Facultative, intracellular bacterial symbionts of arthropods are usually vertically transmitted with great fidelity, and may dramatically affect host biology and reproduction. The length of these symbiont-host associations may be thousands of years, and while symbiont loss is predicted, there have been very few observations of a decline of symbiont infection from high frequencies to low. In a population of the sweetpotato whitefly species (Bemisia tabaci MEAM1) in Arizona, USA, we documented the frequency decline of a strain of Rickettsia in the bellii clade from near-fixation in 2011 to 36% of whiteflies infected in 2017. In previous studies, Rickettsia had been shown to increase from 1% to 97% from 2000 to 2006 and remained at high frequency for at least five years. At that time, Rickettsia infection was associated with both fitness benefits and female bias. In the current study we established matrilines of whiteflies from the field (2016, Rickettsia infection frequency= 58%), and studied Rickettsia vertical transmission, fitness and sex ratios associated with Rickettsia infection, and symbiont titer. The vertical transmission rate was high, approximately 98%. Rickettsia infection in the matrilines was not associated with fitness benefits or sex ratio bias, and appeared to be slightly costly, as more Rickettsia-infected individuals produced non-hatching eggs. Overall, the titer of Rickettsia in the matrilines was lower in 2016 than in the whiteflies collected in 2011, but the titer distribution appeared bimodal, with high and low titer lines, and constancy within lines of the average titer over three generations. We found no association between Rickettsia titer and fitness benefits or sex ratio bias, and this change in the interaction between symbiont and host in 2016 whiteflies may explain the drop in symbiont frequency we observed.

Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile σV is required for lysozyme resistance, and σV is activated in response to lysozyme. Once activated, σV, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here we analyze the contribution of individual genes in the σV regulon to lysozyme resistance. Using CRISPR-Cas9 mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation, but loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also use CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile. Importance: Clostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here we use CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile. We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation and PdaV, encoded as part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile σV is required for lysozyme resistance and σV is activated in response to lysozyme. Once activated σV, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here we analyze the contribution of individual genes in the csfV regulon to lysozyme resistance. Using CRISPR-Cas9 mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also use CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile.

Species of the genera Bacillus and Clostridium form dormant, resistant cells are known as endospores in response to stressful conditions. Endospores form inside of a mother cell, or sporangia, via a modified, asymmetrical cell division pathway followed by a variety of spore‐specific modifications to cell structure and content. Once the spore is formed, it is metabolically dormant and highly resistant to various insults such as heat, noxious chemicals, desiccation and UV irradiation. Several significant pathogens are spore formers, which makes disease prevention via food and environment sterilisation challenging. The resistance properties of spores are derived from chemical and physical alterations such as dehydration and mineralisation of the cytoplasm, protective alteration of DNA structure by specialised proteins and formation of impermeable proteinaceous spore coats. Dormancy is ended when the spore enters an environment where spore germination is favourable. Key Concepts Bacterial endospores can survive for decades and are highly resistant to numerous environmental insults. The resistance properties of bacterial endospores contribute to their roles in food spoilage and in human and animal pathogenesis. Bacterial endospores are cells with specialised modifications to their structure and contents. Bacterial endospores are produced via a simple developmental process involving cooperative and regulated gene expression of two cells. Heat resistance of bacterial endospores is determined by several factors, the most important being the relative dehydration of the spore core. Bacterial endospores possess a unique mode of resistance to UV irradiation involving specialised DNA‐binding proteins.

Bacterial spores exhibit extreme longevity and resistance to many killing agents, and are thus problematic agents of several diseases and of food spoilage. However, to cause disease or spoilage, germination of the spore and return to the vegetative state is necessary.