Cell Envelope Composition Research Articles

FT-IR spectroscopic investigation of bacterial cell envelopes from Zymomonas mobilis which have different surface hydrophobicities

Changes in the cell envelope composition of bacteria Zymomonas mobilis 113S which resulted in varied cell surface hydrophobicity (CSH) were examined using FT-IR-spectroscopy and conventional methods of biochemical analysis. Significant differences in all analyses were detected between hydrophilic and hydrophobic compounds in the envelopes of Z. mobilis. A significant positive linear relationship (P<0.01) was observed between CSH values of Z. mobilis and the ratio of absorbance band intensities assigned to the CH2 and CH3 vibrations from lipids as well as the asymmetric and symmetric stretching vibrations of these methylene and methyl groups. Differences were also seen between the ratio of band absorbances arising from Amide I and Amide II. In addition, the ratio of absorbance intensities assigned to carbohydrates was found to correlate with the CSH of Z. mobilis. Furthermore, a significant inverse relationship (P<0.01) was detected between CSH of Z. mobilis and the band position (frequency) shift of asymmetric and symmetric stretching of methylene groups, alkyl esters (>CO str) and Amide I, as well as a positive linear relationship (P<0.05) between the band position shift of Amide II and carbohydrates and CSH of Z. mobilis. Qualitative and quantitative composition of the cell envelopes was found to correlate with both the CSH of Z. mobilis and the spectral data. The observed multiple correlations between the concentration of proteins, carbohydrates and lipids of cell envelopes and selected indices of FT-IR spectra indicate the complex relationship of Z. mobilis cell envelope constituents. The results of present study suggest that FT-IR spectroscopy can be used as a convenient method to evaluate the properties of cell envelopes from Gram-negative bacteria.

Effect of growth media on cell envelope composition and nitrile hydratase stability in Rhodococcus rhodochrous strain DAP 96253

Rhodococcus is an important industrial microorganism that possesses diverse metabolic capabilities; it also has a cell envelope, composed of an outer layer of mycolic acids and glycolipids. Selected Rhodococcus species when induced are capable of transforming nitriles to the corresponding amide by the enzyme nitrile hydratase (NHase), and subsequently to the corresponding acid via an amidase. This nitrile biochemistry has generated interest in using the rhodococci as biocatalysts. It was hypothesized that altering sugars in the growth medium might impact cell envelope components and have effects on NHase. When the primary carbon source in growth media was changed from glucose to fructose, maltose, or maltodextrin, the NHase activity increased. Cells grown in the presence of maltose and maltodextrin showed the highest activities against propionitrile, 197 and 202 units/mg cdw, respectively. Stability of NHase was also affected as cells grown in the presence of maltose and maltodextrin retained more NHase activity at 55 °C (45 and 23 %, respectively) than cells grown in the presence of glucose or fructose (19 and 10 %, respectively). Supplementation of trehalose in the growth media resulted in increased NHase stability at 55 °C, as cells grown in the presence of glucose retained 40 % NHase activity as opposed to 19 % without the presence of trehalose. Changes in cell envelope components, such mycolic acids and glycolipids, were evaluated by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), respectively. Changing sugars and the addition of inducing components for NHase, such as cobalt and urea in growth media, resulted in changes in mycolic acid profiles. Mycolic acid content increased 5 times when cobalt and urea were added to media with glucose. Glycolipids levels were also affected by the changes in sugars and addition of inducing components. This research demonstrates that carbohydrate selection impacts NHase activity and stability. Cell envelope components such as mycolic acids are also influenced by sugars and inducers such as cobalt and urea. This is information that can be useful when implementing rhodococcal catalysts in industrial applications.

Osmotic stress adaptations in rhizobacteria

Rhizobacteria have been reported to be beneficial to the plants in many different ways. Increasing salinity in the coastal agricultural zones has been shown to be a threat to the plant and microbial life in the area. Exposure of microorganisms to high-osmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm. The microorganisms have developed various adaptations to counteract the outflow of water. The first response to osmotic up shifts and the resulting efflux of cellular water is uptake of K⁺ and cells start to accumulate compatible solutes. Yet another mechanism is by altering the cell envelope composition resulting in changes in proteins, periplasmic glucans, and capsular, exo and lipopolysaccharides. Bacteria also initiate a program of gene expression in response to osmotic stress by high NaCl concentrations, which are manifested as a set of proteins produced in increased amounts in response to the stress. Genomics, transcriptomics and proteomics approaches have revealed the key components in molecular basis of bacteria salt adaptation. Understanding the mechanisms of osmo-adaptation in rhizobacteria would also be relevant from an ecological and an applicative point of view.

A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum – Cellular behavior in adaptation to n-butanol

To gain more insight into the butanol stress response of Clostridium acetobutylicum the transcriptional response of a steady state acidogenic culture to different levels of n-butanol (0.25–1%) was investigated. No effect was observed on the fermentation pattern and expression of typical solvent genes (aad, ctfA/B, adc, bdhA/B, ptb, buk). Elevated levels of butanol mainly affected class I heat-shock genes (hrcA, grpE, dnaK, dnaJ, groES, groEL, hsp90), which were upregulated in a dose- and time-dependent manner, and genes encoding proteins involved in the membrane composition (fab and fad or glycerophospholipid related genes) and various ABC-transporters of unknown specificity. Interestingly, fab and fad genes were embedded in a large, entirely repressed cluster (CAC1988–CAC2019), which inter alia encoded an iron-specific ABC-transporter and molybdenum-cofactor synthesis proteins. Of the glycerophospholipid metabolism, the glycerol-3-phosphate dehydrogenase (glpA) gene was highly upregulated, whereas a glycerophosphodiester ABC-transporter (ugpAEBC) and a phosphodiesterase (ugpC) were repressed. On the megaplasmid, only a few genes showed differential expression, e.g. a rare lipoprotein (CAP0058, repressed) and a membrane protein (CAP0102, upregulated) gene. Observed transcriptional responses suggest that C. acetobutylicum reacts to butanol stress by induction of the general stress response and changing its cell envelope and transporter composition, but leaving the central catabolism unaffected.

Quantitative lipid composition of cell envelopes of Corynebacterium glutamicum elucidated through reverse micelle extraction

Cells of the Corynebacterium-Nocardia-Mycobacterium group of bacteria are surrounded by an outer membrane (OM) containing mycolic acids that are covalently linked to the underlying arabinogalactan-peptidoglycan complex. This OM presumably acts as a permeability barrier that imparts high levels of intrinsic drug resistance to some members of this group, such as Mycobacterium tuberculosis, and its component lipids have been studied intensively in a qualitative manner over the years. However, the quantitative lipid composition of this membrane has remained obscure, mainly because of difficulties in isolating it without contamination from the inner cytoplasmic membrane. Here we use the extraction, with reverse surfactant micelles, of intact cells of Corynebacterium glutamicum and show that this method extracts the free OM lipids quantitatively with no contamination from lipids of the cytoplasmic membrane, such as phosphatidylglycerol. Although only small amounts of corynomycolate were esterified to arabinogalactan, a large amount of cardiolipin was present in a nonextractable form, tightly associated, possibly covalently, with the peptidoglycan-arabinogalactan complex. Furthermore, we show that the OM contains just enough lipid hydrocarbons to produce a bilayer covering the cell surface, with its inner leaflet composed mainly of the aforementioned nonextractable cardiolipin and its outer leaflet composed of trehalose dimycolates, phosphatidylinositol mannosides, and highly apolar lipids, similar to the Minnikin model of 1982. The reverse micelle extraction method is also useful for extracting proteins associated with the OM, such as porins.

Antimicrobial activity of n-alkyltrimethylammonium bromides: influence of specific growth rate and nutrient limitation.

The antimicrobial activity of an homologous series of n-alkyltrimethylammonium bromides has been assessed towards Escherichia coli grown at a variety of specific growth rates and under various conditions of nutrient limitation. For each individual set of growth conditions activity was parabolically related to the n-alkyl chain length of the compounds and thus to compound lipophilicity (log P). The compound that showed optimal activity and thereby optimal lipophilicity (log Po) changed according to growth rate and nutrient limitation. Such changes are related to variations in the gross cell envelope composition of the cultures (phospholipid, lipopolysaccharide, neutral lipids, proteins). The data therefore support the hypothesis that changes in growth rate and nutrient limitation alter the overall lipophilicity of the cell envelope and thereby the optimal value of log P for compounds to traverse it. Additionally, the data suggest that for the compounds examined, the neutral acidic:neutral phospholipid ratios of the cell envelope, also influence the permeation of it.

Overexpression of proinflammatory TLR-2-signalling lipoproteins in hypervirulent mycobacterial variants

Changes in the cell envelope composition of mycobacteria cause major changes in cytokine profiles of infected antigen presenting cells. We describe here the modulation of inflammatory responses by Mycobacterium abscessus, an emerging pathogen in cystic fibrosis. M. abscessus is able to switch from a smooth (S) to a rough (R) morphotype by the loss of a surface glycopeptidolipid. R variants are associated with severe clinical forms and a 'hyper-proinflammatory' response in ex vivo and in vivo models. Using partitioning of cell surface components we found that a complex fraction, more abundant in R variants than in S variants, made a major contribution to the TLR-2-dependent hyper-proinflammatory response induced by R variants. Lipoproteins were the main TLR-2 agonists in this fraction, consistent with the larger amounts of 16 lipoproteins in cell surface extracts from R variants; 15 out of 16 being more strongly induced in R variant than in S variant. Genetic interruption of glycopeptidolipid pathway in wild-type S variant resulted in R phenotype with similar induction of lipoprotein genes. In conclusion, R morphotype in M. abscessus is associated with increased synthesis/exposure at the cell surface of lipoproteins, these changes profoundly modifying the innate immune response through TLR-2-dependent mechanisms.

Cell envelope architecture in the Chloroflexi: a shifting frontline in a phylogenetic turf war

It is important that attempts to understand bacterial phylogeny take into account fundamental bacterial characteristics such as cell envelope composition and organization. Several prominent phylogenetic studies have assumed that the cell envelopes of members of the phylum Chloroflexi are 'gram-negative' (diderm, i.e. defined by both an inner plasma membrane and an outer membrane) and some of these studies have placed the branch leading to the extant Chloroflexi near the root of the bacterial phylogenetic tree. This Correspondence summarizes the compelling evidence that the Chloroflexi are in fact monoderm, i.e. have only a single cellular membrane. The phylogenetic implications of this conclusion are discussed. The data reviewed also shed interesting light on the distribution of protein secretion systems in diderm bacteria.

M. tuberculosis intramembrane protease Rip1 controls transcription through three anti‐sigma factor substrates

Regulated intramembrane proteolysis (RIP) is a mechanism of transmembrane signal transduction that functions through intramembrane proteolysis of substrates. We previously reported that the RIP metalloprotease Rv2869c (Rip1) is a determinant of Mycobacterium tuberculosis (Mtb) cell envelope composition and virulence, but the substrates of Rip1 were undefined. Here we show that Rip1 cleaves three transmembrane anti-sigma factors: anti-SigK, anti-SigL and anti-SigM, negative regulators of Sigma K, L and M. We show that transcriptional activation of katG in response to phenanthroline requires activation of SigK and SigL by Rip1 cleavage of anti-SigK and anti-SigL. We also demonstrate a Rip1-dependent pathway that activates the genes for the mycolic acid biosynthetic enzyme KasA and the resuscitation promoting factor RpfC, but represses the bacterioferritin encoding gene bfrB. Regulation of these three genes by Rip1 is not reproduced by deletion of Sigma K, L or M, either indicating a requirement for multiple Rip1 substrates or additional arms of the Rip1 pathway. These results identify a branched proteolytic signal transduction system in which a single intramembrane protease cleaves three anti-sigma factor substrates to control multiple downstream pathways involved in lipid biosynthesis and defence against oxidative stress.

ExoR is genetically coupled to the ExoS–ChvI two‐component system and located in the periplasm of Sinorhizobium meliloti

Sinorhizobium meliloti enters into a symbiotic relationship with legume host plants, providing fixed nitrogen in exchange for carbon and amino acids. In S. meliloti, exoR and the exoS-chvI two-component system regulate the biosynthesis of succinoglycan, an exopolysaccharide important for host invasion. It was previously reported that a loss-of-function mutation in exoR and a gain-of-function mutation in exoS cause overproduction of succinoglycan and loss of motility, indicating that ExoR negatively regulates and ExoS-ChvI positively regulates downstream genes. However, a relationship between exoR and exoS-chvI has never been clearly established. By identification and detailed characterization of suppressor strains, we provide genetic evidence that exoR and exoS-chvI control many similar phenotypes. These include succinoglycan production, symbiosis, motility, and previously uncharacterized prototrophy and biofilm formation, all of which are co-ordinately restored by suppressors. We further demonstrate that ExoR is located in the periplasm, suggesting that it functions to regulate downstream genes in a novel manner. In pathogenic bacteria closely related to S. meliloti, exoS-chvI homologues are required for virulence and the regulation of cell envelope composition. Our data suggest that periplasmically localized ExoR and ExoS-ChvI function together in a unique and critical regulatory system associated with both free-living and symbiotic states of S. meliloti.

Dominance of sphingomonads in a copper-exposed biofilm community for groundwater treatment

The structure, biological activity and microbial biodiversity of a biofilm used for the removal of copper from groundwater were studied and compared with those of a biofilm grown under copper-free conditions. A laboratory-scale submerged fixed biofilter was fed with groundwater (2.3 l h(-1)) artificially polluted with Cu(II) (15 mg l(-1)) and amended with sucrose (150 mg l(-1)) as carbon source. Between 73 and 90 % of the Cu(II) was removed from water during long-term operation (over 200 days). The biofilm was a complex ecosystem, consisting of eukaryotic and prokaryotic micro-organisms. Scanning electron microscopy revealed marked structural changes in the biofilm induced by Cu(II), compared to the biofilm grown in absence of the heavy metal. Analysis of cell-bound extracellular polymeric substances (EPS) demonstrated a significant modification of the composition of cell envelopes in response to Cu(II). Transmission electron microscopy and energy-dispersive X-ray microanalysis (EDX) showed that copper bioaccumulated in the EPS matrix by becoming bound to phosphates and/or silicates, whereas copper accumulated only intracytoplasmically in cells of eukaryotic microbes. Cu(II) also decreased sucrose consumption, ATP content and alkaline phosphatase activity of the biofilm. A detailed study of the bacterial community composition was conducted by 16S rRNA-based temperature gradient gel electrophoresis (TGGE) profiling, which showed spatial and temporal stability of the species diversity of copper-exposed biofilms during biofilter operation. PCR reamplification and sequencing of 14 TGGE bands showed the prevalence of alphaproteobacteria, with most sequences (78 %) affiliated to the Sphingomonadaceae. The major cultivable colony type in plate counts of the copper-exposed biofilm was also identified as that of Sphingomonas sp. These data confirm a major role of these organisms in the composition of the Cu(II)-removing community.

Effect of hydrophobicity of yeast cell envelopes on the rate of methyl oleate autooxidation

The relationships between the antioxidant and antiperoxide properties and the lipid composition of yeast cell envelopes before and after interaction with a liquid culture medium was studied. Correlations between the hydrophobicity of cell envelopes, their lipid composition, and parameters of kinetic oxidation curves of model methyl oleate in the presence of lipids were found. Irrespective of the total lipid content in yeast cell envelopes, the preparations with a high antiperoxide activity of lipids exhibited a higher hydrophobicity and adsorbed lipophilic prooxidants from the medium more readily, whereas the preparations with a low antiperoxide activity were less hydrophobic and adsorbed predominantly lipophilic inhibitors. It was found that analysis of kinetic oxidation curves in toto provides the most comprehensive information on the physicochemical properties of lipids adsorbed from medium.

The σB regulon in Staphylococcus aureus and its regulation

The Staphylococcus aureus genome codes for a sigma factor that shows close sequence similarity to the alternative sigma factor σ B of Bacillus subtilis. However, of the proteins controlling the activity of σ B in B. subtilis only RsbU, RsbV, and RsbW are encoded in the staphylococcal genome. Therefore, the regulation of the σ B activity must differ between these two bacterial species. The present study was designed (i) to describe the σ B regulon and (ii) to identify stimuli leading to an activation of σ B-dependent transcription. All conditions under which σ B was activated in S. aureus (heat shock, addition of MnCl 2 or NaCl, alkaline shock) required the presence of RsbU, a positive regulator of σ B. In contrast to B. subtilis, a drop in the cellular ATP level caused by the addition of carbonyl cyanide m-chlorophenylhydrazone did not lead to an activation of σ B in S. aureus. Moreover, ethanol, a strong inductor of σ B activity in B. subtilis, also failed to induce σ B in S. aureus. Expression of sigB and σ B-dependent genes was enhanced following entry into stationary phase of cells grown in complex medium (LB medium). Our DNA microarray data indicated that 122 genes are positively regulated by σ B under alkaline stress conditions. Interestingly, only 12% of these genes have an orthologue in the B. subtilis σ B regulon, suggesting that the function of the σ B regulon in S. aureus is different from that in B. subtilis. We could show that σ B of S. aureus, in contrast to B. subtilis, may have a function in more basic cellular processes such as cell envelope composition, membrane transport processes and intermediary metabolism. σ B-dependent genes identified by the DNA microarray approach were subjected to detailed transcriptional analyses using primer extension and Northern blot techniques. These analyses confirmed our DNA microarray data and furthermore revealed different regulatory groups of σ B-dependent genes.

The Virulence-associated Two-component PhoP-PhoR System Controls the Biosynthesis of Polyketide-derived Lipids in Mycobacterium tuberculosis

Two-component regulatory signal transduction systems are important elements of the adaptative response of prokaryotes to a variety of environmental stimuli. Disruption of PhoP-PhoR in Mycobacterium tuberculosis dramatically attenuates virulence, implying that this system directly and/or indirectly coordinates the expression of important virulence factors whose identity remains to be established. Interestingly, in knockingout the PhoP-PhoR two-component system in M. tuberculosis Mt103, dramatic changes in the colonial morphology, cording properties, and reactivity of the mutant strain to the basic dye neutral red, all intrinsic properties of tubercle bacilli known to correlate with virulence, were noted. Because deficiencies in the ability of the mutant to form serpentine cords and stain with the dye are likely the results of alterations of its cell envelope composition, we undertook to analyze the lipid content of phoP and phoP-phoR mutants constructed in two different strains of M. tuberculosis. Our results indicate that PhoP coordinately and positively regulates the synthesis of methyl-branched fatty acid-containing acyltrehaloses known to be restricted to pathogenic species of the M. tuberculosis complex, namely diacyltrehaloses, polyacyltrehaloses, and sulfolipids. Evidence is also provided that PhoP but not PhoR is required for the production of these lipids. This work represents an important step toward the functional characterization of PhoP-PhoR and the understanding of complex lipid synthesis in M. tuberculosis.

Regulation of Mycobacterium tuberculosis cell envelope composition and virulence by intramembrane proteolysis

Mycobacterium tuberculosis infection is a continuing global health crisis that kills 2 million people each year. Although the structurally diverse lipids of the M. tuberculosis cell envelope each have non-redundant roles in virulence or persistence, the molecular mechanisms regulating cell envelope composition in M. tuberculosis are undefined. In higher eukaryotes, membrane composition is controlled by site two protease (S2P)-mediated cleavage of sterol regulatory element binding proteins, membrane-bound transcription factors that control lipid biosynthesis. S2P is the founding member of a widely distributed family of membrane metalloproteases that cleave substrate proteins within transmembrane segments. Here we show that a previously uncharacterized M. tuberculosis S2P homologue (Rv2869c) regulates M. tuberculosis cell envelope composition, growth in vivo and persistence in vivo. These results establish that regulated intramembrane proteolysis is a conserved mechanism controlling membrane composition in prokaryotes and show that this proteolysis is a proximal regulator of cell envelope virulence determinants in M. tuberculosis.

Novel biomarkers for Gram-type differentiation of bacteria by pyrolysis–gas chromatography–mass spectrometry

The pyrolysis–gas chromatography (Py–GC) module of a fielded, briefcase Py–GC–ion mobility spectrometry (Py–GC–IMS) biodetector is shown to generate a set of specific products that reflect the biochemical composition of bacterial cell envelopes. A prominent pyrolysis biomarker derived from the cell walls of Gram-positive bacteria has been identified as pyridine-2-carboxamide (2-picolinamide). Experiments performed using model polymers strongly suggest its origin from the thermal rearrangements of the meso-diaminopimelic acid and/or l-lysine peptide residue cross-linker species in peptidoglycan. Biomarkers produced by pyrolysis from Gram-negative organisms were found to originate from the lipid A portion of the lipopolysaccharide (LPS) cell surface. These biomarkers include pyrolysis products of the 3-hydroxymyristate fatty acid residues such as 1-tridecene, dodecanal and methylundecylketone. A nitrile derivative from amide bound fatty acids and free fatty acids originally present as ester bound moieties in lipid A are also observed upon bacterial pyrolysis. The presence of these biomarkers directly reflect similar information content provided by a classical Gram staining procedure and make them suitable for direct Gram-type differentiation of micro-organisms.

Cellular engineering in a minimal microbe: structure and assembly of the terminal organelle of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a minimal microbe with respect to cell envelope composition, biosynthetic and regulatory capabilities and genome size, yet it possesses a remarkably complex, multifunctional terminal organelle. This membrane-bound extension of the mycoplasma cell is defined by the presence of an electron-dense core that appears as paired, parallel bars oriented longitudinally and enlarging at the distal end to form a terminal button. Most non-cytadhering mutants of M. pneumoniae isolated to date exhibit defects in the architecture of the terminal organelle. Detailed characterization of those mutants has revealed the identities of many component proteins of the terminal organelle as well as the likely order in which some of those components are required. Additional questions regarding the composition of the electron-dense core, the means by which the terminal organelle is duplicated during cell division and the manner in which this process is regulated remain to be answered. Thus, it seems that there is much to be learned about cellular engineering and spatial regulation in these 'simple' cell wall-less bacteria.

Virulence attenuation of two Mas-like polyketide synthase mutants of Mycobacterium tuberculosis.

The cell envelope of pathogenic mycobacteria is highly distinctive in that it contains a large number of structurally related very long multiple methyl-branched fatty acids. These complex molecules are thought to play important roles in cell envelope organization and virulence. The genetic and enzymic characterization of the polyketide synthase Mas, which is responsible for the synthesis of one such family of fatty acids (the mycocerosic acids), paved the way towards the identification of other enzymes involved in the synthesis of methyl-branched fatty acids in M. tuberculosis. In an effort to elucidate the origin of these complex fatty acids and their possible involvement in pathogenesis, the two mas-like polyketide genes pks5 and pks7 were disrupted in M. tuberculosis and the effects of their inactivation on fatty acid composition and virulence were analysed. While the disruption of pks7 resulted in a mutant deficient in the production of phthiocerol dimycocerosates, the cell envelope composition of the pks5 mutant was found to be identical to that of the wild-type parental strain M. tuberculosis H37Rv. Interestingly, both the pks5 and pks7 mutants displayed severe growth defects in mice.

Resistance mechanisms of bacteria to antimicrobial compounds

Abstract A range of antimicrobial compounds (bactericides) commonly termed biocides, microbicides, sanitizers, antiseptics and disinfectants are available, all of which are claimed by their producers to kill bacteria. Resistance has been defined as the temporary or permanent ability of an organism and its progeny to remain viable and/or multiply under conditions that would destroy or inhibit other members of the strain. Bacteria may be defined as resistant when they are not susceptible to a concentration of antibacterial agent used in practice. Traditionally, resistance refers to instances where the basis of increased tolerance is a genetic change, and where the biochemical basis is known. Antimicrobial substances target a range of cellular loci, from the cytoplasmic membrane to respiratory functions, enzymes and the genetic material. However, different bacteria react differently to bactericides, either due to inherent differences such as unique cell envelope composition and non-susceptible proteins, or to the development of resistance, either by adaptation or by genetic exchange. At low concentrations bactericides often act bacteriostatically, and are only bacteriocidal at higher concentrations. For bactericides to be effective, they must attain a sufficiently high concentration at the target site in order to exert their antibacterial action. In order to reach their target site(s), they must traverse the outer membrane of the gram negative bacteria. Bacteria with effective penetration barriers to biocides generally display a higher inherent resistance than those bacteria which are readily penetrated. The rate of penetration is linked to concentration, so that a sufficiently high bactericide concentration will kill bacteria with enhanced penetration barriers. It has been indicated that susceptible bacterial isolates acquire increased tolerance to bactericides following serial transfer in sub-inhibitory concentrations. Whereas the basis of bacterial resistance to antibiotics is well know, that of resistance to antiseptics, disinfectants and food preservatives is less well understood. Three mechanisms of resistance that have been reported include: • limited diffusion of antimicrobial agents through the biofilm matrix, • interaction of the antimicrobial agents with the biofilm matrix (cells and polymer), • enzyme mediated resistance, • level of metabolic activity within the biofilm • genetic adaptation • efflux pumps and • outer membrane structure.

Small proline-rich protein 1 is the major component of the cell envelope of normal human oral keratinocytes

Oral keratinocytes of buccal and gingival tissues undergo a terminal differentiation program to form a protective epithelial barrier as non-keratinized or parakeratinized stratified cells. We have examined the protein composition of cell envelopes (CEs) from normal human buccal and gingival tissues as well as keratinocytes from normal human gingival cells grown in culture. Biochemical and sequencing analyses reveal that the CEs contain 60–70% small proline-rich protein 1a/b (SPR1a/b), together with smaller amounts of involucrin, annexin I and several other known CE proteins. The data imply a specialized role for SPR1 proteins in the unique barrier function requirements of oral epithelia.