Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli

ABSTRACT Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse. IMPORTANCE The integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.


Table S1. E. coli strains
Table S2A.DNA oligomers used to construct the E. coli strains in Table S1.Table S2B.DNA oligomers for verifying single copy prophages.
Table S3.Confirmation of the localization and orientations of prophage in different strains long-read sequencing.

Figure S1 .
Figure S1.Transcript variability of native and inverted orientations of the gal operon in a wild-type background and cI in a recA -background.

Figure S2 .
Figure S2.Schematic configuration of range expansion assays of lysogenic strains with prophages integrated at the wild-type site and its symmetric counterpart.

Figure S3 .
Figure S3.Strains developed and their construction scheme.

Figure S6 .
Figure S6.Analysis of range expansion of a fluorescently-labeled lysogenic strains.

Figure S1 .
Figure S1.Transcript variability of native and inverted orientations of the gal operon in a wild-type background and cI in a recA -background.(A) Distributions of galK transcripts in lysogens integrated in a native (left, XTL856) and inverted (right, XTL855) orientation.(B) Distributions of cI transcripts measured in recA -lysogens integrated with a native (left) and inverted (right) orientations.The distributions, measured by smFISH, represent an average over three experimental repeats and error bars represent standard errors.(C) Distributions of cI transcripts measured in lysogens integrated at 84.19' with a native (left, XTL894) and inverted (right, XTL893) orientations.All distributions represent an average over at least three independent experimental repeats (about 3000 cells for each orientation), while error bars represent standard errors.

Figure S2 .
Figure S2.Schematic configuration of range expansion assays of lysogenic strains withprophages integrated at the wild-type site and its symmetric counterpart.Mixed bacterial colonies (ratio=1:1) of strain pairs with prophages at 17.4' and 50.6' sites, each with the prophage integrated either in inverted or native integration orientations, labelled with CFP and YFP.The ratio of area of the wild-type attB site from the total is shown, determined from two independent experiments, each consisting of nine colonies (mean ± standard error).The schemes represent the chromosomal constructions, the arrows, the prophage integration orientations relative to the replication direction and the blue and yellow colors of the arrows represent CFP and YFP, respectively.

Figure S3 .
Figure S3.Strains developed and their construction scheme.The diagram shows the methods that were used in order to develop the indicated strains including: transformation, recombineering, lambda phage infection and P1 transduction (in blue).The indicated locations of integration at the E. coli chromosome and the phage orientations are indicated for each strain.

Figure S4 .
Figure S4.Bacterial genome sequencing alignment.Bacterial strain sequences of both integration orientations obtained from Nanopore sequence assemblies aligned near the flanking regions to the indicated bacterial genes.Gray shaded regions denote prophage sequences while the integration sites are shown in bold.

Figure S5 .
Figure S5.Bacterial growth curves.The growth in logarithmic scale of strains XTL855 and XTL856 in LB was followed using optical density (OD600).The solid line represents an exponential fit to the data and the doubling times () corresponding to the time constants of the fits are shown.The doubling time of all other strain in the study were determined similarly and are shown in TableS3.

Figure S6 .
Figure S6.Analysis of range expansion of a fluorescently-labeled lysogenic strains.An exemplary range expansion image (top) with Region Of Interest (ROI) is shown, where dark regions correspond to the CFP-labeled strain.After achieving better contrast (see main text), the image threshold was subtracted (middle) and the despeckled ROI was false-colored (bottom).