Vibrios constitute a broad family of gammaproteobacteria with over 100 members classified (NCBI taxonomy browser). They are ubiquitous within marine and estuarine environments and the ecological roles for individual species are diverse. A common characteristic, however, is their ability to adapt and survive within various niches either as free-swimmers or in symbiotic or pathogenic association with diverse aquatic organisms such as plankton, coral, fish and shellfish. Moreover, several Vibrio species are capable of infecting humans with Vibrio cholerae, V. parahaemolyticus and V. vulnificus as the most common causes of disease [1, 2].
A shared trait among vibrios is the presence of two unequally sized chromosomes . The larger shows a more constant size, a lower interspecies sequence variability and harbour many of the genes involved in essential biosynthetic pathways while the smaller Vibrio chromosome is highly variable in size and contains relatively more species specific and unclassified genes [3–6]. This unusual structure and the distinct distribution of genetic content between the replicons has prompted studies on how the system is maintained and has also initiated discussion about fitness benefits with a divided genome [4, 6, 7].
Regarding the maintenance issue, most knowledge about chromosomal replication and partitioning has been gained from studies on V. cholerae. For example, it has been shown that the two chromosomes display different segregation patterns [8–10] and utilise separate sets of partition proteins [11, 12]. It has further been demonstrated that while the large chromosome origin of replication is similar to oriC of Escherichia coli, the small bears resemblance to those of certain plasmids . Nevertheless, the number of initiations for the two replicons remains equal and strictly follows the cell cycle . Therefore, as the difference in size between the replicons theoretically results in differing replication times it was suggested that initiation of replication is synchronised to maintain an equal number of small and large chromosomes . However, more recent studies indicate that inter-chromosomal synchrony between the V. cholerae chromosomes likely occurs at the level of termination [15, 16].
Bacteria with divided genomes must overcome additional obstacles to accurately distribute genetic material to daughter cells, yet the evolutionary success of the broad and diverse Vibrio family implies that split genomes may be beneficial. A possible advantage is that multiple replicons allow faster replication which in turn could lead to faster growth rates . This view is supported by the fact that several Vibrio species display unusually short multiplication times [18, 19]. Another potential benefit may be that multiple chromosomes provide the means to regulate gene expression in a replicon-wide manner by alterations in the 1:1 balance between copy numbers . Such regulation could facilitate large scale adaptations in response to changes in growth conditions , for example when the bacterium associates with or dissociates from a host organism. Considering that the genetic content differs for the small and large Vibrio chromosomes and that the two chromosomes utilise partly different mechanisms for initiation of replication  and partitioning , this is not an unlikely assumption. Consistent with this idea, over-expression of the distinct large or small chromosome replication initiator protein of V. cholerae results in over-initiation of the respective chromosome they control . Also, V. cholerae cells harboring unequal numbers of small and large chromosomes were recently obtained by deleting the small chromosome specific partitioning genes . Although these studies reveal technical possibilities for alterations in chromosome balance, no wild type vibrio with differing numbers of large and small chromosomes has yet been detected.
Interestingly, vibrios may possess an intrinsic mechanism to differentiate gene copy numbers between the chromosomes through gene dosage associated with replication [15, 21]. Such gene dosage occurs as replication always initiates at an origin and proceeds in a bidirectional manner towards the terminus of replication. This mode of replication means that genes located near the origin are duplicated earlier than other genes, which enables higher expression. Therefore, with a given replication speed, there are two main factors that influence gene dosage; initiation rate and replicon size. A higher initiation rate results in an increased average difference in copy numbers between origin proximate and terminus proximate genes as replication takes up a larger proportion of the cell cycle. For the same reason, gene dosage differences are more pronounced for a larger over a smaller replicon assuming equal initiation frequencies .
Although an influence from gene dosage on bacterial gene expression levels has only occasionally been reported [22–24], it is assumed to affect gene positioning . In line with this, altered gene dosage has been used as an explanation for decreased fitness and sometimes deleterious effects that can follow chromosomal rearrangements [26–28] as such events simultaneously change the distance to the origin of replication and thereby average copy numbers for a large number of genes. The impact from gene dosage has also been examined at the scale of genomic conservation, and highly expressed genes tend to locate near the origin of replication, especially for bacteria with fast cell division rates [21, 29]. Furthermore, it has been reported that a high level of gene dosage correlates with a higher degree of genomic stability as fast multiplying bacteria display a stronger conservation of gene positioning . Therefore, despite the fact that direct experimental evidence is scarce, these reports give support for gene dosage as an important factor in the evolution of bacterial genomes, especially for fast growing species.
In the case of vibrios, which have their genomes distributed between two unequally sized chromosomes and also display very short multiplication times, differential gene dosage could have a strong impact. The short multiplication times would result in large gene dosage differences within each chromosome. Also, assuming an equal replication speed for the two replicons, size differences would lead to different gene dosage between the chromosomes. Indeed, a study employing flow cytometry in combination with computer modelling revealed convincing experimental evidence for increased gene dosage differences within the large than within the small chromosome of actively growing V. cholerae . In addition, this examination showed growth rate dependent variations in relative gene copy numbers both within and between the chromosomes and that differing timings of replication initiation creates overall highest copy numbers for genes located near the origin of replication of the large chromosome. A later study employing fluorescence microscopy to detect relative abundances of large and small chromosome origins of replication confirmed these enhanced and growth rate dependent gene dosage differences for the large chromosome relative to the small . However, this report also indicated some question marks regarding an earlier replication start for the large chromosome, at least under certain growth conditions.
In an attempt to extend current knowledge about vibrio replication dynamics and gain insight into how this affect expression and genetic distribution, we here employed real-time PCR to quantify relative abundances of origin and terminus proximate DNA for both chromosomes of actively growing V. parahaemolyticus, V. cholerae and V. vulnificus. For V. parahaemolyticus, microarray analyses at both the genomic and transcription levels were also conducted. We further examined the location of distinct gene types within five sequenced and annotated Vibrionaceae genomes and related this to replication and expression patterns.