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Received: 29 July 2019    Revised: 6 November 2019    Accepted: 13 November 2019

DOI: 10.1111/omi.12273

ORIGINAL ARTICLE

Porphyromonas gingivalis genes conferring fitness in a tobacco-

rich environment

Justin A. Hutcherson1 | Himabindu Gogenini1 | Gwyneth J. Lamont1 | Daniel P. Miller1 |

Zuzanna Nowakowska1,2 | Anna M. Lasica1,3 | Chengcheng Liu1,4 | Jan Potempa1,2 |
Richard J. Lamont1  | Deborah Yoder-Himes5 | David A. Scott1

1
Oral Immunology and Infectious Diseases,
University of Louisville, Louisville, KY, USA Abstract
2
Faculty of Biochemistry, Biophysics and Smokers are more likely than non-smokers to harbour Porphyromonas gingivalis, they
Biotechnology, Jagiellonian University,
are more susceptible to destructive periodontal disease and smokers may, ultimately,
Kraków, Poland
3
Department of Bacterial Genetics, Institute
benefit from tobacco-specific preventive and treatment strategies. A Mariner trans-
of Microbiology, Faculty of Biology, poson insertion library for P. gingivalis ATCC 33277 was exploited to define 256 genes
University of Warsaw, Warsaw, Poland
4
as essential for P. gingivalis survival in a tobacco-rich environment. Genes whose
West China Hospital of Stomatology,
Sichuan University, Chengdu, China products play roles in protein transport and catabolism, nicotinamide processing,
5
Department of Biology, University of protection against oxidative stress, drug resistance, and transcriptional regulation
Louisville, Louisville, KY, USA
have all been identified as essential for CSE survival. Many of these tobacco-essential
Correspondence genes are also requisite for epithelial colonization and abscess formation, suggestive
David A. Scott, Oral Immunology and
of a core stress-related P. gingivalis genome. Single-gene deletions in several of the
Infectious Diseases, University of Louisville
School of Dentistry, 501 S Preston St, TnSeq-implicated genes led to significantly reduced P. gingivalis fitness upon com-
Louisville, KY #40292, USA.
petition with the parent strain, under conditions of cigarette smoke extract-induced
Email: david.scott@louisville.edu
stress (1,000 ng/ml nicotine equivalents). This study identifies, for the first time, a
Funding information
subset of P. gingivalis genes required for surviving the plethora of insults present in
National Institute of General Medical
Sciences, Grant/Award Number: cigarette smoke. Such conditionally essential genes may delineate bacterial persis-
GM125504; National Institute of Dental
tence strategies and represent novel therapeutic foci for the prevention of P. gingi-
and Craniofacial Research, Grant/Award
Number: DE011111, DE012505, DE017921, valis infection and related diseases in smokers and in general.
DE022597, DE023193 and DE026963

KEYWORDS

cigarette smoke, nicotinamide, oral bacteria, periodontitis, proteases, transposon sequencing

library

1 | I NTRO D U C TI O N
Destructive periodontal diseases are infectious diseases of the tis-
sues surrounding the teeth which lead to significant oral debilita-
tion. In addition, they are associated with multiple serious systemic
sequelae and represent a significant economic burden. At the pop-
ulation level, smoking may account for most cases of periodontitis
in adults in developed nations, such as Scandinavia, New Zealand
and the US (Bergstrom, 2014; Haisman-Welsh & Thomson, 2012;
Sanders & Slade, 2013; Tomar & Asma, 2000). Clearly, bacteria are
required to induce disease yet the mechanisms by which smoking
predisposes to periodontal diseases are not well elucidated.
Contemporary data also show that P. gingivalis, a causative
agent of periodontal diseases, and other Bacteroidetes, are more
likely to infect and persist in smokers compared to non-smokers
(Chigasaki et al., 2018; Feres et al., 2014; Ge, Rodriguez, Trinh,

The peer review history for this article is available at https​://publo​ns.com/publo​n/

10.1111/mom.12273​

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10     © 2019 John Wiley & Sons A/S. wileyonlinelibrary.com/journal/omi Mol Oral Microbiol. 2020;35:10–18.
Published by John Wiley & Sons Ltd
HUTCHERSON et al.
Gunsolley, & Xu, 2013; Guglielmetti et al., 2014; Hanioka et al.,
2019; Joshi et al., 2014; Kumar, 2012; Mason et al., 2014). This is
consistent with earlier, more traditional, studies showing P. gingi-
valis to be more prevalent and present in higher numbers in smok-
ers than in non-smokers (Eggert, McLeod, & Flowerdew, 2001;
Haffajee & Socransky, 2001; Kamma, Nakou, & Baehni, 1999;
Zambon et al., 1996). Indeed, P. gingivalis is resistant to very high
doses of cigarette smoke and tobacco constituents (Bagaitkar et
al., 2011, 2009; Cogo et al., 2009).
To induce or exacerbate periodontal diseases in smokers, P.
gingivalis must be able to survive cigarette smoke insults, primar-
ily soluble and systemically distributed cigarette components. We
developed a Mariner transposon insertion library for P. gingivalis
ATCC 33277, a model Gram-negative anaerobic periodontopatho-
gen, and employed it to identify a core genome permitting repli-
cation of this P. gingivalis strain in rich medium (Hutcherson et al.,
2016). We have also assessed the fitness of specific mutants during
epithelial cell colonization and survival in a murine abscess model
(Miller et al., 2017). We have now utilized this tool to test the hy-
pothesis that multiple P. gingivalis genes are essential for survival
upon exposure to physiologically relevant doses of cigarette smoke
extract (CSE). We establish that P. gingivalis strains with mutations
in a subset of these genes exhibit an absolute reduction in fitness
[PGN_1444 (∆cps)], or reduced fitness when competed against the
parent strain [PGN_0088 (∆sinR1), PGN_0287 (∆mfa1), PGN_0388
(∆tpx), PGN_0770 (∆rnz), PGN_1200 (a DNA-dependent ATPase),
and PGN_1524 (∆ptk1)] under tobacco-induced stress, confirming
CSE conditional essentiality.
2 |  M E TH O DS
2.1 | Materials
Gifu anaerobic medium (GAM) was obtained from Nissui
Pharmaceutical. Sheep blood was obtained from Lampire Biological
Laboratories. Gentamicin, erythromycin, tetracycline, ampicillin,
dichloromethane, sodium sulfate, and analytical nicotine standards
were from Sigma-Aldrich. 3R4F research cigarettes were purchased
from the Centre for Tobacco Reference Products (University of
Kentucky, Lexington, KY). All primers were from Biosynthesis Inc.
Lonza flash gels came from Lonza. Wizard SV and PCR clean-up kits
were from Promega. T4 DNA ligase, MmeI; and TAE buffer came
from New England Biolabs. HiFi Hotstart Ready mix was from KAPA
Biosystems and PCR SuperMix came from Invitrogen. Fast digest re-
striction enzymes and ready to use X-gal and plasmid isolation kits
came from Thermo Fisher Scientific.
2.2 | Bacterial culture
P. gingivalis ATCC 33277 and isogenic mutants ∆PGN_0088 (sinR),
∆PGN_0287 (mfa1), ∆PGN_0388 (a putative thiol peroxidase),
|
      11
∆PGN_0491 (ltp1, a tyrosine protein phosphatase), ∆PGN_0770
(rnz), ∆PGN_1200 (putative ATPase), ∆PGN_1444 (carbamoyl
phosphate synthase, cps), ∆PGN_1524 (ptk1), and a non-negatively
selected control mutant, ∆PGN_1753 (2-oxoglutarate oxidoreduc-
tase-related gene) were cultured anaerobically in GAM, cigarette
smoke-conditioned GAM (GAM-CSE) or on GAM agar plates sup-
plemented with defibrinated sheep's blood at 37°C.
2.3 | P. gingivalis transposon library
Full details of the 80,000 colony transposon library employed have
been previously reported (Hutcherson et al., 2016). The P. gingivalis
33277 library was maintained in GAM containing 50 μg/ml gen-
tamicin and 5 μg/ml erythromycin, the input library aliquoted at
1010 CFU and stored at −80°C.
2.4 | Cigarette smoke conditioning
Gifu anaerobic medium was cigarette smoke conditioned, as previ-
ously described (Bagaitkar et al., 2010). Essentially, 3R4F standard
reference cigarette smoke (GAM-CSE) was drawn through GAM
and filtered (0.2 μm). Nicotine content was determined by GLC and
GAM-CSE adjusted to 1,000 ng/ml nicotine, pH 7.2.
2.5 | Construction and sequencing of DNA
libraries and analysis of sequencing data
The P. gingivalis ATCC 33277 TnSeq mutant library was grown to
mid-log phase, as determined by optical density, and passaged
twice in GAM (input) and GAM-CSE (output). Double-stranded
DNA (dsDNA) adapter molecules were created using primers listed
in Table S4. The sequencing platform, sequence sorting, read trim-
ming, normalization, and alignment to the annotated gene list of P.
gingivalis ATCC 33277 were performed as recently reported (Miller
et al., 2017). Significance was determined using CLC Bioinformatics
Workbench v7.2 software. Genes with ≥500 genes in the input pool,
≥50 fold change in input and output pools and a Bonferroni-adjusted
p-value <.05 were considered significant for fitness (Miller et al.,
2017).
2.6 | Mapping and enrichment of CSE-
essential genes
Genes on which P. gingivalis is reliant in a tobacco-rich environment
were mapped to the 33277 genome, along with P. gingivalis TDC60
and W83 orthologues, using the CGViewer software (http://stoth​
ard.afns.ualbe​r ta.ca/cgview_serve​r/) [data not shown]. CSE essen-
tial genes were distributed throughout the bacterial chromosome.
Relationships between essential genes were determined using the
 |
12       HUTCHERSON et al.
KEGG (www.genome.jp/kegg) database. Potential functional anno-
tations for those genes considered hypothetical in the KEGG system
were also examined by processing through the NCBI pipeline (https​
://www.ncbi.nlm.nih.gov/genom​e/annot​ation_prok/).
2.7 | Mutant construction
Mutants employed herein are described in Table S3. The P. gingi-
valis deletion mutants created specifically for the current study
were generated as we have previously described (Nguyen, Travis, &
Potempa, 2007) [PGN_0388] or (Miller et al., 2017; Simionato et al.,
2006) [PGN_0088, PGN_1753], using primers presented in Table S4.
Following anaerobic selection on erythromycin (10 µg/ml) or tetra-
cycline (1 µg/ml) seeded plates, as appropriate (Table S3), colonies
arising from transformed clones were screened for the correct mu-
tations by PCR and sequencing.
2.8 | Competitive fitness assays
In competitive growth assays, P. gingivalis ATCC 33277 and individ-
ual mutant strains were anaerobically co-cultured 1:1 in GAM-CSE
(1,000 ng/ml nicotine equivalents). After 2 days, strain growth was
quantified by CFU counts on GAM agar plates supplemented with
defibrinated sheep's blood, with and without the mutant-specific
resistance antibiotic. All experiments were carried out in triplicate.
Differences in percentage survival rates were determined by paired
t-test using InStat v3.06 (GraphPad).
3 |   R E S U LT S
3.1 | Identification of essential CSE survival genes
A total of 256 genes conditionally essential for survival in the GAM-
CSE model were identified. The genes were distributed throughout
the P. gingivalis chromosome. In silico analyses were used to iden-
tify the functions of these essential genes, as presented in Table S1
(functionally annotated genes) and Table S2 (genes encoding hypo-
thetical proteins).
3.2 | Relationships between essential CSE
survival genes
KEGG analysis revealed predicted functional relationships between
CSE-essential genes, as delineated in Figure S1. Interestingly, 7/13
(54%) of the bacterium's nicotinate and nicotinamide metabolism
genes were essential, as shown in Figure 1. Multiple genes involved
in protection against oxidative stress or in protein and/ or peptide
catabolism were also determined to be essential, as presented in
Tables 1 and 2.
3.3 | Common and unique essential genes in
multiple disease-relevant models
We have previously determined the core essential genome of P. gin-
givalis ATCC 33277, that is, genes required for survival in standard-
rich culture conditions (Hutcherson et al., 2016). More recently,
we have determined a subset of genes required for P. gingivalis to
invade epithelial cells and to produce a subcutaneous abscess in a
mouse model (Miller et al., 2017). We now report that, from a total
of 256 genes essential for surviving CSE-induced stress, a subset
are specific to this particular microenvironment, whereas most are
common between the stress conditions in which this library has
been exploited. Genes uniquely required for surviving in cigarette
smoke extract conditioned medium include PGN_0065 (traG),
PGN_0415 (a restriction endonuclease) and PGN_0584 (topoi-
somerase). Interestingly, PGN_1440 (vanW) is a CSE-essential
gene. PGN_0323, likely an outer membrane protein with some
homology to PorT protein transporter was also CSE-essential.
CSE genes that exhibited the largest differentials between input
and output libraries, as well as those whose functional annota-
tions have clear relevance to CSE survival, are discussed further
below. Essential genes that are uniquely required for CSE survival
as well those commonly requisite for fitness under CSE-induced
stress, epithelial invasion and/or abscess formation are presented
in Tables S1 and S2. The relative distributions of essential genes of
P. gingivalis between the three selection pressures are presented
in Figure 2a (TIGK>abscess>CSE), with epithelial colonization the
environment requiring the largest proportion of the genome. The
distributions are compared to those that may happen by chance
alone, as determined by the generation of randomized integer sets
(Figure 2b).
3.4 | Confirmation of conditionally essential CSE
survival genes
To verify the loss of fitness in strains bearing mutations in TnSeq-
defined essential genes, we conducted in vitro competition assays
between the parental P. gingivalis strain and strains with deletion
mutations in eight different genes. As a control, a strain bearing
a mutation in the PGN_1753 locus, which was shown not to be
essential in the CSE Tn-seq experiments, was employed. First,
we assessed relative growth characteristics to ensure that mu-
tant strains had similar growth rates compared to the parental
strain thus preventing false positives or negatives in our compe-
tition experiments. Growth of the control mutant, ∆PGN_1753,
was not compromised in tobacco-rich environments, either in
CSE-conditioned monoculture compared to non-conditioned me-
dium (p > .05) or in competition with the parent strain (p > .05).
∆PGN_1444 exhibited a reduced fitness phenotype in monocul-
ture, that is, mutant growth was significantly diminished in CSE-
conditioned medium compared to unconditioned GAM (Table 3). In
other words, deletion of PGN_1444 alone results in compromised
HUTCHERSON et al.
F I G U R E 1   Essential nicotinate and
nicotinamide metabolism genes in P.
gingivalis 33277. P. gingivalis 33277 Niconamide
homologues of genes in nicotinate
and nicotinamide metabolic pathways
were enriched by KEGG analysis (www. 3.5.1.19
genome.jp/kegg). Stars denote CSE- PGN_0714
essential genes. PGN_0533, PGN_0534,
Niconate
PGN_0714, PGN_1412 are presented as
their enzyme commission number and
substrates or products. The commission
number 1.6.1.2* is represented by
three CSE-essential P. gingivalis genes,
PGN_1120, PGN_1121, and PGN_1122
growth of CSE-exposed P. gingivalis. Conditional essentiality was
confirmed by competitive fitness assays against parent P. gingivalis
in 6/7 (86%) mutant strains tested, as presented Table 3. Growth
of the ltp1 mutant, ∆PGN_0491, was not compromised by CSE
in monoculture or in competition against wild-type P. gingivalis
ATCC 33277. Nevertheless, most mutants screened exhibited the
library-predicted CSE-related phenotype, suggesting that TnSeq
is a powerful tool for the identification of conditionally essential
bacterial genes.
4 |  D I S CU S S I O N
Using a transposon sequencing library, we have previously estab-
lished a core set of 281 genes, out of a total of 2,155 ORFs, that are
absolutely required to support the growth of P. gingivalis ATCC 33277
in rich medium (Hutcherson et al., 2016), in common with a second,
independently generated and validated P. gingivalis ATCC 33277 li-
brary (Klein et al., 2012). More recently we have determined those
P. gingivalis genes that are essential for P. gingivalis survival in two
disease-related model systems, epithelial cell colonization (n = 541
genes) and subcutaneous abscess formation in mice (n = 496 genes).
Interestingly, 482 of these genes were common to each model, sug-
gesting a high degree of overlap in the fitness determinants required
for these environments. We now identify 256 P. gingivalis genes that
are essential for survival in a tobacco-rich environment. Of these,
the majority are common amongst all disease-relevant models tested
(CSE exposure, abscess formation, and epithelial colonization). Such
commonality amongst datasets may be indicative of a core stress-
related genome in P. gingivalis. As we have recently discussed the
physiological relevance of these common genes, with respect to gin-
gival adhesion, iron acquisition, established stress responses, conju-
gation, and tetratricopeptide repeat (TPR) protein-related virulence
(Miller et al., 2017), we shall not revisit this topic in depth herein.
|
      13
Niconamide
2.4.2.1
PGN_1412 D-ribonucleode NADP+
N-Ribosyl
3.1.3.5 2.7.7.18 2.7.1.23
niconate
1.6.1.2*
3.6.4.21
NAD+
2.4.2.1 3.5.1.42
PGN_1412
6.3.51
2.7.7.18
3.1.3.5 DeaminoNAD+
Niconate D-ribonucleode
2.4.2.19 Quinolate
PGN_0534
nadA
PGN_0533
1.4.3.16
L-Aspartate
The five genes exhibiting the greatest change between input (no
CSE) and output (CSE) libraries were PGN_0407, PGN_0025 (spoU),
PGN_1494 (coproporphyrinogen III oxidase), PGN_0388 (ptx), and
PGN_0604 (ferritin). Unfortunately, efforts to generate mutants in
PGN_0025 (spoU) and PGN_1494 (coproporphyrinogen III oxidase)
proved unsuccessful. There is no functional annotation available for
PGN_0407. P. gingivalis ferritin has been reported to be highly upreg-
ulated under oxidative stress, although it is insufficient to fully pro-
tect against oxygen exposure (Meuric, Gracieux, Tamanai-Shacoori,
Perez-Chaparro, & Bonnaure-Mallet, 2008; Ratnayake et al., 2000).
PGN_0604 mutants have not been evaluated, whereas the CSE-
related phenotype of a PGN_0388 mutant is addressed below.
Nicotinamide is a structural analogue of nicotine, long known to
be toxic to bacteria (Koser & Kasai, 1947; Murray, 2003), that may be
present in cigarette smoke itself (Buyske, Flowers, Hobbs, & Wilder,
1956). Nicotine has also been reported to inhibit the growth of multiple
Gram-negative bacteria, including Escherichia coli, Klebsiella pneumo-
niae, and Borrelia burgdorferi (Gandhi, Athmaram, & Arunkumar, 2016;
Pavia, Pierre, & Nowakowski, 2000; Salman et al., 2016). Therefore, it
is of particular interest that PGN_0714 was identified as CSE-essential
in our TnSeq screen, as well as several genes involved in nicotinate
and nicotinamide metabolism. Some nicotinate- and nicotinamide-re-
lated gene products exhibit functional overlap with an oxidative stress
response. PGN_0714 encodes a nicotinamidase which produces nic-
otinate from nicotinamide. PGN_0533 (quinolate synthetase A) and
PGN_0534 (nicotinate-nucleotide pyrophosphorylase) are involved
in generation of the key redox-related molecule, NAD, whereas
PGN_0533 and PGN_1120 (NADPH-NAD transhydrogenase/alanine
dehydrogenase) have also be shown to be essential for the black pig-
mentation associated with heme binding (Klein et al., 2017).
The precise role in protection against tobacco toxins are not im-
mediately obvious for several of the CSE-specific essential genes.
PGN_0415, PGN_0584, and PGN_1116 encode a restriction endo-
nuclease, a topoisomerase and an aminotransferase, respectively.
 |
14       HUTCHERSON et al.

TA B L E 1   CSE-essential protein catabolism genes of P. gingivalis TA B L E 2   CSE-essential oxidative stress genes of P. gingivalis
33277 33277

Protein catabolism genes Oxidative stress genes

Gene number Annotation Gene number Annotation

PGN_0271 pepO endopeptidase PGN_0168 wbpB, LPS biosynthesis protein/

PGN_0295 Arg-/Lys-gingipain proteinase oxidoreductase
C-terminal domainb PGN_0302 Rubrerythrin
PGN_0303 Zinc protease, M16 family PGN_0373 Thioredoxin
PGN_0335 Zinc carboxypeptidase domain protein PGN_0388 Thiol peroxidase
PGN_0561 prtT, cysteine protease PGN_0533 nadA, Quinolinate synthetase A
PGN_0607 Dipeptidyl peptidase 11 PGN_0534 nicotinate-nucleotide pyrophosphorylase
PGN_0637 htrA, heat shock-related protease PGN_0564 Superoxide dismutase Fe-Mn
PGN_0754 NlpC/P60 peptidase familya PGN_0604 Ferritin
a
PGN_0771 NlpC/P60 peptidase family PGN_0709 Indolepyruvate ferredoxin oxidoreduc-
PGN_0780 prtQ proteasec tase, beta subunit

PGN_0788 Peptidyl dipeptidase PGN_1268 oxidoreductase

PGN_0900 Thiol protease d PGN_2073 oxidoreductasea 

PGN_1335 Hypothetical peptidase PGN_2077 2Fe−2S binding proteina 

PGN_1349 Dipeptidyl aminopeptidase
PGN_1466 rgpB, gingipain B
PGN_1479 S46 family dipeptidyl-peptidasea
PGN_1694 Alanyl dipeptidyl peptidase
PGN_1777 Bleomycin hydrolase
PGN_2064 M48 family peptidase
Note: Annotated functions of CSE-essential genes (>500 reads on input,
>50 fold change, p < .05) were annotated using KEGG.
a
Peptidolytic annotation from the NCBI pipeline.
b
A Blast search of the MEROPS database (https​://www.ebi.ac.uk/
merop​s/) reveals PGN_0295 to be a CTD-secreted protein of unknown
function.
c
No proteolytic motifs were identified upon crystallization (Schacherl,
Montada, Brunstein, & Baumann, 2015).
d
We have previously characterized this protein as periodontain (Nelson,
Potempa, Kordula, & Travis, 1999).
PGN_1440 (vanW) is a vancomycin B-type resistance protein that is,
as yet, understudied. However, it is possible that a detoxification-re-
lated VanW function may be relevant to protection against cigarette
smoke components. The hypothetical protein, PGN_0323, exhibits
some homology to PorT, whereas our TnSeq screen also identifies
another PorT family member, PGN_0156, as CSE-essential. Indeed
PGN_0156 is in the top 10 of input and output library differentials.
As we have previously reported (Nguyen et al., 2009), PorT is im-
portant in the maturation and secretion of gingipains, which are
critical to P. gingivalis colonization and to several other key aspects
of P. gingivalis virulence. Multiple other CSE-essential genes may
provide insight into P. gingivalis robustness in tobacco-rich environ-
ments. Histidine kinases, for example, are key to bacterial signal
transduction and, subsequently, virulence and may represent novel
antibacterial targets (Bem et al., 2015). PGN_0082 is an AraC-family
transcriptional regulator known to be involved in quorum sensing
Note: Annotated functions of CSE-essential genes (>500 reads on input,
>50 fold change, p < .05) were annotated using KEGG.
a
Annotation from the NCBI pipeline.
and stress response in other bacterial species (Romero-Lastra et al.,
2017). Indeed, in the related bacterium, Bacteroides thetaiotaomi-
cron, greater than 80% of a reported 44 hybrid histidine kinases con-
tain AraC-like DNA-binding domains (Galperin, 2006). PGN_0302
encodes rubrerythrin. This is notable, as P. gingivalis, which lacks
catalase, uses rubrerythrin to protect against oxygen- and nitro-
gen-related stresses (Mydel et al., 2006). PGN_0447 is an ABC
transporter permease whose most closely related orthologues are
multidrug and other efflux transporters in Tannerella (BFO_0463 and
BCB71_07875) and Parabacteroides (CI960_00995) spp. PGN_0675
is a DJ-1 family protein. DJ-1 is a multifunctional protein with decly-
case activity in mammals whose bacterial homologues have recently
been shown to be important mediators of genomic stability in bacte-
ria through their nucleotide glycation repair activity (Richarme et al.,
2017). This is likely relevant to the multiple mutagenic compounds
known to be present in cigarette smoke. PGN_0754 and PGN_0771
belong to the NlpC/P60 family of proteins, typically encompassing
papain-like domain endopeptidases with specificity for peptidogly-
can-embedded amino acids (Xu et al., 2015). While such P. gingivalis
genes are poorly characterized, they may be essential for modifica-
tion of the P. gingivalis cell-envelope during stress induced by CSE
components. PGN_0200 is now thought to encode TraB and joins
traA, traG, and traI as stress-related essential conjugation genes.
The dependence of P. gingivalis on proteolysis for survival and sub-
sequent virulence in the oral cavity is a well-established phenomenon.
Those genes involved in protein catabolism that are essential for CSE
survival contain a combination of established proteolytic virulence
factors and a larger set of seemingly more mundane conditionally es-
sential proteolytic genes. The former include pepO, an endopeptidase
HUTCHERSON et al. |
      15

A B
13 313
Abscess “Abscess”
251 113
1 52
231 18
TIGK 18 CSE
6
“TIGK” 44“CSE”
142
41 366

F I G U R E 2   Distribution of essential genes of P. gingivalis between CSE, TIGK, and murine abscess selection pressures. (a) Negatively
selected genes between selection pressures are presented. A total of 231 genes were essential for all conditions. The volume of each sphere
represents the ratio of essential genes per condition (TIGK>abscess>CSE). (b) The Random Integer Set Generator tool (random.org) was
employed to generate random n = 541, n = 496, and n = 256 integer arrays from n = 1874, the total number of genes out with the previously
determined 33277 core genome (Hutcherson et al., 2016)

TA B L E 3   CSE essential genes determined by absolute or
competitive fitness assays
Survival on
Mutant agar [% (SD)]
PGN_0088 (∆sinR1) 22.4 (6.5)*
PGN_0287 (∆fimA) 0.0 (0.0)***
PGN_0388 (∆tpx) 2.7 (2.1)***
PGN_0491 (∆ltp1) 40.3 (26.2)
PGN_0770 (∆rnz) 15.8 (5.8)**
PGN_1200 (∆DNA-dependent ATPase) 17.8 (15.5)*
PGN_1524 (∆ptk1) 1.1 (1.8)***
Note: P. gingivalis ATCC 33277 and mutant strains were anaerobically
cocultured at a 1:1 in GAM-CSE (1,000 ng/ml nicotine equivalents).
Growth was quantified by CFU counts on GAM agar plates
supplemented with defibrinated sheep's blood, with and without the
mutant-specific resistance antibiotic. Differences in percentage survival
rates were determined by paired t-test. The bold values are statistically
significant.
Additionally, ∆PGN_1444 exhibited an absolute reduced fitness
phenotype in monoculture, that is, mutant growth was significantly
diminished in CSE-conditioned medium (1,000 ng/ml nicotine
equivalents) compared to unconditioned GAM (0 ng/ml nicotine
equivalents).
*p < .05, **p = .01, and ***p = .001, respectively.
whose deletion lowers epithelial invasion efficacy (Ansai, Yu, Urnowey,
Barik, & Takehara, 2003), at least in P. gingivalis 381, periodontain and
the archetypal virulence factor, rgpA. However, many of the remaining
CSE-essential protease-related genes are not well characterized. As
with other genes encoding essential but understudied proteins, such
proteolytic genes may represent novel therapeutic targets for the con-
trol of P. gingivalis infection.
Several CSE-essential genes were previously recognized as key
to subcutaneous abscess formation and/or epithelial colonization
that, at the time of discovery, were without functional annotation.
Such stress genome constituents include PGN_1928 (cmr6, type III
CRISPR module RAMP protein), PGN_1930 (cmr4, type III CRISPR
module RAMP protein), and PGN_1932 (cmr2, type III CRISPR-
associated protein). While these genes are considered to be involved
in bacterial innate defense (Wang & Li, 2012), their essential role
in protecting against environmental stress is currently elusive. Of
particular interest is the α2-macroglobulin-encoding PGN_2070.
α2-macroglobulin, as we have also shown, is the only efficient endog-
enous proteinaceous inhibitor of Rgp gingipains described to date
(Gron et al., 1997). Gram-negative prokaryotic α2-macroglobulins are
thought to generally locate to the periplasm (Wong & Dessen, 2014).
It is a possibility, then, that the PGN_2070 product may help control
translocating gingipain activity in a manner that is essential in three
different P. gingivalis environments. Certainly, bacterial α2-macro-
globulins have been shown to sequester exogenous proteases as
part of a rudimentary defense system (Wong & Dessen, 2014). The
type IX secretion system (T9SS) is restricted to a subset of bacteria
of the Bacteroidetes phylum that, as we have recently summarized,
is critical for the transport of over 30 P. gingivalis proteins bearing
a conserved C-terminal domain (CTD), including primary virulence
factors, such as the gingipains (Lasica, Ksiazek, Madej, & Potempa,
2017). It is interesting that multiple CTD-proteins (PGN_0291,
PGN_0295 (MEROPS search), PGN_0335, PGN_0561, PGN_0852,
PGN_0898, PGN_0900, PGN_1116, and PGN_1416) (Lasica et al.,
2017), are CSE-essential, but RgpA and Kgp are not among them.
Klein et al (Klein et al., 2017) have recently identified a set of
P. gingivalis ATCC 33277 genes that are required for heme acquisi-
tion that exhibits only limited overlap with genes that are both func-
tionally annotated and requisite for CSE survival (mfa1, nadA, htrA,
PGN_0862 [type III restriction enzyme], PGN_1116 [aminotrans-
ferase], PGN_1120 [NADPH-NAD transhydrogenase], PGN_1618
[wbpB], PGN_1722 [uridine kinase], PGN_1777 [bleomycin hy-
drolase], and PGN_2017 [YjeF-family], as well as the hypothetical
protein-encoding genes, PGN_1313 and PGN_1591). On the other
 |
16       HUTCHERSON et al.
hand, common CSE-, TIGK,- and abscess-essential genes show con-
siderably more overlap than would be expected by chance alone, as
determined by the generation of random integers, strengthening the
core stress genome hypothesis.
Finally, and critically, we established the tobacco-related pheno-
type of a cross section of CSE-related genes in competitive growth
assays. Interestingly, although compromised growth under CSE-
induced stress was not observed for PGN_0491 (∆ltp1), the other
essential mutants tested [PGN_0088 (∆sinR1), PGN_0287 (∆mfa1),
PGN_0388 (∆tpx), PGN_0770 (∆rnz), PGN_1200 (∆ATPase), and
PGN_1524 (∆ptk1)] were outcompeted by the parent strain of P. gin-
givalis. PGN_1444 (∆cps) exhibited suppressed growth under CSE-
induced stress, an absolute reduction in fitness. PGN_0088 (∆sinR1)
encodes a transcriptional regulator. This protein has been reported
to inhibit exopolysaccharide synthesis (Yamamoto et al., 2013), in
keeping with our own finding that CSE-exposure leads to P. gingiva-
lis capsule diminishment (Bagaitkar et al., 2011, 2009). PGN_1524
encodes the bacterial tyrosine kinase, Ptk1, which is a key compo-
nent of a signaling pathway that controls community development
with Streptococcus gordonii (Wright et al., 2014). We have previously
shown that Ptk1 is also necessary for exopolyscaccharide produc-
tion (Wright et al., 2014). Several CSE essential minor fimbrial genes
were identified. PGN_0287 (mfa1) encodes the minor fimbrial anti-
gen, PGN_0288 encodes FimB/Mfa2, whereas PGN_0291 encodes
Mfa5. It remains unclear how minor fimbrial proteins may abet sur-
vival in CSE-rich batch culture. However, we have previously shown
that smoking augments P. gingivalis monospecies and P. gingivalis-S.
gordonii biofilms in an Mfa1-related manner (Bagaitkar et al., 2011,
2010). Since then, there have been multiple reports of tobacco-en-
hanced biofilm formation in several microbes, as we have recently
summarized (Hutcherson, Scott, & Bagaitkar, 2015). PGN_0388 is
an anti-oxidant thiol peroxidase, thus the conditional essentiality of
an oxidative stress gene is established. The PGN_0770 product is
RNase Z, a 6S RNA-/σ70-related transcriptional regulator, at least in
Escherichia coli (Chen, Dutta, & Deutscher, 2016). The gene targets
of PGN_0770 in P. gingivalis, and their relevance to cigarette smoke,
have yet to be ascertained. PGN_1200 is functionally annotated as
a DNA-dependent ATPase (RarA-related replication-associated re-
combination protein MgsA/RarA) and has been reported to contrib-
ute to genome stability in E. coli (Shibata et al., 2005). PGN_1444,
as we recently hypothesized, may play a key role in P. gingivalis viru-
lence by generating the metabolic cue, arginine (Miller et al., 2017).
In summary, we have determined a subset of genes that delin-
eate, for the first time, essential bacterial strategies relevant to per-
sistence in an environment representing the primary risk factor for
periodontitis—tobacco smoking. Genes whose products play roles
in protein transport and catabolism, nicotinamide processing, pro-
tection against oxidative stress, drug resistance, and transcriptional
regulation have all been identified. Conditional essentiality has been
confirmed using a broad range of bacterial mutants [PGN_0088
(transcriptional regulator, ∆sinR1), PGN_0287 (minor fimbrial anti-
gen, ∆mfa1), PGN_0388 (thiol peroxidase, ∆tpx), PGN_0770 (ribonu-
clease z, ∆rnz), PGN_1200 (DNA-dependent ATPase), and PGN_1524
(tyrosine kinase, ∆ptk1)]. Such essential genes may represent appro-
priate therapeutic targets for the treatment of P. gingivalis infections
specifically in tobacco users. Perhaps more importantly, however, we
have determined a subset of >200 genes that are commonly required
for surviving three different disease relevant conditions, CSE expo-
sure, epithelial colonization, and murine abscess formation. The CSE
model exposes P. gingivalis to a host of environmental toxins. The
epithelial model requires attachment to and/ or passage through a
eukaryotic biological membrane and resistance to the important, but
limited, epithelial defense mechanisms. To facilitate abscess forma-
tion, P. gingivalis strains must withstand a robust mammalian immune
response. This common stress-related gene set contains both estab-
lished and novel potential virulence factors. Interestingly, epithelial
colonization appears to exert the highest selective pressure on this
important periodontal pathogen, whereas CSE survival required the
least number of genes. However, genes with overlapping conditional
essentiality may represent particularly attractive preventative tar-
gets for P. gingivalis-induced disease in general.
AC K N OW L E D G M E N T S
These studies were supported by grants from NIDCR and NIGMS
(DE026963, DE011111, DE012505, DE017921, DE023193,
DE022597, and GM125504).
C O N FL I C T O F I N T E R E S T
The authors declare that they have no conflicted interests.
ORCID
Richard J. Lamont  https://orcid.org/0000-0002-3147-5039
David A. Scott  https://orcid.org/0000-0003-1007-2756
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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found online in the
Supporting Information section.  
How to cite this article: Hutcherson JA, Gogenini H, Lamont
GJ, et al. Porphyromonas gingivalis genes conferring fitness in
a tobacco-rich environment. Mol Oral Microbiol. 2020;35:10–
18. https​://doi.org/10.1111/omi.12273​