CYSH E.Coli

What is the function of the cysH gene in Escherichia coli?

The cysH gene in Escherichia coli K12 encodes phospho-adenylylsulphate reductase (PAPS reductase), a key enzyme in the sulfur assimilation pathway. According to sequencing analysis, the gene consists of an open reading frame 735 nucleotides in length, flanked by a repetitive palindromic sequence centered 36 nucleotides upstream and a terminator-like structure located 20 nucleotides downstream .

The gene product is a homodimeric protein with a molecular weight of 27,927 Da per monomer, comprising 244 amino acids per subunit. This enzyme catalyzes the reduction of phosphoadenosine 5'-phosphosulfate (PAPS) to sulfite, a critical step in the pathway that allows Escherichia coli to utilize inorganic sulfate as a sulfur source for biosynthesis of sulfur-containing compounds .

Notable characteristics of the PAPS reductase enzyme include:

• Contains only one cysteine residue per subunit

• Lacks electron-transferring cofactors

• Undergoes conformational changes upon reduction by dithiols, resulting in a shift of apparent molecular weight from 44,000 to 62,000 without forming an enzyme-thioredoxin complex

This enzyme plays a crucial role in bacterial metabolism by linking inorganic sulfur assimilation to the biosynthesis of organic sulfur compounds essential for cellular function.

How does antibiotic treatment affect cysH expression and sulfur metabolism in Escherichia coli?

Comprehensive transcriptomic analyses have revealed significant relationships between antibiotic exposure and sulfur metabolism in Escherichia coli, with implications for cysH function:

Analysis of Escherichia coli gene expression in response to nine representative classes of antibiotics demonstrated significant reduction of sulfur metabolism pathways across all antibiotic treatments . This suggests that modulation of sulfur metabolism, including pathways involving cysH, represents a conserved response mechanism to antimicrobial stress.

Transcriptomic analysis revealed that antibiotics induce extensive changes in gene expression, with an average of 1,786 genes differentially expressed under antibiotic treatment (approximately 39.7% of the genome) . The magnitude of this response varies substantially between different antibiotics:

• Kanamycin treatment affected 76.4% of the genome

• Polymyxin E affected only 4.7% of the genome

Further analysis of these patterns led researchers to propose four distinct strategies employed by Escherichia coli when responding to antibiotics:

• The pessimistic strategy

• The ignorant strategy

• The defensive strategy

• The invasive strategy

These findings suggest that cysH and related sulfur metabolism genes participate in coordinated stress responses that may contribute to bacterial survival during antibiotic exposure. Understanding these mechanisms could potentially inform new approaches to combat antibiotic resistance.

What methodological approaches are effective for manipulating cysH expression in Escherichia coli?

Several methodological approaches can be employed to manipulate cysH expression in Escherichia coli for research purposes:

Genetic Modification Techniques:

• CRISPR-Cas9 system for precise gene editing

• Homologous recombination using Lambda Red system

• Transposon mutagenesis for random insertional inactivation

• Plasmid-based expression systems for complementation studies

Expression Control Methods:

• Promoter replacement with inducible systems

• Antisense RNA technologies

• Riboswitch engineering

• Small RNA regulation

When performing transformation experiments for genetic manipulation, protocols should include careful preparation of cells, often involving:

• Treatment with ice-cold buffers

• Addition of divalent cations such as calcium chloride

• Heat shock treatment

• Recovery incubation

• Selection on appropriate media

A practical application of cysH manipulation was demonstrated in research on chondroitin sulfate biosynthesis, where researchers employed a "cysH repression or deletion strategy" in an engineered Escherichia coli strain (K4Δ kfoE(DE3)pETM6-S w) . This approach successfully enabled PAPS accumulation and facilitated intracellular sulfation of chondroitin, demonstrating that "PAPS pathway intervention is a necessary condition for GAG biosynthesis in Escherichia coli" .

For researchers designing cysH manipulation experiments, it is critical to consider downstream effects on cellular metabolism, potential growth defects, and the need for supplementation with sulfur-containing compounds depending on the experimental goals.

How can researchers utilize cysH manipulation for biosynthesis of sulfated compounds in Escherichia coli?

Manipulation of cysH expression provides a powerful approach for engineering Escherichia coli strains capable of producing sulfated compounds:

Mechanism and Rationale:

The cysH gene encodes PAPS reductase, which competes with sulfotransferases for phosphoadenosine 5'-phosphosulfate (PAPS) . By repressing or deleting cysH, researchers can increase PAPS availability, redirecting this activated sulfate donor toward sulfotransferase-mediated reactions instead of its reduction to sulfite.

Proven Application - Chondroitin Sulfate Biosynthesis:

A groundbreaking study demonstrated complete microbial synthesis of chondroitin sulfate (CS) in Escherichia coli through this approach . Researchers developed a metabolically engineered strain for the one-step, in vivo production of animal-free CS by:

• Identifying factors controlling CS sulfation levels

• Engineering efficient chondroitin sulfotransferases

• Repressing or deleting cysH to prevent PAPS consumption

• Optimizing expression of pathway components

This methodology represents a significant advancement over previous multi-step chemoenzymatic methods, offering a more sustainable process for producing structurally homogeneous sulfated glycosaminoglycans .

Experimental Considerations:

When applying this approach, researchers should consider:

• PAPS biosynthesis capacity of the engineered strain

• Expression levels of target sulfotransferases

• Potential limitations in cellular export mechanisms for sulfated products

• Metabolic burden of heterologous pathway expression

The successful demonstration of complete CS biosynthesis in Escherichia coli suggests that similar strategies could be applied to produce other valuable sulfated compounds, including pharmaceutically relevant molecules, providing a sustainable alternative to extraction from animal sources.

What is the structure and catalytic mechanism of the cysH gene product in Escherichia coli?

The cysH gene in Escherichia coli encodes phospho-adenylylsulphate reductase (PAPS reductase), with the following structural and mechanistic features:

Structural Characteristics:

• Homodimeric protein structure

• Each monomer consists of 244 amino acids with a molecular weight of 27,927 Da

• Contains only one cysteine residue per subunit

• Lacks electron-transferring cofactors like iron-sulfur clusters or flavins

• Undergoes conformational change upon reduction by dithiols, shifting apparent molecular weight from 44,000 to 62,000

Catalytic Function:

The enzyme catalyzes the reduction of phosphoadenosine 5'-phosphosulfate (PAPS) to phosphoadenosine 5'-phosphate (PAP) and sulfite . This reaction represents a critical step in the assimilatory sulfate reduction pathway, enabling Escherichia coli to utilize inorganic sulfate for biosynthesis of organic sulfur compounds.

Based on the single cysteine per subunit and the absence of electron-transferring cofactors, the catalytic mechanism likely involves thiol chemistry at the active site. The conformational change observed upon reduction suggests structural rearrangements play an important role in the catalytic cycle.

Notably, this conformational change occurs "without formation of an enzyme-thioredoxin complex" , indicating a mechanism distinct from some other redox enzymes that form stable complexes with electron donors.

For researchers interested in studying the structure-function relationship of this enzyme, techniques such as X-ray crystallography, site-directed mutagenesis of the conserved cysteine residue, and enzyme kinetics studies would provide valuable insights into the precise catalytic mechanism.

How does cysH interact with other genes in the sulfur assimilation pathway of Escherichia coli?

The cysH gene operates within a complex network of genes involved in sulfur assimilation and metabolism in Escherichia coli:

Pathway Integration:

The sulfur assimilation pathway involves sequential enzymatic steps:

• ATP sulfurylase (encoded by cysD, cysN) converts sulfate to adenosine 5'-phosphosulfate (APS)

• APS kinase (encoded by cysC) phosphorylates APS to form PAPS

• PAPS reductase (encoded by cysH) reduces PAPS to sulfite

• Sulfite reductase (encoded by cysI, cysJ) further reduces sulfite to sulfide

• O-acetylserine sulfhydrylases (encoded by cysK, cysM) incorporate sulfide into amino acids

Regulatory Interactions:

Analysis of transcription factor-coding genes in Escherichia coli suggests "clustered regulatory patterns implying coregulation" of sulfur metabolism genes. The cysH gene contains a "repetitive palindromic sequence centred 36 nucleotides upstream" that likely serves as a regulatory element .

Competitive Relationship with Sulfotransferases:

In biotechnological applications, cysH has been identified as encoding an enzyme that "competes with sulfotransferases and reduces PAPS to inorganic sulfite" . This competitive relationship represents a critical control point that can be manipulated for metabolic engineering purposes.

Response to Antibiotics:

Transcriptomic analysis revealed that "sulfur metabolism [is] significantly reduced by all antibiotics" , suggesting coordinated regulation of cysH and other sulfur metabolism genes in response to antimicrobial stress. This implies potential regulatory connections between cysH and stress response pathways.

Understanding these interactions is crucial for both fundamental research on bacterial sulfur metabolism and applied studies aiming to manipulate these pathways for biotechnological purposes, such as the production of sulfated compounds or the development of novel antimicrobial strategies.

What diagnostic methods can be used to analyze cysH function and sulfur metabolism in Escherichia coli?

Several experimental approaches can be employed to analyze cysH function and sulfur metabolism in Escherichia coli:

Genetic Analysis:

• Targeted gene knockouts using CRISPR-Cas9 or homologous recombination

• Complementation studies with wild-type and mutant cysH variants

• Suppressor mutation screening to identify functional interactions

• Reporter gene fusions to monitor cysH expression

Biochemical Assays:

• PAPS reductase activity assays measuring conversion of PAPS to sulfite

• Protein purification and characterization using techniques such as:

  • Size exclusion chromatography

  • Enzyme kinetics measurements

  • Substrate binding analysis

Metabolomic Analysis:

• Quantification of sulfur-containing metabolites (PAPS, sulfite, cysteine, etc.)

• Metabolic flux analysis using isotope-labeled sulfur compounds

• Mass spectrometry-based approaches to track sulfur incorporation

Transcriptomic Approaches:

Comprehensive transcriptomic analysis, as demonstrated in research examining Escherichia coli's response to antibiotics, can reveal how cysH expression changes under different conditions . Such studies showed that an average of 1,786 genes were differentially expressed under antibiotic treatment (39.7% of the genome) .

Phenotypic Characterization:

• Growth assays under sulfur-limited conditions

• Antibiotic susceptibility testing

• Stress response analysis

For transformation experiments involved in generating research strains, protocols should follow established methods involving proper cell preparation, DNA introduction, and appropriate selection techniques .

These methodological approaches provide complementary insights into cysH function, allowing researchers to understand its role from genetic, biochemical, and systems-level perspectives.

How does cysH function relate to pathogenicity and infection models of Escherichia coli?

While most types of Escherichia coli are harmless commensals that reside in the intestines of healthy individuals, certain pathogenic strains can cause various infections . The relationship between cysH function and pathogenicity involves several dimensions:

Metabolic Adaptations During Infection:

Pathogenic Escherichia coli must adapt to various environments during infection, including nutrient-limited conditions where efficient sulfur acquisition and metabolism may provide a competitive advantage. The cysH gene's role in sulfur assimilation could be particularly important when infecting tissues where sulfur availability is limited.

Response to Host Defense Mechanisms:

Transcriptomic analysis has shown that sulfur metabolism pathways, including those involving cysH, are significantly affected by antibiotic exposure . Similar regulatory changes may occur in response to host antimicrobial peptides and other defense mechanisms during infection.

Diagnostic Implications:

While standard clinical diagnosis of Escherichia coli infections typically involves culturing samples from infected sites (urine, stool, blood) , research-level analysis might examine expression profiles of metabolic genes, including cysH, to understand adaptation during infection.

Infection Types and Risk Factors:

Escherichia coli can cause various infections, with urinary tract infections being the most common . Other infection types include intestinal infections that can lead to diarrhea (sometimes bloody) and abdominal cramps . Risk factors for infection include:

• Age (children under 5 and adults over 65 at greater risk)

• Consumption of undercooked meats or unpasteurized products

• Decreased stomach acid

• Compromised immune system

Understanding how metabolic pathways, including those involving cysH, contribute to bacterial fitness during infection could potentially inform new approaches for treatment or prevention of Escherichia coli infections.

For researchers using laboratory models to study pathogenic Escherichia coli, careful attention to biosafety protocols is essential, particularly when working with strains that produce Shiga toxins, which can cause serious complications such as hemolytic uremic syndrome .