Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains.

Yersinia enterocolitica 29930 (biogroup 1A; serogroup O:7,8) produces a bacteriocin, designated enterocoliticin, that shows inhibitory activity against enteropathogenic strains of Y. enterocolitica belonging to serogroups O:3, O:5,27 and O:9. Enterocoliticin was purified, and electron micrographs of enterocoliticin preparations revealed the presence of phage tail-like particles. The particles did not contain nucleic acids and showed contraction upon contact with susceptible bacteria. Enterocoliticin addition to logarithmic-phase cultures of susceptible bacterial strains led to a rapid dose-dependent reduction in CFU. Calorimetric measurements of the heat output of cultures of sensitive bacteria showed a complete loss of cellular metabolic activity immediately upon addition of enterocoliticin. Furthermore, a dose-dependent efflux of K(+) ions into the medium was determined, indicating that enterocoliticin has channel-forming activity.

Bacterial dehalorespiration with chlorinated benzenes.

Chlorobenzenes are toxic, highly persistent and ubiquitously distributed environmental contaminants that accumulate in the food chain. The only known microbial transformation of 1,2,3,5-tetrachlorobenzene (TeCB) and higher chlorinated benzenes is the reductive dechlorination to lower chlorinated benzenes under anaerobic conditions observed with mixed bacterial cultures. The lower chlorinated benzenes can subsequently be mineralized by aerobic bacteria. Here we describe the isolation of the oxygen-sensitive strain CBDB1, a pure culture capable of reductive dechlorination of chlorobenzenes. Strain CBDB1 is a highly specialized bacterium that stoichiometrically dechlorinates 1,2,3-trichlorobenzene (TCB), 1,2,4-TCB, 1,2,3,4-TeCB, 1,2,3,5-TeCB and 1,2,4,5-TeCB to dichlorobenzenes or 1,3,5-TCB. The presence of chlorobenzene as an electron acceptor and hydrogen as an electron donor is essential for growth, and indicates that strain CBDB1 meets its energy needs by a dehalorespiratory process. According to their 16S rRNA gene sequences, strain CBDB1, Dehalococcoides ethenogenes and several uncultivated bacteria form a new bacterial cluster, of which strain CBDB1 is the first, so far, to thrive on a purely synthetic medium.

A novel technique for monitoring the development of bacterial biofilms in human periodontal pockets.

A new technique is presented for analyzing subgingival bacterial plaque. Different materials (polytetrafluoroethylene, gold, dentin) kept for several days in periodontal pockets of patients suffering from periodontitis were analyzed by electron microscopy and fluorescence in situ hybridization (FISH). Those parts of the carriers extending into the deepest zone of the pockets were predominantly colonized by spirochetes and Gram-negative bacteria whereas those segments in contact with a shallower region were colonized by streptococci. Independent of the material used, the bacterial colonization of the carriers appears to be similar. FISH using eubacteria- and species-specific oligonucleotides on semi-thin cross-sections of the carriers in combination with confocal laser scanning microscopy allowed detailed analysis of the architecture of biofilms and identification of putative periodontal pathogens with single cell resolution.

Aquabacterium gen. nov., with description of Aquabacterium citratiphilum sp. nov., Aquabacterium parvum sp. nov. and Aquabacterium commune sp. nov., three in situ dominant bacterial species from the Berlin drinking water system.

Three bacterial strains isolated from biofilms of the Berlin drinking water system were characterized with respect to their morphological and physiological properties and their taxonomic position. Phenotypically, the bacteria investigated were motile, Gram-negative rods, oxidase-positive and catalase-negative, and contained polyalkanoates and polyphosphate as storage polymers. They displayed a microaerophilic growth behaviour and used oxygen and nitrate as electron acceptors, but not nitrite, chlorate, sulfate or ferric iron. The substrates metabolized included a broad range of organic acids but no carbohydrates at all. The three species can be distinguished from each other by their substrate utilization, ability to hydrolyse urea and casein, cellular protein patterns and growth on nutrient-rich media as well as their temperature, pH and NaCl tolerances. Phylogenetic analysis, based on 16S rRNA gene sequence comparison, revealed that the isolates are affiliated to the beta 1-subclass of Proteobacteria. The isolates constitute three new species with internal levels of DNA relatedness ranging from 44.9 to 51.3%. It is proposed that a new genus, Aquabacterium gen. nov., should be created, including Aquabacterium citratiphilum sp. nov., Aquabacterium parvum sp. nov. and Aquabacterium commune sp. nov. The type species of the new genus is Aquabacterium commune. The type strain of A. citratiphilum is strain B4T (= DSM 11900T), the type strain of A. parvum is strain B6T (= DSM 11968T) and the type strain of A. commune is strain B8T (= DSM 11901T).

Ultrastructural characterization of Anaerobiospirillum succiniciproducens and its differentiation from Campylobacter species.

The anaerobic spiral-shaped bacterium Anaerobiospirillum succiniciproducens was isolated from the blood of an AIDS patient for the first time in Europe. Electron-microscopical methods, especially negative staining, allowed rapid morphological classification and differentiation from Campylobacter species. While A. succiniciproducens revealed lophotriche flagellation all the investigated Campylobacter species showed monotriche flagellation. The cell diameter of A. succiniciproducens was at least double that of the investigated Campylobacter species. Other ultrastructural features, such as a ring-like structure underneath the flagellar area and fibrils arranged parallel along the axis, were also specific to A. succiniciproducens.

Staphylococcal cell wall: morphogenesis and fatal variations in the presence of penicillin.

The primary goal of this review is to provide a compilation of the complex architectural features of staphylococcal cell walls and of some of their unusual morphogenetic traits including the utilization of murosomes and two different mechanisms of cell separation. Knowledge of these electron microscopic findings may serve as a prerequisite for a better understanding of the sophisticated events which lead to penicillin-induced death. For more than 50 years there have been controversial disputes about the mechanisms by which penicillin kills bacteria. Many hypotheses have tried to explain this fatal event biochemically and mainly via bacteriolysis. However, indications that penicillin-induced death of staphylococci results from overall biochemical defects or from a fatal attack of bacterial cell walls by bacteriolytic murein hydrolases were not been found. Rather, penicillin, claimed to trigger the activity of murein hydrolases, impaired autolytic wall enzymes of staphylococci. Electron microscopic investigations have meanwhile shown that penicillin-mediated induction of seemingly minute cross wall mistakes is the very reason for this killing. Such "morphogenetic death" taking place at predictable cross wall sites and at a predictable time is based on the initiation of normal cell separations in those staphylococci in which the completion of cross walls had been prevented by local penicillin-mediated impairment of the distribution of newly synthesized peptidoglycan; this death occurs because the high internal pressure of the protoplast abruptly kills such cells via ejection of some cytoplasm during attempted cell separation. An analogous fatal onset of cell partition is considered to take place without involvement of a detectable quantity of autolytic wall enzymes ("mechanical cell separation"). The most prominent feature of penicillin, the disintegration of bacterial cells via bacteriolysis, is shown to represent only a postmortem process resulting from shrinkage of dead cells and perturbation of the cytoplasmic membrane. Several schematic drawings have been included in this review to facilitate an understanding of the complex morphogenetic events.

Evidence for a new type of outer membrane lipid in oral spirochete Treponema denticola. Functioning permeation barrier without lipopolysaccharides.

A new class of outer membrane lipid (OML) was isolated from the oral spirochete Treponema denticola strain ATCC 33521 using a phenol/chloroform/light petroleum procedure normally applied for lipopolysaccharide extraction. In addition to chemical analysis, Fourier transform infrared (FTIR) spectroscopy was applied to compare the biophysical properties of OML with lipopolysaccharides (LPS) and lipoteichoic acids (LTA). Isolated OML fractions represent 1.4% of the total dry cell weight, are about 4 kDa in size, and contain 6% amino sugars, 8% neutral sugars, 14% phosphate, 35% carbazol-positive compounds, and 11% fatty acids (containing iso- and anteiso-fatty acyl chains). Rare for outer membrane lipids, OML contains no significant amount of 3-deoxy-D-manno-octulosonic acids, heptoses, and beta-hydroxy fatty acids. The fatty acyl chain composition, being similar to that of the cytoplasmic membrane, is quite heterogeneous with anteiso-pentadecanoic acid (12%), palmitic acid (51%), and iso-palmitic acid (19%) as the predominant fatty acids present. Findings of a glycerol-hexose unit and two glycerol-hexadecanoic acid fragments indicate a glycolipid membrane anchor typically found in LTA. There was also no evidence for the presence of a sphingosine-based lipid structure. The results of FTIR measurements strongly suggest that the reconstituted lipid forms normal bilayer structures (vesicles) expressing a high membrane state of order with a distinct phase transition as typical for isolated LPS. However, in contrast to LPS, OML of T. denticola has a lower Tm near 22 degreesC and a lower cooperativity of the phase transition. The results suggest a different kind of permeation barrier that is built up by this particular OML of T. denticola, which is quite different from LPS normally essential for Gram-negative bacteria.

Two alternative mechanisms of cell separation in staphylococci: one lytic and one mechanical.

Electron microscopy studies revealed two different mechanisms of cell separation in Staphylococcus aureus. Both mechanisms were initiated by the centrifugal lytic action (directed outward from the center) of murosomes, which perforated the peripheral cell wall. Thereafter, during the first type of cell separation, murosomes also lysed large parts of the cross wall proper in the opposite, i.e., centripetal direction, forming spokelike lytic lesions ("separation scars") next to the most prominent structure of the cross wall, the splitting system. This bidirectional lytic action of murosomes revealed that the staphylococcal cross wall is composed of permanent and transitory parts; transitory parts constituted about one-third of the volume of the total cross wall and seemed to be digested during cell separation. The second mechanism of cell separation was encountered within the splitting system, which has been regarded as the main control unit for lytic cell separation for more than 25 years. The splitting system, however, represents mainly a mechanical aid for cell separation and becomes effective when cell-wall autolytic activities are insufficient.

Immunochemical localization of CAMP factor (protein B) in Streptococcus agalactiae.

Biochemical and immunochemical investigations were used in order to study the quantitative and qualitative localization of CAMP factor (protein B) in the cell fractions of Streptococcus agalactiae during the logarithmic growth phase. The dynamic quantitative distribution of CAMP factor activity showed that higher concentrations of CAMP factor were found in the cytoplasm than in the cell envelopes. A maximal intracellular accumulation of CAMP factor activity was observed in the late log phase. Immunoblotting analysis using specific anti-CAMP-IgG showed that CAMP factor could be detected in the different cell fractions of S. agalactiae. During the early log phase, CAMP factor was present as a single 25 kD protein band in the cytoplasm; white it was found together with its 26 and 24 kD satellite proteins in the cytoplasmic membrane and the cell wall as well as in all the cell fractions in the mid- and late log phases. Intracellular CAMP factor exhibited the same antigenic and amphiphilic behaviour as the extracellular species. Additionally, a newly discovered amphiphilic protein of approximately 54 kD which exhibited similar antigenicity with the CAMP factor was present in all the cell fractions. Immunoelectron microscopic examinations using ferritin-labelled antibodies revealed that CAMP factor was mainly found in the cytoplasm, whereas it was associated to a minor extent with the cell envelopes. Interestingly, an accumulation of CAMP factor was also localized either at the sites of cross-wall initiation or at the cell surfaces where the cell wall became autolysed.

Light and electron microscopy study of carbohydrate antigens found in the electron-lucent layer of Pneumocystis carinii cysts.

The localization and biochemical nature of antigens found in the electron-lucent layer (ELL) of Pneumocystis carinii cysts using polyclonal rabbit antibodies are described. These antigens, specific for the cystic stages of the parasite, were shared by organisms from different hosts, suggesting that they represent functionally important components of the cyst cell wall. The binding sites were situated on an interwoven net of fibrils in the ELL produced by mild to strong proteolysis. Degradation of this residue by glucanase and chitinase confirms that this layer contains branched glucan and chitin. In contrast, the prompt susceptibility of the polysaccharide-rich ELL to proteolysis reveals that proteins are also relevant in building up the cyst-wall glucan skeleton. It is therefore concluded that the formation of the Pneumocystis cyst wall shows differences to the typical fungal cell-wall architecture. The taxonomical debate regarding this unique protist is ongoing, and consideration of these immunological and morphological findings may be useful for the study of the biology and phylogeny of Pneumocystis.

Staphylococcal peptidoglycan interpeptide bridge biosynthesis: a novel antistaphylococcal target?

In staphylococci, crosslinking of the peptide moiety of peptidoglycan is mediated via an additional spacer, the interpeptide bridge, consisting of five glycine residues. The femAB operon, coding for two approximately 50-kDa proteins is known to be involved in pentaglycine bridge formation. Using chemical mutagenesis of the beta-lactam-resistant strain BB270 and genetic, biochemical, and biophysical characterization of mutants selected for loss of beta-lactam resistance and reduced lysostaphin sensitivity it is shown that peptide bridge formation proceeds via three intermediate bridge lengths (cell wall peptides with no, one, three, and five glycine units). To proceed from one intermediate to the next, three genes appear necessary: femX, femA, and femB. The drastic loss of beta-lactam resistance after inactivation of FemA or partial impairment of FemX even beyond the level of the sensitive wild-type strains renders these proteins attractive antistaphylococcal targets.

D-alanine deprivation of Bacillus subtilis teichoic acids is without effect on cell growth and morphology but affects the autolytic activity.

Using insertional inactivation of the different genes of the dlt operon in Bacillus subtilis, we searched for metabolic and morphological changes caused by D-alanine ester deprivation of lipoteichoic acid and wall teichoic acid. There were no alterations of cell growth, basic metabolism, cellular content of phosphorus-containing compounds, ultrastructure, cell separation, and surface charge. The only alteration observed was an enhancement of endogenous and beta-lactam-induced cell lysis. Since this enhancement is doubtless correlated with the D-alanine ester deprivation of the teichoic acids, the present view based on in vitro experiments, that negatively charged LTA is inhibitory to autolysins, may be questioned. We propose that negatively charged lipoteichoic acid and/or wall teichoic acid serve in vivo to fix the cationic autolysins within the cell wall-membrane complex by electrostatic interaction. Positively charged D-alanine ester substituents decrease the binding capacity of the teichoic acids for autolysins by charge compensation.

Microcalorimetric and electron microscopic investigation on the effects of essential oil from Cymbopogon densiflorus on Staphylococcus aureus.

Microcalorimetry, optical density measurements and electron microscopy, were used to assess the influence of various amounts of the essential oil of Cymbopogon densiflorus (lemongrass oil) on the metabolic activity, growth and morphology of Staphylococcus aureus. Relatively high concentrations of the oil impaired staphylococcal growth in a bacteriostatic manner (chloramphenicol type), and in low doses metabolism became ineffective due to energy losses in the form of heat. Ultrastructural data revealed morphological changes characteristic for the influence of bactericidal antibiotics inducing bacteriolysis (penicillin type). The essential oil may have antibacterial activity by influencing bacterial targets involved in cytoplasmic and cell wall metabolism.

The occurrence of treponemes and their spherical bodies on polytetrafluoroethylene membranes.

The occurrence of small (diameter 0.125 micrometers) and large (diameter 0.27 micrometers) treponemal species on expanded polytetrafluoroethylene membranes were demonstrated in vivo. In the apical part of the expanded polytetrafluoroethylene membrane only the smaller treponemes were colonizing, while in other parts a mixed population of different bacterial species including large treponemes was seen. Only the smaller treponemes were able to form spherical bodies in the occlusive part of the membrane. All morphological characteristics of spherical bodies such as common outer sheath, randomly distributed axial flagella, sheathless protoplasmic cylinders and central bodies could be differentiated. Possible causes for the formation of spherical bodies are discussed.

A novel, "hidden" penicillin-induced death of staphylococci at high drug concentration, occurring earlier than murosome-mediated killing processes.

In log-phase cells of staphylococci, cultivated under high, "non-lytic" concentrations of penicillin G, there occurred a novel killing process hitherto hidden behind seemingly bacteriostatic effects. Two events are essential for the appearance of this "hidden death": (i) the failure of the dividing cell to deposit enough fibrillar cross-wall material to be welded together, and (ii) a premature ripping up of incomplete cross walls along their splitting system. "Hidden death" started as early as 10-15 min after drug addition, already during the first division cycle. It was the consequence of a loss of cytoplasmic constituents which erupted through peripheral slit-like openings in the incomplete cross walls. The loss resulted either in more or less empty cells or in cell shrinkage. These destructions could be prevented by raising the external osmotic pressure. In contrast, the conventional "non-hidden death" occurred only much later and exclusively during the second division cycle and mainly in those dividing cells, whose nascent cross walls of the first division plane had been welded together. These welding processes at nascent cross walls, resulting in tough connecting bridges between presumptive individual cells, were considered as a morphogenetic tool which protects the cells, so that they can resist the otherwise fatal penicillin-induced damages for at least an additional generation time ("morphogenetic resistance system"). Such welded cells, in the virtual absence of underlying cross-wall material, lost cytoplasm and were killed via ejection through pore-like wall openings or via explosions in the second division plane and after liberation of their murosomes, as it was the case in the presence of low, "lytic" concentrations of penicillin. Bacteriolysis did not cause any of the hitherto known penicillin-induced killing processes.

Formation of multiple treponemes.

The formation of pseudomulticellular treponemes was analyzed using different electron microscopical methods such as thin sectioning, freeze fracturing and scanning electron microscopy. The development of spherical bodies is preceded by the formation of unseparated helical forms and plait-like structures. During this process, the bacteria lose their individual outer sheath as well as their axial flagella and produce a common outer envelope. The possible pathogenetic importance of such multiple structures is discussed.

Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus.

The inactivation of FemB by insertion of Tn551 in the central part of the femB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward beta-lactam antibiotics to the same extent as did inactivation of femA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. Both femA and femB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against beta-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for both femA and femB mutants.

Development of quasi-multicellular bodies of Treponema denticola.

The formation of quasi-multicellular bodies of Treponema denticola was analysed using different electron microscopical methods. These bacteria could develop four different conformations: (i) normal helical forms; (ii) twisted spirochetes, forming plaits; (iii) twisted spirochetes,forming club-like structures; (iv) spherical bodies in different size. Treponemes within spherical bodies, plaits, and clubs proved to be enclosed in a common outer sheath in which the normal arrangement of their axial flagella was lost. The development of the quasi-multicellular bodies starting from the monoforme spirochetes was elucidated and this morphogenetic process is illustrated by a schematic drawing. Factors which might be involved in the induction of the structures are discussed and their possible pathogenetic importance is considered.

Fan-shaped ejections of regularly arranged murosomes involved in penicillin-induced death of staphylococci.

Electron microscopic research into the murosomes of staphylococci has shown that the number of murosomes involved in penicillin-induced death varies depending on the experimental conditions employed. With 0.1 micrograms of penicillin G per ml, only 1 of a total of about 20 murosomes, the "killing murosome," completely perforated the pressure-stabilized peripheral cell wall during a three-step process. This strictly localized event was mainly attributed to a mechanical effect being comparable to the process of aneurysm formation. Wall perforation was also considered to mark the very moment of penicillin-induced death ("nonlytic killing event"), while bacteriolysis started only postmortem. By varying the osmolarity of the growth medium, the number of murosomes involved in penicillin-induced killing increased considerably, which resulted in the ejection of a fan-shaped row of murosomes at the second division plane. These data are compatible with the finding that, in untreated or chloramphenicol-treated staphylococci, the activation of the murosomes resulted in (i) the formation of regularly arranged "blebs" on the cell surface, containing traces of disintegrated wall material, and (ii) the subsequent liberation of the murosomes lying underneath, leaving behind their former sites in the peripheral wall as a row of regularly arranged "pores" in every division plane. The number, distribution, and positioning of these blebs corresponded with those of the pores and the original murosomes. The significance of wall autolysins liberated from the first division plane for penicillin-induced wall perforation at the second division plane is discussed.

The modulation of the bacteriolytic effect of beta-lactam antibiotics by non-antibiotics.

The addition of cationic proteins such as lysozyme, ribonuclease and cytochrome C enhanced the beta-lactam-induced bacteriolysis of staphylococci measured as release of wall label or by optical density. The treatment of staphylococci with penicillin plus cytochrome C resulted in a reduced viability of bacteria compared with those treated with penicillin alone. The wall autolysis and the penicillin-induced bacteriolysis of staphylococci were enhanced by the lysosomal enzyme cathepsin C. The penicillin-induced bacteriolysis was also enhanced by the D-amino acids D-alanine and D-methionine, while the comparable L-amino acids did not reveal any activity. On the other hand, some polyanionic substances were able to suppress the penicillin-induced bacteriolysis. Radiochemical and electron microscopic studies revealed the participation of bacterial wall autolysins in the first steps of degradation processes of staphylococcal walls within murine bone marrow-derived macrophages.