Combining catalyzed reporter deposition-fluorescence in situ hybridization and microautoradiography to detect substrate utilization by bacteria and Archaea in the deep ocean

The recently developed CARD-FISH protocol was refined for the detection of marine Archaea by replacing the lysozyme permeabilization treatment with proteinase K. This modification resulted in about twofold-higher detection rates for Archaea in deep waters. Using this method in combination with microautoradiography, we found that Archaea are more abundant than Bacteria (42% versus 32% of 4',6'-diamidino-2-phenylindole counts) in the deep waters of the North Atlantic and that a larger fraction of Archaea than of Bacteria takes up l-aspartic acid (19% versus 10%).     

An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml(-1)) followed by achromopeptidase (60 U ml(-1)) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.     

Prokaryotic community analysis with CARD-FISH in comparison with FISH in ultra-oligotrophic ground- and drinking water

AIMS: We compared the applicability of catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and FISH to enumerate prokaryotic populations in ultra-oligotrophic alpine groundwaters and bottled mineral water METHODS AND RESULTS: Fluorescent oligonucleotide probes EUB338 and EUB338mix (EUB338/EUB338-II/EUB338-III) were used to enumerate bacteria and probes EURY806 and CREN537 for Euryarchaea and Crenarchaea, respectively. Improved detection of Planctomycetales by probe EUB338-II was tested using a different permeabilization step (proteinase K instead of lysozyme). Total detection efficiency of cells in spring water of four different alpine karst aquifers was on average 83% for CARD-FISH and only 15% for FISH. Applying CARD-FISH on bottled natural mineral waters resulted in an average total hybridization efficiency of 89%, with 78% (range 77-96%) bacteria and 11% (range 3-22%) identified as Archaea. CONCLUSIONS: CARD-FISH resulted in substantially higher recovery efficiency than FISH. Hence, CARD-FISH appears very suitable for the enumeration of specific prokaryotic groups in ground- and drinking water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first evaluation of CARD-FISH on ultra-oligotrophic ground- and drinking water. Results are relevant for basic research and drinking water distributors. Archaea can comprise a significant fraction of the prokaryotic community in bottled mineral water.     

An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml⁻¹) followed by achromopeptidase (60 U ml⁻¹) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.     

Identification of pseudomurein cell wall binding domains

Methanothermobacter thermautotrophicus is a methanogenic Gram-positive microorganism with a cell wall consisting of pseudomurein. Currently, no information is available on extracellular pseudomurein biology and so far only two prophage pseudomurein autolysins, PeiW and PeiP, have been reported. In this paper we show that PeiW and PeiP contain two different N-terminal pseudomurein cell wall binding domains. This finding was used to identify a novel domain, PB007923, on the M. thermautotrophicus genome present in 10 predicted open reading frames. Three homologues were identified in the Methanosphaera stadtmanae genome. Binding studies of fusion constructs of three separate PB007923 domains to green fluorescent protein revealed that it also constituted a cell wall binding domain. Both prophage domains and the PB007923 domain bound to the cell walls of Methanothermobacter species and fluorescence microscopy showed a preference for the septal region. Domain specificities were revealed by binding studies with other pseudomurein-containing archaea. Localized binding was observed for M. stadtmanae and Methanobrevibacter species, while others stained evenly. The identification of the first pseudomurein cell wall binding domains reveals the dynamics of the pseudomurein cell wall and provides marker proteins to study the extracellular pseudomurein biology of M. thermautotrophicus and of other pseudomurein-containing archaea.     

Pathogenic potential of fifty Bacillus weihenstephanensis strains

Sequence comparison of pseudomurein endoisopeptidases PeiW encoded by the defective prophage PsiM100 of Methanothermobacter wolfeii, and PeiP encoded by phage PsiM2 of Methanothermobacter marburgensis, revealed that the two enzymes share only limited similarity. Their amino acid sequences comprise an N-terminal domain characterized by the presence of direct repeats and a C-terminal domain with a catalytic triad C-H-D as in thiol proteases and animal transglutaminases. Both PeiW and PeiP catalyze the in vitro lysis of M. marburgensis cells under reducing conditions and exhibit characteristics of metal-activated peptidases. Optimal temperature and pH were determined to be 63 degrees C and 6.4 for His-tagged PeiP and 71 degrees C and 6.4 for His-tagged PeiW, respectively. Database search results suggest that PeiW and PeiP are the first two experimentally identified members of a novel family of proteases in a superfamily of archaeal, bacterial, and eukaryotic protein homologs of animal transglutaminases.     

Zelloberflächenstrukturen der Bacteria und Archaea

Cell wall types of Bacteria and Archaea The acaryote microorganisms are divided into the two domains Bacteria and Archaea. The third domain represent the Eukarya. There is no universal cell wall polymer found in all Bacteria and Archaea. Due to their morphology several cell wall types can be identified, but the chemical diversity of the individual polymers is considerably greater. Certain cell wall polymers are limited to one of the two domains of Bacteria or Archaea like the murein of the Bacteria or the pseudomurein of some methanogens. Peptidoglycans (murein, pseudomurein) do not occur in eukaryotes. On the other hand individual cell wall polymers possess similarities to polymers of other domains. The structural principle of the methanochondroitin is also implemented in the eukaryotic connective tissue. The cell wall polymers consist frequently of glycoconjugates in which the amino acid content (glycoproteins) or the glycan moiety (proteoglycan‐like polymers) predominate. Both components (carbohydrates, amino acids) can also occur in similar amounts (peptidoglycan). There exist also cell wall polymers, which consist only of glycans (slimes, methanochondroitin) or amino acids (proteins, poly‐γ‐D‐glutamyl polymers). Cell wall‐free species (Mycoplasma) also occur. The chemical composition of the cell surface polymers was one of the first phenotypic characteristics that supported the 16 sRNA concept of Carl Woese to assign acaryote organisms into the two domains Bacteria and Archaea. A common feature of all Archaea is the lack of muramic acid and an outer membrane. The later occurs in the gramnegative Bacteria. During the evolution of Bacteria and Archaea a great variety of chemically different cell wall polymers has been developed which allow the growth and interaction of Bacteria and Archaea in different habitats. In this paper, some important surface polymers of Bacteria and Archaea are presented according to their chemical composition.     

Improved quantification of Dehalococcoides species by fluorescence in situ hybridization and catalyzed reporter deposition

Chlorinated ethenes contamination of soil and groundwater is a widespread problem in most industrialized countries. To date, there is a general consensus in the literature that members of the genus Dehalococcoides are required for complete dechlorination of these compounds. The availability of specific identification tools to track their distribution in the field is therefore a topic of particular relevance in environmental studies. These microorganisms have been successfully visualized by fluorescence in situ hybridization (FISH) in highly active dechlorinating cultures. However, FISH detection of Dehalococcoides under low activity conditions can be strongly hampered by their small cell size and low ribosome content. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative detection method. In a trichloroethene (TCE) dechlorinating enrichment culture, CARD-FISH, using proteinase K as a permeabilization pre-treatment, was found to be significantly superior to conventional FISH in terms of both microscopic visualization and quantification efficiency (about 30%). An application of this method on contaminated aquifer samples is also presented and discussed.     

Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.     

Photosynthetic properties of rapidly permeabilized cells of the cyanobacterium Anacystis nidulans

A study has been made (i) of the effect of the suspension medium composition on the kinetics of permeabilization of Anacystis nidulans to ions by lysozyme, and (ii) of the consequences of permeabilization on the photosynthetic apparatus of the cyanobacterium. Ion-permeable, osmotically resistant cells (permeaplasts) were prepared by a 15 min lysozyme treatment in 0.05 M sodium 4-(2-hydroxyethyl)-1-piperazineethanesulfonate. Permeabilization causes detachment of phycobilisomes from the thylakoid surface and partial dissociation of phycobiliprotein multimers. The permeaplasts contain, however, virtually intact thylakoids, as testified by their fully functional photosynthetic electron-transport chain and their capacity for photosynthetic control. Supplementation of the lysozyme incubation medium with osmotica delays cell permeabilization, while prolongation of the lysozyme treatment inactivates the photosynthetic electron transport at a site preceding electron donation by 1,5-diphenylcarbazide.     

Reliability of CARD-FISH Procedure for Enumeration of Archaea in Deep-Sea Surficial Sediments

The enumeration of Archaea in deep-sea sediment samples is still limited, although different methodological procedures have been applied. Among these, catalysed reporter deposition-fluorescence in situ hybridisation (CARD-FISH) technique is a promising tool for estimation of archaeal abundance in deep-sea sediment samples. Comparing different permeabilisation treatments, the best results obtained both on archaeal pure cultures and on natural assemblages were with hydrochloric acid (0.1 M) and proteinase K (0.004 U/ml) treatments. The application of CARD-FISH on deep-sea sediments revealed that Archaea reach up to 41% of total prokaryotic cells. Specific probes for planktonic Archaea showed that marine Crenarchaea dominated archaeal seafloor communities. No clear bathymetric trends were observed for archaeal abundances and the morphology of continental margin (slope vs. canyon) seems not to have a direct influence on archaeal relative abundances. The site-specific sediment habitat—both abiotic environmental setting and sedimentary organic matter quality—explain up to 65% of variance of archaeal, crenarchaeal and euryarchaeal relative abundance, suggesting a wide ecophysiological adaptation to deep-sea benthic ecosystems. The findings demonstrate that Archaea are an important component of benthic microbial assemblages so far neglected, and hence they lay the groundwork for more focused research on their ecological importance in the functioning of deep-sea benthic ecosystems.     

Methanogens with pseudomurein use diaminopimelate aminotransferase in lysine biosynthesis

Methanothermobacter thermautotrophicus uses lysine for both protein synthesis and cross-linking pseudomurein in its cell wall. A diaminopimelate aminotransferase enzyme from this methanogen (MTH0052) converts tetrahydrodipicolinate to l,l-diaminopimelate, a lysine precursor. This gene complemented an Escherichia coli diaminopimelate auxotrophy, and the purified protein catalyzed the transamination of diaminopimelate to tetrahydrodipicolinate. Phylogenetic analysis indicated this gene was recruited from anaerobic Gram-positive bacteria. These results expand the family of diaminopimelate aminotransferases to a diverse set of plant, bacterial and archaeal homologs. In contrast marine methanogens from the Methanococcales, which lack pseudomurein, appear to use a different diaminopimelate pathway for lysine biosynthesis.     

Catalyzed reported deposition-fluorescence in situ hybridization protocol to evaluate phagotrophy in mixotrophic protists

We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists.     

Stability and Specificity of the Cell Wall-Associated Proteinase from Lactococcus lactis subsp. cremoris H2 Released by Treatment with Lysozyme in the Presence of Calcium Ions

The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris H2 (isolate number 4409) was released from the cells by treatment with lysozyme, even in the presence of 50 mM calcium chloride. Cell lysis during lysozyme treatment was minimal. The proteinase activity released by lysozyme treatment fractionated on ion-exchange chromatography as three main forms, the molecular masses of which were determined by gel exclusion chromatography and polyacrylamide gel electrophoresis. Two of the enzyme forms released, 137 and 145 kDa, were the same as those released by incubation of cells in calcium-free phosphate buffer. In the presence of calcium, lysozyme treatment also resulted in the release of a 180-kDa enzyme molecule. The total proteinase activity released by lysozyme treatment (in the presence or absence of calcium) was not only greater than that released by phosphate buffer but was also greater than that initially detectable on the surface of whole cells, suggesting an unmasking of enzyme on the cell surface. The presence of calcium during release treatment resulted in increased stability of the crude enzyme preparations. For the proteinase preparation released by using lysozyme with 50 mM CaCl₂, the half-life of proteinase activity at 37°C was 39 h, compared with 0.22 h for the calcium-free phosphate buffer-released preparation. In all cases, maximum stability was observed at pH 5.5. Comparison of β-casein hydrolysis by the three forms of the enzyme showed that the products of short-term (5- to 30-min) digestions were very similar, although subtle differences were detected with the 180-kDa form.     

Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml⁻¹ at 37°C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.     

Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml(-1) at 37 degrees C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.     

Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists

We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists.     

Extraction of chromosomal DNA from Staphylococcus and Listeria by a rapid method using achromopeptidase

A rapid and simple method for preparation of chromosomal DNA from Gram-positive bacteria is reported. Susceptibility to lysis with Sodium Dodecyl Sulfate (SDS) increases when undergoing treatment with acetone before being digested by bacteriolytic enzymes. Rapid lysis of Staphylococcus and Listeria cells is obtained through a respective treatment by lysozyme with lysostaphine and by lysozyme with achromopeptidase, adding to that the effect of SDS in Tris-Hcl buffer. This procedure of preparing chromosomal DNA provides 1 to 4 mg of DNA out of 1 g of bacterial cells in a day.     

Transmembrane orientation of the mannose 6-phosphate receptor in isolated clathrin-coated vesicles

The mannose 6-phosphate (Man-6-P) receptor is an integral membrane glycoprotein which mediates intracellular transport and receptor-mediated endocytosis of lysosomal proteins. Clathrin-coated vesicles, which have been shown to be significantly involved in these processes, have also been shown to be a major subcellular site of the receptor. In order to define the orientation of the Man-6-P receptor within the coated vesicle membrane, highly purified preparations of coated vesicles were prepared from bovine brain employing D2O/sucrose gradient centrifugation and Sephacryl S-1000 column chromatography. Using [35S]methionine-labeled lysosomal enzymes secreted by Chinese hamster ovary cells as receptor ligand, significant binding activity was detected only upon permeabilization of the coated vesicle membranes with detergent. Prior treatment of intact vesicles with proteinase K resulted in similar binding activity upon permeabilization. However, examination of the receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with rabbit anti-receptor serum revealed that proteinase K treatment of intact vesicles reduced the size of the receptor by 12,000 daltons. A similar decrease in size was obtained when the vesicles were treated with carboxypeptidase Y. These results suggest that the Man-6-P receptor is a transmembrane protein with its lysosomal enzyme binding site oriented toward the lumen of the coated vesicle and its C-terminal end exposed to the exterior or cytoplasmic portion of the vesicle membrane.     

Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli

Previously we have shown that chicken egg white lysozyme, an efficient bactericidal agent, affects both gram-positive and gram-negative bacteria independently of its muramidase activity. More recently we reported that the digestion of lysozyme by clostripain yielded a pentadecapeptide, IVSDGNGMNAWVAWR (amino acid 98-112 of chicken egg white lysozyme), with moderate bactericidal activity but without muramidase activity. On the basis of this amino acid sequence three polypeptides, in which asparagine 106 was replaced by arginine (IVSDGNGMRAWVAWR, RAWVAWR, RWVAWR), were synthesized which showed to be strongly bactericidal. To elucidate the mechanisms of action of lysozyme and of the modified antimicrobial polypeptides Escherichia coli strain ML-35p was used. It is an ideal organism to study the outer and the inner membrane permeabilization since it is cryptic for periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless the outer or inner membrane becomes damaged. For the first time we present evidence that lysozyme inhibits DNA and RNA synthesis and in contrast to the present view is able to damage the outer membrane of Escherichia coli. Blockage of macromolecular synthesis, outer membrane damage and inner membrane permeabilization bring about bacterial death. Ultrastructural studies indicate that lysozyme does not affect bacterial morphology but impairs stability of the organism. The bactericidal polypeptides derived from lysozyme block at first the synthesis of DNA and RNA which is followed by an increase of the outer membrane permeabilization causing the bacterial death. Inner membrane permeabilization, caused by RAWVAWR and RWVAWR, follows after the blockage of macromolecular synthesis and outer membrane damage, indicating that inner membrane permeabilization is not the deadly event. Escherichia coli bacteria killed by the substituted bactericidal polypeptides appeared, by electron microscopy, with a condensed cytoplasm and undulated bacterial membrane. So the action of lysozyme and its derived peptides is not identical.