The anaerobic bacterium Dehalococcoides ethenogenes is the only known organism that can completely dechlorinate tetrachloroethene or trichloroethene (TCE) to ethene via dehalorespiration. One of two corrinoidcontaining enzymes responsible for this pathway, TCE reductive dehalogenase (TCE-RDase) catalyzes the dechlorination of TCE to ethene. TCE-RDase dehalogenated 1,2-dichloroethane and 1,2-dibromoethane to ethene at rates of 7.5 and 30 mol/min/mg, respectively, similar to the rates for TCE, cis-dichloroethene (DCE), and 1,1-DCE. A variety of other haloalkanes and haloalkenes containing three to five carbon atoms were dehalogenated at lower rates. The gene encoding TCE-RDase, tceA, was cloned and sequenced via an inverse PCR approach. Sequence comparisons of tceA to proteins in the public databases revealed weak sequence similarity confined to the C-terminal region, which contains the eight-iron ferredoxin cluster binding motif, (CXXCXXCXXXCP) 2 . Direct N-terminal sequencing of the mature enzyme indicated that the first 42 amino acids constitute a signal sequence containing the twin-arginine motif, RRXFXK, associated with the Secindependent membrane translocation system. This information coupled with membrane localization studies indicated that TCE-RDase is located on the exterior of the cytoplasmic membrane. Like the case for the two other RDases that have been cloned and sequenced, a small open reading frame, tceB, is proposed to be involved with membrane association of TCE-RDase and is predicted to be cotranscribed with tceA.

An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS.

A recent epiphytotic disease on citrus in Florida nurseries was caused by strains of Xanthomonas campestris with different host specificity and lower pathogenic capacities than those of previously described strains of X. campestris pv. citri. The new strains were classified as X. campestris pv. citri because they were isolated from rutaceous hosts and despite the fact that they caused a different disease than strains previously described in that pathovar. Restriction fragment length polymorphism analyses revealed that the Florida strains comprised a heterogeneous (E) group, interrelated with X. campestris pv. alfalfae, X . campestris pv. cyamopsidis, and X. campestris pv. dieffenbachiae. In contrast, the previously described strains of X. campestris pv. citri formed two highly distinct, homogeneous (A and B/C/D) groups. Furthermore, the strains of X. campestris pv. campestris, X . campestris pv. glycines, X . campestris pv. malvacearum, X . campestris pv. phaseoli, X . campestris pv. pisi, and X. campestris pv. vignicola tested were also distinctive and appeared to be only distantly related to one another and to all X. campestris pv. citri strains. We concluded (i) that some pathovars are sufficiently distinct from the type strain of the species X. campestris to be considered as separate species, (ii) that X. campestris pv. citri group A and X. campestris pv. phaseoli (not including X . campestris pv. phaseoli var. fuscans) represent distinctly separate subbranches of the genus and should be respectively reinstated to species as X. citri (ex Hasse) nom. rev. 3213 and X. phaseoli (ex Smith) nom. rev. 627, and (iii) that the X. campestris pv. citri B/C/D strains and the heterogeneous E strains should be respectively renamed X. campestris pv. aurantifolii pv. nov. and X. campestris pv. citrumelo pv. nov.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to demonstrate the reproducibility of bacterial spectra collected on different days. The reproducibility of analysis by MALDI-MS of intact Escherichia coli and Bacillus atrophaeus is presented as a replicate culture study in which spectra were collected on ten different occasions over a three-month period and by two different operators. The analysis resulted in the detection of specific biomarkers in the m/z 2000-20 000 range. Some of the peaks in the Escherichia coli spectra are identified by comparison with other published work. All of the spectra obtained are reproducible over the course of the experiment, but operator variability does exist. The Escherichia coli spectra show operator variability while the Bacillus atrophaeus spectra do not. This work demonstrates the utility of MALDI in obtaining consistent spectra from bacteria over a period of time.