Deep Rough Strains Research Articles

Sodium polyphosphate and polyethylenimine enhance the antimicrobial activities of plant essential oils

ABSTRACT Plant extracts have been used for millennia for treatment of disease, with much recent interest focusing on the antimicrobial activities of plant essential oils (EOs). Although EOs are active against common microbial pathogens, their effective use as topical, environmental, or food antimicrobials will require EO-based formulations with enhanced antimicrobial activities. In this study, two polyionic compounds, sodium polyphosphate (polyP, a polyanion) and polyethylenimine (PEI, a polycation), were evaluated for their abilities to enhance the antimicrobial activities of six EOs against the human pathogens Escherichia coli O157:H7, Salmonella enterica subsp. enterica ser. Minnesota, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, and Candida albicans. EOs tested were cinnamon, clove, regular and redistilled oregano, and two types of thyme oil. EOs were examined via disk diffusion and broth microdilution, either alone or in the presence of subinhibitory levels of polyP or PEI. Both polyP and PEI were found to be effective enhancers of EO activity against all strains examined, and calculation of fractional inhibitory indices for select EO/organism pairings demonstrated that true synergy was possible with this enhancement approach. Experiments with a deep-rough strain of S. Minnesota probed the role of the outer membrane in both intrinsic resistance to EOs and enhancement by polyions. The use of polyP and PEI for boosting the antimicrobial activities of EOs may eventually facilitate the development of more effective EO-based antimicrobial treatments for use in applications such as wound treatment, surface disinfection, or as generally recognized as safe antimicrobials for use in foods or on food contact surfaces.

Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy

In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotope-labeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region.

Structural and biological characterization of highly purified hepta-acyl lipid A present in the lipopolysaccharide of the Salmonella enterica sv. Minnesota Re deep rough mutant strain R595

One major component of the Salmonella enterica sv. Minnesota Re deep rough mutant (strain R595) lipopolysaccharide is hepta-acyl lipid A (LAhepta). In a recent publication [Tanamoto K-I, Azumi S. Salmonella-type heptaacylated lipid A is inactive and acts as an antagonist of lipopolysaccharide action on human line cells. J Immunol 2000; 164: 3149—3156] the corresponding synthetic hepta-acyl lipid A (compound 516) was reported to be agonistically inactive but to rather suppress pro-inflammatory activation by the endotoxichexa-acyl lipid A (LAhexa, compound 506) and S-form LPS from Escherichia coli in the human macrophage-like cell lines THP-1 and U937. These results, however, were in contrast to previous findings with human mononuclear cells (hMNC) isolated from peripheral blood, in which compound 516 was found to be an agonist, expressing low, but significant,cytokine-inducing activity as compared to LAhexa. We have investigated the structure of natural LA hepta from the S. enterica sv. Minnesota Re deep rough mutant strain (R595) by TLC immunoblot, MALDI-TOF mass spectrometry and NMR spectroscopy. Using these techniques, the structural identity between LAhepta and the synthetic compound 516 was confirmed. In corroboration of previous findings with studies employing compound 516, purified LA hepta was found to induce the production of TNF- , IL-1 and IL-6 in hMNC, thus displaying moderate agonistic activity. Furthermore, we showed that LAhepta agonistically activated nuclear translocation of NF- B in THP-1 cells, thus clearly ruling out the possibility that LAhepta is an antagonist and that its biological activity is influenced by the type of human myeloid cells used for testing endotoxicity(hMNC versus THP-1 cells).

The lipopolysaccharide barrier: correlation of antibiotic susceptibility with antibiotic permeability and fluorescent probe binding kinetics.

Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a permeability barrier, making the outer membrane relatively impermeable to hydrophobic antibiotics, detergents, and host proteins. Mutations in the LPS biosynthetic apparatus increase bacterial susceptibility to such agents. To determine how this increased susceptibility is mediated, we have correlated antibiotic susceptibilities of rough (antibiotic resistant) and deep rough (antibiotic susceptible) bacterial strains with antibiotic permeabilities and fluorescent probe binding kinetics for bilayers composed of LPS purified from the same strains. Bilayer permeabilities of two hydrophobic beta-lactam antibiotics were measured by encapsulating the appropriate beta-lactamases in large unilamellar vesicles. In the presence of MgCl(2), permeabilities of LPS bilayers from rough and deep rough bacteria were similar and significantly lower than those of bacterial phospholipids (BPL). Addition of BPL to the LPS bilayers increased their antibiotic permeability to approximately the level of the BPL bilayers. Binding rates of the fluorescent probe bis-aminonaphthylsulfonic acid (BANS) were 2 orders of magnitude slower for both rough and deep rough LPS bilayers compared to that of bilayers composed of BPL or mixtures of LPS and BPL. On the basis of these results and the observation that deep rough bacteria have higher levels of phospholipid on their surface than do rough bacteria (Kamio, Y., and Nikaido, H. (1976) Biochemistry 15, 2561-2569), we argue that the high susceptibility of deep rough bacteria is due to the presence of phospholipids on their surface. Experiments with phospholipid bilayers showed that the addition of PEG-lipids (containing covalently attached hydrophilic polymers) had little effect on permeability and binding rates, whereas the addition of cholesterol reduced permeability and slowed binding to levels approaching those of LPS. Therefore, we argue that the barrier provided by LPS is primarily due to its tight hydrocarbon chain packing (Snyder et al., (1999) Biochemistry 38, 10758-10767) rather than to its polysaccharide headgroup.

Mechanisms of Action of the Bactericidal/Permeability-Increasing Protein BPI on Endotoxin and Phospholipid Monolayers and Aggregates

We have investigated the mechanisms of interaction of the recombinant N-terminal portion of bactericidal/permeability-increasing protein, rBPI21, with lipopolysaccharide (LPS) isolated from enterobacterial deep rough mutant strains. Experimentally, the ability of rBPI21 to form monolayers at the air/water interface and its action on lipid monolayers were analyzed. We have further studied the interaction of rBPI21 with aggregates from phospholipids and Re mutant LPS by infrared and resonance energy transfer spectroscopy and laser Doppler velocimetry. From monolayer experiments, the molecular area of a single rBPI21 molecule was estimated to be about 12 nm2. At lateral pressures of </=25 mN/m, rBPI21 incorporated into monolayers from negatively charged LPS and phosphatidylglycerol (PG) but not into those from neutral phosphatidylcholine. rBPI21 incorporated not only into monolayers but also into liposomes made from or containing negatively charged phospholipids, reducing the absolute value of the zeta-potential of LPS and PG aggregates. Furthermore, due to intercalation, rBPI21 caused the rigidification of the acyl chains of lipids in the gel as well as in the fluid phase and significantly immobilized their phosphate groups. High concentrations of Mg2+ ions were found to have a protective effect against the action of rBPI21. On the basis of these results, the biophysical characteristics of rBPI21 are discussed and a model is proposed as to how the rBPI21-induced influence on lipid monolayers and bilayers could explain rBPI21-mediated effects on the bacterial membrane.

Lipopolysaccharides and divalent cations are involved in the formation of an assembly-competent intermediate of outer-membrane protein PhoE of E.coli.

To identify the requirements for the biogenesis of outer-membrane proteins in Gram-negative bacteria, the sorting and assembly of the trimeric, pore-forming protein PhoE was studied in vitro. Purified lipopolysaccharide (LPS) in combination with low amounts of Triton X-100 and divalent cations induced the formation of folded monomers. LPS of deep-rough strains was far less efficient in the formation of folded monomers than wild-type LPS was. These folded monomers could be converted into heat-stable trimers upon addition of outer membranes and higher amounts of Triton X-100. Trimerization could precede the insertion step. These in vitro data suggest that the assembly in vivo proceeds sequentially by (i) formation of a folded monomer by interaction with LPS; (ii) sorting of the folded monomers to assembly sites in the outer membrane; (iii) trimerization; and (iv) insertion.

Methanol Extracts LPS from Deep Rough Bacteria

Several rough strains ofEscherichia coli, Salmonella entericaandProteus mirabiliswere cultivated in the presence of (14C)acetate, which incorporated into their phospholipids and lipopolysaccharide. Phospholipids were removed from the cells with ethanol extraction. However, as shown by thin layer chromatography, methanol additionally extracted remarkable quantities of lipopolysaccharide from deep rough strains, but not from bacteria containing complete or nearly complete core.

Isolation and characterization of the tetrasaccharide (bis)phosphate from the glycosyl backbone of Salmonella minnesota and Escherichia coli re-mutant lipopolysaccharides

Intact tetrasaccharide mono- ( 1 ) and bisphosphates ( 2 ) were isolated from deep rough mutant strains of Salmonella minnesota (mutant R595) and Escherichia coli (mutant F515) and their chemical structure was unequivocally characterized by 1H-, 13C- and 31P-NMR spectrometry and FAB-MS and chemical analysis as 1 and 2

Mapping of the binding specificity for five monoclonal antibodies recognizing 3-deoxy-D-manno-octulosonic acid in bacterial lipopolysaccharides.

Abstract Mouse mAb were produced against the deep rough strains Salmonella minnesota R 595, Salmonella typhimurium SL 1102, and Escherichia coli D21f2 and screened by enzyme immunoassay against LPS of several chemotypes. Five antibodies were selected for their ability to bind to chemotype deep rough (Re) LPS which has two 3-deoxy-D-manno-octulosonic acid (Kdo) residues in its nonreducing end. Structurally verified oligosaccharides isolated from rough LPS and synthetic analogues of Kdo were used in an enzyme immunoassay inhibition test to determine the binding epitopes for the antibodies. According to their specificities, the antibodies could be divided into three groups. For two of the groups, the recognized structure was the alpha-Kdo (2----4) Kdo disaccharide and for one group the alpha-Kdo (2----4) alpha-Kdo beta-D-GlcN (1----6) alpha-D-GlcN tetrasaccharide, representing a partial structure of the Re LPS. Inhibition studies with synthetic analogues of Kdo showed that the anomeric configuration and the free carboxyl group of the Kdo residue are important features for antibody binding. Changes in the C-1 to C-6 region of the Kdo molecule do influence the antibody recognition considerably whereas changes in the exocyclic C-7 to C-8 region are of secondary importance. Calculation of the conformation of the inner core region showed that the alpha-Kdo (2----4) alpha-Kdo (2---- disaccharide was free and accessible in chemotype Re LPS, but that linkage of a L-glycero-D-manno-heptose to O-5 of the subterminal Kdo both changes the conformation of the Kdo-disaccharide and covers it thereby making it less accessible.

Partitioning of hydrophobic probes into lipopolysaccharide bilayers

Lipophilic solutes permeate rapidly through lipid bilayer membranes. However, the outer membrane of enteric bacteria, which is composed of a lipopolysaccharide monolayer outer leaflet and the glycerophospholipid inner leaflet, shows extremely low permeability to hydrophobic solutes. In order to examine the cause of this exceptionally low permeability, the lipid/water partition behavior of various lipophilic probes was determined by using lipopolysaccharides of various chemotypes and glycerophospholipids. With all probes, under many different conditions, the lipopolysaccharide/water partition coefficients were generally about an order of magnitude smaller than the phospholipid/water partition coefficients, and this result is consistent with the low permeability of the lipopolysaccharide monolayer, and hence the asymmetric bilayer found in the outer membrane. Furthermore, organic polycations significantly increased the partition of N-phenylnaphthylamine into lipopolysaccharides, a result again consistent with the permeability-increasing effect of such cations on intact outer membrane. Very defective, ‘deep rough’ lipopolysaccharides of chemotypes Rd 2, Rd 1 and Re, had only slightly (20–75%) higher partition coefficients in comparison with the more complete lipopolysaccharides, and this difference is probably not enough to explain the approximately 100-fold increase in lipophile permeability seen in deep rough strains.

Outer membrane vesiculation of acinetobacter calcoaceticus

AbstractFor intact cells of A. calcoaceticus 69V susceptibility to hydrophobic agents (antibiotics, dyes) was established. The composition of its outer membrane and comparison with that of a reference strain, A. calcoaceticus CCM 5593 with a blocked hydrophobic pathway, gave no indication of phospholipid bilayer domains as the structural basis of these permeability characteristics. The outer membrane composition together with the data of time‐resolved fluorescence anisotropy measurements is indicative of a high state of order of the hydrocarbon region.A. calcoaceticus 69V releases lipopolysaccharide (LPS)‐rich membrane vesicles into the growth medium when grown on a hydrophobic carbon source. While the cells contain both R‐form and S‐form LPS, the LPS released with the vesicles is exclusively of the R‐type. The same selectivity with respect to LPS composition was observed when LPS was removed from intact cells by EDTA‐NaCl treatment which leads to a break‐down of the barrier to hydrophobic agents in A. calcoaceticus CCM 5593.We propose that due to its physical properties, R‐form LPS forms tightly packed structures within the membrane which, under certain conditions, become destabilized and liberated into the surrounding medium. As a consequence, a disturbance of the highly ordered lateral molecular arrangement might lead to altered permeability properties of the outer membrane as suggested in one of the two alternative models existing to explain permeability changes observed in deep rough mutant strains of Enterobacteriaceae.

Adsorption of alexidine and chlorhexidine to Escherichia coli and membrane components

Activity, uptake and disposition of two related bisbiguanide antiseptics alexidine and chlorhexidine, were investigated. Rates of onset of membrane damage were faster following alexidine than chlorhexidine treatment. Deep-rough strains of E. coli were less sensitive than their smooth counterparts. Whilst the uptake of both agents to whole cells corresponded to ‘C-type’, alexidine demonstrated additional high affinity uptake (‘H-type’) at low applied drug concentrations. Distribution studies for the absorbed biocide indicated that the agents must saturate a number of envelope targets before penetration to the cytosol is possible. Alexidine possessed a higher affinity towards these sites than chlorhexidine.

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.

Comparative sensitivity of smooth, rough and deep rough strains of Escherichia coli to chlorhexidine, quaternary ammonium compounds and dibromopropamidine isethionate

The effects of chlorhexidine diacetate (CHA) and some quaternary ammonium compounds (QACs) on wild-type strains of Escherichia coli and on mutants (slightly rough, moderately rough and deep rough) are described. CHA was almost equally active in low concentrations against all the strains, whereas the three QACs, cetylpyridinium chloride (CPC), domiphen bromide and benzethonium chloride were most active against the deep rough strains, and least against the wild type and slightly rough strains. Moderately rough strains were consistently found to be slightly less sensitive to the QACs than the deep rough ones. The inhibitory concentration of CPC for any strain was much less in broth than in agar. Studies with ethylenediamine tetraacetate (EDTA) demonstrated that the chelating agent had only a marginal, if any, effect on the activity of CHA, whereas in the presence of EDTA, the activity of CPC against strains with complete or almost complete lipopolysaccharide was enhanced considerably.

The effects of antiseptics, disinfectants and preservatives on smooth, rough and deep rough strains of Salmonella typhimurium

The effects of some antiseptics, disinfectants and preservatives on a smooth strain and rough mutant strains (Ra, Rd 1 and Re) of Salmonella typhimurium are described. The inhibitors consisted of three phenolics, a homologous series (the parabens) of esters of p-hydroxybenzoic (4-hydroxybenzoic) acid, chlorhexidine diacetate and other cationic bactericides and two mercury compounds. The responses of the strains to these agents have been related: (a) to the nature of the bacterial outer membrane; and (b) to the physical properties of the compounds.

Oxidative mutagens specific for A·T base pairs induce forward mutations to L-arabinose resistance in Salmonella typhimurium

Strain TA102 of S. typhimurium is a new histidine-requiring mutant, particularly suited to the detection of oxidative mutagens acting at A·T base pairs. 10 oxidizing chemicals, previously tested in strain TA102, were used to evaluate the mutagenic sensitivity of the L-arabinose forward mutation assay of S. typhimurium with respect to those types of mutagens. The mutagenicity of each compound was determined by liquid test, measuring both the frequency of mutants among the survivors and the absolute number of mutants growing in selective plates with traces of D-glucose. Strain BA13 with a wild-type lipopolysaccharide barrier was used as compared to the deep rough derivative strain BA9. The chemicals studied were: bleomycin, t-butyl hydroperoxide, chromium trioxide, cumeme hydroperoxide, formaldehyde, glyoxal, glutaraldehyde, hydrogen peroxide, paraquat, and phenylhydrazine. Additionally, ultrasonic oscillation was used as a presumable non-mutagenic lethal control treatment. The L-arabinose forward mutation assay detected the mutagenic activity of all the chemicals under study with a high degree of sensitivity, including paraquat which is unable to revert strain TA102. Positive responses were obtained at doses equivalent to or 10 times lower than the doses detected by strain TA102. The results support the idea that the L-arabinose forward mutation assay could replace the set of specific tester strains used by the histidine reverse mutation assay in general screening for genetic toxins.

Sensitivity ofEscherichia coliandPseudomonas aeruginosato avoparcin, flavomycin and virginiamycin

The sensitivities of wild-type, rough, deep-rough and antibiotic-hypersensitive strains of Escherichia coli and Pseudomonas aeruginosa to avoparcin, flavomycin and virginiamycin are described. Avoparcin was least active against these strains. Overall, the results suggested that resistance of wild-type cells to these antibiotics resulted from their exclusion from the cells.

Quantitation of metal cations bound to membranes and extracted lipopolysaccharide of Escherichia coli.

Inductively coupled plasma emission spectroscopy was used to quantitate the metal cations bound to outer and cytoplasmic membranes and to extracted lipopolysaccharide from several Escherichia coli K12 strains. The outer membrane was found to be enriched in both calcium and magnesium relative to the cytoplasmic membrane. Both membranes contained significant levels of iron, aluminum, and zinc. The multivalent cation content of the lipopolysaccharide resembled that of the intact outer membrane. Lipopolysaccharide extracted from wild-type k12 strains contained higher levels of Mg than Ca regardless of the growth medium, but the medium used for growth did affect the relative amounts of bound Mg as well as the levels of the minor cations iron, aluminum, and zinc. In contrast, lipopolysaccharide isolated from a deep rough mutant strain, D21f2, contained more Ca than Mg. Electrodialysis of lipopolysaccharide from wild-type k12 strains removed 1 mol of Mg per mol of lipopolysaccharide but did not significantly affect the level of other bound metal ions. Dialysis of lipopolysaccharide against sodium (ethylenedinitrilo)tetraacetate removed most of the Mg and Ca, resulting in a sodium salt. The equimolar replacement of divalent cations with sodium in the sodium salt resulted in a net loss of counterion change. The sodium salt was dialyzed against either tris(hydroxymethyl)aminomethane hydrochloride, CaCl2, MgCl2, or TbCl3, and the resulting lipopolysaccharide salts were analyzed for their ionic composition. It was shown that tris(hydroxymethyl)aminomethane and Ca can replace some but not all of the Na bound to the sodium salt, but all of the other multivalent cations tested replaced Na, resulting in uniform lipopolysaccharide salts. Lipopolysaccharide isolated from the deep rough mutant strain D21f2 was also converted into a sodium salt. Relative to the wild-type lipopolysaccharide, Na was able to neutralize the anionic charge to a greater extent in the mutant lipopolysaccharide. Our results suggest that the loss of specific groups in the core region of the lipopolysaccharide from the mutant strain results in a more open structure that allows the binding of larger cations and of more monovalent cations.

An intermediate step in translocation of lipopolysaccharide to the outer membrane of Salmonella typhimurium.

Evidence for transient localization of newly synthesized lipopolysaccharide at the periplasmic face of the inner membrane has been obtained by immunoelectron microscopic techniques. Salmonella typhimurium galE mutants in which O-antigen synthesis is dependent on addition of exogenous galactose were employed, and the distribution and fate of pulse-synthesized O antigen was examined by indirect ferritin labeling with anti-O-antigen IgG of spheroplasts prepared by treatment with lysozyme/EDTA. O-reactive lipopolysaccharide appeared rapidly at the exposed periplasmic face of the inner membrane after addition of galactose and was rapidly depleted upon termination of the pulse. Control experiments showed that secondary redistribution of lipopolysaccharide from outer membrane did not occur under the conditions employed for spheroplast formation and immunolabeling, and the pulse-chase kinetics were consistent with those expected for an intermediate in translocation of lipopolysaccharide to the outer membrane. In addition, undecaprenol-linked O antigen was detectable at the periplasmic face of the inner membrane within 30 sec after addition of galactose to a galE deep rough double mutant, and it accumulated stably in that location. The mutation in synthesis of the lipopolysaccharide core in the deep rough strain prevents transfer of O-antigen chains from undecaprenol phosphate to lipopolysaccharide. The result suggests that attachment of O antigen to lipopolysaccharide occurs on the extracytoplasmic side of the inner membrane and supports the conclusion that lipopolysaccharide is translocated to the outer membrane from the periplasmic, rather than the cytoplasmic, face of the inner membrane.

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses.

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.