“…An approximately 3.4-kb fragment of the 5= and 3= regions of ata were amplified by PCR using the primer pairs NdeI-Ata-3.4-F/MluI-Ata-R and MluI-Ata-F/XhoI-Ata-3.4-R, respectively ( Table 2). The fragments were then digested with NdeI/MluI and MluI/XhoI and ligated between the NdeI and XhoI sites of pSSK10 (68). The resulting plasmid, p⌬ata, contained approximately 3.4 kb of DNA flanking regions on both sides of ata.…”

Section: Methodsmentioning

confidence: 99%

Identification of Ata, a Multifunctional Trimeric Autotransporter of Acinetobacter baumannii

et al. 2012

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Acinetobacter baumannii has recently emerged as a highly troublesome nosocomial pathogen, especially in patients in intensive care units and in those undergoing mechanical ventilation. We have identified a surface protein adhesin of A. baumannii, designated the Acinetobacter trimeric autotransporter (Ata), that contains all of the typical features of trimeric autotransporters (TA), including a long signal peptide followed by an N-terminal, surface-exposed passenger domain and a C-terminal domain encoding 4 ␤-strands. To demonstrate that Ata encoded a TA, we created a fusion protein in which we replaced the entire passenger domain of Ata with the epitope tag V5, which can be tracked with specific monoclonal antibodies, and demonstrated that the C-terminal 101 amino acids of Ata were capable of exporting the heterologous V5 tag to the surface of A. baumannii in a trimeric form. We found that Ata played a role in biofilm formation and bound to various extracellular matrix/basal membrane (ECM/ BM) components, including collagen types I, III, IV, and V and laminin. Moreover, Ata mediated the adhesion of whole A. baumannii cells to immobilized collagen type IV and played a role in the survival of A. baumannii in a lethal model of systemic infection in immunocompetent mice. Taken together, these results reveal that Ata is a TA of A. baumannii involved in virulence, including biofilm formation, binding to ECM/BM proteins, mediating the adhesion of A. baumannii cells to collagen type IV, and contributing to the survival of A. baumannii in a mouse model of lethal infection.

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“…Two genes, Bd1802 and Bd3906, encoding potential Tat substrates were knocked out by silent deletion using modifications of the techniques used by Steyert & Pineiro (2007) and Roschanski et al (2011). Approximately 450 bp of flanking DNA from either side of the gene was amplified using primers listed in Supplementary Table S2 and Phusion HighFidelity DNA polymerase (New England Biolabs) and joined together by sequential cloning into vector pUC19 (Vieira & Messing, 1982), giving a deletion construct that still retained the Tat signal peptide and part of the C-terminal peptide coding regions (approximately the last 10 codons of each protein); these constructs were then transferred into the kanamycin-resistant suicide vector pK18mobsacB (Roschanski et al, 2011;Schäfer et al, 1994) and transformed into E. coli S17-1.…”

Section: Methodsmentioning

confidence: 99%

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

et al. 2011

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Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron–sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

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Development of a Novel Genetic System To Create Markerless Deletion Mutants of Bdellovibrio bacteriovorus

Cited by 49 publications

References 31 publications

Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100

Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100

Identification of Ata, a Multifunctional Trimeric Autotransporter of Acinetobacter baumannii

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

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