Temperature-Regulated Formation of Mycelial Mat-Like Biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionellawere examined for biofilm formation on glass, polystyrene, andpolypropylene surfaces in static cultures at 25°C, 37°C,and 42°C. Strains of Legionella pneumophila, the most commoncausative agent of Legionnaires' disease, were found to havethe highest ability to form biofilms among the test strains.The quantity, rate of formation, and adherence stability ofL. pneumophila biofilms showed considerable dependence on bothtemperature and surface material. Glass and polystyrene surfacesgave between two- to sevenfold-higher yields of biofilms at37°C or 42°C than at 25°C; conversely, polypropylenesurface had between 2 to 16 times higher yields at 25°Cthan at 37°C or 42°C. On glass surfaces, the biofilmswere formed faster but attached less stably at 37°C or 42°Cthan at 25°C. Both scanning electron microscopy and confocallaser scanning microscopy revealed that biofilms formed at 37°Cor 42°C were mycelial mat like and were composed of filamentouscells, while at 25°C, cells were rod shaped. Planktoniccells outside of biofilms or in shaken liquid cultures wererod shaped. Notably, the filamentous cells were found to bemultinucleate and lacking septa, but a recA null mutant of L.pneumophila was unaffected in its temperature-regulated filamentationwithin biofilms. Our data also showed that filamentous cellswere able to rapidly give rise to a large number of short rodsin a fresh liquid culture at 37°C. The possibility of thisbiofilm to represent a novel strategy by L. pneumophila to competefor proliferation among the environmental microbiota is discussed.

INTRODUCTION

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Introduction

Legionnaires' disease is a form of pneumonia caused by bacteriaof the genus Legionella, of which Legionella pneumophila isthe type species. This infectious disease involves inhalationof aerosols of water containing Legionella into the lung. Withinthe lung, the bacteria are internalized by alveolar macrophages,replicate within the phagosomes, and eventually lyse the hostmacrophages (27). The genus Legionella currently contains asmany as 48 species, and at least 20 species are known to bepathogenic to humans (22). Despite this, the general perceptionis that the species L. pneumophila is the main causative agentof Legionnaires' disease, due to the much higher incidence ofthe disease reported to be caused by L. pneumophila than bythe other species of the genus (5). One or more of the followingreasons could account for this higher incidence. First, lackof diagnostic reagents sufficiently discriminative to identifynon-L. pneumophila species could lead to inaccurate clinicalstatistics (22). Second, L. pneumophila may be inherently morevirulent than other species. However, only a few aspects ofits pathogenicity e.g., cytopathogenicity and intracellularmultiplication, have currently been shown to be enhanced, comparedto non-L. pneumophila species (1, 23, 30, 42), providing nostrong support for this hypothesis. Third, L. pneumophila maybe easier to transmit than other Legionella species. However,transmission of L. pneumophila from humans to humans has notbeen reported, despite many cases of outbreaks in the recentfew decades. Transmission to humans has reportedly occurredonly via mechanical means, such as air-conditioning units, showerheads,and sprinklers (27, 61); therefore, enhanced human-to-humantransmissibility is an unlikely reason. Fourth and finally,L. pneumophila may have a higher chance than other Legionellaspecies of coming into contact with humans. One speculationis that L. pneumophila has an innate ability to better survivein the water environment, especially man-made ones. Factorsinfluencing survival of L. pneumophila have been studied previously(45), but its survival ability compared to that of other non-L.pneumophila species has not been explored.

The habitats for the genus Legionella are moist soil and aquaticenvironments, and outbreaks of legionellosis are often associatedwith contamination of man-made aquatic environments (for a review,see references 3, 22, and 27). Although Legionella bacteriado exist as free-living planktonic forms in the environment,they are more commonly found as intracellular parasites of protozoanssuch as Acanthamoeba spp., Hartmannella spp., and Tetrahymenaspp. (3, 7) and as inhabitants of mixed-community biofilms (51,62). Biofilms are assemblages of bacteria encased in extracellularpolymeric matrices, attached to surfaces or phase boundaries.Being able to form such an adherent, hydrated, and structuredcommunity confers several advantages on bacteria for survivalin harsh world, e.g., protection from harmful compounds andprevention of desiccation (for a review, see references 15 and18). Legionella has been studied in the context of mixed-communitybiofilm experimental systems (9, 40, 52) or with the purposeof understanding the efficacy of biocides or biocidal treatments(26, 64, 65), but its ability to form biofilms per se has notbeen investigated.

The initial motivation for this study was to address the question:has L. pneumophila better survival ability in terms of biofilmformation than other Legionella species? We were particularlyinterested in differences that may exist at temperatures suchas 37°C and 42°C, often encountered in man-made environments,e.g., hot spring spas, in contrast to the more ambient temperatureof 25°C. In the course of the investigation, we found thata novel form of biofilm composed of filamentous cells of L.pneumophila exists, which may represent another aspect of survivalstrategy by this organism. Here, we shall report on these findings.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and culture conditions.
Legionella spp. were obtained from Gifu Type Culture Collection(GTC) (Table 1). Unless otherwise stated, all strains were grownin buffered yeast extract (BYE) broth (49) at 37°C withshaking or on buffered yeast extract charcoal agar (BYECA; BYEwith 0.2% activated charcoal and 1.5% Bacto agar) (37) at 37°Cfor 3 days. Both types of media were supplemented with 0.4-g/literof L-cysteine and 0.25-g/liter ferric pyrophosphate, and thepH was adjusted to 6.9 with 1 M potassium hydroxide. Kanamycinat 25 µg/ml was added where necessary.

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TABLE 1. Legionella strains used in this study

Biofilm formation and quantification.
Legionella strains were shaken at 37°C for 30 h and thendiluted 1:10 to give a final optical density at 600 nm of 0.2to 0.3. For general observation and quantification, biofilmswere allowed to form from 2.2 ml of this diluted culture inround-bottom test tubes (diameter, 12 mm; height, 75 mm) ofglass (Pyrex, Iwaki Glass, Japan), polystyrene (PS) (Falcon;Becton Dickinson), and polypropylene (PP) (Sarstedt, Germany),statically incubated in water vapor-saturated incubators setat 25°C, 37°C, or 42°C. For confocal laser scanningmicroscope (CLSM) observation, biofilms were allowed to formon Micro glass cover slides of 0.8 mm in thickness (MatsunamiGlass Ind. Ltd., Japan), vertically immersed in the dilutedculture in a thin-layer chromatography tank and similarly incubatedat the indicated temperatures.

Quantification of biofilms were performed based on a protocolmodified from that of O'Toole et al. (46). Briefly, a one-fifthvolume of 0.25% crystal violet solution was added to the biofilmculture, and the biofilm was stained for 15 min. The biofilmwas rinsed three times with deionized water, and the crystalviolet stain was solubilized in 2 ml of 95% ethanol with shakingand then transferred as 200-µl aliquots (in triplicate)to polystyrene 96-well microtiter plates (Costar; Corning, Inc.).Absorbance at 600 nm was measured with a Wallac 1420 ARVOsxmultilabel plate reader (Perkin-Elmer Life Sciences, Japan).

Plasmid construction, genetic manipulation, and recA mutant generation.
The plasmid pPZ1 carrying the green fluorescent protein (GFP)gene gfp-mut3* under the control of the LacI-repressible P_1/04/03promoter (2) was constructed by standard recombinant DNA techniques(57). A NotI fragment from pJBA27 carrying P_1/04/03-gfp-mut3*(2) was inserted into the NotI site of pKM330, an RSF1010-basedbroad-host-range vector derived from pVI397 (58), with the carbenicillinresistance marker replaced by a kanamycin resistance marker(a kind gift from V. Shingler). To visualize L. pneumophiladirectly under CLSM, pPZ1 was introduced via electroporationinto the strain, using a Gene-Pulser (Bio-Rad Laboratories Pte.Ltd., Japan).

For the generation of the recA mutant, the region encoding theL. pneumophila recA gene was PCR amplified with primers 5'-CCAGATTGCTTACTCATCTC-3'and 5'-AGTTGTTCAAGCTCTGCAGC-3', ligated into pGEM-Easy Vector(Promega), and inserted at its internal BamHI site, a 1.7-kbkanamycin resistance cassette from pUT-miniTn5-Km (14). Theinterrupted recA gene was then transferred into the NotI siteof pLAW344, which harbors the sacB gene that confers sucrosesensitivity and allows counterselection for homologous recombinationby double crossover (33). The plasmid was introduced into L.pneumophila JR32 (56) and selected for kanamycin and sucroseresistance, and the recombinants were confirmed for allelicreplacement via PCR and increased UV sensitivity, as would beexpected of a recA mutant (17).

Observation using CSLM, scanning electron microscope (SEM), and transmission electron microscope (TEM).
Glass slides with biofilms formed on both sides by L. pneumophilacarrying pPZ1 were cleaned on one side with an alcohol swaband mounted on an Eclipse TE2000-U inverted microscope (Nikon)attached to a Radiance 2100 Laser Scanning system (Bio-Rad)to observe the biofilm attached on the intact side. Images weretaken under an argon laser source with a dichroic mirror (excitation,488 nm; emission, 515 nm) and processed by LaserSharp 2000 (Bio-Rad)software.

For EM observation, biofilms formed on the glass test tubeswere transferred using a toothpick into glass petri dishes andwashed briefly with deionized water. They were fixed with 2%glutaraldehyde for 2 to 3 h and then additionally with 1% OsO₄in the case of TEM observation. Subsequent dehydration was performedstep wise using 50%, 70%, 90%, 95%, 99.5%, and 100% ethanol.For SEM observation, the ethanol was replaced by isoamyl acetateand then liquid CO₂, which was allowed to vaporize gradually.Following this, the samples were coated with 60% gold-40% palladiumalloy powder. For TEM observation, dehydrated samples were embeddedin Epon resin, ultrathin sectioned, and stained with uranylacetate, followed by lead citrate. The samples were examinedwith or without using a scanning image-observing device attachedto a JEOL JEM 2000EX electron microscope.

Nucleoid staining of filamentous cells.
Filamentous cells from L. pneumophila biofilms were teased onglass slides to separate the strands and stained according toa modified version of HCl-Giemsa staining (50). Briefly, thepreparation was fixed in OsO₄ vapor for 45 min, treated with1 M hydrochloric acid at 60°C for 10 min, rinsed, and stainedwith Giemsa stain for 5 min, followed by phosphate buffer (pH6.4) for 5 min. The air-dried preparation was observed underan oil immersion lens on a light microscope (Olympus).

Monitoring distribution of filamentous and rod-shaped cells.
Biofilms formed on glass test tubes or colonies formed on agarwere picked by toothpicks and spread extensively on glass slidesfor ease of examination of individual cells. For observationof cells in the medium surrounding biofilms, aliquots of themedium were taken with care to avoid disturbing the biofilms,centrifuged briefly to collect the cells in pellets, and thenspread on glass slides. Cells from shaken culture were similarlycollected and spread. Slides with these preparations were flamefixed, stained with Pfeiffer stain, and observed under oil immersionby a light microscope. Images in three different fields wereprocessed with the Image-Pro 3D Suite (Media Cybernetics, Inc.)to quantify the number of pixel units for images of the filamentousand rod-shaped forms. The percentage of biovolume of filamentouscells was calculated as 100 x (pixel units for filamentous cells/pixelunits for all cells).

Growth of cells from biofilm and planktonic culture.
Biofilms cultured for 4 days in glass test tubes were collectedwith Pasteur pipettes and rinsed briefly in sterile deionizedwater. They were then transferred into a test tube containing5 ml of AC buffer [4 mM MgSO₄, 0.4 mM CaCl₂, 3.4 mM sodium citrate,2.5 mM Na₂HPO₄, 2.5 mM KH₂PO₄, 20-mg/liter Fe(NH₄)₂(SO₄)₂ ·6H₂O, pH 6.5] and shaken vigorously for 1 h to physically separatethe filaments. A microscopic check of this suspension revealedthat 90% of the cells were untangled from each other. For the"planktonic control," a 30-h shaken culture in BYE was diluted1:100 in 5 ml AC buffer and shaken similarly for 1 h. Aliquots(each, 4 ml) of the biofilm cell-derived and planktonic cell-derivedsuspensions were then transferred to conical flasks, each containing66 ml of fresh BYE broth. Growth was monitored at 4-h intervalsby counting the bacterial CFU on buffered yeast extract charcoalagar.

RESULTS

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Results

Biofilm formation by Legionella spp.
To assess the ability of Legionella spp. to form biofilms, particularlyto compare L. pneumophila to non-L. pneumophila species, weutilized 13 strains of L. pneumophila and 38 strains of otherLegionella spp. (Table 1). This gives a good representationof the genus Legionella. The experimental system is a staticculture in the BYE broth medium, and three different materials(glass, PS, and PP) were chosen as surfaces for biofilm growthat three temperatures (25°C, 37°C, and 42°C). Thegrowth of all test strains in BYE liquid and agar media at theexperimental temperatures was checked to ensure that any inabilityto form a biofilm was not due to nonoptimal growth conditions.A preliminary experiment, in which biofilm formation was noteddaily by eye over a period of 30 days, indicated that stablebiofilms could be observed from day 11 at 25°C and fromday 3 at 37°C or 42°C. The biofilms formed were thereforequantified by crystal violet staining on day 12 for the 25°Ccultures and day 4 for the 37°C and 42°C cultures. Theresults are presented in Fig. 1. Statistical analysis of thedata indicated that at 37°C and 42°C on glass and PS(Fig. 1A and B) and at 25°C on PP (Fig. 1C), L. pneumophilastrains have significantly greater biofilm-forming capacitythan all other Legionella spp.

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FIG. 1. Quantification of biofilms (as described in Materials and Methods) formed by Legionella spp. on surfaces of glass (A), polystyrene (B), and polypropylene (C) at incubation temperatures of 25°C (black bars), 37°C (white bars), and 42°C (gray bars). Strains or species are indicated as strain numbers and presented in Table 1. Values are presented as a means ± standard deviation (SD) of three to six independent experiments. Statistical analysis was performed using Student's t test to compare the differences between groups, and P values of <0.05 were considered statistically significant.

Quantity, speed of formation, and adherence stability of L. pneumophila biofilms.
In the above data, the quantity of the biofilms formed by L.pneumophila strains (Fig. 1, strains 1 to 13) was observed tobe influenced by temperature. On glass and PS surfaces (Fig.1A and B), biofilms were generally formed more extensively atthe higher temperatures than at 25°C: biofilms at 37°Cwere two- to sevenfold higher (glass) and two- to fourfold higher(PS) in yield and at 42°C were three- to fivefold higher(glass) and two- to fivefold higher (PS) in yield. On PP surfaces,the situation was reversed: at 25°C, 2- to 7-fold and 3-to 16-fold more biofilms were formed than at 37°C and 42°C,respectively.

The temperature dependence could also be observed in the speedof formation and the adherence stability of the biofilms ofL. pneumophila strains. In three to five independent experimentslooking at biofilm formation by the 13 L. pneumophila strainsin glass test tubes, we documented (i) the number of days ittook for the appearance of stable biofilms, as an indicatorof the speed of biofilm formation; and (ii) the day at whichthe biofilms were observed to naturally detach from the wallsof the test tubes, as an indicator of the adherence stability.Statistical analysis revealed that all L. pneumophila strainsshowed similar trends: at 25°C, biofilms were formed moreslowly (12 ± 1 days) but remained stably attached throughoutthe 30-day period of observation. At 37°C and 42°C,biofilms were formed faster (3 ± 1 days), but adherencestability was lower (7 ± 1 days). Similar trends in thespeed of biofilm formation were observed when biofilms weregrown on PS or PP, but the difference in adherence stabilitywas not as apparent (data not shown).

Thickness and structure of L. pneumophila biofilms.
The temperature dependence may have biological significancerelated to possible survival advantages of L. pneumophila inman-made environments over non-L. pneumophila species. To lookfurther into this, L. pneumophila strain Knoxville-1 was chosenas a representative of the 13 L. pneumophila strains for in-depthstructural observation. We introduced pPZ1, which allows constitutiveexpression of the GFP into Knoxville-1 strain for visualizationof its biofilms in the natural hydrated state by CLSM. The GFP-expressingKnoxville-1 strain showed biofilm formation properties at thethree temperatures, similar to those of the wild-type Knoxville-1strain (data not shown), indicating that the expression of GFPdoes not observably affect the gross phenomenon under study.

Biofilms were allowed to form on glass slides immersed in BYEbroth and then observed directly under CLSM on the indicateddays (chosen to arbitrarily represent an early and a maturestage of biofilm formation) to quantify the thickness (Table2). Biofilms at 37°C and 42°C were found to be indeedthicker than that at 25°C by about twofold. In terms ofstructures, biofilms at 25°C (Fig. 2A) possessed featurestypical of biofilms reported to date, i.e., pillar- and mushroom-likestructures and what seemed like water channels within (15).Biofilms at 37°C, however, showed an even and extensivemat of considerably greater cell density without the commonlyobserved water channel structures (Fig. 2B).

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TABLE 2. Thickness of GFP-expressing L. pneumophila Knoxville-1 biofilm

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FIG. 2. Structure of L. pneumophila biofilms. The L. pneumophila Knoxville-1 strain expressing GFP was observed under CSLM (A to D). x-z plane projection of 25°C biofilm at day 18 (A) and 37°C biofilm at day 6 (B); x-y plane projection of 25°C biofilm at day 8 (C) and 37°C biofilm at day 4 (D). The L. pneumophila Knoxville-1 biofilm at 37°C on day 4 was observed under scanning electron microscope (E), and its filamentous cell stained with HCl-Giemsa was observed under the light microscope (F). (G) A section of the filamentous cells of L. pneumophila Philadelphia-1 strain was observed under the transmission electron microscope. Bars, 50 µm (A and B), 10 µm (C and D), 1 µm (E), 5 µm (F), and 1 µm (G). Images were processed and compiled with Adobe Photoshop 7.0 software.

Morphology of L. pneumophila cells in biofilms.
It was apparent that L. pneumophila biofilms at 37°C didnot have the typical biofilm features (16) shown by most bacteria.At higher magnifications, cells within biofilm at 25°C showedthe normal morphology of L. pneumophila, i.e., they were rodshaped (Fig. 2C). In contrast, the morphology of cells in biofilmat 37°C was filamentous (Fig. 2D). The meshwork of filamentsresembled the mycelia of fungi; hence, we tentatively referto it as a "mycelial mat-like biofilm." Filamentation of bacterialcells is sometimes associated with physiological abnormalitiessuch as mutations or overexpression of certain proteins (8,13). To check if the filamentation was an artifact due to gfpoverexpression, we collected the biofilm formed by the wild-typeKnoxville-1 strain at 37°C and prepared it for observationunder the SEM. The electron micrograph (Fig. 2E) showed a filamentousmeshwork similar to that seen by CSLM, ruling out the possibilityof artifacts. Hence, the morphology of L. pneumophila cellsin the biofilms formed on glass (and likewise on PS and PP surfaces)(data not shown) appears to be regulated by temperature.

Distribution of rod-shaped and filamentous cells under different growth conditions.
The distribution of the morphological forms under differinggrowth conditions was next examined. We systematically assessedthe relative contribution of filamentous cells to the totalbiovolume (i) within biofilms grown on glass, (ii) in the surroundingmedia, (iii) in colonies grown on agar, and (iv) in shaken cultures.At 25°C (Fig. 3, black bars), the static culture showeda consistent dominance by rod-shaped cells, both within thebiofilm and outside (i.e., in the medium), throughout the periodof observation. At 37°C and 42°C (Fig. 3, white andgray bars) the biofilms showed that close to 90% of the totalbiovolume consisted of filamentous cells as early as day 2 andcontinued to be dominated by the filamentous form until day7. Upon detachment from the vessel walls (day 11 and day 13),these detached biofilms retained their mycelial characteristics.However, within the surrounding media of biofilms at 37°Cand 42°C, rod-shaped cells were in the majority. Shakencultures were predominantly rod shaped; it should be noted thatat 37°C and 42°C, predominance of rod-shaped cells wasmaintained even up to day 3. Agar plate culture, on the otherhand, showed a profile for distribution of forms similar tothat observed in biofilms at the three temperatures.

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FIG. 3. Percentage of biovolumes represented by filamentous cells within various L. pneumophila Knoxville-1 cultures. Black bars represent growth at 25°C, white bars represent growth at 37°C, and gray bars represent growth at 42°C. The arrow indicates the point at which detachment of biofilms at 37°C and 42°C occurred. Double brackets indicate days when only 25°C biofilms were assessed. Values are presented as means ± SD of triplicate data. Statistical analysis was performed using Student's t test to compare the differences between groups, and P values of <0.05 were considered statistically significant.

Filamentous cells are multinucleate and nonseptate.
The filamentous cells in L. pneumophila's biofilm were stainedfor their nucleoids and found to be clearly multinucleate (Fig.2F). This indicates that the filamentous form within the biofilmis one whereby DNA replication has proceeded in the absenceof cell division. Indeed, observation by TEM showed the totallack of septa within the filamentous cells (Fig. 2G).

Temperature-regulated filamentation in L. pneumophila is not RecA dependent.
The filaments formed as a result of nonseptation, observed withL. pneumophila biofilms when cultured at higher temperatures,is reminiscent of the phenotype of the thermosensitive recAmutants of Escherichia coli. RecA in E. coli is a regulatorinvolved in the SOS response induced by DNA damage, and an abnormalityin this regulator has been reported to result in filamentationat the elevated temperature of 40°C (10, 28). This is dueto the control of RecA on factors involved in cell division,e.g., FtsZ (39) and SulA (20), both of which require well-regulatedexpression levels to ensure normal septation. A RecA homologuein L. pneumophila has been found to be able to functionallycomplement the E. coli counterpart and appears to be similarlyregulated (17). The possible involvement of RecA in temperature-dependentfilamentation by L. pneumophila cells was checked. An insertionalnull mutant of recA was generated in strain JR32, a L. pneumophilaPhiladelphia-1 derivative amenable to genetic manipulation (56)and observed to be able to form biofilms in a manner similarto that of the wild type, showing temperature-regulated morphologicalforms. Thus, the recA gene in L. pneumophila appears not tobe responsible for the temperature-regulated filamentation phenomenon.

Growth of cells from mycelial biofilm.
The fact that the filamentous form of L. pneumophila is multinucleatebut nonseptate suggests that, given favorable conditions, theformation of septa may quickly give rise to more daughter cellsthan rod-shaped cells, since multiple DNA replication couldbe assumed to have already proceeded to completion during thefilamentous state. To test this hypothesis, we mechanically"unraveled" a mature mycelial biofilm from a 37°C day 4culture of Knoxville-1 strain by extensive shaking in a suitablebuffer. We then followed the growth of its cells in fresh BYEmedium shaken at 37°C over 36 h and compared it to thatof the rod-shaped cells, seeded from a 30-h shaken culture (Fig.4). The assumption is that one filamentous cell before septationwill appear as one CFU. The rod-shaped cell culture continuedin its lag phase until 12 h, but the culture from mycelial biofilmentered exponential phase by 8 h and mostly converted to rod-shapedcells. This shortened lag phase was remarkable, consideringthat cells from the biofilm were 4 days old and therefore expectedto have an even longer lag phase than cells from a 30-h-oldshaken culture. The growth rate (gradient at exponential phase)for the mycelial mat-like culture was not significantly higherthan that for the rod-shaped cell culture during their respectiveexponential phases. Hence, the filamentous form of L. pneumophilaseems to allow the organism to proliferate particularly rapidlyin the initial stage, more than does the normal rod-shaped form.

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FIG. 4. Growth of L. pneumophila Knoxville-1 in BYE. The culture was shaken at 37°C and seeded from filamentous cells of a 37°C biofilm at day 4 (open circles) and rod-shaped planktonic cells of a 37°C culture shaken for 30 h (black circles). Data are means ± SD from three independent experiments.

DISCUSSION

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Discussion

The ability to form biofilms provide a bacterium with survivaladvantages in the environment, e.g., anchorage at a locationwhere growth is favorable, protection from desiccation, andresistance to biocides and detergents (15, 16, 18). Under theconditions used in this study, the 13 strains of L. pneumophilatested showed enhanced biofilm formation compared to other non-L.pneumophila strains: at 37°C and 42°C on glass and PSsurfaces (Fig. 1A and B) and at 25°C on PP surfaces (Fig.1C). To some extent, this may be due to the less-robust growthof a few non-L. pneumophila strains at specific temperatures(Table 1, footnotes e to g) in a medium optimized for cultivationof L. pneumophila, but these are minor exceptions. Within thegenus Legionella, L. pneumophila is the species most often associatedwith human clinical cases (22). One speculation is that it isbetter able to survive in man-made environments than non-L.pneumophila species and hence has a greater chance of crossingpaths with the human population. Our finding that L. pneumophilais more proficient at biofilm formation than other Legionellaspecies supports this speculation. Since the temperatures 37°Cand 42°C are often encountered in man-made aquatic environments,e.g., air-conditioning cooling towers in the summer and hotspring spas, the enhanced ability by L. pneumophila to formbiofilms on glass, PS, and possibly other surfaces would likelyincrease its chance of association with humans. On the otherhand, the fact that biofilm formation is favored on PP at 25°Calso suggests that the competitive edge conferred may be dependenton a combination of the temperature and the type of attachmentsurfaces.

It is interesting to note that the properties of L. pneumophilabiofilms formed at 25°C differ considerably from those formedat 37°C and 42°C. At higher temperatures, the biofilmsare formed faster, thicker, and spread wider. Most interestingly,at higher temperatures, they are mycelial mat like in structure.Bacterial biofilms reported to date commonly show structuresof polymeric matrices interspersed with water channels (32).For rod-shaped bacteria, e.g., Pseudomonas spp. and Vibrio cholerae,cells within biofilms remain rod shaped (60), with only a rareappearance of filaments (38). In rivers and on riverbeds, mixed-communitybiofilms may have filamentous components (bacteria or fungi)among rods and cocci (35), but biofilms composed primarily ofextensive meshwork of filaments, to our knowledge, have onlybeen reported for Thiothrix spp. in cases of biofouling (6)and Methanosaeta spp. in anaerobic sludge granules (29). Furthermore,although filamentation in rod-shaped bacteria with physiologicalabnormalities has been reported (13, 28, 48, 66), temperature-regulatedfilamentation in a biofilm context is highly unusual.

One question that may arise with this novel form of biofilmis, can we truly consider it a "biofilm"? The definition givenby Donlan and Costerton (16) states that a biofilm is "a microbiallyderived sessile community characterized by cells that are irreversiblyattached to a substratum or interface or to each other, areembedded in a matrix of extracellular polymeric substances thatthey have produced, and exhibit an altered phenotype with respectto growth rate and gene transcription." For our mycelial mat-likestructure, the criteria of attachment is certainly satisfied;furthermore, a thin layer of ruthenium red-stainable substance(possibly exopolysaccharide) (24) coats the filaments (datanot shown). The filamentous cells have a growth profile (Fig.4) that indicates an altered phenotype from the planktonic,rod-shaped form and are expected to have considerable modificationin gene expression compared to expression in a binary fissionmode. Our current analysis of the transcriptional profiles ofthe biofilm filamentous versus planktonic L. pneumophila cellsalso substantiates this point (Z. Piao, unpublished data). Thus,all the criteria seem to have been met for the mycelial mat-likestructure to be referred to as a biofilm.

L. pneumophila have long been known to be pleomorphic (31),varying its morphology with physiological conditions (36, 43)or infectious cycle phases (61). Species of Legionella havealso been reported to form varied microcolonies within Verocells, some appearing filamentous, although the septation statushas not been addressed (44). L. pneumophila also "differentiates"into a few extracellular or intracellular forms with varyingdegrees of infectivity for HeLa cells (25), which can be distinguishedby the ultrastructural properties of their cell envelopes (21).In our study, the electron micrograph of the filamentous L.pneumophila cell (Fig. 2G) shows cell wall structure that isconsistent with that of the extracellular stationary phase rodforms reported by Faulkner et al. (21), confirming its "extracellular"status.

Extensive filamentation by L. pneumophila has previously beendescribed in conditions of physiological stress such as exposureto antibiotics (19, 59) and nutrient limitation (63). In otherrod-shaped bacteria, filamentation by mutants of global regulator(28) or cell division genes (13, 48) and when exposed to highsalt concentrations (66) has also been reported. We considered,by analogy with the phenotype of an E. coli mutant (10), thatour filamentation phenomenon could be due to the regulatoryaction of the recA gene. The thermosensitive mutation recA441(formerly tif-1) of E. coli is known to convert cells to multinucleate,aseptate filaments at 40°C through the constitutive expressionof the sulA gene that encodes an inhibitor of the cell divisionprotein FtsZ (34). However, this possibility was ruled out forL. pneumophila because disruption of the recA homologue wasfound to be without effect on the phenotype. Another immediatepossibility, suggested by analogy with E. coli ftsZ mutants(28, 47), is that L. pneumophila FtsZ protein might be thermosensitive.We have not yet been successful in substantiating this idea,but we intend to continue to dissect the mechanism of regulationin this novel form of biofilm. The recently completed L. pneumophilagenome sequences of strains Philadelphia-1 (12) and Paris (11)(http://genome3.cpmc.columbia.edu/ $~$ legion/seq_anno.html) indicatethat the organism possesses not only recA and ftsZ but alsohomologues of other E. coli genes, e.g., ftsA and mreB, involvedin cell division and maintenance of cell morphology (48). Weare currently looking into how these genes are regulated andcorrelated with the biofilm phenotype of L. pneumophila.

Filamentation is often a consequence of some stress-induceddisruption of normal cell division functions. However, in rarecases, it serves as a strategy to counter specific types ofstress. Elongation of cells by nutrient-deprived Pseudomonasaeruginosa has been proposed to be a strategic response to enlargetheir nutrient collection surface without substantially changingthe surface area-to-volume ratio (60). Temperature-regulatedfilamentation in L. pneumophila biofilm is unusual in that itis clearly not a simple case of cell division inhibition bysublethal heat stress, e.g., as seen with Listeria monocytogenes(53). Induction of filamentation at 37°C or 42°C wasnot observed for L. pneumophila cells outside of the biofilmsor in a shaken liquid medium. Instead, only surface-associatedcells, i.e., within biofilm or on agar, exhibited filamentation(Fig. 3). Some other signal(s) besides temperature, e.g., attachmentto surfaces, is possibly involved, and it is conceivable thatfilamentation is a strategic regulated response rather thanan accidental by-product of stress. Our growth experiment showedthat cell proliferation of the biofilm filamentous form is fasterat the initial stage than the planktonic rod-shaped form (Fig.4). During this initial stage, the multinucleate filamentouscells have converted into rod-shaped forms, after which no differencein growth rate could be observed. The filament-turned-rod-shapedcells therefore do not differ in terms of growth rate from thenormal rod-shaped cells. Presumably, the time and energy requiredby a filamentous cell to increase in number involve only theformation of cell septa, whereas binary fission involves DNAreplication and biomass generation, in addition to septa formation.Hence, filamentation may represent a novel strategy utilizedby L. pneumophila to rapidly increase its population.

It is worth noting that the experimental system of our studymade use of a rich medium. In low-nutrient medium, very littleformation of biofilm could be observed (C. C. Sze, unpublisheddata). In the environment, the bacterium more often encounterslow-nutrient status, so one may wonder at the relevance of thisparticular study. However, it should not be forgotten that theenvironment is dynamic and that bacteria generally experiencecycles of "feast and famine." The survival of a bacterium, therefore,not only involves living through a famine but also hinges onhow well it exploits the rare but valuable encounter of a feast.L. pneumophila is a slow grower compared to other bacteria suchas E. coli, P. aeruginosa, and V. cholerae and may seem to bein danger of losing out to these fast growers in nature. Ithas, in part, made up for its shortcomings by finding a nicheas an intracellular parasite of free-living protozoans (4, 41,54, 55). By forming mycelial mat-like biofilms, L. pneumophilamay have hit upon another strategy to compete with fast growersin the environment, which allows it to (i) anchor itself ata location transiently flooded with nutrients, (ii) increaserapidly in biomass while able to access the nutrients by eliminatingthe process of septum formation, and then (iii) proliferateand disperse in great numbers when conditions are appropriate.We intend to explore this phenomenon of filamentous biofilmsfurther, from both genetic and ecological points of view.

ACKNOWLEDGMENTS

We thank H. Nakayama for valuable advice, H. Kajiwara and A.Takade for technical assistance, and V. Shingler and S. Molinfor their generous gifts of plasmids.

C.C.S. was supported by grant 13-01132 from the Japan Societyfor Promotion of Science. This work was supported by grant 14370094from the Ministry of Education, Science, Culture and Sportsof Japan and grant H14-047 from the Ministry of Health, Laborand Welfare of Japan.

FOOTNOTES

* Corresponding author. Mailing address: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore. Phone: 65-6316 2872. Fax: 65-6791 3856. E-mail: ccsze{at}ntu.edu.sg.

REFERENCES

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