Question

A bacterial lactose transporter,which is highly specific for lactose,contains a Cys residue that is essential to its transport activity.Covalent reaction of N-cthylmaleimide (NEMwith this Cys residue irreversibly inactivates the transporter.A high concentration of lactosc in the medium prevents inactivation by NEM.presumably by sterically protecting the Cys residue which is in or near the lactose-binding site Cys-containing transporter polypeptide Earlieststep Add lactose to a bacterial suspension at a concentration much greater than K, Add radiolabeled NEM to the bacterial suspension. Remove excess reactants by centrifuging and resuspending bacteria cells. Add nonradiolabeled NEM to the bacterial suspension. Dissolve bacterial membrane proteins in SDS. Separate bacterial membrane protcins using SDS-PAGE Find the radioactive band on the SDS gel. Latestsep

Answer 1

The student question involves a biochemical assay to study lactose transporter activity in bacteria and relates to broader mechanisms of bacterial antibiotic resistance, involving the ykkCD efflux pump and lac operon regulation.

Step-by-step explanation:

The question concerns a biochemical experiment designed to understand the mechanism of action of a bacterial lactose transporter, which is specific for lactose and contains a critical Cys residue necessary for its transport activity. The experiment outlines a process involving the inactivation of this transporter by N-ethylmaleimide (NEM) and the protection of the Cys residue by lactose, suggesting the residue's proximity to the lactose-binding site. Additionally, this question relates to the broader context of bacterial resistance to antibiotics, where the ykkCD multidrug-resistance efflux pump plays a crucial role by expelling antibiotics from the cell, often through a mechanism involving a riboswitch that increases production under specific conditions, such as the presence of tetracycline.

Answer 2

The question is about the biological function of bacterial efflux pumps in antibiotic resistance and the regulation of the lac operon in E. coli, particularly focused on the role of a Cys residue within a lactose transporter.

Step-by-step explanation:

The student's question pertains to the mechanism of action of efflux pumps, specifically the ykkCD multidrug-resistance efflux pump in bacteria, and its role in antibiotic resistance. The ykkCD efflux pump is comprised of two different proteins, ykkC and ykkD, which function together to transport antibiotics out of the bacterial cell, reducing the effectiveness of antibiotic treatment.

Bacteria can employ four major strategies to combat the effects of antibiotics: degradation, alteration of the target site, blocking entry, or active efflux. Efflux pumps like ykkCD are integral in antibiotics resistance by preventing the accumulation of the drug within the bacterial cell at antibacterial levels.

The lac operon's regulation also plays a crucial role in bacterial physiology, as it adapts to available carbon sources. When lactose is present, and glucose is absent, E. coli increases the production of lactose-digesting proteins using an activator protein called catabolite activator protein (CAP), which works when cAMP levels rise due to low glucose concentration. The lactose transporter discussed is part of this adaptive response and the Cys residue mentioned is central to its function, highlighting how structure and function are intricately linked in biological molecules.

Tracking the importance of given residues or proteins within these systems could be achieved using biochemical methods like the one outlined in the question, which involve the protective effect of lactose on the transporter, radiolabeling, and SDS-PAGE analysis to observe which proteins are affected by specific treatments.