Bacterial strains and growth conditions

P. alvei CCM 2051^T was obtained from the Czech Collection of Microorganisms (CCM; Brno, Czech Republic) and was grown at 37°C and 200 rpm in Luria-Bertani (LB) broth or on LB agar plates supplemented with 10 µg/ml chloramphenicol (Cm), when appropriate. Escherichia coli DH5α cells (Invitrogen) were cultivated in selective Luria Bertani (LB) medium (agar and broth) supplemented with 30 μg/ml chloramphenicol (Cm). All strains used in this study are listed in Table 1.

General molecular methods

All enzymes were purchased from Fermentas. Genomic DNA of P. alvei CCM 2051^T was isolated by using a Genomic Tip 100 kit (Qiagen) according to the manufacturer’s instructions, except that cells were broken by repeated freezing and thawing cycles (10 times) [26]. The GeneJET™ Gel Extraction Kit (Fermentas) was used to purify DNA fragments from agarose gels and to purify digested plasmids and oligonucleotides. Plasmid DNA from transformed cells was isolated with the GeneJET™ Plasmid Miniprep kit (Fermentas). Agarose gel electrophoresis was performed as described elsewhere [27]. Primers for PCR and DNA sequencing were purchased from Invitrogen (Table 2). PCR was performed using the Phusion^® High-Fidelity DNA Polymerase (Fermentas) and the thermal cycler My Cycler^TM (Bio-Rad). Transformation of chemically competent E. coli DH5α cells was done according to the manufacturer’s protocol (Invitrogen). Transformation of P. alvei CCM 2051^T wild-type or P. alvei CCM 2051^T ΔslhA cells, with the latter showing a better transformation efficiency, is described elsewhere [26]. Transformants were screened by PCR using RedTaq ReadyMix PCR mix (Sigma-Aldrich), and recombinant clones were analyzed by restriction mapping and confirmed by sequencing (LGC).

Gene knockouts

Disruption of the slhA gene located in the SCWP biosynthesis locus of P. alvei CCM 2051^T and the flagella gene (hag, PAV_2c01710) was performed as described previously [26]. The Ll.LtrB targetron of pTT_wsfA243 was retargeted prior to transformation into P. alvei CCM 2051^T. Identification of potential insertion sites and design of PCR primers for the modification of the intron RNA was accomplished by a computer algorithm (www.Sigma-Aldrich.com/Targetronaccess). The retargeted Ll.LtrB targetron was subsequently digested with HindIII and BsrGI and ligated into pTT_wsfA243 [28] digested with the same restriction enzymes, thereby replacing the wsfA targetron.

To complement the knockout, slhA under its native promoter was amplified from P. alvei CCM 2051^T genomic DNA using the primers P(SlhA) _HindIII_for/ SlhA_KpnI_rev. The amplification product was SphI/KpnI-digested and inserted into the linearized vector pEXALV. The resulting plasmid was named pEXALV_P(SlhA) _SlhA (Table 1) and was transformed into P. alvei CCM 2051^T ΔslhA cells [26].

Plasmid construction for in vivo studies

For analyzing co-display of SlhA and SpaA on P. alvei CCM 2051^T ΔslhA cells in vivo, the vector pSURF was designed based on the pEXALV_EGFP vector [2]. For the construction of a chimera of SlhA and enhanced green fluorescent protein (EGFP) including a GAGGAGGT linker, the slhA gene was PCR-amplified from genomic DNA of P. alvei CCM 2051^T using the primer pair SlhA_SphI_for/ SlhA_w/o Stop_Linker_XbaI_rev. The amplification product was SphI/XbaI-digested and inserted into linearized pEXALV_EGFP. The resulting plasmid was named pEXALV_SlhA_Linker_EGFP (Table 1). Subsequently, the sgsE S‑layer gene promoter of G. stearothermophilus NRS 2004/3a, P(SgsE), and the S‑layer gene spaA of P. alvei CCM 2051^T were amplified from pEXALV_SP_SpaA_6HIS as a template using the primers P(SgsE) _SacI_for/ SpaA_6His_SacI_rev and ligated into pEXALV_SlhA_Linker_EGFP. The resulting plasmid was named pSURF (Table 1).

Recombinant production of His-tagged SlhA variants for in vitro assays

All SlhA variants used for in vitro assays were produced as His₆-tagged constructs for detection purposes. The C-terminal His₆-tag was fused to the sequence of slhA by PCR, using the primers SlhA_ohne SP_NcoI_for/ SlhA_ganz_XhoI_rev for slhA lacking the sequence coding for the signal peptide, SlhA_ohne SP_NcoI_for/ SlhA_His_SLH12_XhoI_rev for slhA lacking the sequences coding for the signal peptide and the third SLH domain, SlhA_ohne SP_NcoI_for/ SlhA_His_SLH1_XhoI_rev for slhA lacking the sequences coding for the signal peptide, the second and third SLH domain, and SlhA_ohne SP_NcoI_for/ SlhA_His_ohne SLH_XhoI_rev for slhA lacking the sequences coding for the signal peptide and all three SLH domains.

All amplifications were done using P. alvei CCM 2051^T genomic DNA as a template. The different His₆-tagged slhA amplification products were digested with NcoI/XhoI and cloned into NcoI/XhoI-linearized pET28a(+) (Novagen). The corresponding plasmids were named pET28a_SlhA_6His (encoding native SlhA lacking the SP; designation: SlhA recombinant), pET28a_SlhA_SLH12_6His (encoding SlhA lacking the SP and SLH domain 3; designation: SlhA₁₂), pET28a_SlhA_SLH1_6His (encoding SpaA lacking the SP and SLH domains 2 and 3; designation: SlhA₁), pET28a_SlhAdSLH_6His (encoding SpaA lacking the SP and SLH domains 1, 2, and 3; designation: SlhA w/o SLH) (Table 1; compare with Figure 1A) and transformed into E. coli BL21 (DE3) cells. Freshly transformed cells were grown in LB medium [27] to the mid exponential growth phase (OD₆₀₀ ~0.6), protein expression was induced with a final concentration of 0.5 mM isopropyl-β-D-thiogalactopyranosid (IPTG) and cultures were grown for additional 4 h at 37°C and 200 rpm. Cells were harvested by centrifugation (4,500 x g, 30 min, 4°C).

Preparation of native peptidoglycan-containing cell wall sacculi

For the preparation of protein-free peptidoglycan (PG)-containing cell wall sacculi including native SCWP of P. alvei CCM 2051^T (PG(+)), cells were grown until the late exponential phase and broken by ten freezing and thawing cycles [26]. After centrifugation (35,000 x g, 30 min, 4°C), the pellet was resuspended in 50 mM Tris-HCl, pH 7.2 (buffer A), containing 0.5% Triton X-100 and incubated for 10 min at 20°C. The pellet was washed four times with buffer A and once with MilliQ-water and finally resuspended in ten pellet volumes (w/v) of 5 M guanidine hydrochloride (GdHCl) in buffer A followed by stirring for 20 min at 20°C to extract the S‑layer protein. The pellet obtained after centrifugation (35,000 x g, 30 min, 4°C) of that solution was washed once with 5 M GdHCl in buffer A and twice with buffer A. 100 mg (wet weight) of that preparation were incubated in a boiling 1% (w/v) SDS solution for 30 min with stirring. The pellet of PG(+) was washed repeatedly with MilliQ-water to remove traces of SDS. SCWP was released from the PG(+) material by treatment with 48% hydrofluoric acid for 96 h at 4°C [29].

Lyophilized PG-containing cell wall sacculi with (PG(+)) and without SCWP (PG(-)) were stored at -20°C.

Interaction studies of SlhA variants and PG-containing cell wall sacculi

The different His₆-SlhA variants were tested for their ability to interact with PG(+) and PG(-) following a recently published protocol [2,8]. Briefly, ~1 µg of SlhA protein (crude cell extract) was incubated at 37°C for 1 h with 0.2 mg of lyophilized PG(+) in 25 mM Tris–HCl, pH 8.0, containing 0.2% (v/v) Tween 20 (incubation buffer), in a total volume of 125 µl. The mixture was centrifuged (16,100 x g, 20 min, 4°C) yielding a supernatant containing unbound protein and a pellet of the insoluble cell wall components including attached protein. The pellet was washed twice, suspended in incubation buffer, and fractions were investigated by SDS-PAGE and by Western-immunoblotting using anti-His₆ mouse antibody (Roche) (see below). Mixtures without PG were used as a control. The pellet fraction and the supernatant fraction of the reaction as well as the control (without PG) were quantified using the Li-Cor Odyssey Infrared Imaging System (Li-Cor; see below) and the value of precipitated protein in the control was subtracted from that in the reaction pellet. For comparability of datasets, the sum of the pellet fraction and the supernatant fraction of each reaction was set to 100%. Experiments were performed in duplicate and standard deviations were calculated.

Fluorescence microscopy

The surface accessibility of displayed SlhA_EGFP and SpaA_His₆ chimeras was analyzed directly by fluorescence microscopy and after immunofluorescence staining, respectively. P. alvei CCM 2051^T ∆slhA cells expressing both proteins from pSURF (see Table 1) were harvested at an optical density of OD₆₀₀ ~0.6 and washed three times with phosphate-buffered saline (PBS). After resuspension in 100 µl of PBS, 5 µl of a penta-His Alexa Fluor 532 conjugate (Qiagen) was used and incubation was done for 3 h at 25°C on a horizontal shaker. After washing for three times, the cells were resuspended in 100 µl of PBS analyzed by fluorescence microscopy. Three μl of that suspension were mixed with 3 μl of a solution containing 0.9% molten agarose, 40 mM Tris-HCl (pH 8.0), 20 mM acetic acid, 1 mM EDTA, pH 8.0, and pre-heated to 55°C. The samples were covered immediately with glass to immobilize the cells in the solidified agarose.

The samples were monitored using a Nikon Eclipse TE2000-S fluorescence microscope at a magnification of 100 x using an oil immersion objective. In the case of the Alexa Fluor 532-labelled SpaA-His₆, the TRITC filter block and for SlhA-EGFP, the GFP filter block were used. Pictures were taken with a Nikon Digital Sight DS-Qi1Mc camera and the NIS-Elements 3.22 imaging software.

General and analytical methods

SDS-PAGE was carried out according to a standard protocol [30] using a Protean II electrophoresis apparatus (Bio-Rad). Protein bands were visualized with Coomassie Brilliant Blue G 250 staining reagent.

For Western-immunoblotting of proteins onto a polyvinylidene difluoride membrane (Bio-Rad) a Mini Trans-Blot Cell (Bio-Rad) was used. Detection of the His₆-tag fused to SlhA-variants was done with the Li-Cor Odyssey Infrared Imaging System using anti-His₆ mouse antibody (Roche) in combination with goat anti-mouse IgGIR Dye 800CW conjugate (Li-Cor). The integrated intensity of the detected bands was determined using the Li‑Cor Odyssey Application Software 3.0.21 applying automatic background subtraction. PG-containing cell wall sacculi (PG (+) and PG(-)) were analyzed for their content of aminosugars and diaminopimelic acid (DAP) using a Biochrom 30 amino acid analyzer as described elsewhere [2].

Colony morphology, swarming and adhesion test

For imaging of the colony morphology of single cells of P. alvei CCM 2051^T wild-type, P. alvei ΔslhA, complemented mutant (P. alvei ΔslhA_comp) and P. alvei Δhag, pictures were taken using a SteREO Discovery. V12 (Zeiss) microscope and an AxioCam MRc5 camera (Zeiss) after growth for 48 h at 37°C on 2% LB-plates.

To compare the adhesion of P. alvei CCM 2051^T wild-type, P. alvei ΔslhA, P. alvei ΔslhA_comp and P. alvei Δhag cells to nutrient plates, an assay based on the adhesion and agar invasion assay for yeast was used [31]. Cells were grown to OD₆₀₀ ~1.0 and 5 µl of the culture were spotted on a 2% LB-agar plate (pH 7.0). Plates were incubated overnight at 37°C. The adhesion of the cells to the agar plates was tested by intense rinsing of the plates with distilled water using a wash bottle. Pictures were taken with a SPImager (S&P Robotics).

To test the different P. alvei variants for their ability to swarm on agar plates, cells were grown to the exponential growth phase (OD₆₀₀ ~1.0) and 5 µl of each culture were spotted on 0.4% (soft agar), 1% (semi-solid agar) and 1.5% (hard agar) LB-agar plates (pH 6.0). P. alvei cells without the pEXALV plasmid were cultivated at 37°C for 24 h, while strains containing pEXALV were incubated at 37°C for 48° h. Pictures were taken using a SPImager (S&P Robotics).

Congo red assay

To quantify EPS production in P. alvei CCM 2051^T wild-type and mutants, cells from 1 ml of an overnight-culture grown in LB medium were pelleted and washed once with 1 ml of LB broth. The pellet was resuspended in 1 ml of broth containing 40 µg/ml Congo red and incubated for 2 h at 37°C with shaking (250 rpm). Congo red bound to P. alvei cells was removed by centrifugation and unbound Congo red was determined by measuring the absorbance of the supernatant at 490 nm [32]. Data represent mean values + SD of at least four independent experiments and were analyzed by the unpaired Student’s T Test. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

To detect EPS production on plates, an overnight-culture was diluted to OD₆₀₀ ~1.0 and 5 µl of that culture were spotted on 2% LB plates containing 40 µg/ml Congo red. Plates were incubated at 37°C for 24 h and 48 h at 25°C, red spots indicated binding of Congo red [32].

Transmission electron microscopy (TEM)

To identify potential differences in flagellation of P. alvei CCM 2051^T wild-type, P. alvei ΔslhA and P. alvei Δhag, cells were grown over-night in LB medium. 30 µl of these cell suspensions were applied to Formvar- and carbon-coated 300-mesh copper grids (Agar Scientific) that were rendered hydrophilic upon glow discharge using a Pelco easiGlow apparatus (Ted Pella) for TEM. The grids were incubated for 10 min face down on the cell suspensions. Samples were fixed with 2.5% glutaraldehyde, washed three times with MilliQ-water, and stained with 1% uranyl acetate solution, pH 4.2, for 40 s [33]. Samples were investigated using a Tecnai G² 20 Twin transmission electron microscope (TEM; FEI), operating at 80 keV. Pictures were taken with an FEI Eagle 4 k CCD camera (4096×4096 pixels).

Microtitre plate assay

The ability of the P. alvei CCM 2051^T variants to form biofilm on an abiotic surface was evaluated according to Yegorenkova et al. [21]. Cultures were grown overnight in LB medium, diluted to an OD₆₀₀ ~0.025 with sterile nutrient medium, and 1-ml aliquots were pipetted into 24-well polystyrene plates (STARLAB). The control received only the sterile nutrient medium. Cultures were incubated at 37°C for 72 h without shaking. Subsequently, planktonic cells were gently removed with a pipette and the plates were washed twice with MilliQ-water. The OD₆₀₀ of planktonic cells was determined. Each well received 1 ml of 1% (w/v) Crystal violet and the plates were allowed to stand for 20 min at 25°C. Crystal violet solution was withdrawn with a pipette and the plates were carefully washed with MilliQ-water. Bound dye was released by adding 2.0 ml of acetone-ethanol (1:4) to the wells. Biofilm forming ability was evaluated by measuring the absorbance at 590 nm (A₅₉₀) of the solution using a Hitachi U-2001 spectrophotometer. Crystal violet values were normalized to the corresponding absorbance of the planktonic cells (A₅₉₀/A₆₀₀). Data represent mean values + SD of at least four independent experiments with each four replicates and were analyzed by the unpaired Student’s T Test. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Confocal laser scanning microscopy (CLSM)

P. alvei CCM 2051^T variants were grown for 72 h in uncoated plastic dishes (µ-Dishes, 35 mm high; Ibidi). Prior to CLMS, the biofilm was washed with 1 ml of PBS buffer to remove planktonic cells and 1 ml of PBS buffer was added.

To visualize the cells of the biofilm, 1 ml of a solution of Hoechst 33258 (Sigma Aldrich) (10 ng/ml) was added to the biofilm and incubated in the dark at room temperature for 25 minutes. Images were taken on an inverted TCS-SP5 confocal microscope (Leica) at excitation of 356 nm and emission of 465 nm [34].

Scanning electron microscopy (SEM)

P. alvei CCM 2051^T variants were grown as standing cultures on glass slides in cell culture dishes (uncoated, PS, 35 mm; Thermo, Fisher) containing 2 ml of LB medium. After 24 h, 48 h and 72 h respectively, the biofilm was washed with PBS to remove planktonic cells, followed by fixation with 2.5% glutaraldehyde in PBS for 4 h at 4°C. Subsequently, a dehydration series in ethanol (25%, 35%, 50%, 60%, 70%, 80%, 90%, 95%, 100% each for 7 min at room temperature) followed by drying with hexamethyldisilazane (HMDS; 33%, 66% and 100% in methanol), the glass slides were carefully removed and air-dried for several hours, were sputter-coated with 2.8-nm Au and examined at 20 keV acceleration voltage using a Inspect S50 scanning electron microscope (FEI) [35].

Localization of SlhA to the cell surface of P. alvei ∆slhA

To localize the SlhA protein on the cell surface of P. alvei ∆slhA in relation to the S‑layer protein SpaA, which is known to completely cover the bacteria as a 2D crystalline array with oblique symmetry (10), a cell surface co-display system was constructed based on a chimera made of SlhA-EGFP and SpaA-His₆.

P. alvei ∆slhA cells simultaneously expressing the two tagged-proteins were analyzed by fluorescence microscopy. The appearance of a continuous fluorescent halo around the cells indicated that SlhA is located on the outermost surface of P. alvei ∆slhA cells. SpaA-His₆ was detected by probing the cells with penta-His Alexa Fluor 532 conjugate (Figure 3, upper panel). In contrast, P. alvei ∆slhA cells expressing the empty vector did not show any fluorescence (Figure 3, lower panel).

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Figure 3. Immunofluorescence microscopy of P. alvei CCM 2051^T ΔslhA cells co-displaying SlhA_EGFP and SpaA_His₆.

For immunofluorescence staining of surface-located SpaA_His₆, a penta-His Alexa Fluor 532 conjugate for direct detection of the His₆-tagged SpaA was used. The TRITC and the GFP Long pass filter blocks were used for detection of Alexa Fluor 532 and EGFP, respectively. The upper three rows show the immunofluorescence microscopy pictures of cells harboring pSURF and co-displaying SlhA_EGFP (upper three rows, second pictures) and SpaA_His₆ (upper three panels, third pictures). P. alvei CCM 2051^T ΔslhA cells harboring pEXALV are shown as a control in the fourth panel. Corresponding brightfield images of the same cells are shown on the very left and overlays are shown on the very right of each panel.

https://doi.org/10.1371/journal.pone.0076566.g003

Knockout of slhA causes changes in colony morphology and bacterial adhesion to agar plates

To determine a putative function of the SlhA surface protein, an slhA knockout was created based on bacterial mobile group II intron-mediated gene disruption [26]. Reconstitution of P. alvei CCM 2051^T slhA::Ll.LtrB (P. alvei ΔslhA) was done by plasmid-based SlhA expression. The ΔslhA mutant clearly differed from the wild-type bacterium in colony morphology when grown on 2% LB-agar plates (Figure 4A). The colonies of the SlhA knockout were non-adhesive, flat and frayed, while those of the wild-type, the complemented bacterium and a flagella knockout (P. alvei Δhag) were sticky, convex and circular (Figure 4A). P. alvei Δhag was included in this study for comparison based on our assumption that SlhA might be involved in bacterial motility.

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Figure 4. Knockout of slhA causes changes in colony morphology and adhesion of P. alvei CCM 2051^T cells to agar plates.

(A) Colony morphology of the wild-type strain (A, first picture), P. alvei CCM 2051^T ΔslhA (A, second picture), P. alvei CCM 2051^T ΔslhA_comp (A, third picture) and P. alvei CCM 2051^T Δhag (A, fourth picture) on LB agar plates. (B) Adhesion test of wild-type, P. alvei ΔslhA, the complemented strain and P. alvei Δhag. The upper panel shows the wild-type (B, upper panel first picture), P. alvei ΔslhA (B, upper panel second picture), the complemented strain ((B, upper panel third picture) and P. alvei Δhag (B, upper panel third picture) before washing, the lower panel shows the same strains after the washing step. The pictures represent one of four independent experiments.

https://doi.org/10.1371/journal.pone.0076566.g004

An adhesion test, in which P. alvei CCM 2051^T wild-type, P. alvei ΔslhA, P. alvei ΔslhA_comp and P. alvei Δhag cultures were spotted onto agar plates, incubated over night at 37°C and washed thoroughly, clearly showed an increased adhesion capability of wild-type, P. alvei ΔslhA_comp and P. alvei Δhag cells compared to P. alvei ΔslhA cells (Figure 4B). Pictures taken immediately before the washing step showed identical spot sizes for all variants, with P. alvei ΔslhA being more transparent, possibly due to different colony morphology (Figure 4B, upper panel). After the washing step, the P. alvei wild-type, P. alvei ΔslhA_comp and P. alvei Δhag spots remained visible, while the P. alvei ΔslhA colony has been completely removed (Figure 4B, lower panel). This clearly indicated differences in the adhesion capability to 2% LB-agar plates of P. alvei CCM 2051^T wild-type, P. alvei ΔslhA_comp and P. alvei Δhag in comparison to P. alvei ΔslhA.

Knock-out of slhA does not affect flagellation of P. alvei

Following up the effect of SlhA on bacterial motility, it was investigated by TEM, if P. alvei CCM 2051^T wild-type, P. alvei ΔslhA and P. alvei Δhag cells grown in LB broth showed differences in flagellation. To avoid shear forces, cell suspensions of the overnight cultures were directly used for TEM analysis. According to the TEM evidence on both P. alvei CCM 2051^T wild-type and P. alvei ΔslhA cells flagella are clearly visible, but are absent in P. alvei Δhag cells. Thus, there is no evidence that SlhA would be directly involved in the production or formation of flagella (Figure 5).

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Figure 5. Knockout of slhA does not alter flagella production of P. alvei CCM 2051^T cells.

Electron microscopic view of P. alvei CCM 2051^T wild-type (first column), ΔslhA (second column) and Δhag cells (third column). Flagella are clearly visible for wild-type and ΔslhA cells but no flagella are present for Δhag cells.

https://doi.org/10.1371/journal.pone.0076566.g005

P. alvei CCM 2051^T ΔslhA cells lose the ability to swarm on LB-agar plates

Since P. alvei cells are known to vigorously swarm on agar plates [13], we were interested to know, if swarming was changed in the ΔslhA mutant. Swarming on 0.4%, 1% and 1.5% LB-agar plates was completely abolished for P. alvei ΔslhA, while the complemented strain P. alvei ΔslhA_comp was able to swarm as effectively as the wild-type bacterium (Figure 6). Considering our observation of the complemented bacterium requiring 48 h instead of 24 h incubation time to swarm as efficiently as the P. alvei wild-type, it was tested, if the pEXALV expression vector had an influence on bacterial swarming. In fact, P. alvei carrying empty pEXALV vector needed longer (48 h) to swarm to the level of the vector-free wild-type bacterium.

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Figure 6. P. alvei CCM 2051^T ΔslhA cells lose the ability to swarm on LB-agar plates.

The upper panel shows swarming cells of wild-type (first column), P. alvei ΔslhA (second column), P. alvei Δhag (third column), wild-type (pEXALV) (fourth column), P. alvei ΔslhA (pEXALV) (fifth column) and the complemented strain P. alvei ΔslhA_comp (sixth column) on 0.4% (upper panel), 1% (middle panel) and 1.5% (lower panel) LB-agar plates. The pictures represent one of three independent experiments.

https://doi.org/10.1371/journal.pone.0076566.g006

Thus, to investigate, if P. alvei cells carrying pEXALV have a slower growth phenotype (extended lag-phase) compared to the wild-type, growth curves were recorded for the P. alvei variants. Comparison of the generation times showed almost identical values for all tested P. alvei variants (data not shown).

Since the expression vector pEXALV affects the lag-phase and, consequently, the swarming ability of P. alvei CCM 2051^T on agar plates (48 h versus 24 h), but not the generation time during exponential growth, loss of swarming motility can be interpreted as a direct consequence of knocking out slhA. Since also, as demonstrated for P. alvei ΔslhA, P. alvei Δhag was not able to swarm on 0.4%, 1% or 1.5% LB agar plates we assume that the flagella might be non- functional in the slhA knockout background under the used conditions (Figure 6).

Knockout of slhA decreases biofilm formation of P. alvei CCM 2051^T cells

In a next step, it was investigated, if SlhA would also affect biofilm formation of P. alvei CCM 2051^T. Biofilm formation of P. alvei wild-type, P. alvei ΔslhA and P. alvei Δhag was monitored at 37°C for 72 h using a crystal violet-based microtiter assay [21]. Since pEXALV was shown to influence bacterial growth (see above), P. alvei wild-type and P. alvei ΔslhA cells, vector-free and carrying pEXALV, respectively, were investigated in two independent experiments. Crystal violet values were normalized to the corresponding absorbance of the planktonic cells (A₅₉₀/A₆₀₀). In both experimental settings, wild-type cells were set to 100% (wild-type: 100% +24.9%, P. alvei wild-type (pEXALV): 100% +8.9%). P. alvei ΔslhA cells showed 31.9% +16.3%, P. alvei Δhag cells 8.0% +5.2% of biofilm formation compared to the wild-type. P. alvei ΔslhA (pEXALV) retained 31.3 +10.8%, while complemented P. alvei ΔslhA_comp showed 109.7% +15.6% of biofilm formation (Figure 7A).

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Figure 7. Knockout of slhA and hag decreases biofilm formation of P. alvei CCM 2051^T cells.

(A) Evaluation of the ability of cells of P. alvei CCM 2051^T wild-type, ΔslhA, Δhag, wild-type (pEXALV), ΔslhA (pEXALV) carrying the pEXALV vector, and the complemented strain P. alvei ΔslhA_comp for biofilm formation using Crystal violet (CV) staining. Data represent mean values + SD of at least four independent experiments with each four replicates and were analyzed by the unpaired Student’s T Test. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) SEM analysis of P. alvei CCM 2051^T wild-type, ΔslhA, Δhag and the complemented strain ΔslhA_comp showing an overview and enlarged view of the biofilm. Size bars are 20 µm for the upper panel and 5 µm for the lower panel. (C) CSLM analysis of P. alvei CCM 2051^T wild-type, ΔslhA, Δhag and the complemented strain ΔslhA_comp stained with Hoechst 33258 showing a diagonally above view (upper panel) and a side view (lower panel) of a three day biofilm. Size bars are 20 µm.

https://doi.org/10.1371/journal.pone.0076566.g007

Thus, knocking out of slhA caused a significant decrease in biofilm formation, being 3.1-fold less for cells without pEXALV and 3.2-fold less for cells carrying pEXALV in comparison to the wild-type bacterium. Plasmid-based expression of SlhA complemented biofilm formation to wild-type levels. Knocking out of hag resulted in 12.5-fold less biofilm formation compared to the wild-type level according to the microtiter assay.

SEM and CLSM confirmed the results of the microtiter assay. For each technique, three biological replicates were analyzed. The SEM pictures taken after 72 h of biofilm development on glass slides revealed a biofilm with a higher cell density for the wild-type and P. alvei ΔslhA_comp compared to P. alvei ΔslhA and P. alvei Δhag (Figure 7B). The P. alvei ΔslhA biofilm showed a reduced content of slimy matrix components, probably due to reduced EPS formation.

The 3D structure of 72 h wild-type, P. alvei ΔslhA, P. alvei ΔslhA_comp and P. alvei Δhag biofilms was visualized by CLSM after staining with Hoechst (Figure 7C). Biofilms of P. alvei ΔslhA and P. alvei Δhag were comparable in height, structure and density. Either strain showed preferentially a carpet-like appearance, with few cell-layers of ~5 µm (ΔslhA) and ~10 µm (Δhag) respectively. Tower-like structures were produced by the wild-type (~3 µm, height) and the P. alvei ΔslhA_comp (~34 µm, height) biofilms. This supported the results of the microtiter assay.

Summarizing, the biofilm of the wild-type is ~6-fold thicker than that of ΔslhA and ~3-fold thicker than that of Δhag, according to CLSM analysis. CLSM analysis of ΔslhA_comp revealed again a slightly increased biofilm formation compared to the microtiter assay. In conclusion, the wild-type and the complemented strain showed by far the best ability for biofilm formation according to three different techniques (microtiter assay, CLSM; SEM). The ΔslhA and Δhag mutant strains both showed significantly reduced biofilm formation.

Knock-out of slhA decreases Congo red-staining of P. alvei CCM 2051^T cells

Given that colonies of the P. alvei CCM 2051^T wild-type were found to be sticky compared to P. alvei ΔslhA, the composition of the extracellular matrix could have been altered in the slhA mutant.

P. alvei CCM 2051^T wild-type, P. alvei ΔslhA, P. alvei ΔslhA_comp and P. alvei Δhag were tested for their EPS production by spotting the cells on Congo red (CR)/LB agar plates. CR accumulates on the colony surface over time only when EPS is produced [36]. Upon incubation of the plates for 24 h at 37°C followed by incubation for 48 h at 25°C, colonies of the wild-type, ΔslhA_comp, and Δhag strain had a reddish appearance, indicative of the presence of CR-reactive EPS on either variant. In comparison, spotted ΔslhA cells showed decreased adsorption of CR (Figure 8A). Quantification of EPS production of planktonic cells showed that the wild-type bound 9.2 +2.3 µg CR/OD₆₀₀, while ΔslhA bound 4.5 +1.7 µg CR/OD₆₀₀ and ΔslhA_comp bound 6.9 +1.7 µg CR/OD₆₀₀ (Figure 6B). CR binding ability of Δhag of 9.0 +2.8 µg compared well to that of the wild-type (Figure 8B). The value of 49% of CR binding of ΔslhA, in relation to 97% CR binding of Δhag compared to the wild-type bacterium, might be indicative of altered EPS production in the ΔslhA mutant.

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Figure 8. Knockout of slhA decreases Congo red staining of P. alvei CCM 2051^T cells.

(A) Congo red plates of P. alvei CCM 2051^T wild-type, ΔslhA, ΔslhA_comp and Δhag cultures. (B) Quantification of Congo red-binding in planktonic cultures at 37°C. Data represent mean values + SD of at least four independent experiments and were analyzed by the unpaired Student’s T Test. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

https://doi.org/10.1371/journal.pone.0076566.g008