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Review
. 2023 Nov 1;47(6):fuac050.
doi: 10.1093/femsre/fuac050.

Illuminating the oral microbiome and its host interactions: tools and approaches for molecular microbiology studies

Justin Merritt  1   2 , Jens Kreth  1   2
Affiliations
Review

Illuminating the oral microbiome and its host interactions: tools and approaches for molecular microbiology studies

Justin Merritt et al. FEMS Microbiol Rev. .

Abstract

Advancements in DNA sequencing technologies within the last decade have stimulated an unprecedented interest in the human microbiome, largely due the broad diversity of human diseases found to correlate with microbiome dysbiosis. As a direct consequence of these studies, a vast number of understudied and uncharacterized microbes have been identified as potential drivers of mucosal health and disease. The looming challenge in the field is to transition these observations into defined molecular mechanistic studies of symbiosis and dysbiosis. In order to meet this challenge, many of these newly identified microbes will need to be adapted for use in experimental models. Consequently, this review presents a comprehensive overview of the molecular microbiology tools and techniques that have played crucial roles in genetic studies of the bacteria found within the human oral microbiota. Here, we will use specific examples from the oral microbiome literature to illustrate the biology supporting these techniques, why they are needed in the field, and how such technologies have been implemented. It is hoped that this information can serve as a useful reference guide to help catalyze molecular microbiology studies of the many new understudied and uncharacterized species identified at different mucosal sites in the body.

Keywords: genetic system; molecular microbiology; mutagenesis; oral microbiome; reporter genes; transformation.

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Conflict of interest statement

None declared.

Figures

Figure 1.
Figure 1.
Targeted mutagenesis using homologous recombination. (A) Insertion duplication mutagenesis. An internal homologous fragment (illustrated in orange stripes) of the target gene is ligated to a suicide vector containing a positive selection marker (‘+’; illustrated in green). Transformation of this construct using selective media will isolate clones containing a plasmid insertion within the target gene, disrupting the function of its encoded protein. Insertion of the plasmid also creates a duplication of the homologous fragment on the chromosome. (B) Allelic replacement mutagenesis. Homologous fragments (illustrated in grey and orange stripes) flanking the intended mutation site of the target gene are ligated to the corresponding 5′ and 3′ ends of a positive selection marker (‘+’; illustrated in green). Following transformation of this construct, both homologous fragments recombine with the chromosome, which deletes all of the intervening chromosomal DNA located between the homologous segments and replaces it with the positive selection marker.
Figure 2.
Figure 2.
Markerless mutagenesis strategies. (A) Counterselection with insertion duplication mutagenesis. Two equally sized homologous fragments (illustrated in grey and orange stripes) flanking the intended mutation site are cloned adjacent to each other on a suicide vector containing both positive and negative selection markers (‘+/–’; illustrated in green). Following transformation of the construct, one of the two fragments will randomly insert to the chromosome via single crossover homologous recombination. The same final outcome is achieved irrespective of which of the two homologous fragments recombines. Therefore, only one option is illustrated. Since the suicide vector contains two homologous fragments, both of these segments will be duplicated on the chromosome after the plasmid has inserted (as indicated by the red and black brackets). Negative selection is used to isolate clones in which these homologous segments have randomly recombined to excise the inserted vector from the chromosome. In this example, a markerless mutant will be created following a recombination event between the homologous segments marked by red brackets. However, if recombination occurs between the homologous segments marked by black brackets, a wild-type genotype will result. Consequently, counterselection with insertion duplication mutagenesis yields a mixed population of clones consisting of 50% mutant and 50% wild-type genotypes. (B) Counterselection with allelic replacement mutagenesis. Two homologous fragments flanking the intended mutation site of the target gene are ligated to the corresponding 5′ and 3′ ends of a counterselection cassette (‘+/–’; illustrated in green). Following transformation of this construct, both homologous fragments recombine with the chromosome, which deletes all of the intervening chromosomal DNA between the homologous segments and replaces it with the counterselection cassette. The resulting strain is then transformed with an unmarked mutagenesis construct and subjected to negative selection to isolate the double crossover recombinants that have deleted the counterselection cassette. All of the resulting transformants should contain the desired markerless mutant genotype. (C) Recombinase-mediated markerless mutagenesis. A typical double crossover allelic replacement construct is assembled using a positive selection cassette (‘+’; illustrated in green) flanked by two Cre recombinase-dependent loxP sites (illustrated in yellow and purple). The allelic replacement mutant is next transformed with a temperature sensitive plasmid encoding the cre gene. Growth at the permissive temperature supports plasmid replication and ectopic production of the Cre recombinase. After a predetermined number of generations, the cells are shifted to the non-permissive temperature to trigger loss of the temperature sensitive cre expression plasmid. Plasmid-free clones are finally screened to identify those that have undergone Cre-mediated excision of the antibiotic cassette. Strains exhibiting the markerless mutant genotype will also retain a hybrid loxP site (illustrated in yellow and purple stripes) created via Cre-mediated recombination between the two original loxP sites.
Figure 3.
Figure 3.
Comparison of the original vs. Theo + theophylline riboswitches. (A) Unmodified theophylline riboswitch. The secondary structure of the ligand-free theophylline riboswitch was calculated using the mFold webserver (http://www.unafold.org/mfold/applications/rna-folding-form.php) (Zuker 2003). In the absence of free theophylline, the Shine-Dalgarno sequence (bold, red font) in the mRNA is sequestered within the secondary structure of the riboswitch, which prevents translation initiation at the downstream initiation codon (bold, green font). Upon binding theophylline, the riboswitch aptamer will adopt an alternate conformation (not pictured) that releases the Shine-Dalgarno sequence from sequestration, thus promoting translation initiation of the mRNA. (B) Theo + riboswitch. The secondary structure of the ligand-free Theo + riboswitch was calculated using the mFold webserver (http://www.unafold.org/mfold/applications/rna-folding-form.php) (Zuker 2003). The predicted secondary structure is nearly identical to the original theophylline riboswitch, except that it contains several point mutations (bold, blue font) that greatly improve its dynamic range of regulation.
Figure 4.
Figure 4.
Tunable proteolysis in S. mutans. To engineer tunable proteolysis onto a target protein, the corresponding target gene (orange) is fused to a chimeric tag encoding the small ubiquitin-like protein NEDD8 (green) followed by an endogenous S. mutans degron (red). The protein chimera will remain stable until the NEDD8-specific endopeptidase NEDP1 (scissor icon) is produced. In this system, NEDP1 is ectopically expressed under the transcriptional control of the Xyl-S1 cassette (brown), which is induced by the sugar xylose. In the presence of xylose, repression of NEDP1 is relieved, NEDP1 is produced, and NEDD8 is subsequently cleaved from the target protein. This exposes the N-degron at the new N-terminus of the protein, which targets the protein for highly efficient Clp- and/or FtsH-mediated proteolysis.
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