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

bNutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75°C heat activation times: 15 min for L-valine germination via the GerA GR and 4 h for germination with the L-asparagine-glucose-fructose-K ؉ mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores' IM and only indirectly affecting GRs. The 150-and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation. Spores of Bacillus species can remain dormant for long periods in the absence of suitable growth conditions (1, 2). However, if specific nutrients are sensed, spores can rapidly become metabolically active in the process of germination followed by outgrowth. The specific nutrients that trigger spore germination are termed germinants, and these molecules are sensed by germinant receptors (GRs) located in spores' inner membrane (IM). Bacillus subtilis spores have three functional GRs: GerA, which responds to L-alanine or L-valine alone, and GerB and GerK, which together are essential for germination with a mixture of L-asparagine, Dglucose, D-fructose, and K ϩ (termed AGFK), with all four components of the mixture required; neither GerB nor GerK alone triggers germination with any nutrient germinant (1, 3). There is also a variant of the GerB GR, termed GerB*, that responds to L-asparagine alone, although GerB* action can be stimulated by glucose via GerK (3). All GRs in B. subtilis spores appear to be located together in a small cluster in the IM termed the germinosome, and formation of this structure is dependent on the GerD protein, which is also in the IM (2, 4). Since gerD spores do not form a germinosome and exhibit extremely slow GR-dependent germination (4), germinosome formation may be essential for rapid GR-dependent germinatio...

The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spore's nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However, like SpoVAD, SpoVAEa is present on the outer surface of the spore's inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel with SpoVAD during proteinase K treatment of germinated spores. SpoVAEa and SpoVAD were also distributed similarly in fractions of disrupted dormant spores. Unlike spoVAD, spoVAEa is absent from the genomes of some spore-forming members of the Bacillales and Clostridiales orders, although SpoVAEa's amino acid sequence is conserved in species containing spoVAEa. B. subtilis strains lacking SpoVAF or SpoVAEa and SpoVAF sporulated normally, and the spores had normal DPA levels. Spores lacking SpoVAF or SpoVAEa and SpoVAF also germinated normally with non-GR-dependent germinants but more slowly than wild-type spores with GR-dependent germinants, and this germination defect was complemented by ectopic expression of the missing proteins.T he spoVA operon of Bacillus subtilis is expressed only in the developing spore during sporulation and encodes seven proteins, with the genes in the order spoVAA, -B, -C, -D, -Eb, -Ea, and -F. At least five of these SpoVA proteins, SpoVAA, -B, -C, -D, and -Eb, are necessary for normal B. subtilis spore formation (1, 2). These proteins are essential for the uptake of dipicolinic acid (DPA) into the dormant spore (3-7); in turn, DPA is essential for spore stability, as DPA-less B. subtilis spores germinate spontaneously after their release from the sporangium and during spore purification (8). At least some SpoVA proteins are also essential for DPA release in spore germination, and one SpoVA protein, SpoVAD, binds DPA specifically, with this binding essential for DPA uptake in sporulation (6). SpoVAD is a soluble protein and has no obvious signal peptide or membrane-spanning or anchor sequences. However, assessment of SpoVAD's location and accessibility in spores indicates that this protein is on the outer surface of the spore's inner membrane (IM) (4, 9, 10).In contrast to SpoVAD, SpoVAA, -B, -C, -Eb, and -F appear to be integral membrane proteins on the basis of predictions from primary sequences and in some cases the localization of proteins expressed in growing bacteria (3,11,12). Although SpoVAF exhibits significant sequence identity to the A subunits of the spore's nutrient germinant receptors (GRs) (2), SpoVAA, -B, -C, -Eb, and -Ea exhibit no significant identity to known proteins. However, SpoVAEa is also predicted to be a soluble protein. Consequently, in this work, we have examined the location an...

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55°C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.

cAs previously reported, gerP Bacillus subtilis spores were defective in nutrient germination triggered via various germinant receptors (GRs), and the defect was eliminated by severe spore coat defects. The gerP spores' GR-dependent germination had a longer lag time between addition of germinants and initiation of rapid release of spores' dipicolinic acid (DPA), but times for release of >90% of DPA from individual spores were identical for wild-type and gerP spores. The gerP spores were also defective in GRindependent germination by DPA with its associated Ca 2؉ divalent cation (CaDPA) but germinated better than wild-type spores with the GR-independent germinant dodecylamine. The gerP spores exhibited no increased sensitivity to hypochlorite, suggesting that these spores have no significant coat defect. Overexpression of GRs in gerP spores did lead to faster germination via the overexpressed GR, but this was still slower than germination of comparable gerP ؉ spores. Unlike wild-type spores, for which maximal nutrient germinant concentrations were between 500 M and 2 mM for L-alanine and <10 mM for L-valine, rates of gerP spore germination increased up to between 200 mM and 1 M L-alanine and 100 mM L-valine, and at 1 M L-alanine, the rates of germination of wild-type and gerP spores with or without all alanine racemases were almost identical. A high pressure of 150 MPa that triggers spore germination by activating GRs also triggered germination of wild-type and gerP spores identically. All these results support the suggestion that GerP proteins facilitate access of nutrient germinants to their cognate GRs in spores' inner membrane.