A variety of nitroaromatic compounds, including 2,4,6-trinitrotoluene (TNT), were reduced by hydrogen in the presence of enzyme preparations from Veillonella alkalescens. Consistent with the proposed reduction pathway, R-NO2 R-NO R-NHOH H, R-NH2, 3 mol of H2 was utilized per mol of nitro group. The rates of reduction of 40 mono-, di-, and trinitroaromatic compounds by V. alkalescens extract were determined. The reactivity of the nitro groups depended on other substituents and on the position of the nitro groups relative to these substituents. In the case of the nitrotoluenes, the para-nitro group was the most readily reduced, the 4-nitro position of 2,4-dinitrotoluene being reduced first. The pattern of reduction of TNT (disappearance of TNT and reduction products formed) depended on the type of preparation (cell-free extract, resting cells, or growing culture), on the species, and on the atmosphere (air or H2). The

No abstract

A 13.5-kilobase Hindlll fragment, bearing an intact mercury resistance (mer) operon, was isolated from chromosomal DNA of broad-spectrum mercury-resistant Bacillus sp. strain RC607 by using as a probe a clone containing the mercury reductase (merA) gene. The new clone, pYW33, expressed broad-spectrum mercury resistance both in Escherlchia coli and in BaciUus subtilis, but only in B. subtilis was the mercuric reductase activity inducible. Sequencing of a 1.8-kilobase mercury hypersensitivity-producing fragment revealed four open reading frames (ORFs). ORF1 may code for a regulatory protein (MerR). ORF2 and ORF4 were associated with cellular transport function and the hypersensitivity phenotype. DNA fragments encompassing the merA and the merB genes were sequenced. The predicted Bacillus sp. strain RC607 MerA (mercuric reductase) and MerB (organomercurial lyase) were similar to those predicted from Staphylococcus aureus plasmid p1258 (67 and 73% amino acid identities, respectively); however, only 40% of the amino acid residues of RC607 MerA were identical to those of the mercuric reductase from gram-negative bacteria. A 69-kilodalton polypeptide was isolated and identified as the merA gene product by examination of its amino-terminal sequence.Continuing environmental heavy-metal pollution has favored the proliferation of microorganisms carrying metal resistance determinants (2, 11, 18, 37). Resistance to mercury encoded by bacterial plasmids and transposons in several gram-negative bacteria (11, 34, 37) and in one grampositive organism (16, 43) has been investigated.We previously described a gram-positive, aerobic sporeformer, Bacillus sp. strain RC607 (18,42), whose mercury resistance determinants were located chromosomally and which showed a broad-spectrum (43) mercury resistance phenotype, i.e., growth both in the presence of such organomnercurials as phenylmercuric acetate (due to the activity of the enzyme organomercurial lyase) and in the presence of inorganic mercury salts (with ability to convert Hg2+ to Hge via the enzyme mercuric reductase). Plasmid pYW40, containing a 6.2-kilobase (kb) fragment of RC607 chromosomal DNA, was transferred to Escherichia coli, in which it produced mercuric reductase constitutively (42). Mercury resistance, however, was produced only in the presence of a second plasmid, pYW22, which encoded a functional mercury transport system (42); i.e., pYW40 did not contain the intact RC607 mer operon. Thus, both transport function and mercuric reductase activity are required for expression of microbial mercury resistance (17, 42).The bacterial transposons Tn2J and Tn501, of gramnegative origin and conferring narrow-spectrum resistance, possess similar mer operons. Essential gene products en-* Corresponding author. t Present address:

An integrated investigation of the sequence of events during the germination of Bacillus megateriwn spores produced on three different media-Liver "B" (LB), synthetic, and Arret and Kirshbaum (A-K)-is reported. Heat-activated spores were germinated in a mixture of glucose and Lalanine. For studies of dipicolinic acid (DPA) release and increase in stainability and phase-darkening, germination levels were stabilized by the addition of 2 mm HgCl2. Heat resistance was measured by conventional plating techniques and by a new microscopic method. The sequence (50% completion time) of LB spore germination events was: loss of resistance to heat and to toxic chemicals (3.0 min); DPA loss (4.7 min); stainability and Klett-measured loss of turbidity (5.5 min); phasedarkening (7.0 min); and Beckman DU-measured loss of turbidity (7.2 min). The time difference between 50% completion of stainability and complete phase darkening was 1.5 min, in excellent agreement with the microgermination time of 1.49 min as determined by observation of spores darkening under phase optics. Alteration of the sporulation medium modified the 50% completion times of these germination events, and, in some cases, their sequence. In the A-K spores, the rates of loss of heat resistance and DPA were substantially higher than those of the other germination events, whereas in spores produced in the LB and synthetic media all germination events followed an approximately parallel time course. This is discussed from the point of view of spore population heterogeneity and germination mechanisms. We present here a less cumbersome and more 1811