Genome-wide dissection reveals diverse pathogenic roles of bacterial Tc toxins.

Tc toxins were originally identified in entomopathogenic bacteria, which are important as biological pest control agents. Tc toxins are heteromeric exotoxins composed of three subunit types, TcA, TcB, and TcC. The C-terminal portion of the TcC protein encodes the actual toxic domain, which is translocated into host cells by an injectosome nanomachine comprising the other subunits. Currently the pathogenic roles and distribution of Tc toxins among different bacterial genera remain unclear. Here we have performed a comprehensive genome-wide analysis, and established a database that includes 1,608 identified Tc loci containing 2,528 TcC proteins in 1,421 Gram-negative and positive bacterial genomes. Our findings indicate that TcCs conform to the architecture of typical polymorphic toxins, with C-terminal hypervariable regions (HVR) encoding more than 100 different classes of putative toxic domains, most of which have not been previously recognized. Based on further analysis of Tc loci in the genomes of all Salmonella and Yersinia strains in EnteroBase, a "two-level" evolutionary dynamics scenario is proposed for TcC homologues. This scenario implies that the conserved TcC RHS core domain plays a critical role in the taxonomical specific distribution of TcC HVRs. This study provides an extensive resource for the future development of Tc toxins as valuable agrochemical tools. It furthermore implies that Tc proteins, which are encoded by a wide range of pathogens, represent an important versatile toxin superfamily with diverse pathogenic mechanisms.

Self-identity barcodes encoded by six expansive polymorphic toxin families discriminate kin in myxobacteria.

Myxobacteria are an example of how single-cell individuals can transition into multicellular life by an aggregation strategy. For these and all organisms that consist of social groups of cells, discrimination against, and exclusion of, nonself is critical. In myxobacteria, TraA is a polymorphic cell surface receptor that identifies kin by homotypic binding, and in so doing exchanges outer membrane (OM) proteins and lipids between cells with compatible receptors. However, TraA variability alone is not sufficient to discriminate against all cells, as traA allele diversity is not necessarily high among local strains. To increase discrimination ability, myxobacteria include polymorphic OM lipoprotein toxins called SitA in their delivered cargo, which poison recipient cells that lack the cognate, allele-specific SitI immunity protein. We previously characterized 3 SitAI toxin/immunity pairs that belong to 2 families. Here, we discover 4 additional SitA families. Each family is unique in sequence, but share the characteristic features of SitA: OM-associated toxins delivered by TraA. We demonstrate that, within a SitA family, C-terminal nuclease domains are polymorphic and often modular. Remarkably, sitA loci are strikingly numerous and diverse, with most genomes possessing >30 and up to 83 distinct sitAI loci. Interestingly, all SitA protein families are serially transferred between cells, allowing a SitA inhibitor cell to poison multiple targets, including cells that never made direct contact. The expansive suites of sitAI loci thus serve as identify barcodes to exquisitely discriminate against nonself to ensure populations are genetically homogenous to conduct cooperative behaviors.

TADB 2.0: an updated database of bacterial type II toxin-antitoxin loci.

TADB2.0 (http://bioinfo-mml.sjtu.edu.cn/TADB2/) is an updated database that provides comprehensive information about bacterial type II toxin-antitoxin (TA) loci. Compared with the previous version, the database refined and the new data schema is employed. With the aid of text mining and manual curation, it recorded 6193 type II TA loci in 870 replicons of bacteria and archaea, including 105 experimentally validated TA loci. In addition, the newly developed tool TAfinder combines the homolog searches and the operon structure detection, allowing the prediction for type II TA pairs in bacterial genome sequences. It also helps to investigate the genomic context of predicted TA loci for putative virulence factors, antimicrobial resistance determinants and mobile genetic elements via alignments to the specific public databases. Additionally, the module TAfinder-Compare allows comparing the presence of the given TA loci across the close relative genomes. With the recent updates, TADB2.0 might provide better support for understanding the important roles of type II TA systems in the prokaryotic life activities.

Uropathogenic Escherichia coli strains harboring tosA gene were associated to high virulence genes and a multidrug-resistant profile.

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to ß-lactam and non-ß-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.

Overexpression of a flower-specific aerolysin-like protein from the dioecious plant Rumex acetosa alters flower development and induces male sterility in transgenic tobacco.

Sex determination in Rumex acetosa, a dioecious plant with a complex XY1 Y2 sex chromosome system (females are XX and males are XY1 Y2 ), is not controlled by an active Y chromosome but depends on the ratio between the number of X chromosomes and autosomes. To gain insight into the molecular mechanisms of sex determination, we generated a subtracted cDNA library enriched in genes specifically or predominantly expressed in female floral buds in early stages of development, when sex determination mechanisms come into play. In the present paper, we report the molecular and functional characterization of FEM32, a gene encoding a protein that shares a common architecture with proteins in different plants, animals, bacteria and fungi of the aerolysin superfamily; many of these function as ß pore-forming toxins. The expression analysis, assessed by northern blot, RT-PCR and in situ hybridization, demonstrates that this gene is specifically expressed in flowers in both early and late stages of development, although its transcripts accumulate much more in female flowers than in male flowers. The ectopic expression of FEM32 under both the constitutive promoter 35S and the flower-specific promoter AP3 in transgenic tobacco showed no obvious alteration in vegetative development but was able to alter floral organ growth and pollen fertility. The 35S::FEM32 and AP3::FEM32 transgenic lines showed a reduction in stamen development and pollen viability, as well as a diminution in fruit set, fruit development and seed production. Compared with other floral organs, pistil development was, however, enhanced in plants overexpressing FEM32. According to these effects, it is likely that FEM32 functions in Rumex by arresting stamen and pollen development during female flower development. The aerolysin-like pore-forming proteins of eukaryotes are mainly involved in defence mechanisms against bacteria, fungi and insects and are also involved in apoptosis and programmed cell death (PCD), a mechanism that could explain the role of FEM32 in Rumex sex determination.

Diversity of Clostridium perfringens toxin-genotypes from dairy farms.

BACKGROUND: Clostridium (C.) perfringens is the causative agent of several diseases in animals and humans, including histotoxic and enteric infections. To gain more insight into the occurrence of its different toxin-genotypes in dairy herds, including those toxin genes previously associated with diseases in cattle or humans, 662 isolates cultivated from feces, rumen content and feed collected from 139 dairy farms were characterized by PCR (detecting cpa, cpb, iap, etx, cpe, and both allelic variants of cpb2). RESULTS: Isolates from feces were assigned to type A (cpa positive, n = 442) and D (cpa and etx positive, n = 2). Those from rumen content (n = 207) and feed (n = 13) were all assigned to type A. The consensus and atypical variants of the cpb2 gene were detected in 64 (14.5 %) and 138 (31.22 %) of all isolates from feces, and 30 (14.5 %) and 54 (26.1 %) of all isolates from rumen content, respectively. CONCLUSION: Both allelic variants of cpb2 occurred frequently in animals without signs of acute enteric disease, whereby the atypical variant dominated. Five (0.8 %) of all type A isolates were positive for the cpe gene. Therefore, the present study indicates that dairy cows are no primary source for potentially human pathogenic enterotoxin gene positive strains.

Sialic acid linkage in glycosphingolipids is a molecular correlate for trafficking and delivery of extracellular cargo.

Gangliosides, glycosphingolipids containing sialic acid moieties, are well known mediators of transmembrane signaling and endocytosis at the plasma membrane. However, little is known about their precise regulatory role at the cell periphery for intracellular sorting of extracellular cargo. Here we inspected published scientific literature for two types of cargoes, namely bacterial toxins and viruses, regarding their usage of gangliosides. We derived a rather simple yet surprisingly consistent framework to classify 20 viruses from 12 different families and five type AB bacterial toxins into two broad categories. We propose that gangliosides with terminally attached sialic acids classify cargo for uptake and trafficking early in the endocytic pathway while gangliosides with internally attached sialic acids associate with uptake and trafficking of cargo late in the endocytic system. Our study provides a testable hypothesis for future investigations into a wide range of trafficking events. It could be utilized as a framework for other intracellular pathogens where lipids are known to be involved in recognition and trafficking. For instance, predictions can be put forward and evaluated based on ganglioside binding patterns and intracellular trafficking routes. Finally, incorporation of our classifier into large scale systems-biology studies could help reveal related molecular determinants in subcellular sorting.

Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes.

Toxin-antitoxin systems are widespread in bacteria and archaea. They perform diverse functional roles, including the generation of persistence, maintenance of genetic loci and resistance to bacteriophages through abortive infection. Toxin-antitoxin systems have been divided into three types, depending on the nature of the interacting macromolecules. The recently discovered Type III toxin-antitoxin systems encode protein toxins that are inhibited by pseudoknots of antitoxic RNA, encoded by short tandem repeats upstream of the toxin gene. Recent studies have identified the range of Type I and Type II systems within current sequence databases. Here, structure-based homology searches were combined with iterative protein sequence comparisons to obtain a current picture of the prevalence of Type III systems. Three independent Type III families were identified, according to toxin sequence similarity. The three families were found to be far more abundant and widespread than previously known, with examples throughout the Firmicutes, Fusobacteria and Proteobacteria. Functional assays confirmed that representatives from all three families act as toxin-antitoxin loci within Escherichia coli and at least two of the families confer resistance to bacteriophages. This study shows that active Type III toxin-antitoxin systems are far more diverse than previously known, and suggests that more remain to be identified.

Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection.

Vibrio vulnificus is a food-borne bacterial pathogen associated with 1% of all food-related deaths, predominantly because of consumption of contaminated seafood. The ability of V. vulnificus to cause disease is linked to the production of a large cytotoxin called the "multifunctional-autoprocessing RTX" (MARTX(Vv)) toxin, a factor shown here to be an important virulence factor by the intragastric route of infection in mice. In this study, we examined genetic variation of the rtxA1 gene that encodes MARTX(Vv) in 40 V. vulnificus Biotype 1 strains and found four distinct variants of rtxA1 that encode toxins with different arrangements of effector domains. We provide evidence that these variants arose by recombination either with rtxA genes carried on plasmids or with the rtxA gene of Vibrio anguillarum. Contrary to expected results, the most common rtxA1 gene variant in clinical-type V. vulnificus encodes a toxin with reduced potency and is distinct from the toxin produced by strains isolated from market oysters. These results indicate that an important virulence factor of V. vulnificus is undergoing significant genetic rearrangement and may be subject to selection for reduced virulence in the environment. This finding would imply further that in the future on-going genetic variation of the MARTX(Vv) toxins could result in the emergence of novel strains with altered virulence in humans.

[Protein toxins of Staphylococcus aureus].

Main scientific-research studies regarding protein bacterial toxins of the most widespread bacteria that belong to Staphylococcus spp. genus and in particular the most pathogenic species for humans--Staphylococcus aureus, are analyzed. Structural and biological properties of protein toxins that have received the name of staphylococcus pyrogenic toxins (PTSAg) are presented. Data regarding genetic regulation of secretion and synthesis of these toxins and 3 main regulatory genetic systems (agr--accessory gene regulator, xpr--extracellular protein regulator, sar--staphylococcal accessory regulator) that coordinate synthesis of the most important protein toxins and enzymes for virulence of S. aureus, are presented.

Toxins-antitoxins: diversity, evolution and function.

Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.

Rapid, multiplex-tandem PCR assay for automated detection and differentiation of toxigenic cyanobacterial blooms.

Cyanobacterial blooms are a major water quality issue and potential public health risk in freshwater, marine and estuarine ecosystems globally, because of their potential to produce cyanotoxins. To date, a significant challenge in the effective management of cyanobacterial has been an inability of classical microscopy-based approaches to consistently and reliably detect and differentiate toxic from non-toxic blooms. The potential of cyanobacteria to produce toxins has been linked to the presence of specific biosynthetic gene clusters. Here, we describe the application of a robotic PCR-based assay for the semi-automated and simultaneous detection of toxin biosynthesis genes of each of the toxin classes characterized to date for cyanobacteria [i.e., microcystins (MCYs), nodularins (NODs), cylindrospermopsins (CYNs) and paralytic shellfish toxins (PSTs)/saxitoxins (SXTs)]. We demonstrated high sensitivity and specificity for each assay using well-characterized, cultured isolates, and establish its utility as a quantitative PCR using DNA, clone and cell-based dilution series. In addition, we used 206 field-collected samples and 100 known negative controls to compare the performance of each assay with conventional PCR and direct toxin detection. We report a diagnostic specificity of 100% and a sensitivity of ≥97.7% for each assay.

Diversity of bacterial type II toxin-antitoxin systems: a comprehensive search and functional analysis of novel families.

Type II toxin-antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as 'addiction', and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the 'guilt by association' principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes.

Intracellular trafficking of Clostridium perfringens iota-toxin b.

Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca(2+) concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes.

Abundance of type I toxin-antitoxin systems in bacteria: searches for new candidates and discovery of novel families.

Small, hydrophobic proteins whose synthesis is repressed by small RNAs (sRNAs), denoted type I toxin-antitoxin modules, were first discovered on plasmids where they regulate plasmid stability, but were subsequently found on a few bacterial chromosomes. We used exhaustive PSI-BLAST and TBLASTN searches across 774 bacterial genomes to identify homologs of known type I toxins. These searches substantially expanded the collection of predicted type I toxins, revealed homology of the Ldr and Fst toxins, and suggested that type I toxin-antitoxin loci are not spread by horizontal gene transfer. To discover novel type I toxin-antitoxin systems, we developed a set of search parameters based on characteristics of known loci including the presence of tandem repeats and clusters of charged and bulky amino acids at the C-termini of short proteins containing predicted transmembrane regions. We detected sRNAs for three predicted toxins from enterohemorrhagic Escherichia coli and Bacillus subtilis, and showed that two of the respective proteins indeed are toxic when overexpressed. We also demonstrated that the local free-energy minima of RNA folding can be used to detect the positions of the sRNA genes. Our results suggest that type I toxin-antitoxin modules are much more widely distributed among bacteria than previously appreciated.

Unraveling cyanobacteria ecology in wastewater treatment plants (WWTP).

Cyanobacteria may be important components of wastewater treatment plants' (WWTP) biological treatment, reaching levels of 100% of the total phytoplankton density in some systems. The occurrence of cyanobacteria and their associated toxins in these systems present a risk to the aquatic environments and to public health, changing drastically the ecology of microbial communities and associated organisms. Many studies reveal that cyanotoxins, namely microcystins may not act as antibacterial compounds but they might have negative impacts on protozoans, inhibiting their growing and respiration rates and leading to changes in cellular morphology, decreasing consequently the treatment efficacy in WWTP. On the other side, flagellates and ciliates may ingest some cyanobacteria species while the formation of colonies by these prokaryotes may be seen as a defense mechanism against predation. Problems regarding the occurrence of cyanobacteria in WWTP are not limited to toxin production. Other cyanobacterial secondary metabolites may act as antibacterial compounds leading to the disruption of bacterial communities that biologically convert organic materials in WWTP being fundamental to the efficacy of the process. Studies reveal that the potential antibacterial capacity differs according to cyanobacteria specie and it seems to be more effective in Gram (+) bacteria. Thus, to understand the effects of cyanobacterial communities in the efficiency of the waste water treatment it will be necessary to unravel the complex interactions between cyanobacterial populations, bacteria, and protozoa in WWTP in situ studies.

Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin.

Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal ß-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a ß-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.

A New Contact Killing Toxin Permeabilizes Cells and Belongs to a Broadly Distributed Protein Family.

Vibrio cholerae is an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts, V. cholerae uses the type VI secretion system (T6SS). The T6SS is a macromolecular contact-dependent weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequenced V. cholerae isolates, strain BGT49 encodes another locus, which we named auxiliary (Aux) cluster 4. The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate both V. cholerae and Escherichia coli cells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (type VI permeabilizing effector Vibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm of E. coli permeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine that tpiV, the gene located immediately downstream of tpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important human, animal, and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins. IMPORTANCE Bacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease. However, the mechanisms by which many T6SS toxins kill or inhibit susceptible target cells are poorly understood. The sequence of the TpeV toxin that we describe here is unlike any previously described protein. We demonstrate that it has antimicrobial activity by permeabilizing cells, eliminating membrane potentials, and causing severe cytotoxicity. TpeV homologs are found near known T6SS genes in human, animal, and plant bacterial pathogens, indicating that the toxin is a representative member of a broadly distributed protein family. We propose that TpeV-like toxins contribute to the fitness of many bacteria. Finally, since antibiotic resistance is a critical global health threat, the discovery of new antimicrobial mechanisms could lead to the development of new treatments against resistant strains.

The Escherichia coli mqsR and ygiT genes encode a new toxin-antitoxin pair.

Toxin-antitoxin (TA) systems are plasmid- or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells.

LT-IIc, a new member of the type II heat-labile enterotoxin family encoded by an Escherichia coli strain obtained from a nonmammalian host.

Two families of bacterial heat-labile enterotoxins (HLTs) have been described: the type I HLTs are comprised of cholera toxin (CT) of Vibrio cholerae, LT-I of Escherichia coli, and several related HLTs; the type II HLTs are comprised of LT-IIa and LT-IIb. Herein, we report LT-IIc, a new type II HLT encoded from an enterotoxigenic E. coli (ETEC) strain isolated from an avian host. Using a mouse Y1 adrenal cell bioassay, LT-IIc was shown to be less cytotoxic than CT, LT-IIa, or LT-IIb. Cytotoxicity of LT-IIc was partially neutralized by antisera recognizing LT-IIa or LT-IIb but not by anti-CT antiserum. Genes encoding putative A polypeptide and B polypeptides of LT-IIc were arranged in an operon which was flanked by potential prophage sequences. Analysis of the nucleotide and predicted amino acid sequences demonstrated that the A polypeptide of LT-IIc has moderate homology to the A polypeptides of CT and LT-I and high homology to the A polypeptides of LT-IIa and LT-IIb. The B polypeptide of LT-IIc exhibited no significant homology to the B polypeptides of CT and LT-I and only moderate homology to the B polypeptides of LT-IIa and LT-IIb. The binding pattern of LT-IIc for gangliosides was distinctive from that of either LT-IIa or LT-IIb. The data suggest that other types of the type II HLT subfamily are circulating in the environment and that host specificity of type II HLT is likely governed by changes in the B polypeptide which mediate binding to receptors.