Aims: To describe, at high resolution, the bacterial population dynamics and chemical transformations during the ensiling of alfalfa and subsequent exposure to air. Methods and Results: Samples of alfalfa, ensiled alfalfa and silage exposed to air were collected and their bacterial population structures compared using 16S rRNA gene libraries containing approximately 1900 sequences each. Cultural and chemical analyses were also performed to complement the 16S gene sequence data. Sequence analysis revealed significant differences (P < 0Á05) in the bacterial populations at each time point. The alfalfa-derived library contained mostly sequences associated with the Gammaproteobacteria (including the genera: Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly sequences associated with the lactic acid bacteria (LAB) (including the genera: Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even greater percentages of LAB, especially among the genus Lactobacillus, and a significant drop in bacterial diversity. Conclusions: In-depth 16S rRNA gene sequence analysis revealed significant bacterial population structure changes during ensiling and again during exposure to air. Significance and Impact of the Study: This in-depth description of the bacterial population dynamics that occurred during ensiling and simulated feed out expands our knowledge of these processes.

Lysobacter enzymogenes strain C3, a biological control agent for plant diseases, produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity against various fungal and oomycetous species. However, little is known about the regulation of these enzymes or their roles in antimicrobial activity and biocontrol. A study was undertaken to identify mutants of strain C3 affected in extracellular enzyme production and to evaluate their biocontrol efficacy. A single mini-Tn5-lacZ 1 -cat transposon mutant of L. enzymogenes strain C3 that was globally affected in a variety of phenotypes was isolated. In this mutant, 5E4, the activities of several extracellular lytic enzymes, gliding motility, and in vitro antimicrobial activity were reduced. Characterization of 5E4 indicated that the transposon inserted in a clp gene homologue belonging to the Crp gene family of regulators. Immediately downstream was a second open reading frame similar to that encoding acetyltransferases belonging to the Gcn5-related N-acetyltransferase superfamily, which reverse transcription-PCR confirmed was cotranscribed with clp. Chromosomal deletion mutants with mutations in clp and between clp and the acetyltransferase gene verified the 5E4 mutant phenotype. The clp gene was chromosomally inserted in mutant 5E4, resulting in complemented strain P1. All mutant phenotypes were restored in P1, although the gliding motility was observed to be excessive compared with that of the wild-type strain. clp mutant strains were significantly affected in biological control of pythium damping-off of sugar beet and bipolaris leaf spot of tall fescue, which was partially or fully restored in the complemented strain P1. These results indicate that clp is a global regulatory gene that controls biocontrol traits expressed by L. enzymogenes C3.

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular ␤-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with ␤-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of ␤-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with ␤-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45°C and that GluB is most active between pH 4.5 and 5.0 at 41°C. Activity of recombinant proteins against various ␤-1,3 glucan substrates indicates that GluA and GluC are most active against linear ␤-1,3 glucans, while GluB is most active against the insoluble ␤-1,3 glucan substrate zymosan A. These data suggest that the contribution of ␤-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of ␤-1,3 glucans from fungal cell walls.Members of the genus Lysobacter typically are found in soil and water habitats and are characterized by gliding motility and the ability to lyse other microorganisms, including fungi and nematodes (4). One species within this group, Lysobacter enzymogenes, produces an impressive repertoire of extracellular degradative enzyme activities. These include chitinase, glucanase, and protease activities, which can degrade components found in fungal cell walls and which are presumed to contribute to the lytic activity of L. enzymogenes. Despite these described traits, relatively little is known about the hydrolytic enzymes that L. enzymogenes produces or the contributing role for each enzyme class in the lytic activity of L. enzymogenes. To date, only proteases (7, 33, 40) from the species have been characterized at the molecular and biochemical levels.␤-1,3-Glucanases, which hydrolyze glucan polymers containing ␤-1,3 linkages (3, 39) from a number of microbial species, have been characterized. Proposed physiological functions for these enzymes vary depending on the source of enzyme. Among bacteria, many ␤-1,3-glucanases have been studied for purposes of fungal and yeast cell wall degradation (see, e.g., references 2, 9, 17, 27, 28, 31, and 41). However, despite obvious associations with fungal antagonism, few ␤-1,3-glucanases from bacteria with demonstrated biological control activity toward fungal plant pathogens have been characterized. The bacterial biocontrol strain N4-7 was isolated originally as a biocont...

Outbreaks of listeriosis and febrile gastroenteritis have been linked to produce contamination by Listeria monocytogenes. In order to begin to understand the physiology of the organism in a produce habitat, the ability of L. monocytogenes to attach to freshly cut radish tissue was examined. All strains tested had the capacity to attach sufficiently well such that they could not be removed during washing of the radish slices. A screen was developed to identify Tn917-LTV3 mutants that were defective in attachment to radish tissue, and three were characterized. Two of the three mutations were in genes with unknown functions. Both of the unknown genes mapped to a region predicted to contain genes necessary for flagellar export; however, only one of the two insertions caused a motility defect. The third insertion was found to be in an operon encoding a phosphoenolpyruvate-sugar phosphotransferase system. All three mutants were defective in attachment when tested at 30°C; the motility mutant had the most severe phenotype. However, not all of the mutants were defective when tested at other temperatures. These results indicate that L. monocytogenes may use different attachment factors at different temperatures and that temperature should be considered an important variable in studies of the molecular mechanisms of Listeria fitness in complex environments.Listeria monocytogenes is a gram-positive pathogen that causes a multitude of symptoms, including septicemia, abortion, liver failure, and meningitis (50). When L. monocytogenes is not living as an intracellular invader, it can be isolated from the feces of animals as well as humans and from the soil, where it can survive as a saprophyte for 10 to 12 years. From these locations, it can contaminate the food supply in many ways. It can survive in sewage and enter the soil, where it may become associated with plants and/or farm animals before entering food-processing plants, grocery stores, and home refrigerators (2, 10). In all of these niches, L. monocytogenes can survive and even thrive, leading to contamination of foods and ingestion of the pathogen. L. monocytogenes is quite hardy under a variety of environmental stresses. It has been reported that it can grow at temperatures of between Ϫ0.4 and 50°C and withstand extremes of osmotic pressure, as evidenced by growth in 10% NaCl, and it displays a pH range of 4.3 to 9 (18, 21).While food contamination with L. monocytogenes is most often associated with smoked meats and dairy products, fresh produce has also been implicated in outbreaks and sporadic cases of listeriosis. Specific produce-related outbreaks of listeriosis and febrile gastroenteritis caused by L. monocytogenes have been associated with contaminated cabbage, corn, and lettuce and/or celery (8,30,51). In addition, this organism has caused the recall of vegetables, including red peppers, sprouts, and lettuce, and potato salad (4-7, 13). L. monocytogenes has been isolated from radishes, potatoes, cucumbers, and cabbage in surveys of produce from U.S. grocery s...