E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis

Accepted Manuscript E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis

Hassan Z.A. Ishag, Qiyan Xiong, Maojun Liu, Zhixin Feng, Guoqing Shao PII: DOI: Reference:

S0167-7012(17)30065-9 doi: 10.1016/j.mimet.2017.03.004 MIMET 5131

To appear in:

Journal of Microbiological Methods

Received date: Revised date: Accepted date:

20 October 2016 2 March 2017 6 March 2017

Please cite this article as: Hassan Z.A. Ishag, Qiyan Xiong, Maojun Liu, Zhixin Feng, Guoqing Shao , E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mimet(2017), doi: 10.1016/ j.mimet.2017.03.004

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT REVISED prominently E. coli recA Gene Improves Gene Targeted Homologous Recombination in

PT

Mycoplasma hyorhinis

1

SC

RI

Hassan Z. A. Ishag1, 2, Qiyan Xiong1*, Maojun Liu1, Zhixin Feng1 and Guoqing Shao1

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key

NU

Laboratory of Veterinary Biological Engineering and Technology, Ministry of

MA

Agriculture, National Research Center for Engineering and Technology of Veterinary Bio-products, Nanjing 210014, China

College of Veterinary Sciences, University of Nyala, Nyala, Sudan

PT E

D

2

AC

CE

*Corresponding author: Qiyan Xiong (E-mail: [email protected])

1

ACCEPTED MANUSCRIPT Abstract Mycoplasma hyorhinis is an opportunistic pathogen of pigs. Recently, it has been shown to transform cell cultures, increasing the attention of the researchers. Studies on the pathogenesis require specific genetic tool that is not yet available for

PT

the pathogen. To address this limitation, we constructed two suicide plasmids

RI

pGEMT-tetM/LR and pGEMT-recA-tetM/LR having a tetracycline resistance marker

SC

flanked by two hemolysin gene arms. The latter plasmid encodes an E. coli recA, a gene involved in DNA recombination, repair and maintenance of DNA. Using

NU

inactivation of the hemolysin gene, which results in a detectable and measurable

MA

phenotype, we found that each plasmid can disrupt the hemolysin gene of M. hyorhinis through a double cross-over homologous recombination. However,

D

inclusion of the E. coli recA gene in the construct resulted in 9-fold increase in the

PT E

frequency of hemolysin gene mutants among the screened tetracycline resistance colonies. The resultant hemolysin mutant strain lacks the ability to lyse mouse bed

CE

blood cells (RBC) when tested in vitro (p<0.001). The host-plasmid system described

AC

in this study, has applications for the genetic manipulation of this pathogen and potentially other mycoplasmas.

Keywords: Mycoplasma hyorhinis, Suicide Vector, recA gene, Hemolysin, Inactivation

2

ACCEPTED MANUSCRIPT 1. Introduction Mycoplasma hyorhinis (M. hyorhinis) is a common commensal inhabitant of pigs but can become an important pathogen of swine that also causes lung lesions and inflammation (Razin et al., 1998), as well as contamination of laboratory tissue

PT

culture cells. It has also been shown to be associated with oncogenic transformation in

RI

vitro (Namiki et al., 2009). These properties of M. hyorhinis have increased the

SC

interest to the researchers.

Following the determination of the first complete genome sequence of M.

NU

hyorhinis HUB-1 (Liu et al., 2010), genomes of several additional strains have been

MA

deposited in GenBank (Calcutt et al., 2012). However, the lack of genetic tools to generate targeted gene knockouts and to conduct complementation studies hinders the

D

study of gene function in this pathogen. Recently, we have described a plasmid

(Ishag et al., 2016).

PT E

expressing GFP to monitor the transient gene transfer and expression in M. hyorhinis

CE

Gene disruption techniques have been developed for other mycoplasmas such

AC

as transposon mutagenesis (Dybvig and Alderete, 1988, Dybvig and Cassell, 1987, Dybvig et al., 2000, Knudtson and Minion, 1993, Maglennon et al., 2013, Mahairas and Minion, 1989). This technique has also been described for the M. hyorhinis (Dybvig and Alderete, 1988). However, transposon mutagenesis produces random insertions into the genome of mycoplasma, where it can be difficult to isolate desired mutants. Replicative plasmids that contain an origin of replication (oriC) of the mycoplasma along with an antimicrobial resistance marker have also been described

3

ACCEPTED MANUSCRIPT for a few mycoplasmas (Chopra-Dewasthaly et al., 2008, Cordova et al., 2002, Duret et al., 2005, Janis et al., 2005, Lartigue et al., 2003, Lee et al., 2008, Li et al., 2015, Maglennon et al., 2013, Nieszner et al., 2013, Shahid et al., 2014, Sharma et al., 2015). Due to the sequence homology between the targeted gene cloned into the replicative

PT

plasmid and the mycoplasma chromosome, these oriC-based plasmids were used in

RI

some cases to induce targeted gene disruption by homologous recombination.

SC

However, the possibility of having oriC-plasmids integrated at the oriC site of the mycoplasma, extrachromosomal forms or mixtures further complicates subsequent

NU

analysis

MA

High-throughput manipulation of natural or synthetic genomes in yeast through the clustered regularly interspaced short palindromic repeats (CRISPR)/

D

CRISPR-associated (Cas) system has recently been applied for the genome

PT E

engineering of Mycoplasma mycoides subsp. capri GM12 (Mmc) (Tsarmpopoulos et al., 2016). However, cloning and assembly of a synthetic bacterial genome in yeast

CE

followed by a back transplantation into the recipient bacterial cells are both expensive

AC

and technically complex.

Using non-replicative (suicide) plasmids to disrupt target genes and to

generate stable mutants through homologous recombination has been described for Mmc (Allam et al., 2010), Mycoplasma gallisepticum (Burgos et al., 2008), Acholeplasma laidlawii (Dybvig and Woodard, 1992) and Mycoplasma genitalium (Dhandayuthapani et al., 2001, Dhandayuthapani et al., 1999) with a low frequency, probably due to the fact that mycoplasmas are generally lacking efficient

4

ACCEPTED MANUSCRIPT recombination systems that in turn reduces allelic exchange. The product of recA gene is a key protein involved in the DNA recombination and repair (Horii et al., 1980). The E. coli recA gene has been previously used to enhance the homologous recombination as well as recovery of the desired mutants in

PT

Mmc (Allam et al., 2010). Therefore, in this study we aimed to construct a suicide

RI

plasmid pGEMT-recA-tetM/LR harboring an E. coli recA gene in addition to tetM as a

SC

tetracycline selection marker, and to test whether the inclusion of recA in the construct could increase the frequency of generating desired mutants in M. hyorhinis

NU

by homologous recombination. The function of this plasmid was also compared to a

MA

plasmid that lacked the recA gene (pGEMT-tetM/LR). It should be noted that although most if not all Mycoplasma species contain a recA gene, the report by Allam et al.,

D

provided the impetus for including a heterologous recA gene during development of

PT E

this method.

In another mycoplasma species, the product of the hemolysin gene was shown

CE

to lyse RBCs (Kannan and Baseman, 2000). Therefore, in this study we selected the

AC

M. hyorhinis hemolysin gene (GI: 504395455) to be inactivated since this gene potentially confers a hemolytic phenotype that can easily be measured. The resultant hemolysin mutant strain was expected to lose the ability to lyse RBCs when tested in vitro.

5

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Bacterial strains and cultural conditions M. hyorhinis strain HUB-1 (GenBank accession CP002170.1) was kindly provided by Prof. Shaobo Xiao (Huazhong Agricultural University, Wuhan, China)

PT

and was cultured at 37 °C in KM2 cell-free liquid medium (a modified Friis medium)

®

G-10; Gene Company Limited, Chi Wan,

SC

medium, 0.7 % Agar (Biowest Agarose

RI

containing 20 % (v/v) swine serum (Xiong et al., 2016). To prepare KM2-solid

Hong Kong) was added to the KM2 liquid medium. For the growth of the mutants,

NU

tetracycline hydrochloride (Sigma-Aldrich, China) was added at 5.0 µg/ml and 2.5

MA

µg/ml to the liquid and solid KM2 media respectively.

D

2.2 Vector construction

PT E

We used the pGEM®-T vector (Promega, China) to construct the suicide vectors. The E. coli XL-10 competent cells (Vazyme, China) were used to sub-clone

CE

and amplify different constructs. The tetM gene (ID: AGI19285.1) was PCR amplified

AC

from pSE-1 vector provided by Prof. Shaobo Xiao (Huazhong Agricultural University, Wuhan, China) and cloned into pGEM®-T vector at SpeI/PstI sites by in-fusion PCR cloning methods (Vazyme, China). The left arm (LA) of the hemolysin gene (amplified from M. hyorhinis DNA) was fused by PCR to the spiralin gene promoter (from Spiroplasma citri, GI: 2384684, amplified from pSE-1 vector) to form a single fragment which was cloned upstream of the tetM gene at the SpeI site while the right arm (RA) of the hemolysin gene was cloned downstream of tetM gene at the PstI site.

6

ACCEPTED MANUSCRIPT Each hemolysin gene arm contains >400 bp homologous sequence to facilitate recombination at the target site (Dhandayuthapani et al., 2001, Dhandayuthapani et al., 1999, Duret et al., 2005) and were cloned to flank the tetM gene as well as to introduce the spiralin gene promoter. The resultant plasmid was designated

PT

pGEMT-tetM/LR. The recombinase gene (recA), GI:446885887 was then PCR

RI

amplified from E. coli BL-21 DNA, ligated by PCR with the spiralin gene promoter to

SC

form a single fragment and was cloned into pGEMT-tetM/LR by in-fusion PCR cloning at the NcoI site. The resulting plasmid was designated pGEMT-recA-tetM/LR.

NU

All cloning steps were verified by colony PCR and analysis of DNA sequencing. The

MA

diagram of the vector constructions is shown in Figure 1, while the primers used to

D

amplify or ligate the fragments into each, are listed in Table-1.

PT E

2.3 Electroporation of M. hyorhinis

Preparation of competent cells of M. hyorhinis HUB-1 and its electroporation

CE

were performed following previously published methods with minor modifications 10 8 CCU)

AC

(Ishag et al., 2016, Maglennon et al., 2013). Briefly, M. hyorhinis

was harvested by centrifugation at 10,000 × g for 10 min at 4 °C, resuspended and washed twice in HEPES–sucrose buffer (272 mM Sucrose, 200 mM HEPES, pH 7.2) by centrifugation as above. The competent cells were finally resuspended in 100 µl HEPES–sucrose buffer and were transformed immediately. Fo h

ansfo ma ion, 20 μg of p asmid DNA was mix d wi h 100 µ omp

n

cells and the DNA–cell mixture was transferred to a chilled 0.2 cm gap-size Gene

7

ACCEPTED MANUSCRIPT Pulser cuvette (Bio-Rad, Hercules, CA, USA). The mixture was incubated on ice for 10 min. Electroporation was performed using an ECM 630 Electroporation System Ha va d Appa a s BTX, Ho is on, MA, USA) a 2.5 KV, 125 Ω, and 25 µF. Immediately following electroporation, 900 µl chilled KM2-medium was added to the

PT

transformed cells. The cells were incubated on ice for 10 min, and recovered at 37 °C

RI

for 3 hours. Transformants were diluted 1:10 in KM2 medium, palted on KM2-agar

SC

containing 2.5 µg/ml tetracycline and grown at 37 °C. After 4-7 days, the tetracycline-resistant colonies were grown in KM2 liquid medium containing 5 µg/ml

MA

NU

tetracycline.

2.4 Analysis of the hemolysin gene inactivation mycoplasma

transformants

containing

pGEMT-tetM/LR

and

D

Putative

PT E

pGEMT-recA-tetM/LR plasmids were plated on KM2-Agar containing 2.5 µg/ml tetracycline. The individual tetracycline resistant colonies were verified for insertion

CE

of tetM in the genome of M. hyorhinis by PCR using the hemolysin gene flanking

AC

primers shown in Table 1 to observe any difference in the size of the PCR product. The large PCR product (expected to contain tetM gene, 1917 bp directed by the spiralin gene promoter and flanked by the hemolysin gene arms) was further analyzed by tetM-PCR using universal tetM gene screening primers (Table-1) (Sharma et al., 2014). This PCR could produce predicted 339 bp amplicon from tetM if tetM insertion had occurred. Finally, the large PCR product (hemolysin mutant) was submitted to the DNA sequencing (GeneScript Company, China) using hemolysin gene flanking

8

ACCEPTED MANUSCRIPT primers shown in (Table-1) to confirm the presence of tetM gene at the desired site.

2.5 Analysis of loss of phenotype in the mutants The mutant and wild-type clones were sub-cultured in KM2 medium containing

PT

5 µg/ml tetracycline and the culture was centrifuged at 10,000 × g for 10 min at 4 °C.

RI

The supernatants were collected and examined for the hemolysis activity using mouse

SC

RBCs collected from BALB/c mice (2 weeks old) purchased from the Animal Center of Nanjing Army Hospital (Nanjing, China). The hemolytic activity was determined

NU

as previously described (Chu and Holt, 1994, Kannan and Baseman, 2000, Takamatsu

MA

et al., 2001) with minor modifications. Briefly, RBCs were incubated with the supernatants at 37 °C for 2 hours. Negative control samples were incubated with PBS.

D

The samples were then centrifuged at 1,500 × g for 10 min and the released

PT E

hemoglobin was measured at 405 nm in a spectrophotometer. All the reactions were

CE

performed in triplicate.

AC

Statistical analysis

Data obtained from three individual experiments, were recorded as Mean ±

SD, and subjected to one-way analysis of variance (ANOVA) using SPSS (version 16.0, SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.

9

ACCEPTED MANUSCRIPT 3. Results 3.1 Vector construction In the current study, DNA fragments containing tetM, recA, hemolysin gene arms and spiralin gene promoter were PCR amplified and individually cloned into the plasmid

to

yield

the

desired

pGEMT-tetM/LR

(Fig.1A).

and

PT

pGEM®-T

SC

RI

pGEMT-recA-tetM/LR suicide vectors (Fig.1B).

3.2 Electroporation of M. hyorhinis and detection of recombinants

NU

The transformation of M. hyorhinis by polyethylene glycol (PEG) has

MA

previously been described (Dybvig and Alderete, 1988). However, in this study the suicide plasmids were introduced into M. hyorhinis competent cells by the

D

electroporation method (Maglennon et al., 2013). The electric field, electric resistance,

PT E

and the amount of the DNA required for the transformation were optimized. With optimal conditions of 20 µg plasmid DNA and an electroporation set at 2.5 kV, 125 Ω

CE

and 25 μF, we obtained average transformation frequencies (transformants/CFU) of

AC

about 3.2×10-4 and 4×10-3 for pGEMT-tetM/LR and pGEMT-recA-tetM/LR respectively. Tetracycline-resistant colonies selected on mycoplasma KM2-Agar plates supplemented with 2.5 µg/ml tetracycline, could be visualized under the microscope on day-3 post-incubation at 37 °C and by naked eye on day-5 (Fig.2). Importantly, we did not observe any significant difference in the growth rate between the hemolysin wild-type and mutant clones of M. hyorhinis which indicates that the hemolysin gene is not an essential gene for bacterial growth and survival in vitro.

10

ACCEPTED MANUSCRIPT 3.3 Targeted hemolysin inactivation Following targeted insertion of the tetM gene along with the spiralin gene promoter into the hemolysin gene site, a predicted fragment of hemolysin (724 bp) will be replaced with 2,230 bp (tetM gene plus spiralin gene promoter) through

PT

homologous recombination event (Fig.3). Thus, the total size of the wild-type

RI

hemolysin gene (no insertion) is 1566 bp while the mutant hemolysin gene locus will

SC

be 3072 bp. To confirm this, we extracted DNA from M. hyorhinis culture transformed with pGEMT-tetM/LR or pGEMT-recA-tetM/LR plasmids and analyzed

NU

the insertion of the tetM gene at the hemolysin gene site (Fig.3).

MA

hemolysin gene flanking primers.

by PCR using

We performed several experiments to evaluate disruption of the hemolysin

D

gene by homologous recombination using our constructs and to evaluate whether

PT E

inclusion of the recA gene in the construct, could improve the targeted gene disruption. In three independent transformation experiments with the basic (or parental) suicide

CE

vector having no recA gene (pGEMT-tetM/LR), only 0 of 30 (0%), 1 of 50 (2%) and 1 on s s

n d, was id n ifi d as a h mo ysin

AC

of 18 (5.5%) tetracycline- sis an

mutant with average 2% (Fig.4A, Fig.4B and Fig.4C). It also reveals that, an about 96 tetracycline resistant colonies appear with no added DNA. However, inclusion of E. coli recA in the construct, improved the generation of targeted hemolysin mutants. Indeed, in three independent transformation experiments using plasmid encoding recA gene (pGEMT-recA-tetM/LR) to create a targeted mutation at the hemolysin gene site, the number of hemolysin mutants detected have increased. Among the

11

ACCEPTED MANUSCRIPT tetracycline-resistant clones screened by PCR using hemolysin gene flanking primers, we have detected 2 of 8 (25%), 2 of 11 (18.2%) and 3 of 22 (13.6%) hemolysin mutant with average 18.9% (Fig.5A and Fig.5B and Fig.5C). Thus, an about 34 tetracycline resistant colonies appear with no added DNA. We conclude that,

PT

inclusion of the E. coli recA gene in the construct resulted in an approximately 9-fold

RI

increase in the percentage of the transformants that had the desired mutation.

SC

Furthermore, in each transformation experiment with pGEMT-recA-tetM/LR, we could detect at least two hemolysin mutant transformants indicating the usefulness of the

NU

recA gene to recover the desired mutant. However, screening of more resistant

MA

colonies is required to fully enumerate the efficacy of recA gene inclusion in this construct.

D

The mutant hemolysin PCR products of 3072 bp (2 with pGEMT-tetM/LR and

PT E

7 for pGEMT-recA-tetM/LR) were further subjected to the tetM-PCR analysis using tetM gene screening primers (Table-1) o onfi m h p s n

of h tetM gene at the

CE

desired site. If the tetM gene had inserted into the hemolysin gene site, the tetM-PCR

AC

could produce a predicted 339 bp. The tetM-PCR analysis of the hemolysin mutant PCR product has confirmed that tetracycline resistance was correlated with the presence of the tetracycline determinant in the hemolysin mutant strain. We used the hemolysin gene flanking primers forward and reverse (mapped in Fig.3) to sequence the mutant PCR product generated by the hemolysin gene flanking primers and to confirm the location, precise site of insertion, and direction of insertion of tetM gene along with spiralin gene promoter into the hemolysin gene site. To cover

12

ACCEPTED MANUSCRIPT the entire insertion, we designed an additional forward primer (tetM-middle-F, Table-1) from the C-terminal of the forward sequencing product and was used to sequence the middle part of the insertion. Assembly of the sequencing reactions and analysis of the resultant sequence showed the presence of the tetM gene along with

PT

the spiralin gene promoter flanked by the hemolysin gene sequence. Two PCR

RI

products that correspond to the wild-type hemolysin gene (prepared with hemolysin

SC

gene flanking primers) were also subjected to the DNA sequencing using hemolysin gene flanking primers. As expected, analysis of the sequencing product revealed that

NU

no insertion occurred at this region (data not shown).

MA

Two colonies containing the hemolysin gene mutation generated by each of pGEMT-tetM/LR and pGEMT-recA-tetM/LR were further passaged three times in the

D

presence of tetracycline to evaluate the stability of the disruption. The tetM-specific

PT E

PCR confirmed the presence of tetM gene in the mutant strain, indicating the stability of the targeted gene disruption.

CE

As an alternative integration event, we considered the possibility that the

AC

whole plasmid could integrate at either arm of the hemolysin gene by a single cross-over recombination. We chose to investigate the integration of pGEMT-tetM/LR by a single cross over at the hemolysin right arm site (Fig.6SA). We designed two primers to investigate this hypothesis. The forward primer (single-cross-F, Table-1) was designed to bind the upstream region of the hemolysin right arm (this region will be deleted in case of the double-cross over homologous recombination but not in single cross-over event), while the reverse primer (single-cross-R,Table-1) was

13

ACCEPTED MANUSCRIPT designed to bind the tetM gene present in the pGEMT-tetM/LR plasmid. If a single cross-over had occurred at this region, we would obtain a PCR fragment of 1017 bp. However, this fragment will be absent in a double-cross over occurrence (the single-cross-F binding site will be deleted). Furthermore, the single cross-over will

PT

retain hemolysin gene function. We extracted the DNA from the hemolysin mutant

RI

samples (2 with pGEMT-tetM/LR and 7 for pGEMT-recA-tetM/LR) and screened for

SC

the single cross-over using single-cross primers. In our experiment, we failed to obtain a positive PCR product (Fig.6SB), indicating that the insertion of tetM gene

NU

along with spiralin gene promoter at the hemolysin gene site is due to the double

MA

cross-over homologous recombination at the hemolysin gene site rather than a single cross-over. However, the presence of the wild-type hemolysin gene could also

D

indicate spontaneous tetracycline resistance colonies or the integration of the full

PT E

plasmid outside the hemolysin gene "the latter scenario is considered to be unlikely to happen due to the absence of sequence homology between the vector and the

AC

CE

M.hyorhinis genome except for the hemolysin gene arms".

3.4 Analysis of the RBCs hemolysis phenotype In our preliminary experiment, we observed that supernatants obtained from the culture of M. hyorhinis could lyse mouse RBCs (data not shown). Therefore, the supernatants collected from the M. hyorhinis harboring the disrupted hemolysin gene were expected to reduce the ability to lyse the RBCs. To test this phenotype, the wild-type (a positive control) and hemolysin mutant M. hyorhinis (2 obtained with

14

ACCEPTED MANUSCRIPT pGEM-tetM/LR and 7 with pGEM-recA-tetM/LR) were grown in KM2 medium and the supernatants were collected and used to assay the hemolysis activity. RBCs were also incubated with PBS as a negative control. We found that the supernatant (un-diluted) collected from all the hemolysin mutant cultures, have a diminished

PT

hemolysis activity compared to the positive control (Fig.7), which indicated the

RI

usefulness of these vectors to precisely inactivate the hemolysin gene of M. hyorhinis

SC

and should be further applicable to the study of additional target genes.

NU

4. Discussion

MA

Currently, there are no available genetic manipulation systems to specifically inactive genes in M. hyorhinis. As a result, studies to identify the virulence factors

D

and the pathogenesis in M. hyorhinis are hindered (Halbedel and Stulke, 2007, Pilo et

PT E

al., 2007). A promising method to study mycoplasma genetics and pathogenesis is to transform the pathogen with plasmids contain regions homologous to the

CE

chromosomal DNA that results in targeted gene inactivation via homologous

AC

recombination. Here, we attempted to develop suicide vectors having small regions of homology with chromosomal DNA (~ 400-450 bp) and to evaluate their potential to induce targeted insertional mutagenesis in the genome of M. hyorhinis. We also compared the effect recA gene incorporation by comparing the mutagenesis frequency between

pGEM-recA-tetM/LR

with

the

plasmid

having

no

recA

gene

(pGEM-tetM/LR). We observed that inclusion of the recA gene in the suicide vector improved the frequency of obtaining mutation among the screened tetracycline

15

ACCEPTED MANUSCRIPT resistance colonies. I ’s w

-know that, the recA gene is the only recombination gene

universally present in mycoplasmas (Allam et al., 2010), and that in at least some species, the gene is not essential (French et al., 2008). However, the recombination event in Mollicutes probably depends on recA gene (Dybvig and Woodard, 1992,

PT

Ogasawara et al., 1991, Rocha et al., 2005). In a previous publication, it has been

RI

suggested that, having a divergent gene sequence (with a constitutive promoter) might

SC

reduce instability issues with the endogenous recA gene and may augment the homologous recombination (Allam et al., 2010). To test this hypothesis with M.

NU

hyorhinis, we first analyzed the similarity index between the E. coli recA gene and

MA

that other candidate recombinase genes of M. hyorhinis such as recA gene (GI: 504896985) or recR gene (GI: 503066962) to exclude the possibility of homologous

D

recombination with a host recombinase gene. Following Blastn analysis, we observed

PT E

that, there is no significant similarity between E. coli recA gene used in our constructs and the recombinase genes of M. hyorhinis. Furthermore, our results are consistent

CE

with the above observation, and indicated that, inclusion of recA gene in the construct

AC

increased the mutation recovery from 3.8% to 18.9% (5 fold-increase). However, this 5 fold-increase in the disruption efficiency obtained by the pGEM-recA-tetM/LR plasmid, is considered low compared to the 140-fold increase obtained with the pExp1-ctpA::tetM-recAec plasmid developed by Allam (Allam et al., 2010) for Mmc. This reduced disruption efficiency observed in our study, may be explained by the difference in the promoters (we used the spiralin gene promoter from another species) used in the constructs and the orientation of the tetM gene. Importantly, all hemolysin

16

ACCEPTED MANUSCRIPT disrupted mutants that were obtained using pGEM-recA-tetM/LR occurred by a double-crossover homologous recombination which suggest that, this disruption may be stable. Compared to the mutant obtained by means of the plasmid integration into the genome of the species (transposon mutagenesis or oriC-plasmids), we assume that,

PT

the mutants obtained with pGEMT-recA-tetM/LR may not contain remnants of the

RI

plasmid backbone, which may also enhance the stability. Absorbance at 405 nm has

SC

been used to evaluate the hemolysis of mouse erythrocytes following previously described methods (Gleibs et al., 1995). In our study, we observed we observed that

MA

mouse RBCs compared to the wild-type.

NU

the M. hyorhinis strains harboring the hemolysin mutation lacked the ability to lyse

Only a few examples of a successful double-crossover homologous

D

recombination using a suicide plasmid in a mycoplasma have been reported, among

PT E

these are M. genitalium (Burgos et al., 2008, Kannan and Baseman, 2006). The novel vector system described here should help manipulate the M. hyorhinis genome and

AC

bacteria.

CE

eventually facilitate studying the gene functions and pathogenic mechanisms of the

Competing interests The authors declare that they have no competing interests.

Acknowledgments This work was supported by the Postdoctoral Fund of Jiangsu Academy of

17

ACCEPTED MANUSCRIPT Agricultural Sciences, China (6511318) National Natural Sciences Foundation of China (Grant No. 31550110211).

References Allam, A.B., Reyes, L., Assad-Garcia, N., Glass, J.I., Brown, M.B., 2010. Enhancement of targeted

PT

homologous recombination in Mycoplasma mycoides subsp. capri by inclusion of heterologous recA. Applied and environmental microbiology. 76, 6951-6954.

RI

Burgos, R., Pich, O.Q., Querol, E., Pinol, J., 2008. Deletion of the Mycoplasma genitalium MG_217 gene modifies cell gliding behaviour by altering terminal organelle curvature. Molecular microbiology.

SC

69, 1029-1040.

Calcutt MJ1, Foecking MF, Rosales RS, Ellis RJ, Nicholas RA. Genome sequence of Mycoplasma hyorhinis strain GDL-1. Journal of

Bacteriology. 194,1848

NU

Chopra-Dewasthaly, R., Citti, C., Glew, M.D., Zimmermann, M., Rosengarten, R., Jechlinger, W., 2008. Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation. Molecular microbiology. 67, 1196-1210.

MA

Chu, L., Holt, S.C., 1994. Purification and characterization of a 45 kDa hemolysin from Treponema denticola ATCC 35404. Microbial pathogenesis. 16, 197-212. Cordova, C.M., Lartigue, C., Sirand-Pugnet, P., Renaudin, J., Cunha, R.A., Blanchard, A., 2002. Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC

D

replicative plasmids. Journal of bacteriology. 184, 5426-5435.

PT E

Dhandayuthapani, S., Blaylock, M.W., Bebear, C.M., Rasmussen, W.G., Baseman, J.B., 2001. Peptide methionine sulfoxide reductase (MsrA) is a virulence determinant in Mycoplasma genitalium. Journal of bacteriology. 183, 5645-5650.

Dhandayuthapani, S., Rasmussen, W.G., Baseman, J.B., 1999. Disruption of gene mg218 of

CE

Mycoplasma genitalium through homologous recombination leads to an adherence-deficient phenotype. Proceedings of the National Academy of Sciences of the United States of America. 96, 5227-5232. Duret, S., Andre, A., Renaudin, J., 2005. Specific gene targeting in Spiroplasma citri: improved vectors

AC

and production of unmarked mutations using site-specific recombination. Microbiology (Reading, England). 151, 2793-2803. Dybvig, K., Alderete, J., 1988. Transformation of Mycoplasma pulmonis and Mycoplasma hyorhinis: transposition of Tn916 and formation of cointegrate structures. Plasmid. 20, 33-41. Dybvig, K., Cassell, G.H., 1987. Transposition of gram-positive transposon Tn916 in Acholeplasma laidlawii and Mycoplasma pulmonis. Science. 235, 1392-1394. Dybvig, K., French, C.T., Voelker, L.L., 2000. Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis. Journal of bacteriology. 182, 4343-4347. Dybvig, K., Woodard, A., 1992. Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment. Plasmid. 28, 262-266. French, C.T., Lao, P., Loraine, A.E., Matthews, B.T., Yu, H., Dybvig, K., 2008. Large-scale transposon mutagenesis of Mycoplasma pulmonis. Molecular microbiology. 69, 67-76. 18

ACCEPTED MANUSCRIPT Gleibs, S., Mebs, D., Werding, B., 1995. Studies on the origin and distribution of palytoxin in a Caribbean coral reef. Toxicon: official journal of the International Society on Toxinology. 33, 1531-1537. Halbedel, S., Stulke, J., 2007. Tools for the genetic analysis of Mycoplasma. International journal of medical microbiology : IJMM. 297, 37-44. Horii, T., Ogawa, T., Ogawa, H., 1980. Organization of the recA gene of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America. 77, 313-317. Ishag, H.Z., Liu, M., Yang, R., Xiong, Q., Feng, Z., Shao, G., 2016. GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis. SpringerPlus. 5, 769.

PT

Janis, C., Lartigue, C., Frey, J., Wroblewski, H., Thiaucourt, F., Blanchard, A., Sirand-Pugnet, P., 2005. Versatile use of oriC plasmids for functional genomics of Mycoplasma capricolum subsp. capricolum.

RI

Applied and environmental microbiology. 71, 2888-2893.

Kannan, T.R., Baseman, J.B., 2000. Hemolytic and hemoxidative activities in Mycoplasma penetrans.

SC

Infection and immunity. 68, 6419-6422.

Kannan, T.R., Baseman, J.B., 2006. ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens. Proceedings of the

NU

National Academy of Sciences of the United States of America. 103, 6724-6729. Knudtson, K.L., Minion, F.C., 1993. Construction of Tn4001lac derivatives to be used as promoter probe vectors in mycoplasmas. Gene. 137, 217-222.

MA

Lartigue, C., Blanchard, A., Renaudin, J., Thiaucourt, F., Sirand-Pugnet, P., 2003. Host specificity of mollicutes oriC plasmids: functional analysis of replication origin. Nucleic acids research. 31, 6610-6618.

Lee, S.W., Browning, G.F., Markham, P.F., 2008. Development of a replicable oriC plasmid for

D

Mycoplasma gallisepticum and Mycoplasma imitans, and gene disruption through homologous recombination in M. gallisepticum. Microbiology (Reading, England). 154, 2571-2580.

PT E

Li, J., Zhang, J., Zhang, N., Zhang, Y., Wu, W., Li, J., 2015. Development of a replicative plasmid for gene expression in Mycoplasma bovis. Journal of microbiological methods. 108, 12-18. Liu, W., Fang, L., Li, S., Li, Q., Zhou, Z., Feng, Z., Luo, R., Shao, G., Wang, L., Chen, H., 2010. Complete genome sequence of Mycoplasma hyorhinis strain HUB-1. J Bacteriol. 192, 5844-5845.

CE

Maglennon, G.A., Cook, B.S., Deeney, A.S., Bosse, J.T., Peters, S.E., Langford, P.R., Maskell, D.J., Tucker, A.W., Wren, B.W., Rycroft, A.N., 2013. Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations. Veterinary

AC

research. 44, 124.

Maglennon, G.A., Cook, B.S., Matthews, D., Deeney, A.S., Bosse, J.T., Langford, P.R., Maskell, D.J., Tucker, A.W., Wren, B.W., Rycroft, A.N., 2013. Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae. Veterinary research. 44, 63. Mahairas, G.G., Minion, F.C., 1989. Random insertion of the gentamicin resistance transposon Tn4001 in Mycoplasma pulmonis. Plasmid. 21, 43-47. Namiki, K., Goodison, S., Porvasnik, S., Allan, R.W., Iczkowski, K.A., Urbanek, C., Reyes, L., Sakamoto, N., Rosser, C.J., Tyson, D., 2009. Persistent exposure to Mycoplasma induces malignant transformation of human prostate cells. PLoS ONE. 4, e6872. Nieszner, I., Vronka, M., Indikova, I., Szostak, M.P., 2013. Development of a site-directed integration plasmid for heterologous gene expression in Mycoplasma gallisepticum. PloS one. 8, e81481. Ogasawara, N., Moriya, S., Yoshikawa, H., 1991. Initiation of chromosome replication: structure and

19

ACCEPTED MANUSCRIPT function of oriC and DnaA protein in eubacteria. Research in microbiology. 142, 851-859. Pilo, P., Frey, J., Vilei, E.M., 2007. Molecular mechanisms of pathogenicity of Mycoplasma mycoides subsp. mycoides SC. Veterinary journal (London, England : 1997). 174, 513-521. Razin, S., Yogev, D., Naot, Y., 1998. Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev. 62, 1094-1156. Rocha, E., Sirand-Pugnet, P., Blanchard, A., 2005. Genome analysis: Recombination, repair and recombinational hotspots. In: Blanchard, G. Browning (Eds.), Mycoplasmas: molecular biology, pathogenicity, and strategies for control. Horizon Bioscience, Hethersett, United Kingdom, pp. 31-73. Shahid, M.A., Marenda, M.S., Markham, P.F., Noormohammadi, A.H., 2014. Development of an oriC

PT

vector for use in Mycoplasma synoviae. Journal of microbiological methods. 103, 70-76. Sharma, S., Citti, C., Sagne, E., Marenda, M.S., Markham, P.F., Browning, G.F., 2015. Development

RI

and host compatibility of plasmids for two important ruminant pathogens, Mycoplasma bovis and Mycoplasma agalactiae. PloS one. 10, e0119000. dispensable in Mycoplasma bovis. PloS one. 9, e97100.

SC

Sharma, S., Markham, P.F., Browning, G.F., 2014. Genes found essential in other mycoplasmas are Takamatsu, D., Osaki, M., Sekizaki, T., 2001. Thermosensitive suicide vectors for gene replacement in

NU

Streptococcus suis. Plasmid. 46, 140-148.

Tsarmpopoulos, I., Gourgues, G., Blanchard, A., Vashee, S., Jores, J., Lartigue, C., Sirand-Pugnet, P., 2016. In-Yeast Engineering of a Bacterial Genome Using CRISPR/Cas9. ACS synthetic biology. 5,

MA

104-109.

Xiong, Q., Wang, J., Ji, Y., Ni, B., Zhang, B., Ma, Q., Wei, Y., Xiao, S., Feng, Z., Liu, M., Shao, G., 2016. The functions of the variable lipoprotein family of Mycoplasma hyorhinis in adherence to host

Figure legends

PT E

D

cells. Veterinary Microbiology. 186, 82-89.

CE

Fig.1 pGEMT-tetM/LR and pGEMT-recA-tetM/LR construction: The tetM gene was cloned into pGEM®-T vector at the SpeI/PstI sites, Hemolysin gene left arm spliced

AC

with spiralin gene promoter (LA/Sp) was cloned at SpeI site, Hemolysin gene right arm (RA) was cloned at the SalI to yield pGEMT-tetM/LR plasmid (A). Finally, the recA gene spliced with spiralin gene promoter was cloned into the pGEMT-tetM/LR plasmid at NcoI site. The resultant vector designated pGEMT-recA-tetM/LR (B).

Fig.2 Phenotype of the colonies of M. hyorhinis cells ( 10 8 CCU) electroporated with pGEMT-tetM/LR (B) and pGEMT-recA-tetM/LR (C) plasmids and grown on 20

ACCEPTED MANUSCRIPT KM2-Agar containing 2.5 µg/ml tetracycline at 37 °C for 5 days. Untransformed control colonies were shown in (A). The mutant colonies harboring the modified DNA were indicated by lines.

at

the

hemolysin

gene

site

using

pGEMT-tetM/LR

and

RI

promoter

PT

Fig.3 Prediction of the possible insertion of tetM gene along with spiralin gene

SC

pGEMT-recA-tetM/LR plasmids. If the insertion at the hemolysin gene site had occurred, a fragment of about 3072-bp could be detected by PCR using hemolysin

NU

gene flanking primers (Table-1). In the absence of insertion, the wild-type hemolysin

MA

gene could be detected (1566-bp). The precise location of the hemolysin gene flanking primers in the genome was mapped in the figure and further indicated by a

PT E

D

black arrow.

Fig.4 Investigation of tetM gene insertion by PCR (using hemolysin gene flanking

CE

primers, Table-1) following transformation with pGEMT-tetM/LR. Wild-type

AC

hemolysin (no insertion) exhibits 1566 bp, while the mutant hemolysin gene produce about 3072 bp. (A), (B) and (C), represent the repeats of the experiments. Lane 1 is control un-transformed; others are the analyzed resistant clones.

Fig.5 Investigation of tetM gene insertion by PCR (using hemolysin gene flanking primers, Table-1) following transformation with pGEMT-recA-tetM/LR, as described in (Fig.4). (A), (B) and (C), represents repeats of the experiments. (A-B): Lane 1 is

21

ACCEPTED MANUSCRIPT control un-transformed, others are the analyzed clones. (C): Lane: 23 control, lanes 1-22 are the analyzed clones.

Fig.6 Prediction and investigation of a single cross-over event. The two suicide

PT

plasmids were shown in (A), the hemolysin gene location in (B) and the prediction of If a

RI

the plasmid integration at the right arm of the hemolysin gene was shown in (C).

SC

single cross-over had occurred at this region, we expected to get a PCR product (using single-cross primer-F and single-cross primer-R, Table-1) of 1017 bp. DNA was

NU

extracted from the 7 hemolysin mutant samples (2 with pGEMT-tetM/LR and 7 for

MA

pGEMT-recA-tetM/LR) and screened by PCR to detect the possible occurrence of a single cross-over. In our experiment, we failed to obtain a positive PCR product (D),

D

indicating the insertion of tetM gene along with spiralin gene promoter is due to the

PT E

double cross-over homologous recombination.

CE

Fig.7 Hemolysis activity of wild-type M. hyorhinis and hemolysin mutant M.

AC

hyorhinis. Supernatant of wild-type M. hyorhinis and hemolysin mutant M. hyorhinis were collected, serially 2-fold diluted and incubated with mouse RBCs. PBS was used as a negative control. The released hemoglobin was determined by measuring the optical density at 405 nm in a spectrophotometer. All reactions were performed in triplicate. P1 and P2 are pGEMT-tetM/LR and pGEMT-recA-tetM/LR plasmids respectively. Data presented as Means ± SD (n= 3). p< 0.001 versus control.

22

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

Fig. 1

23

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

Fig. 2

24

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

Fig. 3

25

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

Fig. 4

26

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

Fig. 5

27

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

Fig. 6

28

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Fig. 7

29

ACCEPTED MANUSCRIPT Table-1 List of primers used to construct pGEM-tetM/LR and pGEM-recA-tetM/LR plasmids

tetM gene

Primer Name

tetM

(no promoter)

Hemolysin-LA-splicing Hemolysin -LA spliced to

Primer sequence (5´-3´)

Product

site

F: GAAATATAAGAAACTAGTATGAAAATTATTAATATTGGAGTTTTAGCTCATGTTGATGC 1917

SpeI/

R: TTCGATTGGTCGACCTGCAGTTATTTTATTGAACATATATCGTACTTTATCTATCCG

PstI

F: GCGGGATATCACTAGTCCAGGCGCACTTACAAAAGATCAC

437 SpeI

R: CTTCACTGTTTTCTTGTTCACTAACATCACAATATCAGCATCTTGCTCGATAG Spiralin gene

F: TGCTGATATTGTGATGTTAGTGAACAAGAAAACAGTGAAGCACCAG

form a single fragment

promoter-splicing-1

R:AATTTTCATACTAGTTTCTTATATTTCCTTTCTCTATTAAGTAGTGTTTTTATTAAA

SC

AGC Hemolysin-RA

313

RI

Spiralin gene promoter to

Hemolysin -RA

Cloning

(bp)

PT

Target Amplicon

F:ATAACTGCAGGTCGACAATCGAAGCTTGATTAGAACATCATAGC

405

SalI

R:CTCCCATATGGTCGACTACTACAATTACTGCTTTCCGAGTTATTAAAATAC

RecA gene spliced to

F: GAAAGGAAATATAAGAAATGGCTATCGACGAAAACAAACAG

NU

RecA- splicing

NcoI

R: TATCCCGCGGCCATGGTTAAAAATCTTCGTTAGTTTCTGCTACGCCT Spiralin gene

F: CTCCCGGCCGCCATGTTAGTGAACAAGAAAACAGTGAAGCACCAG

form a single fragment

promoter-splicing-2

R:TTTCGTCGATAGCCATTTCTTATATTTCCTTTCTCTATTAAGTAGTGTTTTTATTAAAA

1566

-

F: GCAGTTATGGAAGGGATACG

339

-

1017

-

-

-

G Hemolysin flanking

gene product

313

MA

Spiralin gene promoter to

Hemolysin flanking

1062

F: CAATTAGCACGTGAATTAGACACACCG

tetM

tetM

screening

D

R: CCATAATTAGCCTTCATTTTTCTTTGTGATTTGAATTTC

Single cross-over

PT E

R: TTCTTGAATACACCGAGCAG

Single cross-over

predicted fragment

R: GCCCTGTTAGTACCCCAGCAGATTTTC

tetM middle-F

F: GTGTGTGTGACGAACTTTACCGAATCTG

CE

Middle part of the tetM

F: GAGTCAAGGTTTTCAAGGATTCCAGTTAG

AC

TetM = tetracycline hydrochloride, LA = left arm and RA = right arm.

30

ACCEPTED MANUSCRIPT

PT

Highlights  The pGEMT-tetM/LR and pGEMT-recA-tetM/LR suicide vectors have been constructed.  These vectors were used to disrupt Hemolysin gene by inserting the tetM gene at the hemolysin site.  Inclusion of recA gene in the constructs improved the gene targeting efficiency.  Hemolysin mutants have diminished ability to lyse mouse erythrocytes.

AC

CE

PT E

D

MA

NU

SC

RI

]

31