Antigenic Heterology Between Flagellin and Flagella of Bacillus subtilis

The antigenic properties of Bacillus subtilis flagella and periodate-treated flagellin were compared by using immobilization-inhibition and diethylaminoethyl cellulose binding assay. The results suggest that there are both unique and cross-reactive antibodies elicited by B. subtilis flagella and flagellin.     

pH-dependent flagella formation by facultative alkaliphilic Bacillus sp. C-125.

A facultative alkaliphilic strain of Bacillus sp. C-125 grown at alkaline pH had many sinuous peritrichous flagella and was highly motile. However, most of the cells grown initially at pH 7 were non-motile and possessed few straight flagella. The amount of flagellin was low when the organism was grown at pH 7, suggesting that non-motility is due to poor synthesis of flagellin. The molecular mass of the flagellin was 37 kDa and the isoelectric point was pH 5.0. The amino acid composition of the flagellin was similar to that found in the flagellin from neutrophilic Bacillus subtilis 168.     

Antigenic variation of Campylobacter flagella.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of flagella dissociated from strains of Campylobacter coli and Campylobacter jejuni belonging to the heat-labile serogroup LIO 8 showed that some strains were capable of producing flagellin subunits of two different molecular weights (MrS), 59,500 and 61,500. Immunoelectron microscopy of cultures of the type strain of this serogroup, C. coli VC167, showed the presence of two flagellum filaments of different antigenic specificity. Epitopes on the surface of one of these flagella bound antibodies in LIO 8 typing antiserum, and Western blotting (immunoblotting) and immunoprecipitation showed that the flagellum was composed of flagellin of Mr 61,500. The other flagellum antigenic type did not bind LIO 8 antibodies but did possess serospecific epitopes which bound a second polyclonal antiserum, LAH2. This second antigenic flagellum type was composed of the Mr 59,500 flagellin. Cells producing either of the flagellum antigenic types serotyped as LIO 8, indicating that flagella composed of the Mr 61,500 flagellin do not carry the serological determinants for this serogroup. The ability of C. coli VC167 to produce these flagella of different subunit MrS was shown to represent a bidirectional antigenic variation. When measured in culture medium, the phase 1-to-phase 2 transition occurred at a rate of approximately 2.0 x 10(-5) per cell per generation, and the phase 2-to-phase 1 transition occurred at a rate of 1.2 x 10(-6) per cell per generation.     

Identification of flagellin associated with the Bacillus subtilis folded chromosome.

We have analyzed the nature and contents of a major protein, P36, in the nucleoid of the Bacillus subtilis wild type and an isogenic mutant devoid of flagella. It appears that deoxyribonucleic acid-P36 complex is flagellin present as membrane-associated flagella.     

Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by the ifm locus

Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures. No information is available regarding the regulation of flagellar formation in B. subtilis in response to contact with a solid surface. One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates. This study describes the swarming ability of a B. subtilis hypermotile mutant harboring a mutation in the ifm locus that has long been known to affect the degree of flagellation and motility in liquid media. On solid media, the mutant produces elongated and hyperflagellated cells displaying a 10-fold increase in extracellular flagellin. In contrast to the mutant, the parental strain, as well as other laboratory strains carrying a wild-type ifm locus, fails to activate a swarm response. Furthermore, it stops to produce flagella when transferred from liquid to solid medium. Evidence is provided that the absence of flagella is due to the lack of flagellin gene expression. However, restoration of flagellin synthesis in cells overexpressing sigma(D) or carrying a deletion of flgM does not recover the ability to assemble flagella. Thus, the ifm gene plays a determinantal role in the ability of B. subtilis to contact with solid surfaces.     

Flagellin as a biomarker for Bacillus subtilis strains; application to the DB9011 strain and the study of interspecific diversity in amino-acid sequences.

Polymerase chain reaction (PCR) for the flagellin central domain coding region (FCD-PCR) was applied to the detection and discrimination of Bacillus subtilis DB9011, a strain with useful functions in agriculture. Cross-reactions were observed in 4 B. subtilis strains with similar flagellin genes (hag). Alignment of partial amino-acid sequences of flagellin and the results of PCR for the 16S/23S rRNA spacer in 11 B. subtilis strains suggested the presence of a group including strains with antifungal activity (DB9011 and others).     

Flagellin as a Biomarker for Bacillus subtilis Strains; Application to the DB9011 Strain and the Study of Interspecific Diversity in Amino-acid Sequences

Polymerase chain reaction (PCR) for the flagellin central domain coding region (FCD-PCR) was applied to the detection and discrimination of Bacillus subtilis DB9011, a strain with useful functions in agriculture. Cross-reactions were observed in 4 B. subtilis strains with similar flagellin genes (hag). Alignment of partial amino-acid sequences of flagellin and the results of PCR for the 16S/23S rRNA spacer in 11 B. subtilis strains suggested the presence of a group including strains with antifungal activity (DB9011 and others).     

Initiation of deoxyribonucleic acid replication in a temperature-sensitive mutant of B. subtilis: evidence for a transcriptional step

A mutant of Bacillus subtilis unable to initiate a new round of replication at 45 C has been described. Here we show that inhibition of DNA synthesis in this mutant is reversible and that DNA synthesis is resumed at low temperature, even in the presence of chloramphenicol. Initiation of a new replication cycle thus can occur in the absence of protein synthesis. A thermolabile component required for initiation therefore appears to be synthesized at 45 C in an inactive form and can be activated at 30 C in the presence of an inhibitor of protein synthesis. Although resistant to chloramphenicol, the reinitiation of replication occurring after lowering the temperature is sensitive to rifampin and streptolydigin.     

Synthesis of Bacterial Flagella I. Requirement for Protein and Ribonucleic Acid Synthesis During Flagellar Regeneration in Bacillus subtilis

A relatively simple immunochemical procedure for estimating flagellar protein was developed. This procedure involved measuring the binding of purified, radioactively labeled, antiflagellar antibodies to bacteria. The assay was used to determine the requirements for ribonucleic acid (RNA) and protein synthesis during flagellar regeneration in Bacillus subtilis. Immediate inhibition of flagella development was observed when chloramphenical or puromycin was added to cells. This inhibition indicated the absence of a large pool of flagella precursors that could be assembled in the absence of protein synthesis. When the cells were starved for uracil or treated with actinomycin D to inhibit RNA synthesis, the ability of the cells to regenerate flagella decayed with a half-life of 5.5 min. When B. subtilis auxotrophs were starved for tryptophan, they continued to synthesize flagella, although this process was also inhibited by actinomycin D. On the basis of these results, we concluded that (i) the system involved in flagellar regeneration does not have unusual metabolic stability, (ii) regeneration requires both concomitant protein and RNA syntheses, and (iii) B. subtilis continues to synthesize messenger RNA during tryptophan starvation.     

Effects of thermoradiation on bacteria.

A 60Co source was used to determine the effects of thermoradiation on Achromobacter aquamarinus, Staphylococcus aureus, and vegetative and spore cells of Bacillus subtilis var. globigii. The rate of inactivation of these cultures, except vegetative-cell populations of B. subtilis, was exponential and in direct proportion to temperature. The D10 (dose that inactivates 90% of the microbial population) value for A. aquamarinus was 8.0 Krad at 25 degrees C and 4.9 Krad at 35 degrees C. For S. aureus, D10 was 9.8 and 5.3 Krad at 35 and 45 degrees C, respectively. Vegetative cells of B. subtilis demonstrated a rapid initial inactivation followed by a steady but decreased exponential rate. The D10 at 25 degrees C was 10.3 Krad, but at 35 and 45 degrees C this value was 6.2 and 3.8 Krad, respectively. Between 0 and 95 Krad, survival curves for B. subtilis spores at 75 degrees C showed slight inactivation, increasing in rat at and above 85 degrees C. The D10 values for spores at 85 and 90 degrees C were 129 and 92 Krad, respectively. Significant synergism between heat and irradiation was noted at 35 degrees C for A. aquamarinus and 45 degrees C for S. aureus. The presence of 0.1 mM cysteine in suspending media afforded protection to both cultures at these critical temperatures. On the other hand, cysteine sensitized B. subtilis spores at radiation doses greater than 100 Krad. The combined effect of heat and irradiation was more destructive to bacteria than either method alone.     

Regulation of nitrogen catabolic enzymes in Bacillus spp.

The levels of the inducible nitrogen catabolic enzymes arginase (L-arginine amidinohydrolase, EC 3.5.3.1) and alanine dehydrogenase (L-alanine:NAD+ oxidoreductase [deaminating], EC 1.4.1.1) from Bacillus licheniformis and histidase (L-histidine ammonia-lyase, EC 4.3.1.3) from Bacillus subtilis and the ammonia assimilatory enzymes from B. licheniformis were determined in cultures grown in the presence of different nitrogen sources. Although the levels of these enzymes were dependent upon the nitrogen source present, induction of the catabolic enzymes in response to the addition of inducer occurred even in the presence of preferred nitrogen sources. Intracellular pool sizes of ammonia, glutamate, glutamine, and alpha-ketoglutarate were measured in continuous cultures of b. licheniformis growing in the presence of different nitrogen sources. A comparison of the pool sizes of these metabolites with the ammonia assimilatory enzyme levels showed that the pools of the metabolites did not change in a manner consistent with their use as regulators of the synthesis of any of these enzymes.     

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.     

Pleiotropic phenomena in autolytic enzyme(s) content, flagellation, and simultaneous hyperproduction of extracellular alpha-amylase and protease in a Bacillus subtilis mutant.

A mutant of Bacillus subtilis 6160 that had been isolated by its hyperproduction of alpha-amylase and protease lacked flagella and motility, and its content of autolytic enzyme(s) was reduced to one-third to one-fourth that of the parent. These phenotypic differences were completely co-transferred by the deoxyribonucleic acid (DNA) of the mutant when five DNA recipient strains of B. subtilis were transformed. The revertants, isolated by motility with a frequency of approximately 10(-7), recovered a normal level of autolytic activity and showed reduced productivity of alpha-amylase and protease. This point mutation allowed normal flagellin synthesis, spore formation, and rate of growth. The comparison of cell envelope of the mutant with that of the parent indicated that there was no significant difference except loss of flagella. Therefore the association at the cell surface of a group of extracellular proteins consisting of alpha-amylase, proteases, flagellin, and autolytic enzymes(s) seem to be coordinately regulated by the gene or seem to be affected coordinately by certain undetected alterations of the cell envelope.     

Restriction fragments that exert promoter activity during postexponential growth of Bacillus subtilis.

Two restriction fragments of Bacillus subtilis DNA were identified which caused the cat-86 gene present on the promoter cloning plasmid pPL703 to be activated predominantly during postexponential growth of host cells. The postexponential increase was observed in both sporulation-positive strains and in a spoOA mutant of B. subtilis. However, the postexponential increase in the cat-86 gene product, chloramphenicol acetyltransferase, was diminished or not observed when the plasmid-containing cells were grown in the presence of excess glucose. The promoter-containing fragment, designated as 33, was mapped to a site on the B. subtilis chromosome adjacent to hisA. The other fragment, 14, mapped to a site adjacent to ctrA. When present on a high-copy vector, both fragments caused a reduction in the sporulation frequency of host cells. Fragment 33 in high copy number conferred on B. subtilis cells three additional phenotypic changes: brown colony color, intracellular inclusions, and, in a protease-deficient mutant, the production of extracellular protease activity. These activities were observed only in postexponential-phase cultures.     

Protein-lipid-lipopolysaccharide association in the superficial layer of Spirillum serpens cell walls.

The backing layer of the Spirillum serpens VHA cell wall, which supports and is bonded to the outer, structured protein layer, was isolated and shown to be similar in composition to the same elements of the outer membrane. It contained a lipopolysaccharide that was similar, but not identical, to that of the intact wall and the same phospholipids. The interaction of the isolated wall lipopolysaccharide with the loosely bound wall lipids provided lamellae, whose surfaces were an effective template for a lifelike reassembly of the isolated outer-layer hexagonal protein in the presence of Ca2+. Assembly did not take place on pure lipopolysaccharide, which dispersed in differing forms. A lipid-lipopolysaccharide-water interface appeared to be required as a template surface for the assembly. Lipopolysaccharide from Pseudomonas aeruginosa was able to replace that of S. serpens in the template. These observations suggest that lipid-lipopolysaccharide complexes are highly ordered, and this order is important to the nucleation and assembly of the protein array.     

Isolation and characterization of Campylobacter flagellins.

Sequential acid pH dissociation, differential ultracentrifugation, and neutral pH reassociation were used to partially purify serotypically distinct flagella from three strains of Campylobacter jejuni and the two antigenic phases of flagella of Campylobacter coli VC167. Each C. jejuni flagellin and C. coli VC167 antigenic phase 1 flagellin were purified to homogeneity by reverse-phase high-performance liquid chromatography with a C8 Spheri-10 column. C. coli VC167 antigenic phase 2 was purified to homogeneity by ion-exchange chromatography with a Mono-Q column. Amino acid compositional analysis put the C. jejuni flagellin molecular weight in the range 63,200 to 63,800 and the C. coli antigenic phase 1 and 2 flagellins at 61,500 and 59,500, respectively. The amino acid compositions of the C. jejuni were similar to each other and to the C. coli VC167 antigenic phase 1 and phase 2 flagellins. One-dimensional peptide mapping of the C. jejuni flagellins by partial digestion with trypsin or chymotrypsin confirmed the structural similarities of the C. jejuni flagellins and the C. coli VC167 antigenic phase 1 flagellin and showed that C. coli VC167 antigenic phase 2 flagellin was structurally distinct from the phase 1 flagellin. The antigenic phase 2 flagellin was especially sensitive to digestion by chymotrypsin. Amino-terminal sequence analysis showed that the 20 N-terminal amino acids of the Campylobacter flagellins were highly conserved. The Campylobacter flagellins also shared limited sequence homology with the N-terminal sequences reported for Salmonella and Bacillus flagellins.