To examine replication dynamics and gene dosage effects for the two Vibrio chromosomes we first aimed to establish appropriate growth conditions. As larger inter- and intra-chromosomal gene dosage differences should be obtained from quickly multiplying cells, we determined doubling times for three Vibrio species (V. parahaemolyticus, V. cholerae and V. vulnificus, see Table 1) incubated in rich media at 37°C. As comparisons, we determined doubling times of V. parahaemolyticus grown in rich broth at 20°C, which more closely resembles the natural growth temperature for vibrios, and in minimal broth at 37°C. The results are summarised in Table 2 and show shortest doubling times for V. parahaemolyticus (12–14 min) followed by V. cholerae (16–20 min) and V. vulnificus (18–22 min). Doubling times were tripled for V. parahaemolyticus incubated at a lower temperature (36–42 min) and quadrupled for cells grown in minimal broth (50–60 min).

Based on the established maximum replication fork movement of 1000 nt/s for E. coli 30, chromosome size can be used to estimate replication time. With an estimated replication time, knowledge about the doubling time enables predictions about origin/terminus (ori/ter) ratios for a replicon 21. For this, we used the chromosome sizes given for the genomic strain of V. parahaemolyticus RIMD2210633, and employed pulsed-field gel electrophoresis to determine approximate sizes for the chromosomes of V. cholerae RIMD2203577 and V. vulnificus ATCC27562 (Table 1) before calculating ori/ter ratios for each separate chromosome (Table 2). The highest ratio (4.31) is estimated for the large chromosome of the quickly multiplying V. parahaemolyticus. These cells are also supposed to show relatively large inter-chromosomal difference in ori/ter ratios (4.31/2.30 = 1.87). For the large chromosomes of V. cholerae and V. vulnificus, the approximately equal ratios (2.50 and 2.67, respectively) reflect that the faster doubling time for the former is compensated by the larger size for the latter. Similarly, a relatively low ori/ter ratio for the small V. cholerae chromosome (1.47) is explained by its small size. Furthermore, the large size difference between the two V. cholerae chromosomes gives an inter-chromosomal ori/ter ratio (2.50/1.47 = 1.70) that is only slightly lower than for the much faster multiplying V. parahaemolyticus cells. Finally, a comparison between V. parahaemolyticus grown in rich and minimal media suggests that large variations in ori/ter ratios can be expected, especially for the large chromosome.

To experimentally define origin and termini quantities and ratios, RT-qPCR was applied on genomic DNA (gDNA) using primers with target sites located near each origin and terminus of V. parahaemolyticus, V. cholerae and V. vulnificus (Additional file 1). As we wanted to determine relative target abundance in exponentially growing cells, gDNA from non-replicating cells with equal numbers of large and small chromosome origins and termini were used as reference samples (Additional file 2). As is significative for actively replicating chromosomes, the results indicate a relative increase in origin proximate DNA for both chromosomes in all species and under all growth conditions tested (Figure 1Aa, Ba and 1Ca). The figure also shows that whereas relative abundances of origin proximate DNA appear to be higher for the large Vibrio chromosomes than the small, there seem to be similar or only slightly different amounts of termini proximate DNA. In agreement with previous results from V. cholerae 15, this indicates a much earlier replication start for the large chromosome while termination for both chromosomes occurs within a short time span. Therefore, this suggests that such earlier replication start for the large chromosome could be a common trait for vibrios.

To better visualise gene dosage differences, ori/ter ratios were determined for each chromosome (Figure 1Ab, Bb and 1Cb), and in all instances the large chromosomes show a significantly higher ori/ter ratio than the small (P < 0.05). A comparison between V. parahaemolyticus grown in minimal and rich media at 37°C shows a much larger increase in gene dosage for the large than for the small chromosome (Figure 1Ab). This is consistent with doubling time based calculations (Table 2) and agrees with previous results from V. cholerae demonstrating that growth rate has a much larger impact on gene dosage for the large than for the small chromosome 1516. For low temperature cultures in rich media, however, there appear to be only minor differences in ori/ter ratios compared to rich media cultures grown at 37°C (Figure 1Ab, Bb and 1Cb). With consideration to the much faster doubling times at the higher temperature, this could suggest that replication is temporarily blocked or slowed in low temperature grown cells.

A weakness in the above determinations is that they are built on assumptions about an equal and bi-directional replication rate for both chromosomes. To avoid this and get a more detailed and quantitative view of the replication dynamics, we next performed microarray analyses comparing gDNA from exponentially growing V. parahaemolyticus in rich media at 37 and 20°C and poor nutrient broth at 37°C against gDNA from non-replicating cells. The resulting replication patterns are shown in Figure 2 and display gene dosage as a decrease in DNA copy numbers when moving away from the origins of replication. For cells grown in rich broth at 37°C, smooth and similar slopes indicate an even replication progress, both within and between the two chromosomes, which lead to terminations at locations diametrically opposite to the origins of replication (Figure 2A). Also seen is an increase of nearly two orders of magnitude for origin over terminus proximate DNA quantities for the large chromosome, while a similar comparison for the small shows an increase of ~1.2 (Figure 2A). These values correspond to a large chromosome ori/ter ratio slightly below 4 and a small chromosome ori/ter ratio of ~2.3, which is in agreement with the RT-qPCR results (cf. with Figure 1Ab). Moreover, the replication patterns show a higher abundance of large over small chromosome origins while there are approximately equal amounts of termini for both chromosomes. Again, this indicates that termination rather than initiation occurs at a similar time in the cell cycle which is in agreement with previous results from V. cholerae 15. For cells grown in rich media at 20°C (Figure 2B), ori/ter ratios and quantities are also confirmed (cf. with Figure 1A) and the overall replication patterns are very similar to those obtained for cells incubated at the higher temperature (cf. with Figure 2A). However, a closer comparison reveals a slightly more stuttered pattern which suggests that replication is temporarily arrested at 20°C. Although this could partly explain why large gene dosage differences are maintained despite longer doubling times, the relatively continuous decrease in DNA copy numbers emphasises slowed replication kinetics as the major contributor. Indeed, such temperature dependent change in replication speed has previously been detected in E. coli where cultures incubated at 14°C maintained similar replication time/doubling time ratios as cultures grown at 37°C 31. Therefore, this suggests that the maintained gene dosage differences at a lower growth temperature are due to a slower replication kinetics that compensates for the less frequent initiation events. For cultures grown in minimal broth, only very small differences in DNA quantities along and between the chromosomes were seen and no clear locations for replication termination were discernible (Figure 2C). It therefore seems like the assay lack sensitivity for reliable determinations of replication dynamics for cells grown under these conditions. Nevertheless, the RT-qPCR results (Figure 1) and also previous analyses on V. cholerae cells grown in minimal media 15 imply a similar replication dynamics as for cells grown in rich broth.

Previous analyses have indicated a relatively fast replication progress for V. cholerae with replication fork movements around 16 or just below 15 1000 nt/s for cells grown at 37°C. The slightly lower gene dosage differences for the RT-qPCR and microarray analyses compared to the doubling time based estimates (cf. Figure 1 and 2 with Table 2) indicate an average replication speed that may exceed 1000 nt/s. However, it must be considered that determination of a correct replication speed relies on a number of factors and inconsistencies may be due to (i) variations in doubling times estimates, (ii) differences in strains and growth media, (iii) errors in DNA target quantifications, (iv) errors caused by sampling handling and (v) the possibility that reference samples display a certain degree of replicating activity. Nevertheless, our results confirm a fast replication speed for vibrios. In addition, the replication speed seems slowed down to approximately one third for cells grown at 20°C as gene dosage differences were maintained (Figure 1 and 2) while doubling times increased threefold (Table 2).

The above analyses display rather large and growth dependent differences in gene copy numbers within and between the chromosomes. To examine whether this affect expression levels, we compared cDNA derived from V. parahaemolyticus grown in rich media at 37 and 20°C and poor nutrient broth at 37°C against gDNA from non-replicating cells. In addition, ori/ter ratios were determined by linear regression analyses on expression data sorted with respect to distances to the origins of replication. The resulting expression patterns and ori/ter values are shown in Figure 3 and to facilitate comparisons replication patterns are also displayed. As can be observed, the expression data displays very large variations in comparison to DNA quantities. Nevertheless, it is clearly distinguishable that average expression levels are higher from the large chromosome. Also in agreement with the replication patterns is that large chromosome expression levels appear to gradually decrease with an increased distance from the origin of replication, which is also supported by ori/ter ratios larger than 1. For the small chromosome, however, there appears to be no link between gene copy numbers and expression levels. Instead, general expression levels are low and ori/ter ratios indicate decreased rather than increased expression for origin-proximate relative to terminus-proximate genes. Therefore, this suggests that while gene expression from the large chromosome shows a tendency to follow gene dosage, expression from the small is more strictly regulated. A comparison between different growth environments lends further support for this statement in that large chromosome gene dosage effects are more pronounced for the fastest growing cells (cf. Figure 3A with 3B and 3C). Similarly, small chromosome expression seems even more tightly regulated when doubling times are shortened as DNA levels by far exceeds expression levels for cells grown in rich media at 37°C but not at a lower temperature or in minimal media (cf. Figure 3A with 3B and 3C). Also notable is that gene dosage effects are less pronounced for cells grown in rich media at a lower than a higher temperature although relative DNA quantities were comparable (cf. Figure 3A and 3B). This indicates that not only replication but also other cellular processes slow with a decreased temperature.

Although gene dosage and growth environment appear to have an influence on expression levels, the expression patterns also suggest other impacts. Several examinations have pointed out the presence of regularities in bacterial gene expression data where local expression maxima are found at periodicities of around 32 or slightly above 3334 100 kb. These patterns have been explained by higher order nucleoid structuring where the DNA is compacted into one or two large helices containing loops of approximately 100–120 kb lengths 323334. To examine whether periodicities are present in our expression data, grids were fitted to match local peaks. For both chromosomes periodic patterns of approximately 100 kb in length were detected (Additional file 3). Therefore, it appears like, in addition to gene dosage, also higher order chromosomal structuring has an influence on vibrio expression.

The above experiments show that differing size and initiation timing creates higher average gene dosage and gene copy numbers on the large Vibrio chromosome. The results also show that expression levels from the large but not the small chromosome tend to follow gene dosage in a growth rate dependent manner. To gain a better understanding of these replication and expression patterns we next examined the distribution of different gene types within the Vibrio genome.

In a first analysis we wanted to see how genes related to growth were distributed within the genome. As comprehensive information about such genes is lacking for vibrios, we instead used data from a systematic knock-out analysis of all genes in the closely related bacterium Escherichia coli 35 and determined their closest orthologs in the V. cholerae genome using BLASTO 36. Initially, we examined the distribution of orthologs to essential, the most growth contributing (genes whose absence create the largest growth defects for E. coli in LB) and the least growth contributing genes (E. coli genes that disturb growth in LB the least when absent) between the two chromosomes (see the Method section for a more detailed description). The results are displayed in Figure 4A and show a clear over-representation of orthologs to growth essential genes on the large chromosome. This is in agreement with findings from whole genome sequence analyses both for V. cholerae 11 and other Vibrio species 37383940, which have pointed out that many genes involved in essential biosynthetic pathways locate on the large chromosome, while few such genes are found on the small. For orthologs to genes that contribute the most to growth there was also a strong over-representation on the large chromosome while orthologs to the least growth contributing genes did not display a significant deviation from a random distribution between the chromosomes. Therefore, this analysis suggests a tendency where an increased importance for growth parallels a preferential location on the large chromosome. With consideration to the expression patterns this seems logical as only the large chromosome display growth rate dependent variations in expression levels.

We next analysed gene distribution between the early and late replicated parts of the large V. cholerae chromosome. The early replicated part was defined as being replicated before initiation of the small chromosome, assuming an equal bi-directional replication speed for both chromosomes and a synchronous termination. The results show that orthologs to both growth essential and the most growth contributing genes are over-represented within the early replicated part, while orthologs to the least growth contributing genes do not show a significantly biased distribution (Figure 4B). This distribution is in agreement with a previous notion that essential genes, and especially highly expressed such genes, show a clear tendency to locate near the origin of replication in E. coli 29. The explanation given for this distribution was that the highly expressed essential genes benefit from a high gene dosage. With consideration to our expression results, this argument also seems suitable for vibrios. An additional benefit with an early replication for such genes in vibrios could be that this gives the cells more time to build up enough supplies of gene products required later in the cell cycle, including the additional metabolic burden of replicating the small chromosome 15. It is also possible that an early replication of genes important for growth allow the cells to quickly adopt their expression to changes in growth conditions.

We also compared the distribution of orthologs to growth related genes between the late replicating part of the large chromosome and the small chromosome. Essential and most growth contributing genes were clearly under-represented on the small chromosome while genes contributing the least to growth appeared more randomly distributed (Figure 4C). This distribution contrasts with the observation that these two genome parts show a similar gene dosage (Figure 2). However, the disproportionally low number of growth important genes on the small chromosome agrees with the gene dosage and growth rate independent expression observed for this part of the genome (Figure 3). In summary, the results displayed in Figure 4 show that the distribution of genes central for growth is connected to replication timing and gene dosage effects.

To extend the analysis of gene type distribution and attempt to better understand the expression patterns, we next sorted all Vibrio genes into different categories according to the classification system from Clusters of Orthologous Groups of proteins (COGs) 41. Data available for five sequenced and annotated Vibrio genomes (Table 1) were used and relative over- and under-representation of 21 gene categories were examined within the early and late replicated parts of the large and within the small chromosome (see Methods and Additional file 4). Relative abundance of growth essential and most growth contributing genes within each category was also determined (see Methods and Additional file 5). The result is summarised in Figure 5 and display several distinct differences. In agreement with above observations, categories important for proliferation, such as "Translation", "Cell wall/membrane biogenesis", "Intracellular trafficking and secretion", Coenzyme transport and metabolism", "Cell cycle control" and "Nucleotide transport and metabolism" are over-represented on the early replicated part of the large chromosome and concurrently under-represented on the small. A missing category, however, is the "Replication, recombination and repair" genes that should benefit from an early replication and growth rate related gene dosage effects. Indeed, four of the five vibrios show an over-representation of this group on the early replicated part of the large chromosome and it is only P. profundum that, for unknown reasons, deviates from this pattern (Additional file 4). The categories over-represented on the small chromosome are generally of little importance for growth and several show a concurrent under-representation near the origin of replication of the large chromosome (Figure 5). A high abundance of undefined genes and genes lacking orthologs is in line with earlier observations from genome sequencing projects 1137383940 and support speculations that the small chromosome could provide the bacteria with many species specific traits 1142. Furthermore, the over-representation of "Carbohydrate transport and metabolism" genes suggests that the small chromosome could be important for adaptation to different growth environments with varying energy sources, as was previously proposed 40. Also striking is the high over-representation of "Transcription" genes and the concurrent under-representation on both parts of the large. To better understand this distribution it should be considered that transcription genes constitute a very heterogeneous group with regard to expression. It comprises both global regulators required at high concentrations to reach their many targets and much less expressed local regulators that co-localise with their targets either through direct proximity 43 or by three-dimensional proximity created by nucleoid structures 44. This could mean that many transcriptional regulators on the small chromosome exert their action through co-localisation while global regulators gather on the large. Supporting this is that among the 20 transcriptional regulators that regulate the highest number of genes in E. coli 45, 18 have orthologs in V. cholerae and all of these locate on the large chromosome (data not shown). Therefore, a majority of the "Transcription" genes on the small chromosome are likely to regulate nearby genes. Furthermore, their high abundance could provide an explanation to why expression from this part of the genome is more strictly regulated and display independence from gene dosage.

Bacterial strains used for experimental and computational analyses in this study are listed in Table 1. V. parahaemolyticus, V. cholerae and V. vulnificus from frozen glycerol stocks were grown in 2 ml cultures overnight at 20 or 37°C in either M9 media supplemented with 3% NaCl (w/v) and 0.4% glucose (3%M9), Luria-Bertani (LB) broth or LB containing 3% NaCl (3%LB). Cultures were diluted 1:1000 in 2 ml fresh media and incubated at 37 or 20°C with constant shaking at 210 rpm. At harvest, cultures were immediately transferred to – 30°C ice/NaCl/ethanol slurry followed by centrifugation (8000 g, 3 min, 4°C). To obtain non-replicating cells, overnight cultures were either plated onto LB agar followed by a 24 h incubation at 20°C and an additional 24 h incubation at 4°C or incubated in 2 ml liquid media (3%M9, LB or 3%LB) for 24 h at 20 or 37°C. Stationary phase liquid cultures were treated for 2 h with 20 μl rifampicin (50 mg/ml dissolved in DMSO) before harvest to finish ongoing replication rounds without initiating new ones.

Estimates of chromosomal sizes were performed with pulsed-field gel electrophoresis (PFGE) on a CHEF Mapper XA system using reagents provided in the GenePath Univeral Module (Bio-Rad). Samples of V. cholerae, V. vulnificus and V. parahaemolyticus were prepared according to the manufacturers' instructions and loaded along with a molecular marker onto a 0.8% agarose gel prepared with and run in 1xTAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) containing 500 μM thiourea 52. The electrophoresis was run at 14°C for 48 h with a switch time of 500 s at 3 V/cm and an included angle of 106°.

Bacterial cultures were prepared as described above and samples were harvested every 10 minutes for dilution in series and plating onto LB plates for colony counts or every 30 minutes for OD-measurements. Doubling times were determined for mid-exponential phase cultures using the change in colony counts for a one hour time span around OD₆₀₀ ~ 0.5, by first calculating the number of generations (n):

n = log ⁡ b − log ⁡ B log ⁡ 2 MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGaemOBa4Maeyypa0tcfa4aaSaaaeaacyGGSbaBcqGGVbWBcqGGNbWzcqWGIbGycqGHsislcyGGSbaBcqGGVbWBcqGGNbWzcqWGcbGqaeaacyGGSbaBcqGGVbWBcqGGNbWzcqaIYaGmaaaaaa@3FBF@

Here, b is the number of CFU at the end and B the number of CFU at the beginning of the one hour time interval. Doubling times (τ) were given by dividing the time interval (t) with the number of generations (n):

τ = t n MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGaeqiXdqNaeyypa0tcfa4aaSaaaeaacqWG0baDaeaacqWGUbGBaaaaaa@3262@

To estimate the time required to complete one replication round (C) for each chromosome, a bidirectional replication speed of 1000 nt/s was used:

C = C h r o m o s o m e   s i z e 2 × 1000 × 60 MathType@MTEF@5@5@+=feaagaart1ev2aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaGaee4qamKaeyypa0tcfa4aaSaaaeaacqWGdbWqcqWGObaAcqWGYbGCcqWGVbWBcqWGTbqBcqWGVbWBcqWGZbWCcqWGVbWBcqWGTbqBcqWGLbqzcqqGGaaicqWGZbWCcqWGPbqAcqWG6bGEcqWGLbqzaeaacqaIYaGmcqGHxdaTcqaIXaqmcqaIWaamcqaIWaamcqaIWaamcqGHxdaTcqaI2aGncqaIWaamaaaaaa@4D89@

Given that initiation of replication occurs simultaneously for all origins on a replicon 53 it can be assumed that every new required round of replication results in a doubling of the origin/terminus ratio (r_O/T). The following formula, taken from 21, was used to calculate (r_O/T):

r_O/T = 2^C/τ

Calculations are summarised in Table 2.

Genomic DNA (gDNA) was extracted with DNeasy^® Tissue Kit (Qiagen) following the manufacturer's instructions including a prolonged RNase treatment step (15 minutes) and was finally eluted in 2 × 100 μl AE buffer. DNA samples were further purified by addition of equal volumes of phenol and chloroform with subsequent phase separation by centrifugation before precipitation with 1/10 volume 3 M CH₃COONa (pH 5.2) and 2.6 volumes ethanol. DNA was pelleted by centrifugation and further washed with 1 ml 70% ethanol before being dissolved in H₂O.

Total RNA was extracted from bacterial pellets with 1 ml TRIzol^® Reagent (Invitrogen) following the manufacturer's protocol. RNA pellets were re-suspended and treated with DNase I according to instructions that accompany the RNase-free DNaseI set (Qiagen) before purification, precipitation and wash with phenol/chloroform, 3 M CH₃COONa (pH 5.2)/ethanol and 70% ethanol, respectively. RNA was re-dissolved in 100 μl H₂O and further purified using RNeasy^® Mini Kit (Qiagen) before elution in H₂O. RNA was subjected to a further precipitation (3 M CH₃COONa (pH 5.2)/ethanol) and wash (70% ethanol) before finally being dissolved in H₂O.

Relative quantification of origins and termini for both Vibrio chromosomes was carried out with either 10 ng (for V. parahaemolyticus) or 2.5 ng (for V. cholerae and V. vulnificus) gDNA with real time quantitative polymerase chain reaction (RT-qPCR). Reaction volumes were 20 μl and also included 1× Power SYBR^® Green PCR Master Mix (Applied Biosystems) and either of the primer pairs listed in Additional file 1. Importantly, primer pairs were selected to target stably integrated regions not thought capable to excise from their respective places on the genomes. An exception was a primer pair targeting a phage like element present at both V. parahaemolyticus termini that was selected to provide an additional reference for this species. As we aimed to quantify relative numbers of large and small chromosome origins and termini in exponentially growing cultures, reference samples with known relative quantities of these regions were required. To provide such references, gDNA was extracted from non-replicating cells that were pre-grown on LB-plates and incubated for 24 h at 4°C. To verify an equal relationship between large and small chromosome origin and termini, the samples were compared against gDNA from exponentially grown cells along with gDNA from non-replicating cells from liquid media cultures grown under two (V. vulnificus) or three (V. parahaemolyticus and V. cholerae) different conditions (see "Bacterial strains and growth conditions"). Similar ratios between origins and termini were obtained for all non-replicating samples, which confirmed their reliability as reference samples (Additional file 2). In addition, a pyrosequencing based analysis with a 20 fold coverage of the whole genome of V. parahaemolyticus confirmed approximately equal numbers of small and large chromosomes for stationary phase cells in 3%LB (unpublished data from the lab). Amplification efficiencies were validated with gDNA from both non-replicating (three separate experiments with five replicates) and exponentially grown cells (one experiment with five replicates) over a range of template concentrations (2.5–40 ng for V. parahaemolyticus and 0.625–10 ng for V. cholerae and V. vulnificus). Near linear dose responses and similar amplification efficiencies (89–100% for V. parahaemolyticus, 97–104% for V. cholerae, and 98–103% for V. vulnificus) were obtained by analysis with the 2^-ΔΔCT method 54 and indicated that reliable comparisons between the targets could be performed. Experimental analyses were performed in five double samples on gDNA extracted at three or five different occasions.

For generation of aminoallyl-labelled product from gDNA, the procedures described in 55 were followed. For generation of aminoallyl-labelled product from total RNA we used reagents included in SuperScript™ III Reverse Transcriptase Kit (Invitrogen). In brief, 20 μg RNA was mixed with 10 μg random hexamers in a 22 μl reaction that was incubated at 70°C for 5 min before cooling on ice. Addition of 5× First-Strand Buffer (8 μl), 0.1 M DTT (2 μl), SuperScript™ III RT (4 μl) and 10× dNTP-aminoallyl dUTP (4 μl) was followed by a 3 h incubation at 46°C. Samples were precipitated and washed as described earlier.

Coupling of aminoallyl-labelled products with Cy3 or Cy5 monofunctional reactive dyes was performed as described previously 55. Microarray slides and hybridisation procedures are also described in 55, except that human CotI DNA (Invitrogen) replaced yeast tRNA and incubation/hybridisation temperatures were 55°C instead of 60°C. Fluorescence signals were measured and analysed according to previous descriptions 55. Microarray data was submitted to Gene Expression Omnibus (GEO) 56 and has the serial number GSE9968.

To examine the distribution of essential and growth related genes in the Vibrio genome, we used growth and essentiality data from a genome-wide single gene knock-out study performed in E. coli 35 and deduced the nearest orthologs in V. cholerae. E. coli genes were classified into three groups; one containing all essential genes, a second (most growth contributing genes) containing the genes for which knock-outs displayed the slowest growth rates in LB media (OD₆₀₀ < 0.604, see 35), and a third (least growth contributing genes) comprising genes for which knock-outs displays the highest growth rates in LB media (OD₆₀₀ > 0.823, see 35). Next, protein sequences for the essential, most growth contributing and least growth contributing E. coli genes were searched against the NCBI COG database in BLASTO 36 using the default settings (E = 0.001, BLOSUM62). Best hits were sorted into three parts of the V. cholerae genome; an early replicated part of the large chromosome (defined as the part being replicated before initiation of the small chromosome assuming an equal bidirectional replication speed and a simultaneous replication termination), a late replicated part of the large chromosome (the part of the large chromosome that is not defined as being early replicated) and the small chromosome. Deviations from an average distribution were determined by comparison to the total number of genes within the different parts of the genome and significance levels were determined by chi-square tests for comparison of two proportions.

To examine the distribution of different gene categories, the classification system from Clusters of Orthologous Groups of proteins (COGs) 41 was employed. Genes belonging to 21 functional categories were counted within the early and late replicated parts of the large and the small chromosome of five Vibrionaceae species (see Table 1). Relative over- and under-representation was determined by comparing the size of each group against an average distribution of COGs within each genome part. Significance levels for deviations from average distributions were determined by chi-square tests for comparison of two proportions. Categories showing a similar deviation from an average in a specific genome part for all Vibrionaceae species were considered over- or under-represented and categories with a similar and significant deviation for each species were considered highly over- or under-represented.

To examine the relative abundance of growth essential or highly growth contributing genes within the separate COG categories, we first determined the number of essential and highly growth contributing genes for each COG. Proportions of essential genes within each category were compared to the proportion of essential genes in the whole genome while proportions of highly growth contributing genes among the non-essential genes in each category were compared to the proportion highly growth contributing among all non-essential genes. Significance levels for deviations were determined by chi-square tests for comparison of two proportions.