An Extracellular Loop of the Mannose Phosphotransferase System Component IIC Is Responsible for Specific Targeting by Class IIa Bacteriocins

Class IIa bacteriocins target a phylogenetically defined subgroup of mannose-phosphotransferase systems (man-PTS) on sensitive cells. By the use of man-PTS genes of the sensitive Listeria monocytogenes (mpt) and the nonsensitive Lactococcus lactis (ptn) species to rationally design a series of man-PTS chimeras and site-directed mutations, we identified an extracellular loop of the membrane-located protein MptC that was responsible for specific target recognition by the class IIa bacteriocins.Bacteriocins are small, ribosomally synthesized antimicrobial peptides that normally kill bacteria closely related to the bacteriocin producers, but some also target a wider spectrum of bacteria, including a number of pathogens and food spoilage bacterial species (5, 28). Class IIa (pediocin-like) bacteriocins display a broad antimicrobial spectrum, including important pathogens such as Listeria monocytogenes and Enterococcus faecalis. These peptides consist of 37 to 48 nonmodified amino acids, contain a conserved pediocin-box sequence (Y-G-N-G-V/L) in the N-terminal region, and have defined secondary features in their structure: a cationic β sheet at the conserved N terminus and a helix-containing domain at the less-conserved C terminus (16, 30). Class IIa bacteriocins target sensitive cells by using the mannose phosphotransferase system (man-PTS) as a receptor (6, 10, 17, 19, 33). This sugar uptake system is the major glucose transporter for many bacteria, particularly Firmicutes and Gammaproteobacteria (39). Each man-PTS complex consists of four structural domains: IIC and IID, represented by two membrane-located proteins, and IIA and IIB, which are normally represented by a single cytoplasmic protein that can form reversible contacts with its membrane-located partners (31).It has previously been shown that coexpression of the IIC and IID genes is needed to confer sensitivity to class IIa bacteriocins as well as to the lactococcal bacteriocin lactococcin A and that the cytoplasmic IIAB partner is not involved in this process (10). However, while lactococcin A (belonging to class IIc) targets only the lactococcal man-PTS, the class IIa bacteriocins target man-PTSs of species of diverse genera (e.g., Listeria, Enterococcus, and Lactobacillus) but somehow not those of the Lactococcus genus (24). This genus specificity has been recognized for almost 2 decades (20, 23, 26); still, the molecular nature underlying the specificity has remained very enigmatic. In the present report we clarify this issue by demonstrating that these two types of bacteriocins exhibit different binding patterns on their receptors: class IIa bacteriocins specifically interact with a defined region of 40 amino acids in the IIC protein whereas lactococcin A has a more complex interaction involving regions from both IIC and IID.     

Comparative Studies of Class IIa Bacteriocins of Lactic Acid Bacteria

Four class IIa bacteriocins (pediocin PA-1, enterocin A, sakacin P, and curvacin A) were purified to homogeneity and tested for activity toward a variety of indicator strains. Pediocin PA-1 and enterocin A inhibited more strains and had generally lower MICs than sakacin P and curvacin A. The antagonistic activity of pediocin-PA1 and enterocin A was much more sensitive to reduction of disulfide bonds than the antagonistic activity of sakacin P and curvacin A, suggesting that an extra disulfide bond that is present in the former two may contribute to their high levels of activity. The food pathogen Listeria monocytogenes was among the most sensitive indicator strains for all four bacteriocins. Enterocin A was most effective in inhibiting Listeria, having MICs in the range of 0.1 to 1 ng/ml. Sakacin P had the interesting property of being very active toward Listeria but not having concomitant high levels of activity toward lactic acid bacteria. Strains producing class IIa bacteriocins displayed various degrees of resistance toward noncognate class IIa bacteriocins; for the sakacin P producer, it was shown that this resistance is correlated with the expression of immunity genes. It is hypothesized that variation in the presence and/or expression of such immunity genes accounts in part for the remarkably large variation in bacteriocin sensitivity displayed by lactic acid bacteria.Many lactic acid bacteria (LAB), including members of the genera Lactococcus, Lactobacillus, Carnobacterium, Enterococcus, and Pediococcus, are known to secrete small, ribosomally synthesized antimicrobial peptides called bacteriocins (26, 29, 34). Some of these peptides undergo posttranslational modifications (class I bacteriocins), whereas others are not modified (class II bacteriocins) (29, 34). Class II bacteriocins contain between 30 and 60 residues and are usually positively charged at a neutral pH. Studies of a large number of class II bacteriocins have led to subgrouping of these compounds (29, 34). One of the subgroups, class IIa, contains bacteriocins that are characterized by the presence of YGNG and CXXXXCXV sequence motifs in their N-terminal halves as well as by their strong inhibitory effect on Listeria (e.g., 3, 4, 22, 23, 27, 28, 31, 38, 45) (Fig. ​(Fig.1).1). Because of their effectiveness against the food pathogen Listeria, class IIa bacteriocins have potential as antimicrobial agents in food and feed. Open in a separate windowFIG. 1Sequence alignment of class IIa bacteriocins. Residue numbering is according to the sequence of pediocin PA-1. Cysteine residues are printed in boldface; the two known class IIa bacteriocins with four cysteine residues are in the upper group. No attempt was made to optimize the alignment in the C-terminal halves of the peptides. Piscicolin 126 is identical to piscicocin V1a (4). Carnobacteriocin BM1 most probably is identical to piscicocin V1b (4). Sakacin P most probably is identical to bavaricin A (30). Curvacin A is identical to sakacin A (2). The consensus sequence includes residues conserved in at least 8 of the 12 sequences shown; 100% conserved residues are underlined.Class IIa bacteriocins act by permeabilizing the membrane of their target cells (1, 5, 6, 9, 10, 26, 28). The most recent studies on the mode of action of these bacteriocins indicate that antimicrobial activity does not require a specific receptor and is enhanced by (but not fully dependent on) a membrane potential (9, 28). Little is known about bacteriocin structure, and unravelling the relationships between structure and function is one of the great challenges in current bacteriocin research. A logical starting point for structure-function studies is a thorough study of the differences in activity and target cell specificity between naturally occurring homologous bacteriocins. A few such studies have been described, but these suffer from either a very limited number of tested indicator strains or the use of culture supernatants instead of purified bacteriocins (3, 4, 17, 45). The use of purified bacteriocins for comparative analyses is absolutely essential, since it is becoming increasingly evident that bacteriocin producers produce more than one bacteriocin (4, 8, 38, 48; this study).In the present study, the activities of four pure class IIa bacteriocins (pediocin PA-1, enterocin A, curvacin A, and sakacin P) (Fig. ​(Fig.1)1) were tested against a large number of LAB as well as several strains of the food pathogen Listeria monocytogenes. The bacteriocins were purified from their respective producer strains by use of an optimized purification protocol yielding highly pure samples. The contribution of disulfide formation was assessed and found to be important for activity. The effects of the purified bacteriocins on (noncognate) class IIa bacteriocin-producing strains are described, and the implications of our findings for immunity and resistance are discussed.     

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.     

Molecular Ecology of Listeria monocytogenes: Evidence for a Reservoir in Milking Equipment on a Dairy Farm

A longitudinal study aimed to detect Listeria monocytogenes on a New York State dairy farm was conducted between February 2004 and July 2007. Fecal samples were collected every 6 months from all lactating cows. Approximately 20 environmental samples were obtained every 3 months. Bulk tank milk samples and in-line milk filter samples were obtained weekly. Samples from milking equipment and the milking parlor environment were obtained in May 2007. Fifty-one of 715 fecal samples (7.1%) and 22 of 303 environmental samples (7.3%) were positive for L. monocytogenes. A total of 73 of 108 in-line milk filter samples (67.6%) and 34 of 172 bulk tank milk samples (19.7%) were positive for L. monocytogenes. Listeria monocytogenes was isolated from 6 of 40 (15%) sampling sites in the milking parlor and milking equipment. In-line milk filter samples had a greater proportion of L. monocytogenes than did bulk tank milk samples (P < 0.05) and samples from other sources (P < 0.05). The proportion of L. monocytogenes-positive samples was greater among bulk tank milk samples than among fecal or environmental samples (P < 0.05). Analysis of 60 isolates by pulsed-field gel electrophoresis (PFGE) yielded 23 PFGE types after digestion with AscI and ApaI endonucleases. Three PFGE types of L. monocytogenes were repeatedly found in longitudinally collected samples from bulk tank milk and in-line milk filters.Listeria monocytogenes can cause listeriosis in humans. This illness, despite being underreported, is an important public health concern in the United States (23) and worldwide. According to provisional incidence data provided by the Centers for Disease Control and Prevention (CDC), 762 cases of listeriosis were reported in the United States in 2007. In previous years (2003 to 2006), the number of reported annual listeriosis cases in the United States ranged between 696 and 896 cases per year (5).Exposure to food-borne L. monocytogenes may cause fever, muscle aches, and gastroenteritis (30), but does not usually cause septicemic illness in healthy nonpregnant individuals (7, 30). Elderly and immunocompromised people, however, are susceptible to listeriosis (22, 10), and they may develop more-severe symptoms (10). Listeriosis in pregnant women may cause abortion (22, 30) or neonatal death (22).Dairy products have been identified as the source of several human listeriosis outbreaks (4, 7, 10, 22). Listeria is ubiquitous on dairy farms (26), and it has been isolated from cows'' feces, feed (3, 26), and milk (21, 35). In ruminants, L. monocytogenes infections may be asymptomatic or clinical. Clinical cases typically present with encephalitis and uterine infections, often resulting in abortion (26, 39). Both clinically infected and healthy animals have been reported to excrete L. monocytogenes in their feces (20), which could eventually cause contamination of the bulk tank milk or milk-processing premises (39).On-farm epidemiologic research provides science-based information to improve farming and management practices. The Regional Dairy Quality Management Alliance (RDQMA) launched a combined United States Department of Agriculture (USDA)-RDQMA pilot project in January 2004 to scientifically validate intervention strategies in support of recommended best management practices among northeast dairy farms. The primary goal of the project was to track dynamics of infectious microorganisms on well-characterized dairy farms. Target species included Salmonella spp. (6, 36, 37), Mycobacterium avium subsp. paratuberculosis (13, 24), and L. monocytogenes.The objectives of this study were to describe the presence of L. monocytogenes on a dairy farm over time and to perform molecular subtyping by pulsed-field gel electrophoresis (PFGE) on L. monocytogenes isolates obtained from bulk tank milk, milk filters, milking equipment, feces, and the environmental samples to identify diversity among L. monocytogenes strains, persistence, and potential sources of bulk tank milk contamination.     

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.     

Genetic Features of Resident Biofilms Determine Attachment of Listeria monocytogenes

Planktonic Listeria monocytogenes cells in food-processing environments tend most frequently to adhere to solid surfaces. Under these conditions, they are likely to encounter resident biofilms rather than a raw solid surface. Although metabolic interactions between L. monocytogenes and resident microflora have been widely studied, little is known about the biofilm properties that influence the initial fixation of L. monocytogenes to the biofilm interface. To study these properties, we created a set of model resident Lactococcus lactis biofilms with various architectures, types of matrices, and individual cell surface properties. This was achieved using cell wall mutants that affect bacterial chain formation, exopolysaccharide (EPS) synthesis and surface hydrophobicity. The dynamics of the formation of these biofilm structures were analyzed in flow cell chambers using in situ time course confocal laser scanning microscopy imaging. All the L. lactis biofilms tested reduced the initial immobilization of L. monocytogenes compared to the glass substratum of the flow cell. Significant differences were seen in L. monocytogenes settlement as a function of the genetic background of resident lactococcal biofilm cells. In particular, biofilms of the L. lactis chain-forming mutant resulted in a marked increase in L. monocytogenes settlement, while biofilms of the EPS-secreting mutant efficiently prevented pathogen fixation. These results offer new insights into the role of resident biofilms in governing the settlement of pathogens on food chain surfaces and could be of relevance in the field of food safety controls.Listeria monocytogenes is a food pathogen that has been implicated in numerous food-borne disease outbreaks (5, 58). This organism is found not only in food products but also on surfaces in food-processing plants (18). It is well documented that L. monocytogenes is able to adhere and form persistent biofilms on a variety of solid materials, such as stainless steel, glass, or polymers (18, 48, 51, 52). However, in food-manufacturing plants (and particularly in fermented-food-processing environments), it is most likely that the first contact between a pathogen and a surface will concern a resident microbial biofilm covering the solid surface (10, 35, 46). In this context, such a resident biofilm may be regarded as a “conditioning film” that modifies the topographic and physicochemical characteristics of the surface and hence the adhesion capability of planktonic microorganisms coming into contact with this substratum (6).Once the pathogens are immobilized on the surface, interactions between the pathogens and their environment (physiological interactions with resident flora, nutrient availability, pH, water activity, temperature, and cleaning and disinfection procedures) govern the long-term settlement and persistence of the pathogens on the surface. Various studies have demonstrated the inhibition of L. monocytogenes development by natural “protective” biofilms (10, 66). Competition for nutrients has been demonstrated as a major mechanism underlying the inhibition of pathogen development (25, 27). The production of antimicrobial agents (bacteriocins, acids, and hydrogen peroxide) has also been reported as being of importance to such interactions (13, 20, 36). For example, Lactococcus lactis has been described as being exceptionally efficient in controlling the development of L. monocytogenes on food-processing surfaces by means of competitive exclusion (66) or bacteriocin production (35). It has been reported that treating a surface with a bacterial polysaccharide prevented the adhesion of different nosocomial pathogens (60). Furthermore, alginate-overexpressing Pseudomonas aeruginosa biofilms reduced the retention of Cryptosporidium parvum oocysts (54). Other recent studies have shown that the composition and quantity of specific exopolysaccharides (EPS) in Pseudomonas biofilms can inhibit the fixation of Escherichia coli or Erwinia chrysanthemi planktonic cells in porous media (37, 38).The present study investigated those properties of resident biofilms that could affect the settlement of L. monocytogenes. L. lactis was used as a model resident biofilm strain, as this is widely used in dairy fermentations and its cell wall properties have been the subject of considerable study (22, 23). Cell wall mutants of L. lactis MG1363 were used to create a set of model biofilms that differed in terms of their architecture, EPS synthesis, and cell surface hydrophobicity. These biofilms were used to evaluate the attachment of fluorescent inert polystyrene microbeads and of two reference strains of L. monocytogenes (LO28 and EGDe) using in situ confocal fluorescence imaging.     

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).     

Enterocin 96, a Novel Class II Bacteriocin Produced by Enterococcus faecalis WHE 96, Isolated from Munster Cheese

Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.Bacteriocins are a heterogeneous group of ribosomally synthesized antibacterial peptides that inhibit strains and species that are usually, but not always, closely related to the producing bacteria (16). Enterococcal bacteriocins, often termed enterocins, have been widely investigated, mainly because they are active against gram-positive food-borne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. The vast majority of enterocins are active only against gram-positive bacteria (10, 17); however, some exceptions with broad activity spectra described in recent years showed the ability to inhibit the growth of gram-negative microorganisms (5, 11, 13).The increasing number of enterocins reported in the literature and the emergence of novel structures that could not be included in classical bacteriocin classifications (12, 14, 17) prompted the grouping of enterocins into a new four-class scheme by Franz et al. (8). Most enterocins known so far were included in class II (small, nonlantibiotic peptides), which was divided into three subgroups: class II.1, enterocins of the pediocin family; class II.2, enterocins synthesized without a leader peptide; and class II.3, other linear, non-pediocin-like enterocins.The fact that numerous Enterococcus strains found in a variety of fermented and nonfermented foods produce bacteriocins, often more than one per strain, has sparked interest in their use in food preservation (4). Despite the concerns over enterococci as opportunistic pathogens and indicators of fecal contamination, they are indigenous species in the gastrointestinal tract and have long been used as human and/or animal probiotics (1, 2, 7).In this work, we describe and characterize a new class II enterocin produced by Enterococcus faecalis WHE 96, previously isolated from Munster cheese, for its anti-Listeria properties. The amino acid sequence, the structural gene, and the spectrum of activity of this bacteriocin are reported.     

Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pH_i) over time by fluorescence ratio imaging microscopy. pH_i values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pH_i was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pH_i 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.Listeria monocytogenes is a food-borne, human pathogen that has a remarkable ability to colonize food-processing environments (5, 16, 20, 21, 26, 29). Some L. monocytogenes strains can persist for years in food-processing plants (11, 14, 20, 27), and specific molecular subtypes can repeatedly be isolated from the processing environment (29) despite being very infrequent in the outdoor environment (9). This ability to persist has, hitherto, not been linked to any specific genetic or phenotypic trait.It has been suggested that persistent L. monocytogenes strains may be more tolerant or resistant to cleaning and especially disinfectants used in the food industry. Aase et al. (1) found increased tolerance to both benzalkonium chloride and ethidium bromide in L. monocytogenes isolates that had persisted for more than 4 years; however, other studies have not been able to link persistence and tolerance to disinfectants (6, 10, 11, 13). We recently compared disinfection sensitivities of persistent and presumed nonpersistent L. monocytogenes strains using viable-cell counts and did not find the latter group more sensitive to the two disinfectants Triquart SUPER and Incimaxx DES than persistent strains (13). However, we found that for all subtypes of L. monocytogenes, growth with NaCl increased the tolerance of planktonic L. monocytogenes cells to Incimaxx DES, whereas spot-inoculated, dried L. monocytogenes cells were not protected by NaCl against disinfection.There is no doubt that L. monocytogenes will be completely inactivated at the disinfectant concentrations recommended for use in the food industry; however, the efficiency of the disinfectant is very much influenced by the presence of organic material being inactivated by the presence of food debris. Hence, it is likely that the bacterial cell in a food production environment may be exposed to concentrations at a sublethal level. It is currently not known if treatment with a sublethal concentration of disinfectant affects the entire bacterial population or only attacks a fraction of the cell population, leaving another fraction of cells unaffected. In case of the latter, some bacterial cells may be able to survive the disinfection treatment. The potential presence of such tolerant subpopulations could, ultimately, ensure that the genome is propagated, leading to persistence.The presence of a more tolerant subpopulation can be determined on the single-cell level. Flow cytometry is a rapid method useable for measurements at the single-cell resolution (22); however, it cannot monitor the same single cells over time. Optical microscopy combined with microfluidic devices that allow measurement of growth of single cells is a useful technique (2), and in situ analyses of the physiological condition of single cells by the fluorescence ratio imaging microscopy (FRIM) technique represents another elegant approach (25). FRIM enables studies of dynamic changes with high sensitivity and on the single-cell level in important physiological parameters: e.g., intracellular pH (pH_i). Listeria maintains its pH_i within a narrow range of 7.6 to 8 at extracellular pH (pH_ex) values of 5.0 to 8.0 (4, 25) and at pH_ex 4.0 with the presence of glucose (23). It is believed that viable cells need to maintain a transmembrane pH gradient with their pH_i above the pH_ex, and failure to maintain pH_i homeostasis indicates that the bacterial cell is severely stressed and ultimately leads to loss of cell viability. FRIM has been used to determine the pH_i of L. monocytogenes after exposure to osmotic and acid stress (7, 23). Also, the dissipation of the pH gradient in L. monocytogenes after exposure to different bacteriocins has been determined with FRIM (4, 12). Hornbæk et al. (12) found that treatment with subinhibitory concentrations of leucocin and nisin gave rise to two subpopulations: one consisting of cells with a dissipated pH gradient (ΔpH) and the other consisting of cells that maintained ΔpH, which could indicate phenotypic heterogeneity.The aim of the present study was to investigate the physiological effects of the disinfectant Incimaxx DES at sublethal and lethal concentrations on single cells and the population level of a persistent L. monocytogenes strain to study a possible subdivision of sensitivity in the population. We also addressed the potential protective effect of NaCl against disinfection and compared sensitivities in a population of planktonic and attached bacteria. We applied the in situ technique FRIM and compared the pH_i measurements with the traditional viable-cell-count method.(Part of the results have been presented at a poster session at the 95th International Association for Food Protection annual meeting in Columbus, OH, 3 to 6 August 2008.)     

Competition of Listeria monocytogenes Serotype 1/2a and 4b Strains in Mixed-Culture Biofilms

The majority of Listeria monocytogenes isolates recovered from foods and the environment are strains of serogroup 1/2, especially serotypes 1/2a and 1/2b. However, serotype 4b strains cause the majority of human listeriosis outbreaks. Our investigation of L. monocytogenes biofilms used a simulated food-processing system that consisted of repeated cycles of growth, sanitation treatment, and starvation to determine the competitive fitness of strains of serotypes 1/2a and 4b in pure and mixed-culture biofilms. Selective enumeration of strains of a certain serotype in mixed-culture biofilms on stainless steel coupons was accomplished by using serotype-specific quantitative PCR and propidium monoazide treatment to prevent amplification of extracellular DNA or DNA from dead cells. The results showed that the serotype 1/2a strains tested were generally more efficient at forming biofilms and predominated in the mixed-culture biofilms. The growth and survival of strains of one serotype were not inhibited by strains of the other serotype in mixed-culture biofilms. However, we found that a cocktail of serotype 4b strains survived and grew significantly better in mixed-culture biofilms containing a specific strain of serotype 1/2a (strain SK1387), with final cell densities averaging 0.5 log₁₀ CFU/cm² higher than without the serotype 1/2a strain. The methodology used in this study contributed to our understanding of how environmental stresses and microbial competition influence the survival and growth of L. monocytogenes in pure and mixed-culture biofilms.A prominent food-borne pathogen, Listeria monocytogenes can cause severe infections in humans, primarily in high-risk populations, though the disease (listeriosis) is relatively rare (11, 30, 43). Outbreaks of listeriosis have resulted from the contamination of a variety of foods by L. monocytogenes, especially meat and dairy products (27). L. monocytogenes is ubiquitous in the environment, able to grow at refrigeration temperature, and tolerant of the low pHs (3 to 4) typical of acidified foods (28, 32, 44). The capacity to produce biofilms confers protection against stresses common in the food-processing environment (13, 33).Biofilms are characterized by dense clusters of bacterial cells embedded in extracellular polymeric substances which are secreted by cells to aid in adhesion to surfaces and to other cells (4, 5). Strains of L. monocytogenes have been known to persist for years in food-processing environments, presumably in biofilms. Of the 13 known serotypes of L. monocytogenes, three (1/2a, 1/2b, and 4b) account for >95% of the isolates from human illness (21). Serotype 1/2a accounts for >50% of the L. monocytogenes isolates recovered from foods and the environment, while most major outbreaks of human listeriosis have been caused by serotype 4b strains (1, 3, 14, 15, 17, 22, 29, 31, 41, 47, 49,). No correlation between L. monocytogenes strain fitness and serotype has been identified (16, 19). Some studies have reported that strains repeatedly isolated from food and environmental samples (defined as persistent strains) had a higher adherence capacity than strains that were sporadically isolated (2, 36), while this phenomenon was not observed by others (7). Serotype 4b strains exhibited a higher capacity for biofilm formation than did serotype 1/2a strains (36), whereas this was not observed by Di Bonaventura and colleagues (6). It has been suggested that serotype 1/2a strains could be more robust than serotype 4b strains in biofilm formation under a variety of environmental conditions. Furthermore, strains of these serotypes differ in terms of the medium that promotes biofilm formation. Biofilm formation by serotype 4b strains was higher in full-strength tryptic soy broth than in diluted medium, whereas the opposite was observed with serotype 1/2a strains, which produced more biofilm in diluted medium (12).There is limited information on microbial competition between strains of different serotypes in biofilms or on how the environmental stresses present in food-processing environments may affect the biofilm formation and survival of L. monocytogenes of different serotypes. In food-processing plants, the environmental stresses encountered by bacteria are more complex and variable than most laboratory systems used for microbial ecology and biofilm studies. A simulated food-processing (SFP) system has been developed to address this issue (38). The SFP system incorporates several stresses that may affect bacteria in biofilms in the food-processing environment, including exposure to sanitizing agents, dehydration, and starvation. When biofilms were subjected to the SFP regimen over a period of several weeks, the cell numbers of L. monocytogenes strains in the biofilms initially were reduced and then increased as the culture adapted (38). The development of resistance to sanitizing agents was specific to the biofilm-associated cells and was not apparent in the detached cells (38). This suggested that extracellular polymeric substances present in the biofilm matrix were responsible for the resistance to sanitizing agents. It was subsequently found that real-time PCR, in combination with propidium monoazide (PMA) treatment of samples prior to DNA isolation, was an effective method for enumerating viable cells in biofilms (37).The objective of this study was to determine if strains of serotype 1/2a or 4b have a selective advantage under stress conditions. We investigated and compared the initial attachment and biofilm formation capabilities of L. monocytogenes strains of these two serotypes and analyzed the survival and growth of bacteria of each serotype in mixed-serotype biofilms in the SFP system by using PMA with quantitative PCR.     

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.     

Temperature-Dependent Phage Resistance of Listeria monocytogenes Epidemic Clone II

Listeria monocytogenes epidemic clone II (ECII) has been responsible for two multistate outbreaks in the United States in 1998-1999 and in 2002, in which contaminated ready-to-eat meat products (hot dogs and turkey deli meats, respectively) were implicated. However, ecological adaptations of ECII strains in the food-processing plant environment remain unidentified. In this study, we found that broad-host-range phages, including phages isolated from the processing plant environment, produced plaques on ECII strains grown at 37°C but not when the bacteria were grown at lower temperatures (30°C or below). ECII strains grown at lower temperatures were resistant to phage regardless of the temperature during infection and subsequent incubation. In contrast, the phage susceptibility of all other tested strains of serotype 4b (including epidemic clone I) and of strains of other serotypes and Listeria species was independent of the growth temperature of the bacteria. This temperature-dependent phage susceptibility of ECII bacteria was consistently observed with all surveyed ECII strains from outbreaks or from processing plants, regardless of the presence or absence of cadmium resistance plasmids. Phages adsorbed similarly on ECII bacteria grown at 25°C and at 37°C, suggesting that resistance of ECII strains grown at 25°C was not due to failure of the phage to adsorb. Even though the underlying mechanisms remain to be elucidated, temperature-dependent phage resistance may represent an important ecological adaptation of L. monocytogenes ECII in processed, cold-stored foods and in the processing plant environment, where relatively low temperatures prevail.Listeria monocytogenes is responsible for an estimated 2,500 cases of serious food-borne illness (listeriosis) and 500 deaths annually in the United States. It affects primarily pregnant women, newborns, the elderly, and adults with weakened immune systems. L. monocytogenes is frequently found in the environment and can grow at low temperatures, thus representing a serious hazard for cold-stored, ready-to-eat foods (18, 31).Two multistate outbreaks of listeriosis in the United States, in 1998-1999 and in 2002, respectively, were caused by contaminated ready-to-eat meats (hot dogs and turkey deli meats, respectively) contaminated by serotype 4b strains that represented a novel clonal group, designated epidemic clone II (ECII) (3, 4). ECII strains have distinct genotypes as determined by pulsed-field gel electrophoresis and various other subtyping tools, and harbor unique genetic markers (6, 8, 11, 19, 34). The genome sequencing of one of the isolates (L. monocytogenes H7858) from the 1998-1999 outbreak revealed the presence of a plasmid of ca. 80 kb (pLM80), which harbored genes mediating resistance to the heavy metal cadmium as well as genes conferring resistance to the quaternary ammonium disinfectant benzalkonium chloride (10, 29).Listeria phages (listeriaphage) have long been used for subtyping purposes (33), and extensive research has focused on the genomic characterization (2, 24, 26, 35), transducing potential (14), and biotechnological applications of selected phages (25). In addition, applications of listeriaphage as biocontrol agents in foods and the processing plant environment have been investigated (12, 15, 22). However, limited information exists on phages from processing plant environments and on the impact of environmental conditions on susceptibility of L. monocytogenes strains representing the major epidemic-associated clonal groups to such phages. We have found that strains harboring ECII-specific genetic markers can indeed be recovered from the environment of turkey-processing plants (9). Furthermore, environmental samples from such processing plants yielded phages with broad host range, which were able to infect L. monocytogenes strains of various serotypes, and different Listeria species (20). In this study, we describe the impact of growth temperature on susceptibility of L. monocytogenes ECII strains to phages, including phages isolated from turkey-processing plant environmental samples.     

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.     

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.