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Fimbriae are essential virulence factors for many bacterial pathogens. Fimbriae are extracellular structures that attach bacteria to surfaces. Thus, fimbriae mediate a critical step required for any pathogen to establish infection by anchoring a bacterium to host tissue. The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7encodes 16 fimbriae that may be important for EHEC to initiate infection and allow for productive expression of virulence traits important in later stages of infection, including a type III secretion system (T3SS) and Shiga toxin; however, the roles of most EHEC fimbriae are largely uncharacterized. Here, we provide evidence that two EHEC fimbriae, Yad and Yeh, modulate expression of diverse genes including genes encoding T3SS and Shiga toxin and that these fimbriae are required for robust colonization of the gastrointestinal tract. These findings reveal a significant and previously unappreciated role for fimbriae in bacterial pathogenesis as important determinants of virulence gene expression.IMPORTANCEFimbriae are extracellular proteinaceous structures whose defining role is to anchor bacteria to surfaces. This is a fundamental step for bacterial pathogens to establish infection in a host. Here, we show that the contributions of fimbriae to pathogenesis are more complex. Specifically, we demonstrate that fimbriae influence expression of virulence traits essential for disease progression in the intestinal pathogen enterohemorrhagic Escherichia coli . Gram-positive and Gram-negative bacteria express multiple fimbriae; therefore, these findings may have broad implications for understanding how pathogens use fimbriae, beyond adhesion, to initiate infection and coordinate gene expression, which ultimately results in disease., Competing Interests: The authors declare no conflict of interest.

Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments., (© 2022. The Author(s).)

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 relies on sRNAs to coordinate expression of metabolic and virulence factors to colonize the host. Here, we focus on the sRNA, named MavR (metabolism and virulence regulator), that is conserved among pathogenic Enterobacteriaceae. MavR is constitutively expressed under in vitro conditions that promote EHEC virulence gene expression. Using MS2-affinity purification coupled with RNA sequencing, the eutR transcript was identified as a putative target of MavR. EutR is a transcription factor that promotes expression of genes required for ethanolamine metabolism as well as virulence factors important for host colonization. MavR binds to the eutR coding sequence to protect the eutR transcript from RNase E-mediated degradation. Ultimately, MavR promotes EutR expression and in turn ethanolamine utilization and ethanolamine-dependent growth. RNAseq analyses revealed that MavR also affected expression of genes important for other metabolic pathways, motility, oxidative stress and attaching and effacing lesion formation, which contribute to EHEC colonization of the gastrointestinal tract. In support of the idea that MavR-dependent gene expression affects fitness during infection, deletion of mavR resulted in significant (∼10- to 100-fold) attenuation in colonization of the mammalian intestine. Altogether, these studies reveal an important, extensive, and robust phenotype for a bacterial sRNA in host-pathogen interactions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To establish infection, enteric pathogens integrate environmental cues to navigate the gastrointestinal tract and precisely control expression of virulence determinants. Emerging data indicate that post-transcriptional and post-translational gene regulation plays a key role in virulence regulation and pathogen adaptation to the host environment. Here, we highlight recent studies that reveal how physiologically relevant signals initiate post-transcriptional and post-translational regulatory circuits and the impact on virulence gene expression in the attaching and effacing pathogens, enteropathogenic Escherichia coli, enterohemorrhagic E. coli O157:H7, and Citrobacter rodentium., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Resident gut microbes can help to block infection, but the mechanisms involved are not fully understood. It has now been found that changes in the microbial community after infection boost the level of a molecule that combats harmful bacteria.

Regulated macrophage death has emerged as an important mechanism to defend against intracellular pathogens. However, the importance and consequences of macrophage death during bacterial infection are poorly resolved. This is especially true for the recently described RIPK3-dependent lytic cell death, termed necroptosis. Salmonella enterica serovar Typhimurium is an intracellular pathogen that precisely regulates virulence expression within macrophages to evade and manipulate immune responses, which is a key factor in its ability to cause severe systemic infections. We combined genetic and pharmacological approaches to examine the importance of RIPK3 for S. Typhimurium-induced macrophage death using conditions that recapitulate bacterial gene expression during systemic infection in vivo Our findings indicate that noninvasive S. Typhimurium does not naturally induce macrophage necroptosis but does so in the presence of pan-caspase inhibition. Moreover, our data suggest that RIPK3 induction (following caspase inhibition) does not impact host survival following S. Typhimurium infection, which differs from previous findings based on inert lipopolysaccharide (LPS) injections. Finally, although necroptosis is typically characterized as highly inflammatory, our data suggest that RIPK3 skews the peritoneal myeloid population away from an inflammatory profile to that of a classically noninflammatory profile. Collectively, these data improve our understanding of S. Typhimurium-macrophage interactions, highlight the possibility that purified bacterial components may not accurately recapitulate the complexity of host-pathogen interactions, and reveal a potential and unexpected role for RIPK3 in resolving inflammation. IMPORTANCE Macrophages employ multiple strategies to limit pathogen infection. For example, macrophages may undergo regulated cell death, including RIPK3-dependent necroptosis, as a means of combatting intracellular bacterial pathogens. However, bacteria have evolved mechanisms to evade or exploit immune responses. Salmonella is an intracellular pathogen that avoids and manipulates immune detection within macrophages. We examined the contribution of RIPK3 to Salmonella -induced macrophage death. Our findings indicate that noninvasive Salmonella does not naturally induce necroptosis, but it does so when caspases are inhibited. Moreover, RIPK3 induction (following caspase inhibition) does not impact host survival following Salmonella systemic infection. Finally, our data show that RIPK3 induction results in recruitment of low-inflammatory myeloid cells, which was unexpected, as necroptosis is typically described as highly inflammatory. Collectively, these data improve our understanding of pathogen-macrophage interactions, including outcomes of regulated cell death during infection in vivo , and reveal a potential new role for RIPK3 in resolving inflammation., (Copyright © 2020 Satkovich et al.)

Enteric pathogens exploit chemical and nutrient signaling to gauge their location within a host and control expression of traits important for infection. Ethanolamine-containing molecules are essential in host physiology and play important roles in intestinal processes. The transcription factor EutR is conserved in the Enterobacteriaceae and is required for ethanolamine sensing and metabolism. In enterohemorrhagic Escherichia coli (EHEC) O157:H7, EutR responds to ethanolamine to activate expression of traits required for host colonization and disease; however, the importance of EutR to EHEC intestinal infection has not been examined. Because EHEC does not naturally colonize or cause disease in mice, we employed the natural murine pathogen Citrobacter rodentium as a model of EHEC virulence to investigate the importance of EutR in vivo EHEC and C. rodentium possess the locus of enterocyte effacement (LEE), which is the canonical virulence trait of attaching and effacing pathogens. Our findings demonstrate that ethanolamine sensing and EutR-dependent regulation of the LEE are conserved in C. rodentium Moreover, during infection, EutR is required for maximal LEE expression, colonization, and transmission efficiency. These findings reveal that EutR not only is important for persistence during the primary host infection cycle but also is required for maintenance in a host population., (Copyright © 2020 American Society for Microbiology.)

The Gram-negative pathogen Neisseria gonorrhoeae (gonococcus [Gc]) colonizes lysozyme-rich mucosal surfaces. Lysozyme hydrolyzes peptidoglycan, leading to bacterial lysis. Gc expresses two proteins, SliC and NgACP, that bind and inhibit the enzymatic activity of lysozyme. SliC is a surface-exposed lipoprotein, while NgACP is found in the periplasm and also released extracellularly. Purified SliC and NgACP similarly inhibit lysozyme. However, whereas mutation of ngACP increases Gc susceptibility to lysozyme, the sliC mutant is only susceptible to lysozyme when ngACP is inactivated. In this work, we examined how lipidation contributes to SliC expression, cellular localization, and resistance of Gc to killing by lysozyme. To do so, we mutated the conserved cysteine residue (C18) in the N-terminal lipobox motif of SliC, the site for lipid anchor attachment, to alanine. SliC(C18A) localized to soluble rather than membrane fractions in Gc and was not displayed on the bacterial surface. Less SliC(C18A) was detected in Gc lysates compared to the wild-type protein. This was due in part to some release of the C18A mutant, but not wild-type, protein into the extracellular space. Surprisingly, Gc expressing SliC(C18A) survived better than SliC (wild type)-expressing Gc after exposure to lysozyme. We conclude that lipidation is not required for the ability of SliC to inhibit lysozyme, even though the lipidated cysteine is 100% conserved in Gc SliC alleles. These findings shed light on how members of the growing family of lysozyme inhibitors with distinct subcellular localizations contribute to bacterial defense against lysozyme. IMPORTANCE Neisseria gonorrhoeae is one of many bacterial species that express multiple lysozyme inhibitors. It is unclear how inhibitors that differ in their subcellular localization contribute to defense from lysozyme. We investigated how lipidation of SliC, an MliC (membrane-bound lysozyme inhibitor of c-type lysozyme)-type inhibitor, contributes to its localization and lysozyme inhibitory activity. We found that lipidation was required for surface exposure of SliC and yet was dispensable for protecting the gonococcus from killing by lysozyme. To our knowledge, this is the first time the role of lipid anchoring of a lysozyme inhibitor has been investigated. These results help us understand how different lysozyme inhibitors are localized in bacteria and how this impacts resistance to lysozyme., (Copyright © 2020 American Society for Microbiology.)

To establish infection, enteric pathogens integrate environmental cues to navigate the gastrointestinal tract (GIT) and precisely control expression of virulence determinants. During passage through the GIT, pathogens encounter relatively high levels of oxygen in the small intestine before transit to the oxygen-limited environment of the colon. However, how bacterial pathogens sense oxygen availability and coordinate expression of virulence traits is not resolved. Here, we demonstrate that enterohemorrhagic Escherichia coli O157:H7 (EHEC) regulates virulence via the oxygen-responsive small RNA DicF. Under oxygen-limited conditions, DicF enhances global expression of the EHEC type three secretion system, which is a key virulence factor required for host colonization, through the transcriptional activator PchA. Mechanistically, the pchA coding sequence (CDS) base pairs with the 5' untranslated region of the mRNA to sequester the ribosome binding site (RBS) and inhibit translation. DicF disrupts pchA cis -interactions by binding to the pchA CDS, thereby unmasking the pchA RBS and promoting PchA expression. These findings uncover a feed-forward regulatory pathway that involves distinctive mechanisms of RNA-based regulation and that provides spatiotemporal control of EHEC virulence., Competing Interests: The authors declare no conflict of interest.

Competing Interests: The authors have declared that no competing interests exist.

A core principle of bacterial pathogenesis is that pathogens preferentially utilize metabolites that commensal bacteria do not in order to sidestep nutritional competition. The metabolite ethanolamine (EA) is well recognized to play a central role in host adaptation for diverse pathogens. EA promotes growth and influences virulence during host infection. Although genes encoding EA utilization have been identified in diverse bacteria (nonpathogenic and pathogenic), a prevailing idea is that commensal bacteria do not utilize EA to enhance growth, and thus, EA is a noncompetitive metabolite for pathogens. Here, we show that EA augments growth of two human commensal strains of Escherichia coli Significantly, these commensal strains grow more rapidly than, and even outcompete, the pathogen enterohemorrhagic E. coli O157:H7 specifically when EA is provided as the sole nitrogen source. Moreover, EA-dependent signaling is similarly conserved in the human commensal E. coli strain HS and influences expression of adhesins. These findings suggest a more extensive role for EA utilization in bacterial physiology and host-microbiota-pathogen interactions than previously appreciated. IMPORTANCE The microbiota protects the host from invading pathogens by limiting access to nutrients. In turn, bacterial pathogens selectively exploit metabolites not readily used by the microbiota to establish infection. Ethanolamine has been linked to pathogenesis of diverse pathogens by serving as a noncompetitive metabolite that enhances pathogen growth as well as a signal that modulates virulence. Although ethanolamine is abundant in the gastrointestinal tract, the prevailing idea is that commensal bacteria do not utilize EA, and thus, EA utilization has been particularly associated with pathogenesis. Here, we provide evidence that two human commensal Escherichia coli isolates readily utilize ethanolamine to enhance growth, modulate gene expression, and outgrow the pathogen enterohemorrhagic E. coli These data indicate a more complex role for ethanolamine in host-microbiota-pathogen interactions., (Copyright © 2018 Rowley et al.)

To adapt to ever-changing environments, pathogens quickly alter gene expression. This can occur through transcriptional, posttranscriptional, or posttranslational regulation. Historically, transcriptional regulation has been thoroughly studied to understand pathogen niche adaptation, whereas posttranscriptional and posttranslational gene regulation has only relatively recently been appreciated to play a central role in bacterial pathogenesis. Posttranscriptional regulation may involve chaperones, nucleases, and/or noncoding small RNAs (sRNAs) and typically controls gene expression by altering the stability and/or translation of the target mRNA. In this review, we highlight the global importance of posttranscriptional regulation to enterohemorrhagic Escherichia coli (EHEC) gene expression and discuss specific mechanisms of how EHEC regulates expression of virulence factors critical to host colonization and disease progression. The low infectious dose of this intestinal pathogen suggests that EHEC is particularly well adapted to respond to the host environment., (Copyright © 2018 American Society for Microbiology.)

Ethanolamine is a ubiquitous and essential molecule within a host. Significantly, bacterial pathogens exploit ethanolamine during infection to promote growth and regulate virulence. The ethanolamine permease EutH is dispensable for growth in vitro under standard conditions, whereas EutH is required for ethanolamine utilization at low pH. These findings suggested a model in which EutH facilitates diffusion of ethanolamine into the bacterial cell in acidic environments. To date, the ecological significance of this model has not been thoroughly investigated, and the importance of EutH to bacterial growth under physiologically relevant conditions is not known. During infection, immune cells internalize invading bacteria within an acidic, nutrient-depleted vacuole called the phagosome. Here, we investigated the hypothesis that EutH promotes bacterial survival following phagocytosis. Our findings indicate that EutH is important for survival and replication of the facultative intracellular pathogens Salmonella enterica serovar Typhimurium and Listeria monocytogenes during prolonged or transient exposure to the phagosome, respectively. Furthermore, in agreement with EutH being important in the acidic environment, neutralization of the vacuole abolished the requirement for EutH. Significantly, consistent with a role for EutH in promoting intramacrophage survival, EutH was not required during S Typhimurium local intestinal infection but specifically conferred an advantage upon dissemination to peripheral organs. These findings reveal a physiologically relevant and conserved role for EutH in spatiotemporal niche adaptation during infection., (Copyright © 2018 American Society for Microbiology.)

Bacterial pathogens must sense and respond to newly encountered host environments to regulate the expression of critical virulence factors that allow for niche adaptation and successful colonization. Among bacterial pathogens, non-typhoidal serovars of Salmonella enterica , such as serovar Typhimurium ( S. Tm), are a primary cause of foodborne illnesses that lead to hospitalizations and deaths worldwide. S . Tm causes acute inflammatory diarrhea that can progress to invasive systemic disease in susceptible patients. The gastrointestinal tract and intramacrophage environments are two critically important niches during S . Tm infection, and each presents unique challenges to limit S . Tm growth. The intestinal tract is home to billions of commensal microbes, termed the microbiota, which limits the amount of available nutrients for invading pathogens such as S. Tm. Therefore, S. Tm encodes strategies to manipulate the commensal population and side-step this nutritional competition. During subsequent stages of disease, S. Tm resists host immune cell mechanisms of killing. Host cells use antimicrobial peptides, acidification of vacuoles, and nutrient limitation to kill phagocytosed microbes, and yet S. Tm is able to subvert these defense systems. In this review, we discuss recently described molecular mechanisms that S. Tm uses to outcompete the resident microbiota within the gastrointestinal tract. S. Tm directly eliminates close competitors via bacterial cell-to-cell contact as well as by stimulating a host immune response to eliminate specific members of the microbiota. Additionally, S. Tm tightly regulates the expression of key virulence factors that enable S. Tm to withstand host immune defenses within macrophages. Additionally, we highlight the chemical and physical signals that S . Tm senses as cues to adapt to each of these environments. These strategies ultimately allow S. Tm to successfully adapt to these two disparate host environments. It is critical to better understand bacterial adaptation strategies because disruption of these pathways and mechanisms, especially those shared by multiple pathogens, may provide novel therapeutic intervention strategies.

The AraC Negative Regulators (ANR) comprise a large family of virulence regulators distributed among diverse clinically important Gram-negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., and pathogenic E. coli strains. We have previously reported broad effects of the ANR members on regulators of the AraC/XylS family. Here, we interrogate possible broader effects of the ANR members on the bacterial transcriptome. Our studies focused on Aar (AggR-activated regulator), an ANR family archetype in enteroaggregative E. coli (EAEC) isolate 042. Transcriptome analysis of EAEC strain 042, 042aar and 042aar(pAar) identified more than 200 genes that were differentially expressed (+/- 1.5 fold, p<0.05). Most of those genes are located on the bacterial chromosome (195 genes, 92.85%), and are associated with regulation, transport, metabolism, and pathogenesis, based on the predicted annotation; a considerable number of Aar-regulated genes encoded for hypothetical proteins (46 genes, 21.9%) and regulatory proteins (25, 11.9%). Notably, the transcriptional expression of three histone-like regulators, H-NS (orf1292), H-NS homolog (orf2834) and StpA, was down-regulated in the absence of aar and may explain some of the effects of Aar on gene expression. By employing a bacterial two-hybrid system, LacZ reporter assays, pull-down and electrophoretic mobility shift assay (EMSA) analysis, we demonstrated that Aar binds directly to H-NS and modulates H-NS-induced gene silencing. Importantly, Aar was highly expressed in the mouse intestinal tract and was found to be necessary for maximal H-NS expression. In conclusion, this work further extends our knowledge of genes under the control of Aar and its biological relevance in vivo.

The type three secretion system (T3SS) is critical for the virulence of diverse bacterial pathogens. Pathogens use the T3SS to deliver effector proteins into host cells and manipulate host signaling pathways. The prevailing mechanism is that effectors translocate from inside the T3SS directly into the host cell. Recent studies reveal an alternative mechanism of effector translocation, in which an effector protein located outside the bacterial cell relies on the T3SS for delivery into host cells. Tejeda-Dominguez et al. (F. Tejeda-Dominguez, J. Huerta-Cantillo, L. Chavez-Dueñas, and F. Navarro-Garcia, mBio 8:e00184-17, 2017, https://doi.org/10.1128/mBio.00184-17) demonstrate that the EspC effector of enteropathogenic Escherichia coli is translocated by binding to the outside of the T3SS and subsequently gains access to the host cell cytoplasm through the T3SS pore embedded within the host cell membrane. This work reveals a novel mechanism of translocation that is likely relevant for a variety of other pathogens that use the T3SS as part of their virulence arsenal., (Copyright © 2017 Kendall.)

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome throughout the world. A defining feature of EHEC pathogenesis is the formation of attaching and effacing (AE) lesions on colonic epithelial cells. Most of the genes that code for AE lesion formation, including a type three secretion system (T3SS) and effectors, are carried within a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). In this study, we report that a putative regulator, which is encoded in the cryptic E. coli type three secretion system 2 (ETT2) locus and herein renamed EtrB, plays an important role in EHEC pathogenesis. The etrB gene is expressed as a monocistronic transcript, and EtrB autoregulates expression. We provide evidence that EtrB directly interacts with the ler regulatory region to activate LEE expression and promote AE lesion formation. Additionally, we mapped the EtrB regulatory circuit in EHEC to determine a global role for EtrB. EtrB is regulated by the transcription factor QseA, suggesting that these proteins comprise a regulatory circuit important for EHEC colonization of the gastrointestinal tract., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners., (© 2016 The Authors Molecular Microbiology Published by John Wiley & Sons Ltd.)

The gut microbiota plays essential roles in human health and disease. In this review, we focus on the role of the intestinal microbiota in promoting resistance to infection by bacterial pathogens as well as how pathogens overcome this barrier. We discuss how the resident microbiota restricts growth and colonization of invading pathogens by limiting availability of nutrients and through generation of a hostile environment. Additionally, we examine how microbiota-derived signaling molecules interfere with bacterial virulence. In turn, we discuss how pathogens exploit non-competitive metabolites to replicate in vivo as well as to precisely control virulence and cause disease. This bacterial two step of creating and overcoming challenges important in preventing and establishing infection highlights the complexities of elucidating interactions between the commensal bacteria and pathogens. Better understanding of microbiota-pathogen interplay will have significant implications for developing novel therapeutics to treat infectious diseases., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chemical signaling between cells is an effective way to coordinate behavior within a community. Although cell-to-cell signaling has mostly been studied in single species, it is now appreciated that the sensing of chemical signals across kingdoms can be an important regulator of nutrient acquisition, virulence, and host defense. In this review, we focus on the role of interkingdom signaling in the interactions that occur between bacterial pathogens and their mammalian hosts. We discuss the quorum-sensing (QS) systems and other mechanisms used by these bacteria to sense, respond to, and modulate host signals that include hormones, immune factors, and nutrients. We also describe cross talk between these signaling pathways and strategies used by the host to interfere with bacterial signaling, highlighting the complex bidirectional signaling networks that are established across kingdoms., (Copyright © 2016 Kendall and Sperandio.)

Growth of a microorganism in a host is essential for infection, and bacterial pathogens have evolved to utilize specific metabolites to enhance replication in vivo. Now, emerging data demonstrate that pathogens rely on microbiota-derived metabolites as a form of bacterial-bacterial communication to gain information about location within a host and modify virulence gene expression accordingly. Thus, metabolite-sensing is critical for pathogens to establish infection. Here, we highlight recent examples of how the foodborne pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) exploits microbiota-derived metabolites to recognize the host intestinal environment and control gene expression that results in controlled expression of virulence traits., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chemical and nutrient signaling mediate all cellular processes, ensuring survival in response to changing environmental conditions. Ethanolamine is a component of phosphatidylethanolamine, a major phospholipid of mammalian and bacterial cell membranes. Ethanolamine is abundant in the gastrointestinal (GI) tract from dietary sources as well as from the normal turnover of intestinal epithelial and bacterial cells in the gut. Additionally, mammalian cells maintain intracellular ethanolamine concentrations through low and high-affinity uptake systems and the internal recycling of phosphatidylethanolamine; therefore, ethanolamine is ubiquitous throughout the mammalian host. Although ethanolamine has profound signaling activity within mammalian cells by modulating inflammatory responses and intestinal physiology, ethanolamine is best appreciated as a nutrient for bacteria that supports growth. In our recent work (Anderson , et al. PLoS Pathog (2015), 11: e1005278), we demonstrated that Salmonella enterica serovar Typhimurium ( Salmonella ) exploits ethanolamine signaling to adapt to distinct host environments to precisely coordinate expression of genes encoding metabolism and virulence, which ultimately enhances disease progression., Competing Interests: Conflict of interest: The authors declare that no competing interest exists.

Escherichia coli is one of the most-studied species of bacteria due to its frequent incidence in diverse environments and hosts, as well as its use as a tool in molecular biology. Most E. coli strains are commensal, in that they colonize the host without causing disease; however, some strains of E. coli are pathogens and are able to cause diverse illnesses, including urinary tract infections, sepsis/meningitis, as well as intestinal disease that result in diarrhea (Kaper et al. 2004). Six categories of diarrheagenic E. coli are recognized, and these are classified in part based on how they interact with epithelial cells (Kaper et al. 2004). Of these, enterohemorrhagic E. coli O157:H7 (EHEC) is one of the most important pathogenic E. coli strains. EHEC causes major outbreaks of bloody diarrhea that can result in the development of fatal hemorrhagic colitis and hemolytic uremic syndrome (Karmali et al. 1983). EHEC colonizes the colon, where it forms attaching and effacing (AE) lesions on the intestinal epithelial cell. AE lesions are characterized by intimate attachment of EHEC to epithelial cells, effacement of the microvilli and rearrangement of the underlying cytoskeleton, which results in formation of a pedestal-like structure beneath the bacterium (Jerse et al. 1990; Jarvis et al. 1995; Kenny et al. 1997). Most of the genes involved in the formation of AE lesions are encoded within a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE) (McDaniel et al. 1995). The LEE contains 41 genes that are organized in five major operons (LEE1, LEE2, LEE3, LEE5, and LEE4) (Elliott et al. 1998, 1999; Mellies et al. 1999). The LEE encodes a type three secretion system (T3SS) (Jarvis et al. 1995), an adhesin (intimin) (Jerse et al. 1990) and its receptor (Tir) (Kenny et al. 1997), as well as effector proteins (Kenny et al. 1996; Abe et al. 1997; McNamara and Donnenberg 1998; Elliott et al. 2001; Tu et al. 2003; Kanack et al. 2005). EHEC also encodes an arsenal of effector proteins located outside of the LEE that are important in EHEC virulence (Campellone et al. 2004; Deng et al. 2004; Garmendia et al. 2004, 2005; Gruenheid et al. 2004; Tobe et al. 2006).

Chemical and nutrient signaling are fundamental for all cellular processes, including interactions between the mammalian host and the microbiota, which have a significant impact on health and disease. Ethanolamine is an essential component of cell membranes and has profound signaling activity within mammalian cells by modulating inflammatory responses and intestinal physiology. Here, we describe a virulence-regulating pathway in which the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) exploits ethanolamine signaling to recognize and adapt to distinct niches within the host. The bacterial transcription factor EutR promotes ethanolamine metabolism in the intestine, which enables S. Typhimurium to establish infection. Subsequently, EutR directly activates expression of the Salmonella pathogenicity island 2 in the intramacrophage environment, and thus augments intramacrophage survival. Moreover, EutR is critical for robust dissemination during mammalian infection. Our findings reveal that S. Typhimurium co-opts ethanolamine as a signal to coordinate metabolism and then virulence. Because the ability to sense ethanolamine is a conserved trait among pathogenic and commensal bacteria, our work indicates that ethanolamine signaling may be a key step in the localized adaptation of bacteria within their mammalian hosts.

DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.

Bacteria must be able to respond rapidly to changes in the environment to survive. One means of coordinating gene expression relies on tightly regulated and complex signaling systems. One of the first signaling systems that was described in detail is quorum sensing (QS). During QS, a bacterial cell produces and secretes a signaling molecule called an autoinducer (AI). As the density of the bacterial population increases, so does the concentration of secreted AI molecules, thereby allowing a bacterial species to coordinate gene expression based on population density. Subsequent studies have demonstrated that bacteria are also able to detect signal molecules produced by other species of bacteria as well as hormones produced by their mammalian hosts. This type of signaling interaction has been termed cell-to-cell signaling because it does not rely on a threshold concentration of bacterial cells. This review discusses the three main types of cell-to-cell signaling mechanisms used by Escherichia coli and Salmonella: the LuxR process, in which E. coli and Salmonella detect signals produced by other species of bacteria; the LuxS/AI-2 system, in which E. coli and Salmonella participate in intra- and interspecies signaling; and the AI-3/epinephrine/norepinephrine system, in which E. coli and Salmonella recognize self-produced AI, signal produced by other microbes, and/or the human stress hormones epinephrine and/or norepinephrine.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen responsible for disease outbreaks worldwide. In order to colonize the human gastrointestinal (GI) tract and cause disease, EHEC must be able to sense the host environment and promote expression of virulence genes essential for adherence. Ethanolamine (EA) is an important metabolite for EHEC in the GI tract, and EA is also a signal that EHEC uses to activate virulence traits. Here, we report that EA influenced EHEC adherence to epithelial cells and fimbrial gene expression. Quantitative reverse transcriptase PCR indicated that EA promoted the transcription of the genes in characterized and putative fimbrial operons. Moreover, putative fimbrial structures were produced by EHEC cells grown with EA but not in medium lacking EA. Additionally, we defined two previously uncharacterized EA-regulated fimbrial operons, loc10 and loc11. We also tested whether choline or serine, both of which are also components of cell membranes, activated fimbrial gene expression. In addition to EA, choline activated fimbrial gene expression in EHEC. These findings describe for the first time the transcription of several putative fimbrial loci in EHEC. Importantly, the biologically relevant molecules EA and choline, which are abundant in the GI tract, promoted expression of these fimbriae.

Ethanolamine (EA) metabolism is a trait associated with enteric pathogens, including enterohemorrhagic Escherichia coli O157:H7 (EHEC). EHEC causes severe bloody diarrhea and hemolytic uremic syndrome. EHEC encodes the ethanolamine utilization (eut) operon that allows EHEC to metabolize EA and gain a competitive advantage when colonizing the gastrointestinal tract. The eut operon encodes the transcriptional regulator EutR. Genetic studies indicated that EutR expression is induced by EA and vitamin B12 and that EutR promotes expression of the eut operon; however, biochemical evidence for these interactions has been lacking. We performed EA-binding assays and electrophoretic mobility shift assays (EMSAs) to elucidate a mechanism for EutR gene regulation. These studies confirmed EutR interaction with EA, as well as direct binding to the eutS promoter. EutR also contributes to expression of the locus of enterocyte effacement (LEE) in an EA-dependent manner. We performed EMSAs to examine EutR activation of the LEE. The results demonstrated that EutR directly binds the regulatory region of the ler promoter. These results present the first mechanistic description of EutR gene regulation and reveal a novel role for EutR in EHEC pathogenesis.

Unlabelled: Bacterial pathogens must be able to both recognize suitable niches within the host for colonization and successfully compete with commensal flora for nutrients in order to establish infection. Ethanolamine (EA) is a major component of mammalian and bacterial membranes and is used by pathogens as a carbon and/or nitrogen source in the gastrointestinal tract. The deadly human pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) uses EA in the intestine as a nitrogen source as a competitive advantage for colonization over the microbial flora. Here we show that EA is not only important for nitrogen metabolism but that it is also used as a signaling molecule in cell-to-cell signaling to activate virulence gene expression in EHEC. EA in concentrations that cannot promote growth as a nitrogen source can activate expression of EHEC's repertoire of virulence genes. The EutR transcription factor, known to be the receptor of EA, is only partially responsible for this regulation, suggesting that yet another EA receptor exists. This important link of EA with metabolism, cell-to-cell signaling, and pathogenesis, highlights the fact that a fundamental means of communication within microbial communities relies on energy production and processing of metabolites. Here we show for the first time that bacterial pathogens not only exploit EA as a metabolite but also coopt EA as a signaling molecule to recognize the gastrointestinal environment and promote virulence expression., Importance: In order to successfully cause disease, a pathogen must be able to sense a host environment and modulate expression of its virulence genes as well as compete with the indigenous microbiota for nutrients. Ethanolamine (EA) is present in the large intestine due to the turnover of intestinal cells. Here, we show that the human pathogen Escherichia coli O157:H7, which causes bloody diarrhea and hemolytic-uremic syndrome, regulates virulence gene expression through EA metabolism and by responding to EA as a signal. These findings provide the first information directly linking EA with bacterial pathogenesis.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC encodes the sRNA chaperone Hfq, which is important in posttranscriptional regulation. In EHEC strain EDL933, Hfq acts as a negative regulator of the locus of enterocyte effacement (LEE), which encodes most of the proteins involved in type III secretion and attaching and effacing (AE) lesions. Here, we deleted hfq in the EHEC strain 86-24 and compared global transcription profiles of the hfq mutant and wild-type (WT) strains in exponential growth phase. Deletion of hfq affected transcription of genes common to nonpathogenic and pathogenic strains of E. coli as well as pathogen-specific genes. Downregulated genes in the hfq mutant included ler, the transcriptional activator of all the LEE genes, as well as genes encoded in the LEE2 to -5 operons. Decreased expression of the LEE genes in the hfq mutant occurred at middle, late, and stationary growth phases. We also confirmed decreased regulation of the LEE genes by examining the proteins secreted and AE lesion formation by the hfq mutant and WT strains. Deletion of hfq also caused decreased expression of the two-component system qseBC, which is involved in interkingdom signaling and virulence gene regulation in EHEC, as well as an increase in expression of stx(2AB), which encodes the deadly Shiga toxin. Altogether, these data indicate that Hfq plays a regulatory role in EHEC 86-24 that is different from what has been reported for EHEC strain EDL933 and that the role of Hfq in EHEC virulence regulation extends beyond the LEE.

Enterohaemorrhagic Escherichia coli (EHEC) colonizes the large intestine, causing attaching and effacing (AE) lesions. Most of the genes involved in AE lesion formation are encoded within a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). The LysR-type transcriptional factor QseA regulates the LEE by binding to the regulatory region of ler. We performed transcriptome analyses comparing wild-type (WT) EHEC and the qseA mutant to elucidate QseA's role in gene regulation. During both growth phases, several genes carried in O-islands were activated by QseA, whereas genes involved in cell metabolism were repressed. During late-logarithmic growth, QseA activated expression of the LEE genes as well as non-LEE-encoded effector proteins. We also performed electrophoretic mobility shift assays, competition experiments and DNase I footprints. The results demonstrated that QseA directly binds both the ler proximal and distal promoters, its own promoter, as well as promoters of genes encoded in EHEC-specific O-islands. Additionally, we mapped the transcriptional start site of qseA, leading to the identification of two promoter sequences. Taken together, these results indicate that QseA acts as a global regulator in EHEC, co-ordinating expression of virulence genes.

Bacteria must be able to respond rapidly to changes in the environment in order to survive. One means of coordinating gene expression relies on tightly regulated and complex signaling systems. One of the first signaling systems that was described in detail is quorum sensing (QS). During QS, a bacterial cell produces and secretes a signaling molecule called an autoinducer (AI). As the density of the bacterial population increases, so does the concentration of secreted AI molecules, thereby allowing a bacterial species to coordinate gene expression based on population density. Subsequent studies have demonstrated that bacteria are also able to detect signal molecules produced by other species of bacteria as well as hormones produced by their mammalian hosts. These types of signaling interactions have been termed cell-to-cell signaling because the interaction does not rely on a threshold concentration of bacterial cells. This review discusses the three main types of cell-to-cell signaling mechanisms used by E. coli and Salmonella, including the LuxR process, in which E. coli and Salmonella detect signals produced by other species of bacteria; the LuxS/AI-2 system, in which E. coli and Salmonella participate in intra- and interspecies signaling; and the AI-3/ epinephrine/norepinephrine system, in which E. coli and Salmonella recognize self-produced AI, signal produced by other microbes, and/or the human stress hormones epinephrine or norepinephrine.

Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen's adherence to HeLa cells (A. G. Torres and J. B. Kaper, Infect. Immun. 71:4985-4995, 2003). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue-cultured monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. The cadA mutant did not possess lysine decarboxylation activity and was hyperadherent to tissue-cultured cells. Adherence of the cadA mutant was nearly twofold greater than that of the wild type, and the adherence phenotype was independent of pH, lysine, or cadaverine in the media. Additionally, complementation of the cadA defect reduced adherence back to wild-type levels, and it was found that the mutation affected the expression of the intimin protein. Disruption of the eae gene (intimin-encoding gene) in the cadA mutant significantly reduced its adherence to tissue-cultured cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1,332 genes was downregulated and that of 132 genes was upregulated in the mutant compared to the wild-type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins, flagella and F9 fimbria, were upregulated in the cadA mutant, suggestive of their association with adherence in the absence of the Cad regulatory mechanism. In the infant rabbit model, the cadA mutant outcompeted the wild-type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.

Intrakingdom cell-to-cell communication and interkingdom cell-to-cell communication play essential roles in the virulence of enterohemorrhagic Escherichia coli (EHEC). Four signals, autoinducer 2 (AI-2), AI-3, and the human hormones epinephrine and norepinephrine, are important in this communication. The effect of these signaling compounds on the transcriptome of EHEC was examined in this study. We demonstrated that the luxS mutation affects primarily central metabolic genes in both pathogenic and nonpathogenic strains of E. coli and that addition of exogenous AI-2 does not fully restore the expression profile in a luxS-deficient strain lacking the ability to synthesize AI-2. Addition of AI-3 or epinephrine increased expression of the locus of enterocyte effacement regulon, which is known to play a pivotal role in EHEC virulence. Moreover, when epinephrine was added to the culture medium, the greatest number of gene alterations was observed. These alterations included a greater proportion of alterations in EHEC genes than in MG1655 genes, suggesting that epinephrine may be a global virulence signal. Detailed examination with real-time reverse transcriptase PCR (RT-PCR) confirmed the increases in virulence gene expression with addition of AI-3 and epinephrine. Additional studies with real-time RT-PCR examining the EHEC secreted effectors and putative fimbrial gene expression showed a variable expression profile, indicating that there is differential regulation of the secreted molecules. This study began to examine the global signaling networks in EHEC and revealed expression profiles that are signal and pathogen specific.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for worldwide outbreaks of bloody diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. After colonizing the large intestine, EHEC forms attaching and effacing (AE) lesions on intestinal epithelial cells. These lesions cause destruction of the microvilli and elicit actin rearrangement to form pedestals that cup each bacterium individually. EHEC responds to a signal produced by the intestinal microbial flora, autoinducer-3 (AI-3), and the host hormones epinephrine and norepinephrine to activate transcription of the genes involved in AE lesion formation. These three signals, involved in interkingdom communication, are sensed by bacterial sensor kinases. Here we describe a novel two-component system, QseEF (quorum-sensing E. coli regulators E and F), which is part of the AI-3/epinephrine/norepinephrine signaling system. QseE is the sensor kinase and QseF the response regulator. The qseEF genes are cotranscribed, and transcription of qseEF is activated by epinephrine through the QseC sensor. A qseF mutant does not form AE lesions. QseF activates transcription of the gene encoding EspFu, an effector protein translocated to the host cell by the EHEC, which mimics a eukaryotic SH2/SH3 adapter protein to engender actin polymerization during pedestal formation. Expression of the espFu gene from a plasmid restored AE lesion formation to the qseF mutant, suggesting that lack of espFu expression in this mutant was responsible for the loss of pedestal formation. These findings suggest the QseEF is a two-component system involved in the regulation of AE lesion formation by EHEC.

Methanogenesis in cold marine sediments is a globally important process leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. We employed a multidisciplinary approach that combined culture-dependent and -independent analyses with geochemical measurements in the sediments of Skan Bay, Alaska (53 degrees N, 167 degrees W), to investigate methanogenesis there. Cultivation-independent analyses of the archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present at Skan Bay and that methanogens constituted a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO(2)-reducing methanogens: AK-3, AK7, and AK-8. The phylogenetic positions and physiological characteristics of these strains are described. We propose a new species, Methanogenium boonei, with strain AK-7 as the type strain.

Two anaerobic, psychrotolerant, syntrophic strains were enriched from permanently cold, shallow anoxic marine sediments in Skan Bay, Alaska. One strain, AK-B(T), oxidized butyrate syntrophically and was isolated in defined coculture with a H(2)-using methanogen or in a dixenic coculture that also contained an acetate-scavenging methanogen. The other enrichment culture syntrophically oxidized propionate. The growth of these syntrophic cultures was very slow: approximately 1 year for cocultures of strain AK-B(T) to form colonies and >1 year for the propionate-oxidizing enrichment to form colonies. Neither culture grew axenically when supplied with the catabolic substrates crotonate, pyruvate, malate, or sulfate plus butyrate or propionate. Strain AK-B(T) catabolized iso-butyrate in syntrophic coculture but did not catabolize valerate or caproate. Phylogenetic analyses of the 16S rRNA gene sequence suggested that strain AK-B(T) was only distantly related to cultivated sulfate-reducing bacteria, and that this strain represented a new genus. We propose Algorimarina butyrica, with strain AK-B(T) (=OCM 842(T)), as the type strain. This report is the first description of psychrotolerant as well as marine butyrate--and propionate-oxidizing syntrophic organisms.

Little is known about the methanogenic degradation of acetate, the fate of molecular hydrogen and formate or the ability of methanogens to grow and produce methane in cold, anoxic marine sediments. The microbes that produce methane were examined in permanently cold, anoxic marine sediments at Hydrate Ridge (44 degrees 35' N, 125 degrees 10' W, depth 800 m). Sediment samples (15 to 35 cm deep) were collected from areas of active methane ebullition or areas where methane hydrates occurred. The samples were diluted into enrichment medium with formate, acetate or trimethylamine as catabolic substrate. After 2 years of incubation at 4 degrees C to 15 degrees C, enrichment cultures produced methane. PCR amplification and sequencing of the rRNA genes from the highest dilutions with growth suggested that each enrichment culture contained a single strain of methanogen. The level of sequence similarity (91 to 98%) to previously characterized prokaryotes suggested that these methanogens belonged to novel genera or species within the orders Methanomicrobiales and Methanosarcinales. Analysis of the 16S rRNA gene libraries from DNA extracted directly from the sediment samples revealed phylotypes that were either distantly related to cultivated methanogens or possible anaerobic methane oxidizers related to the ANME-1 and ANME-2 groups of the Archaea. However, no methanogenic sequences were detected, suggesting that methanogens represented only a small proportion of the archaeal community.

Three strains of CO(2)-reducing methanogens were isolated from marine sediments. Strain PL-15/H(P) was isolated from marine sediments of the Lipari Islands, near Sicily and the other two strains, Nankai-2 and Nankai-3(T), were isolated from deep marine sediments of the Nankai Trough, about 50 km from the coast of Japan. Analysis of the cellular proteins and 16S rRNA gene sequences indicated that these three strains represented a single novel species that formed a deep branch of the mesophilic methanococci. Phylogenetic analysis indicated that the three strains were most closely related to Methanothermococcus okinawensis (95 % 16S rRNA gene sequence similarity). However, strains PL-15/H(P), Nankai-2 and Nankai-3(T) grew at temperatures that were more similar to those of recognized species within the genus Methanococcus. Strain Nankai-3(T) grew fastest at 46 degrees C. Results of physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strains PL-15/H(P), Nankai-2 and Nankai-3(T) from closely related species. The name Methanococcus aeolicus sp. nov. is proposed, with strain Nankai-3(T) (=OCM 812(T)=DSM 17508(T)) as the type strain.

Three novel strains of methylotrophic methanogens were isolated from Skan Bay, Alaska, by using anaerobic cultivation techniques. The water was 65 m deep at the sampling site. Strains AK-4 (=OCM 774), AK-5T (=OCM 775T=DSM 17273T) and AK-9 (=OCM 793) were isolated from the sulfate-reducing zone of the sediments. Each of the strains was a non-motile coccus and occurred singly. Cells grew with trimethylamine as a catabolic substrate and strain AK-4 could also catabolize methanol. Yeast extract and trypticase peptones were not required for growth, but their addition to the culture medium slightly stimulated growth. Each of the strains grew at temperatures of 5-28 degrees C; they were slight halophiles and grew fastest in the neutral pH range. Analysis of the 16S rRNA gene sequences indicated that strain AK-4 was most closely related to Methanosarcina baltica. DNA-DNA hybridization studies showed 88 % relatedness, suggesting that strain AK-4 represents a novel strain within this species. Strains AK-5T and AK-9 had identical 16S rRNA gene sequences that were most closely related to the sequence of Methanococcoides burtonii (99.8 % sequence similarity). DNA-DNA hybridization studies showed that strains AK-5T and AK-9 are members of the same species (88 % relatedness value), but strain AK-5T had a DNA-DNA relatedness value of only 55 % to Methanococcoides burtonii. This indicates that strains AK-5T and AK-9 should be considered as members of a novel species in the genus Methanococcoides. We propose the name Methanococcoides alaskense sp. nov., with strain AK-5T (=OCM 775T=DSM 17273T) as the type strain.