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The bacterial flagellum is a macromolecular protein complex that enables motility in many species. Bacterial flagella self-assemble a strong, multicomponent drive shaft that couples rotation in the inner membrane to the micrometre-long flagellar filament that powers bacterial swimming in viscous fluids 1-3 . Here, we present structures of the intact Salmonella flagellar basal body 4 , encompassing the inner membrane rotor, drive shaft and outer-membrane bushing, solved using cryo-electron microscopy to resolutions of 2.2-3.7 Å. The structures reveal molecular details of how 173 protein molecules of 13 different types assemble into a complex spanning two membranes and a cell wall. The helical drive shaft at one end is intricately interwoven with the rotor component with both the export gate complex and the proximal rod forming interactions with the MS-ring. At the other end, the drive shaft distal rod passes through the LP-ring bushing complex, which functions as a molecular bearing anchored in the outer membrane through interactions with the lipopolysaccharide. The in situ structure of a protein complex capping the drive shaft provides molecular insights into the assembly process of this molecular machine.

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins-such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)-impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy ([Formula: see text]) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published [Formula: see text] for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force ( F unfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δ x ‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust ( F unfold > 80 pN) and brittle (Δ x ‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion., Competing Interests: The authors declare no competing interest.

The Bacterial flagellar hook is a short supercoiled tubular structure made from a helical assembly of the hook protein FlgE. The hook acts as a universal joint that connects the flagellar basal body and filament, and smoothly transmits torque generated by the rotary motor to the helical filament propeller. In peritrichously flagellated bacteria, the hook allows the filaments to form a bundle behind the cell for swimming, and for the bundle to fall apart for tumbling. Here we report a native supercoiled hook structure at 3.6 Å resolution by cryoEM single particle image analysis of the polyhook. The atomic model built into the three-dimensional (3D) density map reveals the changes in subunit conformation and intersubunit interactions that occur upon compression and extension of the 11 protofilaments during their smoke ring-like rotation. These observations reveal how the hook functions as a dynamic molecular universal joint with high bending flexibility and twisting rigidity.

Pathogens survive and propagate within host cells through a wide array of complex interactions. Tracking the molecular and cellular events by multidimensional fluorescence microscopy has been a widespread tool for research on intracellular pathogens. Through major advancements in 3D electron microscopy, intracellular pathogens can also be visualized in their cellular environment to an unprecedented level of detail within large volumes. Recently, multidimensional fluorescence microscopy has been correlated with volume electron microscopy, combining molecular and functional information with the overall ultrastructure of infection events. In this review, we provide a short introduction to correlative focused ion beam/scanning electron microscopy (c-FIB/SEM) tomography and illustrate its utility for intracellular pathogen research through a series of studies on Shigella, Salmonella, and Brucella cellular invasion. We conclude by discussing current limitations of and prospects for this approach., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Single-cell fluorescence imaging is a powerful technique for studying inherently heterogeneous biological processes. To correlate a genotype or phenotype to a specific cell, images containing a population of cells must first be properly segmented. However, a proper segmentation with minimal user input becomes challenging when cells are clustered or overlapping in three dimensions. We introduce a new analysis package, Seg-3D, for the segmentation of bacterial cells in three-dimensional (3D) images, based on local thresholding, shape analysis, concavity-based cluster splitting, and morphology-based 3D reconstruction. The reconstructed cell volumes allow us to directly quantify the fluorescent signals from biomolecules of interest within individual cells. We demonstrate the application of this analysis package in 3D segmentation of individual bacterial pathogens invading host cells. We believe Seg-3D can be an efficient and simple program that can be used to analyze a wide variety of single-cell images, especially for biological systems involving random 3D orientation and clustering behavior, such as bacterial infection or colonization.

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria by bacteriophage ɸX174 gene E mediated lysis. They represent a novel inactivated vaccine platform; however, the practical application of BGs for human vaccines seems to be limited due to the safety concerns on the presence of viable cells in BGs. Therefore, to improve the lysis efficiency of the gene E, we exploited the peptidoglycan hydrolyzing ability of the λ phage holin-endolysins to expedite the process of current BG production system. In this report, we constructed a novel ghost plasmid encoding protein E and holin-endolysins in tandem. We observed that sequential expressions of the gene E and the holin-endolysins elicited rapid and highly efficient Salmonella lysis compared to the lysis mediated by gene E only. These lysed BGs displayed improved immunogenicity in mice compared to the gene E mediated BGs. Consequently, seventy percent of the mice immunized with these novel ghosts survived against a lethal challenge while all the mice vaccinated with gene E mediated ghosts died by day 9 post-infection. We conclude that this novel strategy has the potential to generate highly efficient inactivated candidate vaccines that could replace the currently available bacterial vaccines.

Medicinal plants are frequently used for the treatment of various infectious diseases. The objective of this study was to evaluate the antibacterial activity and mode of action of Acacia nilotica and the antibiogram patterns of foodborne and clinical strains of Escherichia coli and Salmonella . The mechanism of action of acacia extracts against E. coli and Salmonella was elucidated by observing morphological damages including cell integrity and cell membrane permeability, as well as changes in cell structures and growth patterns in kill-time experiments. The clinical isolates of E. coli and Salmonella were found resistant to more of the tested antibiotics, compared to food isolates. Minimum inhibitory concentration and minimum bactericidal concentration of acacia leaf extracts were in the ranges of 1.56-3.12 mg/mL and 3.12-6.25 mg/mL, respectively, whereas pods and bark extracts showed somewhat higher values of 3.12-6.25 mg/mL and 6.25-12.5 mg/mL, respectively, against all tested pathogens. The release of electrolytes and essential cellular constituents (proteins and nucleic acids) indicated that acacia extracts damaged the cellular membrane of the pathogens. These changes corresponded to simultaneous reduction in the growth of viable bacteria. This study indicates that A. nilotica can be a potential source of new antimicrobials, effective against antibiotic-resistant strains of pathogens.

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity.

Many motile bacteria swim by rotating their motility organ, the flagellum. Rotation of the flagellum is driven by a motor at its base, and torque is generated by the rotor-stator interaction coupled with the specific ion flow through the channel in the stator. Because the stator works as an energy-conversion complex in the motor, understanding the functional mechanism of the stator is critically important. But its characterization has been hampered due to the difficulty in isolating the functional stator complex from the membrane. Recently, successful new approaches for studying the stator have been reported that reveal its novel properties. Two of those, visualization of the in vivo behavior of stator units using fluorescently tagged proteins and structure-guided functional analyses of the soluble region in the stator, are summarized in this short review., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: The objective of this study was to determine the interactions between common spoilage yeast, Candida tropicalis, isolated from ultrafiltration membranes, and Escherichia coli O157:H7 and Salmonella sp. on stainless steel surfaces., Methods and Results: Single and dual-species attachment assays were performed on stainless steel at 25°C using apple juice as culture medium. The growth of Salmonella sp. rose when it was co-cultivated with C. tropicalis in dual biofilms at 16 and 24 h; the same effect was observed for E. coli O157:H7 at 24 h. The colonization of C. tropicalis on stainless steel surfaces was reduced when it was co-cultivated with both pathogenic bacteria, reducing C. tropicalis population by at least 1.0 log unit. Visualization by SEM demonstrated that E. coli O157:H7 and Salmonella sp. adhere closely to hyphal elements using anchorage structures to attach to the surface and other cells., Conclusions: These results suggest a route for potential increased survival of pathogens in juice processing environments. These support the notion that the species involved interact in mixed yeast-bacteria communities favouring the development of bacteria over yeast., Significance and Impact of the Study: This study support the plausibility that pathogen interactions with strong biofilm forming members of spoilage microbiota, such as C. tropicalis, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella sp. in food-processing environments., (© 2014 The Society for Applied Microbiology.)

Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research.

Our studies were undertaken to develop new insights into the function of the Salmonella Stn protein. An analysis of total cell membrane protein fraction suggested the possibility that Stn associates with OmpA. This possibility was confirmed by immunogold labeling using anti-OmpA antibody and far-western blotting. From these results, we conclude that Stn regulates membrane composition and integrity in Salmonella.

Biofilms contain a diverse range of microorganisms and their varying extracellular polysaccharides. The present study has revealed biofilm succession associated with degradative effects on plastic (polypropylene) and contaminants in sludge. The wet weight of biofilm significantly (p < 0.05) increased; from 0.23 ± 0.01 to 0.44 ± 0.01 g. Similarly, the dry weight of the biofilm increased from 0.02 to 0.05 g. Significant reduction in pathogens (E. coli and feacal coliforms) by MPN technique (>80%) and in chemical parameters (decrease in COD, BOD5 of 73.32 and 69.94%) representing diminution of organic pollutants. Energy dispersive X-ray spectroscopy (EDS) of plastic revealed carbon and oxygen contents, further surface analysis of plastic by scanning electron microscopy (SEM) revealed emergence of profound bacterial growth on the surface. Fourier transform infrared (FTIR) spectroscopy conforms its biotransformation under aerobic conditions after 8 weeks. New peaks developed at the region 1050 and 969 cm(-1) indicating CO and CC bond formation. Thus plastic with 6 weeks old aerobic biofilm (free of pathogens, max. weight, and OD, efficient COD & BOD removal ability) is suggested to be maintained in fixed biofilm reactors for wastewater treatment., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus.

The mechanism of action of Salmonella enterotoxin (Stn) as a virulence factor in disease is controversial. Studies of Stn have indicated both positive and negative effects on Salmonella virulence. In this study, we attempted to evaluate Stn function and its effects on Salmonella virulence. To investigate Stn function, we first performed in vitro and in vivo analysis using mammalian cells and a murine ileal loop model. In these systems, we did not observe differences in virulence phenotypes between wild-type Salmonella and an stn gene-deleted mutant. We next characterized the phenotypes and molecular properties of the mutant strain under various in vitro conditions. The proteomic profiles of the total cell membrane protein fraction differed between wild type and mutant in that there was an absence of a protein in the mutant strain, which was identified as OmpA. By far-western blotting, OmpA was found to interact directly with Stn. To verify this result, the morphology of Salmonella was examined by transmission electron microscopy, with OmpA localization being analyzed by immunogold labeling. Compared with wild-type Salmonella, the mutant strain had a different pole structure and a thin periplasmic space; OmpA was not seen in the mutant. These results indicate that Stn, via regulation of OmpA membrane localization, functions in the maintenance of membrane composition and integrity.

This study was conducted to gain insights into the survival of Salmonella on a polypropylene surface in relation to the ability of these bacteria to form a biofilm. We selected Salmonella strains known for the relative ease or difficulty with which they formed biofilms based on microtiter plate assays and studied the survival of these strains on polypropylene discs in a desiccation chamber by sequentially counting CFUs. The biofilm-forming strains survived longer on the plastic disc surface than did biofilm-deficient strains. The biofilm-forming strains remained at over 10(4) CFU per plate until day 175, whereas the biofilm-deficient strains decreased to below 10(2) CFU per plate on day 20 or below 10(4) CFU per plate on day 108. Extracellular materials on the polypropylene surface were observed by scanning electron microscopy and crystal violet staining for the biofilm-forming strains but not for the biofilm-deficient strains. The extracellular polymeric materials on the polypropylene surface may have protected the bacterial cells from dryness, although the possibility of some inherent resistance to environmental stresses linked to biofilm formation could not be excluded. These results indicate that Salmonella strains with high biofilm productivity may be a greater risk to human health via food contamination by surviving for longer periods compared with strains with low biofilm productivity.

The goal of this paper is to introduce a universal method for quantitative control of the particle size of magnetic cellulose microspheres (MCMS) and to produce an optimal antibody absorption capability as an aid in the research of new applications of MCMS in immunomagnetic capture. In this study, "the smallest critical size theory" (TSCS) was proposed, tested, and confirmed by IgG-carrying capability measurements, magnetic response analysis, immunomagnetic capture, and PCR identification of bacteria. A Gaussian expression was proposed and used to guide the preparation of MCMS of the smallest critical size (SCS). The results showed that the diameter of the SCS of MCMS in this study was 5.82 mum, while the IgG absorption capability of the MCMS with SCS was 186.8 mg/mL. In addition, its high sensitivity and the efficiency of immunomagnetic capture of Salmonella bacteria exhibited another new application for MCMS.

A study was conducted to determine if a surface material with antimicrobial properties combined with an effective disinfectant could achieve total clearance of bacterial contamination. Before beginning the project, new rubber picker fingers collected from 3 processing facilities were tested for endogenous microflora. Five species of bacteria common to soil and human handling were present: Bacillus amyloliquefaciens, Bacillus cereus/thuringiensis, Staphylococcus epidermidis, Staphylococcus hominis ssp. novobiosepticus, and Staphylococcus intermedius. In separate experiments, new (unused) rubber picker fingers from 3 manufacturers were exposed to broiler carcass rinses, and the kinetics of bacterial attachment to finger material was determined. Turbidity of the bacterial suspensions at varying dilutions containing picker finger sections was compared hourly with controls to evaluate inhibition. New rubber finger material from the 3 manufacturers significantly inhibited bacterial growth (P < 0.05), without the aid of antibacterial additives. We improved an assay for screening disinfectants against growth of pathogens and determined the activity of 5 disinfectant compounds. Two of the compounds were most effective against Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Salmonella Enteritidis, and Escherichia coli, and one of the compounds was selected for further study with Salmonella Enteritidis. Scanning electron microscopy confirmed the levels of Salmonella Enteritidis before and after treatment. The most effective compound was nontoxic and completely cleared Salmonella Enteritidis contamination from the rubber picker finger surface.

The heat resistance of Salmonella Anatum inoculated on the surface of a model vegetable as affected by hot-air drying temperature (50 to 70 degrees C) and surface characteristics was determined in this study. Cabbage was selected as a model vegetable to demonstrate the effect of topographical feature of vegetable surface on the Salmonella attachment ability. An image analysis technique was developed to monitor the change of cabbage surface during drying and the specific surface characteristics were described in terms of the roughness factor (R). It was found that the water activity of the vegetable decreased while R-value increased with longer drying time and higher drying temperature. However, the changes of both parameters during drying did not show a significant effect on the susceptibility of Salmonella attached on the cabbage surface. Drying temperature was found to be a major factor influencing the heat resistance of Salmonella during drying.

Increasingly, genetically modified Salmonella are being explored as a novel treatment for cancer because Salmonella preferentially replicate within tumors and destroy cancer cells without causing the septic shock that is typically associated with wild-type S. typhimurium infections. However, the mechanisms by which genetically modified Salmonella strains preferentially invade cancer cells have not yet been addressed in cellular detail. Here we present data that show S. typhimurium strains VNP20009, LT2, and CRC1674 invasion of PC-3M prostate cancer cells. S. typhimurium-infected PC-3M human prostate cancer cells were analyzed with immunofluorescence microscopy and transmission electron microscopy (TEM) at various times after inoculation. We analyzed microfilaments, microtubules, and DNA with fluorescence and immunofluorescence microscopy. 3T3 Phi-Yellow-mitochondria mouse 3T3 cells were used to study the effects of Salmonella infestation on mitochondria distribution in live cells. Our TEM results show gradual destruction of mitochondria within the PC-3M prostate cancer cells with complete loss of cristae at 8 h after inoculation. The fluorescence intensity in YFP-mitochondria-transfected mouse 3T3 cells decreased, which indicates loss of mitochondria structure. Interestingly, the nucleus does not appear affected by Salmonella within 8 h. Our data demonstrate that genetically modified S. typhimurium destroy PC-3M prostate cancer cells, perhaps by preferential destruction of mitochondria.

To explore the possible occurrence of sources, other than GlgC, of ADPglucose linked to bacterial glycogen biosynthesis we characterized Escherichia coli and Salmonella DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery. These mutants displayed the expected glycogen-less phenotype but accumulated ADPglucose. Importantly, DeltaglgCAP cells expressing the glycogen synthase encoding glgA gene accumulated glycogen. Protein chromatographic separation of crude extracts of DeltaglgCAP mutants and subsequent activity measurement analyses revealed that these cells possess various proteins catalyzing the conversion of glucose-1-phosphate into ADPglucose. Collectively these findings show that enterobacteria possess more than one important source of ADPglucose linked to glycogen biosynthesis.

Bacterial flagella can adopt several different helical shapes in response to varying environmental conditions. A geometric model by Calladine ascribes these discrete shape changes to cooperative transitions between two stable tertiary structures of the constituent protein, flagellin, and predicts an ordered set of 12 helical states called polymorphic forms. Using long polymers of purified flagellin, we demonstrate controlled, reversible transformations between different polymorphic forms. While pulling on a single filament using an optical tweezer, we record the progressive transformation of the filament and also measure the force-extension curve. Both normal and coiled polymorphic forms stretch elastically with a bending stiffness of 3.5 pN x microm(2). At a force threshold of 4-7 pN or 3-5 pN (for normal and coiled forms, respectively), a fraction of the filament suddenly transforms to the next, longer, polymorphic form. This transformation is not deterministic because the force and amount of transformation vary from pull to pull. In addition, the force is highly dependent on stretching rate, suggesting that polymorphic transformation is associated with an activation energy.

The amplitude contrast of frozen-hydrated biological samples was measured using the bacterial flagellar filament embedded in vitreous ice at an accelerating voltage of 300kV. From the mean radial amplitude spectra of overfocused images, amplitude contrast was estimated to be 6.9+/-1.9% and 2.7+/-1.0% of the whole contrast at the low spatial frequency range with and without energy filtering, respectively, and that of the carbon film to be 9.5+/-2.0% and 5.8+/-1.8%. Energy filtering effectively doubled the signal-to-noise ratio in the images of frozen-hydrated filaments, and substantially improved intensity data statistics of layer lines up to at least approximately 25A resolution in their Fourier transforms. It also markedly improved inter-particle fitting phase residuals of averaged data at resolutions up to approximately 15A. Using the energy filtered data recorded on a new high-performance, lens-coupled CCD camera the three-dimensional map of the flagellar filament was calculated at 8A by applying the amplitude contrast of 6.9%. The map and its mean radial density distribution validated the obtained value of the amplitude contrast.

Salmonella FliI is the flagellar ATPase which converts the energy of ATP hydrolysis into the export of flagellar proteins. It forms a ring-shaped oligomer in the presence of ATP, its analogs, or phospholipids. The extreme N-terminal region of FliI has an unstable conformation and is responsible for the interaction with other components of the export apparatus and for regulation of the catalytic mechanism. To understand the role of this N-terminal region in more detail, we used multi-angle light-scattering, analytical ultracentrifugation, far-UV CD and biochemical methods to characterize a partially functional variant of FliI, missing its first seven amino acid residues (His-FliI(Delta1-7)), whose ATPase activity is about ten times lower than that of wild-type FliI. His-FliI(Delta1-7) is monomeric in solution. The deletion increased the content of alpha-helix, suggesting that the deletion stabilizes the unstable N-terminal region into an alpha-helical conformation. The deletion did not influence the K(m) value for ATP. However, unlike the wild-type, ATP and acidic phospholipids did not induce oligomerization of His-FliI(Delta1-7) or increase its ATPase activity. These results suggest that the deletion suppresses the oligomerization of FliI, and that a conformational change in the unstable N-terminal region is required for FliI oligomerization to effectively couple the energy of ATP hydrolysis to the translocation of flagellar proteins.

The flagellum-specific export system is a specialized type III export machinery. Terminally truncated fragments of flagellin (FliC) were used to identify the secretion signal in the main component of flagellar filaments. The first 13 residues were not essential for export, but removal of 29 or more residues destroyed export ability. When an 8kDa human protein domain was fused to various N-terminal fragments of FliC, the 26-47 sequence alone was sufficient to mediate secretion of this protein module through the flagellum specific export pathway. Neither half of this segment was enough to direct export of the attached protein domain. Our results demonstrate that the 22-residue long 26-47 segment within the disordered N-terminal region of Salmonella flagellin contains the recognition signal for the flagellar export machinery.

It has been demonstrated that horizontal and vertical transmission of Salmonella and Campylobacter can occur in broiler breeder flocks. The mechanism of this transmission is still unclear. Previously negative broiler breeder flocks have been reported to become positive with Salmonella, Campylobacter, or both after the introduction of "spike" roosters at 45 wk of age. To determine whether the rooster semen is a possible source of transmission to hens for colonization, we evaluated the association of both Salmonella and Campylobacter spp. to segments (head, midpiece, and tail) of individual spermatozoa after artificial inoculation. Salmonella typhimurium, Salmonella heidelberg, and Salmonella montevideo, or Campylobacter jejuni (in 0.85% saline) was added to a freshly collected (by abdominal massage) aliquot of pooled semen from roosters housed in individual cages. The semen and bacteria solutions were incubated 1 h at room temperature. Samples were fixed using Karnosvsky and Zamboni fixatives for 24 h prior to centrifuging and rinsing in 0.1 M cacodylate x HCl buffer. Individual aliquot samples were then subjected to both scanning (JSM-5800) and transmission (JEM-1210) electron microscopy. The scanning electron microscopy showed that Salmonella was associated with all 3 segments (head, midpiece, and tail) of the spermatozoa and apparently equally distributed. Campylobacter was mainly associated with the midpiece and tail segments; few isolates were located on the head segment. The transmission electron microscopy showed apparent attachment of Salmonella and Campylobacter to the spermatozoa.

The length of the flagellar hook is regulated; it is 55 +/- 6 nm long in Salmonella. Five genes involved in hook-length regulation are fliK, flhB, fliG, fliM and fliN. The last four genes encode structural components of the protein export apparatus in the flagellar base, whereas FliK is soluble and secreted during flagellar assembly. The role of FliK, however, remains ambiguous. We constructed two kinds of FliK variants: N-terminally truncated FliK protein and FliK N-terminally fused with cyan fluorescent protein (CFP-FliK). Both N-terminally truncated FliK missing the first 99 amino acids (aa) and CFP-FliK fusion variants partially complemented a fliK null (polyhook) mutant to produce cells with filaments, allowing cells to swim; the hooks, however, were not normal but were polyhooks. When the N-terminally defective FliK variants were expressed at high levels, the average polyhook length was shortened coming close to the length of the wild-type hook, independently of the sizes of the FliK variants. These FliK variants were not secreted. CFP-FliK fusion proteins were observed to homogeneously distribute in the cytoplasm. We conclude that FliK does not need to be exported to control hook length and is unlikely to be a ruler; instead, we conclude that FliK controls hook length by the timely switching of secretion modes of the flagellar type III secretion system by the FliK C-terminal domain, and that the N-terminal region is dispensable for hook length control.

To study the effect of processing and storage parameters on the survival of Salmonella on fresh Italian parsley, parsley bunches were dipped for 3 or 15 min in suspensions that were preequilibrated to 5, 25, or 35 degrees C and inoculated with Salmonella transformed to express enhanced green fluorescent protein. Loosely attached and/or associated, strongly attached and/or associated, and internalized and/or entrapped Salmonella cells were enumerated over 0, 1, and 7 days of storage at 25 degrees C and over 0, 1, 7, 14, and 30 days of storage at 4 degrees C using surface-plating procedures. Leaf sections obtained from samples after 0, 1, and 7 days of storage were examined using confocal scanning laser microscopy. Temperature of the dip suspension had little effect on the attachment and survival of Salmonella cells on parsley. Regardless of the temperature or duration of dip, Salmonella was internalized. Immersion for longer times resulted in higher numbers of attached and internalized cells. Microscopic observations supported these results and revealed Salmonella cells near the stomata and within cracks in the cuticle. Storage temperature had the greatest impact on the survival of Salmonella cells on parsley. When stored at 25 degrees C, parsley had a shelf life of 7 days, and Salmonella populations significantly increased over the 7 days of storage. For parsley stored at 4 degrees C, numbers of Salmonella cells decreased over days 0, 1, and 7. After 7 days of storage, there were no viable internalized Salmonella cells detected. Storage temperature represents an important control point for the safety of fresh parsley.

The bacterial flagellum is a supramolecular structure consisting of a basal body, a hook and a filament. Most of the flagellar components are translocated across the cytoplasmic membrane by the flagellar type III protein export apparatus in the vicinity of the flagellar base, diffuse down the narrow channel through the nascent structure and self-assemble at its distal end with the help of a cap structure. Flagellar proteins synthesized in the cytoplasm are targeted to the export apparatus with the help of flagellum-specific chaperones and pushed into the channel by an ATPase, whose activity is controlled by its regulator to enable the energy of ATP hydrolysis to be efficiently coupled to the translocation reaction. The export apparatus switches its substrate specificity by monitoring the state of flagellar assembly in the cell exterior, allowing this huge and complex macromolecular assembly to be built efficiently by a highly ordered and well-regulated assembly process., (Copyright 2004 S. Karger AG, Basel)

The cap at the growing end of the bacterial flagellum is essential for its growth, remaining stably attached while permitting the insertion of flagellin transported from the cytoplasm through the narrow central channel. We analyzed the structure of the isolated cap in its frozen hydrated state by electron cryomicroscopy. The 3D density map now shows detailed features of domains and their connections, giving reliable volumes and masses, making assignment of the domains to the amino acid sequence possible. A model of the cap-filament complex built with an atomic model of the filament allows a quantitative analysis of the cap domain movements on cap binding and rotation that promotes the efficient self assembly of flagellin during the filament growth process.

Trisodium phosphate (TSP) has been reported to have antimicrobial activity and is approved by the U.S. Department of Agriculture for use in food processing applications. A novel (U.S. Patent 6,184,198), antimicrobial solution containing a blend of TSP, sodium bicarbonate, and sodium carbonate (TSP blend) has demonstrated effective inhibition of microbial contamination in a broad spectrum of applications. This high-resolution cold field emission scanning electron microcopy (LVSEM) investigation details structural changes and dry film formation in various classes of microbes as a mechanism for antimicrobial activity of this solution. The results showed that this TSP blend solution completely inhibited the growth of pathogenic E. coli O157:H7, and Salmonella bacteria, the mold Cryptococcus, as well as a Norwalk virus surrogate-feline calicivirus. Results by LVSEM confirmed that the antimicrobial effect was induced when encapsulating the target microbes in a high lubricity film that is formed around these organisms as the solution dries. The thickness of the film was estimated to be approximately 60 nm.

The influence of inoculum populations and environmental factors on attachment of Salmonella Montevideo to the surface of tomatoes and tomatillos was evaluated. To study the effect of inoculum size, red, ripe tomatoes were spot-inoculated with bacterial suspensions (10(5) and 10(8) CFU/fruit) and stored at 22 degrees C under 100% relative humidity. The effects of temperature (12, 22, and 30 degrees C) and relative humidity (75, 85, and 97%) on attachment of the pathogen (10(7) CFU/fruit) to tomatoes (red and green) and ripe tomatillos were also evaluated. Inoculated fruits were stored for 90 min at all combinations of temperature and relative humidity, and after rinsing with water, the number of cells attached to the surface was determined. Salmonella Montevideo attached to the surface of tomatoes within 90 min. A direct correlation between the number of attached cells and the population in the inoculum was observed. The percentage of cells that attached immediately after inoculation was approximately 0.3% for the three test products. After storage for 90 min at various temperature and relative humidity conditions, the number of adhering cells ranged from 4.0 to 5.4 log CFU/fruit (1.2% of inoculum). Both the type of product and the temperature/relative humidity combination had a significant (P < 0.05) effect on attachment of Salmonella Montevideo to the surfaces of tomatoes and tomatillos. Scanning electron micrographs of the cuticles of inoculated washed tomatoes and tomatillos revealed typical skin cell patterns, and only a few randomly dispersed Salmonella Montevideo were observed. Deposition of Salmonella Montevideo on the surface of tomatoes and tomatillos could result in attachment and subsequent colonization under suitable conditions.

Salmonella is an interesting example of how the selective pressure of host environments has led to the evolution of sophisticated bacterial virulence mechanisms. This microbe exploits the first-line of defence, the macrophage, as a crucial tool in the initiation of disease. After invasion of intestinal macrophages, a virulence protein secreted by Salmonella specifically induces apoptotic cell death by activating the cysteine protease caspase-1. The pro-apoptotic capability is necessary for successful pathogenesis. The study of mechanisms by which Salmonella induces programmed cell death offers new insights into how pathogens cause disease and into general mechanisms of activation of the innate immune system.

Aims: To relate the diseases caused by strains of Salmonella paratyphi B and S. java to pathogenic mechanisms expressed by these bacteria for the purpose of organism discrimination., Methods and Results: Epidemiological data relating to cases of disease caused by strains of S. paratyphi B and S. java, isolated over a 10-year period, were analysed with respect to patients' symptoms, particularly those involving enteric fever. Strains of S. paratyphi B and S. java were also examined for a range of known pathogenic mechanisms. Infection with S. paratyphi B involved pyrexia in 12.5% of patients compared with 2.2% of patients infected with S. java. These organisms could not be differentiated based on the pathogenic properties examined., Conclusions: Strains of S. paratyphi B appear not to be a major cause of enteric fever but primarily a cause of gastroenteritis, in common with S. java. Both organisms express similar pathogenic mechanisms, and strains of S. java are probably d-tartrate utilizing variants of S. paratyphi B., Significance and Impact of the Study: Strains of S. paratyphi B are very closely related organisms, primarily causing gastroenteritis. From this study it would appear that strains of S. paratyphi B are not a major cause of enteric fever.

The structure of membranes of Salmonella derby cells both containing R-plasmid and free of plasmid was studied by small- and large-angle X-ray diffraction. Reflections with interplane distances of 8 and 11 A were detected, which are typical of plasmid-carrying S. derby cells. These reflections are assumed to be due to equidistant well ordered positions of the polar groups of phosphatidylcholine and phosphatidylethanolamine molecules on membrane surface. It is also suggested that the formation of these structures is determined by peculiar hydrophilic-hydrophobic interactions of the phospholipid in membranes.

Effects of the pyrimidine derivatives of xymedone and methyluracil upon the induction of point mutations of the base pair substitution type in the Salmonella/microsome test and the frequency of chromosome aberrations in the lymphocytes of patients with the chronic osteomyelitis diagnosis. Xymedone more effectively than methyluracil inhibited the induction of point mutations by nitrosomethylurea. In the case of the cyclophosphane induced mutagenesis, the two preparations exhibited comparable antimutagen effects. Both xymedone and methyluracil (1.5 g/day, 10 days) reduced the (increased) frequency of chromosomal aberrations in the lymphocytes of patients with the chronic osteomyelitis diagnosis. The anticlastogenic effect of methyluracil vanished 5 days after termination of the 10-day administration course, while xymedone exhibited an afteraction anticlastogenic effect over this period. Interrelation of the antimutagen effect and immunomodulating activity of xymedone is discussed.

Attachment and growth of Salmonella Chester on fresh-cut apple disks and in vivo response of attached bacteria to sanitizer treatments were investigated. Apple disks (14 mm in diameter and 3 to 4 mm in thickness) were immersed in a bacterial suspension that contained 8.17 log CFU/ml of Salmonella Chester and air dried at room temperature for 10 min. After two rinses, the population of Salmonella Chester retained on apple disks that contained no skin was 13 to 19% higher than that retained on disks that contained skin, indicating that Salmonella Chester attached more firmly to the surfaces of injured tissue than to the unbroken skin. The number of bacteria attached to the disk was not affected by the immersion time but was directly proportional to the concentration of bacteria in the suspension. The distribution of artificially inoculated Salmonella Chester on the surfaces of three different parts of whole fruit was determined; 94% of attached bacteria was found on the stem and calyx cavity areas and 6% on the skin of the remaining area of the fruit. Despite their acidic pH (4.1), apple disks supported the growth of Salmonella Chester at 20 degrees C but not at 8 degrees C. All four sanitizers tested in the study, including 6% hydrogen peroxide, 2% trisodium phosphate, 0.36% calcium hypochlorite, and 1.76% sodium hypochlorite, were effective in reducing the population of Salmonella Chester on apple disks by 1 to 2 logs. However, 5 to 13% of bacteria survived the sanitizer treatments. Hydrogen peroxide, which reduced the population of Salmonella Chester on skin by 3 to 4 logs and the population of bacteria on stem or calyx by 1 to 2 logs, was the most effective among the four sanitizers tested. Firm attachment of bacteria on calyx, stem, and injured tissue and partial resistance of attached bacteria to sanitizer treatments are two major obstacles to be considered when developing methods for cleaning and decontaminating apple fruits destined for juice production and fresh consumption.

FliE is a flagellar basal body protein of Salmonella whose detailed location and function have not been established. A mutant allele of fliE, which caused extremely poor flagellation and swarming, generated extragenic suppressors, all of which mapped to flgB, one of four genes encoding the basal body rod; the fliE flgB pseudorevertants were better flagellated and swarmed better than the fliE parent, especially when the temperature was reduced from 37 to 30 degrees C. Motility of the pseudorevertants in liquid culture was markedly better than motility on swarm plates; we interpret this to mean that reduced flagellation is less deleterious at low viscous loads. Overproduction of the mutant FliE protein improved the motility of the parental fliE mutant and its pseudorevertants, though not to wild-type levels. Overproduction of suppressor FlgB (but not wild-type FlgB) in the fliE mutant also resulted in improved motility. The second-site FlgB mutation by itself had no phenotype; cells swarmed as well as wild-type cells. When overproduced, wild-type FliE was dominant over FliE-V99G, but the reverse was not true; that is, overproduced FliE-V99G was not negatively dominant over wild-type FliE. We conclude that the mutant protein has reduced probability of assembly but, if assembled, functions relatively well. Export of the flagellar protein FlgD, which is known to be FliE dependent, was severely impaired by the FliE-V99G mutation but was significantly improved in the suppressor strains. The FliE mutation, V99G, was close to the C terminus of the 104-amino-acid sequence; the suppressing mutations in FlgB were all either G119E or G129D, close to the C terminus of its 138-amino-acid sequence. Affinity blotting experiments between FliE as probe and various basal body proteins as targets and vice versa revealed strong interactions between FliE and FlgB; much weaker interactions between FliE and other rod proteins were observed and probably derive from the known similarities among these proteins. We suggest that FliE subunits constitute a junction zone between the MS ring and the rod and also that the proximal rod structure consists of FlgB subunits.

Patterns of invasiveness of Salmonella serotypes Typhimurium, Choleraesuis and Dublin in Caco-2 cells (without centrifugation) were compared with previously published studies of the rabbit ileal invasion assay (RIIA) and (where relevant) a HEp-2 cell invasion assay. Optimal conditions for the use of Caco-2 cell monolayers in bacterial invasion assays were defined. Centrifuge-assisted attachment of bacteria to cells was not used routinely as this increased the invasiveness of known hypo-invasive strains and detachment of Caco-2 cells. Inocula with too high bacterial numbers resulted in rapid acidification of media and detachment of the monolayers. The invasiveness of Typhimurium strains TML, WAKE, WII8, LT7, SL1027 and M206 in Caco-2 cells reflected that seen in the RIIA. The invasiveness of Choleraesuis strain A50 was similar to that in the RIIA except that bacteria grown at 37 degrees C and used without storage at 4 degrees C were slightly more invasive than those grown at 37 degrees C and stored at 4 degrees C before use. Dublin strain 3246 showed no apparent temperature-regulated invasiveness in Caco-2 cells, in contrast to the results observed in the RIIA. Dublin strain 3246 did not cleave tight junctions in the Caco-2 cell monolayer as it did in rabbit ileal epithelia both in vitro and in vivo. Three TnphoA insertion LPS mutants of Typhimurium TML were uniformly hypo-invasive in both Caco-2 cells and the RIIA; in contrast, they were differentially invasive in HEp-2 cells. Three smooth TnphoA insertion mutants of Typhimurium TML (invH, invG and pagC) were hypo-invasive in both the Caco-2 and HEp-2 cell invasion assays but not in the RIIA.

The flagellar hook of Salmonella is a filamentous polymer made up of subunits of the protein FlgE. Hook assembly is terminated when the length reaches about 55 nm. After our recent study of the effect of cellular levels of the hook length control protein FliK, we have now analyzed the effect of cellular levels of FlgE itself. When FlgE was overproduced in a wild-type strain, a fliC (flagellin) mutant, or a fliD (hook-associated protein 2 [HAP2], filament capping protein) mutant, the hooks remained at the wild-type length. In a fliK (hook length control protein) mutant, which produces long hooks (polyhooks), the overproduction of FlgE resulted in extraordinarily long hooks (superpolyhooks). In a flgK (HAP1, first hook-filament junction protein) mutant or a flgL (HAP3, second hook-filament junction protein) mutant, the overproduction of FlgE also resulted in longer than normal hooks. Thus, at elevated hook protein levels not only FliK but also FlgK and FlgL are necessary for the proper termination of hook elongation. When FlgE was severely underproduced, basal bodies without hooks were often observed. However, those hooks that were seen were of wild-type length, demonstrating that FlgE underproduction decreases the probability of the initiation of hook assembly but not the extent of hook elongation.

FliL is one of the least understood proteins in the flagellar systems of Salmonella and Escherichia coli. There is no apparent mutant phenotype associated with it, even when virtually the entire coding sequence has been eliminated. In this study it has been shown that FliL is a cytoplasmic membrane protein associated with the basal body. Although it has a sequence that conforms to the consensus cleavage site for lipoproteins, FliL does not undergo cleavage or modification under physiological conditions.

Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive fliQ or fliR mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.

The peritrichous flagella of Salmonella are synthesized and function through many cell generations. There are two different aspects in the relationship between flagellar biogenesis and cell division. Filament growth is independent from the cell cycle and the length of filaments appear to be locally controlled at each flagellar base, whereas the number of filaments (or flagellar basal bodies) is dependent on cell cycle. We present a model to explain how the number of filaments is maintained through generations. We will also introduce a new direction for research that might directly connect flagellation and cell division; the global communication between flagellar genes and external factors of a complex regulatory network in a cell.

Growth of strains of Salmonella enteritidis and Salmonella pullorum on Hektoen agar has been reported to influence the expression of long-chain lipopolysaccharide and motility respectively. In this study we used a panel of strains of S. enteritidis and S. pullorum to investigate these phenomena. Culture on Hektoen agar did not cause rough strains of S. enteritidis to express long-chain lipopolysaccharide or strains of S. pullorum to become motile. It was concluded that growth of strains of S. enteritidis and S. pullorum on Hektoen agar would not normally affect the expression of somatic or flagellar antigens, and would not influence the interpretation of the Kauffman-White typing scheme.

Antibodies raised against four hybrid Salmonella flagellins carrying amino acid sequences derived from the fimbrial subunit of the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC), i.e. hybrid flagellins Fla I (aa 1-15), Fla II (aa 11-25), Fla III (aa 32-45) and Fla IV (aa 88-102), were not able to inhibit the in vitro binding of CFA/I-expressing ETEC bacteria to enterocyte-like Caco-2 cells. However, one of the hybrid flagellins (Fla II) was recognized by a previously described anti-CFA/I subunit mAb (S-CFA/I 17:8) which was able to block adhesion of CFA/I-expressing bacteria to Caco-2 cells and to bind to the amino acid sequences 15IDLLQ19 of the CFA/I fimbrial subunit. Pepscan analysis of antibodies raised against the hybrid flagellins Fla II and Fla IV showed that they were specific for the sequences 14VIDLL18 and 96FEAAAL101, respectively, of the CFA/I fimbrial subunit. Thus, the discrepancy in the abilities of the anti-Fla II serum and the mAb S-CFA/I 17:8 to block binding might be ascribed to their slightly different fine specificity for epitopes.

In many pathogens, virulence can be conferred by a single region of the genome. In contrast, the facultative intracellular lifestyle of Salmonella demands a large number of genes distributed around the chromosome. The evolution of Salmonella has been marked by the acquisition of several 'pathogenicity islands', each contributing to the unique virulence properties of this microorganism.

The survival and fate of Salmonella enterica serotype Enteritidis in Henle-407 human intestinal epithelial cells was investigated during prolonged incubation to evaluate the persistence of causative microbes and the relationship to patients developing reactive arthritis. Most of the bacteria were killed and degraded quite soon after infection of the cells, but there were still live bacteria inside the cells for up to 14 days. These results suggest that in patients developing reactive arthritis the salmonellae could persist in the epithelial cells and spread within the host to the joint and be present there at the time of the inflammatory response. Production of marked amounts of nitric oxide was observed as a novel response to salmonella infection in human intestinal epithelial cells. The present experimental procedure appears to be a suitable model to further investigate host-bacteria interaction in HLA-B27 positive cells from patients developing reactive arthritis.

A rough (R) Brucella abortus 45/20 mutant was more sensitive to the bactericidal activity of polymyxin B and lactoferricin B than was its smooth (S) counterpart but considerably more resistant than Salmonella montevideo. The outer membrane (OM) and isolated lipopolysaccharide (LPS) of S. montevideo showed a higher affinity for these cationic peptides than did the corresponding B. abortus OM and LPS. We took advantage of the moderate sensitivity of R B. abortus to cationic peptides to construct live R B. abortus-S-LPS chimeras to test the activities of polymyxin B, lactoferricin B, and EDTA. Homogeneous and abundant peripheral distribution of the heterologous S-LPS was observed on the surface of the chimeras, and this coating had no effect on the viability or morphology of the cells. When the heterologous LPS corresponded to the less sensitive bacterium S B. abortus S19, the chimeras were more resistant to cationic peptides; in contrast, when the S-LPS was from the more sensitive bacterium S. montevideo, the chimeras were more susceptible to the action of peptides and EDTA. A direct correlation between the amount of heterologous S-LPS on the surface of chimeric Brucella cells and peptide sensitivity was observed. Whereas the damage produced by polymyxin B in S. montevideo and B. abortus-S. montevideo S-LPS chimeras was manifested mainly as OM blebbing and inner membrane rolling, lactoferricin B caused inner membrane detachment, vacuolization, and the formation of internal electron-dense granules in these cells. Native S and R B. abortus strains were permeable to the hydrophobic probe N-phenyl-1-naphthylamine (NPN). In contrast, only reduced amounts of NPN partitioned into the OMs of the S. montevideo and B. abortus-S. montevideo S-LPS chimeras. Following peptide exposure, accelerated NPN uptake similar to that observed for S. montevideo was detected for the B. abortus-S. montevideo LPS chimeras. The partition of NPN into native or EDTA-, polymyxin B-, or lactoferricin B-treated LPS micelles of S. montevideo or B. abortus mimicked the effects observed with intact cells, and this was confirmed by using micelle hybrids of B. abortus and S. montevideo LPSs. The results showed that LPS is the main cause of B. abortus' resistance to bactericidal cationic peptides, the OM-disturbing action of divalent cationic chelants, and OM permeability to hydrophobic substances. It is proposed that these three features are related to the ability of Brucella bacteria to multiply within phagocytes.