Bacillus Cells Research Articles

Regulating Pathways of Bacillus pumilus Adamalysin-like Metalloendopeptidase Expression.

The minor secreted proteinase of B. pumilus 3-19 MprBp classified as the unique bacillary adamalysin-like enzyme of the metzincin clan. The functional role of this metalloproteinase in the bacilli cells is not clear. Analysis of the regulatory region of the mprBp gene showed the presence of potential binding sites to the transcription regulatory factors Spo0A (sporulation) and DegU (biodegradation). The study of mprBp activity in mutant strains of B. subtilis defective in regulatory proteins of the Spo- and Deg-systems showed that the mprBp gene is partially controlled by the Deg-system of signal transduction and independent from the Spo-system.

Characteristic and activity of cellulolytic bacteria isolated from mangrove soil in Northern Coast of Aceh Province, Indonesia

Abstract. Dewiyanti I, Darmawi D, Muchlisin ZA, Helmi TZ, Arisa II, Rahmiati R, Destri E, Fanisha S. 2022. Characteristic and activity of cellulolytic bacteria isolated from mangrove soil in Northern Coast of Aceh Province, Indonesia. Biodiversitas 23: 6587-6599. The cellulolytic bacteria as decomposing cellulose bacteria is related to the soil fertility and productivity of the mangrove ecosystem, and the presence and activity of enzymes produced can evaluate them. The study aims to analyze the cellulolytic bacteria characterization, cellulase enzyme activity, and production and to evaluate the molecular biology of cellulase bacteria isolated from the mangrove soil on the northern coast of Aceh Province, Indonesia. This study was conducted at six locations, and soil samples were collected randomly with three replications. The results showed that the purified bacteria produced 39 isolates that grew in CMC selective media. The common bacteria discovered have an irregular colony shape, undulate colony edge, raised colony elevation, and cream pigmentation. The bacilli cell is the most common shape, while 22 bacteria out of the 39 isolates showed cellulase activity. The cellulolytic index (CI) ranged from 0.31 to 4.82, and the highest CI was BTM533 (4.82), followed by BTM622 (2.09). The quantitative analysis showed that the highest specific cellulase activity of BTM533 and BTM622 were at 36 hours and 42 hours with the value of 0.042 U mg-1 and 0.129 U mg-1. Among the bacteria isolates, two cellulolytic bacteria with high cellulase activity were identified as Bacillus safensis and B. altitudinis using the 16S rRNA gene molecularly.

Morphological, biochemical and molecular identification of rhizobacteria isolates with potential for biocontrol of fungal plant pathogens

Rhizobacteria have huge potential for biocontrol activity against many plant pathogens. Isolation and identification are therefore crucial to understand their microbial diversity and ecological importance. The study sought to identify eight yam (Dioscorea sp.) rhizobacterial isolates found to be inhibitory in a previous study to several fungal pathogens, using polyphasic approach (morphological, biochemical, and molecular approaches). Morphological and biochemical characterizations were carried out at the Noguchi Memorial Institute for Medical Research (NMIMR), Legon-Accra, Ghana while the Molecular characterization was done at Functional Biosciences Inc., Wisconsin, USA. Morphological and biochemical characters determined were texture, consistency, the morphology of cell growth on nutrient agar, citrate utilization, catalase reaction, Gram reaction, and endospore formation. Molecular identification was achieved through the amplification and sequencing of the 16SrRNA and gyrB regions of the eight rhizobacterial genomic DNA followed by BLAST analysis. Sequencing data were also deposited at GenBank to obtain accession numbers for the rhizobacteria. All the eight bacterial isolates were shown to possess rod-shaped bacilli cells, Gram-positive, and were endospore producers. The amplification of the 16SrRNA and the gyrB regions of the bacterial DNA and subsequent BLAST of these two regions revealed that they were all members of the Bacillus subtilis group at percentage similarity between 99-100%. Four out of the eight isolates were, Bacillus subtilis, three were B. amyloliquefaciens and the last, B. velezensis. Sequence data of both the 16SrRNA and gyrB deposited at the GenBank provided GenBank accession numbers for nucleotide sequence of 16SrRNA as GenBank MT535846-MT535853 and gyrB as GenBank MT908225-MT908232 respectively. This is the first attempt to identify these rhizobacteria, which have huge potential as fungal antagonists of many plant pathogens in Ghana.

Identification of lactic acid bacteria from etawa goat milk kopelma Darussalam Village, Banda Aceh

Identification of Lactic Acid Bacteria (LAB) has been carried out based on the catalase test and Gram staining of Etawa pure Goat Milk samples that obtained from Kopelma Village, Darussalam District, Banda Aceh City, Aceh Province. De Man, Rogosa, Sharpe Agar (MRSA) media was used for LAB isolation. There were 10 isolates of goat milk bacteria that used for microscopic characteristics, morphological observations, catalase test and Gram staining. Based on the microscopic test, it was explained that all colonies had similar characteristics, that having a round shape, convex surface shape, 1-2 mm diameter and cream color. In the catalase test, the results were negative, because all isolates did not produce gas bubbles after dripped with H2O2 solution. The morphological results showed that all isolates were Gram positive, marked by purple cells and bacilli cells. Based on the Gram staining and the shape of the bacillus cells, it were suspected that milk isolates are candidates for the genus Lactobacillus. Microscopic diagnosis is only a provisional presumption. Therefore, it needs a conclusive diagnosis, such as biochemical properties which are important factors that must be carried out for the next stage.

COMPOSITION OF FATTY ACIDS, AMINO ACIDS AND MONOSACCARIDES OF BACILLUS GENUS BACTERIA ISOLATED FROM THE BLACK SEA BOTTOM SEDIMENTS

The aim of this work was to determine the quantitative and qualitative composition of fatty acids, amino acids and, carbohydrates of antagonistically active sporeforming Bacillus genus bacteria isolated from the deep-sea sediments of the Black Sea. Materials and methods. The object of the study was the antagonistically active bacteria of Bacillus velezensis ONU 553, Bacillus pumilus ONU 554, Bacillus subtilis ONU 559, Bacillus megaterium 11, Bacillus pumilus 95 strains. The fatty acid composition was determined by using a gas chromatograph with flame ionization detector Agilent 7890 (Agilent Technologies, USA), monosaccharides – by gas-liquid chromatography-mass spectrometry on Agilent 6890N/5973 inert (Agilent Technologies, USA), amino acids – on liquid chromatograph Agilent 1200 (Agilent Technologies, USA). Results. There were found the characteristic fatty acids for the studied strains: for strain B. velezensis ONU 553 – 17:0, 14:0 anteiso, 11:0 2OH, for B. subtilis ONU 559 – 19:0iso, 19:0anteiso, for B. megaterium 11 – 15:0 2OH and, B. pumilus 95 – 15:1 isow5c and 12:0. The amino acid patterns of the studied strains do not show any significant correlation with the ocean biota pattern and strains B. velezensis ONU 553, B. pumilus ONU 554 and B. pumilus 95 show such characteristic for sandy soil biota. In addition to B. subtilis group bacteria-specific monosaccharides there were revealed arabinose, polyatomic alcohols – L-iditol and myoinositol, which functions are unknown in bacilli cells. Conclusions. It can be concluded that HAI and a15/i15 indices of B. velezensis ONU 553, B. pumilus ONU 554 and B. subtilis ONU 559 strains are characteristic of mesophilic bacteria and B. megaterium 11 and B. pumilus 95 are of moderate thermophiles. The results of the studies support the hypothesis that these bacteria are not autochthonous for the deep-sea bottom sediments of the Black Sea.

Biological activity of the lipopeptide-producing Bacillus amyloliquefaciens PGPBacCA1 on common bean Phaseolus vulgaris L. pathogens

Bacillus amyloliquefaciens PGPBacCA1 was studied regarding its aptitude to protect common bean seeds from their intrinsic pathogens. Also, the inhibition of different environmental phytopathogenic fungi was tested. Two cultivars of Phaseolus vulgaris L. were evaluated: cv. Nag (black bean) and cv. Alubia (white bean). Aspergillus spp., Penicillium spp. and Fusarium spp. constituted the natural fungal biota of both seeds, whereas white bean and black bean also exhibited Cladosporium spp. and Rhizopus spp., respectively. B. amyloliquefaciens PGPBacCA1 prevented the development of the endophytic fungi of black bean, while only Cladosporium spp. survived in the white variety. Growth chamber assays were carried out and bacilli cells were applied on seeds without affecting neither the vigor nor the germination potential of either type of bean. In addition, B. amyloliquefaciens PGPBacCA1, by dual cultures, was able to inhibit the development of the following phytopathogenic fungi: Sclerotium rolfsii (35%), Sclerotinia sclerotiorum (76.5%), Rhizoctonia solani (73%), Fusarium solani (56.5%), and Penicillium spp. (71.5%). The UV-MALDI TOF MS analysis showed that B. amyloliquefaciens PGPBacCA1 co-produces different homologues of the lipopeptides surfactin, iturin and fengycin in the presence of S. sclerotiorum and F. solani. These compounds were identified as the main responsible for the antagonistic effect. SEM analysis confirmed the antifungal effects of the lipopeptides, which also caused damage to chlamydospores and sclerotia of Fusarium and Sclerotinia, respectively. B. amyloliquefaciens PGPBacCA1 can thus be applied to these bean seeds varieties as a potential bioprotection agent.

Microbial Regulation of the Antagonistic Activity of Lactic Acid Bacteria Strains under the Influence of Transient Bacilli of the Bacillus Genus

Aim. To study the effect of metabolites and components of aerobic spore-forming bacilli cells (MCASB) of the Bacillus genus on the antagonistic activity of lactic acid bacteria. Methods. Lactic acid bacteria, sampled from rectal and oral swabs from calves, were used in the study. Strains of conditionally pathogenic Escherichia coli, 055k59 No. 3912, Staphylococcus aureus No. 906, Proteus mirabilis, No. 3177, obtained from the collection of the city sanitary-epidemiological inspection service, were used as test cultures. MCASB were prepared using autoclaved microbial mass of Bacillus subtilis production strain 44-p via treatment with lysozyme and ultra- sound. The antagonistic activity of lactic acid bacteria was determined by agar blocks method. Results. It was demonstrated that the addition of MCASB to the culture medium stimulates the antagonistic activity of lactic acid bacteria to pathogenic and conditionally pathogenic microorganisms of intestinal bacteria. The highest average stimulation index (2.56) of the antagonistic activity of lactic acid bacteria was found for E. coli 055k59 No. 3912. Conclusions. The results obtained may be used to improve the microbiological foundations of elaborating novel probiotic preparations.

Quantitative X-ray phase contrast waveguide imaging of bacterial endospores.

Quantitative waveguide-based X-ray phase contrast imaging has been carried out on the level of single, unstained, unsliced and freeze-dried bacterial cells of Bacillus thuringiensis and Bacillus subtilis using hard X-rays of 7.9 keV photon energy. The cells have been prepared in the metabolically dormant state of an endospore. The quantitative phase maps obtained by iterative phase retrieval using a modified hybrid input-output algorithm allow for mass and mass density determinations on the level of single individual endospores but include also large field of view investigations. Additionally, a direct reconstruction based on the contrast transfer function is investigated, and the two approaches are compared. Depending on the field of view and method, a resolution down to 65 nm was achieved at a maximum applied dose of below 5 × 105 Gy. Masses in the range of about ∼110-190 (20) fg for isolated endospores have been obtained.

Image Recognition and Counting for the Bacilli Cell Based on the Microscopic Image

In order to automatically detect bacilli in sputum image with microscopy, an intelligent recognition method based on machine vision is presented. Firstly, a novel background filter was designed based on the single layer perceptron to realize object segmentation from background. After eliminating the short twig and small area noise, the suspicious goals and the image noise are separated. In the feature extraction, besides the base features of single bacillus two important features are presented to solve the difficult problem of identification and counting for the overlapping and winding bacilli cells. Finally, an EBP neural network classifier is designed for the accurate identification and counting of the bacilli cells. Experimental results verified the effectiveness of the presented method.

Characterization of AmiBA2446, a Novel Bacteriolytic Enzyme Active against Bacillus Species

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.

English

Mycobacterium Tuberculosis(M.TB) bacilli is causative agent of Tuberculosis. Manual detection method for M.TB is time consuming, tedious & sometime it may confuse with some stain residue & non tuberculosis bacilli. For this reason the need of atomization is required for exact identification of tuberculosis. We present a new method for M.TB bacilli cells recognition method using Moment invariant and Gaussian member function. The object i.e. M.TB bacilli cells are extracted using color segmentation method, from Ziehl-Neelsen (ZN) stained sputum smears images. These images have blue color background on which red color M.TB bacilli cells. The objects are extracted using color segmentation (thresholding) method and boundary detection. The various extracted shapes contain M. TB bacilli cells, non TB cells, stain residue etc, these extracted objects are preprocess using morphological dilation and then moment invariant feature extracted. Our main objective is to recognizes M.TB bacilli cells with the help of color segmentation & shape base feature moment invariants using Gaussian member function. The recognition rate of M.TB bacilli cells is obtaining 98.17%.

Comparative assessment of heavy metal removal by immobilized and dead bacterial cells: A biosorption approach

Microorganisms play a vital role in heavy metal contaminated soil and wastewater by the mechanisms of biosorption. In this study, heavy metal resistant bacteria were isolated from an electroplating industrial effluent samples that uses copper, cadmium and lead for plating. These isolates were characterized to evaluate their applicability for heavy metal removal from industrial wastewaters. The physico-chemical parameters of the samples were initially analyzed. The optimum conditions of pH, biomass concentration and heavy metal concentration were determined for the microbial growth on biosorbents and correlated with heavy metal removal. The observed optimum conditions were applied for the biosorption process carried out in immobilized and dead bacterial isolates. The biosorption of immobilized cells of Bacillus sp. was 69.34% of Cu, Pseudomonas sp. was 90.41% of Cd and Micrococcus sp. was 84.27% of Pb, whereas the dead cells of Bacillus sp. was 44.73% of Cu, Pseudomonas sp. was 86.66% of Cd and Micrococcus sp. was 79.22%. Experimental results reveal that all the immobilized isolates have potential application for the removal of Cu, Cd and Pb from industrial wastewater than the dead bacterial cells.

The Redox Regulator Fnr Is Required for Fermentative Growth and Enterotoxin Synthesis in Bacillus cereus F4430/73

Glucose-grown cells of Bacillus cereus respond to anaerobiosis and low extracellular oxidoreduction potentials (ORP), notably by enhancing enterotoxin production. This response involves the ResDE two-component system. We searched the B. cereus genome for other redox response regulators potentially involved in this adaptive process, and we identified one gene encoding a protein predicted to have an amino acid sequence 58% identical (80% similar) to that of the Bacillus subtilis Fnr redox regulator. The fnr gene of the food-borne pathogen B. cereus F4430/73 has been cloned and partially characterized. We showed that fnr was up-regulated during anaerobic fermentation, especially when fermentation occurred at low ORP (under highly reducing conditions). The expression of fnr was down-regulated in the presence of O(2) and nitrate which, unlike fumarate, stimulated the respiratory pathways. The inactivation of B. cereus fnr abolished fermentative growth but only moderately affected aerobic and anaerobic nitrate respiratory growth. Analyses of glucose by-products and the transcription profiles of key catabolic genes confirmed the strong regulatory impact of Fnr on B. cereus fermentative pathways. More importantly, the fnr mutation strongly decreased the expression of PlcR-dependent hbl and nhe genes, leading to the absence of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) secretion by the mutant. These data indicate that fnr is essential for both fermentation and toxinogenesis. The results also suggest that both Fnr and the ResDE two-component system belong to a redox regulatory pathway that functions at least partially independently of the pleiotropic virulence gene regulator PlcR to regulate enterotoxin gene expression.

Production and Validation of the Use of Gamma Phage for Identification of Bacillus anthracis

Gamma phage specifically lyses vegetative cells of Bacillus anthracis and serves as part of the basis for identification of isolates from agar cultures. We report our study to standardize gamma phage production and preparation and to validate the assay for routine use. Unstable phage preparations were largely reduced through propagation of phage on blood agar cultures of the avirulent B. anthracis strain CDC684 and were adequately stable for extended storage beyond 1 to 2 years at 4 degrees C, provided that the preparation initially gave rise to clearly discernible plaques (macroplaques, 5 to 10 mm in diameter) on dilution at 1:8 or greater during potency testing with the Sterne strain or its equivalent. The primary intent of the assay was to test nonhemolytic, ground-glass-appearing bacterial B. anthracis-like colonies arising from culture of clinical or nonclinical samples on 5% sheep blood agar. Specifically, the assay was designed to show clear or primarily clear circular zones of lysis on bacterial lawns at the site of gamma phage inoculation after incubation at 35 degrees C +/- 2 degrees C for 20 h. When tested with 51 B. anthracis strains and 49 similar non-B. anthracis Bacillus species, the analytical specificity was >95%, a value that is intentionally low because our study design included two rare nonsusceptible B. anthracis strains as well as a rare susceptible non-B. anthracis strain, B. cereus ATCC 4342. Repeatability, day-to-day precision, and analyst-to-analyst precision were superior. The assay was rugged to variations among phage lots, phage concentration, amounts of bacterial inoculum, and incubation times as short as 6 to 8 h. System suitability evaluation showed improved robustness when bacterial lawns were tested with high- and low-density inoculum using the first and second quadrants of a serial four-quadrant streak on 5% sheep blood agar plates.

Diversity and redundancy in bacterial chromosome segregation mechanisms

Bacterial cells are much smaller and have a much simpler overall structure and organization than eukaryotes. Several prominent differences in cell organization are relevant to the mechanisms of chromosome segregation, particularly the lack of an overt chromosome condensation/decondensation cycle and the lack of a microtubule-based spindle. Although bacterial chromosomes have a rather dispersed appearance, they nevertheless have an underlying high level of spatial organization. During the DNA replication cycle, early replicated (oriC) regions are localized towards the cell poles, whereas the late replicated terminus (terC) region is medially located. This spatial organization is thought to be driven by an active segregation mechanism that separates the sister chromosomes continuously as replication proceeds. Comparisons of various well-characterized bacteria suggest that the mechanisms of chromosome segregation are likely to be diverse, and that in many bacteria, multiple overlapping mechanisms may contribute to efficient segregation. One system in which the molecular mechanisms of chromosome segregation are beginning to be elucidated is that of sporulating cells of Bacillus subtilis. The key components of this system have been identified, and their functions are understood, in outline. Although this system appears to be specialized, most of the functions are conserved widely throughout the bacteria.

Multicopy plasmids affect replisome positioning in Bacillus subtilis.

The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.

Postdivisional synthesis of the Sporosarcina ureae DNA translocase SpoIIIE either in the mother cell or in the prespore enables Bacillus subtilis to translocate DNA from the mother cell to the prespore.

The differentiation of vegetative cells of Bacillus subtilis into spores involves asymmetric cell division, which precedes complete chromosome partitioning. The DNA translocase SpoIIIE is required to translocate the origin distal 70% of the chromosome from the larger mother cell into the smaller prespore, the two cells that result from the division. We have tested the effect of altering the time and location of SpoIIIE synthesis on spore formation. We have expressed the spoIIIE homologue from Sporosarcina ureae in B. subtilis under the control of different promoters. Expression from either a weak mother cell-specific (sigma(E)) promoter or a weak prespore-specific (sigma(F)) promoter partly complemented the sporulation defect of a spoIIIE36 mutant; however, expression from a strong prespore-specific (sigma(F)) promoter did not. DNA translocation from the mother cell to the prespore was assayed using spoIIQ-lacZ inserted at thrC; transcription of spoIIQ occurs only in the prespore. Translocation of thrC::spoIIQ-lacZ into the prespore occurred efficiently when spoIIIE(Su) was expressed from the weak sigma(E)- or sigma(F)-controlled promoters but not when it was expressed from the strong sigma(F)-controlled promoter. It is speculated that the mechanism directing SpoIIIE insertion into the septum in the correct orientation may accommodate slow postseptational, prespore-specific SpoIIIE synthesis but may be swamped by strong prespore-specific synthesis.

Semicontinuous production of red pigment by immobilized cells ofBacillus sp. BH-99 using column bioreactor

The semicontinuous production of red pigment by immobilized cells ofBacillus sp. BH-99 was investigated in comparison with free cells. The red pigment produced highest productivity under the conditions of aeration of 0.2 mL/min and 2 mm diameter of gel beads by using 3.0% sodium alginate. Semicontinuous production by immobilized cells showed the highest productivity with replacement of fresh production medium in every 72 h for fourth fermentation cycle following the conditions of red pigment productivity.

The spectrum of spontaneous rifampin resistance mutations in the rpoB gene of Bacillus subtilis 168 spores differs from that of vegetative cells and resembles that of Mycobacterium tuberculosis.

Mutations causing rifampin resistance in vegetative cells of Bacillus subtilis 168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the rpoB gene encoding the beta subunit of RNA polymerase. In this study, we demonstrated that spores of B. subtilis 168 exhibit a spectrum of spontaneous rifampin resistance mutations distinct from that of vegetative cells. In addition to the rpoB mutations Q469K, Q469R, and H482Y previously characterized in vegetative cells, we isolated a new mutation of rpoB, H482R, from vegetative cells. Additional new rifampin resistance mutations arising from spores were detected at A478N and most frequently at S487L. The S487L change is the predominant change found in rpoB mutations sequenced from rifampin-resistant clinical isolates of Mycobacterium tuberculosis. The observations are discussed in terms of the underlying differences of the DNA environment within dormant cells and vegetatively growing cells.

Increased killing of Bacillus subtilis on the hair roots of transgenic T4 lysozyme-producing potatoes.

Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium Erwinia carotovora subsp. carotovora have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to associate the cells of Bacillus subtilis with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Désirée and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed B. subtilis cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5- to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growth-sustaining effects of plants on bacteria.