Genetic interaction mapping highlights key roles of the Tol-Pal complex

The conserved Tol-Pal trans-envelope complex is important for outer membrane (OM) stability and cell division in Gram-negative bacteria. It has been proposed to mediate OM constriction during cell division via tethering to the cell wall. Yet, recent studies suggest that the complex has additional roles in OM lipid homeostasis and septal cell wall separation. How the Tol-Pal complex functions to facilitate these many processes is unclear. To gain insights into its role(s), we applied transposon insertion sequencing, and report here a detailed network of genetic interactions with the tol-pal locus in Escherichia coli. We found one positive and >20 negative strong interactions based on fitness. Disruption of genes responsible for osmoregulated periplasmic glucan biosynthesis restores fitness and OM barrier function, but not cell division defects, in tol-pal mutants. In contrast, deletions of genes involved in OM homeostasis and cell wall remodelling give rise to synthetic growth defects in strains lacking Tol-Pal, especially exacerbating OM barrier and/or cell division defects. Notably, the ΔtolA mutant having additional defects in OM protein assembly (ΔbamB) exhibited severe division phenotypes, even under conditions where the single mutants divide normally; this highlights the possibility for OM phenotypes to indirectly influence the cell division process. Overall, our work provides insights into the intricate nature of Tol-Pal function, and reinforces the model that this complex plays crucial roles in cell wall-OM tethering, cell wall remodelling, and in particular, OM homeostasis.

Absence of osmoregulated periplasmic glucan confers antimicrobial resistance and increases virulence in Escherichia coli

Clarifying the molecular mechanisms by which bacteria acquire virulence traits is important toward understanding the bacterial virulence system. In the present study, we utilized a bacterial evolution method in a silkworm-infection model and revealed that deletion of the opgGH operon encoding synthases for osmoregulated periplasmic glucan (OPG) increased the virulence of non-pathogenic laboratory strain of Escherichia coli against silkworms. The opgGH knockout mutant exhibited resistance to the host antimicrobial peptides and antibiotics. Compared with the parent strain, the opgGH knockout mutant produced greater amounts of colanic acid, which is involved in E. coli resistance to antibiotics. RNA sequence analysis revealed that the opgGH knockout altered the expression of various genes, including the evgS/evgA two-component system that functions in antibiotic resistance. In both a colanic acid-negative background and evgS-null background, the opgGH knockout increased E. coli resistance to antibiotics and increased the silkworm killing activity of E. coli. In the null background of the envZ/ompR two-component system, which genetically interacts with opgGH, the opgGH knockout increased the antibiotic resistance and the virulence in silkworms. These findings suggest that the absence of OPG confers antimicrobial resistance and virulence of E. coli in a colanic acid-, evgS/evgA-, and envZ/ompR- independent manner. IMPORTANCE The gene mutation types that increase bacterial virulence of Escherichia coli remain unclear, in part due to the limited number of methods available for isolating bacterial mutants with increased virulence. We utilized a bacterial evolution method in the silkworm infection model, in which silkworms were infected with mutagenized bacteria and highly virulent bacterial mutants were isolated from dead silkworms. We revealed that knockout of OPG synthases increases E. coli virulence against silkworms. The OPG-knockout mutants were resistant to host antimicrobial peptides as well as antibiotics. Our findings not only suggest a novel mechanism for virulence acquisition in E. coli, but also support the usefulness of utilizing the bacterial experimental evolution method in the silkworm infection model.

Deciphering the Components That Coordinately Regulate Virulence Factors of the Soft Rot Pathogen Dickeya dadantii

The bacterial soft rot pathogen Dickeya dadantii utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to disintegrate the plant cell wall. A transposon mutagenesis fluorescence-activated cell sorting screen was used to identify mutants with altered promoter activities of the T3SS pilus gene hrpA. Several insertion mutations, resulting in changes in hrpA expression, were mapped to a new locus, opgGH, which encodes the gene cluster responsible for osmoregulated periplasmic glucan (OPG) synthesis proteins. Our data showed that OPG was involved in T3SS and Pel regulation by altering the expression of the regulatory small RNA RsmB. Through genome searching, the mechanism of two novel regulatory components, the RcsCD-RcsB phosphorelay and CsrD on OPG and the rsmB gene, was further investigated. The Rcs phosphorelay and OPG inversely regulated rsmB at transcriptional and post-transcriptional levels, respectively. CsrD exhibited dual functionality in T3SS and Pel regulation by manipulating levels of RsmB RNA and cyclic diguanylate monophosphate (c-di-GMP). CsrD positively regulated the promoter activity of the rsmB gene but negatively controlled RsmB RNA at the post-transcriptional level via OpgGH. In addition, CsrD contains both GGDEF and EAL domains but acted as a c-di-GMP phosphodiesterase. When the expression of the csrD gene was induced, CsrD regulated T3SS expression and Pel production through controlling intracellular c-di-GMP levels.

Influence of RpoS, cAMP-receptor protein, and ppGpp on expression of the opgGH operon and osmoregulated periplasmic glucan content of Salmonella enterica serovar Typhimurium

A transcriptional fusion (opgG1::MudJ) to the opgGH operon of Salmonella enterica serovar Typhimurium (S. Typhimurium) LT2, isolated by resistance to mecillinam, was used to study the influence of global regulators RpoS, ppGpp, and cAMP/cAMP-receptor protein (CRP) on expression of the opgGH operon and osmoregulated periplasmic glucan (OPG) content. Neither high growth medium osmolarity nor absence of ppGpp or CRP had important effects on opgG1::MudJ expression in exponential cultures. However, under the same conditions, OPG content was strongly decreased by high osmolarity or cAMP/CRP defectiveness, and reduced to a half by lack of ppGpp. In stationary cultures, high osmolarity as well as CRP loss caused significant descents in opgG1::MudJ expression that were compensated by inactivation of RpoS σ factor. No effect of RpoS inactivation on OPG content was observed. It is concluded that opgGH expression in S. Typhimurium is only slightly affected by high osmolarity, but is inversely modulated by RpoS level. On the other hand, osmolarity and the cAMP/CRP global regulatory system appear to control OPG content, either directly or indirectly, mainly at the post-transcriptional level.

Identification of mdoD, an mdoG Paralog Which Encodes a Twin-Arginine-Dependent Periplasmic Protein That Controls Osmoregulated Periplasmic Glucan Backbone Structures

ABSTRACT Osmoregulated periplasmic glucans (OPGs) of Escherichia coli are anionic and highly branched oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. The glucan length, ranging from 5 to 12 glucose residues, is under strict control. Two genes that form an operon, mdoGH, govern glucose backbone synthesis. The new gene mdoD, which appears to be a paralog of mdoG, was characterized in this study. Cassette inactivation of mdoD resulted in production of OPGs with a higher degree of polymerization, indicating that OpgD, the mdoD product (according to the new nomenclature), controls the glucose backbone structures. OpgD secretion depends on the Tat secretory pathway. Orthologs of the mdoG and mdoD genes are found in various proteobacteria. Most of the OpgD orthologs exhibit a Tat-dependent secretion signal, while most of the OpgG orthologs are Sec dependent.

Osmoregulated Periplasmic Glucan Synthesis Is Required for Erwinia chrysanthemi Pathogenicity

ABSTRACT Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGHoperon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgHgene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.