Rhizopus delemar is the leading cause of mucormycosis, a rapidly aggressive and life-threatening invasive fungal disease. Despite its clinical significance, R. delemar and related mucoralean pathogens remain difficult to manipulate genetically because of their coenocytic growth, multinucleate hyphae, difficult transformation, and limited availability of selectable markers. Consequently, many aspects of their pathobiology, including innate stress resistance and mechanisms of infection, remain poorly understood. Previous attempts to express heterologous proteins or implement gene-editing systems using vectors developed for model fungi have been unsuccessful or inefficient, suggesting that gene-expression requirements in R. delemar may differ from those of better-characterized species. To investigate these requirements, we analyzed codon usage and identified a bias involving proline- and arginine-encoding codons, a pattern also observed in the related mucoralean fungus Mucor circinelloides. We then systematically evaluated nuclear-targeted fluorescent protein expression using combinations of endogenous and heterologous regulatory and structural components, including six promoters, two terminators, three histone proteins, two fluorescent reporters, and the presence or absence of a nuclear localization signal. Endogenous promoters orthologous to known high transcriptional activity promoters produced the most robust expression, whereas heterologous regulatory elements remained functional but were generally less efficient. Detectable fluorescent protein expression was also achieved in the absence of a vector-supplied terminator sequence, and an exogenous nuclear localization signal (NLS) was not required when fluorescent proteins were fused to histone H2B. Together, our findings demonstrate that efficient heterologous gene expression in R. delemar requires optimization at multiple levels, including promoter selection, coding-sequence compatibility, and expression-cassette architecture. The expression platform we developed here provides a foundation for molecular genetic studies of R. delemar and the mechanisms underlying mucormycosis.

Rhizopus species are a leading cause of mucormycosis, an aggressive and life-threatening invasive fungal disease that affects immunocompromised individuals. Despite their clinical importance, mucoralean pathogens, especially R. delemar, are difficult to manipulate genetically because of coenocytic growth, multinucleate hyphae, difficulty with transformation, and limited selectable markers. These obstacles hinder the study of innate stress resistance and disease mechanisms. Previous efforts to express heterologous genes or implement gene-editing systems with vectors designed for model fungi have been largely unsuccessful or inefficient in R. delemar, suggesting unique requirements for gene expression in this organism. In this study, we tested nuclear-targeted fluorescent protein expression and identified differences in proline and arginine codon usage between heterologous and endogenous coding sequences. We then evaluated gene expression efficiency using various combinations of heterologous and endogenous regulatory elements and coding sequences, including six promoters, two terminators, three histone fusion proteins, and two fluorescent reporters, with or without an exogenous nuclear localization signal (NLS). Endogenous promoters orthologous to those with high transcriptional activity produced robust fluorescence, in contrast to weak fluorescence from constructs with heterologous sequences. Notably, fluorescence was also observed in a construct lacking an experimentally provided terminator, and the H2B-GFP fusion protein did not require an exogenous NLS for nuclear localization. These results show that efficient heterologous gene expression in R. delemar requires optimization at several levels, including promoter selection, coding sequence compatibility, and expression-cassette design. Although the direct impact of codon usage on expression efficiency remains to be tested, the expression systems developed here provide a platform for molecular analysis of R. delemar biology and pathogenesis.

DNA supercoiling is essential for the developmental cycle of Chlamydia trachomatis , yet its role in shaping antibiotic responses remains poorly understood. We investigated how the fluoroquinolone moxifloxacin (Mox), a potent DNA gyrase inhibitor, affects C. trachomatis by examining developmental progression, DNA replication, topoisomerase expression, and transcriptional response in select genes. Early Mox exposure completely halted bacterial growth, whereas treatment during mid-developmental cycle produced small inclusions with enlarged bacterial forms and abolished formation of infectious progeny. These outcomes coincided with inhibition of DNA replication, apparent loss of detectable DNA gyrase, and repression of ompA and omcB transcription, while the expression of the groESL1 operon was preserved or elevated. Mox also elicited the downregulation of topoisomerase I (TopA), consistent with compensatory rebalancing of DNA supercoiling. Together, these data demonstrate that Mox exerts differential effects across the developmental stages and that disrupted gyrase-TopA coordination drives chlamydial developmental arrest and a noncanonical, DNA replication-limited persistent phenotype IMPORTANCE: C. trachomatis , a clinically significant obligate intracellular bacterial pathogen, can persist under antimicrobial pressure, complicating treatment strategy. This study links supercoiling homeostasis to fluoroquinolone tolerance, providing mechanistic insights into chlamydial responses to topological stress and identifying potential targets to overcome noncanonical persist state.