Ethanolamine metabolism through two genetically distinct loci enables Klebsiella pneumoniae to bypass nutritional competition in the gut.

Successful microbial colonization of the gastrointestinal (GI) tract hinges on an organism's ability to overcome the intense competition for nutrients in the gut between the host and the resident gut microbiome. Enteric pathogens can exploit ethanolamine (EA) in the gut to bypass nutrient competition. However, Klebsiella pneumoniae (K. pneumoniae) is an asymptomatic gut colonizer and, unlike well-studied enteric pathogens, harbors two genetically distinct ethanolamine utilization (eut) loci. Our investigation uncovered unique roles for each eut locus depending on EA utilization as a carbon or nitrogen source. Murine gut colonization studies demonstrated the necessity of both eut loci in the presence of intact gut microbiota for robust GI colonization by K. pneumoniae. Additionally, while some Escherichia coli gut isolates could metabolize EA, other commensals were incapable, suggesting that EA metabolism likely provides K. pneumoniae a selective advantage in gut colonization. Molecular and bioinformatic analyses unveiled the conservation of two eut loci among K. pneumoniae and a subset of the related taxa in the K. pneumoniae species complex, with the NtrC-RpoN regulatory cascade playing a pivotal role in regulation. These findings identify EA metabolism as a critical driver of K. pneumoniae niche establishment in the gut and propose microbial metabolism as a potential therapeutic avenue to combat K. pneumoniae infections.

Deciphering the gastrointestinal carriage of Klebsiella pneumoniae.

Bacterial infections pose a significant global health threat, accounting for an estimated 7.7 million deaths. Hospital outbreaks driven by multi-drug-resistant pathogens, notably Klebsiella pneumoniae (K. pneumoniae), are of grave concern. This opportunistic pathogen causes pneumonia, urinary tract infections, and bacteremia, particularly in immunocompromised individuals. The rise of hypervirulent K. pneumoniae adds complexity, as it increasingly infects healthy individuals. Recent epidemiological data suggest that asymptomatic gastrointestinal carriage serves as a reservoir for infections in the same individual and allows for host-to-host transmission via the fecal-oral route. This review focuses on K. pneumoniae's gastrointestinal colonization, delving into epidemiological evidence, current animal models, molecular colonization mechanisms, and the protective role of the resident gut microbiota. Moreover, the review sheds light on in vivo high-throughput approaches that have been crucial for identifying K. pneumoniae factors in gut colonization. This comprehensive exploration aims to enhance our understanding of K. pneumoniae gut pathogenesis, guiding future intervention and prevention strategies.

The Two-Component System YesMN Promotes Pneumococcal Host-to-Host Transmission and Regulates Genes Involved in Zinc Homeostasis.

The ability to sense and respond rapidly to the dynamic environment of the upper respiratory tract (URT) makes Streptococcus pneumoniae (Spn) a highly successful human pathogen. Two-component systems (TCSs) of Spn sense and respond to multiple signals it encounters allowing Spn to adapt and thrive in various host sites. Spn TCS have been implicated in their ability to promote pneumococcal colonization of the URT and virulence. As the disease state can be a dead-end for a pathogen, we considered whether TCS would contribute to pneumococcal transmission. Herein, we determined the role of YesMN, an understudied TCS of Spn, and observe that YesMN contributes toward pneumococcal shedding and transmission but is not essential for colonization. The YesMN regulon includes genes involved in zinc homeostasis and glycan metabolism, which are upregulated during reduced zinc availability in a YesMN-dependent fashion. Thus, we identified the YesMN regulon and a potential molecular signal it senses that lead to the activation of genes involved in zinc homeostasis and glycan metabolism. Furthermore, in contrast to Spn monoinfection, we demonstrate that YesMN is critical for high pneumococcal density in the URT during influenza A virus (IAV) coinfection. We attribute reduced colonization of the yesMN mutant possibly due to increased association with and clearance by the mucus covering the URT epithelial surface. Thus, our results highlight the dynamic interactions that occur between Spn and IAV in the URT, and the role that TCSs play in modulation of these interactions.

Klebsiella pneumoniae l-Fucose Metabolism Promotes Gastrointestinal Colonization and Modulates Its Virulence Determinants.

Colonization of the gastrointestinal (GI) tract by Klebsiella pneumoniae is generally considered asymptomatic. However, gut colonization allows K. pneumoniae to either translocate to sterile site within the same host or transmit through the fecal-oral route to another host. K. pneumoniae gut colonization is poorly understood, but knowledge of this first step toward infection and spread is critical for combatting its disease manifestations. K. pneumoniae must overcome colonization resistance (CR) provided by the host microbiota to establish itself within the gut. One such mechanism of CR is through nutrient competition. Pathogens that metabolize a broad range of substrates have the ability to bypass nutrient competition and overcome CR. Herein, we demonstrate that in response to mucin-derived fucose, the conserved fucose metabolism operon (fuc) of K. pneumoniae is upregulated in the murine gut, and we subsequently show that fucose metabolism promotes robust gut colonization. Growth studies using cecal filtrate as a proxy for the gut lumen illustrate the growth advantage that the fuc operon provides K. pneumoniae. We further show that fucose metabolism allows K. pneumoniae to be competitive with a commensal Escherichia coli isolate (Nissle). However, Nissle is eventually able to outcompete K. pneumoniae, suggesting that it can be utilized to enhance CR. Finally, we observed that fucose metabolism positively modulates hypermucoviscosity, autoaggregation, and biofilm formation but not capsule biogenesis. Together, these insights enhance our understanding of the role of alternative carbon sources in K. pneumoniae gut colonization and the complex relationship between metabolism and virulence in this species.

Animal Model To Study Klebsiella pneumoniae Gastrointestinal Colonization and Host-to-Host Transmission.

An important yet poorly understood facet of the life cycle of a successful pathogen is host-to-host transmission. Hospital-acquired infections (HAI) resulting from the transmission of drug-resistant pathogens affect hundreds of millions of patients worldwide. Klebsiella pneumoniae, a Gram-negative bacterium, is notorious for causing HAI, with many of these infections difficult to treat, as K. pneumoniae has become multidrug resistant. Epidemiological studies suggest that K. pneumoniae host-to-host transmission requires close contact and generally occurs through the fecal-oral route. Here, we describe a murine model that can be utilized to study mucosal (oropharynx and gastrointestinal [GI]) colonization, shedding within feces, and transmission of K. pneumoniae through the fecal-oral route. Using an oral route of inoculation, and fecal shedding as a marker for GI colonization, we showed that K. pneumoniae can asymptomatically colonize the GI tract in immunocompetent mice and modifies the host GI microbiota. Colonization density within the GI tract and levels of shedding in the feces differed among the clinical isolates tested. A hypervirulent K. pneumoniae isolate was able to translocate from the GI tract and cause hepatic infection that mimicked the route of human infection. Expression of the capsule was required for colonization and, in turn, robust shedding. Furthermore, K. pneumoniae carrier mice were able to transmit to uninfected cohabitating mice. Lastly, treatment with antibiotics led to changes in the host microbiota and development of a transient supershedder phenotype, which enhanced transmission efficiency. Thus, this model can be used to determine the contribution of host and bacterial factors toward K. pneumoniae dissemination.

Capsule Prolongs Survival of Streptococcus pneumoniae during Starvation.

Person-to-person transmission of Streptococcus pneumoniae (the pneumococcus) may occur via environmental sources in close contact with carriers. Pneumococcal polysaccharide capsules, the determinant of serotype (or type), are heterogeneous in structure and amount, and these differences affect rates of transmission. In this study, we examined the contribution of capsule and its variations to the maintenance of pneumococcal viability under starvation conditions. S. pneumoniae retained its ability to colonize infant mice even after incubation for 24 h in phosphate-buffered saline at 25°C. The expression of capsule by the cps locus prolonged survival under these and other nutrient-poor conditions. Analysis of capsule-switch constructs showed that strain-to-strain differences in survival were due to capsule type rather than genetic background. The addition of glucose was sufficient to rescue the survival defect of the capsule-deficient derivative, demonstrating that in the absence of capsule, survival depends upon nutrient availability. During starvation, there was a decrease in capsule size and amount of capsular polysaccharide that was dependent on bacterial viability and the presence of the cps locus. These observations suggest that pneumococci catabolize their own capsular polysaccharide using the genes involved in its biosynthesis to maintain viability when other carbon sources are unavailable. Our findings describe a new role of the pneumococcal capsule: the prolongation of viability under nutrient-limiting conditions as would be encountered during periods when the organism is between hosts.

Single Cell Bottlenecks in the Pathogenesis of Streptococcus pneumoniae.

Herein, we studied a virulent isolate of the leading bacterial pathogen Streptococcus pneumoniae in an infant mouse model of colonization, disease and transmission, both with and without influenza A (IAV) co-infection. To identify vulnerable points in the multiple steps involved in pneumococcal pathogenesis, this model was utilized for a comprehensive analysis of population bottlenecks. Our findings reveal that in the setting of IAV co-infection the organism must pass through single cell bottlenecks during bloodstream invasion from the nasopharynx within the host and in transmission between hosts. Passage through these bottlenecks was not associated with genetic adaptation by the pathogen. The bottleneck in transmission occurred between bacterial exit from one host and establishment in another explaining why the number of shed organisms in secretions is critical to overcoming it. These observations demonstrate how viral infection, and TLR-dependent innate immune responses it stimulates and that are required to control it, drive bacterial contagion.

Transcriptional occlusion caused by overlapping promoters.

RpoS (σ(38)) is required for cell survival under stress conditions, but it can inhibit growth if produced inappropriately and, consequently, its production and activity are elaborately regulated. Crl, a transcriptional activator that does not bind DNA, enhances RpoS activity by stimulating the interaction between RpoS and the core polymerase. The crl gene has two overlapping promoters, a housekeeping, RpoD- (σ(70)) dependent promoter, and an RpoN (σ(54)) promoter that is strongly up-regulated under nitrogen limitation. However, transcription from the RpoN promoter prevents transcription from the RpoD promoter, and the RpoN-dependent transcript lacks a ribosome-binding site. Thus, activation of the RpoN promoter produces a long noncoding RNA that silences crl gene expression simply by being made. This elegant and economical mechanism, which allows a near-instantaneous reduction in Crl synthesis without the need for transacting regulatory factors, restrains the activity of RpoS to allow faster growth under nitrogen-limiting conditions.

Infant Mouse Model for the Study of Shedding and Transmission during Streptococcus pneumoniae Monoinfection.

One of the least understood aspects of the bacterium Streptococcus pneumoniae (pneumococcus) is its transmission from host to host, the critical first step in both the carrier state and the disease state. To date, transmission models have depended on influenza A virus coinfection, which greatly enhances pneumococcal shedding to levels that allow acquisition by a new host. Here, we describe an infant mouse model that can be utilized to study pneumococcal colonization, shedding, and transmission during bacterial monoinfection. Using this model, we demonstrated that the level of bacterial shedding is highest in pups infected intranasally at age 4 days and peaks over the first 4 days postchallenge. Shedding results differed among isolates of five different pneumococcal types. Colonization density was found to be a major factor in the level of pneumococcal shedding and required expression of capsule. Transmission within a litter occurred when there was a high ratio of colonized "index" pups to uncolonized "contact" pups. Transmission was observed for each of the well-colonizing pneumococcal isolates, with the rate of transmission proportional to the level of shedding. This model can be used to examine bacterial and host factors that contribute to pneumococcal transmission without the effects of viral coinfection.

MgrB-Dependent Colistin Resistance in Klebsiella pneumoniae Is Associated with an Increase in Host-to-Host Transmission.

Due to its high transmissibility, Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Here, we studied the biological cost of colistin resistance, an antibiotic of last resort, in this opportunistic pathogen using a murine model of gut colonization and transmission. Colistin resistance in K. pneumoniae is commonly the result of the inactivation of the small regulatory protein MgrB. Without a functional MgrB, the two-component system PhoPQ is constitutively active, leading to an increase in lipid A modifications and subsequent colistin resistance. Using an isogenic mgrB deletion mutant (MgrB-), we demonstrate that the mutant's colistin resistance is not associated with a fitness defect under in vitro growth conditions. However, in our murine model of K. pneumoniae gastrointestinal (GI) colonization, the MgrB- colonizes the gut poorly, allowing us to identify a fitness cost. Moreover, the MgrB- mutant has higher survival outside the host compared with the parental strain. We attribute this enhanced survivability to dysregulation of the PhoPQ two-component system and accumulation of the master stress regulator RpoS. The enhanced survival of MgrB- may be critical for its rapid host-to-host transmission observed in our model. Together, our data using multiple clinical isolates demonstrate that MgrB-dependent colistin resistance in K. pneumoniae comes with a biological cost in gut colonization. However, this cost is mitigated by enhanced survival outside the host and consequently increases its host-to-host transmission. Additionally, it underscores the importance of considering the entire life cycle of a pathogen to determine the actual biological cost associated with antibiotic resistance. IMPORTANCE The biological cost associated with colistin resistance in Klebsiella pneumoniae was examined using a murine model of K. pneumoniae gut colonization and fecal-oral transmission. A common mutation resulting in colistin resistance in K. pneumoniae is a loss-of-function mutation of the small regulatory protein MgrB that regulates the two-component system PhoPQ. Even though colistin resistance in K. pneumoniae comes with a fitness defect in gut colonization, it increases bacterial survival outside the host enabling it to transmit more effectively to a new host. The enhanced survival is dependent upon the accumulation of RpoS and dysregulation of the PhoPQ. Hence, our study expands our understanding of the underlying molecular mechanism contributing to the transmission of colistin-resistant K. pneumoniae.

Pneumococcal capsule blocks protection by immunization with conserved surface proteins.

Vaccines targeting Streptococcus pneumoniae (Spn) are limited by dependence on capsular polysaccharide and its serotype diversity. More broadly-based approaches using common protein antigens have not resulted in a licensed vaccine. Herein, we used an unbiased, genome-wide approach to find novel vaccine antigens to disrupt carriage modeled in mice. A Tn-Seq screen identified 198 genes required for colonization of which 16 are known to express conserved, immunogenic surface proteins. After testing defined mutants for impaired colonization of infant and adult mice, 5 validated candidates (StkP, PenA/Pbp2a, PgdA, HtrA, and LytD/Pce/CbpE) were used as immunogens. Despite induction of antibody recognizing the Spn cell surface, there was no protection against Spn colonization. There was, however, protection against an unencapsulated Spn mutant. This result correlated with increased antibody binding to the bacterial surface in the absence of capsule. Our findings demonstrate how the pneumococcal capsule interferes with mucosal protection by antibody to common protein targets.

Identification of Pneumococcal Factors Affecting Pneumococcal Shedding Shows that the dlt Locus Promotes Inflammation and Transmission.

Host-to-host transmission is a necessary but poorly understood aspect of microbial pathogenesis. Herein, we screened a genomic library of mutants of the leading respiratory pathogen Streptococcus pneumoniae generated by mariner transposon mutagenesis (Tn-Seq) to identify genes contributing to its exit or shedding from the upper respiratory tract (URT), the limiting step in the organism's transmission in an infant mouse model. Our analysis focused on genes affecting the bacterial surface that directly impact interactions with the host. Among the multiple factors identified was the dlt locus, which adds d-alanine onto lipoteichoic acids (LTA) and thereby increases Toll-like receptor 2-mediated inflammation and resistance to antimicrobial peptides. The more robust proinflammatory response in the presence of d-alanylation promotes secretions that facilitate pneumococcal shedding and allows for transmission. Expression of the dlt locus is controlled by the CiaRH system, which senses cell wall stress in response to antimicrobial activity, including in response to lysozyme, the most abundant antimicrobial along the URT mucosa. Accordingly, in a lysM-/- host, there was no longer an effect of the dlt locus on pneumococcal shedding. Thus, our findings demonstrate how a pathogen senses the URT milieu and then modifies its surface characteristics to take advantage of the host response for transit to another host.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is a common cause of respiratory tract and invasive infection. The overall effectiveness of immunization with the organism's capsular polysaccharide depends on its ability to block colonization of the upper respiratory tract and thereby prevent host-to-host transmission. Because of the limited coverage of current pneumococcal vaccines, we carried out an unbiased in vivo transposon mutagenesis screen to identify pneumococcal factors other than its capsular polysaccharide that affect transmission. One such candidate was expressed by the dlt locus, previously shown to add d-alanine onto the pneumococcal lipoteichoic acid present on the bacterial cell surface. This modification protects against host antimicrobials and augments host inflammatory responses. The latter increases secretions and bacterial shedding from the upper respiratory tract to allow for transmission. Thus, this study provides insight into a mechanism employed by the pneumococcus to successfully transit from one host to another.

An Infant Mouse Model of Influenza Virus Transmission Demonstrates the Role of Virus-Specific Shedding, Humoral Immunity, and Sialidase Expression by Colonizing Streptococcus pneumoniae.

The pandemic potential of influenza A viruses (IAV) depends on the infectivity of the host, transmissibility of the virus, and susceptibility of the recipient. While virus traits supporting IAV transmission have been studied in detail using ferret and guinea pig models, there is limited understanding of host traits determining transmissibility and susceptibility because current animal models of transmission are not sufficiently tractable. Although mice remain the primary model to study IAV immunity and pathogenesis, the efficiency of IAV transmission in adult mice has been inconsistent. Here we describe an infant mouse model that supports efficient transmission of IAV. We demonstrate that transmission in this model requires young age, close contact, shedding of virus particles from the upper respiratory tract (URT) of infected pups, the use of a transmissible virus strain, and a susceptible recipient. We characterize shedding as a marker of infectiousness that predicts the efficiency of transmission among different influenza virus strains. We also demonstrate that transmissibility and susceptibility to IAV can be inhibited by humoral immunity via maternal-infant transfer of IAV-specific immunoglobulins and modifications to the URT milieu, via sialidase activity of colonizing Streptococcus pneumoniae Due to its simplicity and efficiency, this model can be used to dissect the host's contribution to IAV transmission and explore new methods to limit contagion.IMPORTANCE This study provides insight into the role of the virus strain, age, immunity, and URT flora on IAV shedding and transmission efficiency. Using the infant mouse model, we found that (i) differences in viral shedding of various IAV strains are dependent on specific hemagglutinin (HA) and/or neuraminidase (NA) proteins, (ii) host age plays a key role in the efficiency of IAV transmission, (iii) levels of IAV-specific immunoglobulins are necessary to limit infectiousness, transmission, and susceptibility to IAV, and (iv) expression of sialidases by colonizing S. pneumoniae antagonizes transmission by limiting the acquisition of IAV in recipient hosts. Our findings highlight the need for strategies that limit IAV shedding and the importance of understanding the function of the URT bacterial composition in IAV transmission. This work reinforces the significance of a tractable animal model to study both viral and host traits affecting IAV contagion and its potential for optimizing vaccines and therapeutics that target disease spread.

Capsule Type and Amount Affect Shedding and Transmission of Streptococcus pneumoniae.

The capsular polysaccharide (CPS) of Streptococcus pneumoniae is characterized by its diversity, as it has over 95 known serotypes, and the variation in its thickness as it surrounds an organism. While within-host effects of CPS have been studied in detail, there is no information about its contribution to host-to-host transmission. In this study, we used an infant mouse model of intralitter transmission, together with isogenic capsule switch and cps promoter switch constructs, to explore the effects of CPS type and amount. The determining factor in the transmission rate in this model is the number of pneumococci shed in nasal secretions by colonized hosts. Two of seven capsule switch constructs showed reduced shedding. These constructs were unimpaired in colonization and expressed capsules similar in size to those of the wild-type strain. A cps promoter switch mutant expressing ~50% of wild-type amounts of CPS also displayed reduced shedding without a defect in colonization. Since shedding from the mucosal surface may require escape from mucus entrapment, a mucin-binding assay was used to compare capsule switch and cps promoter switch mutants. The CPS type or amount constructs that shed poorly were bound more robustly by immobilized mucin. These capsule switch and cps promoter switch constructs with increased mucin-binding affinity and reduced shedding also had lower rates of pup-to-pup transmission. Our results demonstrate that CPS type and amount affect transmission dynamics and may contribute to the marked differences in prevalence among pneumococcal types.IMPORTANCEStreptococcus pneumoniae, a leading cause of morbidity and mortality, is readily transmitted, especially among young children. Its structurally and antigenically diverse capsular polysaccharide is the target of currently licensed pneumococcal vaccines. Epidemiology studies show that only a subset of the >95 distinct serotypes are prevalent in the human population, suggesting that certain capsular polysaccharide types might be more likely to be transmitted within the community. Herein, we used an infant mouse model to show that both capsule type and amount are important determinants in the spread of pneumococci from host to host. Transmission rates correlate with those capsule types that are better at escaping mucus entrapment, a key step in exiting the host upper respiratory tract. Hence, our study provides a better mechanistic understanding of why certain pneumococcal serotypes are more common in the human population.

Host-to-Host Transmission of Streptococcus pneumoniae Is Driven by Its Inflammatory Toxin, Pneumolysin.

Host-to-host transmission is a critical step for infection. Here we studied transmission of the opportunistic pathogen Streptococcus pneumoniae in an infant mouse model. Transmission from nasally colonized pups required high levels of bacterial shedding in nasal secretions and was temporally correlated with, and dependent upon, the acute inflammatory response. Pneumolysin, a pore-forming cytotoxin and major virulence determinant, was both necessary and sufficient to promote inflammation, which increased shedding and allowed for intralitter transmission. Direct contact between pups was not required for transmission indicating the importance of an environmental reservoir. An additional in vivo effect of pneumolysin was to enhance bacterial survival outside of the host. Our findings provide experimental evidence of a microbial strategy for transit to new hosts and explain why an organism expresses a toxin that damages the host upon which it depends.

Genetic evidence for a novel interaction between transcriptional activator SoxS and region 4 of the σ(70) subunit of RNA polymerase at class II SoxS-dependent promoters in Escherichia coli.

Escherichia coli SoxS activates transcription of the genes of the soxRS regulon, which provide the cell's defense against oxidative stress. In response to this stress, SoxS is synthesized de novo. Because the DNA binding site of SoxS is highly degenerate, SoxS efficiently activates transcription by the mechanism of prerecruitment. In prerecruitment, newly synthesized SoxS first forms binary complexes with RNA polymerase. These complexes then scan the chromosome for class I and II SoxS-dependent promoters, using the specific DNA-recognition properties of SoxS and σ(70) to distinguish SoxS-dependent promoters from the vast excess of sequence-equivalent soxboxes that do not reside in promoters. Previously, we determined that SoxS interacts with RNA polymerase in two ways: by making protein-protein interactions with the DNA-binding determinant of the α subunit and by interacting with σ(70) region 4 (σ(70) R4) both "on-DNA" and "off-DNA." Here, we address the question of how SoxS and σ(70) R4 coexist at class II promoters, where the binding site for SoxS either partially or completely overlaps the -35 region of the promoter, which is usually bound by σ(70) R4. To do so, we created a tri-alanine scanning library that covers all of σ(70) R4. We determined that interactions between σ(70) R4 and the DNA in the promoter's -35 region are required for activation of class I promoters, where the binding site lies upstream of the -35 hexamer, but they are not required at class II promoters. In contrast, specific three-amino-acid stretches are required for activation of class I (lac) and class II (galP1) cyclic AMP receptor protein-dependent promoters. We conclude from these data that SoxS and σ(70) R4 interact with each other in a novel way at class II SoxS-dependent promoters such that the two proteins do not accommodate one another in the -35 region but instead SoxS binding there occludes the binding of σ(70) R4.

Protein-protein interactions between sigma(70) region 4 of RNA polymerase and Escherichia coli SoxS, a transcription activator that functions by the prerecruitment mechanism: evidence for "off-DNA" and "on-DNA" interactions.

According to the prerecruitment hypothesis, Escherichia coli SoxS activates the transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase (RNAP) that scan the chromosome for class I and class II SoxS-dependent promoters. We showed previously that the alpha subunit's C-terminal domain plays a role in activating both classes of promoter by making protein-protein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the prerecruitment hypothesis. Here, we identified seven single-alanine substitutions of the region 4 of sigma(70) (sigma(70) R4) of RNAP that reduce SoxS activation of class II promoters. With genetic epistasis tests between these sigma(70) R4 mutants and positive control mutants of SoxS, we identified 10 pairs of amino acids that interact with each other in E. coli. Using the yeast two-hybrid system and affinity immobilization assays, we showed that SoxS and sigma(70) R4 can interact in solution (i.e., "off-DNA"). The interaction requires amino acids of the class I/II (but not the class II) positive control surface of SoxS, and five amino acids of sigma(70) R4 that reduce activation in E. coli also reduce the SoxS-sigma(70) R4 interaction in yeast. One of the epistatic interactions that occur in E. coli also occurs in the yeast two-hybrid system (i.e., off-DNA). Importantly, we infer that the five epistatic interactions occurring in E. coli that require an amino acid of the class II surface occur "on-DNA" at class II promoters. Finding that SoxS contacts sigma(70) R4 both off-DNA and on-DNA is consistent with the prerecruitment hypothesis. Moreover, SoxS is now the first example of an E. coli transcriptional activator that uses a single positive control surface to make specific protein-protein contacts with two different subunits of RNAP.