Ascorbate as an induction inhibitor of β-lactamase in a strain of Enterobacter cloacae

H.A. SHOEB, H.I. AL-SHORA AND T. ABDEL-SALAM. 1995. The effect of ascorbate and anaerobiosis of β-lactamase content (constitutive and inducible) in relation to the susceptibility of a standard strain of Enterobacter cloacae to ampicillin was studied. Enterobacter cloacae ATCC 13047 showed increasing susceptibility to ampicillin when incubated anaerobically in the presence of increasing concentrations of ascorbic acid. The inducible β-lactamase activity in the cell-free extracts of Ent. cloacae decreased when the bacterium was grown aerobically in the presence of ascorbic acid. Under anaerobic growth conditions, however, ascorbic acid abrogated the induction of the enzyme completely. On the other hand, the constitutive enzymatic activity was markedly decreased as the bacterium was grown anaerobically. Thus under these growth conditions, ascorbate-anaerobiosis, the total β-lactamase level in the presence of ampicillin as inducer fell below the basal constitutive activity observed in the absence of ampicillin.     

Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31

Mutations in the genes coding for connexin 26 (Cx26) and connexin 31 (Cx31) cause non-syndromic deafness. Here, we provide evidence that mutations at these two connexin genes can interact to cause hearing loss in digenic heterozygotes in humans. We have screened 108 GJB2 heterozygous Chinese patients for mutations in GJB3 by sequencing. We have excluded the possibility that mutations in exon 1 of GJB2 and the deletion of GJB6 are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in Cx31 was detected in DNA from 200 unrelated Chinese controls. Direct physical interaction of Cx26 with Cx31 is supported by data showing that Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric Cx26/Cx31 connexons. Furthermore, by cotransfection of mCherry-tagged Cx26 and GFP-tagged Cx31 in human embryonic kidney (HEK)-293 cells, we demonstrated that the two connexins were able to co-assemble in vitro in the same junction plaque. Together, our data indicate that a genetic interaction between these two connexin genes can lead to hearing loss.     

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.     

Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism

Mesenchymal cell migration as exhibited by fibroblasts is distinct from amoeboid cell migration and is characterized by dynamic competition among multiple protrusions, which determines directional persistence and responses to spatial cues. Localization of phosphoinositide 3-kinase (PI3K) signaling is thought to play a broadly important role in cell motility, yet the context-dependent functions of this pathway have not been adequately elucidated. By mapping the spatiotemporal dynamics of cell protrusion/retraction and PI3K signaling monitored by total internal reflection fluorescence microscopy, we show that randomly migrating fibroblasts reorient polarity through PI3K-dependent branching and pivoting of protrusions. PI3K inhibition did not affect the initiation of newly branched protrusions, nor did it prevent protrusion induced by photoactivation of Rac. Rather, PI3K signaling increased after, not before, the onset of local protrusion and was required for the lateral spreading and stabilization of nascent branches. During chemotaxis, the branch experiencing the higher chemoattractant concentration was favored, and, thus, the cell reoriented so as to align with the external gradient.     

Data‐driven modeling reconciles kinetics of ERK phosphorylation,localization, and activity states

The extracellular signal‐regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live‐cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK's modification of and release from nuclear substrate anchors. Thus, adaptation of whole‐cell ERK phosphorylation is a by‐product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose‐dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed.     

A nitrate reductase-based colorimetric assay for the study of bacterial adherence

H.A. SHOEB, A.F. TAWFIK AND A.M. SHIBL. 1991. Washed intact cells of Escherichia coli and Streptococcus aureus , grown under partial anaerobic conditions in nitrate media, reduced nitrate quantitatively when formate was used as a reducing substrate. Nitrate reductase was applied as an index for bacterial adherence to different target surfaces including uroepithelial cells, HeLa cells and fibrin clots. Nitrate reduction by adhered as well as control cells was determined by quantitative diazotization reaction for nitrite. Variations in the conditions which affect adherence gave rise to corresponding variations in the nitrate reduction index from which bacterial adherence can be conveniently determined under these conditions. This method is simple, reproducible and easy to perform in a short time.     

Structural basis for the remarkable stability of Bacillus subtilis lipase (Lip A) at low pH

Understanding the structural basis of altered properties of proteins due to changes in temperature or pH provides useful insights in designing proteins with improved stability. Here we report the basis for the pH-dependent thermostability of the Bacillus subtilis lipase (Lip A) using spectroscopic and X-ray crystallographic studies. At pH values above 7, lipase denatures and aggregates when heated at temperatures above 45 degrees C. However, at pH below 6 lipase denatures upon heating but the activity and its native structure is completely recovered upon cooling. In order to obtain the structural basis of this unusual stability of lipase, we determined high-resolution crystal structures of the lipase in two different crystal forms at pH 4.5 and 5. These structures show linear oligomerization of lipase using only two types of dimeric associations and these inter-molecular interactions are completely absent in several crystal forms of wild-type and mutant proteins obtained at basic pH. In accordance with the crystallographic studies, spectroscopic investigations reveal an invariant secondary structure in the pH range of 4-10. Quaternary organization of lipase at low pH resulted in changes in the tryptophan environment and binding of 1-anilino-8-naphthalene sulfate (ANS) at low pH. Low pH stability of the lipase is not observed in the presence of sodium chloride (>0.2 M) indicating the importance of ionic interactions at low pH. Inter- and intra-molecular ionic interactions that occur at pH below 6.0 are proposed to trap the molecule in a conformation that allows its complete refolding upon cooling.     

Thermally denatured state determines refolding in lipase: Mutational analysis

Irreversibility of thermally denatured proteins due to aggregation limits thermodynamic characterization of proteins and also confounds the identification of thermostable mutants in protein populations. Identification of mutations that prevent the aggregation of unfolded proteins provides insights into folding pathways. In a lipase from Bacillus subtilis, evolved by directed evolution procedures, the irreversibility due to temperature‐mediated aggregation was completely prevented by a single mutation, M137P. Though the parent and the mutants unfold completely on heating, mutants having substitutions M137P, along with M134E and S163P, completely or partially prevent the formation of aggregation‐prone intermediate(s) at 75°C. The three mutants show only a marginal increase in free energy of unfolding (ΔG), however, the profiles of the residual activity with temperature shows remarkable shift to higher temperature compared to parent. The intermediate(s) were characterized by enhanced binding of bis‐ANS, a probe to titrate surface hydrophobicity, aggregation profiles and by estimation of soluble protein. Inclusion of salt in the refolding conditions prevents the reversibility of mutant having charge substitution, while the reversibility of mutant with the introduction of proline was unaffected, indicating the role of charge mediated interaction in M134E in preventing aggregation. Partial prevention of thermal aggregation in wild‐type lipase with single substitution, M137P, incorporated by site‐directed mutagenesis, suggests that the affect of M137P is independent of the intrinsic thermostability of lipase. Various effects of the mutations suggest their role is in prevention of the formation of aggregation prone intermediate(s). These mutations, describe yet another strategy to enhance the thermotolerance of proteins, where their influence is observed only on the denatured ensemble.     

A nitrate reductase-based colorimetric assay for the study of bacterial adherence

Washed intact cells of Escherichia coli and Staphylococcus aureus, grown under partial anaerobic conditions in nitrate media, reduced nitrate quantitatively when formate was used as a reducing substrate. Nitrate reductase was applied as an index for bacterial adherence to different target surfaces including uroepithelial cells, HeLa cells and fibrin clots. Nitrate reduction by adhered as well as control cells was determined by quantitative diazotization reaction for nitrite. Variations in the conditions which affect adherence gave rise to corresponding variations in the nitrate reduction index from which bacterial adherence can be conveniently determined under these conditions. This method is simple, reproducible and easy to perform in a short time.