In cells, proteins are synthesized, function, and degraded (dead). Protein synthesis (spring) is important for the life of proteins. However, how proteins die is equally important for organisms. Proteases are secreted from cells and used as nutrients to break down external proteins. Proteases degrade unwanted and harmful cellular proteins. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for cellular protein degradation. Prokaryotes, such as bacteria, have similar protein degradation systems. In this review, we describe the structure and function of the ClpXP complex in the degradation system, which is an ATP-dependent protease in bacterial cells, with a particular focus on ClpP.

Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus, low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including those involved in heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease when the oxygen levels are sufficient, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address whether degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as, along with heme biosynthesis, there is diminished expression of many FixL-activated genes in ∆lon. However, stabilization of FixT in ∆lon strains does not contribute to restoring any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during growth in standard aerobic conditions. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter.
The Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due in part to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.

Bacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order, Caulobacter crescentus, relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such as Brucella abortus, depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation in Caulobacter and showing excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation in B. abortus for the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on the Brucella methylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen.IMPORTANCEDNA methylation plays an important role in bacteria, maintaining genome integrity and regulating gene expression. We used nanopore sequencing to directly measure methylated bases in Caulobacter crescentus and Brucella abortus. In Caulobacter, we showed that stabilization of the CcrM methyltransferase upon loss of the Lon protease results in prolific methylation and discovered that the putative methylase AlkB is unlikely to have a global physiological effect. We measured genome-wide methylation in Brucella for the first time, revealing a similar role for CcrM in cell-cycle methylation but a more complex regulation by the Lon protease than in Caulobacter. Finally, we show how the virulence factor MucR impacts DNA methylation patterns in Brucella.

RNase P is an essential enzyme found across all domains of life that is responsible for the 5\'-end maturation of precursor tRNAs. For decades, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations, especially in the model bacterium, Escherichia coli In E. coli, the temperature-sensitive (ts) rnpA49 mutation in the protein subunit of RNase P has arguably been one of the most well-studied mutations for examining the enzyme\'s activity in vivo. Here, we report for the first time naturally occurring temperature-resistant suppressor mutations of E. coli strains carrying the rnpA49 allele. We find that rnpA49 strains can partially compensate the ts defect via gene amplifications of either RNase P subunit (rnpA49 or rnpB) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies, and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insights into the behavior and complementation of the rnpA49 allele in vivo and provides direction for follow-up studies regarding RNase P regulation and turnover in E. coli.

Limb lengthening relies on the process of distraction osteogenesis. The active periosteal bone formation has been detected in clinical practice with a lengthening and then nail (LATN) technique but has not been confirmed by experimental studies to date. The aim of this study is to compare the tissue regeneration of the distraction regenerate during tibial lengthening in rabbits using a LATN technique. This study was performed on 54 mature rabbits of the Soviet Chinchilla breed, which were divided into three groups of 18 animals. In group 1 (control), the tibia was lengthened in an external fixator. In group 2, the LATN technique was modeled and in group 3, lengthening over nail (LON) was modeled. The total duration of the experiment was 45 days. On the 10th, 15th, 20th, 30th, and 45th day X-ray, computed tomography and morphological studies were performed. In the experimental groups (2 and 3), a more pronounced periosteal bone formation in the area of regenerate was noted when compared to group 1. In group 2 (LATN), wide cortical plates were formed from the intermediate and periosteal areas. In this group, the maximum densitometric density values were noted. Endosteal bone formation was preserved in all groups. The LON and LATN techniques, when compared with the classical Ilizarov lengthening, do not demonstrate any deficiency in the tissue regeneration of the bone tissue at the regenerate sites. The most powerful bone structures are formed with the sequential use of the external fixation and nailing (LATN).

Diapause is a widespread adaptation of insects that enables them to survive during unfavorable seasons and is characterized by suppressed metabolism and increased lifespan. Previous works have demonstrated that high levels of reactive oxygen species (ROS) and hypoxia-inducible factor-1α (HIF-1α) in the pupal brain of the moth Helicoverpa armigera induce diapause and extend lifespan by downregulating mitochondrial transcription factor A (TFAM). However, the molecular mechanisms of ROS-HIF-1α regulating metabolic activity to extend lifespan are still poorly understood. Here, we show that the mitochondrial abundance in diapause-type pupal brains is markedly lower than that in their nondiapause-type pupae, suggesting that ROS-HIF-1α signaling negatively regulates the number of mitochondria. The protease Lon, a major mitochondrial matrix protease, can respond to ROS signals. It is activated by transcription factor HIF-1α, which specifically binds the LON promoter to promote its expression. A high level of LON mediates the degradation of TFAM, which is a crucial factor in regulating mitochondrial abundance and metabolic activity. We believe this is the first report that a previously unrecognized regulatory pathway, ROS-HIF-1α-LON-TFAM, reduces mitochondrial activity to induce diapause, extending insect lifespan.

Mitochondria are multifunctional organelles that play a central role in a wide range of life-sustaining tasks in eukaryotic cells, including adenosine triphosphate (ATP) production, calcium storage and coenzyme generation pathways such as iron-sulfur cluster biosynthesis. The wide range of mitochondrial functions is carried out by a diverse array of proteins comprising approximately 1500 proteins or polypeptides. Degradation of these proteins is mainly performed by four AAA+ proteases localized in mitochondria. These AAA+ proteases play a quality control role in degrading damaged or misfolded proteins and perform various other functions. This chapter describes previously identified roles for these AAA+ proteases that are localized in the mitochondria of animal cells.

OBJECTIVE: The mechanical characteristics of leg lengthening over a nail (LON) using an external fixator are not well known; specifically, the number of rings and K-wires required for this method has not been determined. This study aimed to compare the mechanical characteristics of leg LON using the simplest configuration for a domestic frame and those of leg lengthening using the Ilizarov frame alone.
METHODS: The mechanical characteristics of cow tibial samples for lengthening over an intramedullary nail in combination with a domestic external fixator (LON samples) and for lengthening with the Ilizarov frame (Ilizarov samples) were evaluated by assessing axial compression, bending load, and torsional load. The research indices were compression stiffness, bending stiffness, torsion stiffness, yield axial load, ultimate axial load, yield bending load, and ultimate bending load.
RESULTS: No statistically significant differences were observed in the compression stiffness, ultimate axial load, bending stiffness, and ultimate, yield bending forces between the Ilizarov samples and LON samples. The compressive stiffness, yield axial load, and ultimate axial load of the LON samples were 98 ± 1.31 N/mm, 915 ± 23.89 N, and 1032 ± 29.86 N, respectively. The anterior-posterior bending stiffness and lateral bending stiffness of the LON samples were 122.48 ± 2.92 N/mm and 116.34 ± 3.95 N/mm, respectively. The yield anterior-posterior bending and ultimate anterior-posterior bending forces of the LON samples were 616.4 ± 3.64 N and 753.2 ± 3.49 N, respectively. The yield lateral bending and ultimate lateral bending forces of the LON samples were 624.6 ± 4.04 N and 759.0 ± 3.39 N, respectively. The axial torsional stiffness of the LON samples was 1.73 ± 0.05 N m/°, which was significantly lower than that of the Ilizarov samples (2.63 ± 0.03 N m/°).
CONCLUSIONS: No statistically significant differences were observed in the mechanical fixation characteristics of axial compression and bending between the Ilizarov samples and LON samples. However, the axial torsional stiffness of the Ilizarov samples was statistically greater than that of the LON samples. We recommend using the simplest configuration for domestic frames in combination with LON for limb lengthening. Partial weight-bearing is permitted in the distraction stage.
METHODS: Case-control study.

Horizontal gene transfer advances bacterial evolution. To benefit from horizontally acquired genes, enteric bacteria must overcome silencing caused when the widespread heat-stable nucleoid structuring (H-NS) protein binds to AT-rich horizontally acquired genes. This ability had previously been ascribed to both anti-silencing proteins outcompeting H-NS for binding to AT-rich DNA and RNA polymerase initiating transcription from alternative promoters. However, we now know that pathogenic Salmonella enterica serovar Typhimurium and commensal Escherichia coli break down H-NS when this silencer is not bound to DNA. Curiously, both species use the same protease - Lon - to destroy H-NS in distinct environments. Anti-silencing proteins promote the expression of horizontally acquired genes without binding to them by displacing H-NS from AT-rich DNA, thus leaving H-NS susceptible to proteolysis and decreasing H-NS amounts overall. Conserved amino acid sequences in the Lon protease and H-NS cleavage site suggest that diverse bacteria degrade H-NS to exploit horizontally acquired genes.

Kosakonia radicincitans is a species within the new genus Kosakonia, which is typically a plant pathogen, with rare reports of human infection. The number of human infections may be underestimated because this new genus is under-represented among diagnostic tools. This report describes a case of bloodstream infection caused by K. radicincitans. The pathogen was identified by matrix-assisted laser desorption/ionization-TOF mass spectrometry and 16S rRNA gene sequencing. The hypervirulent human pathogenicity gene LON, which has not been described before, was detected in the bacterial genome by gene annotation. Thus, this discovery provides a new reference for studying the pathogenic mechanism of this rare pathogen.