Rahnella spp. are commonly isolated from onion (Allium cepa) bulbs and are weakly pathogenic.

AIMS: Bacterial decays of onion bulbs have serious economic consequences for growers, but the aetiologies of these diseases are often unclear. We aimed to determine the role of Rahnella, which we commonly isolated from bulbs in the United States and Norway, in onion disease. METHODS AND RESULTS: Isolated bacteria were identified by sequencing of housekeeping genes and/or fatty acid methyl ester analysis. A subset of Rahnella spp. strains was also assessed by multilocus sequence analysis (MLSA); most onion strains belonged to two clades that appear closely related to R. aquatilis. All tested strains from both countries caused mild symptoms in onion bulbs but not leaves. Polymerase chain reaction primers were designed and tested against strains from known species of Rahnella. Amplicons were produced from strains of R. aquatilis, R. victoriana, R. variigena, R. inusitata and R. bruchi, and from one of the two strains of R. woolbedingensis. CONCLUSIONS: Based on binational testing, strains of Rahnella are commonly associated with onions, and they are capable of causing mild symptoms in bulbs. SIGNIFICANCE AND IMPACT OF THE STUDY: While Rahnella strains are commonly found within field-grown onions and they are able to cause mild symptoms, the economic impact of Rahnella-associated symptoms remains unclear.

Survey of nematodes associated with terrestrial slugs in Norway.

A survey of nematodes associated with terrestrial slugs was conducted for the first time in Norway. A total of 611 terrestrial slugs were collected from 32 sample sites. Slugs were identified by means of morphological examination, dissection of genitalia and molecular analysis using mitochondrial DNA. Twelve slug species were identified, representing four different slug families. Internal nematodes were identified by means of morphological analysis and the sequencing of the 18S rRNA gene. Of the sample sites studied, 62.5% were found to be positive for nematode parasites, with 18.7% of all slugs discovered being infected. Five nematode species were identified in this study: Alloionema appendiculatum, Agfa flexilis, Angiostoma limacis, Angiostoma sp. and Phasmarhabditis hermaphrodita. Of these species, only one nematode was previously undescribed (Angiostoma sp.). This is the first record of the presence of A. appendiculatum, A. flexilis and A. limacis in Norway.

First Report of Dollar Spot, Caused by Sclerotinia homoeocarpa, of Creeping Bentgrass in Norway.

In September 2013, symptoms similar to dollar spot caused by Sclerotinia homoeocarpa F.T. Benn., were observed on creeping bentgrass (Agrostis stolonifera L.) fairways at Losby Golf Course, Lørenskog, Akershus County, in Norway (59.8864' N, 10.9862' E). There were small, circular spots and larger irregular patches of sunken, bleached, straw-colored turf. Affected leaves had light-tan lesions with light reddish-brown margins (2). Abundant aerial mycelium was observed in the diseased turf after incubation for 24 h at room temperature in a moist chamber. The mycelium was septate with y-shaped branches. No spores were observed. Diseased leaf segments were washed 30 min in cold running water, surface-sterilized for 60 s using 70% ethanol, placed on water agar, and incubated at room temperature. After 4 days, water agar plugs containing the fungus were transferred to 50% potato dextrose agar (PDA; 19.5 g PDA and 7.5 g agar per 1 liter of media). The fungus colonized the entire 9-cm PDA plates in 6 days. The diameter of the hyphae varied from 2.5 to 12.5 µm. The white, floccose mycelium turned olive green after 7 days and cinnamon brown after 21 days. The cultures became brown from the bottom, forming flat, dark-brown stroma of 0.5 to 5.0 mm in diameter. DNA was extracted from three isolates (from different plants) using a DNeasy Plant Mini Kit (Qiagen). The ribosomal internal transcribed spacer (ITS) region was PCR-amplified using primers ITS1 and ITS4 (3). All three isolates were identical in sequence (GenBank Accession No. KJ775860) and showed up to 97.6% similarity with isolates of S. homoeocarpa of the common type (C-type; e.g., GenBank Accession No. HQ449691) (1). This similarity is considered quite low within a species and indicates that the Norwegian isolates are distinct from other S. homoeocarpa. For Koch's postulates, the fungus was scraped off 21-day-old PDA cultures and chopped, using a sterile scalpel. All three sequenced isolates were pooled and mixed with 200 ml of autoclaved water. Four mature, healthy sod plugs of creeping bentgrass cv. Independence (10-cm-diameter and 10-cm-depth) were taken from an experimental golf green at Landvik, inserted into pots, and inoculated by even distribution of 50 ml of the fungal suspension. Two control pots with creeping bentgrass received 50 ml of sterile water only. All six pots were incubated individually in plastic bags at room temperature and 16-h daylight. After 14 days, 30 to 90% of the inoculated pots of turfgrass exhibited dollar spot symptoms and controls remained healthy. The fungus was recovered from inoculated turf and identified morphologically. This is the first report of dollar spot on any grass species in Norway. For climatic reasons, dollar spot has been considered to be nonexistent in Scandinavia. However, during recent years, symptoms resembling dollar spot have been observed on more than 15 golf courses in Sweden, Denmark, and Norway, and the damage has varied from low to severe. The disease has been given the Norwegian name myntflekk (i.e., coin spot). References: (1) D. Liberti et al. Phytopathology 102:506, 2012. (2) J. D. Smith et al. Fungal diseases of amenity turf grasses. E. & F. E. Spon, London. 1989. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Leaf Spot Caused by Xanthomonas axonopodis pv. poinsettiicola on Poinsettia (Euphorbia pulcherrima) in Norway.

Poinsettia (Euphorbia pulcherrima) is the biggest flowering potted-plant culture in Norway with approximately six million plants produced yearly. A considerable percentage is produced from imported cuttings. In September 2010, we received diseased poinsettias with necrotic leaf spots from a commercial poinsettia grower in Hordaland County. Leaf spots on the upper sides of leaves were brownish, necrotic, irregular in shape, and surrounded by yellow halos. Small, grayish brown, water-soaked spots were observed on the abaxial sides of the leaves from the same plants. Some of the latter carried crusty, dried residues of bacterial exudates. Leaves were surface sterilized and small pieces were excised from the transition area between healthy and diseased tissue. Leaf pieces were soaked in 0.2 ml of sterile phosphate buffered saline (SPBS) for 30 min. The resulting solution was diluted and streaked on several common media suited for the recovery of plant pathogenic bacteria, among them YDC (yeast dextrose chalk agar). The plates were incubated at 25°C in the dark. After 48 to 72 h, pale yellow, smooth, convex, round, and shiny colonies appeared on YDC. On the basis of plant symptoms and colony morphology, the isolated bacteria were expected to be Xanthomonas axonopodis pv poinsettiicola, which is a known pathogen of poinsettia. One isolate was analyzed by fatty acid methyl ester (FAME) analysis according to Sasser (2) and partial gyrase B sequencing as described by Ah-You et al. (1). A strain of X. axonopodis pv. poinsettiicola (NCPPB 581) from the National Collection of Plant Pathogenic Bacteria (UK) was included as a control in both analyses. The isolates were identical to NCPPB581 with respect to the FAME analysis (species level) and the gyrase B sequence. Furthermore, the gyrB sequence was identical to the sequence of strain LMG 849 in GenBank (Accession No. EU015342.1; identities = 774 of 774). Leaf inoculation of disease-free poinsettia was carried out by spraying a solution (approximately 108 CFU ml-1) on the leaves, covering the plants with wetted plastic bags, and placing the plants in a greenhouse maintained at 21°C for 4 weeks. Leaf spot symptoms consistent with the previously observed ones appeared after 2 weeks of incubation. No symptoms were observed on the negative control plant, which was sprayed with SPBS only. The bacterium was successfully reisolated from the induced symptoms and identified by FAME analysis and gyrase B sequencing. In the period following the first detection, Norwegian poinsettia growers were advised to inspect their produce. Suspected samples were sent to us from 28 producers from around the country. The pathogen was detected at 15 production places. Growers were recommended to disinfect their premises and be vigilant with respect to starting up the new season with healthy propagation material. To our knowledge, this is the first report of X. axonopodis pv. poinsettiicola causing bacterial leaf spot on poinsettia in Norway, providing further data on the occurrence of the disease in Europe. References: (1) N. Ah-You et al. Int. J. Syst. Evol. Microbiol. 59:306, 2009. (2) M. J. Sasser. MIDI Tech. Note No. 102. MIDI, 115 Barksdale Prof. Center, Newark, DE, 1990.

First Report of Phytophthora ramorum Causing Shoot Dieback on Bilberry (Vaccinium myrtillus) in Norway.

In the annual Norwegian Phytophthora ramorum survey in 2009, wild bilberry samples, collected during September and October in a semimanaged park (arboretum) in the southwest coast of Norway, tested positive in a P. ramorum-specific real-time PCR test (1). Necrotic lesions were observed in shoot tips, branching points, and around leaf abscission scars. The lesions were of variable dimensions. In the samples collected in October, some lesions were confluent and completely covered some shoots. After direct detection on plant material, P. ramorum was isolated from necrotic lesions of the stems on semiselective media PARP (corn meal agar amended with pimaricin, ampicillin, rifampicin, and pentacloronitrobenzene) (2). The isolates were identified by the production of abundant chlamydospores on agar and deciduous, semipapillate sporangia that is characteristic of P. ramorum (3). Sexual structures were not observed. Three pure cultures obtained from different plant samples also tested positive for P. ramorum by the specific real-time PCR test (1). All positive samples were found in close vicinity of infected rhododendron plants. In this location, P. ramorum had already been detected on rhododendron in 2005. A pathogenicity test was performed with two isolates from bilberry and one from rhododendron. Wild asymptomatic bilberry plants were collected at the end of June in the forest around Oslo. Two shoot tips with 6 to 10 leaves each and one small part of a branch with several shoots and immature berries were used for testing each isolate. The inoculations were made by dipping the shoots in a zoospore suspension (2 to 3 × 104 zoospores ml-1) for 1 min. Inoculated material was placed in moist incubation chambers and incubated at room temperature (19 to 24°C). Controls were obtained by dipping shoots in sterile water. After 2 days, lesions were observed on leaf laminae from all the shoots inoculated with the three different isolates. After 4 days, severe petiole necroses were observed and leaves abscised easily from the stems. Symptoms on the stems were observed in the apical part or areas around the nodes. Some shoots were almost completely necrotic. Heavy sporulation was observed on the berries. P. ramorum was reisolated from leaves and stems of inoculated shoots for all the isolates. P. ramorum was not recovered from control plants. To our knowledge, this is the first report of P. ramorum on bilberry in Norway. References: (1) K. L. Hughes et al. Phytopathology 96:975, 2006. (2) M. E. Kannwischer and D. J. Mitchell. Phytopathology 68:1760, 1978. (3) S. Werres et al. Mycol. Res.105:1155, 2001.

Chalara fraxinea Isolated from Diseased Ash in Norway.

European ash (Fraxinus excelsior), also known as common ash, occurs naturally inland in lower areas of southeastern Norway and along the southern coast of the country. It is important both as a forest and ornamental tree. During the last decade, dieback has become a disastrous disease on F. excelsior in many European countries. The anamorphic fungus Chalara fraxinea T. Kowalski (1), described for the first time from dying ash trees in Poland, is now considered the cause of ash dieback (2). In May of 2008, C. fraxinea was isolated from 1.5 m high diseased F. excelsior in a nursery in Østfold County in southeastern Norway. Symptoms included wilting, necrotic lesions around leaf scars and side branches, and discoloration of the wood. From symptomatic branches, small pieces (approximately 1 cm3) were excised in the transition area between healthy and discolored wood. After surface sterilization (10 s in 70% ethanol + 90 s in NaOCl), the pieces were air dried for 1 min in a safety cabinet, cut into smaller pieces, and placed on media. The fungus was isolated on potato dextrose agar (PDA) and water agar (WA). On PDA, the cultures were tomentose, light orange, and grew slowly (21 mm mean colony diameter after 2 weeks at room temperature). Typical morphological features of C. fraxinea developed in culture. Brownish phialides (14.8 to 30.0 [19.5] × 2.5 to 5.0 [4.1] µm, n = 50) first appeared in the center of the colonies on the agar plugs that had been transferred. The agar plugs were 21 days old when phialides were observed. Abundant sporulation occurred 3 days later. Conidia (phialospores) extruded apically from the phialides and formed droplets. Conidia measured 2.1 to 4.0 (3.0) × 1.4 to 1.9 (1.7) µm (n = 50). The first-formed conidia from each phialide were different in size and shape from the rest by being longer (6 µm, n = 10) and more narrow in the end that first appeared at the opening of the phialide. Internal transcribed spacer sequencing confirmed that the morphological identification was correct (Accession No. EU848544 in GenBank). A pathogenicity test was carried out in June of 2008 by carefully removing one leaf per plant on 10 to 25 cm high F. excelsior trees (18 trees) and placing agar plugs from a 31-day-old C. fraxinea culture (isolate number 10636) on the leaf scars and covering with Parafilm. After 46 days, isolations were carried out as described above from discolored wood that had developed underneath necrotic lesions in the bark and subsequently caused wilting of leaves. All the inoculated plants showed symptoms, and C. fraxinea was successfully reisolated. No symptoms were seen on uninoculated control plants (eight trees) that had received the same treatment except that sterile PDA agar plugs had been used. References: (1) T. Kowalski. For. Pathol. 36:264, 2006. (2) T. Kowalski and O. Holdenrieder, For. Pathol. Online publication, doi: 10.1111/j.1439-0329.2008.00565.x, 2008.

First Report of Crown and Root Rot Caused by Phytophthora capsici on Hydroponically Grown Cucumbers in Norway.

In December 2004, symptoms of root and crown rot were observed on cucumbers (Cucumis sativus L.) in a greenhouse in Norway. Cucumbers were the only crop of the greenhouse that used rockwool as a growing substrate in a hydroponical system. The first symptoms were detected in propagation material. One week after planting, symptoms of root and crown rot were observed and approximately 10% of the plants died. Later, losses of 50% in some greenhouses were observed. A yield reduction as much as 65% was estimated in the winter period (January and February). The two main cucumber cultivars planted were Armada and Lopez. In February 2005, Phytophthora capsici (Leonian) (1) was isolated on potato dextrose agar from a sample of cv. Lopez. The isolate produced deciduous, papillate sporangia (occasionally with two or three papilla) and pedicels that were sometimes longer than the sporangia. Sequencing of amplicons of the internal transcribed spacer region (ITS) rDNA and of the mitochondrial cytochrome c oxidase subunit 1 (Cox1) gene (2) confirmed the identification. Three isolates collected through 2005 from the same greenhouse were crossed with tester strains of P. cryptogea. Formation of oogonia and amphigynous antheridia was always observed in crosses with mating type A2; thus, all isolates were the A1 mating type. All three isolates grew well at 35°C and did not produce chlamydospores. A pathogenicity test was performed with one isolate of P. capsici. Four plants of cucumber cvs. Indira and Jessica were grown in a growth chamber at 24°C. Plants at the two-leaf stage were drenched with 20 ml of a zoospore suspension of 106 zoospores per ml per plant. After 18 days, all plants of both cultivars developed symptoms of crown rot or wilted and died. P. capsici was reisolated from inoculated plants of both cultivars. The pathogenicity test was repeated in the same way, but in a greenhouse with temperatures that ranged between 18 and 29°C. In addition, four plants of both cultivars at the four-leaf stage were inoculated with a suspension of 105 zoospores per ml. After 1 week, all plants developed crown rot or were irreversibly wilted, independently of the plant age or the zoospore concentration. To our knowledge, this is the first report of P. capsici in Norway. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society St. Paul MN, 1996. (2) L. P. N. M. Kroon et al. Phytopathology 94:613, 2004.

The tomato pathogen Clavibacter michiganensis ssp. michiganensis: producer of several antimicrobial substances.

AIM: To purify and analyse antimicrobial substances produced by the tomato pathogen Clavibacter michiganensis ssp. michiganensis (Cmm), with potential application in control of Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. METHODS AND RESULTS: After selection of a suitable producer and indicator strain, antimicrobial compounds were isolated using chromatographic techniques. The resulting preparations were analysed with respect to heat and protease sensitivity, amino acid composition, amino acid sequence and mass. Using this procedure we discovered one post-translationally modified 2145 Da peptide bacteriocin, one 14 kDa antimicrobial protein as well as low molecular weight (<1000 Da) antimicrobial compounds, putatively belonging to the tunicamycin family. CONCLUSIONS: Clavibacter michiganensis ssp. michiganensis produces various antibacterial substances that are active against Cms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first attempt to characterize antimicrobial substances from Cmm at the molecular level. This is an important step towards investigation of the possible use of these compounds to control the potato ring rot pathogen.

Purification, characterization, and gene sequence of michiganin A, an actagardine-like lantibiotic produced by the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

Members of the actinomycete genus Clavibacter are known to produce antimicrobial compounds, but so far none of these compounds has been purified and characterized. We have isolated an antimicrobial peptide, michiganin A, from the tomato pathogen Clavibacter michiganensis subsp. michiganensis, using ammonium sulfate precipitation followed by cation-exchange and reversed-phase chromatography steps. Upon chemical derivatization of putative dehydrated amino acids and lanthionine bridges by alkaline ethanethiol, Edman degradation yielded sequence information that proved to be sufficient for cloning of the gene by a genome-walking strategy. The mature unmodified peptide consists of 21 amino acids, SSSGWLCTLTIECGTIICACR. All of the threonine residues undergo dehydration, and three of them interact with cysteines via thioether bonds to form methyllanthionine bridges. Michiganin A resembles actagardine, a type B lantibiotic with a known three-dimensional structure, produced by Actinoplanes liguriae, which is a filamentous actinomycete. The DNA sequence of the gene showed that the michiganin A precursor contains an unusual putative signal peptide with no similarity to well-known secretion signals and only very limited similarity to the (only two) available leader peptides of other type B lantibiotics. Michiganin A inhibits the growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of ring rot of potatoes, with MICs in the low nanomolar range. Thus, michiganin A may have some potential in biological control of potato ring rot.

Functional analysis of promoters involved in quorum sensing-based regulation of bacteriocin production in Lactobacillus.

Bacteriocin production in Lactobacillus sake LTH673 involves at least four operons: a regulatory operon (sppIPKR); two operons encoding bacteriocins and their immunity proteins (sppAiA and orfX); and an operon needed for secretion (sppTE). We show here that the response regulator encoded by sppR in L. sake LTH673, as well as the homologous response regulators encoded by plnC and plnD in Lactobacillus plantarum C11, bind to characteristic repeats found in the -80 to -40 regions of spp operons. The promoters controlling bacteriocin operons are strictly regulated, and their activity is increased more than 1000-fold upon activation. Constitutive expression for the regulatory and transport operons is driven, at least in part, by promoters upstream of the -80 to -40 regions. Peak promoter activity of the regulatory and transporter operons precedes that of the two bacteriocin operons. The results reveal how promoters involved in quorum sensing-based regulation of bacteriocin production in Lactobacillus differ in strength, leakiness and timing of their activity.

Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution.

In this paper, we describe the structure of chitinase B from Serratia marcescens, which consists of a catalytic domain with a TIM-barrel fold and a 49-residue C-terminal chitin-binding domain. This chitinase is the first structure of a bacterial exochitinase, and it represents one of only a few examples of a glycosyl hydrolase structure having interacting catalytic and substrate-binding domains. The chitin-binding domain has exposed aromatic residues that contribute to a 55-A long continuous aromatic stretch extending into the active site. Binding of chitin oligomers is blocked beyond the -3 subsite, which explains why the enzyme has chitotriosidase activity and degrades the chitin chain from the nonreducing end. Comparison of the chitinase B structure with that of chitinase A explains why these enzymes act synergistically in the degradation of chitin.

A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum.

Several lactic acid bacteria produce so-called pediocin-like bacteriocins that share sequence characteristics, but differ in activity and target cell specificity. The significance of a C-terminal disulfide bridge present in only a few of these bacteriocins was studied by site-directed mutagenesis of pediocin PA-1 (which naturally contains the bridge) and sakacin P (which lacks the bridge). Introduction of the C-terminal bridge into sakacin P broadened the target cell specificity of this bacteriocin, as illustrated by the fact that the mutants were 10 to 20 times more potent than the wild-type toward certain indicator strains, whereas the potency toward other indicator strains remained essentially unchanged. Like pediocin PA-1, disulfide-containing sakacin P mutants had the same potency at 20 and 37 degrees C, whereas wild-type sakacin P was approximately 10 times less potent at 37 degrees C than at 20 degrees C. Reciprocal effects on target cell specificity and the temperature dependence of potency were observed upon studying the effect of removing the C-terminal disulfide bridge from pediocin PA-1 by Cys-->Ser mutations. These results clearly show that a C-terminal disulfide bridge in pediocin-like bacteriocins contributes to widening of the antimicrobial spectrum as well as to higher potency at elevated temperatures. Interestingly, the differences between sakacin P and pediocin PA-1 in terms of the temperature dependency of their activities correlated well with the optimal temperatures for bacteriocin production and growth of the bacteriocin-producing strain.

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.

Pheromone-induced production of antimicrobial peptides in Lactobacillus.

Production of the bacteriocin sakacin P by Lactobacillus sake LTH673 is dependent on a secreted 19-residue peptide pheromone (IP-673). The gene encoding IP-673 (sppIP) was identified and sequenced. SppIP was shown to be co-transcribed with genes encoding a histidine kinase (sppK) and a response regulator (sppR) typical for signal transduction in bacteria. Further sequencing and transcription studies have shown that IP-673 induces transcription of its own gene and of what are often considered to be all genes necessary for bacteriocin production and immunity. Studies with a reporter gene showed that the promoter in front of the sakacin P structural gene (sppA) is strictly regulated. The promoter in front of sppIP turned out to be less strictly regulated, and low basal promoter activity could be detected in uninduced cells. Bacteriocin production in Bac isolates of L. plantarum C11 could be induced by the non-cognate IP-673 only after the introduction of sppK, indicating that sppK encodes the pheromone receptor. These results show that bacteriocin production in lactobacilli is regulated using a short, strain-specific peptide pheromone. Growth conditions were shown to have considerable effects on the functionality of this regulatory mechanism.

Biosynthesis of bacteriocins in lactic acid bacteria.

A large number of new bacteriocins in lactic acid bacteria (LAB) has been characterized in recent years. Most of the new bacteriocins belong to the class II bacteriocins which are small (30-100 amino acids) heat- stable and commonly not post-translationally modified. While most bacteriocin producers synthesize only one bacteriocin, it has been shown that several LAB produce multiple bacteriocins (2-3 bacteriocins). Based on common features, some of the class II bacteriocins can be divided into separate groups such as the pediocin-like and strong anti-listeria bacteriocins, the two-peptide bacteriocins, and bacteriocins with a sec-dependent signal sequence. With the exception of the very few bacteriocins containing a sec-dependent signal sequence, class II bacteriocins are synthesized in a preform containing an N-terminal double-glycine leader. The double-glycine leader-containing bacteriocins are processed concomitant with externalization by a dedicated ABC-transporter which has been shown to possess an N-terminal proteolytic domain. The production of some class II bacteriocins (plantaricins of Lactobacillus plantarum C11 and sakacin P of Lactobacillus sake) have been shown to be transcriptionally regulated through a signal transduction system which consists of three components: an induction factor (IF), histidine protein kinase (HK) and a response regulator (RR). An identical regulatory system is probably regulating the transcription of the sakacin A and carnobacteriocin B2 operons. The regulation of bacteriocin production is unique, since the IF is a bacteriocin-like peptide with a double-glycine leader processed and externalized most probably by the dedicated ABC-transporter associated with the bacteriocin. However, IF is not constituting the bacteriocin activity of the bacterium, IF is only activating the transcription of the regulated class II bacteriocin gene(s). The present review discusses recent findings concerning biosynthesis, genetics, and regulation of class II bacteriocins.

Comparative studies of chitinases A and B from Serratia marcescens.

Serratia marcescens produces several chitinolytic enzymes, including chitinase A (ChiA) and chitinase B (ChiB). In this study, ChiB was purified to homogeneity using a newly developed protocol based on hydrophobic interaction chromatography. Subsequently, characteristics of ChiB and of the hitherto only partly characterized ChiA were determined and compared. Pure ChiA and ChiB shared several characteristics such as a broad pH optimum around pH 5.0-6.0, and a temperature optimum between 50 and 60 degrees C. Both enzymes were fairly stable, with half-lives of more than 10 d at 37 degrees C, pH 6.1. Analyses of the degradation of various N-acetylglucosamine oligomers, fluorogenic substrates and colloidal chitin showed that both enzymes cleave chitobiose [(GlcNAc)2] from (GlcNAc)n and thus possess an exo-N,N'-diacetylchitobiohydrolase activity. Both enzymes were also capable of producing monomers from longer (GlcNAc)n substrates, indicating that they also have an endochitinase (ChiA) or exo-N,N',N"-triacetylchitotriohydrolase (ChiB) activity. Kinetic analyses with 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside, an analogue of (GlcNAc)3, showed cooperative kinetics for ChiA, whereas for ChiB normal hyperbolic kinetics were observed. ChiA had a higher specific activity towards chitin than ChiB and synergistic effects on the chitin degradation rate were observed upon combining the two enzymes. These results, together with the results of sequence comparisons and previous studies of the cellular localization of the two chitinases in S. marcescens indicate possible roles for ChiA and ChiB in chitin breakdown.

Induction of bacteriocin production in Lactobacillus sake by a secreted peptide.

Lactobacillus sake LTH673 is known to produce a bacteriocin called sakacin P. Production of and immunity to sakacin P were found to depend on the presence of a protease-sensitive component that is produced by L. sake LTH673 itself. This component (called inducing factor [IF]) was purified from culture supernatants and shown to be a basic, nonbacteriocin peptide consisting of 19 amino acids, which in principle is capable of forming a highly amphiphilic helical structure. Circular dichroism studies showed that IF indeed could adopt a helical structure, but only in membrane-mimicking environments. Both purified IF and chemically synthesized IF induced expression of the structural gene for sakacin P and concomitant secretion of the gene product. In addition, IF induced its own production and immunity to sakacin P and related bacteriocins. These results indicate that bacteriocin production by L. sake LTH673 is controlled by an autoinduction pathway in which IF may function as a cell density signal.

Chitinase B from Serratia marcescens BJL200 is exported to the periplasm without processing.

A gene encoding a chitinase from Serratia marcescens BJL200 was cloned and expressed in Escherichia coli and S. marcescens. Nucleotide sequencing revealed an open reading frame encoding a 55.5 kDa protein of 499 amino acids without a typical signal peptide for export. The cellular localization of the chitinase was studied, using two types of cell fractionation methods of immunocytochemical techniques. These analyses showed that the chitinase is located in the cytoplasm in E. coli, whereas it is exported to the periplasm in S. marcescens. Analysis of chitinase isolated from periplasmic fractions of S. marcescens carrying the cloned gene showed that export of the enzyme is not accompanied by processing at the N-terminus. The chitinase did not show any of the characteristics that have been proposed to direct the export of other non-processed extracellular proteins such as the E. coli haemolysin and might therefore be secreted via a hitherto unknown mechanism.

Characterization of a chitinase gene (chiA) from Serratia marcescens BJL200 and one-step purification of the gene product.

The nucleotide sequence of the chiA gene from Serratia marcescens strain BJL200 was determined. The gene was found to encode a protein of 563 amino acid residues, with a typical N-terminal signal peptide of 23 residues, that is cleaved off during export. The gene exhibited striking differences with two previously characterized chiA genes of S. marcescens in the region corresponding to amino acid residues 410-467 of the gene product. Periplasmic fractions of an Escherichia coli strain harbouring the cloned gene were used as starting material for the development of a fast, one-step purification protocol for the chitinase that is based on hydrophobic interaction chromatography.