FEMS Microbiology Ecology 48 (2004) 199–207

www.fems-microbiology.org

The use of PCR for the identification and characterisationof bacteriocin genes from bacterial strains isolated from rumen

or caecal contents of cattle and sheep

Adrian L. Cookson *,1, Samantha J. Noel, William J. Kelly, Graeme T. Attwood

Rumen Microbial Functional Genomics, Nutrition and Behaviour Group, AgResearch, Grasslands Research Centre, Tennent Drive,

Palmerston North, New Zealand

Received 16 July 2003; received in revised form 3 November 2003; accepted 16 January 2004

First published online 13 February 2004

Abstract

PCR primers were designed to amplify the gene that encodes bovicin 255 from Streptococcus gallolyticus LRC0255 and the

bacteriocin genes from Butyrivibrio fibrisolvens strains AR10 and OR79A (bviD and bvi79A) in order to screen for their incidence in

rumen and caecal B. fibrisolvens and Streptococcus bovis-like isolates from New Zealand and North American ruminants. None of

the B. fibrisolvens-like strains ðn ¼ 34Þ isolated from New Zealand or North America had the genes encoding for butyrivibriocins

AR10 (bviD) or OR79 (bvi79A). However, seven S. bovis isolates from New Zealand ruminants and three from North American

animals had the bovicin 255 gene. Sequence comparison of cloned bovicin 255 PCR products indicated a 92.9–95.7% similarity to

that of the corresponding bovicin 255 gene sequence of S. gallolyticus. Four of the New Zealand bovicin 255 positive S. bovis isolates

were from the caecal contents of the same sheep and had identical PFGE profiles. Two other S. bovis isolates sharing the same

PFGE profile were isolated from a separate animal from the same flock. PFGE analysis of the North American strains indicated

that all three were closely related as two of three had identical PFGE profiles with the remaining isolate differing only by a single

band position. The 16S rRNA gene sequences of the 10 isolates were at least 99.8% identical to S. bovis. All 10 S. bovis isolates

having the gene for bovicin 255 produced bacteriocin activity that inhibited the growth of Peptostreptococcus anaerobius D1 in a

deferred antagonism plating (DAP) assay. Certain S. bovis isolates obtained from ruminants have bacteriocin activity associated

with a distinct bovicin 255 gene sequence but it appears that bacteriocin production by the rumen anaerobe B. fibrisolvens may be

uncommon in strains isolated from cattle and sheep in New Zealand.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Bacteriocin; Streptococcus bovis; Butyrivibrio fibrisolvens; Bovicin 255; Rumen; Caecum

1. Introduction

The ruminant ecosystem is a complex and diverse

environment where symbiotic bacteria, fungi and pro-tozoa interact with the host in a dynamic relationship to

degrade plant material by anaerobic fermentation [1].

* Corresponding author. Tel.: +64-6-351-8229; fax: +64-6-351-8003.

E-mail address: adrian.cookson@agresearch.co.nz (A.L. Cookson).1 Postal address: Rumen Microbial Functional Genomics, Nutrition

and Behaviour Group, AgResearch, Grasslands Research Centre,

Tennent Drive, Private Bag 11008, Palmerston North, New Zealand.

0168-6496/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsec.2004.01.003

The animal provides the microorganisms with a habitat

for their growth whilst the microorganisms provide the

animal with fermentation acids, microbial protein and

vitamins.The microbial environment within the rumen may

be regulated, in part, by the expression of proteina-

ceous antimicrobial agents such as bacteriocins. These

molecules are a heterogeneous group of peptides and

proteins that are characterised by their ability to inhibit

closely and sometimes more distantly related strains of

bacteria [2–5], thereby potentially playing a key role

in the bacterial population dynamics within the rumen

. Published by Elsevier B.V. All rights reserved.

200 A.L. Cookson et al. / FEMS Microbiology Ecology 48 (2004) 199–207

[6–8]. In particular, where bacteria are attached to feed

particles, the expression of a cell-associated bacteriocin

or the secretion of active molecules into a biofilm

matrix of adherent cells may enhance the chances of

survival, colonisation and retention of the producerorganism by inhibiting sensitive bacteria that compete

for the same resources. If a particular bacterial strain

or species cannot compete effectively with others in a

narrowly defined and exclusive niche, the rate of cell

division may not be sufficient to prevent dilution and

the complete loss from the rumen environment.

Therefore, the diversity and density of the microbial

population may favour the evolution of various in-hibitory molecules, such as bacteriocins, as competitive

factors within the rumen environment. The expression

of bacteriocin molecules may not be confined to a

particular phylogenetic group of bacterial strains and

even closely related isolates may differ in their ability to

produce bacteriocins [8].

Several commonly isolated species of rumen bacte-

ria, such as Streptococcus gallolyticus [9], Streptococcusbovis [10], Butyrivibrio fibrisolvens [6,7,11], Rumino-

coccus albus [12] and Enterococcus faecium [13–16],

produce bacteriocins, but their effect on rumen ecology

has not been clearly defined. S. bovis is a rapidly

growing, acid-tolerant, gram-positive bacterium that

ferments starch to lactic acid. Excessive growth of this

species may lead to lactic acidosis in the rumen of

cattle and sheep fed excess starch [17]. Two inhibitorymolecules, bovicin 255 [9] and bovicin HC5 [10], have

been identified from rumen Streptococcus species. Bo-

vicin 255, isolated from Streptococcus gallolyticus

LRC0255, has an active peptide of 5.97 kDa processed

from a 8.5 kDa prepeptide that contains a protease

processing site [9]. It shares homology with class II

non-lantibiotic bacteriocins such as lactococcin A of

Lactococcus lactis [18]. More recently, the bovicinHC5, a bacteriocin isolated from S. bovis HC5, has

been shown to have a broader antibacterial spectrum

than bovicin 255 [10,19]. Initial analysis indicated that

the active peptide was 2.44 kDa [10] and that the only

other bacteriocin that had significant similarity was the

type A1 lantibiotic streptin (srtA) of Streptococcus

pyogenes [20].

Butyrivibrio fibrisolvens is a frequently isolatedmember of the obligately anaerobic bacterial popula-

tion of the rumen and is important for fibre digestion

with most strains degrading hemicellulose, xylans,

pectin and starch [17]. Several studies indicate that

bacteriocin-like activity is a common characteristic of

B. fibrisolvens [6,7] and inhibitory molecules from B.

fibrisolvens AR10 [7,21] and OR79 [11,22] have been

purified and characterised. The inhibitors identifiedwere small hydrophobic peptides similar to a lantibiotic

(butyrivibriocin OR79A) and non-lantibiotic peptide

(butyrivibriocin AR10) [7,11,21]. The structural gene

encoding butyrivibriocin OR79 (bvi79A) produced a

prepeptide of 47 amino acids and a mature peptide,

butyrivibriocin OR79A, of 25 amino acids [11]. The

prepeptide had significant homology to other lantibi-

otics which contain a double glycine leader peptidasecleavage site, such as variacin from Micrococcus vari-

ans [23] and lacticin 481 from L. lactis [24]. The active

form of butyrivibriocin AR10 was determined to be

approximately 3.9 kDa [7]. This inhibitor molecule

demonstrated significant homology to the bacteriocin

acidocin B from Lactobacillus acidophilus [25]. Both

these butyrivibriocins were able to inhibit a variety of

gram-positive ruminal bacteria including Ruminococcus

spp., S. bovis, other B. fibrisolvens and listerial food

isolates [6,7,11,21].

In this study we used the DNA sequences of the genes

encoding butyrivibriocins OR79A and AR10 and bovi-

cin 255 to design primers for PCR screening of rumen

S. bovis, B. fibrisolvens, or other similar bacterial isolates

from cattle and sheep in New Zealand [26] for the

presence of butyrivibriocins OR79A and AR10 andbovicin 255.

2. Materials and methods

2.1. Bacterial strains and media

Streptococcus gallolyticus LRC0255 was originallyisolated from the rumen of a moose in Alberta, Can-

ada [9] B. fibrisolvens OR79 was originally isolated

from the rumen of a dairy cow [11] and B. fibrisolvens

AR10 was originally isolated from the rumen of a

sheep in Australia [7]. All three strains were kindly

supplied by R.M. Teather (Lethbridge Research Cen-

tre, Alberta, Canada). Peptostreptococcus anaerobius

D1 [27] was obtained from the Rumen MicrobiologyCulture Collection at the AgResearch Grasslands Re-

search Centre, Palmerston North, New Zealand. CC,

L10 and RX media were prepared as described previ-

ously [28–30]. HAP medium was the same as the basal

medium of Chen and Russell [31] with minor modifi-

cations [27]. Purified agar (1.5% or 0.75% [w/v]; Difco)

was used to make agar plates or top agar overlays

respectively.Rumen samples were collected from fistulated ani-

mals as described previously [27], or from the caecal

contents of animals post mortem. One millilitre of

sample was diluted anaerobically in RX medium

(without xylose), serially diluted in both CC and RX

broths and incubated at 39 �C in an anaerobic glove box

(90% CO2, 10% H2). Individual colonies were purified

by repeated streaking onto fresh agar plates and colonypurity was confirmed by Gram stain and microscopic

examination. Presumptive identification of isolates as

B. fibrisolvens or S. bovis was as described previously

A.L. Cookson et al. / FEMS Microbiology Ecology 48 (2004) 199–207 201

[26,32]. Bacterial strains were stored at )85 �C on CC or

L10 agar slopes.

2.2. PCR and sequencing of bacteriocin genes

A single well-spaced colony of the strain to be

tested was resuspended in 50 ll sterile water. Template

DNA was prepared from this sample by placing in a

vigorously boiling water bath for 5 min and placing

on ice until required. PCR primers (Table 1) were

diluted to a final concentration of 5 pM ll�1. The

PCR conditions were an initial denaturation step

(94 �C, 5 min) followed by 30 cycles of denaturation(94 �C, 30 s), annealing (52 �C, 30 s) and extension

(72 �C, 30 s) in a FTS-320 Thermal Sequencer

(Corbett Research) using 23.5 ll Platinum PCR Su-

permix (Invitrogen), 0.5 ll each of forward and re-

verse primers and 0.5 ll of template DNA. After

terminal extension (72 �C, 5 min), samples were kept

at 4 �C. The PCR products were analysed on agarose

gels (1.5%) after staining with ethidium bromide orGel-Star (Bio-Whittaker Molecular Applications,

Rockland, ME, USA).

PCR products were cloned into the pCR2.1 vector

(Invitrogen, Auckland, New Zealand) according to

manufacturer’s instructions and sequenced using the

BigDye Terminator v.3.1 Cycle Sequencing kit (Applied

Biosystems), using M13 Forward ()20) and M13 Re-

verse primers. Two clones were sequenced for each iso-late and aligned with the bovicin 255 gene sequence

from S. gallolyticus LRC0255 retrieved from GenBank,

using the AlignX programme (Vector NTI, InforMax

Inc., MD, USA). The DNA sequences of the bovicin 255

PCR products have been deposited in the GenBank data

library under Accession Nos. AY338260 to AY338269.

2.3. Phylogenetic analysis

Eubacterial primers fD1 and rD1 (5 pM ll�1) [33]

were used to amplify the 16S rRNA genes from bovicin

Table 1

Characteristics of the oligonucleotide sequences used for PCR and sequence

Primer Sequence Am

ALC1 50-AAC AAA TTA TGA GTA ACT GTA-30 bviD

ALC2 50-AAG CTG CAG CTG CTT CTG CTA-30 But

ALC3 50-TTG AAC AAT TTG ATG TAA TGA-30 Bov

ALC4 50-AAT ATC CTC CAC CAA GAG ACG-30

ALC5 50-ATG AAC AAA GAW CTT AAT GCA-30 bvi7

ALC6 50-TAA GAR CAR CAT GTG AAR ATG-30 But

fD1 50-GAG TTT GAT CAT GGC TCA G-30 Eub

rD1 50-AAG GAG GTG ATC CAA CCG-30

F1 50-CTC CTA CGG GAG GCA GCA G-30 Seq

R5 50-GGG TTG CGC TCG TTG-30 Seq

255 PCR positive S. bovis-like isolates (Table 1). The

PCR conditions were 30 s at 94 �C, 30 s at 57 �C and 1

min at 72 �C for 30 cycles with template preparation,

initial denaturation and final extension steps as de-

scribed above. PCR products were cloned and se-quenced as described above using fD1, rD1, F1 and R5

primers and aligned with other related 16S rRNA gene

sequences retrieved from GenBank.

2.4. Pulsed-field gel electrophoresis

The procedure used to prepare genomic DNA in

agarose blocks for pulsed-field gel electrophoresis hasbeen described previously [34]. DNA embedded in aga-

rose was digested with SmaI (New England Biolabs,

Beverly, MA), loaded into the wells of 1% agarose gels

(pulsed field certified agarose, Bio-Rad Laboratories,

Hercules, CA), and run at 5.0 V cm�1 for 20 h at 14 �Cin 0.5� Tris–borate buffer using a CHEF DR III PFGE

apparatus cooled using a model 1000 mini chiller

(Bio-Rad).

2.5. Deferred antagonism plating assay

Screening of bovicin 255 positive S. bovis isolates for

the production of inhibitory molecules was performed

using the deferred antagonism plating (DAP) assay

[7,35]. Strains to be tested for inhibitor production

were spotted onto L10 or CC plates and incubatedanaerobically at 39 �C for 24 h. The plates were re-

moved from the anaerobic chamber and all bacterial

growth was scraped off the agar plate using a sterile

spreader. Excess bacterial debris was washed off using

2 ml sterile water. The plate was then inverted (5 min)

over a filter paper disc that had been bathed in chlo-

roform to sterilise the surface of the agar. Excess

chloroform vapour was then allowed to dissipate (5min) before the agar plate was re-introduced to the

anaerobic chamber to remove oxygen (5 h). Molten

HAP top agar was inoculated with approximately 107

analysis

plicon Length (bp) Accession Nos.

and/or reference

212 AF076529 [21]

yrivibriocin AR10

icin 255 209 AF298196 [9]

9A 146 AF062647 [22]

yrivibriocin OR79A

acterial 16S rRNA 1540 [33]

uencing

uencing

202 A.L. Cookson et al. / FEMS Microbiology Ecology 48 (2004) 199–207

indicator organisms (P. anaerobius D1), mixed gently

and poured over the DAP assay plates. The plates were

re-incubated at 39 �C for 18 h and each isolate was

scored for its ability to create a distinct zone of inhi-

bition in the agar overlay.

Fig. 1. PCR amplification of the genes encoding butyrivibriocins

OR79A and AR10 (lanes 2 and 4) from B. fibrisolvens OR79 and AR10

respectively. Negative controls (lanes 1 and 3).

Fig. 2. PCR amplification of the gene encoding bovicin 255 from

Streptococcus spp. Lane 1, Negative control; 2, S. gallolyticus

LRC0255; 3, S. bovis 7–2; 4, S. bovis 7–25; 5, S. bovis 7–26; 6, S. bovis

GKF1; 7, S. bovis 22/01 F; 8, S. bovis 24/01 B; 9, S. bovis 24/01 H; 10,

S. bovis 4b; 11, S. bovis 13a; and 12, S. bovis 13b.

3. Results

3.1. Isolation of strains

Caecal and ruminal fluid dilutions in RX or CC broth

were spread onto CC agar plates and incubated anaer-obically overnight at 39 �C. Resulting colonies that were

large, and white to orange in colour were Gram stained

and identified as Gram-positive cocci. Slower growing

pale colonies were identified as Gram-negative curved

rods. Based on these characteristics the isolates were

initially identified as S. bovis- and B. fibrisolvens-like

isolates, respectively.

3.2. PCR primer design

PCR primers for the amplification of the genes en-

coding bovicin 255 and butyrivibriocin AR10 (bviD)

were designed using the gene sequences from S. gallo-

lyticus LRC0255 (AF298196) and B. fibrisolvens AR10

(AF076529), respectively. Degenerate bases were in-

cluded in the ALC5 and ALC6 primers to account forthe sequence variation found within the bvi79A genes

encoding the B. fibrisolvens butyrivibriocin OR79A-like

bacteriocins (AF062647 and AF349664 to AF349674)

[22]. Both forward and reverse primers were designed

internally to the gene encoding the bacteriocins to en-

sure that any variations in flanking DNA sequence did

not affect identification of strains possessing the target

gene sequence.

3.3. PCR amplification from bacterial isolates

A PCR product of the expected size was amplified

from both B. fibrisolvens OR79 (146 bp) and AR10 (213

bp) (Fig. 1). However, an additional non-specific am-

plicon slightly larger than the anticipated PCR product

was often seen (Fig. 1), but only from the positivecontrol strains. Attempts were made to eliminate the

non-specific amplification by changing the PCR reaction

parameters, however this resulted in reduced levels of

amplification of the desired amplicon. Thirty four B.

fibrisolvens-like isolates were screened by PCR for the

presence of the genes encoding butyrivibriocins OR79A

(bvi79A) and AR10 (bviD), but apart from the control

B. fibrisolvens OR79 and AR10 strains none of the iso-lates produced any amplified product.

When the S. gallolyticus LRC0255 DNA was used in

the PCR reaction, a PCR amplicon of approximately

209 bp was observed (Fig. 2). Sixty two other S. bovis-like isolates were screened by PCR for the presence of

bovicin 255. Ten strains gave a PCR amplicon of an

indistinguishable size from the product obtained from

S. gallolyticus LRC0255 (Fig. 2). Seven were New

Zealand isolates: four from the caecum of one sheep,

two from the caecum of another sheep and the final

strain was isolated from the rumen contents of a sheep

from a separate flock. All sheep were pasture (ryegrass-clover) fed. The other three strains were isolated from

grain-fed cattle in North America (D. Krause, personal

communication).

3.4. Sequence analysis of bovicin 255 PCR products

All PCR products amplified using the ALC3 and

ALC4 primers, specific for bovicin 255, were cloned andsequenced. The sequence of the PCR product derived

from S. gallolyticus was identical to the GenBank se-

quence, however all other cloned sequences differed

Fig. 3. Alignment of deduced amino acid sequence of bovicin 255 from S. gallolyticus LRC0255 with homologous sequences derived from sequencing

of S. bovis bovicin 255-like PCR products. Conservative substitutions are underlined and 0 indicates the protease processing site of the pre-peptide.

Group 1 contains S. bovis strains 13a, 13b, 7–2, 7–25 and 7–26. Group 2 contains S. bovis strains 4b, 22/01 F, 24/01 B, 24/01 H and GKF1.

Fig. 4. PFGE profiles of Streptococcus spp. possessing the bovicin 255

gene. Lane 1, S. gallolyticus LRC0255; 2, S. bovis 7–2; 3, S. bovis 7–25;

4, S. bovis 7–26; 5, S. bovis GKF1; 6, S. bovis 22/01 F; 7, S. bovis 24/01

B; 8, S. bovis 24/01 H; 9, S. bovis 4b; 10, S. bovis 13a; and 11, S. bovis

13b.

Fig. 5. Deferred antagonism plating (DAP) assay. Inhibitory activity

of isolates possessing the bovicin 255 gene is associated with zones of

inhibition in the P. anaerobius D1 overlay. Strain 3–36 included as

negative control.

A.L. Cookson et al. / FEMS Microbiology Ecology 48 (2004) 199–207 203

from the LRC0255 sequence (209 bp) by between 11 and15 bp substitutions. Isolates may be grouped on the

basis of their amino acid sequences into Group 1, Group

2 and LRC0255 (Fig. 3). Group 1 strains have an

identical processed peptide amino acid sequence to

LRC0255 whilst the Group 2 strains have four base pair

substitutions that result in three amino acid changes

(asparagine to serine, alanine to threonine and aspara-

gine to threonine) in the processed peptide (Fig. 3). Fouradditional base pair changes in Groups 1 and 2 result in

three amino acid changes (aspartic acid to glutamic acid,

glutamic acid to alanine and alanine to glutamic acid) in

the pre-peptide (Fig. 3). The remaining base pair sub-

stitutions are silent.

3.5. Phylogenetic analysis of bovicin 255 producing S.

bovis-like isolates

To determine the identity of the bovicin 255-pro-

ducing isolates, their 16S rRNA genes were sequenced

and aligned against other known streptococcal se-

quences. The comparative sequence analysis confirmed

that all 10 isolates producing bovicin 255 shared almost

identical 16S rRNA genes (99.7–100%) to those of S.

bovis B315 and C14b#1, strains previously isolated fromryegrass-clover fed cattle in New Zealand [36], S. bovis

JB1 and H2-1 [37] and also contained the same PCR

primer sites used by Whitehead and Cotta [37] to iden-

tify ruminal S. bovis isolates.

3.6. Pulsed-field gel electrophoresis of bovicin 255 pro-

ducing S. bovis-like isolates

DNA from each S. bovis-like isolate giving a positive

PCR reaction for the amplification of bovicin 255 was

screened by pulsed-field gel electrophoresis (PFGE)

analysis to determine strain similarity (Fig. 4). Four of

the New Zealand S. bovis isolates from the same sheep

had identical PFGE profiles. Another two isolates from

a different animal within the same flock also had iden-

tical PFGE profiles. The other New Zealand strain,GKF1, isolated in a separate study from the rumen

contents of a ryegrass-fed sheep, had a distinctly differ-

ent profile. Of the three North American isolates, two

had identical PFGE profiles, the other differing by the

position of one DNA band. The PFGE profiles of all the

bovicin 255 positive S. bovis isolates were dissimilar to

204 A.L. Cookson et al. / FEMS Microbiology Ecology 48 (2004) 199–207

the PFGE profile of the bovicin 255 control strain S.

gallolyticus LRC0255 (Fig. 4).

3.7. Measuring inhibitory activity

To determine whether the possession of the bovicin

255 amplicon was associated with inhibitory activity, the

PCR positive isolates were tested in the DAP assay.

When each of the PCR positive streptococci isolates were

grown on L10 agar plates, overlaid with agar containing

P. anaerobius D1 as an indicator strain, zones of inhi-

bition of the indicator organism were observed (Fig. 5).

Strain 3–36 which did not possess the bovicin 255 geneusing the PCR assay was used in the DAP assay as a

negative control and did not give any zones of inhibition

in the indicator overlay (Fig. 5). All S. bovis isolates

possessing the bovicin 255 gene produced inhibition

zones of varying sizes with P. anaerobius D1 overlays.

4. Discussion

Bacteriocins are a large group of microbial com-

pounds that have yet to be fully evaluated for use within

the rumen environment with a view to limiting the loss

of energy and nitrogen through the microbial produc-

tion of methane and ammonia, respectively. Inhibitory

molecules such as monensin are often included as feed

supplements to improve the efficiency of ruminal fer-mentation through reductions of Gram-positive ruminal

strains. The use of novel bacteriocin molecules as feed

additives to increase the overall efficiency of ruminal

fermentation has attracted considerable commercial in-

terest and research focus. In this study we used PCR and

DNA sequence analysis to identify and characterise

genes encoding novel bacteriocin variants from rumen

and caecal bacterial isolates and examined the rela-tionship between bacteriocin DNA sequence PFGE

types.

Previously, phenotypic tests were used to screen for

bacteriocin activity from B. fibrisolvens and S. bovis

strains isolated from the rumen [7,9,11,19]. This is the

first study where PCR amplification, used as a diag-

nostic tool, and bacteriocin sequence diversity from

multiple rumen or caecal S. bovis-like isolates havinginhibitory activity, have been linked to source animal,

and where the bacterial gene profile of the bacteriocin

producer strains has been assessed using PFGE to ex-

amine clonality. In a study by Whitford et al. [22] PCR

amplification and DNA sequence analyses indicated

that 24 of 39 (61%) B. fibrisolvens-like isolates whose

bacteriocin production was identified on the basis of the

DAP assay, had homologues to the bvi79A gene thatencodes the butyrivibriocin OR79. The bvi79A positive

bacteriocin-producing strains could be organised into 3

groups and one variant was found in Butyrivibrio

crossotus [22]. However, no data are available that have

identified further B. fibrisolvens-like isolates other than

strain AR10 that possesses the gene encoding butyri-

vibriocin AR10. In this study none of the B. fibrisolvens-

like isolates screened in the PCR assay were found to bepositive for the butyrivibriocins AR10 or OR79 and as

far as we are aware, butyrivibriocins AR10 or OR79

have not been identified from B. fibrisolvens-like isolates

from the Southern Hemisphere.

Butyrivibrio fibrisolvens-like strains are often assigned

on the basis of their butyrate-production and other

common phenotypic and metabolic characteristics [38–

44], and recent classification has further clarified thecomplex Butyrivibrio group with the identification of

Butyrivibrio hungatei and Pseudobutyrivibrio xylanivo-

rans [45]. However, as none of the field strains in this

study gave amplicons with primers specific for butyri-

vibriocins AR10 or OR79, further characterisation of

the B. fibrisolvens-like isolates to the 16S rRNA gene

level or classification using recently developed methods

[45] were not performed.The identification, cloning and sequencing of bovicin

255 from S. gallolyticus LRC0255 was the first full

characterisation of a bacteriocin from rumen strepto-

coccal spp. [9]. Whilst this isolate was one of seven

strains that inhibited the growth of other streptococci,

the other six strains having inhibitory activity were not

examined for the presence of the bovicin 255 gene. Thus

far no other studies have identified any additionalstreptococcal spp. containing the bovicin 255 gene. Us-

ing PCR, however, this study has identified a further 10

streptococcal strains having a bacteriocin with consid-

erable sequence homology (92.9–95.7%) to that of the S.

gallolyticus LRC0255 from which the bovicin 255 gene

was first sequenced. Whether the sequence variation is a

characteristic of S. bovis isolates is not known as addi-

tional S. gallolyticus isolates having the bovicin 255 genesequence, apart from LRC0255, remain to be identified.

The presence of such similar bacteriocins having un-

dergone sequence divergence in two separate rumen

streptococcal spp. is noteworthy. Similarly, sequence

divergence in the nisin biosynthetic genes found on the

conjugative transposon of L. lactis N8 results in the

substitution of asparagine for histidine at amino acid

position 27 in nisin Z compared to nisin A without anyapparent loss of antimicrobial activity [46,47]. Amino

acid substitutions within the processed bovicin 255

peptide of the S. bovis isolates are relatively conserved

and do not appear to result in any significant change of

inhibitory activity in the DAP assay compared to that of

the homologous peptide from S. gallolyticus LRC0255.

There may, however, be some significance of bovicin 255

amino acid sequence variation within the rumen envi-ronment that is impossible to quantify in vitro.

The extent and diversity of bacteriocin production by

S. bovis-like isolates has not been fully investigated.

A.L. Cookson et al. / FEMS Microbiology Ecology 48 (2004) 199–207 205

However, in a recent study [19] approximately 50% and

60% of S. bovis isolates from hay and grain fed cattle,

respectively, had activities that inhibited the growth of

S. bovis JB1. PCR detection using primers different to

those used in this study, were successfully used to am-plify the bovicin 255 gene from the S. gallolyticus posi-

tive control but indicated that none of the inhibitor

producer isolates had the gene for bovicin 255 [19]. Se-

quence data analysis indicates that at least a single base

pair change is present between the BOV302R primer

sequence [19] and the corresponding PCR annealing site

in 50% (5 of 10) of the S. bovis isolates having bovicin

255 gene homologues sequenced in this study. Thereforeprimer design in conserved areas of the gene sequence

may be important for accurate identification of strep-

tococcal spp. containing bovicin 255-like gene se-

quences. Several other rumen S. bovis isolates, including

HC5, were identified as having bacteriocin activity [19]

but whether sequence divergence or the presence of

other as yet uncharacterised bacteriocin molecules con-

tributes to this activity is unknown.Although the bovicin 255 gene may be found from

diverse geographical locations, including New Zealand

and North America, its distribution is uncertain. On the

basis of bovicin 255 sequence analysis, both Groups 1

and 2 organisms were isolated from New Zealand. The

three isolates from North America may be placed into

Group 1. Only one group type was isolated from any

one animal although strains from both Groups 1 and 2were isolated from separate sheep within the same flock.

The seven New Zealand strains were isolated from only

three ryegrass-clover fed animals and strains within each

animal had a different PFGE profile. It is unclear whe-

ther bovicin 255 positive S. bovis strains preferentially

inhabit ryegrass-fed animals or whether one S. bovis-like

clonal type may predominate in each animal based on

only the few isolates examined from a small number ofanimals. It is interesting to note that 6 of the 7 New

Zealand strains were caecal isolates from two animals

from the same sheep flock. The extent to which S. bovis

colonises the length of the ruminant gastrointestinal

tract and whether the caecum may be an additional site

in which strains producing bovicin 255 may persist has

not been fully established. However, S. bovis isolates are

uncommon in the rectal contents of pre-weaned calves,but an increase in the levels of S. bovis and an associated

reduction in the presence of enterococcal spp. in calves

and dairy cattle [48] may be due, in part, to the colo-

nisation of the rumen through faecally contaminated

forage as calves are weaned. This may indicate that

colonisation is throughout the gastrointestinal tract and

that excretion may be one mechanism by which initial

colonisation with S. bovis occurs.The phylogenetic position of the S. bovis-like isolates

having the bovicin 255 gene was determined using

comparative analysis of the 16S rRNA gene sequences.

Unlike the positive control organism, S. gallolyticus

LRC0255, isolated from the rumen of a moose in Al-

berta, Canada, all isolates described in this study were

almost the same or identical to S. bovis 16S rRNA gene

sequences available in DNA databases. Based on thesedata it appears that the bovicin 255 gene may be more

commonly associated with S. bovis ruminal isolates than

S. gallolyticus.

In summary, our investigations have shown that the

butyrivibriocin genes may not be widely distributed in B.

fibrisolvens-like strains isolated in New Zealand, despite

attempts to overcome sequence variation with the design

of degenerate primers. Any significance in vivo of the S.bovis bovicin 255 gene sequence variants are unknown

but may represent a convenient method of sub-typing

those that have gene sequences homologous to bovicin

255. Careful choice of PCR primers is required to ensure

accurate identification of bacteriocins from those iso-

lates having inhibitory activity. It has become apparent

that the bacterial diversity of the rumen is much greater

than once thought and as examples of bacteriocin-likeactivity among obligate and facultative anaerobes iso-

lated from the rumen of both sheep and cows have been

described, it is conceivable that many other ruminal

bacteriocins remain to be discovered. Future investiga-

tions are required to examine this rumen bacterial strain

diversity and the production of bacteriocin molecules to

assess their impact on the overall rumen ecology and to

begin to understand the interbacterial mechanisms thatinfluence the flux and diversity of microbial species in

the rumen.

Acknowledgements

This study was funded through AgResearch Reposi-

tioning and the New Zealand Lotteries Commission.

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