Single-letter and three-letter codes for the amino acids.
1. Introduction
LAP is a severe form of periodontitis (Genco et al., 1986; Slots & Ting, 1999). For reasons so far unknown, the disease is localized to the premolar and incisor teeth. Infection leads to inflammation and rapid destruction of the periodontal ligament and the alveolar bone and culminates in loss of teeth. The prevalence of LAP has been estimated to be 0.5-1% (Henderson et al., 2002; Löe & Brown, 1991; Rylev & Kilian, 2008). However, prevalence varies considerably with different ethnic groups. For example, in the African-American population, the prevalence is 10-15 times higher than the average. There is evidence that race and socioeconomic status play key roles in determining prevalence.
The molecular mechanisms behind the pathogenesis of
1.1. Non-specific adherence and virulence
A particularly striking property of a fresh clinical isolate of
Studies in our LAP-infection model demonstrated that a wild-type strain is able to colonize and persist in the mouths of rats (Fine et al., 2001). In contrast, an isogenic smooth-colony-forming variant failed to persist. Despite this unequivocal evidence for the importance of tenacious adherence in the colonization of the oral cavity by
1.2. The tad locus
The study of
biosynthesis in
1.3. The tad locus in A. actinomycetemcomitans is a virulence factor
Using our rat model of LAP, we demonstrated that a functional
1.4. tad loci are widespread in prokaryotes
Searches of completed and ongoing microbial sequencing projects have revealed that closely related
2. The Flp pilus
The Flp pili of
After their translocation to the inner membrane, the prepilins are cleaved (processed) to maturity by cognate prepilin peptidases (Giltner et al., 2012). We have shown that the 75-amino acid Flp1 prepilin of
2.1. Alanine-scanning mutagenesis of the coding region for the mature Flp1 pilin
To begin to study the properties of Flp1, we constructed and characterized a series of Flp1 pilin mutants, each with an alanine substitution for a specific non-alanine residue of the mature Flp1 pilin. The codon for each non-alanine residue was changed to a codon for alanine. (The mutant genes were constructed with the fewest possible nucleotide changes.) In this way, translation of the mutant gene would give a mutant Flp1 prepilin, which, after being processed, gave rise to a mutant mature Flp1 pilin. (Alanine was chosen because it is the smallest amino acid that is relatively neutral and can maintain an α-helix in a polypeptide.) We changed the non-alanine residues in the mature Flp1 pilin by overlap extension PCR (polymerase chain reaction) (Ho et al., 1989). In this method, the
2.2. Characterization of mutant Flp1 pilins
The mutant genes were inserted into a plasmid vector downstream of the IPTG (isopropyl β-D-thiogalactopyranoside)-inducible
Each of our Flp1 mutants had one non-alanine residue changed to alanine. (See Table 1 for the single-letter and three-letter codes for the amino acids. Table 2 shows the residue change in the Flp1 pilin for each mutant and the phenotype.) In the mutants, every non-alanine residue of the mature Flp1 pilin was changed to alanine. The Flp1 prepilin has 75 residues; but, after cleavage, the mature Flp1 pilin has 49 residues. Nine residues are already alanine. The other 40 residues of the mature pilin were changed to alanine. The small size of mature Flp1 pilin made it reasonable to create this series of mutant pilins.
alanine | A | ala |
arginine | R | arg |
asparagine | N | asn |
aspartic acid | D | asp |
cysteine | C | cys |
glutamic acid | E | glu |
glutamine | Q | gln |
glycine | G | gly |
histidine | H | his |
isoleucine | I | ile |
leucine | L | leu |
lysine | K | lys |
methionine | M | met |
phenylalanine | F | phe |
proline | P | pro |
serine | S | ser |
threonine | T | thr |
tryptophan | W | try |
tyrosine | Y | tyr |
valine | V | val |
Mutant Residue | Colony Morph.a | Adher.b | Auto-aggreg.c | Protein exp.d | Piliatione | Classf |
V27A | S | - | - | +/- | + | IV |
T28A | S | - | - | + | - | III |
I30A | S | - | - | + | + | IV |
E31A | S | - | - | + | - | III |
Y32A | S | - | - | +/- | - | III |
G33A | R | + | + | ND | ND | I |
L34A | S | - | - | +/- | + | IV |
I35A | S | - | - | - | - | II |
I37A | S | - | - | + | - | III |
V39A | R | + | + | ND | ND | I |
V41A | R | + | + | ND | ND | I |
L42A | R | + | + | ND | ND | I |
I43A | S | - | - | + | + | IV |
V44A | R | + | + | ND | ND | I |
V46A | S | - | - | + | + | IV |
F47A | S | - | - | + | - | III |
Y48A | R | + | + | ND | ND | I |
S49A | R | + | + | ND | ND | I |
N50A | S | - | - | + | - | III |
N51A | R | + | + | ND | ND | I |
G52A | S | - | - | + | + | IV |
F53A | R | + | + | ND | ND | I |
I54A | S | - | - | - | - | II |
N56A | R | + | + | ND | ND | I |
L57A | S | - | - | + | + | IV |
Q58A | R | + | + | ND | ND | I |
S59A | R | + | + | ND | ND | I |
K60A | R | + | + | ND | ND | I |
F61A | S | - | - | + | - | III |
N62A | R | + | + | ND | ND | I |
S63A | R | + | + | ND | ND | I |
L64A | S | - | - | + | - | III |
S66A | R | + | + | ND | ND | I |
T67A | R | + | + | ND | ND | I |
V68A | R | + | + | ND | ND | I |
S70A | R | + | + | ND | ND | I |
N72A | R | + | + | ND | ND | I |
V73A | S | - | - | +/- | - | III |
T74A | R | + | + | ND | ND | I |
K75A | S | - | - | +/- | + | IV |
The mutant pilins were assayed for the phenotypes described above. We divided the mutant pilins into four phenotypic classes (Tables 2 and 3, Fig. 6). Class I Flp1 pilin mutants (21 in number) were indistinguishable from the wild-type pilin in our assays. In other
Because there is no 3D structure yet for Flp1 pilin, we used structure-prediction software with the sequence. Like other type IV pilins, the mature Flp1 pilin was predicted to be divided into a largely hydrophobic N-terminal domain and a distinct C-terminal domain. In Flp1, the C-terminal domain was predicted to contain an 11-residue amphipathic α-helix and a 12-residue, mostly-polar, C-terminal tail. It is thought that pilin subunits interact for polymerization in the hydrophobic region (Giltner et al., 2012). We therefore expected the ”assembly” mutants (Class III) to be caused by alanine substitutions of residues predominantly in the hydrophobic N-terminal region. Conversely, the C-terminal region of a pilin is thought to interact with the environment (Giltner et al., 2012), so we expected Class IV mutants to occur mostly from alterations of C-terminal residues. However, we were somewhat surprised by the number of Class III mutants with a substitution in a C-terminal residue, and by the number of Class IV mutants with a change in an N-terminal residue.
Mutant Class | Adherence | Protein Expression | Piliation | Pilus Morphology |
I | + | ND | ND | ND |
II | - | - | ND | ND |
III | - | + | - | NA |
IV | - | + | + | Normal and altered |
A previous study indicated that seven serine and asparagine residues in the C-terminal region are modified (Inoue et al., 2000). We found that only one substitution of those seven residues, a Class III mutant (N50A), gave a mutant phenotype. We do not know if the defect is due to the loss of modification at this residue or to the change in that amino acid.
Alanine-scanning mutagenesis has been an important step in beginning to understand the Flp1 pilin. We now have a collection of mutant pilins that can be studied for information on pilin stability, pili assembly, pili bundling, and pili adherence. These mutants will guide experiments in which substitutions can be made with amino acids that are of different sizes, have similar properties, or have very different properties. Similar experiments can also be done at the alanine residues.
A 3D structure of the Flp1 pilin is needed. There are a few structures of type IV pilins, most of which were determined from crystals formed after removing the N-terminal hydrophobic domain (Giltner et al., 2012). The Flp1 structure will clearly be different from the few structures of other type IV pilins. For example, Flp1 is 2 to 3-fold smaller than other type IV pilins. Also the mature Flp1 pilin has no cysteine residues, which are thought to be needed to form a disulfide bond to make the D region structure that seems to be conserved in type IV pilins. The phenotypes of the Flp1 mutants have also underscored the differences of Flp1 pilin and the “typical” type IV pilins. A structure would help us to understand (1) what is truly “typical” in type IV pilins, (2) the importance of certain Flp1 residues, and (3) the molecular basis of the phenotypes of the mutants.
3. TadZ
The
We did a large phylogenetic analysis and showed that the
3.1. The atypical Walker-like A box of TadZ proteins
Each of the protein products of the
3.2. Phenotypes of mutants altered in the atypical Walker-like A box of TadZ
We wanted to know the effect of changing the residue in position 6 of the atypical Walker-like A box from alanine in the TadZ protein of
three phenotypes (see Fig. 9 for the adherence result). Therefore, the presence of alanine at position 6 of the Walker-like A box is essential for AaTadZ function.
We noticed that a common feature of the atypical Walker-like A boxes from other TadZ proteins was the absence of lysine at Walker-like box position 6, not the presence of alanine. The
We confirmed this property for AaTadZ. When we mutated the
Even though it seemed that the absence of lysine was the primary requirement for the position 6 residue of the Walker-like A box of TadZ proteins, other residues of the Walker-like A motif are conserved. This observation indicated that the other residues in the Walker-like A boxes of TadZ proteins are important for function. To test this, we made mutants of AaTadZ in which the lysine residue at Walker-like A box position 1 (AaTadZ K150) was changed to arginine or alanine. Likewise, we made mutants at position 7 (AaTadZ S156) with threonine or alanine in place of the conserved serine residue. The mutant AaTadZ proteins did not allow wild-type biofilm formation (Fig. 9). Strains with the K150R, K150A, and the S156T mutants showed some biofilm formation; but it was reduced relative to wild type. The S156A mutant was completely unable to adhere. We concluded that the other residues of the atypical Walker-like A box of TadZ proteins are important to a function leading to tenacious adherence and biofilm formation.
The MinD proteins from
3.3. Mutants altered in the atypical Walker-like A box of TadZ localize properly
We used PCR to amplify the
3.4. Mutants altered in the atypical Walker-like A box of TadZ form dimers
We used a bacterial reporter strain to indicate TadZ dimer formation
We created a chimeric gene that encoded a fusion of the coding regions for λ cIDB and TadZ. The product of the fusion repressed
Protein | % repression of |
Empty vector | 0.0 ± 0.0 |
λcIDB | 9.7 ± 4.0 |
λcI | 66.0 ± 1.4 |
λcIDB-TadZ | 60.1 ± 0.7 |
λcIDB-TadZ K150R | 49.0 ± 1.9 |
λcIDB-TadZ K150A | 60.3 ± 1.5 |
λcIDB-TadZ A155K | 49.7 ± 2.1 |
λcIDB-TadZ S156T | 53.7 ± 0.8 |
λcIDB-TadZ S156A | 57.6 ± 4.2 |
4. Regulation of the tad locus
Logic and evidence indicate that the expression of the
There is evidence for transcriptional regulation of
For
Our studies have indicated that
4.1. Tad+ and Tad- bacteria make different biofilms
Biofilm formation depends on adherence. Biofilms with different characteristics may indicate different modes of adherence. Tad+
4.2. Choosing proteins that may be needed for adherence to inert surfaces
We sought to find a non-
Gene | Function |
Two-component sensor kinase; response to anaerobic and aerobic stress | |
Two-component response regulator; response to outer membrane stress | |
Two-component response regulator; response to nitrate and nitrite stress | |
Two-component response regulator; response to quorum sensing | |
Transcriptional regulator; response to redox stress | |
Integral membrane glycosyltransferase; required for PGA biosynthesis |
4.3. Mutagenesis of possible adherence-required genes by allelic exchange
We constructed a series of six mutant Tad+ strains and six mutant Tad- strains. Each strain had a mutation in one of our six candidates genes (Section 4.2). We asked if any of the mutants was defective in the strain’s biofilm
We used plasmid pMB78 (Bhattacharjee et al., 2007), a “suicide” plasmid,
Adherence of the Tad+ (Fig. 12B) and Tad- (Fig. 12C) mutants was quantified by the crystal violet assay (Section 2.2). One mutation, Δ
5. A strategy for making precise genomic deletions: the new Vector Excision (VEX) method
In Section 4.3, allelic exchange was described as a method of exchanging a wild-type segment of the chromosome with a mutated segment. In the examples given, a single gene was mutated. Allelic exchange can also be used for more than one gene. However, because the technique usually relies on standard molecular cloning methods, allelic exchange often becomes more difficult with larger segments. (See the chapter by Gerlach et al. for a discussion of recombineering – a method of cloning that overcomes the limitation caused by the locations of restriction endonuclease cleavage sites.) Another problem can be caused by the insertion of the marker, which can affect the expression of downstream genes. A method based on site-specific recombination has been developed to remove the marker (Datsenko and Wanner, 2000).
We have developed a straightforward method for making chromosomal deletions that can be any size [one bp to several kb (kilobase pairs)] and do not affect the expression of downstream genes. The strategy is based on the Vector Excision (VEX) method that we developed (Ayres et al., 1993). With VEX, the deleted portion can also be “captured” on a self-transmissible, broad-host-range plasmid. After transfer to another bacterium, the expression and/or functions of the captured genes can be assessed in a different host (see the chapter by Wilson et al.).
The new VEX strategy is illustrated here as a deletion (~11 kb) of all the
The purpose of the two homologous recombination events is to integrate two directly repeated
cloned with the following restriction endonucleases:
After the Cre-mediated deletion (resolution) (Fig. 15), a single
6. Conclusion
The 14-gene
Several proteins are unique products of
Genetic studies of tad loci and the development of new genetic tools are helping us to determine and understand (1) the functions of the individual tad genes, (2) whether and how the various proteins encoded by a tad locus act to colonize a specific niche, and (3) the importance of the tad genes for A. actinomycetemcomitans and for other prokaryotes.
Acknowledgement
We are grateful to David Furgang, Scott Kachlany, Jeff Kaplan, Mari Karched, Paul Planet, Helen Schreiner, Kabilan Velliyagounder, James Wilson, and Gang Yue for discussions and/or technical help. We appreciate the efforts of Oliver Jovanovic on the figures. The work was funded by grants from the National Institutes of Health (NIH), USA. Additional NIH funding supported B.A.P., V.W.G., and K.E.K.
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