The structure of a transposon specifying the biodegradation of chlorobenzoate contaminants is described. Ti5271 is a 17-kilobase (kb)

One hundred Peptostreptococcus isolates from five species were assessed for their ability to hydrolyze gelatin. Most Peptostreptococcus magnus (95.8%) and Peptostreptococcus micros isolates (79.0%) hydrolyzed gelatin in contrast to Peptostreptococcus asaccharolyticus (8.0%), Peptostreptococcus anaerobius (10.0%), and Peptostreptococcus prevotii isolates (16.7%). Gelatin hydrolysis in Peptostreptococcus magnus and Peptostreptococcus micros isolates correlated (r = 0.80; P = 0.0019) with more aminopeptidases produced than Peptostreptococcus asaccharolyticus, Peptostreptococcus anaerobius, or Peptostreptococcus prevotii. The five species were further classified into three groups using the extended Tukey test (P < 0.0001) based on the mean percentage of aminopeptidases produced by each species with Peptostreptococcus magnus and Peptostreptococcus micros belonging to group I, Peptostreptococcus asaccharolyticus and Peptostreptococcus prevotii belonging to group II, and Peptostreptococcus anaerobius forming group III. An analysis of possible proteolytic activity of four selected Peptostreptococcus magnus isolates indicated that only 5 of 11 substrates were hydrolyzed as compared to a control isolate of Porphyromonas gingivalis W83, which had a strong proteolytic profile. Therefore, gelatin hydrolysis by Peptostreptococcus spp., in particular Peptostreptococcus magnus and Peptostreptococcus micros, is probably due to a variety of aminopeptidases rather than proteinases.

The rapid ID32A kit (bioMerieux Vitek, Inc., Hazelwood, Mo.) was evaluated for its ability to identify Peptostreptococcus species compared with conventional biochemical tests and gas-liquid chromatography (Virginia Polytechnic Institute), the current "gold standard" method. A total of 5 Peptostreptococcus American Type Culture Collection strains and 95 clinical isolates comprising Peptostreptococcus anaerobius, P. asaccharolyticus, P. magnus, P. micros, and P. prevotii isolates were included for analysis. Overall, the sensitivity and specificity of the rapid ID32A kit in the identification for five Peptostreptococcus species compared with the Virginia Polytechnic Institute method were 93 and 80%, respectively. All P. anaerobius (n = 20) and P. asaccharolyticus (n = 25) isolates were identified with 100% sensitivity and 100% specificity. For the identification of P. magnus (n = 24) and P. micros (n = 19), the rapid ID32A kit was 100% sensitive for both species; the specificity for P. magnus was 95.8% and that for P. micros was 57.9%. The sensitivity and specificity of the rapid ID32A kit for identification ofP. prevotii (n = 12) were poor (41.7 and 8.3%, respectively). The rapid ID32A kit is a useful method for the rapid differentiation of P. anaerobius and P. asaccharolyticus from other Peptostreptococcus spp. Conventional methods should be used to identify to the species level isolates of P. magnus, P. micros, and P. prevotii.

The distribution of Tn5271-related DNA sequences in samples of groundwater and a groundwater bioremediation system at the Hyde Park (Niagara Falls, N.Y.) chemical landfill site was investigated. PCR amplification of target sequences within the cba genes of Tn5271 revealed similar sequences in the groundwater community and in samples from the sequencing batch reactors treating that groundwater. Cell dilution combined with PCR amplification indicated that cba sequences were carried in about 1 of 10 culturable bacteria from the treatment system. Characterization of isolates involved in chlorobenzoate and toluene biodegradation in the treatment system indicated that two phenotypic clusters, Alcaligenes faecalis type 2 and CDC group IVC-2, contained all of the Tn5271 probe-positive isolates from the community. These two groups differed phenotypically from recipient groups isolated following horizontal transfer of pBRC60 (Tn5271) in pristine freshwater microcosms. A genetic rearrangement in Tn5271 attributable to the intramolecular transposition of the flanking element IS1071R was detected in an isolate from the treatment system. Comparison of the structure of the intramolecular transposition derivative from groundwater isolate OCC13(pBRC13) with a laboratory-derived intramolecular transposition derivative of pBRC60 revealed similarities. The rearrangement was shown to increase the stability of the plasmid under starvation conditions. Class I (insertion element [IS] family) and II (Tn3 family) transposable elements are most often associated with the worldwide spread of antibiotic resistance determinants in bacteria following the widespread use of antibiotics (8,9). However, strong selection for the assimilation of unusual carbon sources has also resulted in the mobilization of catabolic genes by these types of transposable elements. Class II (Tn3 family) elements are represented by toluenecatabolic transposon Tn4653 and the defective naphthalene element Tn4655 of Pseudomonas putida (27,28). Class I elements are also represented, including the composite transposon TnS280, which carries genes for chlorobenzene dioxygenase on P. putida plasmid pP51 (29). The 2,4,5-Tcatabolic genes (chq) of P. cepacia are bracketed by copies of IS931 to form a putative composite transposon (10). Biphenyl-degradative genes have recently been localized to a transposable 59-kb DNA element designated Tn4371 in Alcaligenes eutrophus AS (25). We have reported the structure of an unusual composite transposon, TnS271, found on Alcaligenes sp. strain BR60 plasmid pBRC60 (17). This transposon is flanked by copies of a class II element, designated IS1071, that carries a tnpA transposase gene but lacks resolution functions. The internal region of TnS271 carries cba genes that code for 3-chlorobenzoate-3,4-dioxygenase (18).

IS1071-mediated recombinational equilibrium in Alcaligenes sp. BR60 carrying the 3-chlorobenzoate catabolic transposon Tn5271. In experiments designed to TnS mutagenize the indigenous plasmid pBRC60 of Alcaligenes sp. BR60, kanamycinresistant mutants were isolated that were cured of this plasmid and that exhibited recombination of the plasmid-located chlorobenzoate catabolic transposon Tn5271 into the chromosome. These events were independent of the location of Tn5 insertions into the genome of strain BR60. The chromosomal recombinants carried at least two novel copies of IS1071, the class I1 insertion sequence flanking Tn5271, compared with the parent strain. Recombination of Tn5271 into the chromosome of Alcaligenes sp. BR60 was also detected following mating in of pBRC60-incompatible (IncP1) plasmids, R68 and pGS65. Chromosomal copies of Tn5271 could be mobilized between Alcaligenes strains via plasmids pBRC40 or R68. Conjugation of the incompatible plasmid pGS65 into Alcaligenes strains in the absence of selection for 3-chlorobenzoate catabolism resulted in the recovery of 85010 of transconjugants in which the entire pBRC60 plasmid had integrated into the chromosome. These transconjugants exhibited complex rearrangements in chromosomal IS1071 copies. A model of recombinational equilibrium involving homologous recombination between plasmid and chromosomal copies of IS1071 is presented. The results are discussed in terms of the IS1071 (class 11) transposition mechanism and the observed products of IS1071-mediated recombination in natural recipients of pBRC60 in aquatic environments.