Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log 10 CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log 10 CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log 10 CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibioticresistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log 10 CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reduttase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonasfluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition. Dissimilatory nitrite reductases (dNirs) are pivotal to the fate of combined nitrogen in the environment. They determine the point at which nitrogen is dissimilated instead of assimilated (24). Two distinct types of nitrite reductase are known: one contains a Cu center, and the other contains hemes c and dl. Both seem to carry out the same physiological reaction. NO is typically produced from NO2 during in vitro enzyme assays, but under some conditions, N20 is also produced (6, 32). Cytochrome cd, dNir has been identified in Alcaligenes faecalis (18), Micrococcus (Paracoccus) denitrificans (22), Paracoccus halodenitrificans (5), Pseudomonas aeruginosa (34), Pseudomonas stutzeri (12), and Thiobacillus denitrificans (14). Nonheme Cu dNir has been found in Achromobacter cycloclastes (8), Alcaligenes sp. (Achromobacter xylosoxidans) (17), Alcaligenesfaecalis S6 (10), Nitrosomonas europaea (25), and Rhodopseudomonas sphaeroides forma sp. denitrificans (Rhodobacter sphaeroides) (27). The distribution of the two dNir types in the environment is unknown, although cd, dNir has been identified most often in denitrifying isolates. These strains, which are typically studied in the laboratory, do not reflect the dominant denitrifying populations in nature ...

Three approaches have been used: immunochemical detection, proton translocation measurements, and cell fractionation. Ferritin-conjugated antibody was used by Saraste and Kuronen (20) to locate dNir in Pseudomonas aeruginosa at the inner face of the cytoplasmic membrane. On the basis of proton translocation studies, dNir from Paracoccus denitrificans was placed on the inner face of the cytoplasmic membrane in one study (14) and in the periplasmic space in another (5).Cell fractionation approaches have also given conflicting results. dNir has been identified in the cytoplasm (15, 26) and on the cytoplasmic membrane (15,22,26)

Purpose: The oncofetal antigen, human chorionic gonadotropin ␤ subunit (hCG␤), is expressed by a number of carcinomas and is a prognostic indicator in renal, colorectal, bladder, and pancreatic cancers. We describe the development of a novel antibody-based dendritic cell (DC)-targeted cancer vaccine capable of eliciting cellular immune responses directed against hCG␤.Experimental Design: The tumor-associated antigen hCG␤ was coupled genetically to a human anti-DC antibody (B11). The resulting fusion protein (B11-hCG␤) was evaluated for its ability to promote tumor antigen-specific cellular immune responses in a human in vitro model. Monocytederived human DCs from normal donors were exposed to purified B11-hCG␤, activated with CD40 ligand, mixed with autologous lymphocytes, and tested for their ability to promote hCG␤-specific proliferative and cytotoxic T-lymphocyte responses.Results: B11-hCG␤ was found to be a soluble, welldefined, and readily purified product that specifically recognized the human mannose receptor via the B11 antibody portion of the fusion protein. B11-hCG␤ functionally promoted the uptake and processing of tumor antigen by DCs, which led to the generation of tumor-specific HLA class I and class II-restricted T-cell responses, including CTLs capable of killing human cancer cell lines expressing hCG␤.Conclusions: Although other hCG vaccines have been shown to be capable of eliciting antibody responses to hCG␤, this is the first time that cellular immune responses to hCG␤ have been induced by a vaccine in a human system. This DC-targeted hCG␤ vaccine holds promise for the management of a number of cancers and merits additional clinical development.

Five Tn5 mutants of Pseudomonas fluorescens AK-15 deficient in dissimilatory reduction of nitrite were isolated and characterized. Two insertions occurred inside the nitrite reductase structural gene (nirS) and resulted in no detectable nitrite reductase protein on a Western immunoblot. One mutant had TnS inserted inside nirC, the third gene in the same operon, and produced a defective nitrite reductase protein. Two other mutants had insertions outside of this nir operon and also produced defective proteins. All of the Nir-mutants characterized showed not only loss of nitrite reductase activity but also a significant decrease in nitric oxide reductase activity. When cells were incubated with '5NO in H2180, about 25% of the oxygen found in nitrous oxide exchanged with H20. The extent of exchange remained constant throughout the reaction, indicating the incorporation of 180 from H218O reached equilibrium rapidly. In all nitrite reduction-deficient mutants, less than 4% of the '8O exchange was found, suggesting that the hydration and dehydration step was altered. These results indicate that the factors involved in dissimilatory reduction of nitrite influenced the subsequent NO reduction in this organism.Dissimilatory reduction of nitrite is the key step in the denitrification pathway; it is the point of divergence from assimilatory nitrogen metabolism (24). There are two types of nitrite reductases: one contains the cytochromes cdl, and the other contains copper (6,14,20). Organisms containing the cytochrome cd, nitrite reductases are more frequently isolated from nature, whereas Cu-type nitrite reductases are found in organisms that exhibit more phylogenic diversity and occupy a wider range of ecological niches (6, 9).Nitric oxide is the major product of nitrite reduction by purified nitrite reductases, and nitrous oxide is a minor product (4,14,17,26). NO is generally accepted as one of the free and obligatory intermediates in the denitrification pathway (2,5, 8,10,14,20,28), and NO reductases have been purified from Pseudomonas stutzeri Zobell (13) and Paracoccus denitnificans (4, 7). We recently showed that many denitrifiers containing Cu or cdl nitrite reductases are capable of undergoing 0-atom exchange with H2180 during the reduction of NO to N20 (27) and that a labeled intermediate can be trapped with azide. These results suggest that a nitrosyl complex is formed during the reaction. It was also shown that the extent of the 0-atom exchange reaction depended on the availability of electrons. The dependence of nitrosyl transfer upon the presence of NO during reduction of N02-has been demonstrated (11).TnS was used by Zumft et al. (29) to generate mutants deficient in dissimilatory nitrite reduction (Nir-) in P. stutzeri Zobell. All of the mutants isolated possessed normal NO reduction activity, indicating that NO reduction and nitrite reduction are distinct. The nitrite reductase gene (nirS) from Pseudomonas aeruginosa (21) and from two strains of P. stutzeri (Zobell [15]