Patent Application: US-92279204-A

Abstract:
the invention relates to compositions , methods and kits useful in modulating bacterial cell dna replication and for treating pathogenic bacterial .

Description:
details of various embodiments and examples of the invention are found in the publication entitled , “ distinct replication requirements for the two vibrio cholerae chromosomes ”, by elizabeth s . egan and matthew k . waldor , to appear in ( aug . 22 , 2003 ; cell 114 ( 4 ): 521 – 530 ) and which is hereby incorporated herein in its entirety . dna from v . cholerae n16961 ( heidelberg et al ., 2000 ) was used as template for pcr amplification of fragments for insertion into pgp704 ( miller and mekalanos , 1988 ) to create chri - and chrii - derived minichromosomes . the insert in porici extends from chri bp 2 , 956 , 820 through 1 , 300 according to the published annotation of the v . cholerae genome sequence ( heidelberg et al . 2000 ). the insert in poricii extends from chrii bp 1 , 069 , 696 through bp 3 , 191 . all primer sequences are available upon request . frame - shift and substitution mutations were constructed using the quikchange site - directed mutagenesis kit ( stratagene ), and deletions were constructed using overlap - extension pcr mutagenesis as previously described ( moyer et al ., 2001 ). all mutations were confirmed by dna sequencing . high - copy plasmids containing oricii vc or orici vc sequences were constructed by amplifying sequences from v . cholerae n 16961 and introducing them into the puc - based plasmid pcrii - topo ( invitrogen ). plasmids pee481 and pee482 were constructed by introducing the insert from plasmid p3642 - topo ( see fig4 ) into the vector pgz119 ( lessl et al ., 1992 ) in both orientations . full - length rctb along with upstream sequence including its ribosome binding site was introduced into the expression vector pgz119 ( lessl et al ., 1992 ) to create the complementing plasmid porf2 . the expression plasmid for production of c - terminal his 6 - tagged rctb , porf2 - bad , was constructed by inserting the rctb coding sequence into pbad - topo ( invitrogen ). strain ko1607 is e . coli mc1061 dam13 :: tn9 ( gift of a . wright ). strains mc1061δseqa and ko1607δseqa were constructed by p1 transduction of the seqa allele from strain cm735 seqaδ :: tet ( lu et al ., 1994 ) using standard protocols . conjugation assays to determine the replication capacity of chri and chrii - derived minichromosomes were performed by overlaying approximately equal numbers of donor and recipient cells on lb agar plates and incubating for 5 hr at 37 ° c . dilutions were plated on selective medium to enumerate donor cfu , recipient cfu , and exconjugant cfu . in experiments with a complementing plasmid in trans , iptg was added to the medium to induce gene expression . the frequency of exconjugant formation was calculated by dividing the number of exconjugant by donor cfu . southern hybridization was carried out using horseradish peroxidase - labeled dna probes , prepared and hybridized using the ecl direct nucleic acid labeling and detection system ( amersham pharmacia ). genomic dna was isolated using the gnome dna kit ( bio101 ). nucleotide alignments were performed using vector nti ( informax ). the v . harveyi oricii - region sequence ( genbank ay309011 ) was determined by sequencing the insert of plasmid pnh26 ( zyskind et al ., 1983 ) using dye terminator cycle sequencing at the tufts university school of medicine core facility . rctb - his 6 was affinity purified on ni - nta resin ( qiagen ) from lysates of e . coli rj3236 ( porf2 - bad ) grown in the presence of 0 . 02 % arabinose according to the manufacturer &# 39 ; s protocol . probes for gel shift experiments were amplified by pcr from poricii or porici dna using a 32 p - labeled oligonucleotide . probes were gel purified . gel shift reactions ( 20 μl volume ) were performed by incubating 1 , 000 c . p . m . of each probe with increasing amounts ( 0 ng , 56 ng , 113 ng , 225 ng , 450 ng , and 900 ng ) of purified rctb - his 6 in a reaction buffer containing 25 mm tris - hcl ( ph 7 . 9 ), 110 mm nacl , 5 . 12 mm edta , 2 mm dtt , 0 . 1 mg / ml bsa , and 12 . 5 μg / ml sonicated salmon sperm dna for 30 min at room temperature . reactions were analyzed on 6 % dna retardation gels ( invitrogen ). rna was prepared using the rneasy kit ( qiagen ) from exponentially growing v . cholerae n 16961 , e . coli mc4100 ( poricii ), and e . coli mc4100 without plasmid , and treated with dnase i . yeast rna was provided in the rpa iii kit ( ambion ). riboprobes were synthesized from linearized cloned dna templates with the maxiscript kit ( ambion ) using 40 μci of [ 32 p ] utp for rpob and 50 μci of [ 32 p ] utp for rcta . the expected protected fragment sizes were as follows : 132 bp for rcta , 211 bp for v . cholerae rpob , and 1226 bp for e . coli rpob . the corresponding full - length probes were 233 bp , 341 bp , and 1310 bp , respectively . all riboprobes were gel purified on 6 % denaturing polyacrylamide gels . rpas were conducted using the rpa iii kit ( ambion ) according to manufacturer &# 39 ; s instructions using 15 μg total rna for each sample . products were separated on 6 % denaturing polyacrylamide gels and exposed to film . for the stability tests in v . cholerae , fresh overnight ampicillin - resistant bah - 3 ( pearson et al ., 1993 ) colonies containing the appropriate plasmid were inoculated into lb broth without ampicillin . the percentage of cfu containing plasmid was determined at times t = 0 and t = 6 hr after growth without selection by plating equal volumes of bacteria on selective and nonselective media . transformation experiments were performed with 100 ng of each plasmid using a standard electroporation protocol . electrocompetent e . coli and v . cholerae were prepared using standard protocols . for the incompatibility assays involving two plasmids , the cells were prepared with selection for the ampicillin - resistant plasmid . dilutions were plated on selective medium to determine the number of transformants . unmethylated pjel109 and pmr2 were obtained by isolating the plasmids from the dam e . coli strain ko1607 . we obtained unmethylated poricii by isolating plasmid from a dam pir + e . coli strain ( sm10λpir dam 13 :: tn9 ), where replication initiates from the r6k origin of replication . in various embodiments of the invention herein the origins of replication of the two v . cholerae chromosomes are experimentally defined . novel replicon - specific requirements for each chromosome as well as factors that are required for replication of both chromosomes are provided . to functionally define the genes and sequences required for replication of the two v . cholerae chromosomes , minichromosome derivatives of each chromosome were constructed by introducing each annotated ( heidelberg et al ., 2000 ) replication origin into a mobilizable , conditionally replication - defective vector , pgp704 ( miller and mekalanos , 1988 ). this vector contains the r6k origin of replication , which requires the product of the pir gene to initiate replication . in pir - deficient strains , pgp704 - based plasmids will only replicate if they carry an alternate , functional replication cassette . the ability of the v . cholerae - pgp704 chimeric plasmids to replicate autonomously in both pir e . coli and v . cholerae ( reca strain bah - 3 [ pearson et al ., 1993 ]) was measured by quantifying the frequency of exconjugant formation in conjugation assays in which the plasmids were mobilized from a pir + donor to pir recipients . all of the plasmids tested were able to replicate and form exconjugants in pir + e . coli , showing that their inserts have no negative effect on pgp704 mobility ( data not shown ). the annotated orici vc resides between the genes mioc and gida ( heidelberg et al ., 2000 ). a 447 bp fragment from this intergenic region replicated in both v . cholerae and the surrogate host , e . coli ( fig1 a and 1b , line 1 ). this indicates that this fragment contains the minimal orici vc and demonstrates that e . coli can supply any transacting factors needed for orici vc - based replication . in contrast , a plasmid ( pig1 ) containing the intergenic region previously annotated as oricii vc did not form exconjugants in either v . cholerae or e . coli , indicating that pig1 cannot replicate autonomously , i . e ., that this intergenic region either does not contain oricii vc or requires additional sequences in cis for replication ( fig1 a and 1b , line 3 ). a larger minichromosome ii , poricii , replicated in both species ( fig1 a and 1b , line 2 ), indicating that oricii vc and any other sequences required in cis for replication are contained within the 5 . 8 kb insert of poricii . this minichromosome includes two intergenic regions ( ig1 and ig2 ), two hypothetical genes ( vca0001 and vca0002 ), and two genes with homology to plasmid partitioning genes ( para and parb ). mutational analysis of poricii confirmed that ig1 is not required for replication in e . coli ( fig1 b , line 4 ), indicating that the original annotation of ig1 as oricii vc was incorrect . however , a deletion internal to ig2 , the other intergenic region within poricii , revealed that ig2 includes sequences required for replication in both v . cholerae and e . coli ( fig1 a , line 4 , and fig1 b , line 5 ). furthermore , a minichromosome containing only ig2 sequences could replicate in v . cholerae , indicating that ig2 contains the true oricii vc ( fig1 a , line 5 ). only the right side of ig2 was required for pminig2 to replicate in v . cholerae , demonstrating that the minimal oricii vc is contained within this 406 bp region ( fig1 a , line 6 ). unlike a plasmid containing only orici vc ( pminorici ), pig2 could not replicate in e . coli ( fig1 b , lines 1 and 8 ). instead , derivatives of poricii required inclusion of the two flanking hypothetical genes , designated vca0001 and vca0002 by heidelberg et al . ( 2000 ), to replicate in e . coli ( fig1 b , lines 6 – 10 ). given this requirement , these genes have been herein termed vca0001 , rcta ( replication of chromosome two ; seq id no : 3 ) and vca0002 , rctb . for rctb , a gene ( seq id no : 1 ) encoding a predicted protein product of 658 amino acids ( aa ; seq id no : 2 ), both deletion and frameshift mutations severely compromised the ability of poricii to replicate in e . coli , and in both cases the replication defects could be complemented by expressing rctb in trans on another plasmid ( fig1 b , lines 9 – 10 ). in v . cholerae , the rcta and rctb gene products are provided by the chromosome to facilitate pig2 and pminig2 replication ( fig1 a , lines 5 – 6 ). the dna sequences of both orici vc and oricii vc contain several features also found in e . coli oric . in fact , the sequence of orici vc is very similar to oric ( 58 % identity ). like oric , orici vc has an at - rich region , five apparent dnaa boxes , a likely binding site for ihf ( a protein that bends dna ), and many potential sites for dam methylation ( gatc sequences ) ( fig2 a ). though the minimal oricii vc lacks sequence similarity to e . coli oric , it does contain an at - rich region , a single putative dnaa box , a putative ihf binding site , and an overrepresentation of gatc sequences ( fig2 a ). in addition , within the entire ig2 region , there are two related repeat sequences , an 11 - mer and a 12 - mer , a finding reminiscent of replication origins found in iteron - type plasmids , where small repeat sequences are required for both replication and copy number control ( chattoraj 2000 and del solar et al . 1998 ) ( fig2 a ). the available nucleotide sequence databases were searched and sequences similar to ig2 were found in the complete genomes of vibrio fischeri , vibrio parahaemolyticus , and vibrio vulnificus . in addition , ig2 - like sequences were found in an insert of a plasmid that was isolated as part of a genetic screen for functional v . harveyi origin sequences ( zyskind et al ., 1983 ). alignment of v . cholerae oricii vc with sequences from v . vulnificus , v . fischeri , v . parahaemolyticus , and v . harveyi revealed that the 11 - mer repeats , 12 - mer repeats , the dnaa box , and the at - rich region were all conserved ( fig7 and 8 ). furthermore , in each species , the 12 - mers were spaced 11 bp apart , suggesting that the same dna sequences would be displayed on the same face of the dna . the conservation of the 12 - mer repeats among five vibrio species suggests that they are important for chrii replication . an essential replication function for the 12 - mer repeats was demonstrated by the finding that substitution of six bp in a 12 - mer abolished replication of pminig2 in v . cholerae ( fig1 a , line 7 ). rctb is conserved and encodes an oricii vc binding protein orthologs of rctb were searched and found in v . vulnificus , v . fischeri , v . parahaemolyticus , and v . harveyi ( identity & gt ; 74 %; data not shown ). southern analysis revealed that rctb is present in diverse genera in the family vibrionaceae , suggesting that the presence of two chromosomes may be a defining feature not just of the genus vibrio but of the widespread family vibrionaceae ( fig2 c ). in v . cholerae , all attempts to isolate mutations that inactivate rctb were unsuccessful , consistent with a conclusion that rctb is an essential gene . as rctb is required in replication of poricii , and replication factors often act at origins , rctb binding to oricii vc was investigated . recombinant epitope - tagged rctb was expressed in e . coli , purified , and used in gel shift experiments with probes derived from oricii vc and orici vc . several different probes spanning ig2 were bound by rctb , but an orici vc probe was not ( fig2 b , i – iv ). with a probe derived from the left side of ig2 , multiple shifted species were observed ( fig2 b , i ), indicating that this region contains multiple rctb binding sites . analysis of poricii revealed that the gene vca0001 ( rcta ) is required for oricii vc - based replication ( fig1 b , lines 6 and 7 ). further studies indicated that an rna transcribed from this region was the required replication factor . a substitution mutation in poricii that changed a putative rcta amino acid 25 ( of 44 predicted aa ) to an amber stop codon ( poricii 01amb ) did not affect replication ( fig1 b , line 11 ). similarly , replacement of the putative start codon of rcta with a stop codon did not prevent autonomous replication of the plasmid , poricii 01tag , in e . coli ( fig1 b , line 12 ). these data , together with the fact that there are no other potential start codons within rcta , indicate that a protein is not required for poricii replication translated from rcta . since pig2 and pminig2 , which do not contain rcta in cis , replicate in v . cholerae , rcta is not a required cis - acting sequence . together , these results suggest that a candidate product of this gene is a functional rna . we confirmed that rna is transcribed from rcta in both v . cholerae and poricii - containing e . coli by performing a ribonuclease protection assay ( rpa ), using a probe complementary to rcta ( fig3 ). the entire probe was found to be protected , suggesting that the active species encoded by this region is at least 132 bp , and potentially longer . surprisingly , a single base pair deletion in rcta , at position 21 , did inhibit replication of poricii ( fig1 b , line 13 ). this finding confirms the requirement for rcta , and indicates that this base pair may be important in rna structure . v . cholerae cells containing poricii formed smaller colonies than did cells containing porici or no plasmid ( fig9 ). this phenotype was not attributable to coding regions in poricii , as cells containing pig2 ( which lacks coding sequences ) also formed small colonies ( fig9 ). furthermore , only a portion of ig2 accounts for this phenotype , as cells with pminig2 , a plasmid that only includes the minimal oricii vc , formed normal - sized colonies ( fig9 ). colonies formed by e . coli containing poricii or porici were indistinguishable from each other and from cells without plasmid . these findings suggested that a plasmid - borne copy of a region on the left side of ig2 , outside of the minimal oricii vc , restricted v . cholerae growth in the presence of selection for the plasmid . this impairment may result from instability of plasmids containing this region or from an inhibitory effect of this region on chrii replication . the percentage of v . cholerae cells retaining pig2 fell more than 2000 - fold during a six - hour growth period in the absence of antibiotic selection ( table 1 ). in contrast , pminig2 was relatively stably maintained ( table 1 ). the profound difference in the stability of pminig2 and pig2 suggests that the left side of ig2 renders pig2 unstable in v . cholerae , but is also consistent with ig2 - mediated inhibition of chrii replication leading to a large selective advantage for cells that lose the plasmid . together , these results indicate that the small colony phenotype was attributable to antibiotic - mediated killing of cells without plasmid , likely reflecting incompatibility between pig2 and one or both of the v . cholerae chromosomes . the presence of a chrii incompatibility sequence was confirmed by transforming v . cholerae with a high copy number vector containing chrii or chri sequences and measuring the transformation efficiency compared to vector alone . when high - copy poricii - topo , pig2l - topo , or a 407 bp segment from the left side of ig2 ( p3642 - topo ) were introduced into v . cholerae , the transformation efficiency was more than 90 , 000 - fold lower than the efficiency of transforming vector alone ( fig4 , lines 1 – 3 ). in contrast , high - copy pminig2 ( pminig2 - topo ) transformed v . cholerae almost as efficiently as vector alone ( fig4 , line 4 ), confirming that an incompatibility determinant ( inc ) is contained within the left side of ig2 . additional mapping experiments revealed that incompatibility depends on a short highly conserved sequence present in v . cholerae , v . vulnificus , v . harveyi , v . parahaemolyticus , and v . fischeri ( fig4 , lines 5 – 6 ; supplemental fig . s2 ). while this conserved region clearly contributes to incompatibility , it is not the sole determinant , because a smaller insert ( in prt - topo ) that included the conserved segment transformed v . cholerae efficiently ( fig4 , line 7 ). unlike chrii minichromosomes and high - copy oricii vc sequences , plasmids containing orici vc sequences were not incompatible with v . cholerae . high copy number plasmids containing either a 5 . 2 kb region encompassing orici vc ( porici - topo ) or the minimal orici vc ( pminorici - topo ) were readily introduced into v . cholerae and did not compromise viability ( fig4 , lines 8 – 9 ). in this regard , orici vc is similar to e . coli oric , which in relatively high copy is not toxic in e . coli . to test whether sequence from the left side of ig2 ( termed inc ) specifically interfered with the replication regions of one or both v . cholerae chromosomes , e . coli containing either poricii or porici was used so that the v . cholerae minimal replicons could be studied in relative isolation . the transformation of poricii - containing e . coli by a vector containing the inc region ( pee481 or pee482 ) was & gt ; 10 , 000 - fold lower than by vector alone ( fig5 ). the orientation of inc in the vector did not alter this effect ( fig5 ). when these plasmids were introduced into either porici - containing e . coli or e . coli without a minichromosome , there was virtually no difference in the transformation efficiency between vector alone and vector with insert ( fig5 ). thus , inc renders a vector incompatible with poricii but not porici ; these findings are consistent with a role for inc in control of chrii but not chri replication and / or partitioning . to begin to define the mechanism of incompatibility , frameshift mutations in the putative poricii were created partitioning genes para and parb . the resulting plasmid , pabfs , was still incompatible with pee481 and pee482 in e . coli ( fig5 ), suggesting that incompatibility is not due to interactions of the left side of ig2 with these putative partitioning proteins . instead , incompatibility appears to result from an influence of the inc region on the chrii replication machinery . since pabfs contains both rcta and rctb and depends on them for replication , we hypothesize that the incompatibility region negatively regulates chrii replication by interacting with rcta and / or rctb . replication of many bacterial chromosomes and some plasmids is mediated by dnaa , which binds to the ori and stimulates strand unwinding to initiate replication ( hansen et al . 1986 and messer et al . 1996 ). dnaa proved essential for replication of minichromosomes derived from both v . cholerae chromosomes , as neither of these plasmids could replicate in a dnaa - independent e . coli strain ( table 2 ). thus , dnaa differs from rctb and rcta , which are only required for chromosome ii replication . since the activity of dnaa is regulated in a cell cycle - dependent fashion ( kurokawa et al . 1999 ), this protein may help coordinate replication of the two chromosomes . the roles of dam and dna methylation in replication of the two v . cholerae minichromosomes were compared to its role in replication of an oric minichromosome , since dam methylation sites are overrepresented within all three sequences ( see fig2 a ). in e . coli , dam is not essential for oric replication , but dam methylation regulates the timing of replication initiation at oric ( boye et al . 2000 and marinus 1996 ). as has been observed in several previous studies ( e . g ., lu et al . 1994 and russell et al . 1987 ), a methylated e . coli oric minichromosome was found to transform dam e . coli ˜ 1000 - fold less efficiently than wt e . coli ( fig6 b ). this reduction has been attributed to the binding and sequestration of hemimethylated oric dna by the seqa protein to prevent reinitiation ( campbell et al ., 1990 and lu et al . 1994 ). when methylated poric is used to transform dam e . coli , it is replicated once and then becomes hemimethylated and sequestered . replication cannot be reinitiated because there is no dam methylase in the cell . consistent with this mechanism , dam e . coli can be transformed almost as efficiently as wt e . coli when unmethylated dna is used ( fig6 b ). similarly , methylated poric is able to transform a dam seqa double mutant because seqa - mediated sequestration of oric no longer occurs ( lu et al ., 1994 ) ( fig6 b ). dam plays a different role in replication of the v . cholerae minichromosomes than it does for poric . transformation of dam e . coli by methylated poricii was ˜ 10 , 000 - fold less frequent than transformation of isogenic wt e . coli . unlike poric , unmethylated poricii did not transform dam e . coli , suggesting that replication of poricii absolutely requires dam methylation ( fig6 c ). consistent with this idea , poricii did not transform a dam seqa double mutant ( fig6 c ). this indicates that methylation of oricii vc contributes directly to its replication rather than or in addition to simply being a target for sequestration . dam methylation is required for porici replication as well , since dam e . coli could not be transformed with methylated porici ( fig6 d ). surprisingly , seqa e . coli could not be transformed with porici , suggesting that seqa is required for replication of porici ( fig6 d ). attempts to knock out v . cholerae seqa ( 54 % identity and 69 % similarity to e . coli seqa ) were unsuccessful , indicating that seqa is an essential gene in v . cholerae . in e . coli , several activities of seqa contribute to control of replication . in addition to its role in binding and sequestering hemimethylated origin dna , seqa has been shown to influence transcriptional regulation , dna superhelicity , and nucleoid structure ( slominska et al . 2001 and weitao et al . 2000 ). the data herein indicate that in v . cholerae , seqa plays distinct roles in replication of chri and chrii . given the different effects of a seqa mutation on replication of porici and poric , the roles of seqa in v . cholerae may prove to be different from those described in e . coli . the sequences below were obtained and entered into the public database , genbank accession no . : nc — 002506 . rctb nucleic acid ( nucleotides 1134 - 3110 of nc — 002506 ) is seq id no : 1 as used herein and in the claims has the following nucleotide sequence : atgagctcagaagaaaaacgattgatcaaattgccaagaactcacaaagatggtcatctttttgaagtctctgaagccgcgattgac ( seq id no : 1 ) tggattgaacagtatcaacactttaaaggtgtcacgaaaagcattgttgaacttttgaatctgatctcactgcgtggattacgcagt agagatggcttagtttcaaccacagaactgattgatgcaaccgatgggcagctgacgcgtgcagccatccagcagcgcttgagagca gcggtagctgttggattgttcaaacaaatcccagtgcgttttgaagaggggctggctggcaaaaccatgctccatcgtttcattaac cccaaccaattgatctcggtactcggctcaaccagcttagtcactgaatcggttaagcaaaatgaaaagcaaaagcgctcaaaagca ttagcgcagacgcaagtcaatcaacgtactgcatgagcatggtttaaatacaccgccagccatgaaagatgaggctgatcagtttgt ggtctcaccgactaactgggcagggatcattgatcaagcgttagcgccacccagaacccgcaagagctaccaaaagtctatggtttc gatatcgggtactcgtgctgtgattgaaacacgatcgtctaaaaacatcatgacggtcgacgatctgatgactttgtttgccttatt cactttaacagtgcaataccatgatcatcaccaagatgattaccatttcaatgctaaacaagcaccaaacaaaacgccgctgtatat caccgacattctctctttacgtggcaaaaaagacagcggcccggcacgtgactcgatccgtgacagtattgatcgtattgaatttac cgattttcagttgcatgaactgacgggtcgttggctcagtgagaatatgccagaaggctttaaaagcgatcgttttcgctttttagc gcgcaccatcaccgcttccgaagaggcacctgtggaaggcagtgatggcgagatccgcatcaaacccaatctgtacattttggtgtg ggagccttcgttttttgaagagctattgacgcgagattatttcttcctatttccaccggagatcttgaaacaacataccttggtatt tcagctctactcctatttccgtagccgaatgtctcgtcgtcataccgatgtaatgatgctgagtgaactcaaccaaaaattggccag aaacatcgaatggcgacggttttctatggatctgatccgcgaacttcgtcgtctctccgaagggaaggggagtgaagatctgtttgt ggtcaatctctggggttatcacttgactgtgaaaagcattgaagagaaaggcaaagtggtggattaccaagtcgatatcaaatgtga tgtggaagaggtactgcgctattcacgcgccaaaaccaccaacgcgggtaaacgcaatatggctccaaccttgcctaaccctttacg taacgagctggtttccaagcagaaactggctgagttatcgagcatcatcgatggtgaatttgaaccaatccagcgcaaagccccttc gccgagaggccgcttaggtcggcgcgtgaagctacgtaaacatcttgtcgaaatcaatgctgatgaaatcaccattactctatcgcg ttatacctctccagaagcgctagaacgcagtataacggctttagcggctatgactggacacgccccttcatcaatcaaagaagagtg tgtagagctcatagacaagctagattggctgcgtgttgaaaacgatgtgatccaatacccgacttgagcaagctgcttgagctctac aacagccaaaatgagagtaaacatctgtcgatcgaaaaattgatcgcaggtttagcggtacgccgtaaagtctgtaaattggttcaa gatgggcacattgacgaaacggtgtatcgagccttagatgagatggccgctggagcctaa rctb gene product ( 658 amino acids ) as used herein and in the claims is seq id no : 2 , and has the following amino acid sequence : ( seq id no : 2 ) msseekrliklprthkdghlfevseaaidwieqyqhfkgvtksivellnl islrglrsrdglvsttelidatdgqltraaiqqrlraavavglfkqipvr feeglagktmlhrfinpnqlisvlgstslvtesvkqnekqkrskalaqtq vnqrllhehglntppamkdeadqfvvsptnwagiidqalapprtrksyqk smvsisgtravietrssknimtvddlmtlfalftltvqyhdhhqddyhfn akqapnktplyitdilslrgkkdsgpardsirdsidrieftdfqlheltg rwlsenmpegfksdrfrflartitaseeapvegsdgeirikpnlyilvwe psffeelltrdyfflfppeilkqhtlvfqlysyfrsrmsrrhtdvmmlse lnqklarniewrrfsmdllrelrrlsegkgsedlfvvnlwgyhltvksie ekgkvvdyqvdikcdveevlrysrakttnagkrnmaptlpnplrnelvsk qklaelssiidgefepiqrkapsprgrlgrrvklrkhlveinadeititl srytspealersitalaamtghapssikeecvelidkldwlrvendviqy ptlskllelynsqneskhlsiekliaglavrrkvcklvqdghidetvyra ldemaaga rcta ( nucleotides 112 - 246 of nc — 002506 ) as used herein and in the claims is seq id no : 3 , and has the following nucleotide sequence : the basic features of e . coli oric have , until now , been thought to define chromosomal origins of replication in γ - proteobacteria ( messer and weigel 1996 and zyskind et al . 1983 ). examples herein show that while orici vc largely conforms to this pattern , i . e ., oricii vc shares certain features with oric , including a dnaa box , several sites for dam methylation , and an at - rich region , this origin also has several unusual features for a bacterial chromosome . unlike other known chromosomes , oricii vc - based replication was determined to require , in addition to dnaa , a novel dna binding protein , a repeat sequence , and an rna ; furthermore , a noncoding sequence negatively regulates chrii replication . our assertion that oricii vc represents the true origin of replication of chrii is supported by a genetic screen for origins of replication in v . harveyi ( zyskind et al ., 1983 ). this study led to the isolation of an autonomously replicating sequence that we show herein contains a sequence similar to oricii vc . furthermore , the oricii vc region is conserved among at least three other vibrio species , and rctb was found in many diverse members of the family vibrionaceae , so that these are general among this family . several examples indicate that the rcta gene product is an rna and not a protein . first , neither of two different single base pair changes within rcta affected replication of poricii even though both of these mutations introduced stop codons into the predicted rcta open reading frame ( orf ), one of which was at the start codon ( fig1 b , lines 11 – 12 ). second , rcta is not conserved among related vibrio species . a lack of conservation at the dna sequence level might be expected for a functional rna , which could retain conservation at the structural level . finally , new algorithms for orf identification that account for codon usage suggest that there is no protein - coding gene in the area of rcta ( guo et al ., 2003 ). an rna that spans the annotated vca0001 gene was detected by rpa analysis , but the precise boundaries and function of the rcta rna are not known . transacting rna molecules have been described in plasmid replication ( del solar et al ., 1998 ), and in e . coli , transcriptional activity at oric is believed to influence replication efficiency ( messer and weigel , 1996 ). rcta rna may function directly in replication ( e . g ., as a primer ) or may play a required regulatory role . an incompatibility determinant that was localized to a dna sequence adjacent to the minimal oricii vc region was found to negatively influence oricii vc - but not orici vc - based replication . plasmid replication is often controlled by negative regulators ( del solar et al ., 1998 ), which maintain copy number within a narrow range to avoid overtaxing the host . plasmid inc regions can negatively regulate replication by titration of essential replication factors ( either protein or rna ) ( del solar et al . 1998 and novick 1987 ) or by “ handcuffing ,” in which rep proteins bound to the ori sterically hinder replication initiation ( chattoraj , 2000 ). data herein , without being limited by any particular theory or mechanism , are consistent with either regulatory mechanisms controlling oricii vc - based replication , presumably by influencing of these availability of rcta and / or rctb or by rctb - mediated handcuffing of oricii vc . if this is the case , replication of chrii may at least in part be controlled independently of chri . oricii vc - based replication has four features that characterize certain plasmid replicons : a repeat sequence essential for replication , a dependence on a replicon - specific protein ( rctb ), a requirement for an rna ( rcta ), and an incompatibility determinant ( inc ) that appears to act as a negative regulator . therefore that chrii may originally have been acquired as a plasmid and subsequently captured essential genes ( heidelberg et al . 2000 ). since we found that the v . cholerae replication protein - encoding gene rctb is present in many genera of the family vibrionaceae , the hypothetical plasmid ancestor of chrii must have been acquired prior to diversification of this family . some of the plasmid - like attributes of oricii vc - based replication are different from those of characterized plasmids , and the four novel features have never been described together in a single replicon . the origins of replication of iteron - type plasmids are characterized by the presence of short repeated sequences ( iterons ) to which a plasmid - encoded replication protein ( rep ) binds ( del solar et al ., 1998 ). rep binding to iterons in the ori stimulates strand unwinding , and rep - iteron interactions , both within the ori and in nearby control regions , are involved in controlling copy number through handcuffing ( chattoraj , 2000 ). while rctb may be classified as a rep protein and the 11 - mer and 12 - mer sequences in oricii vc as iterons however , data herein suggest that these repeats do not function as typical plasmid iterons . though the 12 - mers are required for oricii vc - based replication , in gel shift assays a single 12 - mer was not bound by rctb . furthermore , rctb bound to probes containing no apparent repeat sequences ( fig2 b , iii ). the 11 - and 12 - mer repeats in oricii vc also do not appear to function as iterons in replication control . in the iteron plasmid p1 , a single iteron can exert incompatibility because it is bound by rep and can therefore facilitate handcuffing ( papp et al ., 1994 ). in contrast , in v . cholerae a sequence with six 12 - mer repeats was not sufficient to exert incompatibility ( fig4 , line 4 ). even if rctb functions analogously to a plasmid rep protein , it is important to note that rctb has no sequence similarity to known plasmid rep proteins and has no recognizable motifs . how of a bacterium with multiple chromosomes , to ensure that each daughter cell . at least three general scenarios regarding replication , receives a full genome complement at cell division , can be envisioned : ( 1 ) each chromosome replicates using the same factors , ( 2 ) the chromosomes have entirely distinct replication requirements , or ( 3 ) the chromosomes share some common factors yet also maintain some distinct requirements . analysis of the two v . cholerae chromosomes herein revealed that replication of each chromosome involved specific factors ( rctb , rcta , and a control region for chrii and seqa for chri ), and that the chromosomes also shared a requirement for certain factors ( dnaa and dam ). having some common and some distinct factors may be biologically favorable in a multichromosomal bacterium because this mechanism could allow for some degree of coordinated replication while minimizing competition among the replicons . since the two vibrio chromosomes appear to have coexisted throughout vibrionaceae speciation , we presume that there is coordination of their replication ( unlike the unlinked replication of plasmids and their hosts &# 39 ; chromosomes ( del solar et al ., 1998 ). without being bound by any particular mechanism of action , the factors shared by both chromosomes , including dnaa and dam methylation , may mediate this coordination . v . cholerae dnaa is very similar to that of e . coli ( 79 % aa identity ), and e . coli dnaa can enable both orici vc - and oricii vc - based replication , suggesting that v . cholerae dnaa may function and be regulated as in e . coli . sharing this essential and highly regulated ( katayama et al . 1998 and kitagawa et al . 1996 ) initiation factor could ensure that replication of each chromosome is initiated only during a small time window in each cell cycle . the other major shared factor identified , herein , dam methyltransferase , may be essential for replication of both v . cholerae chromosomes . this observation may explain why dam is an essential gene in v . cholerae ( julio et al ., 2001 ). aside from its role in regulation of replication in e . coli , dam to influences dna structure in some origins of replication , such as the p1 plasmid ori ( abeles et al . 1993 ). however , the requirement for dam in v . cholerae may differ from that p1 , in because unmethylated p1 - derived plasmids can transform dam e . coli ( abeles et al . 1993 ). dam may play several roles in v . cholerae chromosome replication . as in e . coli , it may remethylate dna that has been sequestered by seqa due to hemimethylation . however , it is clear that dam must also play additional roles , since poricii could not replicate in a dam host even in the absence of seqa . the methylation state of oricii vc could affect binding of replication factors including rctb or rcta . alternatively , orici vc and oricii vc structures may be influenced by methylation in a manner similar to the ori of p1 ; providing a means by which the two v . cholerae origins are activated by methylation in a cell - cycle dependent and potentially synchronous manner . since the sequences of orici vc and oric are similar , it is surprising that dam appeared essential for orici vc - based replication ( fig6 d ). it is possible that porici competes with the e . coli host chromosome for the available initiator molecules in the absence of dam methylation . such competition is believed to result in integration of oric minichromosomes into the chromosome of dam e . coli ( lobner - olesen and von freiesleben 1996 ); in the absence of sufficient sequence homology for porici integration , competing porici plasmids may not be maintained in dam e . coli . if porici does compete with oric in the absence of dam methylation , then dam must regulate orici vc - based replication . the importance of dam in both oricii vc - and orici vc - based replication is consistent with a role for dam methylation in coordination of replication of the two v . cholerae chromosomes . a bipartite genomic arrangement has persisted throughout vibrionaceae speciation . since there are many duplicated loci present on both v . cholerae chromosomes , it is surprising that the two chromosomes have remained separate replicons throughout evolution . division of the genome into two chromosomes may provide an evolutionary advantage either by facilitating a faster replication time or by allowing for chromosome - specific replication control in certain environmental circumstances . this evolutionary advantage might be eclipsed by competition between two replicons with identical replication initiation factors . the distinct replication requirements of chri and chrii may minimize competition and thereby help ensure the maintenance of the divided genome . abeles , a ., et al ., 1993 . evidence of two levels of control of p1 orir and host oric replication origins by dna adenine methylation . j . bacteriol . 175 , pp . 7801 – 7807 . boye , e . et al ., 1990 . the role of dam methyltransferase in the control of dna replication in e . coli . cell 62 , pp . 981 – 989 . boye , e ., et al ., 2000 . limiting dna replication to once and only once . embo rep . 1 , pp . 479 – 483 . bramhill , d . et al ., 1988 . duplex opening by dnaa protein at novel sequences in initiation of replication at the origin of the e . coli chromosome . cell 52 , pp . 743 – 755 . cabezon , e ., et al ., 1997 . genetic evidence of a coupling role for the trag protein family in bacterial conjugation . mol . gen . genet . 254 , pp . 400 – 406 . campbell , j . et al ., 1990 . e . coli oric and the dnaa gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork . cell 62 , pp . 967 – 979 . chattoraj , d . k ., 2000 . control of plasmid dna replication by iterons : no longer paradoxical . mol . microbiol . 37 , pp . 467 – 476 . del solar , et al ., 1998 . replication and control of circular bacterial plasmids . microbiol . mol . biol . rev . 62 , pp . 434 – 464 . delvecchio , et al . et al ., 2002 . the genome sequence of the facultative intracellular pathogen brucella melitensis . proc . natl . acad . sci . usa 99 , pp . 443 – 448 . fuller , r . s ., et al ., 1984 . the dnaa protein complex with the e . coli chromosomal replication origin ( oric ) and other dna sites . cell 38 , pp . 889 – 900 . guo , f . b ., et al ., 2003 . zcurve : a new system for recognizing protein - 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circle mechanism of phage dna synthesis from both replicative and integrated forms of ctxφ . mol . microbiol . 41 , pp . 311 – 323 . novick , r . p ., 1987 . plasmid incompatibility . microbiol rev . 51 , pp . 381 – 395 . papp , p . p ., et al ., 1994 . negative control of plasmid dna replication by iterons . correlation with initiator binding affinity . j . biol . chem . 269 , pp . 23563 – 23568 . pearson , g . d ., et al ., 1993 . ctx genetic element encodes a site - specific recombination system and an intestinal colonization factor . proc . natl . acad . sci . usa 90 , pp . 3750 – 3754 . russell , d . w . et al ., 1987 . hemimethylation prevents dna replication in e . coli . cell 50 , pp . 1071 – 1079 . slominska , m ., et al ., 2001 . seqa , the escherichia coli origin sequestration protein , is also a specific transcription factor . mol . microbiol . 40 , pp . 1371 – 1379 . trucksis , m ., et al ., 1998 . the vibrio cholerae genome contains two unique circular chromosomes . proc . natl . acad . sci . usa 95 , pp . 14464 – 14469 . weitao , et al ., 2000 . escherichia coli cell cycle control genes affect chromosome superhelicity . embo rep . 1 , pp . 494 – 499 . yamaichi , y ., et al ., 1999 . physical and genetic map of the genome of vibrio parahaemolyticus : presence of two chromosomes in vibrio species . mol . microbiol . 31 , pp . 1513 – 1521 . zyskind , j . w ., et al ., 1983 . chromosomal replication origin from the marine bacterium vibrio harveyi functions in escherichia coli : oric consensus sequence . proc . natl . acad . sci . usa 80 , pp . 1164 – 1168 . those skilled in the art will recognize , or be able to ascertain using no more than routine experimentation , numerous equivalents to the specific procedures described herein . such equivalents are considered to be within the scope of the present invention and are covered by the following claims . the contents of all references , issued patents , and published patent applications cited throughout this application are hereby incorporated by reference . the appropriate components , processes , and methods of those patents , applications and other documents may be selected for the present invention and embodiments thereof .