Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.

Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1-CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.

Outer Membrane Vesicle Production in Escherichia coli Relieves Envelope Stress and is Modulated by Changes in Peptidoglycan

Bacterial outer membrane vesicles (OMVs) are spherical buds of the outer membrane (OM) containing periplasmic lumenal components. OMVs have been demonstrated to play a critical part in the transmission of virulence factors, immunologically active compounds, and bacterial survival, however vesiculation also appears to be a ubiquitous physiological process for Gram-negative bacteria. Despite their characterized biological roles, especially for pathogens, very little is known about their importance for the originating organism as well as regulation and mechanism of production. Only when we have established their biogenesis can we fully uncover their roles in pathogenesis and bacterial physiology. The overall goal of this research was to characterize bacterial mutants which display altered vesiculation phenotypes using genetic and biochemical techniques, and thereby begin to elucidate the mechanism of vesicle production and regulation. One part of this work elucidated a synthetic genetic growth defect for a strain with reduced OMV production (ΔnlpA, inner membrane lipoprotein with a minor role in methionine transport) and envelope stress (ΔdegP, dual function periplasmic chaperone/ protease responsible for managing proteinaceous waste). This research showed that the growth defect of ΔnlpAΔdegP correlated with reduced OMV production with respect to the hyprevesiculator ΔdegP and the accumulation of protein in the periplasm and DegP substrates in the lumen of OMVs. We further demonstrated that OMVs do not solely act as a stress response pathway to rid the periplasm of otherwise damaging misfolded protein but also of accumulated peptidoglycan (PG) fragments and lipopolysaccharide (LPS), elucidating OMVs as a general stress response pathway critical for bacterial well-being. The second part of this work, focused on the role of PG structure, turnover and covalent crosslinks to the OM in vesiculation. We established a direct link between PG degradation and vesiculation: Mutations in the OM lipoprotein nlpI had been previously established as a very strong hypervesiculation phenotype. In the literature NlpI had been associated with another OM lipoprotein, Spr that was recently identified as a PG hydrolase. The data presented here suggest that NlpI acts as a negative regulator of Spr and that the ΔnlpI hypervesiculation phenotype is a result of rampantly degraded PG by Spr. Additionally, we found that changes in PG structure and turnover correlate with altered vesiculation levels, as well as non-canonical D-amino acids, which are secreted by numerous bacteria on the onset of stationary phase, being a natural factor to increase OMV production. Furthermore, we discovered an inverse relationship between the concentration of Lpp-mediated, covalent crosslinks and the level of OMV production under conditions of modulated PG metabolism and structure. In contrast, situations that lead to periplasmic accumulation (protein, PG fragments, and LPS) and consequent hypervesiculation the overall OM-PG crosslink concentration appears to be unchanged. Form this work, we conclude that multiple pathways lead to OMV production: Lpp concentration-dependent and bulk driven, Lpp concentration-independent.

The Ontogeny of Mucosal and Systemic Antibody Responses to HIV-1 Infection

The humoral immune system plays a critical role in the clearance of numerous pathogens. In the setting of HIV-1 infection, the virus infects, integrates its genome into the host's cells, replicates, and establishes a reservoir of virus-infected cells. The initial antibody response to HIV-1 infection is targeted to non-neutralizing epitopes on HIV-1 Env gp41, and when a neutralizing response does develop months after transmission, it is specific for the autologous founder virus and the virus escapes rapidly. After continuous waves of antibody mediated neutralization and viral escape, a small subset of infected individuals eventually develop broad and potent heterologous neutralizing antibodies years after infection. In this dissertation, I have studied the ontogeny of mucosal and systemic antibody responses to HIV-1 infection by means of three distinct aims: 1. Determine the origin of the initial antibody response to HIV-1 infection. 2. Characterize the role of restricted VH and VL gene segment usage in shaping the antibody response to HIV-1 infection. 3. Determine the role of persistence of B cell clonal lineages in shaping the mutation frequencies of HIV-1 reactive antibodies.

After the introduction (Chapter 1) and methods (Chapter 2), Chapter 3 of this dissertation describes a study of the antibody response of terminal ileum B cells to HIV-1 envelope (Env) in early and chronic HIV-1 infection and provides evidence for the role of environmental antigens in shaping the repertoire of B cells that respond to HIV-1 infection. Previous work by Liao et al. demonstrated that the initial plasma cell response in the blood to acute HIV-1 infection is to gp41 and is derived from a polyreactive memory B cell pool. Many of these antibodies cross-reacted with commensal bacteria, Therefore, in Chapter 3, the relationship of intestinal B cell reactivity with commensal bacteria to HIV-1 infection-induced antibody response was probed using single B cell sorting, reverse transcription and nested polymerase chain reaction (RT- PCR) methods, and recombinant antibody technology. The dominant B cell response in the terminal ileum was to HIV-1 envelope (Env) gp41, and 82% of gp41- reactive antibodies cross-reacted with commensal bacteria whole cell lysates. Pyrosequencing of blood B cells revealed HIV-1 antibody clonal lineages shared between ileum and blood. Mutated IgG antibodies cross-reactive with both Env gp41 and commensal bacteria could also be isolated from the terminal ileum of HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 can be shaped by intestinal B cells stimulated by commensal bacteria prior to HIV-1 infection to develop a pre-infection pool of memory B cells cross-reactive with HIV-1 gp41.

Chapter 4 details the study of restricted VH and VL gene segment usage for gp41 and gp120 antibody induction following acute HIV-1 infection; mutations in gp41 lead to virus enhanced neutralization sensitivity. The B cell repertoire of antibodies induced in a HIV-1 infected African individual, CAP206, who developed broadly neutralizing antibodies (bnAbs) directed to the HIV-1 envelope gp41 membrane proximal external region (MPER), is characterized. Understanding the selection of virus mutants by neutralizing antibodies is critical to understanding the role of antibodies in control of HIV-1 replication and prevention from HIV-1 infection. Previously, an MPER neutralizing antibody, CAP206-CH12, with the binding footprint identical to that of MPER broadly neutralizing antibody 4E10, that like 4E10 utilized the VH1-69 and VK3-20 variable gene segments was isolated from this individual (Morris et al., 2011). Using single B cell sorting, RT- PCR methods, and recombinant antibody technology, Chapter 4 describes the isolation of a VH1-69, Vk3-20 glycan-dependent clonal lineage from CAP206, targeted to gp120, that has the property of neutralizing a neutralization sensitive CAP206 transmitted/founder (T/F) and heterologous viruses with mutations at amino acids 680 or 681 in the MPER 4E10/CH12 binding site. These data demonstrate sites within the MPER bnAb epitope (aa 680-681) in which mutations can be selected that lead to viruses with enhanced sensitivity to autologous and heterologous neutralizing antibodies.

In Chapter 5, I have completed a comparison of evolution of B cell clonal lineages in two HIV-1 infected individuals who have a predominant VH1-69 response to HIV-1 infection--one who produces broadly neutralizing MPER-reactive mAbs and one who does not. Autologous neutralization in the plasma takes ~12 weeks to develop (Gray et al., 2007; Tomaras et al., 2008b). Only a small subset of HIV-1 infected individuals develops high plasma levels of broad and potent heterologous neutralization, and when it does occur, it typically takes 3-4 years to develop (Euler et al., 2010; Gray et al., 2007; 2011; Tomaras et al., 2011). The HIV-1 bnAbs that have been isolated to date have a number of unusual characteristics including, autoreactivity and high levels of somatic hypermutations, which are typically tightly regulated by immune control mechanisms (Haynes et al., 2005; 2012b; Kwong and Mascola, 2012; Scheid et al., 2009a). The VH mutation frequencies of bnAbs average ~15% but have been shown to be as high as 32% (reviewed in Mascola and Haynes, 2013; Kwong and Mascola, 2012). The high frequency of somatic hypermutations suggests that the B cell clonal lineages that eventually produce bnAbs undergo high-levels of affinity maturation, implying prolonged germinal center (GC) reactions and high levels of T cell help. To study the duration of HIV-1- reactive B cell clonal persistence, HIV-1 reactive and non HIV-1- reactive B cell clonal lineages were isolated from an HIV-1 infected individual that produces bnAbs, CAP206, and an HIV-1 infected individual who does not produce bnAbs, 004-0. Single B cell sorting, RT-PCR and recombinant antibody technology was used to isolate and produce monoclonal antibodies from multiple time points from each individual. B cell sequences clonally related to mAbs isolated by single cell PCR were identified within pyrosequences of longitudinal samples of these two individuals. Both individuals produced long-lived B cell clones that persisted from 0-232 weeks in CAP206, and 0-238 weeks in 004-0. The average length of persistence of clones containing members isolated from two separate time points was 91.5 weeks both individuals. Examples of the continued evolution of clonal lineages were observed in both the bnAb and non-bnAb individual. These data indicated that the ability to generate persistent and evolving B cell clonal lineages occurs in both bnAb and non-bnAb individuals, suggesting that some alternative host or viral factor is critical for the generation of highly mutated broadly neutralizing antibodies.

Together the studies described in Chapter 3-5 show that multiple factors influence the antibody response to HIV-1 infection. The initial antibody response to HIV-1 Env gp41 can be shaped by a B cell response to intestinal commensal bacteria prior to HIV-1 infection. VH and VL gene segment restriction can impact the B cell response to multiple HIV-1 antigens, and virus escape mutations in the MPER can confer enhanced neutralization sensitivity to autologous and heterologous antibodies. Finally, the ability to generate long-lived HIV-1 clonal lineages in and of itself does not confer on the host the ability to produce bnAbs.

Polymorphisms in the kinesin-like factor 1 B gene and risk of epithelial ovarian cancer in Eastern Chinese women.

The kinesin-like factor 1 B (KIF1B) gene plays an important role in the process of apoptosis and the transformation and progression of malignant cells. Genetic variations in KIF1B may contribute to risk of epithelial ovarian cancer (EOC). In this study of 1,324 EOC patients and 1,386 cancer-free female controls, we investigated associations between two potentially functional single nucleotide polymorphisms in KIF1B and EOC risk by the conditional logistic regression analysis. General linear regression model was used to evaluate the correlation between the number of variant alleles and KIF1B mRNA expression levels. We found that the rs17401966 variant AG/GG genotypes were significantly associated with a decreased risk of EOC (adjusted odds ratio (OR) = 0.81, 95 % confidence interval (CI) = 0.68-0.97), compared with the AA genotype, but no associations were observed for rs1002076. Women who carried both rs17401966 AG/GG and rs1002076 AG/AA genotypes of KIF1B had a 0.82-fold decreased risk (adjusted 95 % CI = 0.69-0.97), compared with others. Additionally, there was no evidence of possible interactions between about-mentioned co-variants. Further genotype-phenotype correlation analysis indicated that the number of rs17401966 variant G allele was significantly associated with KIF1B mRNA expression levels (P for GLM = 0.003 and 0.001 in all and Chinese subjects, respectively), with GG carriers having the lowest level of KIF1B mRNA expression. Taken together, the rs17401966 polymorphism likely regulates KIF1B mRNA expression and thus may be associated with EOC risk in Eastern Chinese women. Larger, independent studies are warranted to validate our findings.

Explicit DNase sequence bias modeling enables high-resolution transcription factor footprint detection.

DNaseI footprinting is an established assay for identifying transcription factor (TF)-DNA interactions with single base pair resolution. High-throughput DNase-seq assays have recently been used to detect in vivo DNase footprints across the genome. Multiple computational approaches have been developed to identify DNase-seq footprints as predictors of TF binding. However, recent studies have pointed to a substantial cleavage bias of DNase and its negative impact on predictive performance of footprinting. To assess the potential for using DNase-seq to identify individual binding sites, we performed DNase-seq on deproteinized genomic DNA and determined sequence cleavage bias. This allowed us to build bias corrected and TF-specific footprint models. The predictive performance of these models demonstrated that predicted footprints corresponded to high-confidence TF-DNA interactions. DNase-seq footprints were absent under a fraction of ChIP-seq peaks, which we show to be indicative of weaker binding, indirect TF-DNA interactions or possible ChIP artifacts. The modeling approach was also able to detect variation in the consensus motifs that TFs bind to. Finally, cell type specific footprints were detected within DNase hypersensitive sites that are present in multiple cell types, further supporting that footprints can identify changes in TF binding that are not detectable using other strategies.

ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation.

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.

Role of DNA binding sites and slow unbinding kinetics in titration-based oscillators.

Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ultrasensitivity that is specific for repression. We then use stochastic simulation to show that multiple binding sites increase the coherence of oscillations by mitigating the binary noise. Slow values of DNA unbinding rate are also effective in alleviating molecular noise due to the increased distance from the bifurcation point. Our work demonstrates how the number of DNA binding sites and slow unbinding kinetics, which are often omitted in biophysical models of gene circuits, can have a significant impact on the temporal and stochastic dynamics of genetic oscillators.

Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma in Older Adults.

Multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are plasma cell disorders of aging. The landscape of the diagnosis and management of MM and MGUS are rapidly changing. This article provides an updated understanding of the clinical presentation, evaluation, diagnosis, and management of older adults with MM and MGUS. Because most oncology providers are not formally trained in geriatric medicine, geriatricians play a key role in providing oncologists with a broader understanding of patient health status in the hope of improving outcomes for older adults with MM.

ROLE OF ICAM-1-MEDIATED ENDOCYTOSIS IN ENDOTHELIAL FUNCTION AND IMPLICATIONS FOR CARRIER-ASSISTED DRUG DELIVERY

Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein found on the surface of vascular endothelial cells (ECs). Its expression is upregulated at inflammatory sites, allowing for targeted delivery of therapeutics using ICAM-1-binding drug carriers. Engagement of multiple copies of ICAM-1 by these drug carriers induces cell adhesion molecule (CAM)-mediated endocytosis, which results in trafficking of carriers to lysosomes and across ECs. Knowledge about the regulation behind CAM-mediated endocytosis can help improve drug delivery, but questions remain about these regulatory mechanisms. Furthermore, little is known about the natural function of this endocytic pathway. To address these gaps in knowledge, we focused on two natural binding partners of ICAM-1 that potentially elicit CAM-mediated endocytosis: leukocytes (which bind ICAM-1 via β₂ integrins) and fibrin polymers (a main component of blood clots which binds ICAM-1 via the γ3 sequence). First, inspired by properties of these natural binding partners, we varied the size and targeting moiety of model drug carriers to determine how these parameters affect CAM-mediated endocytosis. Increasing ICAM-1-targeted carrier size slowed carrier uptake kinetics, reduced carrier trafficking to lysosomes, and increased carrier transport across ECs. Changing targeting moieties from antibodies to peptides decreased particle binding and uptake, lowered trafficking to lysosomes, and increased transport across ECs. Second, using cell culture models of leukocyte/EC interactions, inhibiting regulatory elements of the CAM-mediated pathway disrupted leukocyte sampling, a process crucial to leukocyte crossing of endothelial layers (transmigration). This inhibition also decreased leukocyte transmigration across ECs, specifically through the transcellular route, which occurs through a single EC without disassembly of cell-cell junctions. Third, fibrin meshes, which mimic blood clot fragments/remnants, bound to ECs at ICAM-1-enriched sites and were internalized by the endothelium. Inhibiting the CAM-mediated pathway disrupted this uptake. Following endocytosis, fibrin meshes trafficked to lysosomes where they were degraded. In mouse models, CAM-mediated endocytosis of fibrin meshes appeared to remove fibrin remnants at the endothelial surface, preventing re-initiation of the coagulation cascade. Overall, these results support a link between CAM-mediated endocytosis and leukocyte transmigration as well as uptake of fibrin materials by ECs. Furthermore, these results will guide the future design of ICAM-1-targeted carrier-assisted therapies.

Les facteurs de transcription du groupe PEA3: Régulateurs transcriptionnels dans le processus de cancérisation

Erm, Er81, and Pea3 are the three members of the PEA3 group which belong to the Ets transcription factors family. These proteins regulate transcription of multiple target genes, such as those encoding several matrix metalloproteinases (MMP), which are enzymes degrading the extracellular matrix during cancer metastasis. In fact, PEA3-group genes are often overexpressed in different types of human cancers that also over-express these MMP and display a disseminating phenotype. In experimental models, regulation of PEA3 group member expression has been shown to influence the metastatic process, thus suggesting that these factors play a key role in metastasis. © John Libbey Eurotext.

The Ets transcription factors of the PEA3 group: Transcriptional regulators in metastasis

The PEA3 group is composed of three highly conserved Ets transcription factors: Erm, Er81, and Pea3. These proteins regulate transcription of multiple genes, and their transactivating potential is affected by post-translational modifications. Among their target genes are several matrix metalloproteases (MMPs), which are enzymes degrading the extracellular matrix during normal remodelling events and cancer metastasis. In fact, PEA3-group genes are often over-expressed in different types of cancers that also over-express these MMPs and display a disseminating phenotype. Experimental regulation of the synthesis of PEA3 group members influences the metastatic process. This suggests that these factors play a key role in metastasis. © 2006 Elsevier B.V. All rights reserved.

The 26S proteasome system degrades the ERM transcription factor and regulates its transcription-enhancing activity

ERM is a member of the ETS transcription factor family. High levels of the corresponding mRNA are detected in a variety of human breast cancer cell lines, as well as in aggressive human breast tumors. As ERM protein is almost undetectable in these cells, high degradation of this transcription factor has been postulated. Here we have investigated whether ERM degradation might depend on the proteasome pathway. We show that endogenous and ectopically expressed ERM protein is short-lived protein and undergoes proteasome-dependent degradation. Deletion mutagenesis studies indicate that the 61 C-terminal amino acids of ERM are critical for its proteolysis and serve as a degradation signal. Although ERM conjugates with ubiquitin, this post-translational modification does not depend on the C-terminal domain. We have used an Ets-responsive ICAM-1 reporter plasmid to show that the ubiquitin-proteasome pathway can affect transcriptional function of ERM. Thus, ERM is subject to degradation via the 26S proteasome pathway, and this pathway probably plays an important role in regulating ERM transcriptional activity. © 2007 Nature Publishing Group. All rights reserved.

The evolutionarily conserved transcription factor PRDM12 controls sensory neuron development and pain perception

PR homology domain-containing member 12 (PRDM12) is a highly evolutionary conserved member of the Prdm family of transcription factors that play essential roles in many cell fate decisions. In human, PRDM12 coding mutations have been recently identified in several patients with hereditary sensory and autonomic neuropathy (HSAN) (submitted elsewhere). Here we show that PRDM12 is involved in sensory neurogenesis in Xenopus and that several of the human Prdm12 mutants show altered structure, subcellular localization and function. In Drosophila, we demonstrate that the sensory neuron specific RNAi knockdown of the Prdm12 ortholog Hamlet induces impaired nociception and that a similar phenotype is observed in hypomorph hamlet mutants. In human fibroblasts of patients with PRDM12 mutations, we identified additional possible downstream target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE). Knock-down of fly TRHDE in sensory neurons resulted in altered nociceptive neurons and impaired nociception. Collectively, these findings provide the first evidence showing that Prdm12 plays an important role in sensory neuron development. They also suggest that it has a critical evolutionarily conserved role in pain perception via modulation of the TRH signaling pathway.

Heparin-binding hemagglutinin, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis.

Interactions of Mycobacterium tuberculosis with macrophages have long been recognized to be crucial to the pathogenesis of tuberculosis. The role of non-phagocytic cells is less well known. We have discovered a M. tuberculosis surface protein that interacts specifically with non-phagocytic cells, expresses hemagglutination activity and binds to sulfated glycoconjugates. It is therefore called heparin-binding hemagglutinin (HBHA). HBHA-deficient M. tuberculosis mutant strains are significantly impaired in their ability to disseminate from the lungs to other tissues, suggesting that the interaction with non-phagocytic cells, such as pulmonary epithelial cells, may play an important role in the extrapulmonary dissemination of the tubercle bacillus, one of the key steps that may lead to latency. Latently infected human individuals mount a strong T cell response to HBHA, whereas patients with active disease do not, suggesting that HBHA is a good marker for the immunodiagnosis of latent tuberculosis, and that HBHA-specific Th1 responses may contribute to protective immunity against active tuberculosis. Strong HBHA-mediated immuno-protection was shown in mouse challenge models. HBHA is a methylated protein and its antigenicity in latently infected subjects, as well as its protective immunogenicity strongly depends on the methylation pattern of HBHA. In both mice and man, the HBHA-specific IFN-gamma was produced by both the CD4(+) and the CD8(+) T cells. Furthermore, the HBHA-specific CD8(+) T cells expressed bactericidal and cytotoxic activities to mycobacteria-infected macrophages. This latter activity is most likely perforin mediated. Together, these observations strongly support the potential of methylated HBHA as an important component in future, acellular vaccines against tuberculosis.

Multiple-locus sequence typing analysis of Bacillus thuringiensis recovered from the phylloplane of clover (Trifolium hybridum) in vegetative form

The chromosomal genotype, as judged by multi locus sequence typing, and the episomal genotype, as judged by plasmid profile and cry gene content, were analyzed for a collection of strains of Bacillus thuringiensis. These had been recovered in vegetative form over a period of several months from the leaves of a small plot of clover (Trifolium hybridum). A clonal population structure was indicated, although greater variation in sequence types (STs) was discovered than in previous collections of B. cereus/B. thuringiensis. Isolates taken at the same time had quite different genotypes, whereas those of identical genotypes were recovered at different times. The profiles of plasmid content and cry genes generally bore no relation to each other nor to the STs. Evidently, although relatively little recombination was occurring in the seven chromosomal genes analyzed, a great deal of conjugal transfer, and perhaps recombination, was occurring involving plasmids. A clinical diarrheal isolate of B. cereus and the commercial biopesticide strain HD-1 of B. thuringiensis, both included as out-groups, were found to have very similar STs. This further emphasizes the role of episomal elements in the characteristics and differentiation of these two species.