Cell-wall-less (L-form) bacteria exhibit morphological complexity and heterogeneity, complicating quantitative analysis of them under internal and external stimuli. Stable and efficient labeling is needed for the fluorescence-based quantitative cell analysis of L-forms during growth and proliferation. Here, we evaluated the expression of multiple fluorescent proteins (FPs) under different promoters in the Bacillus subtilis L-form strain LR2 using confocal microscopy and imaging flow cytometry. Among others, Pylb-derived NBP3510 showed a superior performance for inducing several FPs including EGFP and mKO2 in both the wild-type and L-form strains. Moreover, NBP3510 was also active in Escherichia coli and its L-form strain NC-7. Employing these established FP-labeled strains, we demonstrated distinct morphologies in the L-form bacteria in a quantitative manner. Given cell-wall-deficient bacteria are considered protocell and synthetic cell models, the generated cell lines in our work could be valuable for L-form-based research.

Despite the critical role of bacterial cell walls in maintaining cell shapes, certain environmental stressors can induce the transition of many bacterial species into a wall-deficient state called L-form. Long-term induced Escherichia coli L-forms lose their rod shape and usually hold significant mutations that affect cell division and growth. Besides this, the genetic background of L-form bacteria is still poorly understood. In the present study, the genomes of two stable L-form strains of E. coli (NC-7 and LWF+) were sequenced and their gene mutation status was determined and compared with their parental strains. Comparative genomic analysis between two L-forms reveals both unique adaptions and common mutated genes, many of which belong to essential gene categories not involved in cell wall biosynthesis, indicating that L-form genetic adaptation impacts crucial metabolic pathways. Missense variants from L-forms and Lenski\'s long-term evolution experiment (LTEE) were analyzed in parallel using an optimized DeepSequence pipeline to investigate predicted mutation effects (α) on protein functions. We report that the two L-form strains analyzed display a frequency of 6-10% (0% for LTEE) in mutated essential genes where the missense variants have substantial impact on protein functions (α<0.5). This indicates the emergence of different survival strategies in L-forms through changes in essential genes during adaptions to cell wall deficiency. Collectively, our results shed light on the detailed genetic background of two E. coli L-forms and pave the way for further investigations of the gene functions in L-form bacterial models.

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function. In these dynamics, hyperstructures can (1) emit and receive signals or (2) fuse and separate from one another. We ask whether the DnaA-based initiation hyperstructure acts as a logic gate receiving information from the membrane, the chromosome, and metabolism to trigger replication; we try to phrase some of these questions in terms of DNA supercoiling, strand opening, glycolytic enzymes, SeqA, ribonucleotide reductase, the macromolecular synthesis operon, post-translational modifications, and metabolic pools. Finally, we ask whether, underpinning the regulation of the cell cycle, there is a physico-chemical clock inherited from the first protocells, and whether this clock emits a single signal that triggers both chromosome replication and cell division.

Clinical and histological similarities between sarcoidosis and tuberculosis have driven repeated investigations looking for a mycobacterial cause of sarcoidosis. Over 50 years ago, \"anonymous mycobacteria\" were suggested to have a role in the etiology of sarcoidosis. Both tuberculosis and sarcoidosis have a predilection for lung involvement, though each can be found in any area of the body. A key histopathologic feature of both sarcoidosis and tuberculosis is the granuloma-while the tuberculous caseating granuloma has an area of caseous necrosis with a cheesy consistency; the non-caseating granuloma of sarcoidosis does not have this feature. This article reviews and reiterates the complicity of the infectious agent, Mycobacterium avium subsp. paratuberculosis (MAP) as a cause of sarcoidosis. MAP is involved in a parallel story as the putative cause of Crohn\'s disease, another disease featuring noncaseating granulomas. MAP is a zoonotic agent infecting ruminant animals and is found in dairy products and in environmental contamination of water and air. Despite increasing evidence tying MAP to several human diseases, there is a continued resistance to embracing its pleiotropic roles. \"Who Moved My Cheese\" is a simple yet powerful book that explores the ways in which individuals react to change. Extending the metaphor, the \"non-cheesy\" granuloma of sarcoidosis actually contains the difficult-to-detect \"cheese\", MAP; MAP did not move, it was there all along.

Various bacteria can change to a spherical cell-wall-deficient state, called L-from, in the presence of antibiotics that inhibit cell wall synthesis. L-forms are classified into two types: unstable and stable L-forms. Unstable L-forms revert to a normal walled state in the absence of antibiotics, while stable L-forms remain in their wall-deficient state. The conversion from unstable to stable L-forms has been often observed during long-term cultivation. However, the genetic cause for this conversion is not yet fully understood. Here, we obtained stable Bacillus subtilis L-form strains from unstable L-form strains via three independent long-term culturing experiments. The whole genome sequencing of the long-cultured strains identified many mutations, and some mutations were commonly found in all three long-cultured strains. The knockout strain of one of the commonly mutated genes, tagF, in the ancestral strain lost the ability to revert to walled state (rod shape), supporting that eliminating the function of tagF gene is one of the possible methods to convert unstable L forms to a stable state.

Metabolic profiling of the extracts from a library of actinobacteria led to the identification of a novel polyketide, demurilactone A, produced by Streptomyces strain DEM21308. The structure of the compound was assigned based on a detailed investigation of 1D/2D NMR spectra and HR-MS. Whole genome DNA sequencing, followed by bioinformatics analysis and insertional mutagenesis, identified type I polyketide synthases encoded by the dml gene cluster to direct the biosynthesis of this polyene macrolide. While the number of modules is consistent with the carbon backbone of the assigned structure, some discrepancies were identified in the domain organization of five modules. Close investigation of the amino acid sequences identified several mutations in the conserved motifs of nonfunctional domains. Furthermore, the absolute configuration of hydroxy-bearing stereocenters was proposed based on analyses of the ketoreductase domains. Remarkably, although demurilactone A has little detectable activity against normal-walled bacteria, it specifically inhibits the growth of cell wall-deficient \"L-form\" Bacillus subtilis at a minimum inhibitory concentration value of 16 μg/mL. Time-lapse microscopy analyses revealed that demurilactone affects membrane dynamics, probably by reducing membrane fluidity. This compound could be a powerful reagent for studying long-standing questions about the involvement of L-forms in recurrent infection.

Historically, many species of bacteria have been reported to produce viable, cell wall deficient (CWD) variants. A variety of terms have been used to refer to CWD bacteria and a plethora of methods described in which to induce, cultivate and propagate them. In this review, we will examine the long history of scientific research on CWD bacteria examining the methods by which CWD bacteria are generated; the requirements for survival in a CWD state; the replicative processes within a CWD state; and the reversion of CWD bacteria into a walled state, or lack thereof. In doing so, we will present evidence that not all CWD variants are alike and that, at least in some cases, CWD variants arise through an adaptive lifestyle switch that enables them to live and thrive without a cell wall, often to avoid antimicrobial activity. Finally, the implications of CWD bacteria in recurring infections, tolerance to antibiotic therapy and antimicrobial resistance will be examined to illustrate the importance of greater understanding of the CWD bacteria in human health and disease.

Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic resistance genes is well established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as that due to treatment with bacterial cell wall-inhibiting antibiotics such as ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin during the treatment of infectious diseases. This is different from in vitro drug testing, where the organism is exposed to fixed drug concentrations from the beginning until the end. To mimic the mode of antibiotic exposure of microorganisms within host tissues, we cultured the wild-type, ampicillin-sensitive Salmonella enterica serovar Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that displayed several features of the so-called L-form of bacteria, including the absence of the cell wall, altered shape, and lower growth rate compared with the parental form. Studies of the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental form. These results suggested that L-form generation following the initiation of treatment with antibiotics could lead to drug escape of S. Typhi and cell to cell (macrophages) spread of the bacteria, which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through the fecal-oral route, highlighting the need for new approaches to decrease the reservoir of infection.

Streptomyces are soil- and marine-dwelling microbes that need to survive dramatic fluctuations in nutrient levels and environmental conditions. Here, we explore the advances made in understanding how Streptomyces bacteria can thrive in their natural environments. We examine their classical developmental cycle, and the intricate regulatory cascades that govern it. We discuss alternative growth strategies and behaviors, like the rapid expansion and colonization properties associated with exploratory growth, the release of membrane vesicles and S-cells from hyphal tips, and the acquisition of exogenous DNA along the lateral walls. We further investigate Streptomyces interactions with other organisms through the release of volatile compounds that impact nutrient levels, microbial growth, and insect behavior. Finally, we explore the increasingly diverse strategies employed by Streptomyces species in escaping and thwarting phage infections.

Bacterial persisters are a sub-population of phenotypic variants that tolerate high concentrations of antibiotics within the genetically homogeneous cells. They resume division upon the removal of drugs. Bacterial persistence is one of major causes of antibiotic treatment failure and recurrent infection. Cell dormancy, triggered by toxin/antitoxin pair, (p)ppGpp, SOS response and ATP levels, is known to be the mechanistic basis for persistence. However, recent studies have demonstrated that bacteria with active metabolism can maintain persistence by lowering intracellular antibiotic concentration via an efflux pump. Additionally, others and our work have showed that cell wall deficient bacteria (CWDB), including both L-form and spheroplasts that produced by β-lactam antibiotics, are associated with antibiotic persistence. They are not dormant cells as their cell walls have been completely damaged. In this review, we discuss the various types of persisters and highlight the contribution of non-walled bacteria on bacterial persistence.