Cell Wall Teichoic Acids of Two Brevibacterium Strains

Structurally identical teichoic acids were detected in cell walls of two soil isolates assigned to Brevibacterium linens based on phylogenetic data. Both cell walls contain unsubstituted 1,3-poly(glycerol phosphate) and poly(glycosylglycerol phosphate). Repeating units of the latter--alpha-D-GlcpNAc-(1-->4)-beta-D-Galp-(1-->1)-Gro--are bound by phosphodiester bonds including OH-3 of galactose and OH-3 of glycerol. Some of the N-acetylglucosamine residues have 4,6-pyruvic acid acetal, amounts of the latter in the two strains being unequal. Species-specificity of the structures of teichoic acids in the genus Brevibacterium is discussed.     

Lipoidal Components of Bacterial Lipopolysaccharides: Nature and Distribution of Fatty Acids in Aerobacter aerogenes

The fatty acid distribution of Aerobacter aerogenes was studied by comparing the fatty acid composition of the lipoidal component of the endotoxin (lipid A) with the fatty acids of the readily extractable native lipids and total cellular fatty acids. The results for total cellular fatty acids and readily extractable native lipids were generally similar, but both quantitative and qualitative differences exist. In addition, profound differences between these two fractions and lipid A were observed. These differences included fewer fatty acids and lower concentrations of unsaturated and cyclopropane fatty acids in the lipid A. Hydroxy fatty acids persisted in the lipid A. The significance of these differences with respect to mammalian toxicity of endotoxins is discussed.     

Bacterial Decarboxylation of o-Phthalic Acids

The decarboxylation of phthalic acids was studied with Bacillus sp. strain FO, a marine mixed culture ON-7, and Pseudomonas testosteroni. The mixed culture ON-7, when grown anaerobically on phthalate but incubated aerobically with chloramphenicol, quantitatively converted phthalic acid to benzoic acid. Substituted phthalic acids were also decarboxylated: 4,5-dihydroxyphthalic acid to protocatechuic acid; 4-hydroxyphthalic and 4-chlorophthalic acids to 3-hydroxybenzoic and 3-chlorobenzoic acids, respectively; and 3-fluorophthalic acid to 2-and 3-fluorobenzoic acids. Bacillus sp. strain FO gave similar results except that 4,5-dihydroxyphthalic acid was not metabolized, and both 3- and 4-hydroxybenzoic acids were produced from 4-hydroxyphthalic acid. P. testosteroni decarboxylated 4-hydroxyphthalate (to 3-hydroxybenzoate) and 4,5-dihydroxyphthalate but not phthalic acid and halogenated phthalates. Thus, P. testosteroni and the mixed culture ON-7 possessed 4,5-dihydroxyphthalic acid decarboxylase, previously described in P. testosteroni, that metabolized 4,5-dihydroxyphthalic acid and specifically decarboxylated 4-hydroxyphthalic acid to 3-hydroxybenzoic acid. The mixed culture ON-7 and Bacillus sp. strain FO also possessed a novel decarboxylase that metabolized phthalic acid and halogenated phthalates, but not 4,5-dihydroxyphthalate, and randomly decarboxylated 4-hydroxyphthalic acid. The decarboxylation of phthalic acid is suggested to involve an initial reduction to 1,2-dihydrophthalic acid followed by oxidative decarboxylation to benzoic acid.     

Function of Teichoic Acids and Effect of Novobiocin on Control of Mg<Superscript>2+</Superscript> at the Bacterial Membrane

ALTHOUGH the occurrence of both wall and membrane teichoic acids in Gram-positive bacteria has been known for a considerable time and it is believed that they are essential for normal cellular activity, their main function has been somewhat obscure. Confirmatory evidence for the proposal¹ that teichoic acids participate in ion-exchange in the outer regions of the bacterial cell has been described recently². It has been shown that the phosphate groups of the wall teichoic acid are responsible for the capacity of isolated walls to bind magnesium ions; but whole cells of Gram-positive bacteria also invariably contain a poly-glycerol phosphate-teichoic acid located in the region between the wall and the cytoplasmic membrane³ and it is believed that this must be able to bind Mg²⁺ as does the wall polymer. These two regions of anionic polymer might thus constitute an integrated cation-exchange system between the exterior of the cell and the cytoplasmic membrane, where relatively high concentrations of Mg²⁺ are required for a variety of processes. We report here experiments with a membrane-bound enzyme system that requires Mg²⁺, obtained from a broken cell preparation and in which the close contact between the outer layers of the cell is preserved. In this preparation the enzyme system displays maximum activity in the presence of Mg²⁺ bound to the endogenous teichoic acid and is insensitive to changes in the concentration of added Mg²⁺, in marked contrast to the behaviour of the enzyme system in isolated cytoplasmic membrane. These results provide the first direct demonstration of the function of teichoic acids in concentrating Mg²⁺at the cytoplasmic membrane. They lead to the conclusion that failure of teichoic acid biosynthesis in the whole cell would cause inhibition of membrane function through magnesium starvation. In view of this the effect of novobiocin, an antibiotic shown to inhibit teichoic acid biosynthesis in vitro^4–6, is discussed.     

Wall Teichoic Acid Protects Staphylococcus aureus against Antimicrobial Fatty Acids from Human Skin

Skin-colonizing gram-positive bacteria produce wall teichoic acids (WTAs) or related glycopolymers for unclear reasons. Using a WTA-deficient Staphylococcus aureus mutant, we demonstrated that WTA confers resistance to antimicrobial fatty acids from human sebaceous glands by preventing fatty acid binding. Thus, WTA is probably important for bacterial skin colonization.Bacterial life on mammalian skin depends on efficient adaptation strategies to cope with high salt concentrations and dryness. In addition, skin is protected by a variety of antibacterial molecules, such as antimicrobial peptides (16), bacteriolytic enzymes (14), and antibacterial fatty acids (AFAs) (6, 11, 23). The main source of free fatty acids is the sebum, produced by sebaceous glands, and differentiating keratinocytes of the stratum corneum, the outermost layer of the epidermis, which is composed of dead, keratin-filled cells. Sebaceous glands are found in nearly all mammals, and the composition of the sebum is remarkably species specific (12). Up to 47% of human sebum consists of free fatty acids with palmitoleic acid isomer (C_16:1Δ6) as the predominant monoene AFA. Lauric acid (C_12:0) is the most potent saturated AFA (23). Palmitic acid (C_16:0), stearic acid (C_18:0), oleic acid (C_18:1Δ9), and linoleic acid (C_18:2Δ9Δ12) are the main fatty acids in the stratum corneum (9, 23).While most skin-colonizing bacteria are harmless commensals, Staphylococcus aureus frequently causes endogenous infections, ranging from cutaneous infections to life-threatening sepsis and endocarditis (10). S. aureus has developed efficient strategies to survive in its natural niches, the human anterior nares and skin, and to evade the immune system (4, 8). However, only a few studies have previously addressed the molecular basis of staphylococcal resistance to AFA. The major surface protein expressed by S. aureus under iron-limited conditions, IsdA, has recently been shown to confer AFA resistance because it increases the bacterial surface hydrophilicity (2). In addition to proteins, cell wall glycopolymers such as the teichoic acids are thought to govern bacterial surface hydrophobicity. Such polymers are found in most gram-positive bacteria, forming a highly charged mesh within the cell wall (21). They often consist of alternating glycerolphosphate or ribitolphosphate units, which are partially substituted by d-alanine and various glycosyl residues (13, 21). Teichoic acids are anchored in the cytoplasmic membrane via a glycolipid (lipoteichoic acid) or in the peptidoglycan via a phosphodiester linkage (wall teichoic acid [WTA]). A variety of roles in bacterial cell envelope processes and integrity have been assigned to WTA but the major functions of WTA have still remained elusive (21). Our group has recently generated a WTA-deficient S. aureus mutant and demonstrated that WTA is crucial for S. aureus nasal colonization and endovascular infection (19, 20, 22). The tagO gene disrupted in this mutant encodes an N-acetylglucosamine-phosphate transferase catalyzing the first step of WTA biosynthesis (24). The tagO mutant shows a total loss of WTA but seems to be unaffected in growth behavior and susceptibility to different antimicrobial peptides (19). However, the mutant exhibits increased resistance to human beta-defensin 3 (7).In order to study the contribution of WTA to the surface hydrophobicity of S. aureus SA113, a frequently used laboratory strain (5, 19, 22), the affinities of the wild type and the tagO mutant for the hydrophobic solvent dodecan were compared by the microbial adhesion to hydrocarbon test (15). In fact, the hydrophilicity of the WTA-deficient mutant was considerably decreased compared to those of the parental and complemented mutant strains (Fig. ​(Fig.1),1), confirming the crucial impact of WTA on the physicochemical surface properties of S. aureus. Subsequently, the MICs of a variety of saturated and unsaturated fatty acids occurring in human sebum and stratum corneum were determined (Table ​(Table1).1). Twenty-four-well plates with 50%-concentrated Müller-Hinton broth (Sigma) containing increasing concentrations of AFAs were inoculated with the bacterial strains, and the optical density was measured after 48 h of growth at 37°C. The tagO mutant showed a profound increase in susceptibility to all tested AFAs compared to the parental strain and the complemented mutant. The strongest MIC reductions were found for palmitoleic acid (sixfold) and linoleic acid (26-fold). In order to compare potential differences in susceptibility to the bactericidal activities of AFAs, bacteria grown overnight in 50%-concentrated Müller-Hinton broth were resuspended in phosphate-buffered saline (PBS) at an optical density of 0.5 at 578 nm, and 1 ml of each suspension was shaken with increasing concentrations of AFAs at 37°C. Incubation was stopped at different time points by dilution with PBS, and numbers of surviving bacteria were determined by counting CFU. Palmitoleic acid exhibited dose-dependent bactericidal activity to SA113, with the tagO mutant having 26-fold reduced survival compared to that of the wild type at 1.25 mM after 10 min of incubation (Fig. ​(Fig.2A).2A). When different incubation times were used for a given concentration, the tagO mutant was much more rapidly killed than the parental strain, thereby confirming the crucial role of WTA in AFA resistance (Fig. ​(Fig.2B2B).Open in a separate windowFIG. 1.The WTA-deficient ΔtagO mutant has decreased surface hydrophilicity compared to the wild type and the complemented (compl.) mutant strain, as assessed by the microbial adhesion to hydrocarbon test. The percentages of bacteria associated with the hydrophilic phase are shown. Data represent means ± standard errors of the means from three independent experiments. ***, P < 0.001; ns, not significant (in comparison to the wild-type value).Open in a separate windowFIG. 2.The WTA-deficient tagO mutant is more susceptible to the bactericidal activity of AFAs than the wild-type strain. (A) Bacteria were exposed to the indicated concentrations of palmitoleic acid for 10 min. **, P < 0.005; ***, P < 0.001. (B) The wild type (▪) and the tagO mutant (▴) were exposed to lauric acid (5 mM), cis-6-hexadecenoic acid (5 mM), and palmitoleic acid (1.25 mM) for the indicated times. Data represent means ± standard errors of the means from at least three independent experiments.

TABLE 1.

WTA-deficient Sa113 tagO mutant is more susceptible to growth-inhibiting activity of AFAs than wild-type and complemented mutant strains

trans-Monounsaturated Acids in a Marine Bacterial Isolate

A sedimentary bacterial isolate has been shown to contain trans-monounsaturated fatty acids (6% of the total fatty acids). The ratio of trans- to cis-acids in this isolate was in the range 3.2 to 7.6. The identification of trans-monounsaturated acids in a marine bacterium implied that the trans-acids which have been reported in recent sediments could derive, in whole or part, from direct bacterial input.     

trans-Monounsaturated Acids in a Marine Bacterial Isolate

[This corrects the article on p. 849 in vol. 41.].     

Teichoic Acids in the Cell Walls of Microbispora mesophila Ac-1953T and Thermobifida fusca Ac-1952T

The cell walls of Microbispora mesophila strain Ac-1953^T (the family Streptosporangiaceae) and Thermobifida fusca Ac-1952^T (the family Nocardiopsaceae) were found to contain teichoic acids of a poly(glycerol phosphate) nature. The teichoic acid of M. mesophila (formerly Thermomonospora mesophila) represents a 1,3-poly(glycerol phosphate) containing 5% of substituent 2-acetamido-2-deoxy--D-galactosaminyl residues. Teichoic acid of such a kind was found in actinomycetes for the first time. The cell wall of T. fusca (formerly Thermonospora fusca) contains two teichoic acids, namely, unsubstituted 1,3-poly(glycerol phosphate) and -glucosylated 1,3-poly(glycerol phosphate).     

Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δlcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δlcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δlcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes (tagB, tarF, or tarJ2) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δlcp mutant was not relieved by tunicamycin treatment or by mutation of tagO, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δlcp mutant. We propose a model whereby the S. aureus Δlcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.     

L-Rhamnosylation of Listeria monocytogenes Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane

Listeria monocytogenes is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in L. monocytogenes, where WTA modification is achieved solely via glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of L. monocytogenes WTAs relies not only on the rmlACBD locus, which encodes the biosynthetic pathway for L-rhamnose, but also on rmlT encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using in vitro binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of L. monocytogenes delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes L. monocytogenes survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for L. monocytogenes virulence in a mouse model of infection.     

Teichoic acids from streptomyces griseus

Summary During the Schmidt-Thannhauser fractionation procedure at nucleic acid determinations of Streptomyces griseus, a phosphorous-containing compound(s) was found which could not be taken as nucleic acid or polyphosphate. The hydrolisate of the isolated substance proved to have almost the same composition as teichoic acid from Staphylococcus aureus Duncan.Teichoic acid was found both intracellular and in the isolated cell wall of Streptomyces griseus mycelia.In the cell wall only the ribitol type of teichoic acids could be detected but not the glycerol type.     

Selective Inhibition of Bacterial Enzymes by Free Fatty Acids

Octanoic acid inhibits, in vitro, the bacterial enzymes glucose-6-phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, fumarase, lactate dehydrogenase, and the malic enzyme of Arthrobacter crystallopoietes. The free fatty acid appears to act as an inhibitor of lipogenesis, although it does not affect the rate of gluconeogenesis. To demonstrate that this inhibition may be of physiological significance in vivo, those enzymes not involved in lipogenesis, such as fructose-1, 6-diphosphatase, phosphoglucomutase, phosphohexoisomerase, aconitase, nicotinamide adenine dinucleotide phosphate (NADP) isocitrate dehydrogenase, NADP glutamate dehydrogenase, malate dehydrogenase, and isocitrate lyase, were assayed and found not to be inhibited by the free fatty acid.     

The Invasive Nature of an Infectious Bacterial Symbiont

ABSTRACT. Xenosomes are infectious bacterial symbionts that exist exclusively in the cytoplasm of the small philasterine marine ciliate Parauronema acutum. We have used this host-symbiont system as a model to study infection. In the past we postulated that infection took place by a process in which the symbionts escaped digestion and entered into the host's cytoplasm through the food vacuole during phagocytosis. This is clearly not the case. We now present evidence based on electron microscopic observations that the symbionts infect in a manner involving direct penetration of the protozoan's cell membranes. We have obtained additional data that suggest that, following entrance of the symbionts into the cytoplasm, only a single xenosome is required to establish an infection.     

Comparison of Rapid Methods for Analysis of Bacterial Fatty Acids

When rapid gas-liquid chromatography methods for determination of bacterial fatty acids were compared, results showed that saponification was required for total fatty acid analysis. Transesterification with boron-trihalide reagents (BF(3)-CH(3)OH, BCl(3)-CH(3)OH) caused extensive degradation of cyclopropane acids and was less effective than saponification in releasing cellular hydroxy fatty acids. Digestion of cells with tetramethylammonium hydroxide was unsatisfactory because of extraneous gas-liquid chromatography peaks and because of lower recovery of branched-chain and hydroxy fatty acids. A simple, rapid saponification procedure which can be used for total cellular fatty acid analysis of freshly grown cells is described.     

Tanzawaic Acids,a Chemically Novel Set of Bacterial Conjugation Inhibitors

Bacterial conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the conjugation process. A search for conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in bacterial, fungal or human cells, when compared to synthetic conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination.