Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50

A segregationally stable expression and secretion vector for Saccharomyces cerevisiae, named pYABD01, was constructed by cloning the yeast gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1_s) into the S. cerevisiae high-copy-number expression vector pYES2. The structural genes of the two leaderless peptides of enterocin L50 (EntL50A and EntL50B) from Enterococcus faecium L50 were cloned, separately (entL50A or entL50B) and together (entL50AB), into pYABD01 under the control of the galactose-inducible promoter P_GAL1. The generation of recombinant S. cerevisiae strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the MFα1_s-containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the S. cerevisiae Sec system.Lactic acid bacteria (LAB) are widely known for their ability to produce a variety of ribosomally synthesized proteins or peptides, referred to as bacteriocins, displaying antimicrobial activity against a broad range of gram-positive bacteria and, to a lesser extent, gram-negative bacteria, including spoilage and food-borne pathogenic microorganisms (11, 19, 33, 34, 36, 37). These antimicrobials may be classified into three main classes: (i) the lantibiotics, or posttranslationally modified peptides; (ii) the nonmodified, small, heat-stable peptides; and (iii) the large, heat-labile protein bacteriocins. Class II bacteriocins are further grouped into five subclasses: the subclass IIa (pediocin-like bacteriocins containing the N-terminal conserved motif YGNGVxC), the subclass IIb (two-peptide bacteriocins), the subclass IIc (leaderless bacteriocins), the subclass IId (circular bacteriocins), and the subclass IIe (other peptide bacteriocins) (17, 19, 21, 37). All lantibiotics and most class II bacteriocins are synthesized as biologically inactive precursors containing an N-terminal extension (the so-called double-glycine-type leader sequence or the Sec-dependent signal peptide), which is cleaved off concomitantly with externalization of biologically active bacteriocins by a dedicated ATP-binding cassette transporter and its accessory protein or by the Sec system and the signal peptidases, respectively (11, 17). Interestingly, only a few bacteriocins described to date are synthesized without an N-terminal extension, including enterocin L50 (L50A and L50B) (8), enterocin Q (EntQ) (10), enterocin EJ97 (41), and the bacteriocin LsbB (20).In recent years, there has been an increasing interest in the application of bacteriocinogenic microorganisms and/or their bacteriocins as biopreservatives to guarantee the safety and quality of foods and beverages, such as fermented vegetables and meats, dairy and fish products, and wine and beer (12, 15, 16, 39, 47). Three main strategies for the use of bacteriocins as food biopreservatives have been proposed: (i) addition of a purified/semipurified bacteriocin preparation as a food additive; (ii) use of a substrate previously fermented by a bacteriocin-producing strain as a food ingredient; and/or (iii) inoculation of a culture to produce the bacteriocin in situ in fermented foods (13, 15). The lantibiotic nisin A is the most widely characterized bacteriocin and the only one that has been legally approved in more than 48 countries as a food additive for use in certain types of cheeses (13, 16). Likewise, nisin A has been approved as a beer additive in Australia and New Zealand (16). However, the difficulties encountered in addressing the regulatory approval of new bacteriocins as food additives have spurred the development of the other bacteriocin-based food biopreservation strategies (13, 17).Beer is a beverage with a remarkable microbiological stability and is considered as a food substrate difficult to spoil. However, some LAB, such as Lactobacillus brevis, Lactobacillus lindneri, and Pediococcus damnosus, are able to spoil beer and are recognized as the most hazardous bacteria for breweries, being responsible for approximately 70% of microbial beer spoilage incidents (40, 47). The ever-growing consumer demand for less-processed and less chemically preserved foods and beverages is promoting the development of alternative biocontrol strategies, such as those based on the use of bacteriocins as biopreservatives (12, 15, 39, 47). However, beyond the strict requirements to fulfill legal regulations, the commercial application of bacteriocins as beer additives is hindered mainly by low bacteriocin production yields and increases in production costs (44). Considering that Saccharomyces cerevisiae is commonly used as starter culture for brewing (24, 28, 35), a novel beer biopreservation strategy based on the development of bactericidal S. cerevisiae brewing strains has been proposed to overcome the aforementioned challenges (44, 46, 47). In this respect, the heterologous production of LAB bacteriocins, namely, pediocin PA-1 (PedPA-1) from Pediococcus acidilactici PAC1.0 and plantaricin 423 from Lactobacillus plantarum 423, by laboratory strains of S. cerevisiae has been reported (44, 46).Enterocin L50 (EntL50) is a commonly found bacteriocin composed of two highly related leaderless antimicrobial peptides, enterocin L50A (EntL50A) and enterocin L50B (EntL50B), which possesses a broad antimicrobial spectrum against LAB, food-borne pathogenic bacteria, and human and animal clinical pathogens (8, 9, 10, 11). Previous work by our group showed that EntL50 (EntL50A and EntL50B) may be used as a beer biopreservative to inhibit the growth of beer spoilage bacteria (1). Therefore, genetically engineered strains of S. cerevisiae heterologously expressing and secreting EntL50A and EntL50B have been developed in this work. For this purpose, we constructed the segregationally stable expression and secretion vector pYABD01, which allowed the secretion of biologically active EntL50A and EntL50B directed by MFα1_s through the S. cerevisiae Sec system.     

Enterocin X,a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.Bacteriocins are gene-encoded antibacterial peptides and proteins. Because of their natural ability to preserve food, they are of particular interest to researchers in the food industry. Bacteriocins are grouped into three main classes according to their physical properties and compositions (11, 12). Of these, class IIb bacteriocins are thermostable non-lanthionine-containing two-peptide bacteriocins whose full antibacterial activity requires the interaction of two complementary peptides (8, 19). Therefore, two-peptide bacteriocins are considered to function together as one antibacterial entity (14).Enterocins A and B, first discovered and identified about 12 years ago (2, 3), are frequently present in Enterococcus faecium strains from various sources (3, 5, 6, 9, 13, 16). So far, no other bacteriocins have been identified in these strains, except the enterocin P-like bacteriocin from E. faecium JCM 5804^T (18). Here, we describe the characterization and genetic identification of enterocin X in E. faecium KU-B5. Enterocin X (identified after the enterocin P-like bacteriocin was discovered) is a newly found class IIb bacteriocin in E. faecium strains that produce enterocins A and B.     

The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G

Lactococcin G and enterocin 1071 are two homologous two-peptide bacteriocins. Expression vectors containing the gene encoding the putative lactococcin G immunity protein (lagC) or the gene encoding the enterocin 1071 immunity protein (entI) were constructed and introduced into strains sensitive to one or both of the bacteriocins. Strains that were sensitive to lactococcin G became immune to lactococcin G when expressing the putative lactococcin G immunity protein, indicating that the lagC gene in fact encodes a protein involved in lactococcin G immunity. To determine which peptide or parts of the peptide(s) of each bacteriocin that are recognized by the cognate immunity protein, combinations of wild-type peptides and hybrid peptides from the two bacteriocins were assayed against strains expressing either of the two immunity proteins. The lactococcin G immunity protein rendered the enterococcus strain but not the lactococcus strains resistant to enterocin 1071, indicating that the functionality of the immunity protein depends on a cellular component. Moreover, regions important for recognition by the immunity protein were identified in both peptides (Lcn-α and Lcn-β) constituting lactococcin G. These regions include the N-terminal end of Lcn-α (residues 1 to 13) and the C-terminal part of Lcn-β (residues 14 to 24). According to a previously proposed structural model of lactococcin G, these regions will be positioned adjacent to each other in the transmembrane helix-helix structure, and the model thus accommodates the present results.Lactic acid bacteria (LAB) produce ribosomally synthesized antimicrobial peptides, generally referred to as bacteriocins. There are two main classes of these bacteriocins (8, 22): the class I bacteriocins (often referred to as lantibiotics) that contain the modified amino acid residues lanthionine and/or β-methyllanthionine and the class II bacteriocins that lack modified residues (8). The class II bacteriocins are further divided into four subclasses, IIa, IIb, IIc, and IId (8). Class IIa contains the pediocin-like bacteriocins, which have very similar amino acid sequences, class IIc consists of the cyclic bacteriocins, and the one-peptide, noncyclic bacteriocins that show no sequence similarity to the pediocin-like bacteriocins are placed in class IId (8). The unmodified two-peptide bacteriocins are placed in class IIb. They are unique in that they consist of two different peptides, both of which must be present, in about equal amounts, to obtain optimal antimicrobial activity (25). More than 10 two-peptide bacteriocins have been isolated and characterized (see reference 25 for original references) since the first isolation of such a bacteriocin (lactococcin G) in 1992 (21). For the two-peptide bacteriocins that have been genetically characterized, the genes encoding the two bacteriocin peptides are always found next to each other in the same operon, along with the gene encoding the immunity protein that protects the bacteriocin producer from being killed by its own bacteriocin.Lactococcin G is perhaps the best-characterized two-peptide bacteriocin (12, 18, 19, 21, 24, 26, 27). It consists of the 39-residue α peptide (termed Lcn-α) and the 35-residue β peptide (termed Lcn-β) (Fig. ​(Fig.1A).1A). Like all two-peptide bacteriocins whose mode of action has been studied, lactococcin G causes cell death by rendering the membranes of target cells permeable to various ions (18, 19). Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have revealed that the two lactococcin G peptides adopt mainly α-helical structures when they are individually exposed to membrane-like entities (12, 27). Based on the NMR structures and findings from site-directed mutagenesis studies, a structural model of lactococcin G has recently been proposed (23, 26, 27). In this model, the two complementary peptides form parallel helices that span the target cell membrane. The helix-helix segment consists of the N-terminal region of Lcn-α (from about Trp-3 to Gly-22) and the C-terminal region of Lcn-β (from about Tyr-13 to Trp-32). The model also proposes that the cationic C-terminal end (residues 35 to 39, R-K-K-K-H) of Lcn-α is unstructured and forced through the target cell membrane by the membrane potential, thereby positioning the C termini of the two peptides inside the target-cell (Fig. ​(Fig.2).2). The tryptophan-rich N-terminal end of Lcn-β is also proposed to be relatively unstructured and to position itself in the outer membrane interface, thus forcing the N termini of the two peptides to remain on the outer side of the target cell membrane and the helix-helix segment to transverse the membrane (Fig. ​(Fig.2).2). This proposed structure is presumably also valid for the two-peptide bacteriocins enterocin 1071 (4, 5, 11), enterocin C (17), and lactococcin Q (32), since their sequence similarities to lactococcin G (lactococcin G has about 88 and 57% sequence identity to lactococcin Q and enterocin 1071, respectively; enterocin 1071 and enterocin C are identical except for one residue) indicate that these four bacteriocins have similar three-dimensional structures.Open in a separate windowFIG. 1.(A) Amino acid sequence alignment of enterocin 1071 (peptides Ent1071A and Ent1071B) and lactococcin G (peptides Lcn-α and Lcn-β) and the cognate immunity proteins (Ent1071im and LcnGim, respectively). Ent1071A and Lcn-α show 59% sequence identity, whereas Ent1071B and Lcn-β show 54% sequence identity. The immunity proteins consist of 110 amino acid residues each and show 38% sequence identity. Identical amino acid residues are colored in red. (B) Amino acid sequences of the two hybrid peptides. The Lcn-α-Ent1071A hybrid peptide (α-hybrid) is termed α[1-16]/A[14-39] in this study (it is designated α2-4 in reference 24). Residues are numbered according to the corresponding amino acid positions in Lcn-α (Fig. 1A). Residues in orange are derived from Lcn-α, and residues in blue are derived from Ent1071A. The overlapping region (i.e., residues 14 to 16) is marked in red, and this region consists of residues that are identical in Lcn-α and Ent1071A. The α-hybrid peptide contains an additional lysine residue in the C-terminal end derived from Lcn-α (see reference 24 for the construction of the hybrid peptide). The Lcn-β-Ent1071B hybrid peptide (β-hybrid) is termed β[1-13]/B[11-35] in this study (it is designated β1-6 in reference 24). Residues in orange are derived from Lcn-β, and residues in blue are derived from Ent1071B. The overlapping region (i.e., residues 11 to 13) is marked in red, and residues in this region are identical in Lcn-β and Ent1071B.Open in a separate windowFIG. 2.Proposed structural model of lactococcin G. The two peptides (Lcn-α and Lcn-β) form a transmembrane helix-helix structure, with the flexible tryptophan-rich N-terminal end of Lcn-β positioned in the outer membrane interface and the unstructured, highly cationic C-terminal end of Lcn-α inside the target cell membrane. The transmembrane helix-helix segment consists of the N-terminal region of Lcn-α (from about Trp-3 to Gly-22) and the C-terminal region of Lcn-β (from about Tyr-13 to Trp-32). (Adapted from reference 26 with permission of the publisher. Copyright 2008 American Chemical Society.)The sequence of the lactococcin G operon (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ938036","term_id":"238480353","term_text":"FJ938036"}}FJ938036) has been determined, and a gene (lagC) encoding the putative lactococcin G immunity protein has been identified downstream of the two genes encoding the two lactococcin G peptides. Downstream of the two genes encoding the two enterocin 1071 peptides, a gene (entI) encoding the enterocin 1071 immunity protein has been identified (4, 11). The putative lactococcin G immunity protein shows 38% amino acid sequence identity to the enterocin 1071 immunity protein (the sequence of which was obtained from Franz et al. [11]), and both proteins consist of 110 amino acid residues (Fig. ​(Fig.1A).1A). The aim of this study was to identify which peptides or which parts of the peptides of the two-peptide bacteriocins lactococcin G and enterocin 1071 are recognized by these immunity proteins. To achieve this, combinations of wild-type lactococcin G peptides, wild-type enterocin 1071 peptides, and hybrid lactococcin-enterocin peptides were assayed against sensitive strains that were transformed with an expression plasmid carrying either the lactococcin G or the enterocin 1071 immunity gene.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

DNA Sequencing and Homologous Expression of a Small Peptide Conferring Immunity to Gassericin A,a Circular Bacteriocin Produced by Lactobacillus gasseri LA39

Gassericin A, produced by Lactobacillus gasseri LA39, is a hydrophobic circular bacteriocin. The DNA region surrounding the gassericin A structural gene, gaaA, was sequenced, and seven open reading frames (ORFs) of 3.5 kbp (gaaBCADITE) were found with possible functions in gassericin A production, secretion, and immunity. The deduced products of the five consecutive ORFs gaaADITE have homology to those of genes involved in butyrivibriocin AR10 production, although the genetic arrangements are different in the two circular bacteriocin genes. GaaI is a small, positively charged hydrophobic peptide of 53 amino acids containing a putative transmembrane segment. Heterologous expression and homologous expression of GaaI in Lactococcus lactis subsp. cremoris MG1363 and L. gasseri JCM1131^T, respectively, were studied. GaaI-expressing strains exhibited at least sevenfold-higher resistance to gassericin A than corresponding control strains, indicating that gaaI encodes an immunity peptide for gassericin A. Comparison of GaaI to peptides with similar characteristics found in the circular bacteriocin gene loci is discussed.Bacteriocins are antimicrobial peptides that act primarily against related bacterial species. The classification of bacteriocins remains controversial. Here, we use the classification of Maqueda et al. (30): class I (lantibiotics); class II (nonlantibiotics) with subclasses IIa (antilisteral pediocin-like bacteriocins), IIb (two-peptide bacteriocins), and IIc (leaderless bacteriocins); class III (large heat-labile bacteriocins); and class IV (circular bacteriocins linked at the N- and C-terminal amino acids).Nine class IV circular bacteriocins have been reported to date. They can be further divided into two major groups by using their primary structures, biochemical characteristics, and genetic arrangements. One group is the family of enterocin AS-48 (32), the first circular bacteriocin described (in 1994), which includes circularin A (25) and uberolysin (40). The other group is the family of gassericin A (19, 21), the second bacteriocin found (in 1998), which includes acidocin B (28), reutericin 6 (with a primary structure 100% identical to that of gassericin A) (22, 23), butyrivibriocin AR10 (17), and carnocyclin A, from Carnobacterium maltaromaticum UAL307 (33). The lantibiotic-like subtilosin A produced by Bacillus subtilis subsp. subtilis strain 168 (24) is an orphan member of the class IV bacteriocins. The gassericin A family of bacteriocins have been isolated from various bacterial species in several countries, suggesting the bacteriocin genes may be associated with transferable genetic elements.The bacteriocins of lactic acid bacteria (LAB) and bacteriocin-producing LAB strains isolated from foods are promising food preservative candidates, and strains of human origin are expected to be probiotics that could help to prevent the growth of harmful bacteria in food and the human intestine. Lactobacillus gasseri belongs to the Lactobacillus acidophilus group of LAB, which are natural inhabitants of the human intestinal tract (35), and many L. gasseri strains have been shown to produce bacteriocins (16, 20). Gassericin A was produced by L. gasseri LA39 isolated from the feces of a human infant; it has bactericidal activity against the food-borne pathogens Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus (16). Recently, using proteose peptone, some strains of L. gasseri containing LA39 were successfully cultured in reconstituted skim milk and cheese whey, where L. gasseri LA39 produced gassericin A; these low-cost, safe media could be used to improve the safety of biopreservation (1). Gassericin A has been purified and characterized, and its structural gene (gaaA) has been cloned and sequenced (21, 22). Determination of the complete chemical structure of gassericin A showed that the bacteriocin belongs to class IV and consists of 58 amino acid residues linked at the N and C termini (19). Little is known about the mechanisms of secretion and circularization of gassericin A and immunity to the circular bacteriocin.Here, we sequenced six genes surrounding gaaA thought to be related to production of and immunity to gassericin A and examined the homologous and heterologous expression of a small hydrophobic peptide, GaaI; we found that gaaI is an immunity gene providing protection against gassericin A.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Specific Degradation of the Mucus Adhesion-Promoting Protein (MapA) of Lactobacillus reuteri to an Antimicrobial Peptide

The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota.Mammals have a microbiota in their digestive tract that contains lactic acid bacteria (LAB). It has been increasingly evident that some of these lactic acid bacteria produce antimicrobial peptides that may contribute to the positive effect on their host. Such bacteria are often referred to as probiotics, and one of their important beneficial effects is their ability to produce antimicrobial compounds that prevent or interfere with the growth of pathogenic bacteria in the host.It is known that the fecal microflora of pigs/piglets is large and diverse and develops rapidly after birth. Lactobacillus reuteri is among the very first lactic acid bacteria that colonize the intestine of new-born piglets, and their numbers gradually increase until they become the most dominant LAB in pigs (5, 17, 28). Other lactobacilli that are also part of the gut microbiota of pigs include L. amylovorus, L. acidophilus, L. salivarius, and L. casei (4, 8). Probiotic isolates have been identified within all these species, and many of them are today used as food/feed supplements to support good health (4, 11, 27). An important part of the antimicrobial arsenal produced by lactic acid bacteria (LAB) is a group of peptides called bacteriocins, which are ribosomally synthesized antibiotic-like peptides (antimicrobial peptides [AMPs]) (3, 7, 19). The bacteriocins constitute a wide range of structurally different peptides that are divided into different classes and subclasses. Some are modified (the lantibiotics, or class I), while others are basically unmodified (class II) (3, 6, 19).Most bacteriocins are derived from prepeptides, each containing a short leader sequence (14 to 30 amino acids [aa]) which is cleaved off during the secretion of the mature peptide (19). In recent years, a new group of AMPs have been recognized (18); these are different from regular bacteriocins in that they are derived from larger proteins through specific degradations, leading to a defined peptide possessing antimicrobial activity. Such antimicrobial peptides have been known for a long time in mammalian systems. For instance, lactoferrin, a protein in milk, is readily degraded to a specific antimicrobial peptide through heat, acid treatment, or pepsin digestion (14, 24, 26). Defined histone fragments with antimicrobial properties have been isolated from different eukaryotic species (1, 2, 15, 21, 23), and a few antimicrobial peptides derived from larger proteins have been isolated in bacteria, including Helicobacter pylori (22), propionic acid bacteria (9, 10), and Clostridium beijerinckii (13). Such antimicrobial peptides are most likely formed by proteolytic degradation during cell proliferation or death.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.