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Clostridium perfringens type F food poisoning (FP) strains produce C. perfringens enterotoxin (CPE) to cause a common bacterial food-borne illness in the United States. During FP, CPE is synthesized in the intestines when C. perfringens sporulates. Besides CPE, FP strains also produce sialidases. Most FP strains carry their cpe gene on the chromosome and all surveyed chromosomal cpe (c-cpe) FP strains produce NanH sialidase or both NanJ and NanH sialidases. NanR has been shown previously to regulate sialidase activity in non-FP strains. The current study investigated whether NanR also regulates sialidase activity or influences sporulation and CPE production for c-cpe FP strains SM101 and 01E809. In sporulation medium, the SM101 nanR null mutant showed lower sialidase activity, sporulation, and CPE production than its wild-type parent, while the 01E809 nanR null mutant showed roughly similar sialidase activity, sporulation, and CPE production as its parent. In vegetative medium, the nanR null mutants of both strains produced more spores than their parents while NanR repressed sialidase activity in SM101 but positively regulated sialidase activity in 01E809. These results demonstrate that NanR regulates important virulence functions of c-cpe strains, with this control varying depending on strain and culture conditions. [ABSTRACT FROM AUTHOR]

Clostridium perfringens type F food poisoning (FP) strains produce C. perfringens enterotoxin (CPE) to cause a common bacterial food-borne illness in the United States. During FP, CPE is synthesized in the intestines when C. perfringens sporulates. Besides CPE, FP strains also produce sialidases. Most FP strains carry their cpe gene on the chromosome and all surveyed chromosomal cpe (c-cpe) FP strains produce NanH sialidase or both NanJ and NanH sialidases. NanR has been shown previously to regulate sialidase activity in non-FP strains. The current study investigated whether NanR also regulates sialidase activity or influences sporulation and CPE production for c-cpe FP strains SM101 and 01E809. In sporulation medium, the SM101 nanR null mutant showed lower sialidase activity, sporulation, and CPE production than its wild-type parent, while the 01E809 nanR null mutant showed roughly similar sialidase activity, sporulation, and CPE production as its parent. In vegetative medium, the nanR null mutants of both strains produced more spores than their parents while NanR repressed sialidase activity in SM101 but positively regulated sialidase activity in 01E809. These results demonstrate that NanR regulates important virulence functions of c-cpe strains, with this control varying depending on strain and culture conditions.

Understanding how proteins interact with DNA, and particularly the stoichiometry of a protein–DNA complex, is key information needed to elucidate the biological role of the interaction, e.g. transcriptional regulation. Here, we present an emerging analytical ultracentrifugation method that features multi-wavelength detection to characterise complex mixtures by deconvoluting the spectral signals of the interaction partners into separate sedimentation profiles. The spectral information obtained in this experiment provides direct access to the molar stoichiometry of the interacting system to complement traditional hydrodynamic information. We demonstrate this approach by characterising a multimeric assembly process between the transcriptional repressor of bacterial sialic acid metabolism, NanR and its DNA-binding sequence. The method introduced in this study can be extended to quantitatively analyse any complex interaction in solution, providing the interaction partners have different optical properties. [ABSTRACT FROM AUTHOR]

Abstract: Transcriptional biosensors have various applications in metabolic engineering, including dynamic pathway control and high‐throughput screening of combinatorial strain libraries. Previously, various biosensors have been created from naturally occurring transcription factors (TFs), largely relying on native sequences without the possibility to modularly optimize their response curve. The lack of design and engineering techniques thus greatly hinders the development of custom biosensors. In view of the intended application this is detrimental. In contrast, a bottom‐up approach to design tailor‐made biosensors was pursued here. Novel biosensors were created that respond to N‐acetylneuraminic acid (Neu5Ac), an important sugar moiety with various biological functions, by employing native and engineered promoters that interact with the TF NanR. This bottom‐up approach, whereby various tuned modules, e.g., the ribosome binding site (RBS) controlling NanR translation can be combined, enabled the reliable engineering of various response curve characteristics. The latter was validated by testing these biosensors in combination with various Neu5Ac‐producing pathways, which allowed to produce up to 1.4 ± 0.4 g/L extracellular Neu5Ac. In this way, the repertoire of biosensors was expanded with seven novel functional Neu5Ac‐responsive biosensors. [ABSTRACT FROM AUTHOR]

Clostridium perfringens type F strains, which produce C. perfringens enterotoxin (CPE), are a major cause of gastrointestinal infections, including the second most prevalent bacterial foodborne illness and 5 to 10% cases of antibiotic-associated diarrhea. Virulence of type F strains is primarily ascribable to CPE, which is synthesized only during sporulation. Many type F strains also produce NanI sialidase and carry a nan operon that likely facilitates uptake and metabolism of sialic acid liberated from glycoconjugates by NanI. During vegetative growth of type F strain F4969, NanR can regulate expression of nanI Given their importance for type F disease, the current study investigated whether NanR can also influence sporulation and CPE production when F4969 or isogenic derivatives are cultured in modified Duncan-Strong sporulation (MDS) medium. An isogenic F4969 nanR null mutant displayed much less sporulation and CPE production but more NanI production than wild-type F4969, indicating that NanR positively regulates sporulation and CPE production but represses NanI production in MDS. Results for the nanR mutant also demonstrated that NanR regulates expression of the nan operon. A nanI nanR double null mutant mirrored the outcome of the nanR null mutant strain but with a stronger inhibition of sporulation and CPE production, even after overnight incubation. Coupled with results using a nanI null mutant, which had no impairment of sporulation or CPE production, NanR appears to carefully modulate the availability of NanI, nan operon-encoded proteins and sialic acid to provide sufficient nutrients to sustain sporulation and CPE production when F4969 is cultured in MDS medium., (Copyright © 2018 American Society for Microbiology.)

Clostridium perfringens can produce up to three different sialidases, including NanI, its major exosialidase. The current study first showed that human intestinal strains of C. perfringens can grow by utilizing either glucose or sialic acids, such as N -acetylneuraminic acid (Neu5Ac), which are the end products of sialidase activity. For the human enteropathogenic strain F4969, it was then determined that culture supernatant sialidase activity and expression of exosialidase genes, particularly nanI , are influenced by the presence of Neu5Ac or glucose. Low Neu5Ac concentrations increased culture supernatant sialidase activity, largely by stimulating nanI transcription. In contrast, low glucose concentrations did not affect exosialidase activity or nanI transcription. However, either high Neu5Ac or high glucose concentrations repressed F4969 culture supernatant sialidase activity and nanI transcription levels. Furthermore, high glucose levels repressed F4969 culture sialidase activity and nanI expression even in the presence of low Neu5AC concentrations. To begin to evaluate the mechanistic basis for nanI expression, a nanR null mutant was used to demonstrate that NanR, a member of the RpiR family of regulatory proteins, decreases exosialidase activity and nanI transcription in the absence of sialic acid. The ability of C. perfringens to regulate its exosialidase activity, largely by controlling nanI expression, may affect intestinal pathogenesis by affecting the production of NanI, which may affect C. perfringens growth, adhesion, and toxin binding in vivo ., (Copyright © 2017 American Society for Microbiology.)