Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer. We report the identification and characterization of a gene, herein called cotH, located at 300؇ on the B. subtilis genetic map between two divergent cot genes, cotB and cotG. The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa. Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination. The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes. On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat.Endospores of the gram-positive bacterium Bacillus subtilis are encased in a thick protein shell known as the coat (2). The coat is composed of 15 or more polypeptides arranged in an electron-dense outer layer and a lamellar inner layer. These layers protect the spore from bactericidal enzymes and chemicals, such as lysozyme and chloroform. So far, the genes for 13 of these coat proteins have been identified. These are located at diverse positions on the chromosome and code for polypeptides of 65 (CotA), 59 (CotB), 10 (CotC), 9 (CotD), 24 (CotE), 19 (CotF), 24 (CotG and CotJ), 41 (CotS), 10 (CotT), 19 (CotX), 26 (CotY), and 18 (CotZ) kDa (1,3,6,7,10,18,22,24). Certain proteins, such as CotD, are located in the inner coat, and others, such as CotA, CotB, CotC, and CotG, are located in the outer coat (18, 24). The coat is produced at a relatively late stage in the process of sporulation, when the developing spore (or forespore) is present as a free protoplast within the mother cell compartment of the sporangium (14). Coat proteins are organized on the outer surface of the membrane surrounding the forespore by the sporulation protein SpoIVA (8,17,21). SpoIVA controls the assembly of a ring of CotE proteins around the forespore (8). The CotE ring is thought to regulate the assembly of the proteins of the outer coat and is separated from the outer surface of the forespore membrane by a small gap, which is believed to be the site at which the inner coat will be assembled (8). The production of coat proteins is governed by a regulatory cascade of four transcription factors acting in the mother cell compartment of the sporangium in the sequence E , SpoIIID, K , and GerE (26). E and K are RNA polymerase sigma factors, whereas SpoIIID and GerE are DNA-binding proteins that act in conjunction with E -and K -containing forms of RNA polymerase, respectively (4,9,13,23,25).We report the identification of a gene, herein called cotH, located at 300Њ on the B. subtilis chromosomal map, where it is clustered with two previously described cot genes, cotB and cotG (7,18). cotH codes for a 42.8-kDa protein, apparently located in the inner laye...

Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

A bacteriocin-producing BaciUlus cereus strain was isolated. The bacteriocin, here called cerein, was shown to be active specifically against other B. cereus strains and inactive against all other bacterial species tested. Cerein was detected in the culture supernatants of stationary-phase cells, and its appearance was inhibited by induction of sporulation. The bactericidal activity of cerein was insensitive to organic solvents and nonproteolytic enzymes, partially stable to heat, and active over a wide range of pH values. Direct detection of antimicrobial activity on sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of about 9 kDa.

Limestone aquifers provide the main drinking water resources of southern Italy. The groundwater is often contaminated by fecal bacteria because of the interaction between rocks having high permeability and microbial pollutants introduced into the environment by grazing and/or manure spreading. The microbial contamination of springwater in picnic areas located in high mountains can cause gastrointestinal illness. This study was carried out in order to analyze the interaction between Enterococcus faecalis and the soil of a limestone aquifer and to verify the influence of this interaction on the time dependence of groundwater contamination. E. faecalis was chosen because, in the study area involved, it represents a better indicator than Escherichia coli. The research was carried out through field (springwater monitoring) and laboratory experiments (column tests with intact soil blocks). The transport of bacterial cells through soil samples was analyzed by simulating an infiltration event that was monitored in the study area. Comparison of laboratory results with data acquired in the field showed that discontinuous precipitation caused an intermittent migration of microorganisms through the soil and produced, together with dispersion in the fractured medium (unsaturated and saturated zones), an articulated breakthrough at the spring. The short distances of bacterial transport in the study area produced a significant daily variability of bacterial contamination at the field scale.