COLLOIDS AND SURFACES

ELSEVIER Colloids and Surfaces B: Biointerfaces 7 11996)2(17 214

Bacterial wetting agents working in colonization of bacteria on surface environments

Tohey Matsuyama a,,, Yoji Nakagawa b Department of Bacteriology, Niigata Unicersity School ~f Medicine. Niigata 95I, Japan

b Department (!/'Applied Biological Chemistry, Faculty ~]Agriculture. Nii.~ata Unit,ersity. Niigata 950-21. Japan

Received 13 May 1996: accepted 10 June 1996

Abstract

Bacteria are very small and highly susceptible to the effects of intermolecular forces. Especially, bacteria living on solid-air interfaces are under a strong influence from the surface tension of water. During studies of bacteria which prefer to live on surfaces, we noted that some species of bacteria (e.g. Serratia marcescens) secrete large amounts of wetting agents (e.g. serrawettins). Therefore, we isolated mutants defective in the production of wetting agents and examined the physiological functions of these wetting agents by comparing the behavior of wild types and mutants on surface environments. In terms of accessibility to the water-repelling surfaces and spreading growth on solid media. mutants demonstrated inferior abilities in comparison with wild types. Thus, the ability of S. marcescens to form a giant fractal colony through nutrient-diffusion-limited growth processes was shown to be defective in the serrawettin- less mutants. In the locomotion of flagellated bacteria on surfaces, the bacteria seem to overcome various restrictive intermolecular forces. In contrast to swimming in a liquid, a single bacterium alone was unable to translocate on a surface. By video-microscopic analyses, the cooperative multicellular behavior of bacteria was clearly demonstrated. The remarkable effects of wetting agents on such microbial swarming behavior on surfaces were also disclosed.

Kevwords: Bacterial wetting agents: Colony: Fractal; Multicellular behavior: Surface environments

1. Introduction

For opt ica l microscopic examina t ion of bacteria, a small a m o u n t of bacter ia l suspension is dr ied and fixed on the surface of a glass slide. In this rout ine step of prac t ica l bac ter io logy, we not iced that a d r o p of Serratia marcescens suspension ins tan taneous ly spread over the whole glass sur- face. As a suspension of Escherichia coli did not spread like this, remain ing as a round drople t on

* Corresponding author. 1 Presented at the 1995 International Chemical Congress of

Pacific Basin Societies in the Symposium on Biosurfactants and Biosurfaces, Honolulu, HI, USA, December 17 22, 1995.

0927-7765/96 $15.00 ~c~ 1996 Elsevier Science B.V. All rights reserved PII S0927-7765(96)111300-8

the glass surface, we concluded that S. marcescens is endowed with a novel type of act ivi ty called "wett ing act ivi ty" [1 ] . The wett ing act ivi ty of S. marcescens was p rominen t when the bacter ia were grown at 30 C and absent when grown at 37 C. Therefore, specific c o m p o u n d s of S. marcescens which are p roduced at 30 C but not at 3 7 C were surveyed. On a th in- layer ch roma tog - raphy (TLC) plate, 2 3 3 0 C culture-specific spots were identified. One spot was red-colored and identified as prodig ios in (water- insoluble p igment of S. marcescens), the o ther spots were unknown exolipids. As S, marcescens strains showed wett ing act ivi ty irrespective of p igment p roduc t ion , these exol ipids were isolated and examined for wett ing

208 T. Matsuyama, Y. Nakagawa/ColloMs Surjaces B: BiointerJaces 7 (1996) 20Z~214

activity. Surface activities of the isolated exolipids were shown to be strong enough to explain the wetting activity of these bacteria on surfaces of glass and polystyrene [ 1,2].

We thought that wetting activity was an impor- tant property of bacteria and therefore examined many bacterial strains for this activity. Three different wetting agents were identified from different strains of S. marcescens and denoted serra- wettin W1, W2 and W3 [3]. Each S. marcescens

strain produces only one type of serrawettin. Serratia rubidaea is another species showing strong wetting activity. Two different wetting agents, denoted rubiwettin R1 and RG1, were the products from a single S. rubidaea strain [4]. The chemical structures of these wetting agents were investigated. Serrawettin W1 is a cyclic lipopeptide with the chemical formula cyclo(D-3-hydroxydecanoyl-L- seryl)2 [1] and is identical to serratamolide which was reported previously as an antibiotic [5,6]. Serrawettin W2 and W3 are also cyclic lipopeptides containing five amino acids and give protonated molecular ions at m/z 732 and 684, respectively in positive secondary ion mass spectra. Recently we elucidated the chemical structure of serrawettin W2 completely [7,8]. The main component of serrawettin W2 is D-3-hydroxydecanoyl-D-leucyl- L-seryl-L-threonyl-D-phenylalanyl-L-isoleucyl lac- tone. The chemical structures of rubiwettin R1 and RG1 have also been clarified [4,9]. Rubiwettin R1 is a mixture of linked 3-hydroxy fatty acids [D-3-(D-3'-hydroxytetradecanoyloxy) decanoate, D- 3-(3'-hydroxyhexadecenoyloxy} decanoate, and minor molecular isomers]. The main component of rubiwettin RG 1 is shown to be fi-D-glucopyrano- syl D-3-(D-3'-hydroxytetradecanoyloxy) decanoate. Bacillus subtilis and Pseudomonas aeruginosa,

which are well-known for the production of sur- face-active exolipid surfactin and rhamnolipid respectively, also demonstrated wetting activity [10].

The extracellular production of serrawettins and rubiwettins was confirmed by chemical and electron-microscopic examination of cellular and cell-free fractions [2,4]. Since mutants defective in the production of these exolipids were unable to show wetting activity, these lipids were identified as actual wetting agents of these bacteria [ 3,7,11 ].

For analyses of the physiological functions of these bacterial wetting agents, many mutants defective in the production of these wetting agents were isolated [3,11,12]. Differences clarified between wild types and mutants indicated the importance of wetting agents in the bacterial world in surface environments. Herein, we describe how small organisms, such as bacteria exist by overcom- ing intermolecular forces and how wetting agents are useful in such a micro-scale world.

2. Materials and Methods

2.1. Bacteria and wet t ing agents

Bacterial strains and growth conditions were described previously [ 1,2,13]. Wetting agents and the producing bacterial strains are listed in Table 1. Serrawettins were prepared as described previously [7] . Rhamnolipid was a gift from Y. Ishigami (National Institute of Materials and Chemical Research, Tsukuba, Japan).

Table 1 Bacterial wetting agents and producing strains

Wetting agent Surface Bacterial strain Ref. activity a

Serrawettin Wl 32.2 Serratia marcescens [2] ATCC 13880, NS 38 ATCC 274

Serrawettin W2 3 3 . 9 Serratia marcescens [2] NS 25

Serrawettin W3 2 8 . 8 Serratia marcescens [2] NS 45, NS 50

Rubiwettin RI 25.5 Serratia rubidaea [4] ATCC 27593

Rubiwettin RG1 2 5 . 8 Serratia rubidaea [4] ATCC 27593

Rhamnolipid 28.0 Pseudomonas aeruginosa [-27] ATCC 27853, BOP 100

Surfactin 27.0 Bacillus subtilis [28] ATCC 21331, OG-01

" Lowest surface tension (mN m ~) reported in the indicated reference.

T. Matsuyama, Y. Nakagawa/Colloi& Surl~tccs B." BiointetTl~we,s 7 i' 1996) 207 214 209

2.2. Mutant isolation 3. Results and discussion

S. marcescens mutants defective in the pro- duction of wetting agents were isolated after muta- genesis with UV, nitrosoguanidine, or transposon mini-Mu lac, kan. Defective production of the specific wetting agents was examined by direct colony TLC [11,12].

2.3. Assay O/wetting activity

A bacterial suspension (1 mg [dry weight] per l ml of saline) was examined for wetting activity at 30 :'C. In the canvas disk method, the time taken for a cotton disk (diameter, 30mm; previously dried on a hot block at 120':C for 1 h) dipped in a suspension to sink was determined. In the absorp- tion method, the length of the wetted area of a suspended cotton strip ( 10 mm width) maintaining contact with the suspension at its lower end for 5 min was measured.

2.4. Fractal analysis

Photographs of colonies were analyzed with a charge-coupled device (CCD) camera (Ikegami, Tokyo, Japan) and a digital image analyzer (EXCEL: Nippon Avionics, Tokyo} using a per- sonal computer. Fractal analysis was carried out by box-counting methods as described previously [13]. In an alternative conventional method, images taken by a Photo CD system (Kodak, Tokyo) were analyzed by Public Domain Software NIH lmageFractal (version 1.2}.

3.1. Surface accessibility of bacteria

in nature, most bacteria prefer to live on the surfaces of biotic or non-biotic objects [14]. Delivery of bacteria to these surfaces is mostly dependent on water, which is suitable as a carrier. However, surfaces in environments are not always water-wettable. Especially, many living creatures have developed water-repelling outer surfaces with hydrophobic substrates and structured unevenness. Skins of animals and surfaces of plants are known to have well-designed micro-structures. We think that such non-wettability of the outer biosurfaces is some sort of bio-defense mechanism against microbial infections. To overcome the difficulty in accessing the water-repelling surfaces, some bacte- ria seem to have evolved to produce wetting agents. As shown in Fig. 1, a 5.0~1 suspension of S. marcescens wild type successfully spread over the surface of a pea pod. However, a suspension of a mutant defective in the production of serrawet- tin Wl failed to spread on the pod surface and remained as a distinct droplet.

An S. marcescens wild type and a serrawettin- less mutant were quantitatively compared for wet- ting activity on cotton. As shown in Table 2, S. marcescens NS 38 grown at 30:'C (suitable temperature for the production of serrawettin W 1 ) demonstrated strong wetting activity in assays with the canvas disk and absorption methods. S. marcescens NS 38 grown at 37 C (unsuitable temperature for the production of serrawettin Wl l

2.5. Video microscope

For the analysis of the spreading behavior of bacteria, images from the C C D camera connected to a microscope were video recorded. An objective lens with a long working distance (NCF Plan LWD DL100 x C, Nikon) was used for phase contrast microscopic tracing of bacteria in locomo- tion. Neither an immersion liquid nor a cover glass slip was placed between the lens and the bacteria.

Fig. I. Suspensions of S. marcescens on a water- repel l ing surface of a pea pod. A suspension ( 5.0 ~tl of salinel con ta in ing

a wild type NS 38 i a ) a n d a serrawett in- less mu tan t NS 38-09 (b) was placed on a pea (Cassia mmtame) pod.

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Table 2 Wetting activity of an S. marcescens wild type NS 38 and a serrawettin-less mutant NS 38 09

Method Bacteria a

NS 38 NS 38-09

Temperature ("C)

30 37 30

Canvas disk (min b) 9.1 > 60 > 60 Absorption (mm c) 11.2 < 0.5 < 0.1

" Each suspension contains 1 mg of bacterial dry weight per 1 ml of distilled water. b Time (mean of four determinations) until sinking of a canvas disk at 30"C. c Wetted area length (mean of four determinations) of a cotton strip maintaining contact at its end with the bacterial suspension for 5 min at 30~'C.

and serrawet t in- less m u t a n t NS 38 09 showed m a r k e d l y reduced wett ing act ivi ty in bo th assays.

3.2. Bacterial growth on a surface

Bacter ia mul t ip ly ing at a fixed site of a solid surface increase the to ta l popu l a t i on vo lume and form a visible mass k n o w n as a "colony". Mos t colonies first appea r as a r o u n d mass with a d iamete r of 1-2 mm. Since such colonies are large enough for conven t iona l bac te r io log ica l examina- tions, no further cu l t iva t ion has been t r ied so far. I t is, however, no t ewor thy tha t co lony growth is a representa t ive process of surface inhab i t a t ion by bac te r ia in nature. By cul t ivat ing for a longer time, we were surpr ised at the r emarkab le divers i ty in the morpho log ie s of wel l -grown colonies. Each bacter ia l species seemed to have a specific s t ra tegy for surface occupa t ion fol lowing cell mul t ip l i ca t ion [13,15] . Thus, S. marcescens po in t - inocu la t ed at the center of an agar m e d i u m deve loped a charac- terist ic g iant co lony after more than 1 week of cu l t iva t ion at 30°C (Fig. 2A). In contras t , a serra- wett in-less m u t a n t of the same bacter ia fo rmed a s imple r o u n d co lony (Fig. 2B). Since an external supply of a purif ied ser rawet t in enabled the charac- terist ic sp read ing g rowth of serrawett in- less m u t an t s (Fig. 3), the cri t ical role of the ser rawet t in in the co lony g rowth of S. marcescens is evident.

Fig. 2. Colonies of S. marcescens NS 38 (A) and serrawettin- less mutant NS 38 09 (B) on a hard agar medium (Vogel Bonner, 1.5% agar). The bacteria were point-inoculated at the center of the plate (diameter, 85 ram) and cultivated at 30 C for 12 days.

Wild types of S. rubidaea, P. aeruginosa and B. subtilis which p roduce species-specific wett ing agents also deve loped a giant co lony with a f jord- l ike out l ine (data not shown).

3.3. Fractal colony growth and underlying principles

The giant colonies made by S. marcescens and o ther species of bacter ia are composed of r a n d o m

T. Malsu),ama, Y. Naka#awalColloids Sur/itces B." Biointerlaces 7,1996) 207 214 211

Fig. 3• Effects of serrawettin W2 on the spreading growth of scrrawettin-less mutants NS 25 03. Sterile paper disks soaked with 5 ~tl of ethanol containing 100 lag of serrawettin W2 (right) or no serrawenin W2 (control, left} were dried and placed next to the mutanl which was point-inoculated on Vogel Bonner medium. The plate was incubated at 30 C for 2 weeks•

branching patterns. These patterns, however, seemed to have a common feature, i .e. self- similarity or scale invariance, a well-known charac- teristic feature of self-similar fractals [16]. Therefore the fractal nature of these bacterial colo- nies was exainined by the box-counting method. The number N(p) of boxes (with a pixel size, p) covering a colony pattern was determined for each covering. If the pattern really has a self-similar property, a log log plot will give a straight line indicating an N(p)~p ~ relationship. The Dr value obtained from the slope of the regression line indicates the fractal dimension of the pattern. For example, the fractal nature of the colony pattern shown in Fig, 2A was ascertained by using a compttter analysis program NIH ImageFractal [Fig. 4). From fractal analyses of many giant colo-

N(p)

104 Df = 1.79

1000

100

10

q

10 100 P

Fig. 4. Fraclal analysis of a colon), (shown in Fig. 3A) by the box-counting inet hod.

nies, Dr values of most colony patterns were deter- mined to be in the range 1.6 1.8 [10,17,18].

Fractal colony growth performed by living bac- teria is a morphogenic process observable in a Petri dish under controllable conditions. Therefore, bacterial colony growth is identified as an appro- priate experimental system for the analysis of basic principles of fractal morphogenesis. Self-similar fractal patterns made by bacteria are similar to familiar patterns in nature (e.g. patterns on a dendrite} [ 19], and are quite similar to the pattern produced by a computer simulation adopting the two-dimensional diffusion-limited aggregation (DLAt model proposed by Witten and Sander [20]. The Dr value of the DLA pattern is 1.71, and close to the Dr values of colony fractals. Actually, diffusion of nutrients in the two-dimensional agar field has been shown to be critical in the fractal morphogenesis by bacteria [21]. In addition, two characteristic phenomena, repulsion and screening effects, have been predicted to occur froln computer simulations of the DLA model [22]. We have experimentally shown that the two effects really occur during fractal colony growth [ 18,21]. Thus. as a result of our studies, the morphogenic mecha- nisms of fractal colony growth was revealed to be a process dependent on physicochemical principles rather than genetic blueprints, it is also intriguing that a theoretically predicted morphogenic process (DLA m o d e l ) i s shown to be really occurring in nature.

The effect of wetting agents on the fractal growth of a bacterial colony was then examined. Since bacterial cells are always associated with water. surface tension was expected to function as the limiting force acting against branch extension at the growing front of the colony. Such a limiting factor is not taken into account in the original DLA model. In a modified DLA model proposed by Vicsek [23], a surface tension parameter was introduced as a curwiture-dependent sticking prob- ability, P~(k) [Ps(k) = Ak + B, where k is the local surface curwtture, and A and B are constants]. By increasing the value of A, a computer simulation gives a pattern similar to the pattern made by' mutants defective in the production of wetting agents (Fig. 5). Thus, the effects of surface tension

212 T. Matsuyama, Y. Nakagawa/Colloids Surfaces B: Biointerjitces 7 (1996) 20~214

(a) (b)

Fig. 5. Computer simulation of a DLA model with a surface tension parameter which was introduced as a curvature (k)- dependent sticking probability [Ps(k) - Ak + B]: (a) A = 2 , B = 0.5; (b) A - 1 2 , B=0.5.

and the role of wetting agents in bacterial colony growth are brought to light in this simulation.

3.4. Surface locomotion of bacteria

Most bacteria having peritrichous flagella are able to swim in liquid and swarm on a surface. Both S. marcescens and B. subtilis are flagellated and belong to such locomotive bacteria. For sur- face locomotion (swarming), however, the surface must be under semi-solid condition (0.9-0.5% agar concentration). On a hard agar medium containing more than 1.0% agar, these flagellated bacteria, with some exceptions (e.g. Bacillus alvei), are not able to move. When the agar concentration is lower than 0.5%, the bacteria do not swarm on the surface but instead they swim through water channels in the agar medium. The effects of wetting agents on bacterial locomotion were recognized only on the semi-solid surface.

The effects of wetting agent on bacterial surface locomotion can be seen in Figs. 6 and 7. After point inoculation of the bacteria onto the central surface of the agar medium, the flagellated bacteria (wild types and mutants) swarm over the surface in a short time (mostly in a day or two). However, swarming behavior varies between wild types and mutants. In contrast to the thin well-branched spreading pattern of the wild types (Figs. 6A and 7A), mutants defective in the production of serra- wettin or surfactin spread over the surface rather

Fig. 6. Swarming colonies of S. marcescens NS 38 (At and serrawettin-less mutant NS 39 09 (B). Bacteria were point- inoculated onto the center of semi-solid nutrient agar {0.5% agar) and cultivated at 30 'C for 12 h.

slowly and form thick branches with a remarkable tendency to bend to the right (Figs. 6B and 7B). Since the purified wetting agent exerts the same effect on the swarming of the mutants irrespective of the bacterial species (effect of rhamnolipid from P. aeruginosa on surfactin-less B. subtilis is repre- sentatively shown in Fig. 8), the effect is directly attributable to the surface activity of the bacterial wetting agent.

T. MatmLvama, Y. Nakagawa/Colloid~ Sur/aces B: BiomtelT/aces 7 ~ 1996) 207 214 213

Fig. 7. Swarming colonies of B. subtilis ATCC 21331 (A) and surfactin-less mutant 21331-b3 (B). Bacteria were point- inoculated onto the center of semi-solid nutrient agar and cultivated at 37 (" for 24 h.

Fig. 8. Effect of rhamnolipid on swarming of surfactin-less B. suhtilis 21331-b3. A paper disk containing 100Bg of rhamnolipid was placed next to the bacteria which had been point-inoculated onto the semi-solid nutrient agar and incu- bated at 37 C for 12h.

3.5. S m J h c e bac ter ia in coopera t ion

Analyses of v ideo- recorded microscopic figures revealed the dynamic processes of bacter ia l beha- vior on surfaces. As shown in Fig. 9A, the t ip of the extending thin branch is encased by diffusely d is t r ibu ted B. subt i l i s cells which seem to be reluc- tant to move by themselves but are easily pushed to the outs ide by the active mot ion of inner cells (b lu r red images in Fig. 9A). In contrast , the grow- ing front of the branch made by surfactin-less B. . suh t i l i s seems to be even and is c rowded with many cells. Marg ina l cells are also inactive and form a t ight ou te r shell. Just behind this immobi le cell layer, act ively swirling cells are recognizable as b lurred images (Fig. 9B). Cells loca ted in the

Fig. 9. Photomicrographs of the end of a swarming colony o1 B. suhtilis ATCC 21331 (A) and surfactin-less mutant 21331 -b3 (B). The snapshot photographs were taken with a 1:4 s shutter speed. The extending cell population is composed of inactive marginal cells Iclear images) and actively swirling inner cells ( blurred imagesL The bars indicate 5 ~tm.

marg ina l shell seemed to be pushed by these active inner cells and the extension of the branch seems to be dependent on such pressure- l ike popu la t ion forces. In the absence of bacter ial wett ing agents. swarming bacter ia are conta ined in a large branch p resumably by the surface tension effect of water. Bacter ia are very small and are under the direct influence of in te rmolecular forces [24] . However , the surface tension of water is very strong. Consequent ly , bacter ia in tending to t rans locate freely in the surface envi ronment must overcome such forces by some means. A single bacter ium is not powerful enough to overcome these forces on

214 T. Matsuyama, Y. Nakagawa/Colloids Surfaces B: Biointerfaces 7 (1996) 20~214

surfaces. By making a cooperative population com- posed of cells exerting different roles [9,10,25,26], bacteria seem to be able to move on surfaces. Wetting agents may be useful for translocating such bacterial populations by lessening the restricting force of the surface tension of water.

Acknowledgments

We are grateful to M. Matsushita, A. Nakahara, M. Ohgiwari and J. Wakita for valuable discussions and assistance with the experiments. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (No. 07670303).

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