Identification of Plasmids in the Genus Marinococcus and Complete

Nucleotide Sequence of Plasmid pPL1 from Marinococcus halophilus

Petra Louis and Erwin A. Galinski
Instituí fur Mikrobiologie & Biotechnologie, Rheinische Friedrich-Wilhelms-Universitüt Bonn,
Meckenheimer Altee 168, 53115 Bonn, Germany
Received April 4, 1997; revised July 2, 1997

Several plasmids were detected in the Gram-positive halophilic eubacterium Marinococcus

halophilus and in the related strain M52. The complete nucleotide sequence (3874 bp) of one
of these plasmids, pPLl, was determined. Four major open reading frames were identified.
Whereas orf3 and orf4 showed no sequence similarities to known proteins, rep displayed a high
sequence similarity to replication proteins of rolling circle plasmids. Upstream of this ORF, a
sequence resembling the double-strand origin was detected. A región probably constituting the
single-strand origin was identified. The ORF mob showed sequence similarity with Mob proteins
of rolling circle plasmids. The observed characteristics suggest that pPLl replicates according
to the rolling circle mechanism. © 1997 Academic Press

Moderately halophilic eubacteria grow op timally under conditions of elevated salinity.

They are of biotechnological interest as potential producers of compatible solutes, which can
beapplied for the stabilization of enzymes and whole cells (Lippert and Galinski, 1992;
Louis et al, 1994). However, genetic studies on these organisms are scarce at present.
Kunte and Galinski (1995) established a transposon mutagenesis system in the Gram

negative halophile Halomonas elongata. Several plasmids were identified and used for
construction of shuttle vectors in Gram negative halophiles (Fernandez Castillo et al,
1992; Mellado et al, 1995a, b; Vargas et al,1995). The genes for the biosynthesis of the
compatible solute ectoine from the Gram positive organism Marinococcus halophilus were
cloned and sequenced (Louis and Galinski,1997); however, no vectors for this organism
are available at present.

The genus Marinococcus currently consists of

two species, M. halophilus and M. albus. M.
hispanicus has been transferred to the genus
Salinicoccus(Ventosa et al, 1992).
The isolate M52 (Wohlfarth, 1993)) probably
also belongs to the genus Marinococcus, as
has been suggested on the basis of
physiological tests and DNA hybridization
studies (Jahnke, 1994;

A. Ventosa and B. J. Tindall, personal communication). A variety of plasmids, primarily of

Gram positive origin, have been shown to replicate by a rolling circle mechanism (Espinosa et
al, 1995; Gruss and Ehrlich, 1989). These plasmids harbour a gene encoding a plasmid
replication initiation-termination protein (Rep), a doublé strand origin which is the target site for
Rep, and a singlestrand origin for the conversión of single strand intermediates into double-
strand plasmids. In this study, we report the isolation of plasmids from the genus Marinococcus
as well as cloning and sequencing of the cryptic plasmid pPLl from M. halophilus. The resulting
sequence was analyzed for open reading frames and sequence similarities to other published
sequences. To our knowledge, this is the first report of plasmids from a Gram-positive
moderately halophilic eubacterium.
MATERIALES Y METODOS
Bacterial Strains, Growth Conditions, and
Plasmid M. halophilus DSM 20408T, M. albus DSM 20748T, and strain M52 (Wohlfarth, 199
3
were grown aerobically at 37°C in LB médium (Miller, 1972) containing 0.3% (w/v) artificial sea
salt and 3.7% (w/v) NaCl.
Escherichia coli XLl-Blue (Stratagene, Heidelberg, Germany) was grown aerobically at
37°C in LB médium. Vector pUC18 (Pharmacia, Uppsala, Sweden) was used for the cloning of
plasmid pPLl. To select for cells bearing recombinant derivatives of pUC18, the médium
contained ampicillin at a final concentration of 100 /¿g mi"1, isopropyl /?-D-
thiogalactopyranoside (0.5 im), and 5-bromo-4-chloro-3-indolyl /?-D-galactopyrano side (40 ¡i
% mi"1).

Plasmid DNA Identification, Isolation, and Manipulation

Plasmids were detected using a modified method of Eckhardt (1978). Cell cultures (1.5 mi)
were harvested and resuspended in 50 fú Tes buffer (5 mM EDTA, 50 mM Tris, 50 mM NaCl, pH
8.0). After adding 75 ¡A lysozyme solution (450 mM Tris, 50 mM EDTA, 8% sucrose, 2%
Ficoll 400, pH 8.0, 0.5 /¿g mi"1 RNase (Qiagen, Hilden, Germany), 4 mg mi"1 lysozyme (Merck,
Darmstadt, Germany)) the suspensión was incubated for 30 min at 25°C. The cells were loaded
onto an agarose gel containing 1% agarose and 0.5% sodium do-decyl sulfate (SDS) in TBE
buffer (90 mM Tris, 90 mM borate, 2.5 mM EDTA, pH 8.3). After 30 min at 30 V the voltage
was raised to 60 V. After the run the gel was washed in aqua demin to remove excess SDS,
stained with ethidium bromide, and analyzed under UV light.
Plasmids from M. halophüus and M52 were isolated using the Plasmid Midi Kit from Qiagen
(Hilden, Germany). Prior to lysis, the cells were incubated for 30 min at 25°C in buffer Pl,
which contained 3.75 mg mi"1 lysozyme (Merck).
Small- and large-scale plasmid preparations of E. coli XLl-Blue were performed using
Qiaprep spin columns and the Plasmid Midi Kit from Qiagen, respectively.
Restriction analyses were carried out as specified by the manufacturer (Boehringer
Mannheim, Germany). Analytical gel electro
phoresis was performed according to standard techniques (Ausubel et al, 1991). Two-di-
mensional electrophoresis to distinguish CCC and OC forms of plasmid DNA was carried out
as described by Hintermann et al. (1981).

DNA Cloning and Sequencing

Plasmid pPLl was cloned at its unique restriction site BamHl into pUC18 according to
standard techniques. Transformation of E. coli XLl Blue was performed by the CaCl2 method.
Sequencing by primer walking was performed by Sequiserve in both directions. The región
containing the BamHI restriction site was sequenced in both directions by Sequiserve after
cloning pPLl at its unique Sacl restriction site into pUC18. The nucleotide sequence data have
been deposited in GenBank under Accession number U75508.
DNA sequences were analyzed with the programs GENEPRO, DNASIS, and MACAW.
Databank searches were carried out through the NCBI with the BLAST program and current
versions of the available databases.

RESULTS AND DISCUSSION

Detection of Plasmids in M. halophüus and Strain M52
By means of Eckhardt analysis, two-dimen-sional electrophoresis, and restriction analysis two
plasmids, pPLl (3.8 kb) and pPL2 (7.3 kb), were identified in M. halophüus. A larger plasmid of
about 20 kb seemed to be present, but it was not examined in detail. M52 harbours plasmid
pTLl of 7.8 kb. In M. albus, no plasmid could be detected with the Eckhardt analysis. Attempts
to isolate plasmids from M. albus were not successful either. Therefore, we concluded that M.
albus does not contain any plasmids that are detectable with the techniques applied.

Marinococcus halophilus PLASMID pPLl 109

Nucleotide Sequence ofpPLl

The complete nucleotide sequence ofpPLl was determined (Fig. 1A). pPLl has a length of
3874 bp and a GC contení of 38.9% (GC contení of toíal DNA of M. halophilus ac-cording to
Hao et al, 1984: 46.4%). Three open reading frames (mob, rep, orfS) greaíer íhan 100 codons,
wiíh puíaíive ribosome bind-ing siles, could be deíecíed in íhe same orien-íaíion (Fig. IB). They
are predicíed ío encode proíeins of 453, 308, and 196 residues, wiíh deduced molecular masses
of 54,279, 36,910, and 23,158 Da, respecíively. One ORF of 101 codons was presení in íhe
opposite direction; however, no ribosome binding site could be deíecíed. A second síarí codon
24 bp down-síream of the first one displayed a possible ribosome binding siíe. This ORF
(orf4, Fig. IB) encodes 93 residues, wiíh a deduced molecular mass of 10,806 Da. No
significaní sequence similarities of or/3 and orf4 to known protein sequences could be found
using íhe blasí algorithm (Altschul et al, 1990).

Replication Functions ofpPLl

Daíabank searches using íhe blasí algorithm (Alíschul et al, 1990) revealed a high similariíy
of ORF rep ío replicaíion proteins of rolling circle plasmids. Rolling circle plasmids have been
classified into four families based on genetic organization and homologies ai the doublesírand
origin, the degree of Rep proíein conservaíion, and similarities ai the replicaíion control región
(Espinosa et al, 1995). The highest homology of the deduced amino acid sequence oí rep
ofpPLl was found to the Rep proteins of plasmids pSK89 (Littlejohn et al, 1990) and pC194
(Horinouchi and Weisblum, 1982) from Staphylococcus aureus belonging to the pUBUO
family, with 59 and 61% identiíy ai the protein level, respecíively. An alignmení is shown in
Fig. 2A. Ilyina and Koonin (1992) ideníified íhree conserved sequence moíifs in Rep proíeins.
The reactions caíalyzed by Rep proíeins require Mg2+ or Mn2+. Two hisíidine residues
embedded in a highly hydrophobic sequence are believed ío ací as ligands to metal cenlers.
Another moíif coníains a íyrosine, believed ío form íhe covalení link wiíh nicked DNA. No
funclion has been proposed for the íhird conserved moíif. All of íhese motifs were detected in
íhe deduced amino acid sequence of rep of pPLl (Fig. 2A).
The doublesírand origin íypically lies upstream of or within the rep open reading frame
(Gruss and Ehrlich, 1989). It could be ideníified by sequence comparisons abouí 200 bp
upsíream of rep. An alignmení wiíh íhe double-sírand origins of pSK89 and pC194 is shown
in Fig. 2B.
The single sírand origin of rolling circle plasmids is less conserved íhan íhe double-strand
origin. This site is normally localed ouíside the minimal replicón and consisís of palindromic
sequences of up to 200-300 bp (Gruss and Ehrlich, 1989; Coffey et al, 1994). The single strand
origins of several rolling circle plasmids have been divided inío dislincí families based on
sequence comparisons (Espinosa et al, 1995; Meijer et al, 1995). Addilional singlestrand
origins could not be classified in one of íhese families (De Rossi et al, 1992; Fremaux et al,
1993; Meijer et al, 1995). A región wiíh several inverted repeaís could be found beíween bp
3650 and 3850 in pPLl. Furthermore, íhis región coníained three streíches wiíh a high
homology lo the palT-íype singlesírand origin of pTX14-3 from Bacillus thuringiensis
(Andrup et al, 1994; Meijer et al, 1995): bp 3638-3654, 3671-3689, and 3829-3874 from pPLl
wiíh 94, 100, and 83% identiíy ío bp 6444-6460, 6466-6494, and 6643-6689 from pTX14-3
(marked by a dashed line in Fig. 1A). Thus, that región of pPLl probably represenís the single-
strand origin of replication.
Different mechanisms seem lo be involved in íhe copy number control of rolling circle
plasmids. In some of these plasmids, an antisense RNA (countertranscribed or ct RNA) has
been deíecíed, which inlerferes with the transcription or translation of rep (Espinosa et al,
1995). Furthermore, a small repressor proíein Cop, which binds in íhe promoíer región of rep
and cop, has been found in several rolling circle plasmids (del Solar et al, 1993; Espinosa et al,
1995). However, none of íhes
e
FIG. 1. Sequence of plasmid pPLl. (A) Complete nucleotide sequence, beginning at the unique Haell site. Deduced
amino acid sequences of ORFs designated mob, or/4, rep, and orf3 are shown below the sequence. Stop codons are
marked with astensks. Putative ribosome binding sites are boxed. Palindromic sequences are marked by arrows. The
double-strand origin is underlined, and the putative single-strand origin is marked by a dotted line (regions with
homology to the single-strand origin of pTX14-3 (Andrup et al, 1994) are dashed). (B) Physical map and genetic
organization of pPLl. ORFs are indicated by arrows, the double-strand origin is represented by a box, and the single-
strand origin is marked by a dashed line. Unique restriction sites are given.

components could be identified on pPLl using sequence comparisons.Open Reading Frame

mob The open reading frame mob is preceded two putative ribosome binding sites, so that

Fia 2. Alignment of the replication functions of pPLl with plasmids pSK89 and pC194 (LitÜejohn et
al, 1990; Horinouchi and Weisblum, 1982). (A) Deduced amino acid sequences of Rep proteins. Identical
positions are shown against a dark background and conservative replacements are boxed. The following
were regarded as conservative replacements: R-K-H, D-E, N-Q, S-T, G-A, F-Y-W, I-L-V-M. Bars above
the sequences indícate conserved regions, and triangles mark residues conserved in all examined sequences
according to llyina and Koonin (1992). (B) Nucleotide sequence of double-strand origins. Identical
nucleotides are shown against a dark background. The arrow marks the nicking site of the Rep protein.

of rolling circle plasmids. The Mob protein seems to be required for the mobilization of the respective
plasmid with the aid of a conjugative plasmid (Josson et al, 1990). The different Mob proteins show
extensive sequence homology only in their amino terminal portions. The same holds true for Mob of
pPLl. The highest similarity was found to the Mob proteins of pTX14-l and pTX14-3 from B.
thioingiensis (GenBank Accession No. U67921; Andrup et al, 1994). The first 228 amino acids of the
deduced Mob protein of pPLl showed 61% identity to the first 227 amino acids of the Mob protein of
pTX14-l. An alignment is shown in Fig. 3. As during conjugation Mob proteins act in a similar way on
the plasmid
sites seem to
DNA as do Rep proteins of rolling circle plasmids, similar exist.
conserved motifs can be found (llyina and Koonin, 1992). Furthermore,
All of these motifs were also identified in Mob of pPLl the DNA
(Fig. 3). The Mob protein acts at a site in the promoter región
región of mob containing an inverted repeat, the RSA site upstream of
(Josson et al, 1990; van der Lelie et al, 1989). In the mob of pPLl
sequence of pPLl, no sequence similarity to known RS A exhib
sites could be detected, but two inverted repeats were Marinococcus
present upstream of mob. Similarly, Fremaux et al. (1993) halophilus
identified a mob gene in pLol3 from Leuconostoc anos PLASMID pPLl
with no apparent RSA site. Thus, as yet unidentified RS A

FIG. 3. Alignment of the deduced amino acid sequences of the amino-terminal regions of ORFs
mob of pPLl and pTX14-l (GenBank Accession No. U67921). Identical positions are shown against a
dark back-ground and conservatíve replacements are boxed. Conservative replacements are defined in
the legend to Fig. 2. Bars above the sequences indicate conserved regions, and triangles mark residues
conserved in all examined sequences according to llyina and Koonin (1992).
its some sequence similarity to the DNA región upstream oí mob of pTX14-l (73% identity of
71 bp of pPLl located 29 bp upstream of mob to a DNA región 32 bp upstream of mob of
pTX14~l), which at present has not been examined in detail.

pPLl Is Organized into Different Cassettes

The organization of different rolling circle plasmids as interchangeable cassettes of genes
(Projan and Novick, 1988; Gruss and Ehrlich, 1989; del Solar et al, 1993) could also be
observed with pPLl. Whereas the open reading frame rep and the doublestrand origin of pPLl
showed the highest homology to plasmids of S. awreus, the singlestrand origin as well as the
mob gene revealed the highest similarity to plasmids of B. thuringiensis.

CONCLUSIONS
An examination of the nucleotide sequence revealed that pPLl belongs to the group of rolling
circle plasmids and can be classined to the pUBUO family according to Rep protein and
doublestrand origin homologies. Furthermore, it encodes a putative Mob protein as well as
two additional open reading frames.
Further studies will be concemed with the use of pPLl for the construction of useful cloning
vectors.