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Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics
Journal of Biotechnology 104 (2003) 27 /40 www.elsevier.com/locate/jbiotec
Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics Andreas Tauch a,*, Alfred Pu¨hler b, Jo¨rn Kalinowski a, Georg Thierbach c a
Institut fu ¨ r Genomforschung, Universita ¨ t Bielefeld, Universita ¨ tsstraße 25, D-33615 Bielefeld, Germany b Lehrstuhl fu ¨ r Genetik, Universita ¨ t Bielefeld, Universita ¨ tsstraße 25, D-33615 Bielefeld, Germany c Degussa AG, Kantstraße 2, D-33788 Halle-Ku ¨ nsebeck, Germany
Received 18 November 2002; received in revised form 17 January 2003; accepted 11 March 2003
Abstract Endogenous plasmids and selectable resistance markers are a fundamental prerequisite for the development of efficient recombinant DNA techniques in industrial microorganisms. In this article, we therefore summarize the current knowledge about endogenous plasmids in amino acid-producing Corynebacterium glutamicum isolates. Screening studies identified a total of 24 different plasmids ranging in size from 2.4 to 95 kb. Although most of the C. glutamicum plasmids were cryptic, four plasmids carried resistance determinants against the antibiotics chloramphenicol, tetracycline, streptomycin /spectinomycin, and sulfonamides. Considerable information is now available on the molecular genetic organization of 12 completely sequenced plasmid genomes from C. glutamicum . The deduced mechanism of plasmid DNA replication and the degree of amino acid sequence similarity among replication initiator proteins was the basis for performing a classification of the plasmids into four distinct C. glutamicum plasmid families. # 2003 Elsevier B.V. All rights reserved. Keywords: Corynebacterium glutamicum ; Plasmid family; Antibiotic resistance gene; Plasmid replication; Rolling circle replication; Theta replication
1. Introduction A prerequisite for the development of recombinant DNA techniques for amino acid-producing Corynebacterium glutamicum was the identification of endogenous plasmids (Santamaria et al., 1984; Miwa et al., 1984). The subsequent construction of cloning vectors carrying antibiotic
* Corresponding author. Fax: /49-521-106-5626. E-mail address: [email protected] (A. Tauch).
resistance genes as selectable markers and the development of methods enabling an efficient DNA transfer provided a fundamental basis for the genetic manipulation of the biochemical pathways in C. glutamicum . Meanwhile, a large number of C. glutamicum isolates was screened for the presence of plasmid replicons and 24 endogenous plasmids have been discovered ranging in size from 2.4 to 95 kb (Table 1). Most of the small plasmids were cryptic with the exception of pXZ10145 from C. glutamicum 1014, which was described to carry a transposon-encoded chloram-
0168-1656/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1656(03)00157-3
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Table 1 Plasmids identified in C. glutamicum Plasmid
Size
C. glutamicum
Former species designation
Reference
pAG1 pAG3 pBD12a pBL1 pBL770b,c pBI68 pBT40 pBY502 pCG1 pCG2 pCG4 pCGL500 pCL1d pCRY4 pGA1 pGA2 pGX1906 pHM1520e,f pMA54 pMA90 pTET3 pXZ608 pXZ10142 pXZ10145
20.4 kb 4.5 kb 36 kb 4.3 kb 55 kb 48 kb 40 kb 45 kb 3.2 kb 6.8 kb 29 kb 29 kb 4.1 kb 48 kb 4.9 kb 20 kb 95 kb /25 kb 40 kb 24 kb 27.8 kb 5.9 kb 2.4 kb 5.3 kb
22243 22220 ATCC 14020 ATCC 13869 ATCC 13869 ATCC 14068 ATCC 19240 MJ-233 ATCC 31808 ATCC 31832 ATCC 31830 ATCC 17965 ATCC 15990 LP-6 LP-6 LP-6 ATCC 13869 ATCC 13058 ATCC 15354 ATCC 21490 LP-6 227 1014 1014
C. melassecola C. melassecola Brevibacterium divaricatum B. lactofermentum B. lactofermentum Brevibacterium immariophilum Brevibacterium thiogenitalis Brevibacterium flavum / / / C. melassecola Corynebacterium lilium / / / B. lactofermentum / Microbacterium ammoniaphilum M. ammoniaphilum / / / /
Takeda et al., 1990 Takeda et al., 1990 Sandoval et al., 1985 Santamaria et al., 1984 Sandoval et al., 1985 Sandoval et al., 1985 Sandoval et al., 1985 Satoh et al., 1990 Ozaki et al., 1984 Ozaki et al., 1984 Katsumata et al., 1984 Bonamy et al., 1990 Chan Kwo Chion et al., 1991 Tauch et al., 2002a Sonnen et al., 1991 Sonnen et al., 1991 Smith et al., 1986 Yoshihama et al., 1985 Sandoval et al., 1985 Sandoval et al., 1985 Tauch et al., 2002a Lei et al., 2002 GenBank X72691 Zheng et al., 1987
a b c d e f
Also identified as pBD14 with a calculated size of 33.6 kb (Kato et al., 1989). Also identified as unnamed plasmid with a calculated size of 55.5 kb (Kaneko et al., 1979). Also identified as unnamed plasmid with a calculated size of more than 45 kb (Yeh et al., 1986). The existence of pCL1 could not be confirmed by Sandoval et al. (1985) and Tauch et al. (2002a). The plasmid was originally not named by Yoshihama et al. (1985). A plasmid with similar size was detected in C. glutamicum ATCC 19223 (Yoshihama et al., 1985).
phenicol resistance gene (Shen et al., 1993). Very recently, a systematic search for the presence of plasmid-encoded antibiotic resistance determinants was performed in C. glutamicum isolates and gave very similar results (Tauch et al., 2002a). Among the large C. glutamicum plasmids, only pAG1, pCG4, and pTET3 (Table 1) were shown to encode antibiotic resistance determinants against tetracycline, streptomycin /spectinomycin, and sulfonamides (Takeda et al., 1990; Katsumata et al., 1984; Nesvera et al., 1998; Tauch et al., 2002a). In addition, plasmid pCGL500 from C. melassecola ATCC 17965 (Table 1) was found to carry a restriction /modification system responsible for a decrease in transformation efficiency of the host strain (Bonamy et al., 1990). As shown by in vitro experiments with protein extracts from C. melas-
secola and l phage DNA, pCGL500 encodes the restriction /modification system Cme I representing an isoschizomer of the well-characterized Eco RI system (A. Guyonvarch, unpublished data). Since smaller plasmids are generally more useful as vector systems for recombinant DNA technology, small cryptic plasmids from C. glutamicum were initially chosen for a detailed genetic analysis and for subsequent nucleotide sequence determination (Table 2). In addition, the complete nucleotide sequences of the large antibiotic resistance plasmids pAG1, pCG4, and pTET3 were determined (Tauch et al., 2000, 2002a). Therefore, considerable information is now available on the genetic organization of plasmids from C. glutamicum . Comparative genetic analyses can be applied
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Table 2 Plasmid families in C. glutamicum deduced from comparative genomics Plasmid family
Plasmids
Group A: rolling circle replication pBL1 family pBL1 pAG3 pCG2 pXZ608 pCG1 family pCG1 pCG4 pAG1 pGA1 pGA2 pTET3 Group B: theta replication pXZ10142 family pCRY4 family a b c d
pXZ10142 pXZ10145 pCRY4
Plasmid size
C. glutamicum strain
GenBank number
4447 bpa,b 4603 bp 6758 bp 5949 bp 3069 bpc 29 371 bp 19 751 bp 4826 bp 19 218 bp 27 856 bp
ATCC 22220 ATCC 227 ATCC ATCC 22243 LP-6 LP-6 LP-6
AF092037 AY172684 AY172685 AF479770 AB027714 AF164956 AF121000 X90817 AY172687 AJ420072
2444 bp 4885 bp 48 kbd
1014 1014 LP-6
13869 31832 31808 31830
X72691 U85507 AY172686
The length of the pAM330 sequence is 4448 bp (GenBank D00038). The length of the pGX1901 sequence is 4457 bp (GenBank X03987). The length of the pSR1 sequence is 3054 bp (GenBank Z22927). Only the 1856 bp replication region of pCRY4 was sequenced.
to identify conserved nucleotide sequence motifs within non-coding regions of the plasmid genomes and to characterize conserved amino acid sequence motifs within the plasmid-encoded proteins. Both types of conserved features may be relevant for plasmid DNA replication, stable plasmid maintenance, incompatibility, and copy number control in C. glutamicum . The knowledge on the basic principles of stable plasmid inheritance is of great relevance in the context of establishing powerful genetic systems for a further manipulation of the C. glutamicum genome. The data presented in this paper focus on the molecular genetic classification of sequenced plasmid genomes from C. glutamicum (Table 2). Circular bacterial plasmids mainly use two modes of DNA replication, known as rolling circle replication and theta type replication (del Solar et al., 1998). The mechanism of plasmid DNA replication and the degree of amino acid sequence similarity between replication initiator proteins are criteria generally applied for the classification of plasmids (Ilyina and Koonin, 1992). Using these genetic criteria, the set of sequenced C. glutamicum plasmids was divided into four distinct plasmid families, which are described in more detail below.
2. The pBL1 family of Corynebacterium glutamicum plasmids Plasmid pBL1 is a small cryptic plasmid, which was isolated from C. glutamicum ATCC 13869 (formerly Brevibacterium lactofermentum ) and its derivatives (Santamaria et al., 1984). The plasmid was later also described as pAM330 (Miwa et al., 1984), pBL25 (Kato et al., 1989), pBL100 (Shaw and Hartley, 1988), pGX1901 (Smith et al., 1986), pWS101 (Yoshihama et al., 1985), and pX18 (Yeh et al., 1986). The complete nucleotide sequences of pAM330 (Yamaguchi et al., 1986), pGX1901 (Filpula et al., 1986), and pBL1 (GenBank AF092037) were determined and revealed only minor differences (Table 2). Several aspects of the molecular biology of pAM330 have been analyzed and described in detail by Yamaguchi et al. (1986). The minimal region for autonomous replication of pBL1 in C. glutamicum was identified by Fernandez-Gonzalez et al. (1994) on a 1.8 kb Hin dII-Sph I DNA fragment (Fig. 1). This DNA region contains two ORFs (ORF1 and ORF5), which are essential for pBL1 replication, since deletion of either coding region led to the inability to transform C. glutamicum with hybrid plasmids.
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Fig. 1
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ORF1 seems to encode the replication protein of pBL1, which shows homology to the replication initiator proteins of the Streptomyces plasmids pIJ101 and pJV1 (Fernandez-Gonzalez et al., 1994). These plasmids represent a distinct subfamily of the pC194 family, which is one of the four basic groups of plasmids replicating via the rolling circle mechanism (del Solar et al., 1993). The accumulation of single-strand intermediates in C. glutamicum indicated that pBL1 also replicated via the rolling circle replication model (FernandezGonzalez et al., 1994). This hypothesis was further confirmed by the identification of both a homologous stretch of DNA resembling the nick site within the double-stranded origin of pIJ101 and a potential secondary structure comprising the putative single-stranded origin of pBL1 (FernandezGonzalez et al., 1994). This stem-loop region of pBL1 contains four out of six bases, which appear in the hexanucleotide consensus sequence (5?TAGCGT-3?) of single-stranded origins from several plasmids using the rolling circle mode of replication (Zaman et al., 1993). Functional singlestranded origins are important for maintaining the structural stability of cloning vectors by preventing the accumulation of large amounts of singlestranded vector DNA. Another finding regarding the construction of C. glutamicum /Escherichia coli shuttle vectors based on pBL1 was described by Goyal et al. (1996). Bifunctional vectors containing pBL1 DNA segments were found to inhibit growth and to cause extensive cell filamentation in E. coli . Chromosome segregation was also severely affected. Deletion analysis showed that a 1.23 kb Acc I-Hin dIII DNA fragment comprising ORF3 of pBL1 (Fig. 1) was ultimately responsible for this effect. The deduced ORF3 protein exhibited no homology with any existing protein in databases (Goyal et al., 1996). Interestingly, the growth
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inhibition of E. coli was dependent on the genedose of ORF3 since the inhibitory effect was negligible in the case of using hybrid plasmids with a low copy number. ORF3 is located outside the minimal replication region of pBL1 and is thus dispensable for plasmid replication and stable maintenance of pBL1 in C. glutamicum . The observation of Goyal et al. (1996) should be taken into consideration when constructing bifunctional vectors by means of the pBL1 replicon. Recently, the complete nucleotide sequences of the small C. glutamicum plasmids pAG3, pCG2, and pXZ608 were determined (Table 1; Fig. 1). The putative replication initiator proteins deduced from the DNA sequence annotation revealed homology to the replication protein of pBL1 suggesting that the four plasmids are members of a distinct plasmid family, designated pBL1 family of C. glutamicum plasmids (Table 2). The extended set of plasmid sequences enabled a comparative analysis of the pBL1 plasmid family and allowed the prediction of amino acid residues essentially involved in the catalytic process of plasmid DNA replication (Fig. 2A). Global amino acid sequence alignments with the deduced replication proteins identified conserved residues resembling the ‘two His’ motif and the Yuxk motif of initiator proteins for rolling circle DNA replication (Ilyina and Koonin, 1992). The conserved Yuxk motif includes the DNA-linking tyrosine residue, which forms a covalent link with the nicked plasmid DNA whereas the conserved histidine residues of the ‘two His motif’ might be involved in metal ion coordination required for the activity of the replication protein (Ilyina and Koonin, 1992). Both the ‘two His’ motif and the Yuxk motif thus represent characteristic features for replication initiator proteins of the pBL1 family of C. glutamicum plasmids.
Fig. 1. The pBL1 family of C. glutamicum plasmids. Genetic maps of the small cryptic plasmids pBL1, pAG3, pXZ608, and pCG2 are shown. Predicted coding regions are marked by arrows indicating the direction of transcription. The positions of double-stranded origins (DSO) and single-stranded origins (SSO) in pBL1 and pXZ608 were deduced from nucleotide sequence annotations (Fernandez-Gonzalez et al., 1994; Lei et al., 2002).
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Fig. 2. Conserved amino acid sequence motifs of replication proteins from C. glutamicum plasmids using the rolling circle mode of DNA replication. (A) Motifs present in RepA proteins of the pBL1 family of C. glutamicum plasmids. Amino acid sequences were aligned with the CLUSTAL W program (Thompson et al., 1994). Sequence information was obtained from the GenBank database using accession numbers listed in Table 2. The GenBank accession number of the pIJ101 sequence is M21778. Only the conserved sequence motifs of replication initiator proteins are shown (Ilyina and Koonin, 1992). Identical residues in all sequences (*), conserved substitutions (:), and semi-conserved substitutions (.) are specifically marked. Numbers in parentheses correspond to the start of the sequence motif relative to the start of each protein. (B) Conserved motif present in RepA proteins of the pCG1 family of C. glutamicum plasmids. The Yux3k motif, which is characteristic of the pNG2 family of corynebacterial plasmids, is indicated (Tauch et al., 2003). The GenBank accession number of the pNG2 sequence is AF492560.
3. The pCG1 family of Corynebacterium glutamicum plasmids The small cryptic plasmid pCG1 was originally identified in C. glutamicum ATCC 31808 by Ozaki et al. (1984). Plasmids with restriction maps virtually identical to that of pCG1 (Fig. 3) were also identified in other C. glutamicum isolates and designated pHM1519 (Miwa et al., 1984), pCG100 (Shaw and Hartley, 1988; Trautwetter and Blanco, 1991), and pSR1 (Yoshihama et al., 1985). The complete nucleotide sequences of pCG1 (GenBank AB027714) and pSR1 (Archer and Sinskey, 1993) were reported and provided the basis for an intensive use of these plasmids in cloning vector design. Trautwetter and Blanco (1991) identified the minimal region for autonomous replication of pCG100 on a 1.9 kb Nco I-Bgl II DNA fragment and provided experimental evidence that a 380 bp Hin dIII-Sph I fragment is able to replicate in the presence of the parental plasmid, which presumably provided a trans -acting replication factor. Likewise, transposon insertion and deletion mu-
tants of pSR1 located the minimal replicon within a 2.1 kb Nco I-Bcl I restriction fragment containing a single coding region (Archer and Sinskey, 1993). The predicted protein exhibited significant amino acid sequence similarity to the replication-associated protein of the C. diphtheriae plasmid pNG2 indicating that both plasmids from environmentally separated species are members of a conserved family of corynebacterial plasmids (Archer and Sinskey, 1993; Tauch et al., 2003). A derivative of the C. diphtheriae plasmid pNG2 was already shown to replicate via the rolling circle mechanism (Zhang et al., 1994) suggesting the same mode of DNA replication for the pCG1 family of C. glutamicum plasmids. Meanwhile, the cryptic plasmid pGA1 from C. glutamicum LP-6 (Table 1; Fig. 3) is the best studied member of the pCG1 family of C. glutamicum plasmids. The complete nucleotide sequence of pGA1 was determined and the function of the repA gene was confirmed by deletion mapping of a minimal replicating fragment (Nesvera et al., 1997). Very recently, the
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Fig. 3. Members of the pCG1 family of C. glutamicum plasmids. Genetic maps of the small cryptic plasmids pCG1 from C. glutamicum ATCC 31808 and pGA1 from C. glutamicum LP-6 are shown. Predicted coding regions are marked by arrows indicating the direction of transcription. Re-annotation of the pCG1 plasmid sequence is based on its homology with pGA1 (Nesvera et al., 1997). The position of the double-stranded origin of replication (DSO) of pGA1 was experimentally localized by Abrhamova et al. (2002).
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double-stranded origin of replication of pGA1 was precisely localized in the distal part of the repA gene (Abrhamova et al., 2002). The site- and strand-specific breakage of double-stranded pGA1 DNA occurred within the nucleotide sequence 5?-CTGG¡/AT-3? (nic site). This location of the double stranded origin of pGA1 differs from origin positions identified in other plasmids using the rolling circle mode of DNA replication. Deletion derivatives of pGA1 devoid of the per gene, encoding a positive effector of replication, exhibited a significant effect on plasmid copy number in C. glutamicum (Nesvera et al., 1997). The per gene product was found to be dispensable for replication, but it positively influenced the copy number and the segregational stability of pGA1. A similar gene product was identified on pSR1 by comparative analysis and shown to act in trans on pGA1 derivatives (Nesvera et al., 1997). In addition, the small aes gene, encoding an accessory effector of stable maintenance, was shown to increase the segregational stability of pGA1 derivatives in the presence of the main stability determinant per (Venkova et al., 2001). The experimental work on the segregational stability of pGA1 (and of pSR1) can be considered as basis for designing highly stable cloning vectors for C. glutamicum . In contrast to the stability mechanisms encoded by pGA1 and pCG1, faithful segregation during cell division of the larger plasmids belonging to the pCG1 family (Table 2) might be mediated by a class Ib partitioning system comprising conserved parA and parB genes (Tauch et al., 2000, 2002a). This hypothesis can be deduced from the nucleotide sequence annotations (Fig. 4), but it remains to be functionally proven. Comparative genomic studies among the members of the pCG1 family of C. glutamicum plasmids identified a conserved Yux3k motif within the predicted replication proteins (Fig. 2B). This conserved amino acid sequence strongly resembles the Yuxk motif of replication initiator proteins for rolling circle DNA replication (Ilyina and Koonin, 1992) as it was already observed in the pBL1 family of C. glutamicum plasmids (Fig. 2A). The original Yuxk motif is only present in the replication proteins of pCG1 and pGA1 suggesting that the pCG1 family of C. glutamicum
plasmids can be divided into two distinct subfamilies (Fig. 2B). A further characteristic feature of replication proteins from the pCG1 plasmid family is the absence of the conserved ‘two His’ motif (Ilyina and Koonin, 1992). Interestingly, the RepA proteins encoded by the pCG1 family of C. glutamicum plasmids revealed no homology with other replication proteins indicating that the corresponding replicons represent a novel group of plasmids using the rolling circle mode of DNA replication (Zhang et al., 1994; Nesvera et al., 1997). This observation was confirmed very recently since the pCG1 family of C. glutamicum plasmids was shown to be part of a much larger plasmid family, which solely includes replicons from Corynebacterium species (Tauch et al., 2003). Furthermore, comparative nucleotide sequence analysis revealed that the pCG1 family of C. glutamicum plasmids can be characterized by a novel non-coding feature, termed 22 bp box of corynebacterial plasmids (Tauch et al., 2003). This highly conserved nucleotide sequence motif (5?CrTAAGCArwAhACGGTTCCCC-3?) is located downstream of the repA gene and present in one or two copies per plasmid genome. Two 22 bp boxes are present in the minimal replicon fragment of pCG100, which was identified by Trautwetter and Blanco (1991). Derivatives of pCG100 deleted of a small Hin dIII fragment containing both 22 bp boxes could still replicate with the help of the parental plasmid. However, the location of the 22 bp box and its conservation make it an ideal candidate for sequences playing a critical role in plasmid replication.
4. The pXZ10142 family of Corynebacterium glutamicum plasmids Another C. glutamicum plasmid family consists of two members, designated pXZ10142 and pXZ10145 (Fig. 5), which are genetically closely related since they were identified in the same host strain, C. glutamicum 1014 (Table 1). Complete nucleotide sequence determination of both plasmids revealed that pXZ10142 is obviously a spontaneous deletion derivative of the chloramphenicol resistance plasmid pXZ10145 (Zheng et
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Fig. 4. Physical and genetic map of the streptomycin-spectinomycin resistance plasmid pCG4 from C. glutamicum ATCC 31830. Coding regions deduced from the complete nucleotide sequence of pCG4 are shown by arrows indicating the direction of transcription. The positions of insertion sequences (IS) are shown by boxes. A detailed annotation of the nucleotide sequence has been deposited in the GenBank database with accession number AF164956. Blue, plasmid replication and partitioning functions; red, components of a class I integron carrying the aadA2 streptomycin /spectinomycin resistance gene cassette (Nesvera et al., 1998; Tauch et al., 2002a); orange; coding regions with predicted functions; yellow, hypothetical coding regions; grey, transposase genes and transposase gene fragments (tnpNA , tnpNB , tnpDF ).
al., 1987), which had lost the complete chloramphenicol resistance transposon Tn45 by a precise excision event at the duplicated insertion site (Shen et al., 1993). The cryptic plasmid pXZ10142 is the smallest plasmid identified in C. glutamicum to date, comprising a genome of only 2444 bp (Tables 1 and 2).
Two overlapping coding regions, now designated repA and repB , were identified on pXZ10142 and shown to be essential for autonomous replication in C. glutamicum (Na et al., 1991). The deduced RepA protein showed significant amino acid sequence similarity to a number of replicases from small cryptic plasmids, including
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Fig. 5. The pXZ10142 family of C. glutamicum plasmids. Genetic maps of the cryptic plasmid pXZ10142 and of the chloramphenicol resistance plasmid pXZ10145 from C. glutamicum 1014 are presented. Predicted coding regions are marked by arrows indicating the direction of transcription. The chloramphenicol resistance transposon Tn45 is specifically marked. The terminal inverted repeats of Tn45 are shown by black boxes. The insertion site of Tn45 is located within orf1 , which is therefore, disrupted into a 5? and a 3? segment. The position of the origin of replication (ori ) was deduced from homology analyses with pAL5000-related plasmids (de Mot et al., 1997).
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Fig. 6. Conserved amino acid sequence motifs of replication proteins from C. glutamicum plasmids using theta replication. (A) Motifs 1 and 2 (Ankri et al., 1996) present in the RepA protein from pXZ10142 and from pAL5000-related plasmids. Amino acid sequences were aligned with the CLUSTAL W program (Thompson et al., 1994). Identical residues in all sequences (*), conserved substitutions (:), and semi-conserved substitutions (.) are marked. Numbers in parentheses correspond to the start of the conserved sequence motif relative to the start of each protein. (B) The conserved amino acid sequence motif identified in the replication protein of pCRY4 and related plasmids is shown. Plasmids pMOL98, pSB102, and pIPO2 were obtained from uncultured bacteria. Sequence information was used from GenBank; B. linens pRBL1 (U39878), Bifidobacterium longum pMB1 (X84655), R. erythropolis pFAJ2600 (AF015088) and pRC4 (AB040101), Rhodococcus rhodochrous pKA22 (AF165152), M. fortuitum pAL5000 (M23557), E. coli ColE2-P9 (D30054), Corynebacterium striatum pTP10 (AF024666), Corynebacterium jeikeium pKW4 (AF401314), Rhodothermus marinus pRM21 (U10426), pMOL98 (AJ345055), pSB102 (AJ304453), pIPO2 (AJ297913), and Shigella flexneri pSa (U30471).
pAL5000 from Mycobacterium fortuitum (Stolt and Stoker, 1996a), pRBL1 from Brevibacterium linens (Ankri et al., 1996), and pFAJ2600 from Rhodococcus erythropolis (de Mot et al., 1997). The RepA protein of pXZ10142 and its counterparts on other pAL5000-related plasmids are similar to replication proteins encoded by thetareplicating ColE2-type plasmids (Hiraga et al., 1994). Two amino acid sequence motifs typical for replication proteins of theta-replicating plasmids (Ankri et al., 1996) are conserved in the RepA proteins of pXZ10142 and pAL5000-related plasmids (Fig. 6A). This protein homology suggests that the RepA protein of pXZ10142 may also act as plasmid-specific primase, which synthesizes a specific primer RNA at the replication origin (Takechi et al., 1995). In addition, the predicted RepB proteins are conserved within a family of pAL5000-related plasmids from various bacteria
(Rossi et al., 1996; de Mot et al., 1997; Hirasawa et al., 2001). Two putative DNA-binding domains were predicted in the RepB proteins whereas possible DNA-binding regions could not be identified in the different RepA proteins (Stolt and Stoker, 1996b; de Mot et al., 1997). Therefore, both the primase RepA and the DNA-binding protein RepB of pXZ10142 and of pAL5000related plasmids may act together when initiating DNA replication in a way similar to the replication protein of ColE2-type plasmids, which possess both activities (Takechi et al., 1995). When comparing the putative promoter regions of the repAB genes from pXZ10142 and pAL5000related plasmids, a conserved 15 bp DNA element was noticed approximately 100 bp upstream of the repA start codon (de Mot et al., 1997). The deduced nucleotide consensus sequence 5?-AAATATCTGACTTGG-3? resembles the putative ori-
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gin motif between the low- and high-affinity RepB binding sites in the incompatibility (inc ) region of pAL5000 (Stolt and Stoker, 1996a,b) and the core sequence of the ori region in ColE2-type plasmids (Hiraga et al., 1994; del Solar et al., 1998). During replication of ColE2-type plasmids, leading strand synthesis is initiated at this site following synthesis of a specific primer RNA by the DNA-bound replication protein (Takechi et al., 1995). The conserved genetic arrangement of the repA and repB genes and the presence of theta-type motifs in the RepA protein of pXZ10142 strongly indicated that pXZ10142 replicates via a theta-type mechanism in C. glutamicum . Since theta replication generally results in better plasmid stability than the rolling circle mode of replication, this plasmid classification should encourage the use of pXZ10142 for the development of alternative cloning vectors in C. glutamicum .
5. The pCRY4 family of Corynebacterium glutamicum plasmids The cryptic plasmid pCRY4 was recently identified in C. glutamicum LP-6 (Tauch et al., 2002a) and represents the prototype of a new C. glutamicum plasmid family (Tables 1 and 2). Plasmid pCRY4 has a genome size of approximately 48 kb and coexists in C. glutamicum LP-6 together with pGA1, pGA2, and pTET3 (Sonnen et al., 1991; Tauch et al., 2002a), which are members of the pCG1 family of C. glutamicum plasmids (Table 2). A minimal region capable of autonomous replication in C. glutamicum was cloned as 1856 bp Sph I DNA fragment and used for the construction of a cloning vector system (Tauch et al., 1999). The copy number of the recombinant plasmid pCRY4-Rep was calculated with three plasmid copies per chromosome in C. glutamicum indicating that pCRY4 is a low-copy-number plasmid. Nucleotide sequence analysis of the cloned DNA fragment from pCRY4 revealed only one coding region, which obviously encodes the putative replication protein RepA. As a specific feature of the partial pCRY4 nucleotide sequence, a cluster of five 22 bp direct repeats is present downstream of the repA coding region. Clusters of direct repeats (iterons)
occur in the replication origin region of several plasmids (del Solar et al., 1998). It has been suggested that these repeat sequences constitute binding sites of the replication protein and play an important role in the control of plasmid replication. The deduced RepA protein of pCRY4 exhibited different degrees of global amino acid sequence similarity (32 /45%) to RepA proteins from various plasmids. This set of plasmids includes the thetareplicating IncW plasmid pSa (Okumura and Kado, 1992) and thus suggests the same mode of DNA replication for pCRY4. Weak similarity was also observed to replication proteins from a recently defined family of broad-host-range plasmids from uncultured bacteria, comprising pSB102 (Schneiker et al., 2001) and pIPO2 (Tauch et al., 2002b). Multiple amino acid sequence alignments identified a conserved amino acid sequence motif within the replication proteins (Fig. 6B). This conserved amino acid sequence motif of RepA can be used as a characteristic feature for the molecular classification of C. glutamicum plasmids into the pCRY4 plasmid family. The genetic data deduced from the replication region of pCRY4 indicated that the analysis of large C. glutamicum plasmids may not only extend the current spectrum of plasmid families, but may also provide novel replicon types for an efficient genetic investigation of the C. glutamicum genome.
6. Concluding remarks Considerable progress has been made during the last years in understanding the genetic organization and molecular biology of plasmids from amino acid-producing C. glutamicum . This progress was mainly achieved by systematic sequencing studies, which resulted in the determination of a number of complete plasmid sequences. Subsequent annotation of the plasmid genomes provided a wealth of genetic information and enabled efficient comparative genomic studies. The molecular genetic classification of C. glutamicum plasmids into four distinct plasmid families not only identified conserved features critical for a proper functioning of the plasmid replicons in C. glutamicum , but will also allow the design of targeted
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experiments to further elucidate plasmid functions. The improved knowledge on the genetic organization of C. glutamicum plasmids may also enable the construction of new generations of cloning vectors with specific features optimally adjusted to each genetic experiment. Since cloning vectors are a fundamental basis for an expert investigation of a genome sequence, a substantial effort is required to more fully analyze the identified C. glutamiucm plasmids, especially the larger ones, which may provide novel replicon types for genetic engineering of C. glutamicum .
Acknowledgements The authors thank A. Guyonvarch (Universite Paris-Sud, France) for providing unpublished data on plasmid pCGL500 from C. melassecola ATCC 17965.
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Fernandez-Gonzalez, C., Cadenas, R.F., Noirot-Gros, M.F., Martin, J.F., Gil, J.A., 1994. Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model. J. Bacteriol. 176, 3154 /3161. Filpula, D., Ally, A.H., Nagle, J., 1986. Complete nucleotide sequence of a native plasmid of Brevibacterium lactofermentum . Nucleic Acids Res. 14, 5114. Goyal, D., Wachi, M., Kijima, N., Kobayashi, M., Yukawa, H., Nagai, K., 1996. A cryptic plasmid pBL1 from Brevibacterium lactofermentum causes growth inhibition and filamentation in Escherichia coli . Plasmid 36, 62 /66. Hiraga, S.-I., Sugiyama, T., Itoh, T., 1994. Comparative analysis of the replication regions of eleven ColE2-related plasmids. J. Bacteriol. 176, 7233 /7243. Hirasawa, K., Ishii, Y., Kobayashi, M., Koizumi, K., Maruhashi, K., 2001. Improvement of desulfurization in Rhodococcus erythropolis KA2-5-1 by genetic engineering. Biosci. Biotechnol. Biochem. 65, 239 /246. Ilyina, T., Koonin, E.V., 1992. Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res. 20, 3279 /3285. Kaneko, H., Tanaka, T., Sakaguchi, K., 1979. Isolation and characterization of a plasmid from Brevibacterium lactofermentum . Agric. Biol. Chem. 43, 867 /868. Kato, F., Hara, N., Matsuyama, K., Hattori, K., Ishii, M., Marata, A., 1989. Isolation of plasmids from Brevibacterium . Agric. Biol. Chem. 53, 879 /881. Katsumata, R., Ozaki, A., Oka, T., Furuya, A., 1984. Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J. Bacteriol. 159, 306 /311. Lei, C., Ren, Z., Yang, W., Chen, Y., Chen, D., Liu, M., Yan, W., Zheng, Z., 2002. Characterization of a novel plasmid pXZ608 from Corynebacterium glutamicum . FEMS Microbiol. Lett. 216, 71 /75. Miwa, K., Matsui, H., Terabe, M., Nakamori, S., Sano, K., Momose, H., 1984. Cryptic plasmids in glutamic acidproducing bacteria. Agric. Biol. Chem. 48, 2901 /2903. Na, S., Shen, T., Jia, P., Men, D., Chen, Q., 1991. Characterization of the natural deletion mutant of plasmid pXZ10145 in Corynebacterium glutamicum and construction of a recombinant plasmid. Chin. J. Biotechnol. 7, 271 /277. Nesvera, J., Patek, M., Hochmannova, J., Abrhamova, Z., Becvarova, V., Jelinkova, M., Vohradsky, J., 1997. Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number. J. Bacteriol. 179, 1525 /1532. Nesvera, J., Hochmannova, J., Patek, M., 1998. An integron of class 1 is present on the plasmid pCG4 from gram-positive bacterium Corynebacterium glutamicum . FEMS Microbiol. Lett. 169, 391 /395. Okumura, M.S., Kado, C.I., 1992. The region essential for efficient autonomous replication of pSa in Escherichia coli . Mol. Gen. Genet. 235, 55 /63. Ozaki, A., Katsumata, R., Oka, T., Furuya, A., 1984. Functional expression of the genes of Escherichia coli in gram-
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positive Corynebacterium glutamicum . Mol. Gen. Genet. 196, 175 /178. Rossi, M., Brigidi, P., Gonzalez Vara y Rodriguez, A., Matteuzzi, D., 1996. Characterization of the plasmid pMB1 from Bifidobacterium longum and its use for shuttle vector construction. Res. Microbiol. 147, 133 /143. Sandoval, H., del Real, G., Mateos, L.M., Aguilar, A., Martin, J.F., 1985. Screening of plasmids in non-pathogenic corynebacteria. FEMS Microbiol. Lett. 27, 93 /98. Santamaria, R., Gil, J.A., Mesas, J.M., Martin, J.F., 1984. Characterization of an endogenous plasmid and development of cloning vectors and a transformation system in Brevibacterium lactofermentum . J. Gen. Microbiol. 130, 2237 /2246. Satoh, Y., Hatakeyama, K., Kohama, K., Kobayashi, M., Kurusu, Y., Yukawa., H., 1990. Electrotransformation of intact cells of Brevibacterium flavum MJ-233. J. Ind. Microbiol. 5, 159 /166. Schneiker, S., Keller, M., Dro¨ge, M., Lanka, E., Pu¨hler, A., Selbitschka, W., 2001. The genetic organization and evolution of the broad-host-range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa. Nucleic Acids Res. 29, 5169 /5181. Shaw, P.-C., Hartley, B.S., 1988. A host /vector system for an Arthrobacter species. J. Gen. Microbiol. 134, 903 /911. Shen, T., Jia, P., Na, S., Men, D., 1993. Determination of nucleotide sequence of Corynebacterium glutamicum plasmid pXZ10145. Chin. J. Biotechnol. 9, 171 /178. Smith, M.D., Flickinger, J.L., Lineberger, D.W., Schmidt, B., 1986. Protoplast transformation in coryneform bacteria and introduction of an a-amylase gene from Bacillus amyloliquefaciens into Brevibacterium lactofermentum . Appl. Environ. Microbiol. 51, 634 /639. Sonnen, H., Thierbach, G., Kautz, S., Kalinowski, J., Schneider, J., Pu¨hler, A., Kutzner, H.J., 1991. Characterization of pGA1, a new plasmid from Corynebacterium glutamicum LP-6. Gene 107, 69 /74. Stolt, P., Stoker, N.G., 1996a. Functional definition of regions necessary for replication and incompatibility in the Mycobacterium fortuitum plasmid pAL5000. Microbiology 142, 2795 /2802. Stolt, P., Stoker, N.G., 1996b. Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000. J. Bacteriol. 178, 6693 /6700. Takechi, S., Matsui, H., Itoh, T., 1995. Primer RNA synthesis by plasmid-specific Rep protein for initiation of ColE2 DNA replication. EMBO J. 14, 5141 /5147. Takeda, Y., Fujii, M., Nakajyoh, Y., Nishimura, T., Isshiki, S., 1990. Isolation of a tetracycline resistance plasmid from a glutamate-producing corynebacterium, Corynebacterium melassecola . J. Ferment. Bioeng. 70, 177 /179. Tauch, A., Kalinowski, J., Pu¨hler, A., Thierbach, G., 1999. Plasmide aus Corynebacterium glutamicum und deren Verwendung. German Patent Application DE 19953206. Tauch, A., Pu¨hler, A., Kalinowski, J., Thierbach, G., 2000. TetZ, a new tetracycline resistance determinant discovered
in gram-positive bacteria, shows high homology to gramnegative regulated efflux systems. Plasmid 44, 285 /291. Tauch, A., Go¨tker, S., Pu¨hler, A., Kalinowski, J., Thierbach, G., 2002a. The 27.8-kb R-plasmid pTET3 from Corynebacterium glutamicum encodes the aminoglycoside adenyltransferase gene cassette aadA9 and the regulated tetracycline efflux system Tet 33 flanked by active copies of the widespread insertion sequence IS6100 . Plasmid 48, 117 /129. Tauch, A., Schneiker, S., Selbitschka, W., Pu¨hler, A., van Overbeek, L.S., Smalla, K., Thomas, C.M., Bailey, M.J., Forney, L.J., Weightman, A., Ceglowski, P., Pembroke, T., Tietze, E., Schro¨der, G., Lanka, E., van Elsas, J.D., 2002b. The complete nucleotide sequence and environmental distribution of the cryptic, conjugative, broad-host-range plasmid pIPO2 isolated from bacteria of the wheat rhizosphere. Microbiology 148, 1637 /1653. Tauch, A., Bischoff, N., Brune, I., Kalinowski, J., 2003. Insights into the genetic organization of the Corynebacterium diphtheriae erythromycin resistance plasmid pNG2 deduced from its complete nucleotide sequence. Plasmid 49, 63 /74. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673 /4680. Trautwetter, A., Blanco, C., 1991. Structural organization of the Corynebacterium glutamicum plasmid pCG100. J. Gen. Microbiol. 137, 2093 /2101. Venkova, T., Patek, M., Nesvera, J., 2001. Identification of a novel gene involved in stable maintenance of plasmid pGA1 from Corynebacterium glutamicum . Plasmid 46, 153 /162. Yamaguchi, R., Terabe, M., Miwa, K., Tsuchiya, M., Takagi, H., Morinaga, Y., Nakamori, S., Sano, K., Momose, H., Yamazaki, A., 1986. Determination of the complete nucleotide sequence of Brevibacterium lactofermentum plasmid pAM330 and analysis of its genetic information. Agric. Biol. Chem. 50, 2771 /2778. Yeh, P., Oreglia, J., Prevots, F., Sicard, A.M., 1986. A shuttle vector system for Brevibacterium lactofermentum . Gene 47, 301 /306. Yoshihama, M., Higashiro, K., Rao, E.A., Akedo, M., Shanabruch, W.G., Follettie, M.T., Walker, G.C., Sinskey, A.J., 1985. Cloning vector system for Corynebacterium glutamicum . J. Bacteriol. 162, 591 /597. Zaman, S., Radnedge, L., Richards, H., Ward, J.M., 1993. Analysis of the site for second-strand initiation during replication of the Streptomyces plasmid pIJ101. J. Gen. Microbiol. 139, 669 /676. Zhang, Y., Praszkier, J., Hodgson, A., Pittard, A.J., 1994. Molecular analysis and characterization of a broad-hostrange plasmid, pEP2. J. Bacteriol. 176, 5718 /5728. Zheng, Z.-X., Ma, C.-P., Yan, W.-Y., He, P.-F., Mao, Y.-X., Sun, W., Lei, Z.-Z., Zhu, P., Wu, J.-F., 1987. Restriction map of plasmid pXZ10145 of Corynebacterium glutamicum and construction of an integrated plasmid. Chin. J. Biotechnol. 3, 183 /188.
Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics Andreas Tauch a,*, Alfred Pu¨hler b, Jo¨rn Kalinowski a, Georg Thierbach c a
Institut fu ¨ r Genomforschung, Universita ¨ t Bielefeld, Universita ¨ tsstraße 25, D-33615 Bielefeld, Germany b Lehrstuhl fu ¨ r Genetik, Universita ¨ t Bielefeld, Universita ¨ tsstraße 25, D-33615 Bielefeld, Germany c Degussa AG, Kantstraße 2, D-33788 Halle-Ku ¨ nsebeck, Germany
Received 18 November 2002; received in revised form 17 January 2003; accepted 11 March 2003
Abstract Endogenous plasmids and selectable resistance markers are a fundamental prerequisite for the development of efficient recombinant DNA techniques in industrial microorganisms. In this article, we therefore summarize the current knowledge about endogenous plasmids in amino acid-producing Corynebacterium glutamicum isolates. Screening studies identified a total of 24 different plasmids ranging in size from 2.4 to 95 kb. Although most of the C. glutamicum plasmids were cryptic, four plasmids carried resistance determinants against the antibiotics chloramphenicol, tetracycline, streptomycin /spectinomycin, and sulfonamides. Considerable information is now available on the molecular genetic organization of 12 completely sequenced plasmid genomes from C. glutamicum . The deduced mechanism of plasmid DNA replication and the degree of amino acid sequence similarity among replication initiator proteins was the basis for performing a classification of the plasmids into four distinct C. glutamicum plasmid families. # 2003 Elsevier B.V. All rights reserved. Keywords: Corynebacterium glutamicum ; Plasmid family; Antibiotic resistance gene; Plasmid replication; Rolling circle replication; Theta replication
1. Introduction A prerequisite for the development of recombinant DNA techniques for amino acid-producing Corynebacterium glutamicum was the identification of endogenous plasmids (Santamaria et al., 1984; Miwa et al., 1984). The subsequent construction of cloning vectors carrying antibiotic
* Corresponding author. Fax: /49-521-106-5626. E-mail address: [email protected] (A. Tauch).
resistance genes as selectable markers and the development of methods enabling an efficient DNA transfer provided a fundamental basis for the genetic manipulation of the biochemical pathways in C. glutamicum . Meanwhile, a large number of C. glutamicum isolates was screened for the presence of plasmid replicons and 24 endogenous plasmids have been discovered ranging in size from 2.4 to 95 kb (Table 1). Most of the small plasmids were cryptic with the exception of pXZ10145 from C. glutamicum 1014, which was described to carry a transposon-encoded chloram-
0168-1656/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1656(03)00157-3
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Table 1 Plasmids identified in C. glutamicum Plasmid
Size
C. glutamicum
Former species designation
Reference
pAG1 pAG3 pBD12a pBL1 pBL770b,c pBI68 pBT40 pBY502 pCG1 pCG2 pCG4 pCGL500 pCL1d pCRY4 pGA1 pGA2 pGX1906 pHM1520e,f pMA54 pMA90 pTET3 pXZ608 pXZ10142 pXZ10145
20.4 kb 4.5 kb 36 kb 4.3 kb 55 kb 48 kb 40 kb 45 kb 3.2 kb 6.8 kb 29 kb 29 kb 4.1 kb 48 kb 4.9 kb 20 kb 95 kb /25 kb 40 kb 24 kb 27.8 kb 5.9 kb 2.4 kb 5.3 kb
22243 22220 ATCC 14020 ATCC 13869 ATCC 13869 ATCC 14068 ATCC 19240 MJ-233 ATCC 31808 ATCC 31832 ATCC 31830 ATCC 17965 ATCC 15990 LP-6 LP-6 LP-6 ATCC 13869 ATCC 13058 ATCC 15354 ATCC 21490 LP-6 227 1014 1014
C. melassecola C. melassecola Brevibacterium divaricatum B. lactofermentum B. lactofermentum Brevibacterium immariophilum Brevibacterium thiogenitalis Brevibacterium flavum / / / C. melassecola Corynebacterium lilium / / / B. lactofermentum / Microbacterium ammoniaphilum M. ammoniaphilum / / / /
Takeda et al., 1990 Takeda et al., 1990 Sandoval et al., 1985 Santamaria et al., 1984 Sandoval et al., 1985 Sandoval et al., 1985 Sandoval et al., 1985 Satoh et al., 1990 Ozaki et al., 1984 Ozaki et al., 1984 Katsumata et al., 1984 Bonamy et al., 1990 Chan Kwo Chion et al., 1991 Tauch et al., 2002a Sonnen et al., 1991 Sonnen et al., 1991 Smith et al., 1986 Yoshihama et al., 1985 Sandoval et al., 1985 Sandoval et al., 1985 Tauch et al., 2002a Lei et al., 2002 GenBank X72691 Zheng et al., 1987
a b c d e f
Also identified as pBD14 with a calculated size of 33.6 kb (Kato et al., 1989). Also identified as unnamed plasmid with a calculated size of 55.5 kb (Kaneko et al., 1979). Also identified as unnamed plasmid with a calculated size of more than 45 kb (Yeh et al., 1986). The existence of pCL1 could not be confirmed by Sandoval et al. (1985) and Tauch et al. (2002a). The plasmid was originally not named by Yoshihama et al. (1985). A plasmid with similar size was detected in C. glutamicum ATCC 19223 (Yoshihama et al., 1985).
phenicol resistance gene (Shen et al., 1993). Very recently, a systematic search for the presence of plasmid-encoded antibiotic resistance determinants was performed in C. glutamicum isolates and gave very similar results (Tauch et al., 2002a). Among the large C. glutamicum plasmids, only pAG1, pCG4, and pTET3 (Table 1) were shown to encode antibiotic resistance determinants against tetracycline, streptomycin /spectinomycin, and sulfonamides (Takeda et al., 1990; Katsumata et al., 1984; Nesvera et al., 1998; Tauch et al., 2002a). In addition, plasmid pCGL500 from C. melassecola ATCC 17965 (Table 1) was found to carry a restriction /modification system responsible for a decrease in transformation efficiency of the host strain (Bonamy et al., 1990). As shown by in vitro experiments with protein extracts from C. melas-
secola and l phage DNA, pCGL500 encodes the restriction /modification system Cme I representing an isoschizomer of the well-characterized Eco RI system (A. Guyonvarch, unpublished data). Since smaller plasmids are generally more useful as vector systems for recombinant DNA technology, small cryptic plasmids from C. glutamicum were initially chosen for a detailed genetic analysis and for subsequent nucleotide sequence determination (Table 2). In addition, the complete nucleotide sequences of the large antibiotic resistance plasmids pAG1, pCG4, and pTET3 were determined (Tauch et al., 2000, 2002a). Therefore, considerable information is now available on the genetic organization of plasmids from C. glutamicum . Comparative genetic analyses can be applied
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Table 2 Plasmid families in C. glutamicum deduced from comparative genomics Plasmid family
Plasmids
Group A: rolling circle replication pBL1 family pBL1 pAG3 pCG2 pXZ608 pCG1 family pCG1 pCG4 pAG1 pGA1 pGA2 pTET3 Group B: theta replication pXZ10142 family pCRY4 family a b c d
pXZ10142 pXZ10145 pCRY4
Plasmid size
C. glutamicum strain
GenBank number
4447 bpa,b 4603 bp 6758 bp 5949 bp 3069 bpc 29 371 bp 19 751 bp 4826 bp 19 218 bp 27 856 bp
ATCC 22220 ATCC 227 ATCC ATCC 22243 LP-6 LP-6 LP-6
AF092037 AY172684 AY172685 AF479770 AB027714 AF164956 AF121000 X90817 AY172687 AJ420072
2444 bp 4885 bp 48 kbd
1014 1014 LP-6
13869 31832 31808 31830
X72691 U85507 AY172686
The length of the pAM330 sequence is 4448 bp (GenBank D00038). The length of the pGX1901 sequence is 4457 bp (GenBank X03987). The length of the pSR1 sequence is 3054 bp (GenBank Z22927). Only the 1856 bp replication region of pCRY4 was sequenced.
to identify conserved nucleotide sequence motifs within non-coding regions of the plasmid genomes and to characterize conserved amino acid sequence motifs within the plasmid-encoded proteins. Both types of conserved features may be relevant for plasmid DNA replication, stable plasmid maintenance, incompatibility, and copy number control in C. glutamicum . The knowledge on the basic principles of stable plasmid inheritance is of great relevance in the context of establishing powerful genetic systems for a further manipulation of the C. glutamicum genome. The data presented in this paper focus on the molecular genetic classification of sequenced plasmid genomes from C. glutamicum (Table 2). Circular bacterial plasmids mainly use two modes of DNA replication, known as rolling circle replication and theta type replication (del Solar et al., 1998). The mechanism of plasmid DNA replication and the degree of amino acid sequence similarity between replication initiator proteins are criteria generally applied for the classification of plasmids (Ilyina and Koonin, 1992). Using these genetic criteria, the set of sequenced C. glutamicum plasmids was divided into four distinct plasmid families, which are described in more detail below.
2. The pBL1 family of Corynebacterium glutamicum plasmids Plasmid pBL1 is a small cryptic plasmid, which was isolated from C. glutamicum ATCC 13869 (formerly Brevibacterium lactofermentum ) and its derivatives (Santamaria et al., 1984). The plasmid was later also described as pAM330 (Miwa et al., 1984), pBL25 (Kato et al., 1989), pBL100 (Shaw and Hartley, 1988), pGX1901 (Smith et al., 1986), pWS101 (Yoshihama et al., 1985), and pX18 (Yeh et al., 1986). The complete nucleotide sequences of pAM330 (Yamaguchi et al., 1986), pGX1901 (Filpula et al., 1986), and pBL1 (GenBank AF092037) were determined and revealed only minor differences (Table 2). Several aspects of the molecular biology of pAM330 have been analyzed and described in detail by Yamaguchi et al. (1986). The minimal region for autonomous replication of pBL1 in C. glutamicum was identified by Fernandez-Gonzalez et al. (1994) on a 1.8 kb Hin dII-Sph I DNA fragment (Fig. 1). This DNA region contains two ORFs (ORF1 and ORF5), which are essential for pBL1 replication, since deletion of either coding region led to the inability to transform C. glutamicum with hybrid plasmids.
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Fig. 1
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ORF1 seems to encode the replication protein of pBL1, which shows homology to the replication initiator proteins of the Streptomyces plasmids pIJ101 and pJV1 (Fernandez-Gonzalez et al., 1994). These plasmids represent a distinct subfamily of the pC194 family, which is one of the four basic groups of plasmids replicating via the rolling circle mechanism (del Solar et al., 1993). The accumulation of single-strand intermediates in C. glutamicum indicated that pBL1 also replicated via the rolling circle replication model (FernandezGonzalez et al., 1994). This hypothesis was further confirmed by the identification of both a homologous stretch of DNA resembling the nick site within the double-stranded origin of pIJ101 and a potential secondary structure comprising the putative single-stranded origin of pBL1 (FernandezGonzalez et al., 1994). This stem-loop region of pBL1 contains four out of six bases, which appear in the hexanucleotide consensus sequence (5?TAGCGT-3?) of single-stranded origins from several plasmids using the rolling circle mode of replication (Zaman et al., 1993). Functional singlestranded origins are important for maintaining the structural stability of cloning vectors by preventing the accumulation of large amounts of singlestranded vector DNA. Another finding regarding the construction of C. glutamicum /Escherichia coli shuttle vectors based on pBL1 was described by Goyal et al. (1996). Bifunctional vectors containing pBL1 DNA segments were found to inhibit growth and to cause extensive cell filamentation in E. coli . Chromosome segregation was also severely affected. Deletion analysis showed that a 1.23 kb Acc I-Hin dIII DNA fragment comprising ORF3 of pBL1 (Fig. 1) was ultimately responsible for this effect. The deduced ORF3 protein exhibited no homology with any existing protein in databases (Goyal et al., 1996). Interestingly, the growth
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inhibition of E. coli was dependent on the genedose of ORF3 since the inhibitory effect was negligible in the case of using hybrid plasmids with a low copy number. ORF3 is located outside the minimal replication region of pBL1 and is thus dispensable for plasmid replication and stable maintenance of pBL1 in C. glutamicum . The observation of Goyal et al. (1996) should be taken into consideration when constructing bifunctional vectors by means of the pBL1 replicon. Recently, the complete nucleotide sequences of the small C. glutamicum plasmids pAG3, pCG2, and pXZ608 were determined (Table 1; Fig. 1). The putative replication initiator proteins deduced from the DNA sequence annotation revealed homology to the replication protein of pBL1 suggesting that the four plasmids are members of a distinct plasmid family, designated pBL1 family of C. glutamicum plasmids (Table 2). The extended set of plasmid sequences enabled a comparative analysis of the pBL1 plasmid family and allowed the prediction of amino acid residues essentially involved in the catalytic process of plasmid DNA replication (Fig. 2A). Global amino acid sequence alignments with the deduced replication proteins identified conserved residues resembling the ‘two His’ motif and the Yuxk motif of initiator proteins for rolling circle DNA replication (Ilyina and Koonin, 1992). The conserved Yuxk motif includes the DNA-linking tyrosine residue, which forms a covalent link with the nicked plasmid DNA whereas the conserved histidine residues of the ‘two His motif’ might be involved in metal ion coordination required for the activity of the replication protein (Ilyina and Koonin, 1992). Both the ‘two His’ motif and the Yuxk motif thus represent characteristic features for replication initiator proteins of the pBL1 family of C. glutamicum plasmids.
Fig. 1. The pBL1 family of C. glutamicum plasmids. Genetic maps of the small cryptic plasmids pBL1, pAG3, pXZ608, and pCG2 are shown. Predicted coding regions are marked by arrows indicating the direction of transcription. The positions of double-stranded origins (DSO) and single-stranded origins (SSO) in pBL1 and pXZ608 were deduced from nucleotide sequence annotations (Fernandez-Gonzalez et al., 1994; Lei et al., 2002).
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Fig. 2. Conserved amino acid sequence motifs of replication proteins from C. glutamicum plasmids using the rolling circle mode of DNA replication. (A) Motifs present in RepA proteins of the pBL1 family of C. glutamicum plasmids. Amino acid sequences were aligned with the CLUSTAL W program (Thompson et al., 1994). Sequence information was obtained from the GenBank database using accession numbers listed in Table 2. The GenBank accession number of the pIJ101 sequence is M21778. Only the conserved sequence motifs of replication initiator proteins are shown (Ilyina and Koonin, 1992). Identical residues in all sequences (*), conserved substitutions (:), and semi-conserved substitutions (.) are specifically marked. Numbers in parentheses correspond to the start of the sequence motif relative to the start of each protein. (B) Conserved motif present in RepA proteins of the pCG1 family of C. glutamicum plasmids. The Yux3k motif, which is characteristic of the pNG2 family of corynebacterial plasmids, is indicated (Tauch et al., 2003). The GenBank accession number of the pNG2 sequence is AF492560.
3. The pCG1 family of Corynebacterium glutamicum plasmids The small cryptic plasmid pCG1 was originally identified in C. glutamicum ATCC 31808 by Ozaki et al. (1984). Plasmids with restriction maps virtually identical to that of pCG1 (Fig. 3) were also identified in other C. glutamicum isolates and designated pHM1519 (Miwa et al., 1984), pCG100 (Shaw and Hartley, 1988; Trautwetter and Blanco, 1991), and pSR1 (Yoshihama et al., 1985). The complete nucleotide sequences of pCG1 (GenBank AB027714) and pSR1 (Archer and Sinskey, 1993) were reported and provided the basis for an intensive use of these plasmids in cloning vector design. Trautwetter and Blanco (1991) identified the minimal region for autonomous replication of pCG100 on a 1.9 kb Nco I-Bgl II DNA fragment and provided experimental evidence that a 380 bp Hin dIII-Sph I fragment is able to replicate in the presence of the parental plasmid, which presumably provided a trans -acting replication factor. Likewise, transposon insertion and deletion mu-
tants of pSR1 located the minimal replicon within a 2.1 kb Nco I-Bcl I restriction fragment containing a single coding region (Archer and Sinskey, 1993). The predicted protein exhibited significant amino acid sequence similarity to the replication-associated protein of the C. diphtheriae plasmid pNG2 indicating that both plasmids from environmentally separated species are members of a conserved family of corynebacterial plasmids (Archer and Sinskey, 1993; Tauch et al., 2003). A derivative of the C. diphtheriae plasmid pNG2 was already shown to replicate via the rolling circle mechanism (Zhang et al., 1994) suggesting the same mode of DNA replication for the pCG1 family of C. glutamicum plasmids. Meanwhile, the cryptic plasmid pGA1 from C. glutamicum LP-6 (Table 1; Fig. 3) is the best studied member of the pCG1 family of C. glutamicum plasmids. The complete nucleotide sequence of pGA1 was determined and the function of the repA gene was confirmed by deletion mapping of a minimal replicating fragment (Nesvera et al., 1997). Very recently, the
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Fig. 3. Members of the pCG1 family of C. glutamicum plasmids. Genetic maps of the small cryptic plasmids pCG1 from C. glutamicum ATCC 31808 and pGA1 from C. glutamicum LP-6 are shown. Predicted coding regions are marked by arrows indicating the direction of transcription. Re-annotation of the pCG1 plasmid sequence is based on its homology with pGA1 (Nesvera et al., 1997). The position of the double-stranded origin of replication (DSO) of pGA1 was experimentally localized by Abrhamova et al. (2002).
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double-stranded origin of replication of pGA1 was precisely localized in the distal part of the repA gene (Abrhamova et al., 2002). The site- and strand-specific breakage of double-stranded pGA1 DNA occurred within the nucleotide sequence 5?-CTGG¡/AT-3? (nic site). This location of the double stranded origin of pGA1 differs from origin positions identified in other plasmids using the rolling circle mode of DNA replication. Deletion derivatives of pGA1 devoid of the per gene, encoding a positive effector of replication, exhibited a significant effect on plasmid copy number in C. glutamicum (Nesvera et al., 1997). The per gene product was found to be dispensable for replication, but it positively influenced the copy number and the segregational stability of pGA1. A similar gene product was identified on pSR1 by comparative analysis and shown to act in trans on pGA1 derivatives (Nesvera et al., 1997). In addition, the small aes gene, encoding an accessory effector of stable maintenance, was shown to increase the segregational stability of pGA1 derivatives in the presence of the main stability determinant per (Venkova et al., 2001). The experimental work on the segregational stability of pGA1 (and of pSR1) can be considered as basis for designing highly stable cloning vectors for C. glutamicum . In contrast to the stability mechanisms encoded by pGA1 and pCG1, faithful segregation during cell division of the larger plasmids belonging to the pCG1 family (Table 2) might be mediated by a class Ib partitioning system comprising conserved parA and parB genes (Tauch et al., 2000, 2002a). This hypothesis can be deduced from the nucleotide sequence annotations (Fig. 4), but it remains to be functionally proven. Comparative genomic studies among the members of the pCG1 family of C. glutamicum plasmids identified a conserved Yux3k motif within the predicted replication proteins (Fig. 2B). This conserved amino acid sequence strongly resembles the Yuxk motif of replication initiator proteins for rolling circle DNA replication (Ilyina and Koonin, 1992) as it was already observed in the pBL1 family of C. glutamicum plasmids (Fig. 2A). The original Yuxk motif is only present in the replication proteins of pCG1 and pGA1 suggesting that the pCG1 family of C. glutamicum
plasmids can be divided into two distinct subfamilies (Fig. 2B). A further characteristic feature of replication proteins from the pCG1 plasmid family is the absence of the conserved ‘two His’ motif (Ilyina and Koonin, 1992). Interestingly, the RepA proteins encoded by the pCG1 family of C. glutamicum plasmids revealed no homology with other replication proteins indicating that the corresponding replicons represent a novel group of plasmids using the rolling circle mode of DNA replication (Zhang et al., 1994; Nesvera et al., 1997). This observation was confirmed very recently since the pCG1 family of C. glutamicum plasmids was shown to be part of a much larger plasmid family, which solely includes replicons from Corynebacterium species (Tauch et al., 2003). Furthermore, comparative nucleotide sequence analysis revealed that the pCG1 family of C. glutamicum plasmids can be characterized by a novel non-coding feature, termed 22 bp box of corynebacterial plasmids (Tauch et al., 2003). This highly conserved nucleotide sequence motif (5?CrTAAGCArwAhACGGTTCCCC-3?) is located downstream of the repA gene and present in one or two copies per plasmid genome. Two 22 bp boxes are present in the minimal replicon fragment of pCG100, which was identified by Trautwetter and Blanco (1991). Derivatives of pCG100 deleted of a small Hin dIII fragment containing both 22 bp boxes could still replicate with the help of the parental plasmid. However, the location of the 22 bp box and its conservation make it an ideal candidate for sequences playing a critical role in plasmid replication.
4. The pXZ10142 family of Corynebacterium glutamicum plasmids Another C. glutamicum plasmid family consists of two members, designated pXZ10142 and pXZ10145 (Fig. 5), which are genetically closely related since they were identified in the same host strain, C. glutamicum 1014 (Table 1). Complete nucleotide sequence determination of both plasmids revealed that pXZ10142 is obviously a spontaneous deletion derivative of the chloramphenicol resistance plasmid pXZ10145 (Zheng et
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Fig. 4. Physical and genetic map of the streptomycin-spectinomycin resistance plasmid pCG4 from C. glutamicum ATCC 31830. Coding regions deduced from the complete nucleotide sequence of pCG4 are shown by arrows indicating the direction of transcription. The positions of insertion sequences (IS) are shown by boxes. A detailed annotation of the nucleotide sequence has been deposited in the GenBank database with accession number AF164956. Blue, plasmid replication and partitioning functions; red, components of a class I integron carrying the aadA2 streptomycin /spectinomycin resistance gene cassette (Nesvera et al., 1998; Tauch et al., 2002a); orange; coding regions with predicted functions; yellow, hypothetical coding regions; grey, transposase genes and transposase gene fragments (tnpNA , tnpNB , tnpDF ).
al., 1987), which had lost the complete chloramphenicol resistance transposon Tn45 by a precise excision event at the duplicated insertion site (Shen et al., 1993). The cryptic plasmid pXZ10142 is the smallest plasmid identified in C. glutamicum to date, comprising a genome of only 2444 bp (Tables 1 and 2).
Two overlapping coding regions, now designated repA and repB , were identified on pXZ10142 and shown to be essential for autonomous replication in C. glutamicum (Na et al., 1991). The deduced RepA protein showed significant amino acid sequence similarity to a number of replicases from small cryptic plasmids, including
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Fig. 5. The pXZ10142 family of C. glutamicum plasmids. Genetic maps of the cryptic plasmid pXZ10142 and of the chloramphenicol resistance plasmid pXZ10145 from C. glutamicum 1014 are presented. Predicted coding regions are marked by arrows indicating the direction of transcription. The chloramphenicol resistance transposon Tn45 is specifically marked. The terminal inverted repeats of Tn45 are shown by black boxes. The insertion site of Tn45 is located within orf1 , which is therefore, disrupted into a 5? and a 3? segment. The position of the origin of replication (ori ) was deduced from homology analyses with pAL5000-related plasmids (de Mot et al., 1997).
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Fig. 6. Conserved amino acid sequence motifs of replication proteins from C. glutamicum plasmids using theta replication. (A) Motifs 1 and 2 (Ankri et al., 1996) present in the RepA protein from pXZ10142 and from pAL5000-related plasmids. Amino acid sequences were aligned with the CLUSTAL W program (Thompson et al., 1994). Identical residues in all sequences (*), conserved substitutions (:), and semi-conserved substitutions (.) are marked. Numbers in parentheses correspond to the start of the conserved sequence motif relative to the start of each protein. (B) The conserved amino acid sequence motif identified in the replication protein of pCRY4 and related plasmids is shown. Plasmids pMOL98, pSB102, and pIPO2 were obtained from uncultured bacteria. Sequence information was used from GenBank; B. linens pRBL1 (U39878), Bifidobacterium longum pMB1 (X84655), R. erythropolis pFAJ2600 (AF015088) and pRC4 (AB040101), Rhodococcus rhodochrous pKA22 (AF165152), M. fortuitum pAL5000 (M23557), E. coli ColE2-P9 (D30054), Corynebacterium striatum pTP10 (AF024666), Corynebacterium jeikeium pKW4 (AF401314), Rhodothermus marinus pRM21 (U10426), pMOL98 (AJ345055), pSB102 (AJ304453), pIPO2 (AJ297913), and Shigella flexneri pSa (U30471).
pAL5000 from Mycobacterium fortuitum (Stolt and Stoker, 1996a), pRBL1 from Brevibacterium linens (Ankri et al., 1996), and pFAJ2600 from Rhodococcus erythropolis (de Mot et al., 1997). The RepA protein of pXZ10142 and its counterparts on other pAL5000-related plasmids are similar to replication proteins encoded by thetareplicating ColE2-type plasmids (Hiraga et al., 1994). Two amino acid sequence motifs typical for replication proteins of theta-replicating plasmids (Ankri et al., 1996) are conserved in the RepA proteins of pXZ10142 and pAL5000-related plasmids (Fig. 6A). This protein homology suggests that the RepA protein of pXZ10142 may also act as plasmid-specific primase, which synthesizes a specific primer RNA at the replication origin (Takechi et al., 1995). In addition, the predicted RepB proteins are conserved within a family of pAL5000-related plasmids from various bacteria
(Rossi et al., 1996; de Mot et al., 1997; Hirasawa et al., 2001). Two putative DNA-binding domains were predicted in the RepB proteins whereas possible DNA-binding regions could not be identified in the different RepA proteins (Stolt and Stoker, 1996b; de Mot et al., 1997). Therefore, both the primase RepA and the DNA-binding protein RepB of pXZ10142 and of pAL5000related plasmids may act together when initiating DNA replication in a way similar to the replication protein of ColE2-type plasmids, which possess both activities (Takechi et al., 1995). When comparing the putative promoter regions of the repAB genes from pXZ10142 and pAL5000related plasmids, a conserved 15 bp DNA element was noticed approximately 100 bp upstream of the repA start codon (de Mot et al., 1997). The deduced nucleotide consensus sequence 5?-AAATATCTGACTTGG-3? resembles the putative ori-
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gin motif between the low- and high-affinity RepB binding sites in the incompatibility (inc ) region of pAL5000 (Stolt and Stoker, 1996a,b) and the core sequence of the ori region in ColE2-type plasmids (Hiraga et al., 1994; del Solar et al., 1998). During replication of ColE2-type plasmids, leading strand synthesis is initiated at this site following synthesis of a specific primer RNA by the DNA-bound replication protein (Takechi et al., 1995). The conserved genetic arrangement of the repA and repB genes and the presence of theta-type motifs in the RepA protein of pXZ10142 strongly indicated that pXZ10142 replicates via a theta-type mechanism in C. glutamicum . Since theta replication generally results in better plasmid stability than the rolling circle mode of replication, this plasmid classification should encourage the use of pXZ10142 for the development of alternative cloning vectors in C. glutamicum .
5. The pCRY4 family of Corynebacterium glutamicum plasmids The cryptic plasmid pCRY4 was recently identified in C. glutamicum LP-6 (Tauch et al., 2002a) and represents the prototype of a new C. glutamicum plasmid family (Tables 1 and 2). Plasmid pCRY4 has a genome size of approximately 48 kb and coexists in C. glutamicum LP-6 together with pGA1, pGA2, and pTET3 (Sonnen et al., 1991; Tauch et al., 2002a), which are members of the pCG1 family of C. glutamicum plasmids (Table 2). A minimal region capable of autonomous replication in C. glutamicum was cloned as 1856 bp Sph I DNA fragment and used for the construction of a cloning vector system (Tauch et al., 1999). The copy number of the recombinant plasmid pCRY4-Rep was calculated with three plasmid copies per chromosome in C. glutamicum indicating that pCRY4 is a low-copy-number plasmid. Nucleotide sequence analysis of the cloned DNA fragment from pCRY4 revealed only one coding region, which obviously encodes the putative replication protein RepA. As a specific feature of the partial pCRY4 nucleotide sequence, a cluster of five 22 bp direct repeats is present downstream of the repA coding region. Clusters of direct repeats (iterons)
occur in the replication origin region of several plasmids (del Solar et al., 1998). It has been suggested that these repeat sequences constitute binding sites of the replication protein and play an important role in the control of plasmid replication. The deduced RepA protein of pCRY4 exhibited different degrees of global amino acid sequence similarity (32 /45%) to RepA proteins from various plasmids. This set of plasmids includes the thetareplicating IncW plasmid pSa (Okumura and Kado, 1992) and thus suggests the same mode of DNA replication for pCRY4. Weak similarity was also observed to replication proteins from a recently defined family of broad-host-range plasmids from uncultured bacteria, comprising pSB102 (Schneiker et al., 2001) and pIPO2 (Tauch et al., 2002b). Multiple amino acid sequence alignments identified a conserved amino acid sequence motif within the replication proteins (Fig. 6B). This conserved amino acid sequence motif of RepA can be used as a characteristic feature for the molecular classification of C. glutamicum plasmids into the pCRY4 plasmid family. The genetic data deduced from the replication region of pCRY4 indicated that the analysis of large C. glutamicum plasmids may not only extend the current spectrum of plasmid families, but may also provide novel replicon types for an efficient genetic investigation of the C. glutamicum genome.
6. Concluding remarks Considerable progress has been made during the last years in understanding the genetic organization and molecular biology of plasmids from amino acid-producing C. glutamicum . This progress was mainly achieved by systematic sequencing studies, which resulted in the determination of a number of complete plasmid sequences. Subsequent annotation of the plasmid genomes provided a wealth of genetic information and enabled efficient comparative genomic studies. The molecular genetic classification of C. glutamicum plasmids into four distinct plasmid families not only identified conserved features critical for a proper functioning of the plasmid replicons in C. glutamicum , but will also allow the design of targeted
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experiments to further elucidate plasmid functions. The improved knowledge on the genetic organization of C. glutamicum plasmids may also enable the construction of new generations of cloning vectors with specific features optimally adjusted to each genetic experiment. Since cloning vectors are a fundamental basis for an expert investigation of a genome sequence, a substantial effort is required to more fully analyze the identified C. glutamiucm plasmids, especially the larger ones, which may provide novel replicon types for genetic engineering of C. glutamicum .
Acknowledgements The authors thank A. Guyonvarch (Universite Paris-Sud, France) for providing unpublished data on plasmid pCGL500 from C. melassecola ATCC 17965.
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