G-quadruplex (G4), formed by repetitive guanosine-rich sequences, is known to play various key regulatory roles in cells. Herpesviruses containing a large double-stranded DNA genome show relatively higher density of G4-forming sequences in their genomes compared to human and mouse. However, it remains poorly understood whether all of these sequences form G4 and how they play a role in the virus life cycle. In this study, we performed genome-wide analyses of G4s present in the putative promoter or gene regulatory regions of a 235-kb human cytomegalovirus (HCMV) genome and investigated their roles in viral gene expression. We evaluated 36 putative G4-forming sequences associated with 20 genes for their ability to form G4 and for the stability of G4s in the presence or absence of G4-stabilizing ligands, by circular dichroism and melting temperature analyses. Most identified sequences formed a stable G4; 28 sequences formed parallel G4s, one formed an antiparallel G4, and four showed mixed conformations. However, when we assessed the effect of G4 on viral promoters by cloning the 20 putative viral promoter regions containing 36 G4-forming sequences into the luciferase reporter and monitoring the expression of luciferase reporter gene in the presence of G4-stabilizing chemicals, we found that only 9 genes were affected by G4 formation. These results revealed promoter context-dependent gene suppression by G4 formation. Mutational analysis of two potential regulatory G4s also demonstrated gene suppression by the sequence-specific G4 formation. Furthermore, the analysis of a mutant virus incapable of G4 formation in the UL35 promoter confirmed promoter regulation by G4 in the context of virus infection. Our analyses provide a platform for assessing G4 functions at the genomic level and demonstrate the properties of the HCMV G4s and their regulatory roles in viral gene expression. Although a number of G4-forming sequences are predicted in the herpesviral genomes and some G4s are characterized individually, why so many G4s are present in the genome and whether they are all functional are not yet clear. Our genome-wide bioinformatic sequence analyses and biophysical stability and structure assays of G4s present in the HCMV genome revealed that most G4s present in the HCMV gene regulatory regions form a stable G4 structure. However, it was found that only a few HCMV G4s suppress viral gene expression when the effect of G4 on gene expression was examined by in vivo reporter assay. Therefore, this study demonstrates that G4 activity relies on the promoter context, providing a new insight into understanding gene regulation by G4 structures. This study also provides evidence that G4 plays a regulatory role in gene expression during HCMV infection.

Vibrio cholerae strains of the O1 serogroup that typically cause epidemic cholera can be classified into two biotypes, classical and El Tor. The El Tor biotype emerged in 1961 and subsequently displaced the classical biotype as a cause of cholera throughout the world. In this study we demonstrate that when strains of the El Tor and classical biotypes were cocultured in standard LB medium, the El Tor strains clearly had a competitive growth advantage over the classical biotype starting from the late stationary phase and could eventually take over the population. The classical biotype produces extracellular protease(s) in the stationary phase, and the amounts of amino acids and small peptides in the late stationary and death phase culture filtrates of the classical biotype were higher than those in the corresponding culture filtrates of the El Tor biotype. The El Tor biotype cells could utilize the amino acids more efficiently than the classical biotype under the alkaline pH of the stationary phase cultures but not in medium buffered to neutral pH. The growth advantage of the El Tor biotype was also observed in vivo using the ligated rabbit ileal loop and infant mouse animal models.

A unique event in bacterial epidemiology was the emergence of the El Tor biotype of Vibrio cholerae O1 and the subsequent rapid displacement of the existing classical biotype as the predominant cause of epidemic cholera. We demonstrate that when the El Tor and classical biotypes were cocultured in standard laboratory medium a precipitous decline in colony forming units (CFU) of the classical biotype occurred in a contact dependent manner. Several lines of evidence including DNA release, microscopy and flow cytometric analysis indicated that the drastic reduction in CFU of the classical biotype in cocultures was not accompanied by lysis, although when the classical biotype was grown individually in monocultures, lysis of the cells occurred concomitant with decrease in CFU starting from late stationary phase. Furthermore, uptake of a membrane potential sensitive dye and protection of genomic DNA from extracellular DNase strongly suggested that the classical biotype cells in cocultures retained viability in spite of loss of culturability. These results suggest that coculturing the classical biotype with the El Tor biotype protects the former from lysis allowing the cells to remain viable in spite of the loss of culturability. The stationary phase sigma factor RpoS may have a role in the loss of culturability of the classical biotype in cocultures. Although competitive exclusion of closely related strains has been reported for several bacterial species, conversion of the target bacterial population to the viable non-culturable state has not been demonstrated previously and may have important implications in the evolution of bacterial strains.

The emergence of antimicrobial resistance and rapid acclimation allows Vibrio vulnificus to rapidly propagate in the host. This problematic pathological scenario can be circumvented by employing an antivirulence strategy, treating Vibrio infections without hindering the bacterial growth. We developed a genome-integrated orthogonal inhibitor screening platform in E. coli to identify antivirulence agents targeting a master virulence regulator of V. vulnificus. We identified 2′,4′-dihydroxychalcone (DHC) from the natural compound library and verified that it decreases the expression of the major toxin network which is equivalent to the ∆hlyU deletion mutant. 2′,4′-DHC also reduced the hemolytic activity of V. vulnificus which was tested as an example of virulence phenotype. The electrophoretic mobility shift assay confirmed that 2′,4′-DHC specifically targeted HlyU and inhibited its binding to P_rtxA1 promoter. Under in vivo conditions, a single dose of 2′,4′-DHC protected ~50% wax-worm larvae from V. vulnificus infection at a non-toxic concentration to both V. vulnificus and wax-worm larvae. In the current study, we demonstrated that an orthogonal reporter system is suitable for the identification of antivirulence compounds with accuracy, and identified 2′,4′-DHC as a potent antivirulence agent that specifically targets the HlyU virulence transcriptional regulator and significantly reduces the virulence and infection potential of V. vulnificus.