Patterns of abundance, chromosomal localization, and domain organization among c-di-GMP-metabolizing genes revealed by comparative genomics of five alphaproteobacterial orders

Abstract

Background: Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a bacterial second messenger that affects diverse processes in different bacteria, including the cell cycle, motility, and biofilm formation. Its cellular levels are controlled by the opposing activities of two types of enzymes, with synthesis by diguanylate cyclases containing a GGDEF domain and degradation by phosphodiesterases containing either an HD-GYP or an EAL domain. These enzymes are ubiquitous in bacteria with up to 50 encoded in some genomes, the specific functions of which are mostly unknown.

Results: We used comparative analyses to identify genomic patterns among genes encoding proteins with GGDEF, EAL, and HD-GYP domains in five orders of the class Alphaproteobacteria. GGDEF-containing sequences and GGDEF-EAL hybrids were the most abundant and had the highest diversity of co-occurring auxiliary domains while EAL and HD-GYP containing sequences were less abundant and less diverse with respect to auxiliary domains. There were striking patterns in the chromosomal localizations of the genes found in two of the orders. The Rhodobacterales' EAL-encoding genes and Rhizobiales' GGDEF-EAL-encoding genes showed opposing patterns of distribution compared to the GGDEF-encoding genes. In the Rhodobacterales, the GGDEF-encoding genes showed a tri-modal distribution with peaks mid-way between the origin (ori) and terminus (ter) of replication and at ter while the EAL-encoding genes peaked near ori. The patterns were more complex in the Rhizobiales, but the GGDEF-encoding genes were biased for localization near ter.

Conclusions: The observed patterns in the chromosomal localizations of these genes suggest a coupling of synthesis and hydrolysis of c-di-GMP with the cell cycle. Moreover, the higher proportions and diversities of auxiliary domains associated with GGDEF domains and GGDEF-EAL hybrids compared to EAL or HD-GYP domains could indicate that more stimuli affect synthesis compared to hydrolysis of c-di-GMP.

Keywords: Cell cycle; DNA replication; Diguanylate cyclase; EAL; GGDEF; HD-GYP; Phosphodiesterase.

Fig. 1

Numbers of sequences with GGDEF, EAL or HD-GYP sequences in the five orders. The number of genomes and the total number of sequences for each order are above the diagrams. The Venn diagrams show the numbers of sequences with both GGDEF and EAL domains in the corresponding overlapping circles. The coloration is a gradient from the highest (red) to lowest (white) values within each order

Fig. 2

Mean number of c-di-GMP-metabolizing sequences per genome per genus in the different orders. The number of c-di-GMP-metabolizing genes in a genus was divided by the number of strains considered in the respective genus. The mean values from all genera of each order were used to make the box plot

Fig. 3

Numerical relationships among c-di-GMP-metabolizing sequences. A. Ratios for GGDEF:EAL, GGDEF:GGDEF_EAL, and GGDEF:HD_GYP sequences for the orders Rhodobacterales and Rhizobiales. The ratios were calculated per genome and the mean per genus was plotted. The median is indicated by the black dot. B. Counts of sequences with only a GGDEF domain or only an EAL domain per genome. The median value (50% quantile) is given on top of each box

Fig. 4

Proportions of genomes with one, two or more than two replicons > 800 kb in the five orders. The total numbers of genomes in each order are above the plot

Fig. 5

Chromosomal locations of c-di-GMP-metabolizing genes. Cumulative distributions of c-di-GMP-metabolizing genes on the chromosomes of Rhodobacterales and Rhizobiales, with lengths normalized to 100% where ori is at 0% and ter is at 50%. The color-coded lines represent the estimate of the kernel density. Only closed genomes with one unambiguously determined ori were used in this analysis

Fig. 6

Occurrence of auxiliary domains on c-di-GMP-metabolizing proteins of the different enzyme groups. A. Numbers of different auxiliary domains that can be found for each group and shared among groups. The first number below the group identification (EAL, GGDEF, GGDEF_EAL, HD-GYP) indicates the number of auxiliary domains in the respective group and the second number indicates the number of sequences these domains are found in. The c-di-GMP-metabolizing domains themselves are not included in this analysis. The color code of the Venn diagram represents the domain counts from the highest (red) to zero (white). B. Percentage of sequences with none, one, or more than one auxiliary domain. The number of sequences included in this analysis is given above the group identification. Repeated occurrence of a domain in a sequence was counted as one

Fig. 7

Weighted graphs representing the co-occurrences of auxiliary domains with c-di-GMP-metabolizing sequences. Auxiliary domains occurring together are connected by lines with the size and red color of the node indicating higher frequency of co-occurrence with other domains. Lengths of edges represent the number of times the connected domains co-occur, and the sizes of the points indicate the number of times these domains occur. All full domain names are provided in Table S8