The Xyl Operon and Xylose Metabolism in Bacillus megaterium D-xylose is a pentose (a five-carbon sugar) which is commonly found in nature, especially in the hemicellulose of plant’s cell walls. It is processed by many living organisms, including bacteria, as a carbon source.

Gene function and metabolic transformation of the xylose

In lots of bacteria, including Bacillus megaterium, the metabolism of xylose involves a well-coordinated set of genes and proteins organized within the Xyl operon. This operon includes xylA and xylB genes, which respectively encode for xylose isomerase and xylulokinase.

XylA (Xylose Isomerase) catalyzes the isomerization of D-xylose to D-xylulose, which is the first step in the xylose utilization pathway. This enzyme plays a crucial role in converting xylose into a form that can be further metabolized. ^[1]

The structure of XylA has been extensively studied, revealing a tetrameric protein with a highly conserved active site that catalyzes the conversion of xylose to xylulose. This enzyme also presents the ability to convert glucose to fructose, highlighting its dual functionality. ^[2]

XylB is a kinase that phosphorylates D-xylulose to produce D-xylulose-5-phosphate, allowing it to take part in the pentose phosphate pathway. This phosphorylation step is essential for the integration of xylose into central metabolic pathways. Xylulokinase is a monomeric enzyme that binds ATP and D-xylulose, catalyzing the transfer of a phosphate group to the sugar molecule. ^[2]

In addition to these two proteins, a transport system is mediated thanks to a protein encoded by xylT, as the inactivation of this gene leads to a deficiency in xylose uptake. ^[1] This protein has very high homology with XylE, a membrane-bound transporter responsible for the uptake of xylose into the bacterial cell. It operates as a proton symporter, coupling the transport of xylose with protons to facilitate its import against a concentration gradient ^[3]. The protein consists of multiple transmembrane helixes that form a channel through which xylose is transported. The binding and translocation of xylose involve conformational changes in the transporter. ^[4,5]

As mentioned, all these enzymes allow the formation of xylulose-5-phosphate which can then enter the pentose phosphate pathway, leading to intermediates that can be utilized in glycolysis.

Regulation of the Xyl Operon

The Xyl operon in B. megaterium is tightly regulated to ensure efficient utilization of xylose only when needed. Two main regulatory mechanisms control this operon: repression by XylR and carbon catabolite repression (CCR).

XylR is a DNA-binding protein that negatively regulates the Xyl operon. In the absence of xylose, XylR binds to the operator region of the Xyl operon, preventing transcription. When xylose is present, it binds to XylR, causing a conformational change that releases the repressor from the DNA, allowing transcription to proceed [6]. The structural similarity between xylose and glucose allows these sugars to use the same binding site on several enzymes. The Xyl repressor in B. megaterium can bind to glucose and glucose-6-phosphate, creating competition with xylose ^[7]. The structure of XylR includes a DNA-binding domain and an effector-binding domain. The effector-binding domain interacts with xylose, altering the other part affinity for DNA.

The CCR (Carbon Catabolite Repression) pathway is mainly mediated by the protein CcpA and Hpr. When glycolytic activity is high, ATP and pyruvate are being produced, indicating abundant glucose or other carbohydrates. In glycolysis the step of production of pyruvate is the transformation of phosphoenolpyruvate (PEP) creating an ATP. At this point a key protein, E1, transfers a phosphoryl group from PEP to HPr with the help of a kinase, HprK on the serine residue (Ser-46) of HPr. ^[8,9].
Phosphorylated HPr interacts with CcpA, changing its tridimensional structure and enabling its binding to the catabolite response element (cre) within the xylA coding sequence, thereby blocking the transcription of the xyl operon. This mechanism ensures that xylose metabolism is repressed when glucose, a preferred carbon source, is available. The interaction between CcpA and the cre sites is a key regulatory point for balancing the utilization of different sugars.

Regulation and structure of the Cad operon in Escherichia coli

The Cad operon of Escherichia coli plays a crucial role in the acidic stress response, allowing the bacteria to survive in low pH environments. This adaptive response allows E. coli to neutralize external acidity and maintain a stable intracellular pH, thereby enabling bacterial survival and growth under unfavorable conditions.

Description and regulation of the Cad Operon

The Cad Operon is a genetic system containing the genes allowing E.Coli to cope with acid stresses by converting lysine into cadaverine. This process uses one proton and helps in neutralizing extreme acidity.
CadC becomes an active receptor when forming stable dimers depending on stimuli such as pH conditions and/or the presence of lysine. Those two elements are essential to lead to the rearrangement of the receptor, which then facilitates its dimerization and activation. In this form, CadC then interacts with two regions of the CadBA promoter: CAd1 and CAd2. The products of these two genes in the operon are essential for the synthesis and export of cadaverine ^[1,2].The first site, Cad1, contains repeated and palindromic sequences, which are ideal for the fixation of transcription factors such as CadC.
On the other hand, Cad2 site is extremely rich in AT nucleotides, which is ideal for RNA polymerases when they break apart the DNA strands. Those two types of sequences are essential for the good activation of the cadBA operon ^[3,4].

CadC is tightly regulated by various intermediaries, including inducers such as Fnr and MlrA, which bind to well-characterized regions of the promoter (pCadC), triggering its transcription ^[5-8]. More generally it’s the H-NS protein (Histone-like Nucleoid Structuring Protein) which negatively regulate the production of CadC by preventing the fixation of the RNA polymerase in non inducible conditions, such as a neutral pH or anaerobic conditions ^[9].
Under stress conditions, H-NS synthesis is inhibited by ppGpp and as a result, it is not produced in sufficient quantities to bind to the DNA of many promoters that regulate stress response genes, making them now accessible.
OmpR is a transcriptional regulator that is part of a two-component system (EnvZ/OmpR) which responds to osmotic changes and other environmental stresses, such as a decrease in pH to 6.55 or lower. In response, OmpR is phosphorylated by EnvZ, which detects the pH changes. Once activated, OmpR binds to the promoters of certain genes, including CadC, and proceeds to repress their transcription. The repression of CadC leads to an increase in the intracellular pH, allowing the cell to modulate its osmolarity and adapt to the acidic environment ^[10].

Structure and Function of CadC operon’s proteins

CadA is a cytoplasmic enzyme that catalyzes the decarboxylation of lysine into cadaverine and CO2, taking a proton in the process. CadA forms a homodecamer, consisting of five dimers associated into a double-ring with fivefold symmetry. This means that ten CadA units assemble into two stacked rings, each composed of five dimers, providing a stable and functional structure for the enzyme ^[11,12]. This organization is essential for the enzyme's functionality.


CadB is a transmembrane protein that functions as an antiporter, simultaneously exchanging cadaverine produced by CadA for extracellular lysine. CadB is anchored in the plasma membrane of E. coli and has several transmembrane alpha helices that form a channel through the membrane. The transmembrane domains of CadB contain specific binding sites for lysine and cadaverine ^[15]. Under normal conditions, there is a higher concentration of lysine outside the cell and a higher concentration of cadaverine inside the cell, which favors the entry of lysine and the exit of cadaverine.