Plasmid-Based System for High-Level Gene Expression and Antisense Gene Knockdown in
            <i>Bartonella henselae</i>

Gillaspie, Devin B.; Perkins, Izabella; Larsen, Kellie; McCord, Amy M.; Pangonis, Scott; Sweger, Debra; Seleem, Mohamed N.; Sriranganathan, Nammalwar; Anderson, Burt

doi:10.1128/aem.00949-09

Cited by 16 publications

(19 citation statements)

References 13 publications

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“…Bacteria were either cultured on heart infusion agar (Remel, Lenexa, KS) supplemented with 1% bovine hemoglobin (Remel, Lenexa, KS) (also known as chocolate agar) or in Schneider’s insect medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences, Pittsburgh, PA) and incubated for 3–4 days at 37°C in the presence of 5% CO 2 [24]. The pNS2-derived expression vector, which has been shown previously to replicate in Bh [25], was used as a backbone for construction of promoter reporter plasmids and introduced into Bh via electroporation [21]. For bacterial strains carrying the pNS2 plasmids, 50 µg/ml of kanamycin was supplemented to select for bacteria colonies that harbor the plasmid.…”

Section: Methodsmentioning

confidence: 99%

“…The lacZ reporter gene encoding the beta-galactosidase enzyme was previously ligated into the pNS2 plasmid downstream of the strong trc promoter [25]. The divergent nepR and phyR promoters were PCR amplified from Bh genomic DNA using specific primers (Table S1) and ligated into the pNS2 plasmid to replace the trc promoter at the Sal I and Bam HI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of the general stress response in Bartonella henselae

Lima

Bandeali

et al. 2016

Microbial Pathogenesis

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Bacteria utilize a general stress response system to combat stresses from their surrounding environments. In alpha-proteobacteria, the general stress response uses an alternate sigma factor as the main regulator and incorporates it with a two-component system into a unique regulatory circuit. This system has been described in several alpha-proteobacterial species, including the pathogens Bartonella quintana and Brucella abortus. Most of the studies have focused on characterizing the PhyR anti-anti-sigma factor, the NepR anti-sigma factor, and the alternate sigma factor. However, not enough attention is directed toward studying the role of histidine kinases in the general stress response. Our study identifies the general stress response system in Bartonella henselae, where the gene synteny is conserved and both the PhyR and alternate sigma factor have similar sequence and domain structures with other alpha-proteobacteria. Our data showed that the general stress response genes are up-regulated under conditions that mimic the cat flea vector. Furthermore, we showed that both RpoE and PhyR positively regulate this system and that RpoE also affects transcription of genes encoding heme-binding proteins and the gene encoding the BadA adhesin. Finally, we identified a histidine kinase, annotated as BH13820 that can potentially phosphorylate PhyR.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Characterization of the general stress response in Bartonella henselae

Lima

Bandeali

et al. 2016

Microbial Pathogenesis

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“…fied as the major regulatory factor for transcriptional osmotic regulation in E. coli (34,53). Furthermore, ompR was shown to be involved in the virulence and survival of Brucella melitensis during macrophage infection (59), and a recent study suggested the involvement of ompR in the ability of B. henselae to invade HEC (23). Gene cluster 2 thus seems to reflect the B. henselae response to the drastic change in environment encountered at the onset of the in vitro infection and may comprise factors important for the establishment of contact with the HEC.…”

Section: Discussionmentioning

confidence: 99%

The BatR/BatS Two-Component Regulatory System Controls the Adaptive Response ofBartonella henselaeduring Human Endothelial Cell Infection

et al. 2010

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Here, we report the first comprehensive study of Bartonella henselae gene expression during infection of human endothelial cells. Expression of the main cluster of upregulated genes, comprising the VirB type IV secretion system and its secreted protein substrates, is shown to be under the positive control of the transcriptional regulator BatR. We demonstrate binding of BatR to the promoters of the virB operon and a substrate-encoding gene and provide biochemical evidence that BatR and BatS constitute a functional twocomponent regulatory system. Moreover, in contrast to the acid-inducible (pH 5.5) homologs ChvG/ChvI of Agrobacterium tumefaciens, BatR/BatS are optimally activated at the physiological pH of blood (pH 7.4). By conservation analysis of the BatR regulon, we show that BatR/BatS are uniquely adapted to upregulate a genus-specific virulence regulon during hemotropic infection in mammals. Thus, we propose that BatR/BatS two-component system homologs represent vertically inherited pH sensors that control the expression of horizontally transmitted gene sets critical for the diverse host-associated life styles of the alphaproteobacteria.

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“…Our experiments demonstrated that expression of NrnC from B. henselae can rescue the growth defect of strain UM341 in the absence of Atc (data not shown). We cloned nrnC of B. henselae into a vector that allowed low-level expression, pNS2Amp, and a vector that allowed high-level expression, pNS2Trc (Gillaspie et al, 2009). The gene was in the reverse orientation such that the antisense strand was transcribed.…”

Section: Nrnc Activity Is Important For Growth In B Henselaementioning

confidence: 99%