virulence of pasteurella multocida reca mutants

Virulence of Pasteurella multocida recA mutants

Maribel CaÂrdenasa, Antonio R. FernaÂndez de Henestrosaa,Susana Campoya, Ana M. Perez de Rozasb, Jordi BarbeÂa,

Ignacio Badiolab, Montserrat Llagosteraa,*

Molecular Microbiology Group.aDepartament de GeneÁtica i de Microbiologia, Edi®ci Cn, Universitat AutoÂnoma de Barcelona,

08193 Bellaterra, Barcelona, SpainbUnitat de Sanitat Animal, Institut de Recerca i Tecnologia Agroalimentaria Barcelona, Barcelona, Spain

Received 20 June 2000; received in revised form 22 September 2000; accepted 20 October 2000

Abstract

In order to determine the role of the RecA protein in the virulence of Pasteurella multocida, a

recA mutant was constructed and used in studies of virulence and competition in relation to wild-

type strain. To achieve this, ®rstly, the recA gene was isolated and sequenced, showing an

Escherichia coli-like SOS box and encoding a protein of 354 amino acids which has the closest

identity with the Haemophilus in¯uenzae RecA protein. Further, the recA mutant was constructed,

by inactivating this gene by single recombination of a suicide plasmid containing an internal region

of the P. multocida recA gene, and shown to be more sensitive to UV radiation than the parental

strain. The P. multocida mutant was slightly attenuated in virulence, as indicated by the LD50, the

time of death of infected animals, and a failure to compete with the wild-type strain in mixed

infections. Compared to the parent strain, the mutant had a similar growth rate but a longer lag

phase. These data suggest that the diminished virulence of the recA mutant as well as its failure in

competition were more a consequence of the long lag phase rather than a direct effect of the

inactivation of the recA gene on genes involved in virulence. # 2001 Elsevier Science B.V. All

rights reserved.

Keywords: Pasteurella multocida; recA gene; Virulence genes; DNA repair

1. Introduction

Pasteurella multocida is responsible for infectious diseases in many species of

mammals and birds, producing important economic losses. For this reason, development

Veterinary Microbiology 80 (2001) 53±61

* Corresponding author. Tel.: �34-93-5812615; fax: �34-93-5812387.

E-mail address: [email protected] (M. Llagostera).

0378-1135/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 3 7 2 - 2

of vaccine strains against this organism is an important goal. It has been largely

demonstrated that live attenuated strains are more effective as vaccines than inactivated

strains (Dougan, 1994; Marsden et al., 1996; Simmons et al., 1998; MaÈkelaÈ, 2000). It is

essential that attenuated vaccine strains do not revert to virulence. To accomplish this

objective, these vaccine strains usually present either deletion or insertion mutations in

one gene of virulence, giving rise to attenuation. Nevertheless, these attenuated mutants

are not totally safe since several recombinational processes (transduction, transformation

and conjugation) could restore the wild-type phenotype. To avoid this, an important

safety measurement is the introduction of mutations in the recA gene of vaccine strains

(Fuchs et al., 1999).

The RecA protein is a key component of homologous recombination in bacteria

because it is involved in the early steps of this process, allowing the alignment of DNA

molecules before strand exchange (Walker, 1984). This protein is also a positive regulator

of the SOS system, one of the most important DNA repair-systems of bacteria. This

network is made up of at least 20 genes whose products enable the cell to survive after

DNA damage (Walker, 1984). The LexA protein is the repressor of all genes comprising

the SOS response. It has been shown that, in E coli, the LexA repressor binds at a speci®c

site at the 50-ends of SOS genes. This site, known as the E. coli SOS box, is an imperfect

palindrome comprising the sequence CTGTN8ACAG (Walker, 1984).

Similar DNA repair-systems have been described in other bacteria, and several recA

genes have been isolated and characterized (Miller and Kokjohn, 1990). Likewise, the SOS

boxes of two other bacterial phylogenetic groups have been identi®ed (Gram-positive

bacteria and alpha group of the Proteobacteria), with the sequences GAACN4GTTC and

GTTCN7GTTC, respectively (Winterling et al., 1998; Labazi et al., 1999).

The aim of this work was to isolate and sequence the P. multocida recA gene

to facilitate the construction of RecAÿ derivatives, and to analyze the role of the

P. multocida recA gene in the virulence of the organism.

2. Materials and methods

2.1. Bacterial strains and growth conditions

The bacterial strains and plasmids used are listed in Table 1. E coli strains were grown

in LB broth (Miller, 1992). P. multocida strains were cultured on brain-heart infusion

(BHI) or on sheep blood agar (SBA) plates. Antibiotics were added to the culture media

at the concentrations previously reported (Fernandez de Henestrosa et al., 1997).

2.2. Genetic methods

The suicide plasmid pUA1002 was used to obtain the P. multocida RecAÿ mutant. This

plasmid is a derivative of pGY2, which is unable to replicate in host strains devoid of the

R6K-speci®ed p-protein product of the pir gene (Young and Miller, 1997) and which

must be maintained in lysogenic strains for the lpir bacteriophage. Triparental mating,

using pRK2013 as the mobilizing plasmid, was used to obtain the P. multocida RecAÿ

54 M. CaÂrdenas et al. / Veterinary Microbiology 80 (2001) 53±61

mutant as previously reported (Fernandez de Henestrosa et al., 1997). Brie¯y, conjugation

was performed by mixing 1 ml aliquots of early-stationary-phase cultures of donor

(E. coli MC1061 (lpir) carrying the plasmid pUA1002), recipient (P. multocida PM1002)

and E. coli HB101, carrying pRK2013 plasmid. This mixture was ®ltered through a

sterile swinex ®lter unit equipped with a 0.45 mm Millipore ®lter, which was put on a BHI

agar plate and incubated overnight at 308C. Afterwards, the ®lter was suspended in 3 ml

of BHI liquid medium and dilutions were plated on BHI agar plates, supplemented with

streptomycin, rifampicin and ampicillin.

2.3. Biochemical methods and DNA techniques

DNA methodology and sequence computer analyses were as published (Fernandez de

Henestrosa et al., 1997). A library of P. multocida PM25 chromosomal DNA was

constructed by inserting size-fractionated, Sau3AI restriction fragments of 4 kb average

size from a partial digest into the BamHI site of the pUA520 broad host-range plasmid,

and transforming the RecAÿ E. coli strain DH5a. From this library, a clone carrying a

2.2 kb SacII±KpnI fragment putatively containing the P. multocida recA gene was

Table 1

Bacterial strains and plasmids used in this work

Relevant features Source or reference

Organism

E. coli

AB2463 recA13, supE44, thr1, leu6, proA2, his4, argE3, thi1, galK2,

ara14, xyl5, mtl1, tsx33, rpsL31

ECGSCa

DH5a supE4, DlacU169 (f80 lacZDM15), hsdR17, recA1, endA1,

gyrA96, thi1, relA1

Clontech

HB101 supE4, hsdS20 (rÿBmÿB), recA13, ara14, proA2, lacY1,

galK2, rpsL20, xyl5, mtl1

J. Frey

MC1061 (lpir) hsdR, mcrB, araD139, D (araABC-leu) 7679, DlacX74, gal1,

galK, rpsL, thi, lysogenized with lpir bacteriophage

This laboratory

P. multocida

PM25 Wild-type This laboratory

PM1002 As PM25, but RifR, SpcR This laboratory

PM1028 As PM1002, but recA::pUA1002 This work

Plasmids

pRK2013 Tra� of RK2, ColE1 replicon, KmR D.R. Helinski

pBBR1MCS A broad-host-range cloning vector, CmR Kovach et al., 1994

pUA520 As pBBR1MCS, but KmR This work

pUA811 As pUA520 but carrying a 2.2 kb chromosomal fragment

of P. multocida containing the recA gene

This work

pGY2 Mob�, R6K replicon, ApR StrR SpcR Young and Miller,

1997

pUA826 As pGY2, but lacking the 800 bp SalI fragment containing

the cat gene

This laboratory

pUA1002 As pUA826, but carrying a 548 bp internal fragment of

the P. multocida recA gene

This work

a ECGSC, E. coli Genetic Stock Center.

M. CaÂrdenas et al. / Veterinary Microbiology 80 (2001) 53±61 55

identi®ed through its ability to confer methyl methanesulfonate resistance to the DH5acells. The plasmid carrying this 2.2 kb SacII±KpnI fragment was designated pUA811 and

was transformed into E. coli AB2463, which carries the mutant recA13 allele. The

presence of the plasmid was found to restore UV resistance in AB2463 to parental strain

levels (data not shown).

The entire nucleotide sequence of P. multocida recA gene was determined for both

DNA strands by the dideoxy method on an ALF Sequencer (Pharmacia Biotech). The

nucleotide sequence of this gene and its ¯anking regions appears in the EMBL/GenBank/

DDBJ Nucleotide Sequence Data Libraries under Accession Number X99294.

Oligonucleotide primers used in this work are listed in Table 2.

2.4. Virulence and competition assays

Swiss mice, 3±5 weeks old, were used for studies of virulence. The LD50 of strains was

determined by triplicate as reported (Fernandez de Henestrosa et al., 1997). Basically,

groups of four mice were injected with serial 10-fold dilutions of bacteria in buffered

peptone water. The concentration of the original bacterial suspensions was determined by

the plate-count method. For competition assays, PM1002 and PM1028 strains were grown

separately on SBA, and mixed before injection to obtained the desired concentration of

each. Three mice were inoculated intraperitoneally with 0.1 ml of a suspension containing

121 CFU of PM1002 and 150 CFU of PM1028 as determined by plating. In both assays,

bacteria were recovered of the hearts of dead animals to study their characteristics.

3. Results

The ®rst experimental approach of this work was the isolation and sequence of the recA

gene of P. multocida. Following procedures above mentioned (Section 2.3), a 2.2 kb

Table 2

Oligonucleotide primers used in this work

Primer Sequence Position

PosRec1a 50-TTTGCGATGCGTTAGTGC-30 �386b

PosRec2a 50-TTCTAACCATTTCATCGCG-30 �933b

Rec1c 50-GCTCTATTATGAAATTGGGCG-30 �74b

Rec2c 50-AAGGGTTAAGGTGGTTTTACCG-30 �235b

RecA2d 50-CGCGAGCAACTCATTGCG-30 �990b

Aadd 50-CGGCGATCACCGCTTCCC-30 �22e

a Primers used to obtain the 548 bp internal fragment of the P. multocida recA gene.b Position of the 50-end of the oligonucleotide with respect of the translational starting point of the P.

multocida recA gene.c Primers used to obtain the 161 bp internal fragment of the P. multocida recA gene, used as probe.d Primers used to determine the presence of the 548 bp internal fragment of P. multocida recA gene into the

plasmid pUA1002.e Position of the 50-end of the oligonucleotide with respect the translational starting point of aad gene of the

plasmid pGY2.


SacII±KpnI fragment of the plasmid pUA811 was identi®ed to contain the recA gene of

P. multocida. The nucleotide sequence of this fragment revealed one large open-reading

frame, which was located seven nucleotides downstream of a typical Shine-Dalgarno

sequence (GAGGA). The P. multocida recA ORF of 1065 nucleotides codes for a

polypeptide of 354 amino acids whose calculated molecular mass is 37,912 Da.

P. multocida RecA showed the highest similarity with the RecA protein of

Haemophilus in¯uenzae (79%). The greatest degree of differences between both proteins

was found primarily at the C-termini. Amino acid residues in E. coli RecA known to be

associated with functional activities which include protease activity, and homologous

recombination (Karlin and Brocchieri, 1996), were all highly conserved in the RecA

protein of P. multocida. Likewise, upstream of the P. multocida recA gene was the

sequence CTGTN8ACAG, which was identical to the E. coli LexA binding site (SOS

box), suggesting that the regulation of this P. multocida gene is the same as that of the

E. coli gene. This fact is in agreement with the high resistance against UV irradiation

shown by the E. coli RecAÿ cells carrying the pUA811 plasmid (data not shown).

The second purpose of this work was to obtain a RecAÿ mutant of P. multocida for

studying the role of this gene in the virulence of this organism. Firstly the recA gene was

disrupted by cloning a kanamycin-resistance cassette in an internal point of the recA

gene. However, all attempts to introduce this mutated copy of P. multocida recA by

marker exchange into the P. multocida chromosome were unsuccessful. For this reason,

we decided to use an alternative approach inactivating the P. multocida recA gene by

single recombination of a suicide plasmid containing an internal region of this gene. A

548 bp internal region of the P. multocida recA gene was isolated through PCR-

ampli®cation using PosRec1 and PosRec2 oligonucleotide primers (Table 2), correspond-

ing to nucleotides 386±403 and 933±915 of this gene, respectively. This 548 bp fragment

was afterwards cloned in the pUA826 suicide-plasmid, giving rise to the pUA1002

plasmid. The presence of this fragment in pUA1002 plasmid was con®rmed through

PCR-ampli®cation using the oligonucleotide primers RecA2 and Aad (Table 2). In

addition, the fact that an ampli®cation product was obtained with these primers (data not

shown) allowed us to predict the result of the single recombination with the recA

chromosomal gene of P. multocida (Fig. 1). The pUA1002 plasmid was introduced by

triparental matting into P. multocida PM1002. Eighteen ampicillin-resistant transconju-

gants were obtained and screened for UV sensitivity. Among these clones only one, the

PM1028 strain, showed a higher UV sensitivity than the wild-type strain. Thus, at a

dosage of 6 Jmÿ2, the fraction surviving of PM1028 was lower than 4� 10ÿ4. Moreover,

wild-type cells UV irradiated at the same doses presented a 105-fold higher survival. In

addition, a product of PCR-ampli®cation was obtained with oligonucleotide primers

RecA2 and Aad (data not shown).

To con®rm that P. multocida PM1028 was a recA mutant, chromosomal DNA from this

strain and also from the parental cells was digested with EcoRI and probed by Southern

blotting with a 161 bp internal fragment of the recA gene, obtained by PCR-ampli®cation

with oligonucleotide primers Rec1 and Rec2 (Table 2). Fig. 2 shows that the probe

hybridized to a 22 kb fragment in the parental strain PM1002 (lane 1) and to a 3 kb

fragment in the PM1028 strain (lane 2), indicating that the recA gene had been disrupted

in this transconjugant.


The role of recA gene in virulence of the P. multocida was studied by determining the

LD50 of both the recA mutant and the parental strain in a mouse model. Values (CFU/

animal) obtained were 54.9 (S:D: � 5) and 11.75 (S:D: � 2:6), respectively, with a

statistical signi®cance P < 0:05. To determine stability during the infection with the P.

multocida recA cells, several clones recovered from the heart of intraperitoneally infected

mice were analyzed by both UV sensitivity and PCR ampli®cation with the

oligonucleotide primers RecA2 and Aad. It is worth noting that all of these clones

showed the same pattern for both parameters, as did the recA mutant (data not shown).

In the course of virulence assays, we observed that mortality produced by the recA

mutant was slightly delayed (72 h after infection) in relation to the parental strain (48 h

after infection). This delay could re¯ect differences in growth parameters between both

strains. When growth kinetics (data not shown) were studied, a similar growth rate for

both strains was found, but signi®cant differences in the duration of the lag phase (10 min

for PM1002 versus 90 min for PM1028) were seen. The next step was to perform

competition assays between PM1002 and PM1028 strains in mixed infections. Death of

all animals was at 48 h from infection. Clones (500), recovered from the heart of each

animal, were replicated in the absence and in the presence of streptomycin and

ampicillin. All clones were sensitive to both antibiotics indicating that all were the wild-

type strain.

Fig. 1. Schematic representation of single recombination between the 548 bp internal fragment of the

P. multocida recA, cloned in pUA1002 plasmid, and the P. multocida chromosomal recA gene. Primers used to

con®rm the presence of the 548 bp internal fragment of P. multocida recA gene into the plasmid pUA1002, and

in the RecAÿ strain are shown.


4. Discussion

Little is known about the importance of the recA gene in bacterial virulence. Some

studies indicate that this gene is involved in the infection process by several bacteria by

either: (i) increasing the expression of phage encoded-virulence genes as in

enterohemorrhagic E. coli O157:H7 and Vibrio cholerae strains (Fuchs et al., 1999;

Faruque et al., 2000) (ii) promoting DNA rearrangements as has been shown in

enteroinvasive strains of E. coli and Shigella ¯exnerii (Zagaglia et al., 1991), and in

Campylobacter fetus and Neisseria gonorrhoeae (Dworkin et al., 1997; Ilver et al., 1998)

or (iii) enhancing cell survival against host-factors damaging bacterial DNA (Buchmeier

et al., 1995). Thus, it has been shown that the presence of a recA mutation gives rise to an

attenuated phenotype in Vibrio cholerae, Staphylococcus aureus and Salmonella

typhimurium (Buchmeier et al., 1993; Kumar et al., 1994; Mei et al., 1997). Nevertheless,

this mutation did not seem to modify the virulence of Brucella abortus and

Corynebacterium pseudotuberculosis (Tatum et al., 1993; Pogson et al., 1996). These

opposite behaviors indicate that the role of the recA gene is not the same for all

pathogenic bacteria.

Values of LD50 obtained in this work clearly indicate that the recA mutation only

provokes a marginal reduction of the P. multocida virulence, especially in comparison

Fig. 2. Southern analysis of chromosomal DNA of wild-type (lane 1) and RecAÿ (lane 2) strains of P. multocida

PM1002. A 161 bp fragment containing an internal region of the P. multocida recA gene was used to hybridize

with the EcoRI-digested genomic DNA. Molecular size markers corresponding to HindIII-digested lDNA are

shown in the left.


with the effect on virulence described for the galE mutation (Fernandez de Henestrosa

et al., 1997). However, concerning competition assays, the recA mutant has a clear

disadvantage in front to wild-type strain. We hypothesize that non-success in competition

assays could be more a consequence of a long lag phase than a direct effect of recA gene

on genes related to virulence. Additionally, this delay in growing could also explain the

marginally diminished virulence of the P. multocida recA mutant. In fact it is known that

E. coli recA mutants produce anucleate cells disturbing signi®cantly the culture growth

(Zyskind et al., 1992). This behavior seems to be attributed to the role of RecA protein in

the partition of the bacterial chromosome between daughter cells (Mao et al., 1991). In

this line, our hypothesis also agrees with the relationship found between generation time

and virulence in E. coli, S. typhimurium and Actinobacillus pleuropneumoniae pathogenic

strains (Byrd and Hooke, 1997; Linde et al., 1998).

In summary, we conclude that recA gene has not an speci®c role in the virulence of P.

multocida, although its de®ciency gives rise to a delay in the process of infection and to a

marginal reduction of virulence. The P. multocida recA mutant obtained in this work will

be useful in both the development of a stable host/vector system which will facilitate

genetic studies to characterize virulence factors of this organism as well as in the

construction of safe live vaccines against it.

Acknowledgements

This work was partially funded by grant BIO99-0779 of the ComisioÂn Interdeparta-

mental de Ciencia y TecnologõÂa of Spain (CICYT), and by the Comissionat per

Universitats i Recerca de la Generalitat de Catalunya (1999SGR-106). Susana Campoy

was a recipient of a predoctoral fellowship from the DireccioÂ General d'Universitats de la

Generalitat de Catalunya. We are deeply indebted to Joan Ruiz and M. Mar LoÂpez for

their excellent technical assistance.

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virulence of pasteurella multocida reca mutants

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