While the genetic setup in eukaryotic cells comprises multiple linear chromosomes, the standard in prokaryotes is a single circular chromosome [
1]. The number of replication start sites is also different, with eukaryotic chromosomes starting replication at multiple origins, while all known bacterial chromosomes are replicated from a single origin of replication. However, in bacteria, some interesting exceptions occur in alternative genetic setups, including linear chromosomes and separation of the genetic information onto multiple chromosomes [
2,
3]. When two chromosomes exist in one bacterial cell, new questions arise about, for example, the timing of initiation and coordination of segregation in comparison to single-chromosome bacteria. The best studied two-chromosome bacterium is
Vibrio cholerae, the causative agent of the cholera disease [
4,
5]. Chr1 of
V. cholerae strain El Tor N16961 has a size of about 3 M bps and Chr2 of about 1 M bps [
6]. Each of the two chromosomes has its own initiator protein to start replication at each single replication origin [
7]; for Chr1, the initiator is DnaA, known to be the standard from studies in other model bacteria [
8]. Meanwhile, the initiator for Chr2 is the protein RctB [
8,
9]. Although RctB is unique within the phylogenetic group of
Vibrionaceae, it shows structural similarity to plasmid initiators [
10,
11]. This fits the common idea that Chr2 originates from a plasmid that was acquired by the cell early in evolution and then developed into a secondary chromosome [
12]. One chromosome-like characteristic of Chr2 is its regulation in a cell-cycle dependent manner, attributed to the participation of SeqA and Dam methyltransferase in regulation of
ori2 [
13,
14,
15]. Another feature of Chr2, further distinguishing it from plasmids, is that it encodes essential genes, although they are less frequent than when compared to the primary chromosome [
16,
17]. However, the plasmid ancestry of Chr2 is shown by the similarity of its replication initiation mechanism to plasmid systems; one such shared feature of both is the binding of the initiator protein to an array of specific binding sites, the so-called iterons [
18]. In addition, handcuffing is involved in negative regulation of
ori2 as has also been shown for plasmid origins [
12]. Although RctB alone is sufficient to melt the DNA double strand at
ori2, this replication origin has been found to also depend on DnaA [
8], the experimental evidence being the inability of the transfer of an
ori2-based minichromosome to an
E. coli strain lacking DnaA [
5]. DnaA activity at
ori2 is probably linked to a conserved DnaA box some base pairs away from the iteron array [
6,
19]. Coordination of replication in the two-chromosome system of
V. cholerae appears to work through the
crtS site (=chromosome II replication triggering site), located on Chr1 and found to positively regulate initiation at
ori2 by binding RctB [
20]. Regulation is thought to include physical contacts between
crtS and
ori2, as these two chromosome parts appeared to be coupled in chromatin conformation capture experiments [
21]. Genetic changes of the
crtS position on Chr1, either closer to
ori1 or further away, resulted in a corresponding shift of Chr2 initiation time as seen by a changed copy number [
21], showing that the native position of the
crtS sets the timing of Chr2 replication to finish in synchrony with Chr1 replication [
21,
22,
23]. An important tool in studies on
V. cholerae DNA replication was and is the use of
E. coli as a heterologous host. First evidence of which sequences function as replication origins in
V. cholerae came from testing their ability to replicate a corresponding minichromosome in
E. coli [
5]. Later, the native replication origin of
E. coli was replaced by the very similar
ori1 of the primary
V. cholerae chromosome [
13,
14], which was used to show that the Dam methyltransferase is not essential for
ori1 replication and can thus not be responsible for Dam being essential in
V. cholerae (and not in
E. coli). The conclusion was therefore that Dam-dependent methylation of
ori2 is crucial for initiation of replication; this assumption was confirmed by showing firstly that RctB binding sites need to be methylated in order to be bound by RctB, and secondly, that Dam loss selects for chromosome fusion in
V. cholerae, omitting the need for a functional
ori2 since all genetic material can be replicated by
ori1 [
13,
24]. Attempts to construct an
E. coli strain with
V. cholerae ori2 driving DNA replication instead of
oriC as an important tool to study related questions had previously been unsuccessful [
13]. Here, we study replication of such a strain with an insertion of
ori2 including the genes encoding
parAB and
rctB at position 4,422,941 of the
E. coli chromosome and an
oriC deletion that was constructed on the way toward developing an
E. coli strain with two chromosomes [
25]. We show that chromosomes over-replicate in
E. coli with an
ori2 origin and that its replication is indeed dependent on Dam. Further experiments assess the relationship of
crtS to
ori2 copy number, the role of DnaA in
ori2 initiation and make use of the genetic system to study a chemical compound that was described to act specifically on RctB. Finally, we show that
ori2 can replace
oriC at its native location by constructing a corresponding strain.