Bacteriocins are the most abundant and verse of the microbial defense systems. Theyproduced by all major groups of Bacteria aArchaea (Riley, 1998) (Dykes, 1995; Torrblanca et al., 1989), are often produced at vehigh frequencies within a population, and ehibit extraordinary levels of protein diversi(Riley, 1998; Tagg et al., 1976). In Escherichiacoli, bacteriocins are exclusively encoded plasmid replicons.
The colicins of E. colihave served as a modsystem for investigating the evolutionary mecanisms involved in bacteriocin diversificatio(Riley, 1993a, 1993b, 1998; Riley and Gordo1995; Pilsl et al., 1999; Tan and Riley, 19951997; Braun et al., 1994; Roos et al., 1989).Phylogenetic studies distinguish two major cicin families, the pore-former and nuclease cicins (Riley, 1998). These two families appeardiffer in their modes of diversification. Thpore-former colicins, which kill by formingchannels in the cytoplasmic membrane, arrelatively ancient family that has diversified pmarily through the action of recombination (rviewed in Riley, 1998). The nuclease coliciare a more recent family of proteins that kill nonspecific digestion of DNA or specific cleaage of RNA (James et al., 1991). The nucleascolicins investigated have diversified primarthrough positive selection acting on point mu
209
tions that result in novel immunity phenotyp(reviewed in Riley, 1998).
Two questions arise from studies of colicevolution. First, why do the two families of coicins experience such different evolutionaforces, as described above? Second, are theterns of sequence divergence observed foE.coli plasmid-encoded colicins representativebacteriocin evolution? We provide an evolutioary investigation of a bacteriocin plasmid islated from Klebsiella pneumoniaethat shedslight on both of these issues.
The bacteriocin plasmid pKlebB-K17/8(pKlebB) was isolated from K. pneumoniaeandidentified as a non-self-transmissible plasmencoding a bacteriocin-like killing phenotyp(James, 1988). James further localized the bteriocin gene cluster using a combination subcloning and transposon mutagenesis. Frestriction site analysis and mapping studiJames noted several structural and organtional similarities between the bacteriocin gecluster of klebicin B and those of the E-coliciof E. coli.
We report here the nucleotide sequence ofentire pKlebB plasmid. The location of the bateriocin gene cluster was inferred from DNand protein sequence comparisons with all chacterized bacteriocins. These comparisons sgest that klebicin B functions as a DNase bac
riocin. Further, the klebicin B plasmid is showto be a chimera, composed of regions similathose of several members of the colicin plasmfamily of E coli, the chromosomally encodenuclease pyocins of Psuedomonas aeruginosand several additional ColE1-like plasmids islated from Klebsiella oxytocaand K. pneumo-niae. The klebicin B plasmid provides one of t
first indications that recombinational diversifi
t
i
e.,a
ni
le
ei
m
nyrd,were All forumree.sen-sentod in-n-
fidence in the inferred tree topologies was as-00
eirsig-Bof
-terhese
n-ti-de-llthee
cation is not restricted to the pore-former bacriocins.
MATERIALS AND METHODS
Plasmid
Richard James kindly provided the plasmpRJ180. This plasmid was constructed by serting pKlebB-K17/80 into the HindIII site ofpUC19 (James, 1988).
DNA Preparation and Sequencing
pRJ180 was transformed into competDH5a E. coli cells (Life Technologies IncGaithersburg, MD) using standard methods, ampicillin-resistant colonies were selected LB agar containing ampicillin (50 mg/ml)(Ausubel et al., 1992). Cells were growovernight in 5 ml of LB broth containing ampcillin (50 mg/ml) at 37°C, and plasmid DNAwas isolated using a QIAprep kit (QiageChatsworth, CA).
Approximately 300 ng of plasmid DNA waused for cycle sequencing according to the ptocol of the ABI PRISM Dye Terminator CycSequencing Ready Reaction Kit (Perkin–ElmPalo Alto, CA). Primers were designed to annto the pUC19 vector and were employed to inate DNA sequencing. Additional primers weconstructed at approximately 250- to 300-bptervals. Primer locations and sequences available upon request. The nucleotide sequeof the plasmid pKlebB-K17/80 is available froGeneBank (Accession No. AF156893).
Computer Analysis
DNA sequence data were assembled andited with DNASTAR programs (DNASTAR
Inc., Madison, WI). The nucleotide and proteisequences were submitted to the National Ce
T AL.
n toid
-
e-e-
idn-
ter for Biological Information and the BLAS(Altschul et al., 1990) network service was employed to search for similar sequences in database.
Nucleotide and protein sequence alignmewere made with the Clustal-W algorithm of tLASERGENE program (DNASTAR Inc1999). Gene trees were inferred using neighjoining and maximum likelihood and parsimoalgorithms (Saitou and Nei, 1987; Swoffo1993). Heuristic searches were used. Gaps not treated as characters in any analysis.methods produced a single, congruent treeeach gene, with the exception of the maximparsimony applied to the nuclease region tIn that case, two trees were found and a consus tree was compiled. Figures 2 and 3 repregene trees inferred with a maximum likelihomodel. Figures 4 and 7 report gene treesferred with a maximum parsimony model. Co
nt
ndon
-
n,
sro-
er,al
ti-rein-arence
ed-
sessed by application of a bootstrap, with 10replications (Felsenstein, 1985).
RESULTS AND DISCUSSION
Klebicin B Plasmid
The entire pKlebB-K17/80 DNA sequencwas determined. The plasmid is 5258 base paand encodes seven recognizable functions. Fure 1 illustrates the organization of the pKlebplasmid and Table 1 provides the position each predicted function.
Bacteriocin Gene Cluster
Three bacteriocin-related genes (klebicin, immunity, and lysis) were detected as a clus(Fig. 1). The three genes are oriented in tsame direction, a pattern typical for nucleacolicin gene clusters.
The klebicin B lysis gene is 156 bp and ecodes a protein of 51 amino acids with an esmated molecular weight of 5 kDa (Fig. 1 anTable 1). Lysis proteins are involved in the rlease of bacteriocin from the producing cell. Acharacterized lysis genes were aligned and gene tree inferred is shown in Fig. 2. Th
nn-klebicin B lysis gene is most similar to the lysisgenes of colicin gene clusters A and S4, two
o-
nd4,
ted toS4nce
he arrows represent functional regions detected by DNA and pro- a vertical
Rom 5181–105Excll 105–506
members of the colicin pore-former family islated from Citrobacter freundii (A) (Morlon,1983) and E. coli (S4) (Pilsl et al., 1999). Levelsof lysis protein sequence identity are 61 a62% between klebicin B and colicins A and Srespectively.
The lysis and immunity genes are separaby 107 bp (Fig. 1). This region is most similarthe corresponding region of the colicin A and gene clusters, with 50 and 41% DNA seque
gene expression. The origin of replication is indicated byre indicated.
KLEBICIN B PLASMID EVOLUTION 211
identity, respectively. The 38 flanking region
FIG. 1. Physical and genetic map of pKlebB-k17/80. Ttein sequence comparisons and the inferred direction of line, labeled Ori. Several restriction endonuclease sites a
TABLE 1
Nucleotide Positions of Inferred Functional Regions ofpKlebB-K17/80
Gene Location (bp)
Klebicin 1628–3925Immunity 3927–4184Lysis 4292–4447RNA I 4601–4496RNA II 4494–5001Ori 5002
(110 bp) of the klebicin B lysis gene is also mostsimilar to the corresponding region of the col-
E
2
n elee-
en
alseof
forene-
ty
212 RILEY
icin A and S4 gene clusters, with 89 and 9DNA sequence identity, respectively.
The klebicin B immunity gene is 258 bp aencodes a protein of 85 amino acids with antimated molecular weight of 9.6 kDa. A singbp separates the 58 end of the immunity genfrom the 38 end of the klebicin gene (Fig. 1). Immunity proteins interact with the C-terminal rgion of their corresponding bacteriocin protei
conferring immunity to the host cell or othe
FIG. 2. A maximum likelihood network inferred for lysisthe appropriate branches.
T AL.
%
ds-
-s,
with all characterized immunity proteins revelow levels of sequence similarity, save for thoassociated with the family of DNase colicins E. coli (E2, E7–E9) and the pyocins of P. aeru-ginosa(S1, S2, and AP41).
Immunity gene sequences were aligned this subset of sequences and the inferred gtree is provided in Fig. 3. The klebicin B immunity gene is most similar to the pyocin immuni
rgenes. The topology of this tree is not well sup-
eset to
cells encoding the same bacteriocin. Sequencecomparisons of the klebicin B immunity protein
ported by bootstrap analysis, implying that thimmunity protein sequences are too divergen
DNA sequences. Bootstrap values above 80% are indicated on
triynu6ee
rn
d65 oftnslln-
r-re,
FIG. 3.A maximum likelihood network inferred for immunity DNA sequences. Bootstrap values above 70% are indicated
allow precise reconstruction of their ancesrelationships. The klebicin B immunity proteis most similar to the immunity proteins of pocins S1 and S2 with 57% protein sequeidentity. It is slightly less similar to the immnity proteins of colicins E2 and E7–E9, with 552, 51, and 52% sequence identity, respectivThe klebicin B immunity protein shows no dtectable similarity to colicins A and S4.
As noted above, the region 38 to the immunitygene is most similar in sequence to the cosponding region of the colicin A and S4 ge
on the appropriate branches.
clusters. This region could not be aligned btween klebicin B and the nuclease pyocins colicins.
aln-ce-,ly.-
re-e
The klebicin B bacteriocin gene is predicteto be 2298 bp and encodes a protein of 7amino acids. The estimated molecular weightklebicin B is 79.7 kDa, which agrees with thareported by James (1988). Bacteriocin proteicontain at least four functional domains: cesurface receptor recognition, translocatioacross the cell membrane, cell killing, and immunity protein binding (reviewed in James etal., 1996). In colicins, these functions are odered as follows: translocation functions afound in the N-terminal portion of the protein
KLEBICIN B PLASMID EVOLUTION 213
e-orreceptor recognition comprises the middle ofthe protein, and the killing domain and immu-nity binding region are located in the carboxy-
E
rnath
r-.
g-
214 RILEY
terminal (C-terminal) region (James et al.,1996).
A BLAST search reveals similarity with othebacteriocin proteins restricted to the C-termihalf of the klebicin B protein. The most similbacteriocins with respect to this region are DNase colicins (E2, E7–E9) and pyocins (S
FIG. 4. A maximum parsimony network inferred for the CBootstrap values above 80% are indicated on the appropr
T AL.
alre
1,
ure 4 provides a gene tree inferred for the C-teminal regions of the DNase bacteriocinsKlebicin B is intermediate in position betweenthe DNase colicins and pyocins.
The DNA sequence immediately 58 to theklebicin B bacteriocin gene encodes a set of reulatory elements. All colicin gene clusters
ed
S2, and AP41). Levels of protein sequence iden-tity with the final 132 amino acid residues ofklebicin B range from 43 (S1) to 29% (E9). Fig-
whose regulatory regions have been sequenccontain a s70 promoter, a LexA binding site, anda Shine-Dalgarno box (Fig. 5). The klebicin B
-terminal half of the nuclease colicins, pyocins, and klebicin B.iate branches.
ei
n
ys,
Ia
nt
tifs.
(Akutsu et al., 1989), ColE7 (Soong et al., 1992), Col10 (Pilsl and Braun, 1995a), Col5 (Pilsl and Braun, 1995b), ColS4(Pilsl et al., 1999), ColB (Schramm et al., 1987), ColU (Smajs et al., 1997), ColN (Pugsley, 1987), ColY (Riley, 2000), ColK
a .
gene cluster contains all of these common ments and shares high levels of sequence slarity throughout this control region with thosassociated with the characterized colicin geclusters. s70 promoter sequences of colicin geclusters are highly conserved; in fact, the 235
(Kuhar and Zgur-Bertok, 1999), ColA (Morlon et al., 1983),
sequences exactly match the consensus quence. All colicin gene clusters contain a Lexbinding site 3–4 bases downstream of the Pr
le-mi-enee
now box. The LexA binding site is actuallcomposed of two overlapping binding sitewith the exception of colicin gene clusters forand Ib (Mankovich et al., 1986), which have asingle site.
The colicin A gene cluster shows significa
nd KlebB (this work). Asterisks designate 10 unit intervals
KLEBICIN B PLASMID EVOLUTION 215
FIG. 5. An alignment of the regulatory regions of bacteriocin gene clusters annotated to show identified regulatory moSequences are from ColE1 (Tomizawa, et al., 1977), ColE2 (Cole et al., 1985), ColE3 (Masaki and Ohta, 1985), ColE6
se-Aib-
deviation from the typical colicin regulatory re-gion relative to the majority of other colicin geneclusters. The colicin A gene cluster has an inser-
E
b
r
d
ao
e
u
s
s
8
p
i
t
ii
e-dst-i-
ataser-ald
-
ay
edi-n-t-m
peor
fod-
fsly
d
216 RILEY
tion of about 60 bp located between the douLexA binding site and the Shine-Dalgarno boThe colicin A gene cluster regulatory regioalso lacks the highly conserved thymine-rich gion of unknown function found just downstream of the LexA binding site (at nucleotipositions 87–100, Fig. 5).
Overall, the klebicin B gene cluster regultory region most closely resembles that of cicin A. It contains a slightly larger insert (,72bp), it lacks the thymine-rich region conservin all other colicin regulatory regions (Fig. 5and it shares the highest degree of sequeidentity (69%) with the Col A gene cluster reglatory region.
Klebicin B Killing Function
The DNase killing domain is containewithin a short stretch (132 aa) of the C-terminregions of DNase colicins and pyocins (Jameetal., 1991; Sano et al., 1990). An alignment ofthis killing domain among all nuclease colicinpyocins, and klebicin B reveals that the DNaand RNase colicins share little detectable quence similarity. Five residues (amino acid psitions 10, 44, 101, 125, and 126; Fig. 6) ashared by all nuclease colicins and an additiofour residues (amino acid positions 47, 52, and 97; Fig. 6) are shared by all but one memof the entire nuclease group. In contrast, residues are shared by all DNase colicins, ocins and klebicin B and an additional 2residues are shared by all but one member ofDNase group. The RNase colicins are most silar in this region, sharing all but 21 of the 13residues.
Klebicin B is identical to the consensus squence of the DNase killing domain at all buof the 44 sites shared by other members ofDNase group (Fig. 6). Such high levels of squence similarity argue that klebicin B is member of the nonspecific endonuclease faily of nuclease bacteriocins. A gene tree ferred for the nuclease region clearly distguishes the RNase and DNase bacterioc(Fig. 7) and reveals that klebicin B clustewithin the DNase branch of this tree and sha
greater similarity with the nuclease pyocins P. aeruginosa.
T AL.
lex.ne--e
-l-
d),nce-
dal
s,see-o-renal3,
ber37y-0 them-2
e- 7thee-am-n-n-insrsres
There is no detectable sequence similarity btween the N-terminal region of klebicin B anthose of any other bacteriocin proteins, suggeing that klebicin B has unique receptor recogntion and translocation systems.
Klebicin B Plasmid Maintenance Functions
BLAST searches reveal several regions thare involved in pKlebB maintenance, such replication control and mobilization. Thesfunctions were inferred from sequence similaity with the corresponding regions of severcolicin plasmids, including pCol A (Zverev anKhmel, 1985), pCol E1 (Chan et al., 1985),pClo DF13 (Nijkamp et al., 1986), pCol Y(Riley et al., 2000) and pBERT from Salmonellabertii (Hanes et al., unpublished), and nonbacteriocin plasmids from Klebsiellaand Salmonella,K. pneumoniaeplasmid pJHCMWI (Dery et al.,1997), K. oxytoca pNBL63 (Wu, 1999) andpTKHII (Wu, 1999), and Salmonella ty-phimurium cryptic plasmid pIMVSI (Astill,1993).
pKlebB appears to replicate in the same was ColE1 (Chan et al., 1985). This form of repli-cation is mediated by chromosomally encodproteins, and the frequency of initiation of replcation is regulated by two plasmid-derived trascripts, an RNA primer, RNA II, and a modulaing antisense RNA, RNA I, encoded upstreaof the point of initiation of DNA synthesis(Polisky, 1988). Some plasmids using this tyof replicon also encode a small protein (Rom Rop) involved in the modulation of the initiationprocess (Mikiewicz, 1997).
The antisense RNA I and RNA II region opKlebB (Fig. 1 and Table 1), although similar tall ColE1-type plasmids, is most closely relateto the corresponding region of pJHCMWI (isolated from K. pneumoniae), pIMVSI (isolatedfrom S. typhimurium), and p15A (isolated fromE. coli). The inferred secondary structure oRNA I from pKlebB has the three hairpin loopcharacteristic of ColE1-type plasmids (typicaldesignated I8, II8, and III8) (Tomizawa and Itoh,1981). Most mutations, which result in altereincompatibility, are located in loops I8 and II8
of(Davison, 1984). The RNA I sequences fromp15A (Selzer, 1983), pJHCMWI, and pIMVSI
KLEBICIN B PLASMID EVOLUTION 217
FIG. 6. An alignment of the C-terminal 132 amino acids of the nuclease colicins, pyocins, and klebicin B. Gray shadingindicates those residues conserved among all RNase colicins. Dark gray shading indicates those residues shared among allDNase colicins. Stars indicate those residues shared by all nuclease colicins, pyocins, and klebcin B.
tituituThth
nc8ar5
leed
oathereemison
FIG. 7.A maximum parsimony network inferred for the C-terminal 132 amino acids of the nuclease colicins, pyocins, andn t
are all more similar to each other (2–5 substions in the region containing secondary strture) than they are to pKlebB (10–12 substtions and two insertions in the same region). majority of sequence variations between KlebB sequence and the other RNA I sequeoccurs in loops I8 and II8 (one insertion and 6–substitutions). Since pJHCMWI and p15A compatible (Dery et al., 1997) and differ by
klebicin B. Bootstrap values above 60% are indicated o
substitutions, only one of which is in loop I8, itis reasonable to assume that pKlebB would bcompatible with the other three plasmids.
is-
u-c--eees
e
The pKlebB Rom protein (Fig. 1 and Tab1) is most similar to the Rom protein encodby plasmid pNBL63 isolated from K. oxytoca.These two Rom proteins differ at only twsites. Mikiewicz et al. (1997) proposed that family of Rom proteins existed, based upon sequence similarity observed among thcharacterized Rom proteins. Additional Roproteins have been described and a comparof these proteins firmly establishes the ex
he appropriate branches.
218 RILEY ET AL.
etence of this protein family (Riley, data notshown).
M
ioBx3r
eitnenay
htod
se
sta
ddss
re
son,ion
I)t
rss,s a
ri-ds.areort
an- of
edneirlyta-e-
-cin.in-lu-ionin
ap-
KLEBICIN B PLAS
A region similar in sequence to the exclusregion of ColE1 is also detected in pKlebBLAST searches reveal that the encoded Eprotein is most similar to that found in pNBL6The Exc1 proteins of pKlebB and pNBL63 aquite similar to each other and highly divergfrom the corresponding protein in ColE1, w95% sequence identity between pKlebB apNBL63 and 27% sequence identity betwepKlebB and ColE1. In ColE1 this region ecodes two proteins (Exc1 and Exc2), which involved in a form of plasmid incompatibilitknown as entry exclusion (Chan et al., 1985).The mechanism of incompatibility involves tinhibition of conjugal transfer of a plasmid ina host cell. A second Exc protein was not tected in either pKlebB or pNBL63.
Recombination in pKlebB
pKlebB is a chimeric plasmid. Figure 8 illutrates the regions of pKlebB that differ in squence origin. Based upon DNA and proteinquence comparisons, we deduce that bacteriocin gene cluster is the product of at lethree recombination events that have joinepore-former type of 58 regulatory region anlysis gene (most similar to colicin A), a DNatype of bacteriocin and immunity region (mosimilar to pyocin S1 and colicin E9), and a ceptor recognition and translocation region
FIG. 8. The chimeric nature of the pKlebB sequence is icestry of each region.
ID EVOLUTION 219
n.c1.enthdn-re
e
e-
--e-hest
a
et-
of
functions, also based on sequence compariappears to be the product of recombinatevents that have joined a K. pneumoniaeRNA Iand RNA II region (most similar to pJHCMWand a K. oxytocaRom and Exc1 region (mossimilar to pNBL63). Although this region beathe signature ColE1-like replication functionlevels of sequence similarity suggest that it iKlebsiella-specific plasmid backbone.
Recombination has been implicated in the ogin and evolution of numerous native plasmiIndeed, it has been argued that plasmids merely shuttle vectors that serve to transpgenes from one chromosomal background toother. Thus, it was surprising to find a classplasmids, the nuclease colicin plasmids of E. coli,in which recombination appears to have playlittle or no role in their origin and diversificatio(Riley, 1993a). In fact, nuclease colicins and thplasmids were shown to diversify primarithrough the action of positive selection on mutions that result in novel immunity functions (rviewed in Riley, 1998). pKlebB is the first example of a plasmid-encoded nuclease bacteriooriginating from multiple recombination events
It is possible that a two-step process is volved in nuclease bacteriocin plasmid evotion. Recombination may serve in the creatof novel plasmids that combine bacteriocfunctions from multiple ancestries, such that
unknown origin. The plasmid “backbone,”which is composed of plasmid maintenance
propriate receptor recognition functions arecombined with existing killing and immunity
ndicated by alternate shadings. The key indicates the probable an-
of
ol-d
-
l-nl-
-
An
of
bi-
ri-
220 RILEY
functions and these are coupled with a replicthat can survive in a particular chromosombackground. Those recombinants that survmay then experience the force of strong potive, diversifying selection detected for the nclease colicins as they subsequently diverswithin that species.
A prediction of this two-step hypothesis that nuclease bacteriocins within a species wbe closely related and show the pattern of quence divergence typical of diversifying seletion [as observed for the nuclease colicin plmids (Riley, 1998)]. When compared betwespecies, the chimeric origins of these same bteriocins will be revealed. With respect to tnuclease bacteriocins of Klebsiella, this ques-tion can only be addressed with the charactzation of additional nuclease klebicin plasmidAnalysis of pKlebB has revealed that the plamid-encoded nuclease bacteriocins are likcreated by recombination, as was seen for
pore-former colicins of E. coli. How the family
-
n-
of nuclease klebicins diversifies remains toaddressed.
ACKNOWLEDGMENTS
We thank Richard James for supplying t
klebicin B plasmid and acknowledge supp
i
p
im-
-
,-
z-oft a
.,teid
-nt
from the NIH and NSF (GM58433 and DEB9458247).
REFERENCESAkutsu, A., Masaki, H., and Ohta, T. (1989). Molecu
structure and immunity specificity of colicin E6, aevolutionary intermediate between E-group colicand cloacin DF13. J. Bacteriol.171(12), 6430–6436.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Liman, D. J. (1990). Basic local alignment search tooJ.Mol. Biol. 215,403–410.
Astill, D., Manning, P., and Heuzenroeder, M. (1993). Chacterization of the small cryptic plasmid, pIMVS1 Salmonella enterica ser. Typhimurium. Plasmid30,258–267, doi:10.1006/plas.1993.1057.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. DSeidman, J. G., Smith, J. A., and Struhl, K. (199“Current Protocols in Molecular Biology.” J. WileyNew York.
Braun, V., Pilsl, H., and Gro , P. (1994). Colicins: Stru
tures, modes of actions, transfer through membraand evolution. Arch. Microbiol.161,199–206.
Chan, P. T., Ohmor, H., Tomizawa, J., and Lebowitz,
ET AL.
onalivesi-u-ify
isill
se-c-
as-enac-
he
eri-s.s-
elythe
be
heort-
larnns
-l.
ar-of
.,2).,
c-
(1985). Nucleotide Sequence and Gene OrganizationcolE1 DNA*. J. Biol. Chem.260(15), 8925–8935.
Cole, S. T., Saint-Joanis, B., and Pugsley, A. P. (1985). Mecular characterisation of the colicin E2 operon anidenification of its products. Mol. Gen. Genet.198,465–472.
Davison, J. (1984). Mechanism of control of DNA replication and incompatibility in ColE1-type plasmids—Areview. Gene28,1–15.
Dery, K. J., Chavideh, R., Waters, V., Chamorro, R., Tomasky, L., and Tolmasky, M. (1997). Characterizatioof the replication and mobilization regions of the mutiresistance Klebsiella pneumoniae plasmidpJHCMW1. Plasmid 38, 97–105, doi:10.1006/plas.1997.1303.
DNASTAR Inc. (1999). “LASERGENE,” Fourth Ed.DNASTAR Inc., Madison, WI.
Dykes, C. A. (1995). Bacteriocins: Ecological and evolutionary significance. Trends Ecol. Evol.10,186–189.
Felsenstein, J. (1985). Confidence limits on phylogenies: approach using the bootstrap. Evolution39,783–791.
James, R. (1988). Molecular cloning and purification klebicin B. J. Gen. Microbiol.134,2525–2533.
James, R., Kleanthous, C., and Moore, G. R. (1996). Theology of E colicins: Paradigms and paradoxes. Micro-biology142,1569–1580.
James, R., Lazdunski, C., and Pattus, F., (1991). “Bacteocins, Microcins, and Lantibiotics,” Vol. 65, NATO ASIseries. Springer-Verlag, New York.
Kuhar, I., and Zgur-Bertok, D. (1999). Transcription regulation of the colicin K ckagene reveals induction of col-icin synthesis by differential responses to environmetal signals. J. Bacteriol.181(23), 7373–7380.
Mankovich, J. A., Hsu, C.-H., and Konisky, J. (1986). DNAand amino acid sequence analysis of structural and munity genes of colicins Ia and Ib. J. Bacteriol.168(1),228–236.
Masaki, H., and Ohta, T. (1985). Colicin E3 and its immunity genes. J. Mol. Biol.182,217–227.
Mikiewicz, D., Wrobel, B., Wegrzyn, G., and PlucienniczakA. (1997). Isolation and characterization of a Col E1like plasmid from Enterobacter agglomeranswith anovel variant of rom gene. Plasmid 38, 210–219,doi:10.1006/plas.1997.1312.
Morlon, J., Lloubes, R., Varenne, S., Chartier, M., and Ladunski, C. (1983). Complete nucleotide sequence the structural gene for colicin A, a gene translated anon-uniform rate. J. Mol. Biol.170,271–285.
Nijkamp, H., deLang, R., Stuitje, A., Elzen, P. v. dVeltkamp, E., and Putten, A. v. (1986). The complenucleotide sequence of the bacteriocinogenic plasmCloDF13. Plasmid16,135–160.
Pilsl, H., and Braun, V. (1995a). Novel colicin 10: Assignment of four domains to TonB- and TonC-dependeuptake via the Tsx receptor and to pore formation. Mol.Microbiol. 16 (1), 57–67.
nes,
J.
Pilsl, H., and Braun, V. (1995b). Strong function-related ho-mology between the pore-forming colicins K and 5. J.Bacteriol.177(23), 6973–6977.
KLEBICIN B PLASMID EVOLUTION 221
Pilsl, H., Smajs, D., and Braun, V. (1999). Characterizationof colicin S4 and its receptor, OmpW, a minor proteinof the Escherichia coliouter membrane. J. Bacteriol.181(11), 3578–81.
Polisky, B. (1988). ColE1 replication control circuitry:
trt
a
v
it
c
io
u
ne
ro
i
T
origin of replication of plasmid p15A and comparativestudies on the nucleotide sequences around the originof related plasmids. Cell 32,119–129.
Smajs, D., Pilsl, H., and Braun, V. (1997). Colicin U, a novelcolicin produced by Shigella boydii. J. Bacteriol.179
of
s
6).
-
mral
m-
in
F.
.f a
di-
e-lA
Sense from antisense. Cell 55 (6), 929–32.Pugsley, A. P. (1987). Nucleotide sequencing of the s
tural gene for colicin N reveals homology between catalytic, C-terminal domains of colicins A and N. Mol.Microbiol. 1 (3), 317–325.
Riley, M., Cadavid, L., Collett, M., Neely, M., Adams, MPhillips, C., Neel, J., and Friedman, D. (2000). Tnewly characterized colicin Y provides evidencepositive selection in pore-former colicin diversifiction. Microbiology146,1671–1677.
Riley, M. A. (1993a). Molecular mechanisms of colicin elution. Mol. Biol. Evol.10,1380–1395.
Riley, M. A. (1993b). Positive selection for colicin diversin bacteria. Mol. Biol. Evol.10,1048–1059.
Riley, M. A. (1998). Molecular mechanisms of bacterioevolution. Ann. Rev. Genet.32,255–278.
Riley, M. A., and Gordon, D. (1995). Ecology and evolutof bacteriocins. J. Ind. Microbiol.17,155–158.
Roos, U., Harkness, R. E., and Braun, V. (1989). Assemof colicin genes from a few DNA fragments: Ncleotide sequence of colicin D. Mol. Microbiol. 3 (7),891–902.
Saitou, N., and Nei, M. (1987). The neighbor-joinimethod: A new method for reconstructing phylogentrees. Mol. Biol. Evol.4 (4), 406–425.
Sano, Y., Matsui, H., Kobayashi, M., and Kageyama,(1990). Pyocins S1 and S2, bacteriocins Pseudomonas aeruginosa. In “Pseudomonas:Bio-transformations, Pathogenesis, and Evolving Biotenology” S., Silver, Ed.), pp. 352–358. Am. Soc. Micbiology, Washington, DC.
Schramm, E., Mende, J., Braun, V., and Kamp, R. (1987). Nucleotide sequence of the colicin B activgene cba: Consensus pentapeptide among TonBpendent colicins and receptors. J. Bacteriol.169 (7),3350–3357.
Selzer, G., Som, T., Itoh, T., and Tomizawa, J. (1983).
uc-he
.,heof-
o-
y
in
n
bly-
gtic
M.of
ch--
M.ty-de-
he
(15), 4919–4928.Soong, B., Lu, F., and Chak, K. (1992). Characterization
the caegene of the ColE7 plasmid. Mol. Gen. Genet.233,177–183.
Swofford, D. L. (1993). “PAUP: Phylogenetic AnalysiUsing Parsimony,” Version 3.01. Champaign, IL.
Tagg, J. R., Dajani, A. S., and Wannamaker, L. W. (197Bacteriocins of gram positive bacteria. Bacterial Rev.40,722–756.
Tan, Y., and Riley, M. A. (1995). Rapid invasion of colicinogenic bacteria with novel immunity functions. Microbi-ology142,2175–2180.
Tan, Y., and Riley, M. A. (1997). Nucleotide polymorphisin colicin E2 gene clusters: Evidence for nonneutevolution. Mol. Biol. Evol.14, (6), 666–673.
Tomizawa, J. I., and Itoh, T. (1981). Plasmid ColE1 incopatibility determined by interaction of RNA I withprimer transcript. Proc. Natl. Acad. Sci. USA78,6096–6100.
Tomizawa, J. I., Ohmori, H., and Bird, R. E. (1977). Origof replication of colicin E1 plasmid DNA. Proc. Natl.Acad. Sci. USA74 (5), 1865–1869.
Torreblanca, M., Meseguer, I., and Rodriguez-Valera,(1989). Halocin H6, a bacteriocin from Haloferax gib-bonsii. J. Gen. Microbiol.135,2655–2661.
Wu, S., Dornbusch, K., Kronvall, G., and Norgren, M(1999). Characterization and nucleotide sequence oKlebsiella oxytocacryptic plasmid encoding a CMY-type B-lactamase: Confirmation that the plasmid-meated cephamycinase originated from the Citrobacterfreundii amp C B-lactamase. Antimicrob. AgentsChemother.43, (6), 1350–1357.
Zverev, V. V., and Khmel, I. A. (1985). The nucleotide squences of the replication origins of plasmids Coand ColD. Plasmid14,192–199.
