Comparison of transformation protocols in <i>Streptococcus gordonii</i> and evaluation of native promoter strength using a multiple-copy plasmid

Warren, Travis K.; Lund, Susan Amanda; Jones, Kevin F.; Hruby, Dennis E.

doi:10.1139/w07-004

Cited by 4 publications

(3 citation statements)

References 26 publications

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“…To construct the mCherry-expressing S. gordonii strain BL98, we transformed plasmid pVA8912 (Vickerman, Mansfield, Zhu, Walters, & Banas, 2015) into the wild-type S. gordonii V288 according to previously published protocol (Warren, Lund, Jones, & Hruby, 2007), utilizing competence-stimulating peptide N-DVRSNKIRLWWENIFFNKK (Pepmic, Suzhou, China). The mCherry encoding gene was inserted into S. gordonii attB site and is expressed under the control of the ldh promoter (for a full description of the construct, see Vickerman et al, 2015).…”

Section: Mcherry + S Gordoniimentioning

confidence: 99%

Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii

Lima¹,

Shi

Lux

2017

MicrobiologyOpen

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To successfully colonize the oral cavity, bacteria must directly or indirectly adhere to available oral surfaces. Fusobacterium nucleatum plays an important role in oral biofilm community development due to its broad adherence abilities, serving as a bridge between members of the oral biofilm that cannot directly bind to each other. In our efforts to characterize the molecular mechanisms utilized by F. nucleatum to physically bind to key members of the oral community, we investigated the involvement of F. nucleatum outer membrane proteins in its ability to bind to the pioneer biofilm colonizer, Streptococcus gordonii. Here, we present evidence that in addition to the previously characterized fusobacterial adhesin RadD, the interaction between F. nucleatum ATCC 23726 and S. gordonii V288 involves a second outer membrane protein, which we named coaggregation mediating protein A (CmpA). We also characterized the role of CmpA in dual‐species biofilm formation with S. gordonii V288, evaluated growth‐phase‐dependent as well as biofilm expression profiles of radD and cmpA, and confirmed an important role for CmpA, especially under biofilm growth conditions. Our findings underscore the complex set of specific interactions involved in physical binding and thus community integration of interacting bacterial species. This complex set of interactions could have critical implications for the formation and maturation of the oral biofilms in vivo, and could provide clues to the mechanism behind the distribution of organisms inside the human oral cavity.

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Section: Mcherry + S Gordoniimentioning

confidence: 99%

Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii

Lima¹,

Shi

Lux

2017

MicrobiologyOpen

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“…The following modified assay that allows S. gordonii stocks to be stored in frozen aliquots, and directly applied in competence experiments has been suggested [21]:…”

Section: Other Oral Streptococcimentioning

confidence: 99%

Natural Transformation of Oral Streptococci by Use of Synthetic Pheromones

Salvadori

Junges

Khan

et al. 2016

Methods in Molecular Biology

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The discovery that Streptococcus pneumoniae uses a competence-stimulating peptide (CSP) to induce competence for natural transformation, and that other species of the mitis and the anginosus streptococcal groups use a similar system, has expanded the tools to explore gene function and regulatory pathways in streptococci. Two other classes of pheromones have been discovered since then, comprising the bacteriocin-inducing peptide class found in Streptococcus mutans (also named CSP, although different from the former) and the SigX-inducing peptides (XIP), in the mutans, salivarius, bovis, and pyogenes groups of streptococci. The three classes of peptide pheromones can be ordered from peptide synthesis services at affordable prices, and used in transformation assays to obtain competent cultures consistently at levels usually higher than those achieved during spontaneous competence. In this chapter, we present protocols for natural transformation of oral streptococci that are based on the use of synthetic pheromones, with examples of conditions optimized for transformation of S. mutans and Streptococcus mitis.

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“…Often times it is necessary to express a gene in trans, which necessitates cloning constructs onto E. coli – Streptococcus shuttle vectors. Since streptococcal promoters are typically active in E. coli , cloned genes tend to be highly expressed due to the plasmid copy number and frequently exhibit toxicity (Warren et al, 2007). Consequently, certain constructs may be difficult or impossible to assemble.…”

Section: Introductionmentioning

confidence: 99%

Cloning-independent plasmid construction for genetic studies in streptococci

Xie

Merritt

2013

Journal of Microbiological Methods

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Shuttle plasmids are among the few routinely utilized tools in the Streptococcus mutans genetic system that still require the use of classical cloning methodologies and intermediate hosts for genetic manipulation. Accordingly, it typically requires considerably less time and effort to introduce mutations onto the S. mutans chromosome than it does to construct shuttle vectors for expressing genes in trans. Occasionally, shuttle vector constructs also exhibit toxicity in E. coli, which prevents their proper assembly. To circumvent these limitations, we modified a prolonged overlap extension PCR (POE-PCR) protocol to facilitate direct plasmid assembly in S. mutans. Using solely PCR, we created the reporter vector pZX7, which contains a single minimal streptococcal replication origin and harbors a spectinomycin resistance cassette and the gusA gene encoding β-glucuronidase. We compared the efficiency of pZX7 assembly using multiple strains of S. mutans and were able to obtain from 5×103 – 2×105 CFU/μg PCR product. Likewise, we used pZX7 to further demonstrate that Streptococcus sanguinis and Streptococcus gordonii are also excellent hosts for cloning-independent plasmid assembly, which suggests that this system is likely to function in numerous other streptococci. Consequently, it should be possible to completely forgo the use of E. coli – Streptococcus shuttle vectors in many streptococcal species, thereby decreasing the time and effort required to assemble constructs and eliminating any toxicity issues associated with intermediate hosts.

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Comparison of transformation protocols in Streptococcus gordonii and evaluation of native promoter strength using a multiple-copy plasmid

Cited by 4 publications

References 26 publications

Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii

Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii

Natural Transformation of Oral Streptococci by Use of Synthetic Pheromones

Cloning-independent plasmid construction for genetic studies in streptococci

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