scholarly journals The Actinobacillus actinomycetemcomitans Autotransporter Adhesin Aae Exhibits Specificity for Buccal Epithelial Cells from Humans and Old World Primates

2005 ◽  
Vol 73 (4) ◽  
pp. 1947-1953 ◽  
Author(s):  
Daniel H. Fine ◽  
Kabilan Velliyagounder ◽  
David Furgang ◽  
Jeffrey B. Kaplan
Keyword(s):  
Epithelial Cells ◽  
Host Range ◽  
Mutant Strain ◽  
Actinobacillus Actinomycetemcomitans ◽  
Broad Host Range ◽  
Wild Type ◽  
New World Primates ◽  
Old World ◽  
Broad Host Range Plasmid ◽  
Buccal Epithelial Cells

ABSTRACT Cells of the gram-negative periodontopathogen Actinobacillus actinomycetemcomitans express a surface-exposed, outer membrane autotransporter protein, designated Aae, which has been implicated in epithelial cell binding. We constructed a mutant strain of A. actinomycetemcomitans that contained a transposon insertion in the Aae structural gene (aae) and tested the mutant to determine its ability to bind to buccal epithelial cells (BECs) isolated from healthy volunteers. Significantly fewer mutant cells than wild-type cells bound to BECs. A broad-host-range plasmid that contained an intact aae gene driven by a heterologous tac promoter restored the ability of the mutant strain to bind to BECs at wild-type levels. This plasmid also conferred upon Escherichia coli the ability to express the Aae protein on its surface and to bind to human BECs. Aae-expressing E. coli also bound to BECs isolated from six Old World primates but not to BECs isolated from four New World primates or nine other nonprimate mammals, as well as to human gingival epithelial cells but not to human pharyngeal, palatal, tongue, bronchial, or cervical epithelial cells. Our findings indicate that Aae mediates binding of A. actinomycetemcomitans to BECs from humans and Old World primates and that this process may contribute to the host range specificity and tissue tropism exhibited by this bacterium.

scholarly journals Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range PromoterPm

2011 ◽  
Vol 77 (8) ◽  
pp. 2648-2655 ◽  
Author(s):  
Rahmi Lale ◽  
Laila Berg ◽  
Friederike Stüttgen ◽  
Roman Netzer ◽  
Marit Stafsnes ◽  
...  
Keyword(s):  
Host Range ◽  
Untranslated Region ◽  
Micrococcus Luteus ◽  
Background Level ◽  
Broad Host Range ◽  
Continuous Control ◽  
Wild Type ◽  
Content Type ◽  
Broad Host Range Plasmid ◽  
Genes Encoding

ABSTRACTThe induciblePmpromoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in thePmDNA region corresponding to the 5′ untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding β-lactamase, luciferase, and phosphoglucomutase) inEscherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter,P1(Pantitet), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C50carotenoid sarcinaxanthin in an engineered strain ofE. colithat produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing threeMicrococcus luteusderived genes from the promoterPm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.

scholarly journals A Second Aggregatibacter actinomycetemcomitans Autotransporter Adhesin Exhibits Specificity for Buccal Epithelial Cells in Humans and Old World Primates

2007 ◽  
Vol 75 (9) ◽  
pp. 4440-4448 ◽  
Author(s):  
Gang Yue ◽  
Jeffrey B. Kaplan ◽  
David Furgang ◽  
Keith G. Mansfield ◽  
Daniel H. Fine
Keyword(s):  
Epithelial Cells ◽  
Double Mutant ◽  
Aggregatibacter Actinomycetemcomitans ◽  
Species Specificity ◽  
Targeted Mutagenesis ◽  
Tissue Tropism ◽  
Dependent Manner ◽  
Wild Type ◽  
Old World ◽  
Buccal Epithelial Cells

ABSTRACT Previous work showed that the Aggregatibacter actinomycetemcomitans adhesin Aae demonstrated species specificity and tissue tropism to buccal epithelial cells (BECs) derived from humans and Old World primates, but a second, lower-affinity adhesin was noted. This study was designed to determine if Omp100 (also known as ApiA), a surface-expressed A. actinomycetemcomitans adhesin, is that second adhesin and if so to investigate its tissue tropism and species specificity. A targeted mutagenesis protocol was used to construct an isogenic apiA mutant and an aae apiA double mutant with wild-type A. actinomycetemcomitans. In addition, Escherichia coli strain DH5α was used to express apiA to further assess binding parameters. Results indicated that the apiA mutant strain showed significantly less binding to BECs than its parent strain (P ≤ 0.05). Further, binding mediated by ApiA was specific to BECs from humans and Old World primates, as seen in both wild-type A. actinomycetemcomitans and E. coli expressing ApiA (P ≤ 0.05). Pretreatment of wild-type A. actinomycetemcomitans cells with anti-ApiA antiserum reduced binding in a dose-dependent manner. The aae apiA double mutant completely abrogated A. actinomycetemcomitans binding to both human and Old World primate BECs. Taken together, these studies indicate that ApiA and Aae, in concert, modulate binding of A. actinomycetemcomitans to human BECs. Since the BEC is a prominent reservoir for A. actinomycetemcomitans, identification of this second adhesin could lead to important therapeutic strategies.

Broad Host-Range Plasmid R1162: Replication, Incompatibility, and Copy-Number Control

1985 ◽  
pp. 173-188 ◽  
Author(s):  
Richard J. Meyer ◽  
Lung-Shen Lin ◽  
Kyunghoon Kim ◽  
Michael A. Brasch
Keyword(s):  
Host Range ◽  
Copy Number ◽  
Broad Host Range ◽  
Copy Number Control ◽  
Broad Host Range Plasmid

scholarly journals Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

Genetics ◽  
1992 ◽  
Vol 130 (1) ◽  
pp. 27-36 ◽  
Author(s):  
A Greener ◽  
S M Lehman ◽  
D R Helinski
Keyword(s):  
Host Range ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Firefly Luciferase ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Broad Host Range Plasmid ◽  
Transcriptional Start Sites ◽  
Plasmid Rk2

Abstract A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

scholarly journals A broad host range plasmid vector that does not encode replication proteins

2002 ◽  
Vol 211 (1) ◽  
pp. 91-95 ◽  
Author(s):  
Eza Kalyaeva ◽  
Irina Bass ◽  
Gennady Kholodii ◽  
Vadim Nikiforov
Keyword(s):  
Host Range ◽  
Plasmid Vector ◽  
Broad Host Range ◽  
Replication Proteins ◽  
Broad Host Range Plasmid
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