The ply genes encoding the endolysin proteins from Bacillus cereus phages Bastille, TP21, and 12826 were identified, cloned, and sequenced. The endolysins could be overproduced in Escherichia coli (up to 20% of total cellular protein), and the recombinant proteins were purified by a two-step chromatographical procedure. All three enzymes induced rapid and specific lysis of viable cells of several Bacillus species, with highest activity on B. cereus and B. thuringiensis. Ply12 and Ply21 were experimentally shown to be N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). No apparent holin genes were found adjacent to the ply genes. However, Ply21 may be endowed with a signal peptide which could play a role in timing of cell lysis by the cytoplasmic phage endolysin. The individual lytic enzymes (PlyBa, 41.1 kDa; Ply21, 29.5 kDa, Ply12, 27.7 kDa) show remarkable heterogeneity, i.e., their amino acid sequences reveal only little homology. The N-terminal part of Ply21 was found to be almost identical to the catalytic domains of a Bacillus sp. cell wall hydrolase (CwlSP) and an autolysin of B. subtilis (CwlA). The C terminus of PlyBa contains a 77-amino-acid sequence repeat which is also homologous to the binding domain of CwlSP. Ply12 shows homology to the major autolysins from B. subtilis and E. coli. Comparison with database sequences indicated a modular organization of the phage lysis proteins where the enzymatic activity is located in the N-terminal region and the C-termini are responsible for specific recognition and binding of Bacillus peptidoglycan. We speculate that the close relationship of the phage enzymes and cell wall autolysins is based upon horizontal gene transfer among different Bacillus phages and their hosts.Numerous bacteriophages for the genus Bacillus have been isolated, and at present, they are grouped into 33 phage species. With one exception, all of them belong to the tailed phages (1). Lytic activity is not necessarily restricted to a single species, especially with respect to the phages used for typing strains of Bacillus cereus and Bacillus thuringiensis (2, 35). B. cereus is genotypically closely related to B. thuringiensis, and it has been suggested that they be grouped into a single species (4). In contrast to the well-studied phages infecting B. subtilis (29,40), no information is available on the molecular biology and relationships of viruses for the B. cereus-B. thuringiensis group.At the end of their multiplication cycle, most bacteriophages are released from the cells through the action of endogenous cell wall hydrolases, termed endolysins or phage lysins (for a review, see reference 38). Several genes encoding lysins from phages infecting both gram-negative and gram-positive hosts have been cloned and sequenced. In some cases, the catalytic mechanisms have been determined, which place the phage lysins into three distinct groups: amidases, muramidases (glycosidases and transglycosylases), and endopeptidases (38). Recently, a fourth type of enzyme was described (L-alanoyl-Dglutamate pe...

We investigated the cellular mechanisms that led to growth inhibition, morphological changes, and lysis of Bacillus cereus WSBC 10030 when it was challenged with a long-chain polyphosphate (polyP). At a concentration of 0.1% or higher, polyP had a bacteriocidal effect on log-phase cells, in which it induced rapid lysis and reductions in viable cell counts of up to 3 log units. The cellular debris consisted of empty cell wall cylinders and polar caps, suggesting that polyP-induced lysis was spatially specific. This activity was strictly dependent on active growth and cell division, since polyP failed to induce lysis in cells treated with chloramphenicol and in stationary-phase cells, which were, however, bacteriostatically inhibited by polyP. Similar observations were made with B. cereus spores; 0.1% polyP inhibited spore germination and outgrowth, and a higher concentration (1.0%) was even sporocidal. Supplemental divalent metal ions (Mg2+ and Ca2+) could almost completely block and reverse the antimicrobial activity of polyP; i.e., they could immediately stop lysis and reinitiate rapid cell division and multiplication. Interestingly, a sublethal polyP concentration (0.05%) led to the formation of elongated cells (average length, 70 μm) after 4 h of incubation. While DNA replication and chromosome segregation were undisturbed, electron microscopy revealed a complete lack of septum formation within the filaments. Exposure to divalent cations resulted in instantaneous formation and growth of ring-shaped edges of invaginating septal walls. After approximately 30 min, septation was complete, and cell division resumed. We frequently observed a minicell-like phenotype and other septation defects, which were probably due to hyperdivision activity after cation supplementation. We propose that polyP may have an effect on the ubiquitous bacterial cell division protein FtsZ, whose GTPase activity is known to be strictly dependent on divalent metal ions. It is tempting to speculate that polyP, because of its metal ion-chelating nature, indirectly blocks the dynamic formation (polymerization) of the Z ring, which would explain the aseptate phenotype.

The effect of novel food-grade long-chain polyphosphate formulations (JOHA HBS sodium polyphosphate glassy, 69 ± 1% P2O5, and two similar salts (HBS-1 and HBS-9) on the growth of Clostridium tyrobutyricum ATCC 25755 in liquid culture and in pasteurized, processed cheese spreads was evaluated. In broth, 0.1 % polyphosphate was sufficient to inhibit vegetative growth of the organism. In addition, a panel of 21 other gram-positive and 11 gram-negative bacteria were tested for their sensitivity against the polyphosphates. Whereas 17 of the gram-positives could be inhibited by 0.05 to 0.3% polyphosphate, none of the tested gram-negatives were affected. Two different cheese spread formulations (cheese blend A: 55% moisture, 47.2% fat in dry matter; cheese blend B: 55% moisture, 57% fat in dry matter) were fortified with 0.1 % to 1.0% polyphosphates, inoculated with 5 × 105 (cheese blend A) or 2.5 × 106 (cheese blend B) C. tyrobutyricum spores per gram, and incubated at 35°C for up to 7 weeks. Determination of viable cell counts was carried out at days 1, 9, 19, and 49 (cheese blend A) and 8, 16, 27, 35, and 50 (cheese blend B). While 0.1 % polyphosphate had little effect, higher concentrations were increasingly inhibitory to growth from a spore inoculum, to cell multiplication, and to gas formation. With 0.5% polyphosphate, onset of growth was delayed for about 3 weeks in cheese blend A, while this concentration was able to inhibit the organism in cheese blend B. In view of the experimental parameters selected (high initial contamination level; intrinsic and extrinsic parameters optimized for growth of clostridia), 0.5% polyphosphate may be sufficient to control C. tyrobutyricum growth under “normal” conditions, where initial spore counts are rather low, and storage temperatures are usually at or below 20°C. Moreover, clostridia were completely inhibited by 1.0% polyphosphate, which clearly indicated the usefulness of these polyphosphates for prevention of butyric blowing in pasteurized processed cheese spreads.

The lysis genes of the virulent Staphylococcus aureus bacteriophage Twort were cloned and their nucleotide sequences determined. The endolysin gene plyTW encodes a 53.3-kDa protein, whose catalytic site is located in the amino-terminal domain. An enzymatically active fragment (N-terminal 271 amino acids) was overexpressed in Escherichia coli and partially purified. The enzyme rapidly cleaves staphylococcal peptidoglycan, and was shown to act as N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28). Significant sequence homology to the specific cell wall targeting domain of lysostaphin was observed in a 101-amino acid C-terminal overlap. However, we found that the large C-terminal portion (63%, 295 aa) of PlyTW is not required for staphylolytic activity. Located upstream of and overlapping plyTW by 35 bp in a different reading frame (+1), we identified holTW, which starts with a single TTG triplet. The gene specifies a 185-amino acid (20.5 kDa) holin protein, which features two potential hydrophobic, antiparallel transmembrane domains, and a highly charged, acidic C-terminus. HolTW is the largest class II holin described to date. It can substitute for the defective allele in phase lambda S' amber mutants, both in trans from an expression plasmid, and from within gt11::holTW. The proposed function is the formation of unspecific membrane lesions to promote access of the endolysin to the bacterial peptidoglycan.

Studies of the trophic relationships in microbial reduction of sulfate established the need for three physiological types of organisms: cellulosedigesters (Cellulomonas, Cytophaga or Micromonospora), a lactic acid fermenter (Enterobacter), and a sulfate reducer (Desulfovibrio). Microcosms based on cellulose mineral salts medium were inoculated with varying combinations of mixed cultures and incubated at 30°C for 18 days. The extent of sulfate reduction varied in the microcosms. A 5-member culture which contained the three cellulolytic organisms reduced the most sulfate (52%). Cellulomonas was the most efficient cellulolytic partner. Unless all three physiological types were present, sulfate was not reduced.In nature, the formation of sulfides through sulfate reduction is enhanced by low redox potential (Eh), availability of organic matter and a suitable temperature (1). When sulfate is reduced, the hydrogen sulfide (H2S) evolved may combine with ferrous salts to form black sulfide deposits which appear in the profiles of boggy soils and aquatic mulls. Sulfate-reducing bacteria may use organic substrates such as lactate (2), choline and acetate (3, 4) as electron donors for sulfate reduction. In some strains, a reductive pathway through hydrogen oxidation has been observed (5, 6). Species of Desulfovibrio have limited substrate range so trophic interactions with other organisms is necessary to release nutrients from decomposing organic matter and to produce a reducing microenvironment for sulfate reduction. The degradation of plant detritus is through the activities of cellulolytic organisms. It is therefore clear that the generation and maintenance of various nutrient cycles in nature necessitate complex syntrophic relationships among microbial species. TEZUKA et al. (7) in an ecological study of the Sumida River ' Present Address: