Virus particle production in lysogenic bacteria exposed to protozoan grazing

MICROBIOLOGY LETTERS

ELSEVIER

FEMS

Microbiology

Letters

166 (1998) 177-180

Virus particle production in lysogenic bacteria exposed to protozoan grazing Ken J. Clarke * Institute of Freshwater Ecology. Windermere Laboratory,

Fur Sawrey. Ambleside, Cumhricr LA22 OLP, IIK

Received 16 June 1998; revised 22 July 1998; accepted 22 July 1998

Abstract Electron microscopy was used to investigate the apparent induction of virus particle production in bacteria undergoing digestion by ciliates. Results showed that numbers of bacteria containing virus particles increased by a factor of 25 when enclosed within ciliate food vacuoles. It was also found that 10% of these particles survived the digestion process to be released back into the aquatic habitat within faecal pellets. The possibility of virus gene transfer occurring between lysogenically infected bacteria that survive the ciliate digestive processes, is also considered. 0 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords:

Freshwater;

Bacterium;

Virus; Ciliate; Ingestion;

Faecal pellet

1. Introduction

2. Materials and methods

There is much current interest in the occurrence and impact of viruses in the aquatic, mainly marine, environment [1,2], with high numbers of free viruslike particles (vlps) reported and their significance discussed. Interactive aspects of virus activity have been described notably on the grazing of viruses by nano-flagellates [3], and on the occurrence of bacteriophage virus particles present in marine bacteria [4]. The present paper reports the apparent induction of bacteriophage particle production in bacteria ingested by ciliates and other phagotrophic microbes in the freshwater environment.

The 3.5-m deep water-column of a small hypereutrophic lake in the English Lake District [5] was sampled regularly through each season of a year (1996) and the microbial community examined by transmission electron microscopy (TEM). The August 1996 samples collected from a depth of 1.5 m, within the oxycline, revealed a high level of virus activity, and aquatic free-virus numbers approaching log vlps ml-’ were recorded. To study the physical inter-relationships between the microorganisms and viruses present at this depth, 20-ml water samples were collected, concentrated, fixed with glutaraldehyde and osmium tetroxide and embedded and sectioned for TEM examination [6]. Observations were made from ultra-thin sections < 60 nm thick, recording all cells and their associa-

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Fax:

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0378.1097/98 /$19.00 0 1998 Federation PII: SO378-1097(98)00328-O

of European

Microbiological

Societies. Published by Elsevier Science B.V. All rights reserced

tions in 160 mm” of sectioned sample, and analysed to determine and compare the percentage population of each relevant microbial inhabitant. The sections were examined in a JEOL 1OOCX TEMSCAN electron microscope.

tragellate contained 54 loosely concentrated, morphologically identical virus particles within its remains. Of 6 14 ( IOO%) free-living, non-ingested bacteria examined. two (0.3%) contained the discrete particles of an infecting virus in lytic phase. However. 30 of 42 Euplotrs cells examined contained one or two food vacuoles, 32 in total, and 19 (60%) of these vacuoles contained groups of virus particles. In 15 of these 19 food vacuoles the virus particles were contained within partially digested but still recognisable bacteria (Fig. 1). In the remaining four of the 19, the food vacuoles were packed with morphologically identical virus particles amongst lysed bacterial remains (Fig. 2). A total of 107 virus particles were counted within Euphtes food vacuoles. A further 246 partially-digested, but virus-free, bacteria were also recorded from within the vacuoles; the bacteria that contained virus particles representing roughly 6% of all recognisable bacteria enclosed within the food vacuoles. No Euplotrs cell examined showed virus particles within its organelles or cytoplasm. In cells of a Rlzodomona.~ species, also present in the examined sample, two out of 12 cells contained

3. Results TEM revealed a frequent concentration of vlps within the food vacuoles of certain ciliates and heterotrophic flagellates. It also appeared that recognisable faecal pellets expelled from the ciliated protozoa contained vlps, although at a lower concentration than observed in ciliate food vacuoles. In wholewater samples. bacteria were present in concentrations greater than 10” ml .’ and ciliates in densities greater than 200 ml ‘. of which those in the genus Euphtes predominated. The investigation focussed on this ciliate, its bacterial food and its associated free virus particles. The survey of sectioned material recorded 12 morphologically diverse free vlps (255200 nm in size) in 80 sections examined, although one ruptured dino-

Fig.

I.

Ciliate/virus

associations.

Virus

particles

within

a bacterium.

virus

particles

within

the food

ingested

withm

the

food

vacuole

Bar = 2.50 nm.

Fig.

2. Morphologically

Fig.

4. A

faaecal pellet

identical

of Eu/J/o~s\ containing

a digestwn-resistant

vacuok

of k.@/~/e.\.

bacterium.

Bar = 2.50 nm.

Bar = 250

nm

of the

phagotrophic

ciliate

Eupb~cw

K. J. Clarke/ FEMS Microbiology

recognisable bacteria, and these possessed virus particles. Although bacteria containing virus particles accounted for only 6% of the recognisable bacterial population in Euplotes food vacuoles, this was some 25 x higher than in bacteria living free within the surrounding water. Sections through a number of ciliates showed waste food material in the process of expulsion as faecal pellets from the cell. Within 80 faecal pellets examined (1.5-3 urn in diameter), 69 apparently undamaged bacteria and 41 ‘free’ virus particles were recorded (Figs. 3 and 4). In most cases these were well embedded in the matrix of the pellet and had apparently survived the ingestive and digestive processes of the ciliate. None of the surviving bacteria contained virus particles and no pellet showed more than three bacteria or virus particles present in any one section.

4. Discussion Bacteriophages can replicate in two ways, lytically and lysogenically. In the lytic process, a free phage contacting a potential bacterial host infects it, and causes it to replicate the virus. The cell wall of the bacterium then lyses releasing a burst of the infective virus particles into the aquatic environment. In the lysogenic process, an infected bacterium can carry the virus encoded as part of its own genome from generation to generation until a specific environmental or physiological condition affecting the bacterium triggers virus particle production within the cell. Subsequent bacterial lysis releases the infective virus particles into the water. A study of the infection of bacteria by bacteriophages in the marine environment [4], estimated that at any time, up to 27% of rods, 79% of cocci and 100% of spirillae are infected by viruses. It is probable, therefore, that a high percentage of the bacteria surveyed in the present study were infected by lysogenie viruses. It appears that when these infected bacteria are ingested by a phagotroph such as Euplotes, the digestive process triggers particle production in the encoded virus, enhancing its chance of surviving otherwise certain destruction as the bacterium and its virus-containing genome are digested.

Letters 166 (1998) 177-180

179

Fig. 5. Flow diagram showing the course of virus infection, particle production and re-infection, vectored through the ciliate Euplates and its bacteria1 food. A: Lysogenic infection of a bacterium by a single virus particle. B: The infected bacterium divides through several generations. C: The infected bacterium is ingested by a ciliate. D: The infected bacterium enters the ciliate’s food vacuole. E: The virus within the ingested bacterium enters lytic phase triggered by the stresses of digestion. F: The bacterium and most of the virus particles are digested by the ciliate. G: The surviving virus particles are expelled from the ciliate within a faecal pellet. H: The pellet disintegrates, releasing the virus particles to re-infect.

Even so, the ultimate fate of the majority of these virus particles generated within the ciliate’s food vacuole appears to be digestion, with roughly 10% surviving to appear in faecal pellets (Fig. 5). Viability of these survivors has not been tested and may have been impaired whilst progressing through the ciliate’s digestive processes. The particles’ eventual release as free viruses is dependent on the disintegration of the pellet. None of the bacteria in ciliate faecal pellets showed signs of ultrastructural damage or of virus particle production. It is possibile that these cells were not infected with virus, but it is most likely that a majority contained an encoded bacteriophage. If the bacteria were resistant to the digestive processes in the ciliate, and were therefore not submitted to stress, virus particle production would not be triggered in the encoded virus. Non-digested bacteria in ciliate faecal pellets have

also been reported elsewhere, but especially by Schlimme and co-workers [7] who showed that these bacteria did not survive re-ingestion. Certain resistant bacteria, such as species of Legionrlb, are not affected by digestion in amoebae at normal growth temperatures [S]. and will continue to reproduce within the food vacuoles of the cells. However. at lower temperatures these bacteria are digested 01 ejected from the feeding amoebae. These workers proposed that bacteria such as Lrgiotwllu. Listwb and Vibr-io species have evolved to survive ingestion and the digestive processes of protozoan grazers. It has also been suggested [9] that resistance to digestion by predatory protozoa is an evolved survival mechanism. None of these workers report the presence of virus particles within the ingested bacteria 01 within the food vacuoles or faecal pellets associated with phagotrophic species. It remains to be demonstrated whether lysogenically infected bacteria, resistant to digestion at ambient temperatures, will be lytitally triggered if they arc ingested at lower temperatures. Further evidence of the apparent induction of lytic phase in a virus within an ingested lysogenic bacterium is provided by four Euplotcs cells where food vacuoles were found to be packed with virus particles of one morphotype. It is unlikely. in view of the morphological diversity of free viruses present in the immediate environment, that the ciliate had packed its food vacuole with identical virus particles. by normal feeding. Either the virus-infected bacterial food was induced into rapid particle production bethe particles then filling fore the bacterium lysed the food vacuole ~~or lytic phase had been induced in material leaking and filling the vacuole from an infected bacterium previously lysed by the ciliate’s digestive process. Although the figure of 10% for ultimate survival ot virus particles originally generated within the food vacuoles of a micro-phagotroph. might appear small in terms of absolute numbers, it represents an extraordinarily large number of virus particles. It also underlines the complex nature of the mechanisms by which viruses can interact with microorganisms to enhance their chances of survival in extreme adverse conditions. A recent report [7] demonstrated the process of conjugational gene transfer between

bacteria within the food vacuoles and fdecal pellets of ciliated protozoa. It follows from the present report that viruses existing lysogenically within ingested bacteria should also be recognised as a component of the genetic material transferred during the same process. The report [7] recognised the implications of this transfer process for biosafety: its importance is stressed still further by the results of the present study.

Acknowledgments

This work was supported by the Centre for Ecology and Hydrology (Integrating Fund), Natural Environment Research Council, UK. The author would like to thank Dr. Bland J. Finlay, a collegue at IFE. Windermere, for reading the manuscript and providing helpful comment.

References Bcrgh.

0..

(1989)

High

Borsheim.

men&.

Nature

Suttle.

C.A..

of

340.

A.M. by

Nature

Go~vilu. on

gestion.

and

viruses

Mar.

Weinbauer.

M.G. of

morphotypes. Finlay. 209

and

Cottrell, and

and

Heldal,

in aquatic

M.T.

M.

envirotl-

(1990)

reduction

CA.

(1993)

virus-sized

Prog.

Ser.

of

InfectIon

primary

Peduzi.

Ecol.

pro-

in

and

by

nanotla-

ingestion

and

di-

I IO.

P. (19Y4)

Prog.

S.C.

Grazing

particles:

94.

bacteriophages

Maberly.

Frequency,

different

Ser.

108,

I I

Cooper,

size and

marine

J.I.

bacterial

20. (1997)

Oikos

LlorCns,

H.

80.

213.

Clarke.

K.J..

Miracle.

M.R.

prokaryote

Finlaq.

W.,

King,

Marchiani. transfer

J. and

microbial gen>

Microbial.

of protozoa.

Barker.

world:

(19X8)

Surwval

protozoa 3033.

during

M..

FEMS

Brown,

of

E.B.

and

coliforms

chlorination.

K.

and

23.

Trojan

the survival

R.E.

bacterial

Appl.

Jenni.

digestive

Environ.

1253 and

B. \a-

239-247. horses

of bacterial

140.

Wooley,

C/~wvw

and

within Ecol.

(1994)

Muobiology Jr..

bacterium 505.

bacteria

Microbial.

and

of a polymorphic

Hanselmann.

M.R.W.

protozoa

Shotts.

E..

life-cycle

159. 498

between

in the environment. C.H.,

Vicente,

complex

of the photosynthetic

Arch.

Gent

cuoles

The

epibiont

Schlimme. (1997)

B.J..

(1993)

riurr~ wiwci.

3023

and

Suttle.

Mar.

B.J..

G.

found

467~469.

and

Ecol.

distribution

viruses

viruses

347.

J.M.

gellates

Bratbak.

of

467 -468.

Ghan.

phytoplankton

ductivity.

K.\i..

abundance

of

the

patho-

1259. Porter,

pathogens Microbial.

K.G. within 54.