The bacterial cytoskeleton: in vivo dynamics of the actin-like protein Mbl of Bacillus subtilis

Mbl is a bacterial actin homolog that controls cell morphogenesis in Bacillus subtilis. A functional GFP-Mbl fusion protein was used to examine the behavior of the helical cables formed by Mbl protein in live B. subtilis cells. The cables undergo dynamic changes during cell cycle progression. They are stable but not rigid while elongating in parallel with cell growth, and they require septum formation to divide/cleave. Fluorescence recovery after photobleaching (FRAP) analysis showed that the cables are continuously remodeled during cell elongation. Turnover occurs along the length of the helical Mbl filaments, with no obvious polarity and a recovery half-time of about 8 min. These findings have important implications for the nature of bacterial cell wall architecture and synthesis.     

Intracellular growth of Listeria monocytogenes insertional mutant deprived of protein p60

The study of the mutant strain described here demonstrates that several characteristics contribute to maximal virulence of pathogenic strains of L. monocytogenes. The invasion levels of L. monocytogenes JB1115, a p60-deficient strain, were the same as for the parent strain L. monocytogenes 1043S in J774 macrophage-like cells. The invasion level of Listeria strains in Int407 cells was 100 times lower than in J774 cells. In epithelial Int407 cells, the time of division of p60- strain L. monocytogenes JB1115 was 43% slower than for the parent strain. In this study, two lisosomotrophic agents, ammonium chloride and chlorquinoline were tested in experimental L. monocytogenes 1043S and p60-deprived mutant JB1115 infection in both cell lines. The presence of ammonium chloride increased the level of infection (calculated as number of gentamicin-resistant cells) of both Listeria strains, but in the case of infection by p60 mutant, the increased amount of ammonium chloride showed only a minimal effect on the number of isolated bacteria. In both cell lines treated with chlorquinoline we observed a decrease in the number of viable intracellular bacteria isolated from infected monolayers. Our observation of parental and mutated strains of Listeria showed that phospholipase activity also depends on the presence of p60 protein. Mutated strain showed 31.46% reduction of PI-PLC activity measured in normal growth conditions. Protein p60 plays a role not only in listeriolysin O mediated haemolytic activity but full phospholipase C activity is also dependent on the presence of the Iap protein.     

Production of human papillomavirus type 33 L1 major capsid protein and virus-like particles from Bacillus subtilis to develop a prophylactic vaccine against cervical cancer

We developed a bacterial expression system to produce human papillomavirus (HPV) type 33 L1 major capsid protein and virus-like particles from a recombinant Bacillus subtilis strain. For the first time, we have isolated self-assembled virus-like particles (VLPs) of HPV type 33 from B. subtilis, a strain generally recognized as safe (GRAS). The gene encoding the major capsid protein L1 of HPV type 33 was amplified from viral DNA isolated from a Korean patient and expressed in B. subtilis; a xylose-induction system was used to control gene activity. HPV33 L1 protein was partially purified by 40% (w/v) sucrose cushion centrifugation and strong cation exchange column chromatography. Eluted samples exhibited immunosignaling in fractions of 0.5-1.0 M NaCl. The HPV33 L1 protein was shown to be approximately 56 kDa in size by SDS-PAGE and Western blotting; recovery and purity were quantified by indirect immuno-ELISA assay. The final yield and purity were approximately 20.4% and 10.3%, respectively. Transmission electron microscopic analysis of fractions immunoactive by ELISA revealed that the L1 protein formed self-assembled VLPs with a diameter of approximately 20-40 nm. Humoral and cellular immune responses provoked by the B. subtilis/HPV33 L1 strain were approximately 100- and 3-fold higher than those of the empty B. subtilis strain as a negative control, respectively. Development of a VLP production and delivery system using B. subtilis will be helpful, in that the vaccine may be convenient production as an antigen delivery system. VLPs thus produced will be safer for human use than those purified from Gram-negative strains such as Escherichia coli. Also, use of B. subtilis as a host may aid in the development of either live or whole cell vaccines administered by antigen delivery system.     

The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity.

Expression of the iap gene of Listeria monocytogenes in the L. monocytogenes rough mutant RIII and in Bacillus subtilis DB104 caused the disruption of the cell chains which these two strains normally form under exponential growth conditions. The p60 protein produced by L. monocytogenes and B. subtilis DB104 also exhibited bacteriolytic activity detected in denaturing polyacrylamide gels containing heat-killed Micrococcus lysodeikticus. Purification of the p60 protein led to aggregation of p60 and loss of the cell chain disruption and bacteriolytic activities. A cysteine residue in the C-terminal part of p60 which is conserved in all p60-like proteins from the other Listeria species seems to be essential for both activities. The iap gene could not be inactivated without a loss of cell viability, indicating that p60 is an essential housekeeping protein for L. monocytogenes and probably also for other Listeria species. These data suggest that p60 possesses a murein hydrolase activity required for a late step in cell division.     

The safety of Bacillus subtilis and Bacillus indicus as food probiotics

Aims:  To conduct in vitro and in vivo assessments of the safety of two species of Bacillus , one of which, Bacillus subtilis , is in current use as a food supplement.
Methods and Results:  Cultured cell lines, Caco-2, HEp-2 and the mucus-producing HT29-16E cell line, were used to evaluate adhesion, invasion and cytotoxicity. The Natto strain of B. subtilis was shown to be able to invade and lyse cells. Neither species was able to adhere significantly to any cell line. The Natto strain was also shown to form biofilms. No strain produced any of the known Bacillus enterotoxins. Disc-diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that only Bacillus indicus carried resistance to clindamycin at a level above the minimum inhibitory concentration breakpoints set by the EFSA. In vivo assessments of acute and chronic dosing in guinea pigs and rabbits were made. No toxicity was observed in animals under these conditions.
Conclusions:  Bacillus indicus and B. subtilis should be considered safe for oral use although the resistance of B. indicus to clindamycin requires further study.
Significance and Impact of the Study:  The results support the use of B. subtilis and B. indicus strains as food supplements.     

Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis.

The structural gene for a thermostable protease from Bacillus stearothermophilus was cloned in plasmid pTB90. It is expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about 15-fold more protease (310 U/mg of cell dry weight) than did the wild-type strain of B. stearothermophilus. Some properties of the proteases that have been purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant difference was observed among the enzyme properties studied here despite the difference in host cells. We found that the protease, neutral in pH characteristics and with a molecular weight of 36,000, retained about 80% of its activity even after treatment of 65 degrees C for 30 min.     

Regulation of cell wall morphogenesis in Bacillus subtilis by recruitment of PBP1 to the MreB helix

The bacterial actin homologue MreB plays a key role in cell morphogenesis. In Bacillus subtilis MreB is essential under normal growth conditions and mreB mutants are defective in the control of cell diameter. However, the precise role of MreB is still unclear. Analysis of the lethal phenotypic consequences of mreB disruption revealed an unusual bulging phenotype that precedes cell death. A similar phenotype was seen in wild-type cells at very low Mg²⁺ concentrations. We found that inactivation of the major bi-functional penicillin-binding protein (PBP) PBP1 of B. subtilis restored the viability of an mreB null mutant as well as preventing bulging in both mutant and wild-type backgrounds. Bulging was associated with delocalization of PBP1. We show that the normal pattern of localization of PBP1 is dependent on MreB and that the proteins can physically interact using in vivo pull-down and bacterial two-hybrid approaches. Interactions between MreB and several other PBPs were also detected. Our results suggest that MreB filaments associate directly with the peptidoglycan biosynthetic machinery in B. subtilis as part of the mechanism that brings about controlled cell elongation.     

Establishment of monoclonal antibodies specific for Bacillus subtilis DB9011

Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.     

Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure

Microwave radiation in Escherichia coli and Bacillus subtilis cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions. However, no significant reduction of cell density was observed in either cell suspension. It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed. Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the B. subtilis cells. Microwave-injured E. coli cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet B. subtilis cells were resistant to SDS.     

Adherence of Bilophila wadsworthia to cultured human embryonic intestinal cells

Adherence of Bilophila wadsworthia to the cultured human embryonic intestinal cell line, Intestine 407 (Int 407), varied among the strains tested from strongly adherent (76-100% cells positive for one or more adherent bacteria) to non- or weakly adherent (0-25% positive cells). Although negative staining revealed that infrequent cells of an adherent strain, WAL 9077, the adherent type-strain, WAL 7959, and a non-adherent strain, WAL 8448, expressed loosely associated fimbrial structures, a role for these structures in adhesion could not be confirmed with either scanning or thin-section electron micrography. Ruthenium red staining of thin-section preparations and subsequent electron microscopy failed to reveal an extensive extracellular polysaccharide layer. SDS-PAGE analysis of crude outer membrane fractions of WAL 9077 and WAL 8448 demonstrated clear differences in their major and minor outer membrane protein components. Thus, we postulate that the adherence of B. wadsworthia to Int 407 cells is mediated by an outer membrane or cell wall component.     

Function of heterologous and truncated RNase P proteins in Bacillus subtilis

Bacterial RNase P is composed of an RNA subunit and a single protein (encoded by the rnpB and rnpA genes respectively). The Bacillus subtilis rnpA knockdown strain d7 was used to screen for functional conservation among bacterial RNase P proteins from a representative spectrum of bacterial subphyla. We demonstrate conserved function of bacterial RNase P (RnpA) proteins despite low sequence conservation. Even rnpA genes from psychrophilic and thermophilic bacteria rescued growth of B. subtilis d7 bacteria; likewise, terminal extensions and insertions between beta strands 2 and 3, in the so-called metal binding loop, were compatible with RnpA function in B. subtilis. A deletion analysis of B. subtilis RnpA defined the structural elements essential for bacterial RNase P function in vivo. We further extended our complementation analysis in B. subtilis strain d7 to the four individual RNase P protein subunits from three different Archaea, as well as to human Rpp21 and Rpp29 as representatives of eukaryal RNase P. None of these non-bacterial RNase P proteins showed any evidence of being able to replace the B. subtilis RNase P protein in vivo, supporting the notion that archaeal/eukaryal RNase P proteins are evolutionary unrelated to the bacterial RnpA protein.     

Influence of electrical properties on the evaluation of the surface hydrophobicity of Bacillus subtilis

The surface hydrophobicity of nine Bacillus subtilis strains in different states (spores, vegetative cells, and dead cells) was assessed by water contact angle measurements, hydrophobic interaction chromatography (HIC) and bacterial adhesion to hydrocarbon (BATH). Electrokinetic properties of B. subtilis strains were characterized by zeta potential measurements and found to differ appreciably according to the strain. Correlations between HIC data, BATH data and zeta potential showed that HIC and BATH are influenced by electrostatic interactions. Water contact angle measurements thus provide a better estimate of cell surface hydrophobicity. The water contact angle of B. subtilis varied according to the strain and the state, the spores tending to be more hydrophobic than vegetative cells.     

Bacillus thuringiensis pulmonary infection: critical role for bacterial membrane-damaging toxins and host neutrophils

The occurrence of Bacillus thuringiensis bacteremia in a neutropenic patient suffering from severe pulmonary disease addressed the question of whether the aggressive behavior of B. thuringiensis depended on the host status and/or on the membrane-damaging toxins the isolate produced. After intratracheal injection, BALB/c mice developed pneumonia followed by fatal dissemination into deep organs, with mice rendered neutropenic by cyclophosphamide injection being extremely more susceptible to infection than normal animals. In animals infected with isogenic strains of B. thuringiensis progressively more defective in membrane-damaging toxins (407 Cry->IP2>MP02), an increase in the 50% lethal dose was registered (3.9x10(5), 1.1x10(6), 1.2x10(7)CFU). Consistently, after non-lethal dose application, only 407 Cry- replicated intrapulmonary, reaching a bacterial burden 4.7-fold and 40.9-fold higher than IP2 (P=0.018) and MP02 (P=0.008) at 48h post-inoculation. Notably, the time-course of infection was similar in animals infected with viable bacilli or spores, with neutropenic mice always being more susceptible to infection. The overall results indicate that B. thuringiensis may be responsible for opportunistic infections and strongly suggest that membrane-damaging toxins contribute to intrapulmonary bacterial persistence favoring dissemination.     

Correlation between the growth inhibitory effects, partition coefficients and teratogenic effects of lipophilic acids.

The inhibition of cell duplication by many lipophilic acids was measured in Bacillus subtilis and in the following mammalian cell lines, the human epithelial-type cell lines HeLa, strain R and strain L-132, the human fibroblast cell line VA-13, and the rat glial cell line C. The results were correlated to the partition coefficient and the distribution coefficient (= apparent partition coefficient at pH 7.2) of the compounds, using octanol/water partition coefficients and pKa values either from the literature or measured for this work. For B. subtilis, the logarithm of the inhibitory potency of most compounds increases linearly with the logarithm of the partition coefficient. Exceptional high potencies were observed for compounds that can efficiently delocalize the charge of the negative ion over the whole molecule. Most compounds inhibit tissue cultures at least as potently as they inhibit B. subtilis. But some compounds are significantly more potent in tissue cultures than would have been expected from the B. subtilis data; such compounds (analgesics/antipyretics, anti-inflammatory compounds, butyrate, norepinephrine) presumably inhibits mammalian cells by specific reactions with certain cell components. However, most compounds inhibit the different cell lines to a similar degree, indicating no cellular specificity; exceptions to this rule are chlorambucil, chlortetracycline and dexamethasone. Many of the lipophilic acids that are potent inhibitors of mammalian cell replication are also teratogenic. Exceptional compounds may not reach the embryo. We propose that a number of other lipophilic acids that are potenta inhibitors and to which humans are frequently exposed should be tested for their teratogenic effect.     

Levels of glycine betaine in growing cells and spores of Bacillus species and lack of effect of glycine betaine on dormant spore resistance

Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased approximately 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and > or =20-fold lower in B. cereus and B. megaterium spores. Although GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.     

Metalloproteinase from Bacillus subtilis: - "intracellular" and extracellular enzymes

"Intracellular" metalloproteinase was purified to homogeneity from Bacillus subtilis 103 crude cell extract, using affinity chromatography on bacitracin-Sepharose 4B. The degree of purification and the yield of the enzyme were about 260-fold and 3%, respectively. In its physico-chemical properties and the amino acid composition the enzyme is very similar, if not identical, to the extracellular metalloproteinase isolated from the culture filtrate of the same strain. Extracellular metalloproteinase-deficient mutant strain Bacillus subtilis SMY-512 does not produce the "intracellular" enzyme either. THe activity of "intracellular" metalloproteinase in the periplasmic space of the cells is about 70% of that in the cytoplasm, thus being indicative of a rather regular distribution of the enzyme throughout the cell compartment.     

Actin homolog MreBH governs cell morphogenesis by localization of the cell wall hydrolase LytE

MreB proteins are bacterial actin homologs involved in cell morphogenesis and various other cellular processes. However, the effector proteins used by MreBs remain largely unknown. Bacillus subtilis has three MreB isoforms. Mbl and possibly MreB have previously been shown to be implicated in cell wall synthesis. We have now found that the third isoform, MreBH, colocalizes with the two other MreB isoforms in B. subtilis and also has an important role in cell morphogenesis. MreBH can physically interact with a cell wall hydrolase, LytE, and is required for its helical pattern of extracellular localization. Moreover, lytE and mreBH mutants exhibit similar cell-wall-related defects. We propose that controlled elongation of rod-shaped B. subtilis depends on the coordination of cell wall synthesis and hydrolysis in helical tracts defined by MreB proteins. Our data also suggest that physical interactions with intracellular actin bundles can influence the later localization pattern of extracellular effectors.     

Expression of Escherichia coli Min system in Bacillus subtilis and its effect on cell division

In both rod-shaped Bacillus subtilis and Escherichia coli cells, Min proteins are involved in the regulation of division septa formation. In E. coli , dynamic oscillation of MinCD inhibitory complex and MinE, a topological specificity protein, prevents improper polar septation. However, in B. subtilis no MinE is present and no oscillation of Min proteins can be observed. The function of MinE is substituted by that of an unrelated DivIVA protein, which targets MinCD to division sites and retains them at the cell poles. We inspected cell division when the E. coli Min system was introduced into B. subtilis cells. Expression of these heterologous Min proteins resulted in cell elongation. We demonstrate here that E. coli MinD can partially substitute for the function of its B. subtilis protein counterpart. Moreover, E. coli MinD was observed to have similar helical localization as B. subtilis MinD.