FEMS MicrobiologyLetters 93 (1992) 167-172
© 1992 Federation of European MicrobiologicalSocieties 0378-1097/92/$05.00
Published by Elsevier
167
FEMSLE (14895
Phylogenetic analysis of the genera Planococcus, Marinococcus
and Sporosarcina and their relationships to members
of the genus Bacillus
J.A.E. F a r r o w , C a r o l A s h , Sally W a l l b a n k s a n d M . D . C o l l i n s
Department of Microbiolo~,9..AFRC bJstitute of F¢nMRt;~earch.Reading Laln~ratory. Shb~flehl. Reading. UK
Received 9 March 1992
Accepted 13 March 1992
Key words: Phylogeny; Planococcus; Marinococcus: Sporosarchm; Bacillus
1. S U M M A R Y
2. I N T R O D U C T I O N
A phylogenetic analysis based on 16S r R N A
was performed on the genera Planococcus,
Marinococcus, Sporosarcina and endospore-forruing rods. In agreement with earlier 16S r R N A
c a t a l o g u i n g data, Planococcus citre,~s a n d
Sporosarcina ureae clustered with Bacillus pasteurii and other bacilli containing lysine in their
cell walls. Sporosarcina halophila was shown tc be
genetically distinct from S. ureae and formed a
loose association with the main Bacillus subtilis
grouping. Marinococcus halophilus (formerly
Planococcus halophilus) exhibited low levels of
relatedness to all reference species examined and
formed a distinct line of descent.
T h e taxonomic histories of the g e n e r a
Planococcus and Sporosarcb~a have been full of
turbulence and controversy. Historically, pianococci have been considered to be taxonomically
related to micrococci. Comparative 16S r R N A
cataloguing studies over the past few years, however, have demonstrated that Planococcus citreus
(type species of the genus) is only remotely related to micrococci and shows a specific relationship to certain round-sporeforming bacilli [1,2].
Planococcus citreus does not form endospores and
the possible association of this asporogenous coccus with certain endosporeforming bacilli is controversial. The taxonomy of planococci is further
complicated by some evidence of heterogeneity
within the genus. In particular, the placement of
P. h,:lophilus in the genus Planococcus is equivocal [3]. P. halophilus differs significantly in cell
wall murein composition from P. citreus and P.
kocurii in containing meso-diaminopimelic acid
Correspondence to: M.D. Collins, Department of Microbiology, AFRC Institute of Food Research, Reading Laboratory.
Shinfield, Reading RG6 2EF, UK.
168
ences, Hao and associates [4] have proposed P.
[4]. By contrast, the murein of the latter two
species contains L-lysine as the dibasic amino acid
t5]. Primarily on the basis of these murein differ-
halophihts be placed in a separate genus,
Marinococctts. Similar uncertainty surrounds the
A
MHAL
PCIT
PKOC
SHAL
SURE
GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGCGGGAAGCCGGCGGAG
........... GAAACCGGTGG
GAGAGTTTGATCCTGGcTCAGGACGAACGCTGGCGGCGTG~CTAATA~ATGCAAGTCGAGCG---GAGA-TAGTGGAGCTTGCTC~ATT--ATCTTC--GAGAGT~TGAT~CTGG~T~AGGA~GAA~GCTGG~GGCGTGCCTAATACATGCAAGTCGAG~G---GAAG-ANGTGAAcGTTGCTC~CAT---TGTTC~-GAGAGTTTGATCcTGGCTCA•GACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGCGGGAAGcAAGCGGATCCTT•GGGGGTGAAGCTTGTGG
GAGAGTTNGATCCTGGCTCAGGACGAACGCTNGCGGCATGCCTAATACATGCAAGTCGAGCG .... AACTGTTGGAAGCTTGCTTCCTT .... CN-GTT-
PKOC
SH~L
SURE
AACGAGCGGCGGACGGGTGAGTAACACGTGGGCAACcTGC•GGACTGATGGGAATAAC•CCGGGAAA•CGGGG•TAATG•CCAATA•GC•CTGA•CT•GC
.... AGCGGCGGACGGGTGAGTAA•ACGTGGGCAA•CTG•CCTGcAGATCGGGATAA•TCCGGGAAACCGGTGcTAATAC•GN•TAGTTTGCGGCcTcTc
.... AGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCCTGCAGAT~GGGATAACTC~GGGAAAC~GGTGcTAATAC~GRATAGTTTGCGGCC~fCTC
AA•GAG•GG•GGA•GGGTGAGTAACA•GTGGGCAACCTGCCTGTAAGACCGGAATAACCCCGGGA•ACCGGGGCTAATG•CGGATAAcACCTACCTTCAC
.... A G C G G C G G A C G G G T G A G T A A ~ A ~ G T G G G ~ A A ~ C T G ~ N N N N A G A N N N ~ N A T A A ~ T ~ G G G A A N c N G G N G ~ T A A T A ~ G N N N N N N N N N N N N N N N N N N
PCIT
PKOC
SHAL
SURE
MHAL
PCIT
PKOC
SHAL
SURE
MHAL
PCIT
PKOC
SHAL
SURE
7
CTGAGGTCAG~GGT~AAGCAGGGATCTTCGGATCcTTG~AcAGTccGATGGG~ccG~GG~Gc~TTAGCTAGTTC~GA~GTAAGGGcTcC~AAGGCG~c
ATGAGG-CTGCACGGAAAGACGGTTTCG-G .... CT N TCACTNACAGAATGG CCCG CGGCG CATTAG CTAGTTGGTGAGGTAACGG CTCACCAAGG CCAC
CTGAGG-CTGCACGGAAAGACGGTTTCG-G .... CTTTCACTTNCAGAATGGCCCGCGGCGCATTAG CTAGTTGGTGGGGTAATGG CCTACCAAGG CGAC
CTGAAG - GAAGG TTAAAAGATGG CTTCTCG .... CTATCACTTACAGATGGG CC CG CGG CGCATTAGCTAGTTGGTGAGGTAATAGCTCACCAAGGCGAC
NNNNNNNNNNNNNNNNNNNNNNNNNN--NGG- - - CTATCACTNAAGGATGGG CCCG eGG CGCATTAG CTAGTTGGTGGGGTAATGGCTCACCA~GGCGAC
GATGC~TAGcCGA~CTGAGAGGGTGATCGG~CAcACTGGGAcTGAGA~ACGGCCCAGACT~CTACGGGAGGCAGCAGTAGGG~ATcATCCG~AATGGGCG
GATGCGTAGC~GA~CTGAGAGGGTGAT~GGCCACACTGGGACTGAGACACGGCC~A~AcTC~TACGGGAGGCAG~AGTAGGG~ATcTTCcG~AATGGACG
GATGCGTAG~CGAC~TGAGAGGGTGATCGGCCACACTGGGAcTGAGACACGG~CAGACTCCTACGGGAGGCAGCAGTAGGGRATcTTcCG~AATGGACG
GATGcGTAG~GA~cTGAGAGGGTGAT~GG~ACACTGGGACTGAGACACGGC~CAGACT~CTACGGGAGGCAGCAGTAGGG~ATCTT~CGCAATGGACG
GATGCGTAGCCGACcTGAGAGGGTNATCGGccA•NcTGGGA•TGAGA•ACGG••cAGACTccTA•GGGAGGcAGcAGTAGGGAATcTT•cA•AATGGAcG
RAAG~TGACGGTG~AACG~GCGTGAGTGATGAAGGTTTTCGGATcGTAAAGCT~TGTTGAcAGGGAAGAAC~CACGTGAGTcGANcAG~CTGGCG~N
AAAGTCTGA~GGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAA~TCTGTTGTGAGGGAAGAACAAGTACCA-AGTAACT-ACTGGTAC~
AAAGTCTGACGGAGCAACGCCGCG GAGT ACG
GG
CGGA CGTAA]L%
G
G CAA ACC&-AGTAA
-A
AC
AAAGT CTGATGGAG CNATG CCG CGTGAGTGAAGAAGGTTTT CGGATCGTAAAG~TNTGTTGTAAGGGAAGAACAAGTACAGG~GTNACTN- CCNNTACCT
MHAL
PCIT
PKOC
SHAL
SURE
MHAL
PCIT
PKOC
SHAL
SURE
TNA A GT C G T G G C N A G C G T T G T C C G G A A T T ~ G G C G T N A A G C G C G
CG~AGGCGGTTTCGT~AGT~CGATGTGA~AGG~CACGGCT~AA~cGTGGAAGGCCATTGGAAAL~G~GA~ACTTGAGGACAGAAGAGGAGAGTGGAATTc
CGCAGGCGGTCcTTTAAGTCTGATGTGAAAGCCCACGGCTCAAC~GTGGAGGGT~ATTGGAAAC~GGGGGACTTGAGTG~AGAAGAGGAAAGTGGAATTC
~GcAGGcGTTcCTTAAAGT~GATGTGAAAG~A~GGCTCAAC~GTGGAGGGTCATTGGAAACTGGGGAA~TTGAGTG~AGAAGAGGA~AGTC~AATT~
CGCAGGCGGTTCTTTAAGTCTGATGTGA~AGCC~A~GGcT~AAc~GTGGAGGGT~ATTGGAAACTC~G~AACTTGAGGAcAGAAGAGGAGAGTGGAATTc
~Gc~GG~GGT~TTTAAGTcTNATGTGAAAGc~A~GGCTNAA~GTGGAGGGTCATTGGAAACTGGAGGACTTGAGTAC~GAAGAGGA~AGCGGAATTC
MHAL
PCIT
PKOC
SHAL
SURE
~AcGTGTAGc~GTGAAATGcGTAGATATGTGGAGGAAcA~AGTGGcGAAGG~GACTcT~TGGTCTGTA~TGACGCTGAGGTGcGAAAGcATGGGGAG~
cA~GTGTAG~GGTGAAATG~GTAGAGATGTGGAGGAACACCAGTGGCGAAGG~GACTTTCTGGTcTGTAACTGACGCTGAGGCGCGA~AGCGTC~GGAGc
CATGTGTAGCGGTGAAATG•GTAGAGATGTGGAGGAAcA•CAGTGGCGAAGG•GACTTTcTGGTcTGTAACTGA•G•TGAGGCGCGAAAGCGTGGGGAG•
CA•GTGTAGcGGTGAAATGCGTAGATATGTGGAGGAA•A••AGAGGCGAAGGCGACTCTCTGGTCTGTTTCTGA•GCTGAGGTGCGARAGCGTGGGTAGC
•ACGTGTAGCGGTNAAATG•GTAGAGATGTGGAGGAACN•CAGTGGCGAAGG•GGcTNTcTNGTcTGTAA•TGACGcTGAGG•GCGARAG•GTGGGGAGc
T G ~.~G
O ~ T C A CTCACCAGAAAGcCACGGcTAACTACGTGCcAGCAG~CGCGGTAATACGTAGGTGGCAAG~GT~GT~GGAATTATTGGGCGTRAAG~G~G
AAACAGGA
AGA ACCCTGGTA
CCA
CCG
CG
GAGTG
AGG G
A
G
TACC
A
C
AA C
PKOC
SHAL
SURE
AAAcAGGATTAGATA~TGGTAGT~A~G~GTAAA~GATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCANT-AACGCATT~AGCACT
AAACAG GATTAGATA CCCTGGTAGTCCACGCCGTAAACGATGAGTG CTAGGTGTTAGG GGG CTTCCACCCCTTAGTGCTGAAGTTAACGCATTAAGCACT
AAACAG GATTAGATAC CCTNGTNGTCCACG CCGTAAACGATGAGTG C T N A G T G T T A G G G G G T T T C ~ CC CCTTAGTG CTGCAN - T A A ~ C A T T A A G CACT
MHAL
PCIT
PKOC
SHAL
SURE
CCGCCTG G GGAGTACGACCGCAAGGTTGAAACT CAAAG GAATTGACGGGG G CCCG CACAAG C G G T G G A G C A T G T G G T T T A A T T C G A C G C A A ~ G ~
ccGc~TGGGGAGTA~GG~GCAAGGCTGAAACTCAAAGGAATTGACGGGGG~CCGCACAAGCGGTGGAG~ATGTGGTTT~ATTCGAAGCAACGCGAAGAA
C~GC~TGGGGAGTACGGcCG~AAGGcTGAAAcTcAAAGGAATTGAcGGGGG~G~AcAAGcGGTGGAG~ATGTGGTTTAATTCGAAGCAAcGCGAAGAA
~G~cTGGGGAGTAcGG~G~AAGGNTGAAACTCAAAGGAATTGACGGGGGCC~GCACAAG~GGTGGAGCATGTGGTTTAATT~GA]~AA~CGAAGAA
N~G~CTGGG~AGTACGG~CG~AAGGCTNAAA~T~AAAGGAATNGA~GGGGAc~-GcA~AAG~GGTc~AGCATGTc~TTTAATT~3AAGNAA~G~G~
MHAL
PCIT
PKOC
SHAL
SURE
CCTTACCAGATCTTGACATCTTCCCTACGCCTCGAGAGA- - -GGCNNTTCCTTCGGGGGACC~AATGACAGGTGGTGCATC~TTGTt~TC&C~t~TGTC
CCTTACCAGGTCTTGACATCCCG CTGACCGC CTAGGAGACTAG GCTTTTCCTTCGGGGACAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTC
CCTTACCAGGTCTTGACATCC CACTGAC CGGTGTAGAGA-TACG CTTTTCCTTCGGGGACAGTGGTGACAGGTGGTG CATGGTTGTCGTCAGCTCGTGTC
~CTTA~AGGTCTTGA~ATCCTTG-GACcAc~CTAGAGA-TAGG-TGTT~CTT~GGG~ACcAAGGTGAcAGGTG~'~ATGGTTGT~TCA~TCGTGTc
GGGGN CACTNGTGACAGGTGGNG CATNGTTGT CGTCAG CTCGTGTCCCTTACCAGGTCTTGACATCCCAGTNACCGTCATGGAGAC -ATC~TNTTC-TTC
PKOC
SHAL
SURE
GTGAGATGTTGGGT~AAGTCCCGCAACGAGCG CAAC CCCTAATCTTAGTTG CCAG CATTCAGTTGC~ C A C T C T A A G G T G A ~ C ~ A ~ C C ~ A G
GTGAGATGTTGG GTTAAGTCC CG CAACGAGeGCAACCCTTGATCTTAGTTG CCAG CATTCAGTTGGG CACTCT]L%GGTGACTG C C G G T G A ~ C ~ A G
GTGAGATGTTGGGTTAAGTCCCG CAACGAG CG CAACC CTTGATCTTAGTTGCCAGCATTCAGTTGGGCAt'TCTAAGGTGACTGCCGGTGACAAACCC~AG
GTGAGATGTTGGGTTAAGTCCCG CAACGAG CG CAACCCCTAATCTTAGTTGC CAGCATTCAGTTGGGCACTCTAAGGTGACTG C C G G T G A C ~ C ~ A G
GTGAGATGTTGGGTNAAGTCCCG CAACGAG CG CAACCCTTAATGTTAGTTGCCATCATTCAGTTGC~ C A C T C T N A T G T G A C T G C ~ A ~ C ~ A G
MHAL
PCIT
PKOC
SHAL
SURE
GAAGG
MHAL
PCXT
PKOC
SHAL
SURE
C~%TCCAGAAAAGc~ATTcTcAGTTcGGATTGCAGGcTGCAACTCGcCTGcATGAAGCC~GAATCGCTAGTAAT~ATCAG~C~G~TA
C
CC AGAAAACCGTTCTCAGTTCGGATTG CAG G CTG CAACTCGCCTG CATGAAG CCGGAATCGCTAGTAATCG CGGATCAG CATGCCGCGGTGAATA
CT ATGAAG CCGGAATCGCTAGTAATCG CGGATCAGCATGCCGCGGTGAATA
CAATCCCACAR~ATCGTTCCCAGTTCGGATTGCAGG CTG CAACTNG CCTN CATGAAG CCGGAATCG CTNGTAATCGTGGATCAGCATG CCACGGTNAATA
CGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCC
CGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCC
CGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACNCCC
CGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTGGCAACACCC
CGTTCCCG GCTCTN GTACACACCGCNCGTCACACCACGAGAGTT"~GTAACACC C
MHAL
PCIT
PKOC
SHAL
SURE
GGGATGACGTCAAATCATCATGCTC
A
AT
GG
ACACACG A
ACAA
AC
G GA
AA
C
ACe
T
GA AC T c A A A T ~ A T ~ A T G ~ C T T A T G A ~ C T G G G C T A C A ~ A ~ G ~ G C T A C A A T G G A C G N T A C A A A G G G C T G ~ A A C C C c G C G A G G G C A A G ~
GAAGG CG GGGATGACG TCAAATCATCATG CCCCTTATGACCTGG GCTACACACGTG CTACAATGGATGGTACAAAGGG CAGCGAAGCCGCGAGGTGTAGC
GAAGGTGGGGATGACGTCAAAT CATCATG CCCCTTATGACCTN G GCTACACACGTG CTACAATGGACGATACAGAGGGCTN CNAACCCG C G A G C ~ G A G C
Fig. |. Primary structures of 16S rRNAs of PCIT, P. citreus NCIMB 1493x; PKOC, P. kocurii NCIMB 629T; MHAL, 114. hah~phiht~
NCIMB 2178T; SHAL, S. halophila NC1MB 2269T: SURE, S. ~¢reae NCIMB 9251T as determined using reverse transcription. N,
undetermined nucleotide.
169
taxonomy of the genus Sporosarcb~a [61. Despite
marked morphological differences. S. ur,'ae (type
species of the genus) has been shown to be phylogenetically related to a number of species of the
genus Bacillus which contain mureins based on
l,-lysine (e.g.B. pasteurii, B. sphaericus ) [ I,7,g]. A
second species of the genus, S. halophila, however, differs from S. ureae in possessing a cell
wall murein based on ornithine [9]. The cell walls
of S. halopltila also contain high amounts of a
T-o-glutamyl polymer, which is absent from S.
ureae [9]. The genetic interrelationships of S.
halophila and S. ureae, however, have not been
investigated. Although comparative 16S r R N A
cataloguing indicates a specific relationship between P. citreus, S. ureae and certain bacilli,
these investigations haw to date, included relatively few members of the genus Bacillus (nine
species). Consequently it has been difficult to
draw conclusions regarding the precise placement
of Planococcus and Sporosarcitla within the overall structure/radiation of the cndosporeforming
rods. In addition, these studies did not include
the 'atypical' species P. halophilus and 5.
h:dophila. We recently reported a comprehensive
phylogenetic analysis of the genus Bacillus using
almost complete 16S rRNA sequences determined by reverse transcription [10]. This study
included over 50 species of bacilli and revealed
the presence of five major phylogenetic groups
within the genus [10]. in the present study, wc
have extended this analysis to include members
of Planococcus and Sporo,varcitla in an attempt to
determine (i) the precise intra- and inter-generic
relationships of these taxa, and (ii) the phylogcnetic validity of the genus Marinococcus.
3. M A T E R I A L S A N D M E T H O D S
3.1. Cultures and cultit'ation
The type strains of S. ureae, S. halophila, P.
eilreus, P. kocurii and M. halophilus were obtained from the National Collection of Industrial
and Marine Bacteria (NCIMB) Ltd. (Aberdeen,
UK). S. ureae was cultivated on Nutrient agar
(Oxoid CM3, Basingstoke, U.K.), whereas S.
halophila was grown on Marine agar (Difco
2216E) at 3I}°C. Strains of P. citreus, P. kocurii
and hi. halophilus were cultivated on Sea water
yeast pcptone agar (yeast extract 3 g, peptone 5 g,
fihcrcd sea water 0.75 I, agar 15 g, distilled water
0.25 I, pH 7.3) at 30°C.
3.2. Extraction and sequence determb:ation of 16S
rRNA
Cells (approx. I g wet weight) were mechanically broken using a Braun homogenizer and
rRNA was purified according to the procedure of
Embley ct al. till. Nucleotide sequences were
determined by the Sanger dideoxTnucleotide
c.~ain termination method from directly rRNAusing avian myeloblastosis virus reverse transcriptase [I I].
3.3. Analysis of sequence data
The sequences were aligned, and homology
values and nuclcotide substitution rates (K ......
values) wcrc determined using the Wisconsin
Molecular Biology package [12]. An unrooted
phylogcnetic trcc was produced using the algorithm of Fitch and Margoliash [13]. The 16S
rRNA sequences of 51 reference Bacillus strains
wcrc obtained from Ash et al. fill], and that of B.
o'chduT~tanicus from Wisotzkcy ct al. [14].
4. RESULTS
The 16S rRNA scqucnccs of P. citreus, P.
kocurii, I: halophihls, S. urcae and S. halophila
wcrc determined by reverse transcription. Thc
determined sequences arc shown in Fig. I and
have been deposited with the EMBL data base
(accession numbcrs X62171 to X62176). Between
1463 and 1481 nucleotides were determined, corresponding to about 95% of the whole 16S r R N A
molecule. The sequences were aligned with those
of 50 previously reported 16S r R N A Bacillus
sequcnces [!0] and the 16S rDNA sequence of B.
cycloheptanicus [14]. Sequence similarity calculations were based on a comparison of a continuous
stretch of about 1330 nucleotides ranging from
positions 107 to 1410 of the E. coli numbering
system [15]. Approximately 100 nucleotides at the
5' end of the rRNA molecule were not included
170
in the calculation t o eliminate possible alignment
errors due t o the extremely variable Vl region
and to ensure that only homologous positions
wcrc compared. A phylogenclic trcc calculated
from a matrix of derived K . . c wdues using the
Fitch and Margoliash [13] algorithm is depicted in
Fig. 2.
Comparative analysis of the RT sequence data
confirmed the presence of five major clusters of
Bacillus species [10]. Considerable phylogenetic
heterogeneity was, however, also apparent within
the genera Phmococcus and Sporosarcina. The
distance matrix analysis (Fig. 2) clearly demonstrates that S. ureae and P. citreus arc distinct
Group 2
p,~st,'u.,
~ tus,tor.,,s
t~ ,..vso~,ou,t.,,',,'.,',
e,, ,mot t,s
B mroohaeus
Group 1
//~
.
'.....................
M.hatophdus /
Group 4
:~..........J
t,.,a,. >
.
s,,,, ,,,
~ B~.,,,o,,,,.,,,.....
.
[qt,.,L,::
Group 3
/
,,m,,o~t,o,,,s
/
E~ c,,clon,,t,t.v,,c,,s
........
..°,°°,,/>.. pulv,t,lc~ons
'
El
/
...........
~ p 'v,,;v,,~
B latv.'le
Fig. 2. L::¢oll.llioilal3' distance tree showing II1¢ position of I ) h m o c o c ( ' t t ~ . M a r o m c o c c u . ~ - a n d Sporo.~archm species ~ithin a radiation
of endosporeti)rming rods. K , , , valt[cs x~erc based on a comparison of 1332 nuclcolides, ranging from positions 1117 1o 14111of the
I:'. c o l t numhcrhlg system The ~:volulionary dislance b¢lweell two species is tllu sum of Ihe I~ranch lengths between them.
from the main Bacillus group (designated Bacilh+s sensu stricto; group I. [10]) and cluster with
B. sphaericus, B. fits(form(s, B. gdobi,spon+s, B.
htsolitus, B. pasteurii and B. p.sTclzropltilu.s" (group
2, [10]). In addition, the sequence analysis clearly
identified P. kocurii as another member of this
group. The remaining species examined, S.
halophila and P. halophilus, were found to bc
phylogenetically distinct from the type species of
their respective genera. S. halophila exhibited a
loose association with Bacillus scnsu stricto
(group I), although no specific relationship to any
species within this group was apparent. By contrast, P. halophihls formed a distinct line of descent exhibiting low levels of relatedness to all of
the other bacilli and reference strains examined.
5. DISCUSSION
Earlier rRNA cataloguing studies rcveulcd a
specific relationship between the non-sporefl~rming coccus P. citreus and several sporeforming
rod-shaped species of the genus Bacillus [!,2,7].
These cataloguing studies, however, were vet3,'
restricted in the number of bacilli 19 species)
examined (over 4t) validly published Bacillus
species arc rccognised in Bergcy's Manual of
Systematic Bacteriology [6] and numerous other
species have subsequently been described). Wc
recently rermrted a comprche~isivc phylogenctic
analysis {'~t over 50 Bacillus species, based on RT
16S rRNA sequences [10]. In the present study,
we have extended this analysis to include several
representatives of the genera Phlnocot't'us and
$'porosar~,::l,d. The comparative analysis of almost
complete RT primary 16S rRNA sequences revealed S. to't'ae and P. citreus were specifically
related to 13. sldzaericus and related species (group
2, Fig. 2) thereby confirming the results of
oligonucleotide cataloguing. P, kocurii (a species
not included in the earlier cataloguing studies)
was shown to be genetically closely related to i:
citreus (98.2% sequence similarity) and was clearly
identifiable as a member of the B. sphaericus
group of species. By contrast, P. halophilus
formed a distinct line of descent and was only
distantly related to the other planococci and ref-
erence species examined. The recovery , f P.
halophilus as a separate phylogenetic lineage supports the creation of the new genus Marinococcus
to accommodate this species [4]. The rRNA sequence analysis also indicates that S. halophila is
not genetically closely related to S. ureae and the
other species of rRNA group 2. The type strain
of S. halophila exhibited a loose assciation with
B. mhtilis and relatives (group I). It is worth
noting that this main B. suhtilis group shows
considerable phylogcnetic depth, with many of
the lineages radiating from almost the same position. Although the distances between branching
points is subject to some statistical uncertainty,
:he branching order in the tree clearly indicates
that S. halophiht is peripheral t o this main group
and is not a member of Bacilha" sensu stricto.
The distinctiveness of S. halophila is in accordance with cell wall murein coml~sition. S.
holophiht contains the murein type orn-o-asp [9],
whereas B. suhtili~ and other species of rRNA
group I invariably have walls based on meso-diaminopimclic acid [51.
There is now a growing recognition that the
taxonomy of the genus Bacillus is in urgent need
of revision..-\ltlaough rRNA sequencing has done
much to clarify the genetic interrelationships of
the endospore-forming rods and related taxa
[10.16], it is evident that the considerable complexity revealed within this group suggests the
need for further studies before formal proposals
l o r the division of the genus Bacillus into several
genera may bc made with confidence.
ACKNOWLEDGEMENTS
Wc arc grateful to M A F F for supporting this
work.
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