Complete nucleotide sequence of plasmid pST-III from Lactobacillus plantarum ST-III Chen Chen a,b , Lianzhong Ai b , Fangfang Zhou b , Jing Ren b , Kejie Sun b , Hao Zhang c , Wei Chen a,⇑ , Benheng Guo b,⇑ a State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China b State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co. Ltd., 1518 West of Jiangchang Road, Shanghai 200436, China c School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China article info Article history: Received 23 July 2011 Accepted 15 December 2011 Available online 24 December 2011 Communicated by Dr. W.F. Fricke Keywords: Kdp system Lactobacillus plantarum Osmolyte transport Theta-type mechanism abstract The complete nucleotide sequence of the 53,560-bp plasmid pST-III from Lactobacillus plan- tarum ST-III has been determined. The plasmid contains 42 predicted protein-coding sequences, and the functions of 34 coding sequences could be assigned. Homology analysis for the replication protein and the typical features of the origin of replication suggested that pST-III replicates via the theta-type mechanism. Among the predicted genes, we iden- tified a kdp gene cluster (a high-affinity K + -transport system) for the first time in the Lac- tobacillus genus and a system for osmolyte transport. Analysis of the plasmid-encoded functions and the plasmid-cured experiment showed that the genes of pST-III could serve for the niche adaptations of L. plantarum ST-III and make significant contributions to its via- bility under hyperosmotic conditions. Furthermore, the relative copy number of pST-III was determined to be 6.79 ± 1.55 copies per cell. Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved. 1. Introduction Lactobacillus plantarum, an important lactic acid bacte- rium (LAB), is widely used as starter cultures for produc- tion of food and feed materials. It is a flexible and versatile species that is encountered in a variety of envi- ronmental niches, including some dairy, meat, and many vegetable or fermenting plants as well as the gastrointesti- nal tracts of humans and animals (Kleerebezem et al., 2003). The ecological flexibility of L. plantarum is reflected by the observation that this species has one of the largest chromosome known among LAB and many strains harbor one or more natural plasmids. In recent years, plenty of plasmids of L. plantarum have been sequenced and characterized. Analysis of the results have shown that some important properties, such as bacte- riocin synthesis, toxin–antitoxin systems, antibiotic resis- tance and bacteriophage resistance (Danielsen, 2002; Eguchi et al., 2000; Feld et al., 2009; Sorvig et al., 2005; Van Reenen et al., 2006) are encoded on plasmids. These properties are always related to niche adaptation of the strains under extreme conditions and on the other hand, have much application value such as bioactive metabolites production or use as selectable markers. L. plantarum ST-III, originally isolated from Chinese pickle, exhibits many probiotic properties such as choles- terol removal (Liu et al., 2008) and strong adhesion to Caco-2 cells (Chen et al., 2007). The complete nucleotide se- quence of the genome of L. plantarum ST-III has been deter- mined and a plasmid named pST-III was first discovered (Wang et al., 2011). The genes carried by pST-III were found to be related to the adaptation to the pickle environment and have attracted our great interest. Here, we present 0147-619X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2011.12.005 ⇑ Corresponding authors. Address: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China. Fax: +86 510 85913583 (W. Chen), +86 21 66553572 (B. Guo). E-mail addresses: [email protected](W. Chen), awy7677@ hotmail.com (B. Guo). Plasmid 67 (2012) 236–244 Contents lists available at SciVerse ScienceDirect Plasmid journal homepage: www.elsevier.com/locate/yplas
Transcript
Plasmid 67 (2012) 236–244
Contents lists available at SciVerse ScienceDirect
The complete nucleotide sequence of the 53,560-bp plasmid pST-III from Lactobacillus plan-tarum ST-III has been determined. The plasmid contains 42 predicted protein-codingsequences, and the functions of 34 coding sequences could be assigned. Homology analysisfor the replication protein and the typical features of the origin of replication suggestedthat pST-III replicates via the theta-type mechanism. Among the predicted genes, we iden-tified a kdp gene cluster (a high-affinity K+-transport system) for the first time in the Lac-tobacillus genus and a system for osmolyte transport. Analysis of the plasmid-encodedfunctions and the plasmid-cured experiment showed that the genes of pST-III could servefor the niche adaptations of L. plantarum ST-III and make significant contributions to its via-bility under hyperosmotic conditions. Furthermore, the relative copy number of pST-III wasdetermined to be 6.79 ± 1.55 copies per cell.
Crown Copyright � 2011 Published by Elsevier Inc. All rights reserved.
1. Introduction
Lactobacillus plantarum, an important lactic acid bacte-rium (LAB), is widely used as starter cultures for produc-tion of food and feed materials. It is a flexible andversatile species that is encountered in a variety of envi-ronmental niches, including some dairy, meat, and manyvegetable or fermenting plants as well as the gastrointesti-nal tracts of humans and animals (Kleerebezem et al.,2003). The ecological flexibility of L. plantarum is reflectedby the observation that this species has one of the largestchromosome known among LAB and many strains harborone or more natural plasmids.
2011 Published by Elsevier
atory of Food Scienceechnology, Jiangnanina. Fax: +86 510
. Chen), awy7677@
In recent years, plenty of plasmids of L. plantarum havebeen sequenced and characterized. Analysis of the resultshave shown that some important properties, such as bacte-riocin synthesis, toxin–antitoxin systems, antibiotic resis-tance and bacteriophage resistance (Danielsen, 2002;Eguchi et al., 2000; Feld et al., 2009; Sorvig et al., 2005;Van Reenen et al., 2006) are encoded on plasmids. Theseproperties are always related to niche adaptation of thestrains under extreme conditions and on the other hand,have much application value such as bioactive metabolitesproduction or use as selectable markers.
L. plantarum ST-III, originally isolated from Chinesepickle, exhibits many probiotic properties such as choles-terol removal (Liu et al., 2008) and strong adhesion toCaco-2 cells (Chen et al., 2007). The complete nucleotide se-quence of the genome of L. plantarum ST-III has been deter-mined and a plasmid named pST-III was first discovered(Wang et al., 2011). The genes carried by pST-III were foundto be related to the adaptation to the pickle environmentand have attracted our great interest. Here, we present
the nucleotide sequence analysis and characterization ofthe plasmid.
2. Materials and methods
2.1. Bacteria and growth conditions
L. Plantarum ST-III had been isolated from samples ofhomemade pickle in China and been deposited in the ChinaGeneral Microbiological Culture Collection Center with thenumber CGMCC No. 0847. It was routinely cultured anaer-obically in MRS broth (Merck KGaA, Darmstadt, Germany)at 37 �C for 12–14 h.
2.2. Nucleotide sequence analysis
The nucleotide sequence of pST-III had been obtainedfrom the whole-genome shotgun sequencing of L. planta-rum ST-III (Wang et al., 2011). Open reading frames (ORFs)containing more than 30 amino acid residues were pre-dicted using Glimmer 3.0 (http://cbcb.umd.edu/software/glimmer/) and verified by comparison with other closelyrelated genome sequences. Potential protein-coding se-quences were subsequently manually analyzed by data-base searches using the BLAST suite of programs (http://www.ncbi.nlm.nih.gov/, including blastp, blastn, clustersof orthologous groups [COG] and conserved domain data-base [CDD]). Interproscan (http://www.ebi.ac.uk/interpro/) was used for domain characterization and the Interpronumber is listed in Table 1. Functional information forthe predicted proteins was obtained from the UniProtKnowledgebase (http://www.uniprot.org). Phylogenetictrees were constructed by the Neighbor-Joining methodwith the Mega 4.0 program after aligning the sequencesusing the computer program ClustalW. The completenucleotide sequence of pST-III has been deposited in theGenBank database under Accession No. CP002223.
2.3. Plasmid extraction and curing
Overnight culture of L. plantarum ST-III was used for plas-mid extraction by the alkaline lysis method as describedpreviously (O’Sullivan and Klaenhammer, 1993). Plasmidcuring was done as described previously (Ruiz-Barba et al.,1991) with some modification. Briefly, L. plantarum ST-IIIwas incubated 72 h at 42 �C in MRS containing 0.25 lg/mlnovobiocin. Then treated cells were diluted in saline and ali-quots (0.1 ml) of the appropriate dilution were plated ontononselective MRS agar plates that were incubated at 37 �C.After 16 h of incubation, isolated colonies were randomlyselected and analyzed for the lack of plasmids by electro-phoresis in a 0.7% agarose gel. The result was confirmed byPCR analysis using one pair of the pST-III sequencing prim-ers. The plasmid-cured strain was named as ST-III-PC.
2.4. Hyperosmotic resistance by the wild-type and plasmid-cured strains of ST-III
Three hundred milliliters of fresh MRS broth with 0%, 2%,4% (m/v) NaCl were inoculated with 1.0% (v/v) overnight cell
suspensions of ST-III or ST-III-PC, respectively (OD600nm =0.06). The hyperosmotic resistance was measured by thelength of incubation time for OD600nm value to reach 0.5(mid-log phase, approximately 3 � 108 cfu/mL) from inocu-lation. Each assay was conducted in triplicate. Statisticalanalysis of data was performed using Student’s t test pro-vided by SPSS 11.0 software (SPSS Inc., Chicago, USA), inwhich P < 0.05 was considered to be significantly different.
2.5. Relative copy number determination
The relative copy number of pST-III was determined byreal-time PCR according to the relative quantification meth-od (Lee et al., 2006; Providenti et al., 2006) with some mod-ification. Total DNA was extracted as described previously(te Riele et al., 1986). Amplification and detection were car-ried out in an ABI 7300 Real-time PCR System (Applied Bio-systems Inc., Carlsbad, USA) using a SYBR Green PCR MasterMix (Applied Biosystems Inc.). PCR primers were designedusing Primer Premier 5.0 software. The elongation factorTu gene tuf (GenBank Accession No. YP_003925060.1), a sin-gle-copy gene in the chromosome of L. plantarum ST-III, andthe repB gene in pST-III were used as reference gene and tar-get gene, respectively. A 162-bp fragment of the tuf gene wasamplified with primers tuf-F (50-CTTCAACAGGCATTAA-GAAAGGC-30) and tuf-R (50-TCCCTGGTGACGATATTCCTGT-30), and a 156-bp fragment of the rep gene used as a probewas amplified with primers rep-F (50-GGATAACCTTGACC-TAATTCCTTCA-30) and rep-R (50-TGCACATCCAACAGAATTA-CATCA-30). Then real-time PCR was conducted as describedand each assay was performed in triplicate. The relativecopy number of pST-III was calculated from the formula:Nrelative ¼ E�DCT , where E and DCT represent the PCR amplifi-cation factor and the difference between the threshold cyclenumbers CT of the repB and tuf reactions, respectively. Ecould be calculated according to the slope of the standardcurve by the equation: E ¼ 10�1=slope and equals two if theproduct amplifies at 100% efficiency (Pfaffl, 2001).
3. Results
3.1. General features
pST-III is a circular plasmid of 53,560 bp (Fig. 1), which isone of the largest completely sequenced plasmids isolatedfrom L. plantarum. The average G + C content of pST-III is38.69%, lower than that of the L. plantarum ST-III chromo-some (44.58%). Forty-five potential genes (containing morethan 30 amino-acid residues) were identified including threepseudogenes. All but 13 of the putative genes were tran-scribed in the lagging replication strand (Fig. 2, Table 1).Among the 42 predicted protein-coding sequences, 33 ofthem were assigned by homologous sequence search and do-main characterization, whereas nine genes could only beannotated as hypothetical proteins (more details in Table 1).
3.2. Replicons
The first ORF, repB, encodes a 286-amino acid proteinthat shows homology with replication proteins from
IPR008250, IPR01830330 YP_003927894.1 kdpA 590 Potassium-transporting ATPase subunit A(KdpA) IPR00462331 YP_003927895.1 orf31 276 Putative mononucleotidyl cyclase IPR00105432 YP_003927896.1 uvrX 433 ImpB/MucB/SamB family protein(UvrX) IPR001126, IPR01796333 YP_003927897.1 orf33 106 Hypothetical protein –34 YP_003927898.1 orf34 131 Hypothetical protein –35 YP_003927899.1 orf35 381 Hypothetical protein –36 YP_003927900.1 orf36 374 Transposase –37 YP_003927901.1 orf37 686 Nickase IPR00505338 YP_003927902.1 orf38 69 Hypothetical protein –39 YP_003927903.1 orf39 92 Hypothetical protein –40 YP_003927904.1 orf40 104 Hypothetical protein –41 YP_003927905.1 orf41 93 DNA-damage-inducible protein IPR00733742 YP_003927906.1 orf42 110 Hypothetical protein –
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theta-type replicating plasmids, such as RepB from pNP40of Lactococcus lactis subsp. diacetylactis DRC3 (67% identity)and RepB from pWCFS103 of L. plantarum WCFS1 (37%identity). RepB contains the SoJ (COG1192, from positions18 to 276) and ParA (cd02042, from positions 18 to 56and from positions 142 to 207) domains indicating that itmay also be involved in plasmid and chromosome parti-tioning (O’Driscoll et al., 2006).
In addition to the replication protein, The region up-stream of repB contains typical sequences characteristic
of the replication origins of theta-type replicons (Fig. 3).These include: an AT-rich region that may be the recogni-tion site for host-encoded functions involved in replication(Seegers et al., 1994); a 11-bp sequence directly repeatedthree times (termed iteron), which interacts directly withthe RepB protein to initiate replication (del Solar et al.,1998); and a DnaA box (50-TTATCCCAA-30, two mismatchescompared with the consensus DnaA box 50-TTATCCACA-30),where the host DnaA initiator protein binds (Bramhill andKornberg, 1988). Like many other theta replicons, a pair of
Fig. 1. Plasmid profiles of wild-type strains of L. plantarum ST-III and itscured derivative ST-III-PC. Plasmid DNA was isolated by alkaline lysismethod and electrophoresed in a 0.7% (w/v) agarose gel. Lane M, LambdaMix Marker. Lane 1, wild-type strain of L. plantarum ST-III, pST-III wasindicated by the dark arrows. Lane 2, the plasmid-cured strain ofLactobacillus plantarum ST-III, ST-III-PC.
Fig. 3. Nucleotide sequence of the putative origin of replication and the 50
end of the repB gene. The AT-rich region is displayed on the DNAsequence. The DNA iteron (DR1) and the 11-bp direct repeats (DR2) areindicated by solid arrows, while the 7-bp inverted repeats (IR) areindicated by dashed arrows. The putative DnaA box is underlined with adashed line. The presumed start codon of repB is depicted in bold whereasthe putative �10 promoter region and a possible RBS are also marked.
C. Chen et al. / Plasmid 67 (2012) 236–244 239
11-bp direct repeats (DR) was found between the DnaAbox and the RBS sequence of pST-III, but its function is stilluncharacterized. A pair of inverted repeats (IR) was found
Fig. 2. General features of plasmid pST-III. The outer circle represents the scale(red, leading replication strand; blue, lagging replication strand). The third arespectively.
to overlap the putative �35 regions of the repB promoter,and they probably serve as a RepB protein binding site toregulate its own transcription (Foley et al., 1996).
To elucidate the replication mechanism of pST-III, aphylogenetic tree was constructed using the bacterial plas-mid replicons that replicate via either the double-stranded
in kb. The first two inner circles show the putative protein-coding genesnd the fourth inner circles represent the G + C content and GC-skew,
240 C. Chen et al. / Plasmid 67 (2012) 236–244
theta mechanism or the RC mechanism (Fig. 4). The resultsshowed that the selected plasmids could be divided intotwo groups according to the two replication mechanisms,and pST-III belonged to the theta group. Together, the ge-netic organization, similarities, and iteron structure of thepST-III replication region strongly suggest that this largeplasmid replicates via the theta-type mechanism.
3.3. Plasmid-encoded functions
The plasmid includes 44 ORFs (one ORF is RepB), andamong of them, 33 ORFs have been assigned to biologicalfunctions. The majority of these genes display the highestsimilarity with genes located on other lactobacilli plas-mids, but some biological functions have never been dem-onstrated in this genus before. The most interestingfunctions are presented below.
3.3.1. K+-transport systemIn this study, a typical kdpABCDE locus (orf26-orf30) was
identified in pST-III which showed high degree of similaritywith the genes in the chromosome of Clostridium perfrin-gens ATCC 13124 (CPF_1210 – CPF_1214). The Kdp systemis an inducible high-affinity K+-transport P-type ATPasewhich was first discovered in Escherichia coli (Laiminset al., 1981). The sensor kinase KdpD and the response reg-ulator KdpE control induction of the kdpABC operon underK+ limiting growth conditions and in response to an osmo-tic upshift (Epstein, 2003; Jung and Altendorf, 2002). UnderK+ limiting conditions the Kdp system restores the intracel-
Fig. 4. Phylogenetic tree (Neighbor-Joining) for bacterial plasmid replicons that raccession number of the replication-associated protein of each plasmid is presencategories corresponding to the replication mechanisms. The RepB of pST-III (sh
lular K+ concentration while, in response to osmotic upshift,K+ is accumulated far above the normal content as the pri-mary response (Heermann et al., 2009).
Many experimental results have demonstrated that theKdp system is present in enterobacteria, cyanobacteria,and Pseudomonas aeruginosa (Jung and Altendorf, 2002;Treuner-Lange et al., 1997). However through a greedysearch, we found this had been the first report of a Kdp sys-tem in the Lactobacillus genus. In neighboring genus, theKdp system has also been discovered in Lactococcus lactissubsp. lactis strains KF147 and KF282. In fact, both of themwere also isolated from plant materials (Siezen et al.,2008).
In order to investigate the origin and relationships ofthe Kdp system in pST-III, a phylogenetic tree (Neighbor-Joining) was built. Bacteria with Kdp systems were se-lected with the demand that each component of the Kdpsystem must has >40% identity to that of pST-III (Fig. 5).It was shown that the Kdp system in pST-III has a closerrelationship with that of Clostridium spp. and Desulfovibriodesulfuricans. Moreover, this entire region has a lower GCcontent (36.13%) than the rest of the plasmid. Together,these results suggested that the genes may have been ac-quired by horizontal gene transfer (HGT).
On the other hand, the kdp locus of pST-III lacked a KdpFsubunit, compared with other Kdp systems. KdpF is a smallhydrophobic peptide, probably important for stability butnot essential for function (Gassel et al., 1999). In order todetermine in-depth the physiological functions of the KdpFsubunit, related research is currently being carried out.
eplicate via theta and RC mechanisms according to the published data. Theted in brackets. The replication-associated proteins are clustering into twoown in bold) apparently belongs to the theta mechanism.
Fig. 5. Phylogenetic tree (Neighbor-Joining) for the Kdp systems of different bacteria, of which each component has >40% identity to that of pST-III. The Kdpsystem of pST-III is shown in bold.
C. Chen et al. / Plasmid 67 (2012) 236–244 241
Downstream of the kdp cluster in pST-III, another K+-transport system named Kup, was found. The Kup systemwas composed of 678 amino acids and showed 86% iden-tity to Kup from Lactobacillus brevis subsp. gravesensis ATCC27305 (HMPREF0496_1314). Compared with Kdp, Kup is alow affinity potassium transport system with Km = 0.5 mM(Rhoads et al., 1976). In E. coli, Kup is the major K+ uptakesystem under hyperosmotic stress and low pH conditions(Trchounian and Kobayashi, 1999; Zakharyan and Trchou-nian, 2001). For strain ST-III, Kup may act as the major K+
uptake system under normal growth conditions.
3.3.2. Osmolyte transport systemThree genes (orf5–orf7) encoding the glycine/betaine/
carnitine ABC transporters (ProX, ProW, ProV) were inden-tified in plasmid pST-III. They are thought to be involved ina multi-component binding-protein-dependent transportsystem for glycine betaine and camitine, which entailstheir accumulation to high levels inside the cell in responseto increased external osmolarity (Sleator and Hill, 2002;Yancey et al., 1982). A Blast search revealed the transport-ers share the greatest similarity with the gene in Lactoba-cillus sakei 23 K (LSA1694-LSA1696), which is capable ofgrowing on meat during refrigeration and in the presenceof curing salts (3–9% NaCl) (Chaillou et al., 2005). Thesegenes, together with orf4, are flanked by two IS elements,suggesting the possible dissemination of this region byHGT. In the ST-III chromosome, there are at least threeosmolyte transport systems (ChoS–ChoQ, OpuA–OpuB–OpuC–OpuD, LPST_C2736). The existence of many trans-port systems in the genome of L. plantarum ST-III will en-hance the accumulation of osmolyte to cope withenvironments of elevated osmolarity.
3.3.3. DNA synthesisFour genes (orf17–orf20) encoding putative ribonucleo-
tide reductases were identified in pST-III. Ribonucleotide
reductases can catalyze the reduction of four kinds of ribo-nucleotides to deoxyribonucleotides, which is necessary forDNA replication and repair. According to their metal cofac-tors and sensitivity to molecular oxygen, these ribonucleo-tide reductases can be divided into three major classes(classes I, II, and III) (Jordan and Reichard, 1998; Kolberget al., 2004). The activities of class I enzymes (subdividedinto two subclasses, Ia and Ib) are limited to aerobic condi-tions (Fontecave et al., 1992) whereas class II enzymes canexhibit physiological functions in both aerobic and anaero-bic organisms (Jordan and Reichard, 1998). Class III en-zymes are strictly anaerobic (King and Reichard, 1995). Ingeneral, facultative anaerobic bacteria contain two or morekinds of reductases to synthesize DNA under both aerobicand anaerobic conditions. For example, L. lactis and E. colicontain both aerobic class I and anaerobic class III reduc-tases (Jordan et al., 1997, 1996). In addition to the clusterin the plasmid, L. plantarum ST-III also contains nrdHEF(LPST_C0526- LPST_C0528) and nrdDG (LPST_C2414-LPST_C2415) operons in its chromosome, which belong toclass Ib and class III, respectively (Wang et al., 2011).
Specifically, orf17, orf18, and orf19 were predicted to en-code the beta, beta, and alpha subunits of ribonucleosidediphosphate reductase, respectively, whereas orf20 be-longs to the NrdI family. Sequence analysis showed thatORF17 and ORF19 exhibit 81% similarity to the NrdF2(HMPREF0511_1437) and NrdE2 (HMPREF0511_1435),respectively, in Lactobacillus fermentum ATCC 14931, indi-cating the ribonucleotide reductases encoded by the fourgenes may belong to the class I enzymes. The existenceof another ribonucleotide reductase system of class I mayenhance DNA synthesis in the aerobic environment for L.plantarum ST-III.
3.3.4. Inorganic ion transporterpST-III was found to possess two genes, orf12 and orf13,
which were predicted to encode proteins involved in
Table 2The hyperosmotic resistance compared between the wild-type andplasmid-cured strains of ST-III.
NaCl(%) The length of incubation time for OD600nm value to reach0.5 (h)
Statistical analysis was performed with Student’s t test.* Means significantly different from the value of ST-III in the same row(P < 0.05).
Fig. 6. The standard curves of CT values vs template concentration forrepB and tuf. The total DNA of L. plantarum ST-III was serial 10-folddiluted, ranging from 10�1 to 10�5, and the CT values of each gene wereplotted against the logarithm of concentration (n = 3). A standard curvewas generated by linear regression through these points for each gene.
242 C. Chen et al. / Plasmid 67 (2012) 236–244
inorganic ion transport. The result of BLAST searches ofORF12 revealed 91% identity to Eric (one kind of chloridechannel proteins) of Lactobacillus casei ATCC 334(LSEI_0973). Eric belongs to the chloride channel family (TC2.A.49) and exchanges two chloride ions for one proton withan antiport pattern. It could mediate the extreme acid resis-tance response that allows bacteria to survive in acidicenvironments by decarboxylation-linked proton utilization(Iyer et al., 2002). For ORF13, the Na–H_exchange domain(IPR006153) was characterized (from positions 8 to 376)whereas a similar Kef-type K+ transport system(COG0475) was also identified (from positions 3 to 356).Regardless of whether it transports Na+ or K+, the cation/H+ antiporter is responsible for the counter-transport of pro-tons and cations across lipid bilayers, which are key to main-taining the optimum pH range. These exchangers could alsoprovide increased salt tolerance by removing cations in ex-change for extracellular protons (Padan et al., 2001).
3.3.5. DNA repairThe orf32 gene, designated uvrX, encodes the DNA re-
pair protein, which contains a conserved domain UmuC(cd01700, from positions 12 to 360). The UmuC family ofproteins constitute the catalytic subunit of DNA polymer-ase V. DNA polymerase V is a bacterial translation synthe-sis (TLS) polymerase that is responsible for inducing theSOS response in bacteria that invokes repair of UV-inducedDNA damage (Pham et al., 2001; Schlacher et al., 2006). Itpossesses a translation DNA synthesis activity at the ex-pense of normal replicative fidelity (Wang, 2001). Apartfrom uvrX, another DNA repair protein, the DNA-damage-inducible protein (orf25), was also found in pST-III.
3.4. The effect of plasmid curing on the hyperosmoticresistance for ST-III
A curing experiment was performed to examine theeffect of plasmid-encoded functions on hyperosmotic resis-tance. Treatment with a sublethal concentration of novobi-ocin and cultivation at a high temperature promoted adrop-out of the native plasmid from ST-III (Fig. 1). The plas-mid-cured strain, ST-III-PC, was studied in comparison toits wild-type strain of ST-III in the hyperosmotic environ-ment imposed by MRS broth containing NaCl (0%, 2%, 4%)(Table 2). ST-III-PC showed lower hyperosmotic resistancecompared with native ST-III in the same concentration ofNaCl (All P < 0.05) indicating that genes of pST-III play a sig-nificant role in hyperosmotic resistance for ST-III.
3.5. Relative copy number of pST-III
The copy number of pST-III in L. plantarum ST-III cellswas determined by real-time PCR. A 10-fold serial dilutionof total DNA from L. plantarum ST-III was used as a tem-plate and the standard curves for both the repB and tufgenes were constructed (Fig. 6). Theoretically, for a 10-folddilution template DNA, the slope of standard curve valueshould be �3.322 (Lee et al., 2006). The two standardcurves were both linear (R2 > 0.99) in the range testedand the slopes were 3.326 and 3.342 (less than 1% differentfrom each other, only slightly different from the theoretical
value), respectively, indicating almost the same amplifica-tion efficiency for the two reactions. Analysis of the resultsrevealed that the copy number of pST-III was 6.79 ± 1.55copies per cell, which can be classified as a low-copy num-ber plasmid (1–10 copies) (Providenti et al., 2006).
4. Discussion
Plasmids are generally dispensable for host survival, butthey often carry genes that might be essential for survivalunder harsh conditions. The analysis of the plasmid-en-coded functions of pST-III confirmed this theory. L. planta-rum ST-III was isolated from pickle, which provides theanaerobic environment of high acidity and osmolarity. Cor-respondingly, the K+-transport system, osmolyte transportsystem, and the inorganic ion transporters are related to itsacidic and hyperosmotic environment, and the existence ofclass I ribonucleotide reductases may enhance DNA syn-thesis under aerobic conditions.
The analysis of the replication protein and origin of rep-lication suggest that pST-III replicates via the theta-typemechanism. In recent years, a large number of theta-repli-cating plasmids have been characterized from Gram-posi-
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tive bacteria, especially from LAB, such as pWCFS103,pRV500, and PCD4. In contrast to RC plasmids, which arerather unstable and small (<12 kb) (Kiewiet et al., 1993),theta-type plasmids replicate by means of a double-stranded rather than a single-stranded replication interme-diate, which results in better structural stability, allowingfor the insertion of large DNA fragments (Alpert et al.,2003). This property is useful for the construction of cloningvectors from pST-III.
A previous study has shown that L. plantarum ST-IIIcould live in MRS medium with 7.5% NaCl and was moreresistant to salinity than tested lactobacilli from othersources (Ai et al., 2005). Analysis of the plasmid-encodedfunctions and the plasmid-cured experiment showed thatthe genes of pST-III could make significant contributionsto its hyperosmotic resistance. According to the generalstrategies for coping with elevated osmolarity of bacteria,the most rapid response to osmotic up-shock, both inGram-positive and Gram-negative bacteria, is a stimula-tion of K+ uptake (Sleator and Hill, 2002). For L. plantarumST-III, the Kdp system may activate its expression via KdpDand KdpE and accumulate K+ content, as the primary re-sponse for osmotic up-shock. Increase in the salt concen-tration above a certain level triggers the secondaryresponse: accumulation of the osmolytes in the cytoplasm(either by synthesis and/or uptake) (Sleator and Hill, 2002).For L. plantarum ST-III, the osmolytes might be mainlyuptaken by its four putative osmolyte transport systems.The two responses can maintain cell turgor and tune theintracellular osmolarity within certain limits. From theabove, the mechanism of hyperosmotic resistance for L.plantarum ST-III can be partly explained by theory; furtherresearch is required to elucidate its mechanisms underexperimental conditions.
HGT is one of the major driving forces of bacterial evo-lution for lifestyle adaptation. In pST-III, some genes, espe-cially the kdp gene clusters, may be transferred by HGTfrom other bacteria in its niche. Once acquired, thesenew genes may be maintained and passed onto succeedinggenerations under selective pressure. Such additions cansignificantly expand the potential of a bacterium to adaptto new niches, such as to the hyperosmotic conditions inthis example.
Acknowledgments
This work was supported by National Basic ResearchProgram of China (2010CB735705) and Shanghai Scienceand Technology Commission Program (09DZ2251400 and10XD1420400).
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