Probing protein stability and proteolytic resistance by loop scanning: a comprehensive mutational analysis.

Improvement in protein thermostability was often found to be associated with increase in its proteolytic resistance as revealed by comparative studies of homologous proteins from extremophiles or mutational studies. Structural elements of protein responsible for this association are not firmly established although loops are implicated indirectly due to their structural role in protein stability. To get a better insight, a detailed study of protein wide mutants and their influence on stability and proteolytic resistance would be helpful. To generate such a data set, a model protein, Bacillus subtilis lipase was subjected to loop scanning site-saturation mutagenesis on 86 positions spanning all loops including termini. Upon screening of ~16,000 clones, 17 single mutants with improved thermostability were identified with increment in apparent melting temperature (Tm(app) ) by 1-6°C resulting in an increase in free energy of unfolding (ΔG(unf) ) by 0.04-1.16 kcal/mol. Proteolytic resistance of all single mutants upon incubation with nonspecific protease, Subtilisin A, was determined. Upon comparison, post-proteolysis residual activities as well as kinetics of proteolysis of mutants showed excellent correlation with ΔG(unf) , (r > 0.9), suggesting that proteolysis was strongly correlated with the global stability of this protein. This significant correlation in this set, with least possible sequence changes (single aa substitution), while covering >60% of protein surface strongly argues for the covariance of these two variables. Compared to studies from extremophiles, with large sequence heterogeneity, the observed correlation in such a narrow sequence space (ΔΔG(unf) = 1.57 kcal⁻¹) justifies the robustness of this relation.

Anaerobic degradation of m-cresol in anoxic aquifer slurries: carboxylation reactions in a sulfate-reducing bacterial enrichment.

The anaerobic biodegradation of m-cresol was observed in anoxic aquifer slurries kept under both sulfate-reducing and nitrate-reducing but not methanogenic conditions. More than 85% of the parent substrate (300 microM) was consumed in less than 6 days in slurries kept under the former two conditions. No appreciable loss of the compound from the corresponding autoclaved controls was measurable. A bacterial consortium was enriched from the slurries for its ability to metabolize m-cresol under sulfate-reducing conditions. Metabolism in this enrichment culture was inhibited in the presence of oxygen or molybdate (500 microM) and in the absence of sulfate but was unaffected by bromoethanesulfonic acid. The consortium consumed 3.63 mol of sulfate per mol of m-cresol degraded. This stoichiometry is about 87% of that theoretically expected and suggests that m-cresol was largely mineralized. Resting-cell experiments demonstrated that the degradation of m-cresol proceeded only in the presence of bicarbonate. 4-Hydroxy-2-methylbenzoic acid and acetate were detected as transient intermediates. Thus, the parent substrate was initially carboxylated as the primary degradative event. The sulfate-reducing consortium could also decarboxylate p- but not m-hydroxybenzoate to near stoichiometric amounts of phenol, but this reaction was not sulfate dependent. The presence of p-hydroxybenzoate in the medium temporarily inhibited m-cresol metabolism such that the former compound was metabolized prior to the latter and phenol was degraded in a sequential manner. These findings help clarify the fate of a common groundwater contaminant under sulfate-reducing conditions.

Mineralization of carbofuran by a soil bacterium.

A bacterium, tentatively identified as an Arthrobacter sp., was isolated from flooded soil that was incubated at 35 degrees C and repeatedly treated with carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methylcarbamate). This bacterium exhibited an exceptional capacity to completely mineralize the ring-labeled C in carbofuran to CO(2) within 72 to 120 h in a mineral salts medium as a sole source of carbon and nitrogen under aerobic conditions. Mineralization was more rapid at 35 degrees C than at 20 degrees C. No degradation of carbofuran occurred even after prolonged incubation under anaerobic conditions. The predicted metabolites of carbofuran, 7-phenol (2,3-dihydro-2,2-dimethyl-7-benzofuranol) and 3-hydroxycarbofuran, were also metabolized rapidly. 7-Phenol, although formed during carbofuran degradation, never accumulated in large amounts, evidently because of its further metabolism through ring cleavage. The bacterium readily hydrolyzed carbaryl (1-naphthyl N-methylcarbamate), but its hydrolysis product, 1-naphthol, resisted further degradation by this bacterium.

Reductive dehalogenation of chlorinated benzenes and toluenes under methanogenic conditions.

The anaerobic metabolism of chlorinated benzenes and toluenes was evaluated in soil slurry microcosms under methanogenic conditions. A mixture of hexachlorobenzene, pentachlorobenzene, and 1,2,4-trichlorobenzene (TCB) in soil slurries was biotransformed through sequential reductive dechlorination to chlorobenzene (CB). The metabolic pathway for hexachlorobenzene and pentachlorobenzene decay proceeded via 1,2,3,4-tetrachlorobenzene (TTCB)-->1,2,3-TCB + 1,2,4-TCB-->1,2-dichlorobenzene (DCB) + 1,4-DCB-->CB. In a mineral salts medium, the CB-adapted soil microorganisms dehalogenated individual 1,2,4,5-TTCB, 1,2,3,4-TTCB, 1,2,3-TCB, and 1,2,4-TCB but not 1,2,3,5-TTCB or 1,3,5-TCB. Similarly, a mixture of 2,3,6-trichlorotoluene (TCT), 2,5-dichlorotoluene (DCT), and 3,4-DCT was reductively dechlorinated in soil slurries to predominantly toluene and small amounts of 2-, 3-, and 4-chlorotoluene (CT). Toluene was further degraded. When tested individually in a mineral salts medium, the CT-adapted soil microorganisms dechlorinated several TCT and DCT isomers. Key metabolic routes for TCTs followed: 2,3,6-TCT-->2,5-DCT-->2-CT-->toluene; 2,4,5-TCT-->2,5-DCT + 3,4-DCT-->3-CT + 4-CT-->toluene. Among DCTs tested, 2,4-DCT and 3,4-DCT were dechlorinated via the removal of o- and m-chlorine, respectively, to 4-CT and subsequently to toluene via p-chlorine removal. Likewise, 2,5-DCT was dechlorinated via 2-CT to toluene. Evidently, microorganisms capable of removing o-, m-, and p-chlorines are present in the soil system, as reflected by the dechlorination of different isomers of CBs and CTs to CB and toluene, respectively. These findings help clarify the metabolic fate of chlorinated benzenes and toluenes in anaerobic environments.

Reductive dechlorination of the nitrogen heterocyclic herbicide picloram.

The anaerobic biodegradation of picloram (3,5,6-trichloro-4-amino-2-pyridinecarboxylic acid) in freshwater sediment was favored under methanogenic conditions but not when sulfate or nitrate was available as a terminal electron acceptor. Under the former conditions, more than 85% of the parent substrate (340 muM) was removed from nonsterile incubations in 30 days, following a 50-day acclimation period. Concomitant with substrate decay, an intermediate transiently accumulated in the sediment slurries. By liquid chromatography-mass spectrometry, the intermediate was identified as an isomer of dichloro-4-amino-2-pyridinecarboxylic acid. Proton nuclear magnetic resonance evidence suggested that a chlorine was reductively removed from the parent substrate at the position meta to the nitrogen heteroatom. Upon continued incubation, the dechlorinated product was transformed into an unidentified compound which accumulated and resisted further decay. The addition of sulfate or bromoethanesulfonic acid to sediment slurries inhibited picloram dehalogenation, but molybdate reversed the inhibitory effect of sulfate on pesticide metabolism. These findings help clarify the fate of a halogenated nitrogen heterocyclic herbicide in anaerobic environments.

Reductive dehalogenation and mineralization of 3-chlorobenzoate in the presence of sulfate by microorganisms from a methanogenic aquifer.

We investigated the anaerobic biodegradation of 3-chlorobenzoate (3CBz) by microorganisms from an aquifer where chloroaromatic compounds were previously found to resist decay in the presence of sulfate. After a lengthy lag period, 3CBz was degraded in the presence of sulfate and concurrently with sulfate reduction. Chlorine removal from 2,5- or 3,5-dichlorobenzoates and the transient appearance of benzoate from 3CBz confirmed that reductive dehalogenation was the initial fate process for these substrates. Sulfate did not influence 3CBz degradation rates in acclimated enrichment cultures but accelerated the development of 3CBz degradation activity in fresh transfers. Benzoate degradation was more rapid in the presence of sulfate regardless of the enrichment history. Nitrate, sulfite, and a headspace of air inhibited 3CBz dehalogenation, while thiosulfate had no effect. Mass balance determinations revealed that 71 to 107% of the theoretically expected amount of methane was produced from 3CBz and benzoate oxidation in the absence of sulfate. In parallel cultures containing 15 mM sulfate, methanogenesis was reduced to 48 to 71% of that theoretically expected, while sulfate reduction accounted for 12 to 50% of the reducing equivalents. In either the presence or absence of sulfate, steady-state dissolved hydrogen concentrations were similar to those reported for sulfate-reducing or methanogenic environments, respectively. Molybdate inhibited sulfate reduction and 3CBz dehalogenation to a similar extent but did not affect benzoate biodegradation. Sulfate-dependent 3CBz biodegradation was not observed. We conclude that reductive dehalogenation and sulfate reduction occur concurrently in these enrichments and that the sulfate-dependent stimulation in fresh transfers was likely due to the acceleration of benzoate oxidation.