1 Expanding the genetic toolbox for *Neisseria meningitidis* with efficient tools for

2 unmarked gene editing, complementation and labelling

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4 Morgane WUCKELT¹,², Audrey LAURENT¹,², Clémence MOUVILLE¹,², Julie MEYER¹,²,

5 Anne JAMET¹,², Hervé LECUYER¹,², Xavier NASSIF¹,², Emmanuelle BILLE¹,², Vladimir

6 PELICIC³, Mathieu COUREUIL¹,² §

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8 ¹Université Paris Cité, UFR de Médecine, Paris, France

9 ²Inserm U1151, Institut Necker-Enfants Malades, CNRS UMR 8253, Paris, France

10 ³Laboratoire de Chimie Bactérienne, Aix-Marseille Université-CNRS (UMR 7283), Institut de

11 Microbiologie de la Méditerranée, Marseille, France

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13 $corresponding author: mathieu.coureuil@inserm.fr
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ABSTRACT

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The efficient natural transformation of *Neisseria meningitidis* allows the rapid construction of bacterial mutants in which the genes of interest are interrupted or replaced by antibiotic resistance cassettes. However, this proved to be a double-edged sword, *i.e.*, although facilitating the genetic characterization of this important human pathogen, it has limited the development of strategies for constructing markerless mutants without antibiotic resistance markers. In addition, efficient tools for complementation or labelling are also lacking in *N. meningitidis*. In this study, we significantly expand the meningococcal genetic toolbox by developing new and efficient tools for the construction of markerless mutants (using a dual counterselection strategy), genetic complementation (using integrative vectors) and cell labelling (using a self-labeling protein tag). This expanded toolbox paves the way for more in-depth genetic characterization of *N. meningitidis* and might also be useful in other *Neisseria* species.
26 **IMPORTANCE**

27 *Neisseria meningitidis* and *Neisseria gonorrhoeae* are two important human pathogens.

28 Research focusing on these bacteria requires genetic engineering, which is facilitated by their

29 natural ability to undergo transformation. However, the ease of mutant engineering has led the

30 *Neisseria* community to neglect the development of more sophisticated tools for gene editing,

31 particularly for *N. meningitidis*. In this study, we have significantly expanded the

32 meningococcal genetic toolbox by developing novel and efficient tools for markerless mutant

33 construction, genetic complementation and cell tagging. This expanded toolbox paves the way

34 for more in-depth genetic characterization of *N. meningitidis* and might also be useful in other

35 *Neisseria* species.
36 INTRODUCTION

37 *Neisseria meningitidis* and *Neisseria gonorrhoeae* are two important human pathogens
38 belonging to the genus *Neisseria*. The first causes meningitis and acute septicemia, while the
39 second is responsible for sexually transmitted infections and has developed multiple resistance
40 mechanisms against antibiotics. The frequent overlap of *Neisseria* species niches and the ease
41 with which they can exchange genetic material by natural transformation are likely to drive the
42 emergence of pathogenic strains and/or strains more resistant to antibiotics [1], [2], [3].

43 Research priorities for these pathogens include deciphering the molecular mechanisms of host-
44 pathogen interaction or acquisition of new genetic material, and developing improved vaccines.

45 These objectives require genetic manipulation of *N. meningitidis* and *N. gonorrhoeae*, which is
46 facilitated by their natural competence for transformation. Both pathogens efficiently take up
47 exogenous DNA, which is mediated by their type IV pili [4], [5], [6]. As a result, it is possible
48 to rapidly construct bacterial mutants and even complete libraries of mutants using classical
49 molecular biology methods [7]. On the other hand, the ease of PCR-based construction of
50 mutants using antibiotic resistance cassettes to interrupt or replace genes – although it hinders
51 the construction of strains with multiple mutations due to the limited number of cassettes – has
52 led the *Neisseria* community to neglect the development of more sophisticated tools for gene
53 editing. Only few methods for constructing markerless mutants in *Neisseria* species have been
54 published [8], [9], [10], [11]. The methods by Johnston and Cannon and Nyongesa *et al*. use
55 *rpsL* as a counterselectable marker, while the method by Seow *et al*. is based on the co-
56 transformation of a resistance cassette and a markerless mutation of interest. Unfortunately,
57 these two strategies generate mutants resistant to antibiotics. This is not the case for the strategy
58 developed by Jones *et al.*, which is based on the use of *galK* as a counterselectable marker [12].

59 This gene encodes a galactokinase (GalK), which phosphorylates 2-deoxy-galactose (2-DOG)
60 into 2-deoxy-galactose-1-phosphate, a toxic intermediate in many bacterial species [13]. Jones
et al. successfully obtained markerless mutants on plates containing 6% 2-DOG, a concentration that could be considered a limitation given its retail price. In addition, all three strategies are subject to spontaneous mutation of the counterselection markers, which generates false-positive mutants and limits the use of these tools in bacteria with high mutation rates such as the *N. meningitidis* Z5463 model strain [14]. Finally, Geslewitz et al. proposed recently to use a CRISPR interference strategy to inhibit the expression of genes in *N. gonorrhoeae* [15].

Another limitation is the paucity of genetic complementation tools. Two sets of complementation plasmids were developed in *N. gonorrhoeae* by the Seifert lab (Mehr and Seifert, 1998) by introducing a *lacI* sequence, a *lac* promoter (Plac) and an erythromycin or chloramphenicol resistance gene between the *aspC* and *lctP* genes. This locus is less attractive for complementation in *N. meningitidis* because of the presence of two small open reading frames (ORFs) between *aspC* and *lctP* (NMV_1885 and NMV_1887 in strain NEM8013). More recently, the Dillard lab developed new complementation plasmids which introduce either a *lacI-Plac* or a tetracycline repressor and the tetracycline-inducible *Ptet* promoter/operator between the *iga* and *trpB* genes [17].

Lastly, bacteria and/or protein labelling is also poorly developed in *Neisseria* species. GFP has been used to make bacteria fluorescent by introducing the *gfp* gene on a plasmid [18], [19], but GFP generates free radicals and induces oxidative stress [20]. Recently, Nyongesa *et al.* demonstrated that mCherry can be used as a bioluminescence marker in *Neisseria* species [10].

Here, we report the development of a set of new tools for unmarked mutagenesis, complementation, and labelling in *N. meningitidis*. We first adapted the strategy from Jones *et al.* by generating a new cassette containing one antibiotic selection marker and two counterselection markers (*galK* and *pheS*\*). We show that the resulting cassette enables rapid and low-cost selection of markerless meningococcal mutants, even in strains with high mutation
rates. We then designed complementation plasmids for integration of complementing genes at

the genomic locus between NMV_2142 and NMV_2143 ORFs, which is conserved in *N.*

*meningitidis* and *N. gonorrhoeae*. Finally, we adapted for use in *N. meningitidis* the versatile

labelling technology based on human O6-alkylguanine-DNA-alkyltransferase (hAGT) [21],

which enables labelling of bacteria or proteins of interest with a wide range of probes.
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# RESULTS

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## *Easy construction of markerless mutations*

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Our strategy for obtaining markerless mutations in an affordable and efficient way was to design a cassette carrying a kanamycin resistance marker (*aphA-3*) and two counter-selectable markers genes (*pheS*\* and *galK*). Our rationale was that this cassette, named APG (for *aphA-3 pheS*\* *galK*), should be less affected by high spontaneous mutation rates thanks to its two counterselection markers expressed from two different promoters. We assembled the APG cassette as follows. We first used as an example the cassette designed by Gurung *et al.* for use in *Streptococcus sanguinis* [22] where the *aphA-3* gene, which encodes an aminoglycoside phosphotransferase conferring resistance to kanamycin [23], was combined into one operon with the mutated *pheS*\* gene encoding a PheS<sub>A316G</sub> variant. This variant in one of the two subunits of the phenylalanyl-tRNA synthetase renders bacteria sensitive to the phenylalanine analogue *p*-chloro-phenylalanine (Cl-Phe) [24]. We therefore designed a similar synthetic APG cassette using the *pheS* gene from *N. meningitidis* strain NEM8013 as a template, with one mutation to encode a PheS<sub>A318G</sub> variant equivalent to PheS<sub>A316G</sub> in *S. sanguinis*. This APG cassette was then fused to the *galK* counterselectable marker used by Jones *et al.* – where *galK* is under the control of the *opaB* promoter – conferring sensitivity to 2-DOG [9]. We assessed the sensitivity to Cl-Phe and 2-DOG conferred by the APG cassette after inserting it into the same genomic location in two model meningococcal strains used in our laboratory: NEM8013 clone 2C4.3 which is a serogroup C isolate [25] and Z5463 which is a hypermutator serogroup A strain [14] (Figure 1A). In both cases, the APG cassette was fused to the genomic regions upstream and downstream of the *pilV* gene, enabling its integration in the genome upon homologous recombination. After transformation and selection for kanamycin resistance, the two resulting *pilV::APG* strains were named 2C4.3 APG and Z5463 APG. Wild-type (WT) and APG mutant strains grown overnight on plates with or without kanamycin were resuspended in
GC Broth (GCB) and serial dilution were spotted on plain GCB plates or plates containing increasing concentrations of Cl-Phe or 2-DOG (Figure 1B). Although Cl-Phe at 20 mM or 40 mM appeared to strongly impair the growth rate of *pilV::APG* mutants, it also reduced growth of WT meningococci (especially at 40 mM). In contrast, 2-DOG even at 1% had no effect on the growth of WT meningococci, whereas it dramatically reduced the growth of *pilV::APG* mutants. Strikingly, when counter-selection was performed by combining 1% 2-DOG and 20 mM Cl-Phe, the residual growth of mutants carrying the APG cassette was completely abolished (Figure 1B boxed).

Next, we aimed to obtain a markerless *ΔpilV* gene deletion mutant by removing the APG cassette during a second step of homologous recombination. This strategy can also be used to insert a mutated gene or another marker in place of the APG cassette (Figure 1A). Strains 2C4.3 APG and Z5463 APG were directly transformed with PCR products in which the upstream and downstream sequences of *pilV* were fused. After one night of growth on selective plates – containing either 1% 2-DOG, or 20 mM Cl-Phe, or a combination of both – twelve colonies were picked and grown on GCB agar plates containing either the same counter-selective compounds or kanamycin (Figure 2A). Transformant colonies that no longer carry the APG cassette – in which the *pilV* gene is deleted – should be resistant to both 2-DOG and Cl-Phe, and sensitive to kanamycin (Figure 1A). The use of Cl-Phe alone did not allow us to recover kanamycin-sensitive clones in either 2C4.3 or Z5463 APG background, since all the clones were resistant to both Cl-Phe and kanamycin (Figure 2A). Even a second round of counterselection by picking and isolating a colony from each of the twelve clones did not allow the recovery of kanamycin-sensitive clones. In contrast, in the presence of 2-DOG alone, 8 out of 12 clones in the 2C4.3 background were sensitive to kanamycin after one round of counterselection, and are therefore likely to be markerless *ΔpilV* deletion mutants. This was less efficient in the Z5463 APG background, with only two mutants out of 12 isolated after two
rounds of counterselection (Figure 2A). Strikingly, the combined use of 2-DOG and Cl-Phe dramatically improved isolation of markerless mutants. After a single round of counterselection 12 out of 12 2C4.3 clones and 11 out of 12 Z5463 clones analyzed were markerless mutants (Figure 2A), with very few kanamycin-resistant clones. To confirm that kanamycin-sensitive clones corresponded to markerless *ΔpilV* deletion mutant, genomic DNA was obtained from the clones selected on 2-DOG + Cl-Phe and analyzed by PCR in the 2C4.3 (Figure 2B) and Z5463 (Figure 2C) backgrounds. This confirmed that the *pilV* gene was deleted in all kanamycin-sensitive clones. Our strategy does not impact bacterial fitness as the growth of *ΔpilV* mutants was similar to that of the WT or the *pilV::APG* derivatives (Supplementary Figure 1). In conclusion, while Cl-Phe on its own is not sufficient for the selection of markerless meningococcal mutants, its combination with 2-DOG was highly effective and can be used for the routine selection of markerless mutants, including in hypermutator strains such as Z5463.

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Construction of complementation and expression vectors for N. meningitidis

Once a mutant has been designed, confirmation that a mutant phenotype is due to the introduced mutation often needs to be confirmed by showing that complementation of the mutant by a WT copy of the gene restores a WT phenotype. The use of an inducible promoter to modulate the expression of the complementing allele may be beneficial in certain circumstances, and it may often allow to overexpress a gene of interest in a WT context. In the absence of small vectors that can replicate in *Neisseria* species, complementation is mostly done by integrating the complementing gene in the genome of the mutants via homologous recombination. To date, two *N. gonorrhoeae* loci have been mostly used as genomic insertion sites. Although the corresponding plasmids were also used in *N. meningitidis*, this is far from ideal because the first locus *lctP-aspC* contains two additional ORFs in *N. meningitidis*, and the *iga* gene in the second

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locus *iga-trpB* is not well conserved between gonococci ad meningococci (Supplementary Dataset 1). We thus decided to generate integrative complementation vectors for the meningococcus by replacing the *iga-trpB* locus from the gonococcus in pMR33 and pMR68 from Ramsey *et al.* [17] by the same locus from meningococcal strain NEM8013, which conveniently contains a DNA uptake sequence (DUS) motif. The two resulting plasmids were named pNM99<sub>Ptet</sub> (Figure 3A) and pNM99<sub>Plac</sub> (Figure 3B), and they allow expression of complementing genes from *Ptet* or *Plac* promoters inducible by anhydrotetracycline (aTC) or isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. In order to be able to perform double complementation/expression experiments with two genes simultaneously, we selected a second suitable locus in the meningococcal genome, between the NMV_2142 and NMV_2143 ORFs in NEM8013. These two genes are conserved in *N. meningitidis* and *N. gonorrhoeae* (Supplementary Dataset 1), transcribed in opposite directions, and each followed by a terminator with two DUS motifs. The *Ptet* promoter was thus added between NMV_2142 and NMV_2143, resulting in the plasmid named pNM42<sub>Ptet</sub> (Figure 3C). Finally, we replaced the *ermC* gene conferring resistance to erythromycin by the *aadA* gene conferring resistance to spectinomycin in each pNM99 and pNM42 plasmid, making their combined use possible in the same strain.

Finally, to assess the expression of protein using these constructions, we inserted the SNAP-tag sequence obtained from the pSNAP-tag (T7)-2 vector (New England Biolabs) after *Ptet* or *Plac*. This sequence encodes a 20 kDa mutant of the human DNA repair protein hAGT that reacts with benzylguanine derivatives, resulting in irreversible covalent labelling of the protein tag with dedicated synthetic probes [21]. This tag is particularly interesting as it allows the tagged protein to be labelled with different fluorophores, or even biotin. In addition, when expressed alone, this tag can be used to label bacteria fluorescently. Therefore, 2C4.3 was transformed with pNM99<sub>Ptet</sub>-SNAP, pNM99<sub>Plac</sub>-SNAP or pNM42<sub>Ptet</sub>-SNAP and erythromycin-
resistant clones with the tag at either the *iga-trpB* or NMV_2142-NMV_2143 locus were
selected. Bacteria were plated on GCB agar plates supplemented with increasing concentrations
of aTC or IPTG, lysed and processed for SNAP-tag detection by immunoblotting. A strong
expression of the SNAP tag was observed with 2 ng/ml aTC (Figure 3D, F) or 0.05 mM IPTG
(Figure 3E). These results demonstrate that the complementation plasmids are functional in *N.
meningitidis* and allow strong expression of genes under the control of *Ptet* or *Plac* promoters.

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198 Covalent labelling of bacteria using the hAGT-based technology

We then asked whether the SNAP-tag could be used in *N. meningitidis* to label live or fixed
bacteria. To easily detect and observe bacteria, we decided to work on meningococci that form
colonies on human cells. WT bacteria or bacteria expressing the SNAP-tag grown in the
presence of aTC were used to infect endothelial cells - grown on glass coverslips - for 2 h and
fixed with 4% paraformaldehyde (PFA). Labelling was performed in a final volume of 30 µl
using Dapi and 0.12 µM of red fluorescent membrane-permeable SNAP-Cell TMR-Star
(Figure 4A). At this substrate concentration the bacteria expressing the SNAP-tag were clearly
labelled, with a faint background fluorescence for the endothelial cells, while the WT bacteria
were not.

Next, we asked whether this tag could be used as a marker to follow the growth of living
bacteria in a fluorescence dilution assay [26]. In theory, living bacteria labelled with the
substrate should have their fluorescence reduced 2 fold per division, which takes place
approximately every 40 min in *Neisseria* species grown in rich media [27]. Bacteria were grown
in culture media, incubated with the TMR-Star substrate for 30 min and washed. Then, bacteria
were either fixed with 4% PFA immediately, or grown again for 30, 60 or 180 min before
fixation. The fluorescence of bacteria was then measured by flow cytometry. While bacteria
that were fixed just after labelling were highly fluorescent, the fluorescence decreased very
rapidly, with a drop of approximately 90% in signal intensity 30 min after labelling **(Figure 4B)**. The decrease in fluorescence did thus not follow the theoretical decrease in fluorescence caused by bacterial division every 40 min. Therefore while this strategy allows efficient fluorescent labelling of fixed or living bacteria, it could not be used to monitor the growth of *N. meningitidis*.
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DISCUSSION

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Pathogenic *Neisseria* species represent an important area of research, particularly given the severity of diseases caused by meningococci or the antibiotic resistance of gonococci. These species are naturally transformable and relatively easy to handle, although the level of biosecurity must be taken into account. However, the toolbox at our disposal for the genetic characterization of *Neisseria* species is limited, in particular for the meningococcus. In this work, we have designed three new tools for the study of *N. meningitidis* to (i) engineer markerless mutations rapidly and cost-effectively, (ii) conditionally express one or two genes inserted at chosen genomic loci, and (iii) label bacteria using hAGT-based technology. The close relationship between *Neisseria* species suggests that these tools should be useful in the gonococcus. It should also be possible to use these tools in non-pathogenic *Neisseria* species, although this could be challenging in poorly transformable ones [28].

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Our strategy for constructing markerless mutants is based on the simultaneous use of two counterselectable markers, *i.e.*, *galK* that sensitizes bacteria to 2-DOG already used in gonococci [9] and *pheS*\* that sensitizes bacteria to Cl-Phe, not tested in *Neisseria* before. The combined use of two counterselectable markers proved more effective than when they were used separately, pointing to a synergistic effect. In our hands, *pheS*\* is an inefficient counterselectable marker in the meningococcus as the use of Cl-Phe – to which the meningococcus appears slightly sensitive – does not allow selection of markerless mutants in the two genetic backgrounds tested. On the other hand, *galK* is a much better counterselectable marker in the meningococcus. The use of 2-DOG – that is not toxic to meningococci – enabled the selection of markerless mutants with a good success rate of 67% in strain 2C4.3, although the mutants were not perfectly pure after a single isolation. Unfortunately, *galK* efficiency was much lower in the hypermutator strain Z5463, with a success rate of merely 17% after two isolation steps. The combined use of 2-DOG and Cl-Phe proved to be very effective even after
246 a single isolation step, with success rates of 100% in 2C4.3 and 92% in Z5463. Exposure of the
247 mutants to 2-DOG and Cl-Phe did not lastingly impact the mutants since their growth was
248 comparable to that of the WT strain. Finally, the use of a cassette combining two
249 counterselectable markers makes our strategy cost-effective by reducing the concentration of
250 2-DOG to 1%, from 6% as previously described in the gonococcus [9].

251 The integrative plasmids we have designed in this work were developed to express
252 recombinant genes in meningococci, especially during complementation experiments. Our
253 plasmids allow the conditional expression of recombinant genes in meningococci from two
254 different promoters – inducible with either aTC or IPTG – at two distinct genomic locations
255 (the *iga-trpB* or NMV_2142-NMV_2143 loci). The second locus has the advantage of being
256 highly conserved in *N. gonorrhoeae*, suggesting that the corresponding plasmids could be used
257 in this human pathogen as well. Although we have not tested this, swapping of the antibiotic
258 resistance genes used for selection in the above plasmids ensures that pairs of pNM99 and
259 pNM42 plasmids promoting resistance to different antibiotics are compatible and could be used
260 to conditionally express two different genes in the same meningococcal strain.

261 We have used these plasmids to evaluate the hAGT technology for labelling bacteria in
262 *N. meningitidis*. We have shown that the SNAP-tag (designed by New England Biolabs) allows
263 fluorescent labelling of meningococcal cells under *in vivo* conditions or after fixation, which
264 can then be observed by microscopy or flow cytometry. However, our results show that *in vivo*
265 labelling requires rapid fixation, as fluorescence intensity fades rapidly in living bacteria.
266 Although this remains to be tested, it is likely that this labelling strategy can also be considered
267 for the expression and detection of SNAP-tagged recombinant proteins. One of the main
268 advantages of the SNAP-tag is its flexibility. It can be used for both visualization and
269 purification from the same sample. It allows the use of a marker with a 1:1 stoichiometry
270 between target and marker. Labelling is performed at the end of the experiment, which makes
the SNAP-tag compatible with high-resolution microscopy strategies such as dSTORM or STED, as well as immunogold electron microscopy especially when coupled with streptavidin. In addition, cellular compartmentalization of the SNAP-tag may allow the development of recombinant markers for inner or outer membranes, which would represent a major advantage for the study of outer membrane vesicles for example. Finally, another version of the tag is available from New England Biolabs – called CLIP-tag – allowing for dual labelling.

In summary, we have significantly expanded the meningococcal genetic toolbox by (i) developing an efficient and cost-effective method for markerless gene modification, (ii) constructing two new sets of compatible complementation plasmids, and (iii) demonstrating the feasibility of using hAGT-based technology for cell labelling in *N. meningitidis*. We believe that our tools pave the way for tackling some of the research priorities in the meningococcal field, and should be readily applicable to several other *Neisseria* species.
283 METHODS

284 Strains and growth conditions

285 *N. meningitidis* 2C4.3 strain is a piliated and encapsulated Opa⁻ Opc⁻ variant of the serogroup
286 C meningococcal clinical isolate 8013 [25]. *N. meningitidis* Z5463 is a piliated strain isolated
287 from the throat of a patient with meningitis in the Gambia in 1983 [14]. All strains and mutants
288 used in this study are listed in **Table 1**. Bacterial strains were stored frozen at -80°C and
289 routinely grown at 37°C in a moist atmosphere with 5% CO₂ on gonococcal base (GCB) agar
290 (Difco) plates containing Kellogg's supplements [29]. Liquid cultures were performed in GCB
291 liquid medium containing 1.5 % proteose peptone (Difco), 0.4 % K₂HPO₄, 0.1 % KH₂PO₄, 0.1
292 % NaCl, and Kellogg's supplements. For (counter)selection, we used kanamycin at 100 µg/ml,
293 erythromycin at 2 µg/ml, spectinomycin at 62.5 µg/ml, *p*-chloro-phenylalanine (Sigma) at 20
294 mM or 2-deoxy-d-galactose (Sigma) at 1% when necessary. Competent cells of *Escherichia
295 coli* DH5-α (New England Biolabs) were used for DNA cloning and plasmid propagation. They
296 were grown on Lysogeny Broth (LB) agar plates at 37°C or in liquid LB medium with
297 kanamycin at 50 µg/ml.

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299 Cell line and growth conditions

300 EA Hy926 cells (ATCC # CRL-2922) are hybrid endothelial cells. They were grown in
301 Dulbecco's modified Eagle's medium (DMEM) high glucose (Gibco), supplemented with 10%
302 decomplemented fetal calf serum (FCS), at 37°C in a moist atmosphere with 5% CO2.

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304 Transformation of N. meningitidis

305 Strains of interest were grown overnight on GCB agar plates and resuspended to OD₆₀₀=1 in
306 transformation GCB liquid media containing MgCl₂ at 2.5 mM and MgSO₄ at 2.5 mM. Five µl
307 of PCR product or 150 ng of plasmid were added to this bacterial suspension and incubated
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during 30 min at 37°C, 5% CO₂ with agitation. Then, bacteria were grown for 3 h after addition
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of 1 ml of transformation GCB liquid media. Transformants were selected on suitable GCB
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agar plates. Genomic DNA was extracted from overnight cultures on plates using Wizard
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Genomic DNA purification kit (Promega) following the manufacturer's instructions. Mutations
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were verified by PCR using OneTaq polymerase (New England Biolabs) with the appropriate
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primers (Supplementary Methods) and analyzed by agarose gel electrophoresis.

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**Drop sensitivity assay**

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Strains of interest grown overnight on GCB agar plates were resuspended to OD₆₀₀=1 in GCB
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liquid media. Drops of successive dilutions were deposited and grown overnight on plates
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containing different concentrations of Cl-Phe and/or 2-DOG.

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**Generation of markerless mutants in *N. meningitidis***

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The *aphA-3 pheS*\* *galK* (APG) cassette was designed as follows. We first synthesized an AP
cassette combining into an operon the *aphA-3* kanamycin resistance gene from [23]
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*Streptococcus faecalis* under its own promoter and the *pheS* gene from *N. meningitidis* strain
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NEM8013 with one mutation to encode a PheS*A318G* variant (GeneArt), equivalent to PheS*A316G*
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in *S. sanguinis*. We fused this cassette with *galK* under the control of the *opaB* promoter [9] to
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generate the APG cassette, which was used as follows to generate markerless mutants. The
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sequence of the APG cassette is available in **Supplementary Methods**.
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In the first step, we introduced the APG cassette instead of the target gene (from START
to STOP codons) in *N. meningitidis* by homologous recombination. The PCR product carrying
the cassette flanking by regions upstream and downstream the target gene was obtained by
overlap PCR using Q5 polymerase (New England Biolabs) using primers detailed in
**Supplementary Methods**. For each target gene, DNA fragments ranging from 500 to 1,000
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333 base pairs (bp) upstream and downstream of the gene were amplified using two pairs of primers (5'Fw/Rv and 3'Fw/Rv). Another pair of primers (APGFw/Rv) was used to amplify the APG cassette. To enable fragment assembly, the 5'Rv/APGFw primers and the APGRv/3'Fw primers were designed to overlap on 30 nucleotides. Following the amplification of the three fragments, they were purified using Monarch DNA Gel Extraction kit (New England Biolabs) and assembled by two-step PCR using Q5 polymerase. First, fragments were mixed in stoichiometric quantities and assembled by PCR using "overlap" program: 98°C 30 s, 20 cycles (98°C 20 s, 46°C 1 min, 72°C 1 min/kb), 12°C ∞. Then, the assembled fragment was amplified by PCR with external primers (5'Fw and 3'Rv) and the PCR product was introduced into N. meningitidis 2C4.3 or Z5463 by transformation. Transformants were selected on GCB agar plates containing kanamycin. Transformants sensitivity to Cl-Phe and/or 2-DOG was confirmed on GCB agar containing 20 mM Cl-Phe, 1% 2-DOG or both compounds. Genomic DNA was extracted from 12 transformants, the presence of the APG cassette and the length of fragments bounded by external primers was tested by PCR to confirm the allelic exchange.

347 In the second step, a new PCR product generated by bringing together fragments upstream and downstream the target gene was used to replace the APG cassette by homologous recombination. The same PCR strategy than above was used. For this, primers 5'RH\_Rv and 3'RH\_Fw have been modified to overlap on 30 nucleotides. Transformation in a N. meningitidis strain in which the target gene has been replaced by the APG cassette was performed as described above, except that the strain was resuspended at OD₆₀₀=0.01. Transformants were selected on GCB agar containing 20 mM Cl-Phe and 1% 2-DOG. The absence of the APG cassette was checked by plating on GCB agar + kanamycin and on GCB agar + 20 mM Cl-Phe and 1% 2-DOG, and verified by PCR after genomic DNA extraction.

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357 Liquid growth assays
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Strains of interest were grown overnight on GCB agar plates and resuspended to OD₆₀₀=0.1 in GCB liquid media. Two hundred µl of each bacterial suspension was added into the wells of a 96-well plate and the OD₆₀₀ was monitored for 24 h at 37°C in 5% CO₂ using a microplate reader (Tecan Spark). Four independent repeats were carried out for each strain. Curves were obtained using GraphPad Prism (version 8.2) software.

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## Construction of pNM99 and pNM42 plasmids

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The genomic loci comprised between *iga* and *trpB*, or NMV_2142 and NMV_2143, in *N. meningitidis* 2C4.3 were inserted in pMR68 [17] instead of the existing *N. gonorrhoeae* sequence as follows. The resulting plasmids were named pNM42 and pNM99. Complementary primers were designed to overlap on 30 bp (primers used are described in the **Supplementary Methods**) and the two PCR products (inserts and pMR backbone) were extracted and assembled using the NEBuilder HiFi DNA Assembly kit (New England Biolabs). The assembly product was transformed into *E. coli* DH5-α, plated on LB agar plates containing 50 µg/ml kanamycin. The following day, eight clones were verified by PCR and four positive clones were cultured overnight in liquid LB in the presence of 50 µg/ml kanamycin. Plasmid DNA was extracted (Monarch Plasmid Miniprep, New England Biolabs) and verified by Sanger sequencing (Eurofins Genomics). Following the same strategy, these plasmids were used to insert the aTC/IPTG-inducible promoters from pMR33 or pMR68 [17] between *iga* and *trpB*, or NMV_2142 and NMV_2143. Finally, the erythromycin resistance gene was exchanged for *aad* conferring resistance to spectinomycin using the same strategy. The sequences of the plasmids and of the *aad* cassette are available in **Supplementary Methods**.

## SNAP-tag expression in *N. meningitidis* 2C4.3
382

To enable SNAP-tag expression in *N. meningitidis*, plasmids pNM99<sub>Ptet</sub>, pNM42<sub>Ptet</sub>, pNM99<sub>Plac</sub> were used. The plasmids were constructed following the strategy mentioned above, by inserting the SNAP-tag (New England Biolabs #N9183S) after the *Ptet* or *Plac* promoters. Plasmids were verified by sequencing. The different SNAP-expressing plasmids were introduced into *N. meningitidis* 2C4.3 by transformation. Transformants were selected on GCB agar plates containing erythromycin. The presence of the construct of interest was verified by PCR after genomic DNA extraction and the absence of full plasmid integration was verified by sensitivity to kanamycin.

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## Immunoblotting

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To detect tag expression by immunoblotting, one bacterial loop of each strain of interest – grown overnight in GCB agar + erythromycin + aTC or IPTG at different concentrations to induce tag expression – was resuspended in 500 µl RIPA buffer (50 mM Tris pH 7.5, 150 nM NaCl, 25 µM HEPES, 2 mM EDTA, 1% w/vol SDS). This suspension was heated at 95°C for 5 min. Proteins were quantified using Bradford method and 10 µg of protein was deposited on a 12% acrylamide SDS-PAGE gel. After transfer to nitrocellulose membrane, the membrane was incubated for 20 min in a blocking solution (PBS + 0.1% tween-20 + 4% milk) and washed twice with PBS + 0.1% tween-20 (PBST). The membrane was incubated for 1 h in the presence of anti-SNAP-tag primary antibody (New England Biolabs #P9310S) diluted 1:2,000 in PBST, washed three times for 5 min with PBST and then incubated for 45 min with a rabbit anti-IgG secondary antibody coupled to horseradish peroxidase diluted at 1:10,000 in PBST. Proteins were detected by chemiluminescence using the Clarity Western ECL substrate detection kit (Bio-Rad) and a Chemidoc imaging system (Bio-rad). Whole proteins were detected on gel using the stain-free technology from Bio-Rad.
407 **Immunofluorescence**

408 Four days before infection, 50,000 cells/well were cultured on 2 cm² circular coverslips coated
409 with rat tail type I collagen (Corning). On the day of infection, bacteria grown from an overnight
410 culture on GCB agar plate were adjusted to OD₆₀₀=0.1 and incubated for 2 h at 37 °C in pre-
411 warmed cell culture medium, at 37°C with 5% CO₂ under agitation. OD₆₀₀ was measured and
412 a total of 10⁷ bacteria were added to cells grown on coverslips and incubated for 40 min at 37°C
413 in 5% CO₂. After 40 min, the cells were washed twice with DMEM and incubated for 2 h. Then,
414 cells were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min,
415 rinsed, incubated 5 min with 50 mM NH₄Cl and rinsed twice with PBS. The coverslips were
416 then incubated for 30 min at 37°C with DMEM supplemented with 10% FCS containing 0.12
417 µM SNAP-Cell TMR-Star (New England Biolabs #S9105S), rinsed twice with PBS and
418 incubated for 45 min with Dapi (1 µg/ml). After 2 additional washes, coverslips were mounted
419 in Mowiol. Image acquisition was performed on Zeiss Apotome fluorescence microscope.
420 Images were collected and processed using the ZEN (ZEISS Efficient Navigation) software.

421

422 **Flow cytometry**

423 For quantitative fluorescence analysis, bacteria grown from an overnight culture on GCB agar
424 plates were adjusted to OD₆₀₀=0.1 and incubated for 45 min at 37 °C in 5% CO₂ under agitation
425 in 5 ml pre-warmed GCB liquid media. Bacteria were then incubated with 0.12 µM SNAP-Cell
426 TMR-Star for 30 min at 37°C in 5% CO₂. A control with no added SNAP-Cell TMR-Star was
427 performed. The suspensions were centrifuged for 5 min at 2,600 g and resuspended in 40 µl of
428 liquid GCB. Ten µl was reintroduced in three flasks containing 5 ml of liquid GCB with
429 Kellogg's supplements, and incubated respectively for 30 min, 60 min, and 180 min. The
430 remaining 10 µl were washed twice by centrifugation for 5 min at 1,200 g, then fixed with 500
431 µl of 4% PFA for 20 min, centrifuged, incubated for 5 min with 50 mM NH₄Cl, centrifuged,
432 washed three times and resuspended in PBS. Bacteria that were allowed to grow were
433 centrifuged and fixed after 30 min, 60 min, or 180 min in the same way as above. Two µl of
434 SNAP-Cell Block (New England Biolabs #S9106S) were added to strain 2C4.3 WT. The
435 samples were analyzed on FACS (ID700 Spectral Cell Analyzer, Sony) with FlowJo software.
436 ACKNOWLEDGEMENTS

437 This work was supported by the research grant ANR-19-CE14-0045-002 (to MC) and funding

438 from INSERM. We thank C. Tang and A. Cehovin for the *galK* cassette. Image acquisition and

439 image analysis were performed at the Imaging Facility of Structure Fédérative de Recherche

440 (SFR) Necker, INSERM US24/CNRS UAR3633. Flow cytometry and analysis was performed

441 at the Cytometry facility of SFR Necker, INSERM US24/CNRS UAR3633.
442

FIGURE AND SUPPLEMENTARY FIGURES LEGENDS

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**Figure 1.** Markerless mutagenesis strategy using 2-deoxy-galactose and *p*-chloro-*phenylalanine for counterselection. (A) Selection strategy of markerless mutants. In the first step (STEP 1), the gene of interest (gene X) is replaced by the APG cassette using homologous recombination. Mutants carrying the APG cassette are selected by plating on kanamycin. In the second step (STEP 2), the APG cassette is removed or replaced by a mutated gene X or another gene Y. Markerless mutants are enriched on plates containing both 2-DOG and Cl-Phe. (B) Growth of meningococci on different concentrations of 2-deoxy-galactose (2-DOG) or *p*-chloro-phenylalanine (Cl-Phe). Wild-type strains (2C4.3 and Z5463), and their derivatives carrying the APG cassette (2C4.3 APG and Z5463 APG) were adjusted to 10⁹ CFU/ml and tenfold dilutions were spotted on plain GCB agar plates, or plates supplemented with increasing concentrations of 2-DOG (from 0.1 to 1%) or Cl-Phe (from 10 to 40 mM). Two representative experiments were performed. In the boxed panel, the bacteria were spotted on GCB or GCB supplemented with both 2-DOG (1%) and Cl-Phe (20mM).

**Figure 2.** Selection of markerless mutants using 2-deoxy-galactose, *p*-chloro-*phenylalanine or both. (A) Selection of markerless mutants on 20 mM Cl-Phe, 1% 2-DOG or both. Mutants carrying the APG cassette in place of the gene *pilV* (*pilV::APG*) were transformed with the fusion PCR product of the sequences upstream and downstream of the gene *pilV* to allow homologous recombination, and plated on GCB agar plates supplemented with 20 mM Cl-Phe, 1% 2-DOG or both. For each bacterial background (boxed panel), 12 colonies were picked from each plate (Cl-Phe, 2-DOG, or 2-DOG+ Cl-Phe), resuspended and plated again on the same plates with or without kanamycin. Pictures of plates were then taken after 24 h or 48 h (1st isolation). A second round of selection was carried out for the Z5463 background (2nd isolation). For each of the twelve isolated clones, one colony was picked and isolated again on
the same plates with or without kanamycin. (B, C) Characterization of markerless mutants by PCR in the 2C4.3 (B) or Z5463 (C) backgrounds. WT and derivatives carrying the APG cassette in place of the gene *pilV* (*pilV::APG*) and *pilV* ML mutants obtained by selection on 2-DOG + Cl-Phe were checked by PCR using the two primer pairs (see Fig. 1A): 5'Fw-3'Rv (upper gels) and APG Fw-APG Rv (lower gels). An asterisk indicates the faint band corresponding to the 5'Fw-3'Rv amplicon of the *pilV::APG* in the 2C4.3 background. The largest band on the ladder corresponds to 3 kb.

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**Figure 3. Complementation plasmids.** Circular maps of (A) pNM99<sub>Ptet</sub>, (B) pNM99<sub>Plac</sub> and (C) pNM42<sub>Ptet</sub>. Regions of homology with *N. meningitidis* chromosome are shown in purple. Antibiotic selection cassettes are shown in yellow. *Plac*: lac promoter and operator. LacI: lac repressor. *Ptet*: anhydrotetracycline-inducible promoter. TetR: *tet* repressor. The inserted sequence is indicated in red. (D, E, F) Expression of the SNAP-tag using the different plasmids. Expression of the SNAP tag under the control of *Ptet* or *Plac* was assessed in WT 2C4.3 transformed with (D) pNM99<sub>Ptet</sub>-SNAP, (E) pNM99<sub>Plac</sub>-SNAP or (F) pNM42<sub>Ptet</sub>-SNAP. Bacteria were incubated overnight on GCB agar plates containing increasing concentrations of aTC or IPTG inducers. Expression of the tag was assessed by western blotting using anti-SNAP tag antibodies. Total proteins were detected in the gel by Stain-Free technology.

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**Figure 4. Labelling of fixed or alive bacteria expressing the SNAP-tag (SNAP) using the hAGT-based technology.** (A) Imaging of bacteria labelled with the red fluorescent membrane-permeable SNAP substrate TMR-Star after fixation. 2C4.3 or a derivative expressing the SNAP-tag (2C4.3 SNAP) were used to infect endothelial cells and then fixed and processed for staining with Dapi and labelling with 0.12 µM of TMR-Star. Bar: 10 µm. (B) Flow cytometry analysis of TMR-Star fluorescence labelling in living bacteria. Bacteria grown in GCB liquid
media were incubated 30 min with the TMR-Star substrate. They were then washed and fixed

in 4% PFA (T0 min) or grown again in fresh broth and fixed after 30 min (T30 min), 60 min

(T60 min), or 180 min (T180 min). To visualize the decrease in fluorescence, the observed

mean (red) fluorescence and the theoretical mean (blue) fluorescence, calculated for a division

rate of 40 min, were plotted against time. Representative experiment.
<table><caption>**Table 1. Strains and mutants used in this study**</caption><thead><tr><th>Strain</th><th>Description</th><th>Antibiotic resistance</th><th>Reference</th></tr></thead><tbody><tr><td>2C4.3</td><td>Serogroup C <i>N. meningitidis</i> isolate</td><td></td><td>Nassif et al., 1993</td></tr><tr><td>Z5463</td><td>Serogroup A <i>N. meningitidis</i> isolate</td><td></td><td>Achtman et al., 1988</td></tr><tr><td>2C4.3 <i>pilV</i>::<i>apha3</i>'<i>pheS</i>*<i>galk</i></td><td><i>pilV</i> replaced by APG cassette</td><td>Kanamycin</td><td>This study</td></tr><tr><td>2C4.3 Δ<i>pilV</i></td><td>Markerless deletion of <i>pilV</i></td><td></td><td>This study</td></tr><tr><td>Z5463 <i>pilV</i>::<i>apha3</i>'<i>pheS</i>*<i>galk</i></td><td><i>pilV</i> replaced by APG cassette</td><td>Kanamycin</td><td>This study</td></tr><tr><td>Z5463 Δ<i>pilV</i></td><td>Markerless deletion of <i>pilV</i></td><td></td><td>This study</td></tr><tr><td>2C4.3 99_tet_SNAP-tag</td><td>Sureexpression of SNAP-tag by transformation of 2C4.3 with pNM99<sub>Ptet</sub>-SNAP-tag</td><td>Erythromycin</td><td>This study</td></tr><tr><td>2C4.3 42_tet_SNAP-tag</td><td>Sureexpression of SNAP-tag by transformation of 2C4.3 with pNM42<sub>Ptet</sub>-SNAP-tag</td><td>Erythromycin</td><td>This study</td></tr><tr><td>2C4.3 99_lac_SNAP-tag</td><td>Sureexpression of SNAP-tag by transformation of 2C4.3 with pNM99<sub>Plac</sub>-SNAP-tag</td><td>Erythromycin</td><td>This study</td></tr></tbody></table>
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<figure><img src="image_1.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_2.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Supplementary Figure 1</figcaption></figure>

**Supplementary Figure 1. Growth of selected deletion mutants in 2C4.3 (A) and Z5463 (B) backgrounds is not impaired.** The growth of WT bacteria, a clone of the selected derivative carrying the APG cassette (*pilV::APG*) and a selected markerless Δ*pilV* clone was assessed in liquid GCB.