Isolation and characterization of a Cytophagales

Kacie DeOms, Ben Ruddle

MICRB 421W, Section 2Department of Biochemistry & Molecular Biology

The Pennsylvania State University

Abstract

The purpose of this experiment was to isolate and characterize a Cytophagales from nature.

Specifically, gliding motility and presence of a lytic enzyme were to be demonstrated. Bacterial

colonies, isolated from a soil sample, were tested to determine if they were a member of the

order Cytophagales. One colony that showed potential gliding was isolated and visualized with a

camera microscope to confirm gliding motility and cell shape. Casein agar was used to show

lytic activity of the isolate. A Gram stain and a flexirubin test were also performed on the isolate

to verify typical Cytophagales characteristics. These tests showed that a rod-shaped, Gram-

negative strain was isolated. This isolate showed gliding motility and the presence of lytic

enzymes and flexirubin-type pigments. These results indicate that a member of the order

Cytophagales was isolated.

Introduction

The purpose of this experiment was to isolate and characterize a Cytophagales species,

from nature. Evidence of gliding motility and the presence of a lytic enzyme was specifically

desired for this study.

Cytophagales bacteria have distinct characteristics. All members of the Cytophagales

order are Gram negative, rod-shaped, bacteria (3). Cytophagales are the most common gliding

bacteria, but usually do not display gliding motility on high nutrient media. Most Cytophagales

have brightly colored colonies in shades of yellow, orange, pink, or brick red (3). Many

Cytophagales (yellow and orange colonies) possess flexirubin-type pigments. This characteristic

distinguishes Cytophagales species from Lysobacter, another Gram-negative, rod-shaped, gliding

order (9). All cytophagales are organotrophs. Many are able to degrade biomacromolecules,

including proteins, chitin, pectin, starch, cellulose, DNA, and RNA (3).

Cytophagales are found in almost all aerobic and microaerobic environments. They are

ubiquitous in habitats that are rich in organic material (3). This includes soils, decaying plant

material, and animal dung (especially that of herbivores). Cytophagales can also be found in low

nutrient environments and aquatic environments (fresh and saltwater) (3).

Cytophagales species have both beneficial and undesirable functions. Cytophagales

species play a vital role in the breakdown of matter in nature, sewage treatment plants, and cattle

manure composts (10,1,6). While some minor applications of Cytophagales properties have been

utilized, no significant commercial use for Cytophagales, or their enzymes, has been identified

(3). As for adverse effects, multiple cytophagales species have been shown to cause disease in

cultivated fish populations. The most common of these diseases are columnaris, caused by Cy.

columnaris, and cold water disease, caused by Cy. psychrophilia (4). The conditions of fish

hatcheries are seen to intensify the pervasiveness of Cytophagales-linked fish diseases (11).

Cytophagales species have also been implicated in the spoilage of milk, fresh vegetables, and

fish (5,7,2).

In this study, a Cytophagales (“Isolate K1”) was isolated from a garden soil sample. The

K1 strain was isolated using VY/2 media. Presence of a lytic enzyme was shown using casein

agar. A Nikon E600 microscope was used to document gliding motility and cell morphology.

This experiment also involved a Gram stain and a flexirubin test (using 20% KOH solution) to

determine if Isolate K1 exhibited typical Cytophagales qualities.

Materials and MethodsIsolation. Isolate K1 was found in a soil sample. The soil sample used was obtained from a

residential garden. Serial dilutions of the soil sample were made. The dilutions were plated onto

VY/2 agar plates. These VY/2 agar plates were used to isolate and cultivate Isolate K1. All

inoculated media was incubated at 28° C.

Characterization. Several tests were performed on Isolate K1. A standard Gram stain was

performed on Isolate K1. Also, a flexirubin test was executed on Isolate K1. A flexirubin test

consists of applying several drops of 20% KOH solution to yellow or orange colonies. A color

change to red or brown constitutes a positive result (9).

The gliding motility of Isolate K1 was demonstrated by visualizing an agar slice on a

Nikon E600 Nomorsky phase contrast microscope. Differential interference contrast (DIC) was

used specifically. The motility of specific cells was evidenced by capturing images of the same

cells at varying time points.

Isolate K1 was streaked on casein agar plates to show the presence of a lytic enzyme.

Media. VY/2 agar was used to isolate the colony. VY/2 consists of Bakers’ yeast (0.5%), CaCl2-

2H20 (0.1%), and agar (1.5%). The pH of the media was adjusted to 7.2. The media was

autoclaved at 121°C for 20min.

Casein agar was used for the characterization of the Isolate K1. Casein agar was made

with NaCl (250%), agar (20%), MgCl2 (20%), Casein hydrolysate (7.5%), KCl (2%), and CaCl2

(0.2%). The pH of the media was adjusted to 7.2. The media was autoclaved at 121°C for 20min.

Results

Isolate K1 was obtained from a sample of garden soil. When the isolate was grown on

minimal nutrient VY/2 agar, a thin film with a slight yellow color were seen. When Isolate K1

was grown on casein agar, bright yellow colonies were seen. The Gram stain of Isolate K1

produced a Gram-negative result. The flexirubin test revealed a positive result. This positive

result was evidenced by a color change of Isolate K1, from yellow to brick red. Figure 1 shows

this color change. Figure 1a shows Isolate K1 before the 20% KOH was added. Figure 1b shows

the same colony immediately after 20% KOH was added to the bottom half of the colony.

Gliding motility of Isolate K1 was visualized and recorded. Figures 2a and 2b displays

this movement. Specific cells are outlined to draw attention to prominent movement. The cell

morphology of Isolate K1 can also be seen in Figure 2.

The presence of proteases is evidenced by Figure 1. These images show an Isolate K1

colony plated on casein agar media.

Discussion

Christensen showed that Cytophagales are always gliding, rod-shaped, Gram-negative bacteria,

with yellow, orange, or red colony color (3). Isolate K1 is rod-shaped (Figure 2), Gram-negative,

gliding (Figure 2), and has a yellow colony color (Figure 1). Christensen also wrote that

Cytophagales are found in large numbers in soil, because many Cytophagales contain lytic

enzymes. Isolate K1 was obtained from a soil sample. Isolate K1 was also shown to contain

proteases (Figure 1). Isolate K1 demonstrated all of the prescribed characteristics of a typical

Cytophagales, indicating that it is a Cytophagales itself.

Reichenbach, et al. wrote that Cytophagales give a positive flexirubin test result. If a

negative flexirubin result was obtained for a yellow, gliding, Gram-negative bacteria, it would

indicate a Lysobacter (9). Isolate K1 produced a positive result for the flexirubin test (Figure 1b).

This further indicates that Isolate K1 is a Cytophagales.

All evidence obtained in this study supports the hypothesis that a Cytophagales was

isolated as Isolate K1.

References

1. Bauer, L. 1962. Untersuchungen an Spaeromyxa xanthochlora, n. sp., einer auf Tropfkorpern vorkommenden Myxobakterienart. Arch. Hyg. Bakteriol. 146: 392-400.

2. Cho, Y., Sinano, H., Akiba, M. 1984. Studies on the microbiological ecology of mackerel stored by the method of partial freezing. Bull. Faculty Fish. (Hokkaido Univ.) 35:271-285.

3. Christensen, P.J. 1977. The history, biology, and taxonomy of the Cytophaga group. Can. J. Microbiol. 23: 1599-1653.

4. Collins, V. G. 1970. Recent studies of bacterial pathogens of freshwater fish. Water Treatm. Exam. 19:3-31.

5. Cousin, M.A. 1982. Presence and activity of psychrotrophic microorganisms in milk and dairy products: a review. J. Food Protect. 45: 172-207.

6. Godden, G., Penninckx, M. J. 1984. Identification and evolution of the cellulolytic microflora present during composting of cattle manure: On the role of actinomycetes sp. Ann. Microbiol. (Inst. Pasteur) 135B: 69-78.

7. Liao, C.H., Wells, J.M. 1986. Properties of Cytophaga johnsonae strains causing spoilage of fresh produce at food markets. Appl. Environm. Microbiol. 52: 1261-1265.

8. Reichenbach, H. 2006. The Order Cytophagales, p 549-590. In Dworkin, M (ed), The Prokaryotes, 3rd ed, New York, NY.

9. Reichenbach, H., et al. 1980. Flexirubin-type Pigments in Flavobacterium. Arch. Microbiol. 126: 291-293.

10. Ruschke, R. 1968. Die Bedeutung von Wassermyxobakterien fur den Abbau organischen Materials. Mitt. Internat. Limnol. 14:164-167.

11. Snieszko, S.F. 1974. The effects of environmental stress on outbreaks of infectious diseases of fishes. J. Fish Biol. 6: 197-208

Legends to Figures

Figure 1 a. Isolate K1 plated on casein agar, incubated at 28°C for 48hr. b. Isolate K1 plated on casein agar, incubated at 28°C for 48hr. Red/orange area shows where the flexirubin test was performed (20% KOH).

Figure 2 a. Isolate K1 imaged on a Nikon E600 camera microscope, at t=0. b. Isolate K1, imaged on a Nikon E600 camera microscope, less than 1 minute later. Agar squash prep as described in M&M.

Figure 1a

Figure 1b.

Figure 2a

Figure 2b