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The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Antimicrobial resistance (AMR) is a serious global threat demanding innovations for effective control of pathogens. The bacterial SOS response, regulated by the master regulators, LexA and RecA, contributes to AMR through advantageous mutations. Targeting the LexA/RecA system with a novel inhibitor could suppress the SOS response and potentially reduce the occurrence of AMR. RecA presents a challenge as a therapeutic target due to its conserved structure and function across species, including humans. Conversely, LexA which is absent in eukaryotes, can be potentially targeted, due to its involvement in SOS response which is majorly responsible for adaptive mutagenesis and AMR. Our studies combining bioinformatic, biochemical, biophysical, molecular, and cell-based assays present a unique inhibitor of mycobacterial LexA, wherein we show that the inhibitor interacts directly with the catalytic site residues of LexA of Mycobacterium tuberculosis (Mtb), consequently hindering its cleavage, suppressing SOS response thereby reducing mutation frequency and AMR., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of the article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Enterococcus faecalis is a troublesome nosocomial pathogen that acquired resistance to most available antimicrobial agents. Antivirulence agents represent an unconventional treatment approach. Here, transcription factor decoy (TFD)-loaded cationic liposomes (TLL) were developed as an inhibitor of the Fsr quorum-sensing system and its associated virulence traits, in E. faecalis. The consensus sequence of the FsrA binding site was found conserved among 651 E. faecalis annotated genomes. The TFD was synthesized as an 82 bp DNA duplex, containing the conserved binding sequence, and loaded onto cationic liposomes. The optimum loading capacity, mean particle size, and zeta potential of the TLL were characterized. The developed TLL lacked any effect on E. faecalis growth and significantly inhibited the in vitro production of the proteolytic enzymes controlled by the Fsr system; gelatinase and serine protease, in a concentration-dependent manner. This inhibition was accompanied by a significant reduction in the transcription levels of FsrA-regulated genes (fsrB, gelE, and sprE). The developed TLL were safe as evidenced by the nonhemolytic effect on human RBCs and the negligible cytotoxicity on human skin fibroblast cells. Moreover, in the larvae infection model, TLL displayed a significant abolish in the mortality rates of Galleria mellonella larvae infected with E. faecalis. In conclusion, the developed TLL offer a new safe strategy for combating E. faecalis infection through the inhibition of quorum-sensing-mediated virulence; providing a platform for the development of similar agents to combat many other pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus signal peptidase IB (SpsB) is an essential enzyme for protein secretion. While inhibition of its activity by small molecules is a well-precedented mechanism to kill bacteria, the mode of activation is however less understood. We here investigate the activation mechanism of a recently introduced activator, the antibiotic compound PK150, and demonstrate by combined experimental and Molecular Dynamics (MD) simulation studies a unique principle of enzyme stimulation. Mass spectrometric studies with an affinity-based probe of PK150 unravel the binding site of PK150 in SpsB which is used as a starting point for MD simulations. Our model shows the localization of the molecule in an allosteric pocket next to the active site which shields the catalytic dyad from excess water that destabilizes the catalytic geometry. This mechanism is validated by the placement of mutations aligning the binding pocket of PK150. While the mutants retain turnover of the SpsB substrate, no stimulation of activity is observed upon PK150 addition. Overall, our study elucidates a previously little investigated mechanism of enzyme activation and serves as a starting point for the development of future enzyme activators., (© 2024. The Author(s).)

Aim: Recombinase RecA and its homologs play a key role in homologous recombination DNA repair and revive stalled replication fork DNA synthesis. RecA plays an essential role in the evolution of antibiotic-resistant strains via stress-induced DNA repair mechanisms during the SOS response. Accordingly, RecA has become an attractive target to slow down antibiotic resistance rates and prevent mutations in pathogenic bacterial species., Methods and Results: We employed RecA conserved activities: DNA binding, displacement loop formation, strand exchange, ATP hydrolysis, and LexA cleavage, to elucidate the inhibitory role of gallic acid on Staphylococcus aureus RecA functions. Gallic acid inhibition of the SOS response by western blot analysis and its antibacterial activity were measured. The gallic acid inhibited all the canonical activities of S. aureus RecA protein., Conclusion: The natural phenolic compound gallic acid interferes with RecA protein DNA complex formation and inhibits activities such as displacementloop formation, strand exchange reaction, ATP hydrolysis, and coprotease activity of S. aureus. Additionally, gallic acid can obstruct ciprofloxacin-induced RecA expression and eventually confer the inhibitory role of gallic acid in the SOS survival mechanism in S. aureus., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Importance: In the expanding market of recombinant proteins, microbial cell factories such as Bacillus subtilis are key players. Microbial cell factories experience secretion stress during high-level production of secreted proteins, which can negatively impact product yield and cell viability. The CssRS two-component system and CssRS-regulated quality control proteases HtrA and HtrB play critical roles in the secretion stress response. HtrA has a presumptive dual function in protein quality control by exerting both chaperone-like and protease activities. However, its potential role as a chaperone has not been explored in B. subtilis . Here, we describe for the first time the beneficial effects of proteolytically inactive HtrA on α-amylase yields and overall bacterial fitness., Competing Interests: Jonathan Walgraeve and Michael Seefried are employees of AB Enzymes. All other authors declare no competing interests.

Having previously shown that soluble E-cadherin (sE-cad) is found in sera of Q fever patients and that infection of BeWo cells by C. burnetii leads to modulation of the E-cad/β-cat pathway, our purpose was to identify which sheddase(s) might catalyze the cleavage of E-cad. Here, we searched for a direct mechanism of cleavage initiated by the bacterium itself, assuming the possible synthesis of a sheddase encoded in the genome of C. burnetii or an indirect mechanism based on the activation of a human sheddase. Using a straightforward bioinformatics approach to scan the complete genomes of four laboratory strains of C. burnetii , we demonstrate that C. burnetii encodes a 451 amino acid sheddase (CbHtrA) belonging to the HtrA family that is differently expressed according to the bacterial virulence. An artificial CbHtrA gene (CoxbHtrA) was expressed, and the CoxbHtrA recombinant protein was found to have sheddase activity. We also found evidence that the C. burnetii infection triggers an over-induction of the human HuHtrA gene expression. Finally, we demonstrate that cleavage of E-cad by CoxbHtrA on macrophages-THP-1 cells leads to an M2 polarization of the target cells and the induction of their secretion of IL-10, which "disarms" the target cells and improves C. burnetii replication. Taken together, these results demonstrate that the genome of C . burnetii encodes a functional HtrA sheddase and establishes a link between the HtrA sheddase-induced cleavage of E-cad, the M2 polarization of the target cells and their secretion of IL-10, and the intracellular replication of C. burnetii .

DNA damage triggers a widely conserved stress response in bacteria called the SOS response, which involves two key regulators, the activator RecA and the transcriptional repressor LexA. Despite the wide conservation of the SOS response, the number of genes controlled by LexA varies considerably between different organisms. The filamentous soil-dwelling bacteria of the genus Streptomyces contain LexA and RecA homologs, but their roles in Streptomyces have not been systematically studied. Here, we demonstrate that RecA and LexA are required for the survival of Streptomyces venezuelae during DNA-damaging conditions and for normal development during unperturbed growth. Monitoring the activity of a fluorescent recA promoter fusion and LexA protein levels revealed that the activation of the SOS response is delayed in S. venezuelae . By combining global transcriptional profiling and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we determined the LexA regulon and defined the core set of DNA damage repair genes that are expressed in response to treatment with the DNA-alkylating agent mitomycin C. Our results show that DNA damage-induced degradation of LexA results in the differential regulation of LexA target genes. Using surface plasmon resonance, we further confirmed the LexA DNA binding motif (SOS box) and demonstrated that LexA displays tight but distinct binding affinities to its target promoters, indicating a graded response to DNA damage. IMPORTANCE The transcriptional regulator LexA functions as a repressor of the bacterial SOS response, which is induced under DNA-damaging conditions. This results in the expression of genes important for survival and adaptation. Here, we report the regulatory network controlled by LexA in the filamentous antibiotic-producing Streptomyces bacteria and establish the existence of the SOS response in Streptomyces . Collectively, our work reveals significant insights into the DNA damage response in Streptomyces that will promote further studies to understand how these important bacteria adapt to their environment.

The transcription factor RpoS of Escherichia coli controls many genes important for tolerance of a variety of stress conditions. IraD promotes the post-translation stability of RpoS by inhibition of RssB, an adaptor protein for ClpXP degradation. We have previously documented DNA damage induction of iraD expression, independent of the SOS response. Both iraD and rpoS are required for tolerance to DNA damaging treatments such as H2O2 and the replication inhibitor azidothymidine in the log phase of growth. Using luciferase gene fusions to the 672 bp iraD upstream region, we show here that both promoters of iraD are induced by azidothymidine. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at -81 bp and are regulated by regulatory inactivation of DnaA, dependent on the DnaN processivity clamp. By electrophoretic mobility shift assays, we show that purified DnaA protein binds to the iraD upstream region, so DnaA regulation of IraD is likely to be direct. DNA damage induction of iraD during log phase growth is abolished in the dnaA-T174P mutant, suggesting that DNA damage, in some way, relieves DnaA repression, possibly through the accumulation of replication clamps and enhanced regulatory inactivation of DnaA. We also demonstrate that the RNA-polymerase associated factor, stringent starvation protein A, induced by the accumulation of ppGpp, also affects iraD expression, with a positive effect on constitutive expression and a negative effect on azidothymidine-induced expression., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus , which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA ( lexA -asRNA) is generated by a TRE on the intrinsic terminator (TT sbrB ) of the sbrB gene, which is located downstream of lexA , in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TT sbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TT sbrB prevented lexA -asRNA transcription. The level of lexA -asRNA directly correlated with cellular stress since the expressions of sbrB and lexA -asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TT sbrB , suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.

DNA repair systems allow microbes to survive in diverse environments that compromise chromosomal integrity. Pathogens such as Mycobacterium tuberculosis must contend with the genotoxic host environment, which generates the mutations that underlie antibiotic resistance. Mycobacteria encode the widely distributed SOS pathway, governed by the LexA repressor, but also encode PafBC, a positive regulator of the transcriptional DNA damage response (DDR). Although the transcriptional outputs of these systems have been characterized, their full functional division of labor in survival and mutagenesis is unknown. Here, we specifically ablate the PafBC or SOS pathways, alone and in combination, and test their relative contributions to repair. We find that SOS and PafBC have both distinct and overlapping roles that depend on the type of DNA damage. Most notably, we find that quinolone antibiotics and replication fork perturbation are inducers of the PafBC pathway, and that chromosomal mutagenesis is codependent on PafBC and SOS, through shared regulation of the DnaE2/ImuA/B mutasome. These studies define the complex transcriptional regulatory network of the DDR in mycobacteria and provide new insight into the regulatory mechanisms controlling the genesis of antibiotic resistance in M. tuberculosis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacillus subtilis has five type I signal peptidases, one of these, SipW, is an archaeal-like peptidase. SipW is expressed in an operon (tapA-sipW-tasA) and is responsible for removing the signal peptide from two proteins: TapA and TasA. It is unclear from the signal peptide sequence of TasA and TapA, why an archaeal-like signal peptidase is required for their processing. Bioinformatic analysis of TasA and TapA indicates that both contain highly similar signal peptide cleavage sites, both predicted to be cleaved by Escherichia coli signal peptidase I, LepB. We show that expressing full length TasA in E. coli is toxic and leads to cell death. To determine if this phenotype is due to the inability of the E. coli LepB to process the TasA signal peptide, we fused the TasA signal peptide and two amino acids of mature TasA (up to P2') to both maltose binding protein (MBP) and β-lactamase (Bla). We observed a defect in secretion, indicated by an abundance of unprocessed protein with both TasA-MBP and TasA-Bla fusions. A series of mutations in both TasA-MBP and TasA-Bla were made around the junction of the TasA signal peptide and the fusion protein. Both of these studies indicate that residues around the predicted TasA signal sequence cleavage site, particularly the sequence from P3 to P2', inhibit processing by LepB. The cell death observed when TasA and TasA signal sequence fusion proteins are expressed is likely due to the TasA signal peptide blocking LepB and thereby the general secretion pathway., (Copyright © 2021. Published by Elsevier B.V.)

High Temperature Requirement A (HtrA) was identified as a secreted virulence factor in many pathogenic bacteria, including Listeria monocytogenes. Recently, it was discovered that Helicobacter pylori and Campylobacter jejuni HtrAs can directly cleave the human cell-adhesion molecule E-cadherin, which facilitates bacterial transmigration. HtrAs also interact with extracellular matrix (ECM) molecules. However, only a limited number of studies have been carried out in this regard. In the present study, the protease and ECM binding properties of L. monocytogenes HtrA (LmHtrA) were studied using native rLmHtrA, catalytically inactive rLmHtrA(S343A) and rLmHtrA lacking the PDZ domain (∆PDZ) to gain more insights into HtrA-ECM molecule interaction. The results show that (1) native rLmHtrA cleaves fibrinogen, fibronectin, plasminogen and casein in a time and temperature dependent manner, (2) interaction of rLmHtrA with various host proteins was found in the micromolar to nanomolar range, (3) in the absence of PDZ domain, rLmHtrA exhibits no drastic change in binding affinity toward the host molecules when compared with native rLmHtrA and (4) the PDZ domain plays an important role in the substrate cleavage as rLmHtrA1-394∆PDZ cleaves the substrates only under certain conditions. The proteolysis of various ECM molecules by rLmHtrA possibly highlights the role of HtrA in L. monocytogenes pathogenesis involving ECM degradation., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Transcriptional repressor, LexA, regulates the 'SOS' response, an indispensable bacterial DNA damage repair machinery. Compared with its Escherichia coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA-binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in 'SOS' regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA-DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different 'SOS' boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, the present study provides insights into the kinetics of the interaction between Mtb LexA and its target 'SOS' boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of 'SOS' regulation and activation in Mtb., (© 2021 The Author(s).)

Lesions to DNA compromise chromosome integrity, posing a direct threat to cell survival. The bacterial SOS response is a widespread transcriptional regulatory mechanism to address DNA damage. This response is coordinated by the LexA transcriptional repressor, which controls genes involved in DNA repair, mutagenesis and cell-cycle control. To date, the SOS response has been characterized in most major bacterial groups, with the notable exception of the Bacteroidetes. No LexA homologs had been identified in this large, diverse and ecologically important phylum, suggesting that it lacked an inducible mechanism to address DNA damage. Here, we report the identification of a novel family of transcriptional repressors in the Bacteroidetes that orchestrate a canonical response to DNA damage in this phylum. These proteins belong to the S24 peptidase family, but are structurally different from LexA. Their N-terminal domain is most closely related to CI-type bacteriophage repressors, suggesting that they may have originated from phage lytic phase repressors. Given their role as SOS regulators, however, we propose to designate them as non-canonical LexA proteins. The identification of a new class of repressors orchestrating the SOS response illuminates long-standing questions regarding the origin and plasticity of this transcriptional network., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Lasso peptides are members of the natural product superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we describe the first lasso peptide originating from a biosynthetic gene cluster belonging to a unique lasso peptide subclade defined by the presence of a bifunctional protein harboring both a leader peptidase (B2) and an ABC transporter (D) domain. Bioinformatic analysis revealed that these clusters also encode homologues of the NisR/NisK regulatory system and the NisF/NisE/NisG immunity factors, which are usually associated with the clusters of antimicrobial class I lanthipeptides, such as nisin, another distinct RiPP subfamily. The cluster enabling the heterologous production of the lasso peptide cochonodin I in E. coli originated from Streptococcus suis LSS65, and the threaded structure of cochonodin I was evidenced through extensive MS/MS analysis and stability assays. It was shown that the ABC transporter domain from SsuB2/D is not essential for lasso peptide maturation. By extensive genome mining dedicated exclusively to other lasso peptide biosynthetic gene clusters featuring bifunctional B2/D proteins, it was furthermore revealed that many bacteria associated with human or animal microbiota hold the biosynthetic potential to produce cochonodin-like lasso peptides, implying that these natural products might play roles in human and animal health.

Porphyromonas gingivalis is a keystone pathogen of the human dysbiotic oral microbiome that causes severe periodontitis. It employs a type-IX secretion system (T9SS) to shuttle proteins across the outer membrane (OM) for virulence. Uniquely, T9SS cargoes carry a C-terminal domain (CTD) as a secretion signal, which is cleaved and replaced with anionic lipopolysaccharide by transpeptidation for extracellular anchorage to the OM. Both reactions are carried out by PorU, the only known dual-function, C-terminal signal peptidase and sortase. PorU is itself secreted by the T9SS, but its CTD is not removed; instead, intact PorU combines with PorQ, PorV, and PorZ in the OM-inserted "attachment complex." Herein, we revealed that PorU transits between active monomers and latent dimers and solved the crystal structure of the ∼260-kDa dimer. PorU has an elongated shape ∼130 Å in length and consists of seven domains. The first three form an intertwined N-terminal cluster likely engaged in substrate binding. They are followed by a gingipain-type catalytic domain (CD), two immunoglobulin-like domains (IGL), and the CTD. In the first IGL, a long "latency β-hairpin" protrudes ∼30 Å from the surface to form an intermolecular β-barrel with β-strands from the symmetric CD, which is in a latent conformation. Homology modeling of the competent CD followed by in vivo validation through a cohort of mutant strains revealed that PorU is transported and functions as a monomer through a C 690 /H 657 catalytic dyad. Thus, dimerization is an intermolecular mechanism for PorU regulation to prevent untimely activity until joining the attachment complex., Competing Interests: The authors declare no competing interest.

The bacterial SOS response represents a paradigm of gene networks controlled by a master transcriptional regulator. Self-cleavage of the SOS repressor LexA induces a wide range of cell functions that are critical for survival and adaptation when bacteria experience stress conditions 1 including DNA repair 2 , mutagenesis 3,4 , horizontal gene transfer 5-7 , filamentous growth and the induction of bacterial toxins 8-12 , toxin-antitoxin systems 13 , virulence factors 6,14 and prophages 15-17 . SOS induction is also implicated in biofilm formation and antibiotic persistence 11,18-20 . Considering the fitness burden of these functions, it is surprising that the expression of LexA-regulated genes is highly variable across cells 10,21-23 and that cell subpopulations induce the SOS response spontaneously even in the absence of stress exposure 9,11,12,16,24,25 . Whether this reflects a population survival strategy or a regulatory inaccuracy is unclear, as are the mechanisms underlying SOS heterogeneity. Here, we developed a single-molecule imaging approach based on a HaloTag fusion to directly monitor LexA in live Escherichia coli cells, demonstrating the existence of three main states of LexA: DNA-bound stationary molecules, free LexA and degraded LexA species. These analyses elucidate the mechanisms by which DNA binding and degradation of LexA regulate the SOS response in vivo. We show that self-cleavage of LexA occurs frequently throughout the population during unperturbed growth, rather than being restricted to a subpopulation of cells. This causes substantial cell-to-cell variation in LexA abundances. LexA variability underlies SOS gene-expression heterogeneity and triggers spontaneous SOS pulses, which enhance bacterial survival in anticipation of stress., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

SOS response is a conserved response to DNA damage in prokaryotes and is negatively regulated by LexA protein, which recognizes specifically an "SOS-box" motif present in the promoter region of SOS genes. Myxococcus xanthus DK1622 possesses a lexA gene, and while the deletion of lexA had no significant effect on either bacterial morphology, UV-C resistance, or sporulation, it did delay growth. UV-C radiation resulted in 651 upregulated genes in M. xanthus , including the typical SOS genes lexA , recA , uvrA , recN and so on, mostly enriched in the pathways of DNA replication and repair, secondary metabolism, and signal transduction. The UV-irradiated lexA mutant also showed the induced expression of SOS genes and these SOS genes enriched into a similar pathway profile to that of wild-type strain. Without irradiation treatment, the absence of LexA enhanced the expression of 122 genes that were not enriched in any pathway. Further analysis of the promoter sequence revealed that in the 122 genes, only the promoters of recA2 , lexA and an operon composed of three genes ( pafB , pafC and cyaA ) had SOS box sequence to which the LexA protein is bound directly. These results update our current understanding of SOS response in M. xanthus and show that UV induces more genes involved in secondary metabolism and signal transduction in addition to DNA replication and repair; and while the canonical LexA-dependent regulation on SOS response has shrunk, only 5 SOS genes are directly repressed by LexA.

Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP-seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP-seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Glutamic endopeptidases (Glu), belonging to the class of serine proteases, are a subfamily of chymotrypsin-like proteolytic enzymes, which are regarded as important virulence factors in bacteria. However, the roles of glutamic endopeptidases of Nocardia seriolae in pathogenic process still remain uncertain. Here, a glutamic endopeptidase homolog from N. seriolae (GluNS) was cloned and its function was elucidated. GluNS encoded a 414-aa protein which shared 93% identity to N. concava. In the phylogenetic tree, the glutamic endopeptidases of genus Nocardia clustered together firstly and then clustered with Streptomyces species. Moreover, GluNS was identified to be a secreted protein of N. seriolae and localized in the mitochondria of FHM cells. The transient overexpression of GluNS significantly induced increase in caspase-3 activity and decrease in ΔΨm values in FHM cells. The number of apoptotic bodies was remarkably higher than that in control group. Taken together, GluNS overexpression induced apoptotic characteristics in FHM cells. This study provided new insights into the function of glutamic endopeptidase from N. seriolae., (© 2020 John Wiley & Sons Ltd.)

Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double-ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen Pseudomonas aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP1 7 P2 7 tetradecamer that is required for proper biofilm development. PaClpP1 14 and PaClpP1 7 P2 7 complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non-canonical features in its N- and C-terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late-stage P. aeruginosa development., (© 2020 John Wiley & Sons Ltd.)

The multidrug-resistant pathogen Acinetobacter baumannii displays unusual control of its SOS mutagenesis genes, as it does not encode a LexA repressor, but instead employs the UmuDAb repressor and a small protein, DdrR, that is uniquely found in Acinetobacter species. We used bacterial adenylate cyclase two-hybrid analyses to determine if UmuDAb and DdrR coregulation might involve physical interactions. Neither quantitative nor qualitative assays showed UmuDAb interaction with DdrR. DdrR hybrid proteins, however, demonstrated modest head-to-tail interactions in a qualitative assay. The similarity of UmuDAb to the homodimer-forming polymerase manager UmuD and LexA repressor proteins suggested that it may form dimers, which we observed. UmuDAb homodimerization required a free C terminus, and either small truncations or addition of a histidine tag at the C terminus abolished this homodimerization. The amino acid N100, crucial for UmuD dimer formation, was dispensable if both C termini were free to interact. However, mutation of the amino acid G124, necessary for LexA dimerization, yielded significantly less UmuDAb dimerization, even if both C termini were free. This suggests that UmuDAb forms dimers like LexA does, but may not coregulate gene expression involving a physical association with DdrR. The homodimerization of these coregulators provides insight into a LexA-independent, coregulatory process of controlling a conserved bacterial action such as the mutagenic DNA damage response.

Compartmentalization of single genes in water-in-oil emulsion droplets is a powerful approach to create millions of reactors for enzyme library selections. When these droplets are formed at ultrahigh throughput in microfluidic devices, their perfect monodispersity allows quantitative enzyme assays with a high precision readout. However, despite its potential for high quality cell-free screening experiments, previous demonstrations of enrichment have never been successfully followed up by actual enzyme library selections in monodisperse microfluidic droplets. Here we develop a three-step workflow separating three previously incompatible steps that thus far could not be carried out at once: first droplet-compartmentalized DNA is amplified by rolling circle amplification; only after completion of this step are reagents for in vitro protein expression and, finally, substrate added via picoinjection. The segmented workflow is robust enough to allow the first in vitro evolution in droplets, improving the protease Savinase that is toxic to E. coli for higher activity and identifying a 5-fold faster enzyme.

The use of plant growth-promoting bacteria represents an alternative to the massive use of mineral fertilizers in agriculture. However, some abiotic stresses commonly found in the environment, like salinity, can affect the efficiency of this approach. Here, we investigated the key mechanisms involved in the response of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus to salt stress by using morphological and cell viability analyses, comparative proteomics, and reverse genetics. Our results revealed that the bacteria produce filamentous cells in response to salt at 100 mM and 150 mM NaCl. However, such a response was not observed at higher concentrations, where cell viability was severely affected. Proteomic analysis showed that salt stress modulates proteins involved in several pathways, including iron uptake, outer membrane efflux, osmotic adjustment, cell division and elongation, and protein transport and quality control. Proteomic data also revealed the repression of several extracytoplasmic proteins, especially those located at periplasm and outer membrane. The role of such pathways in the tolerance to salt stress was analyzed by the use of mutant defectives for Δtbdr (iron uptake), ΔmtlK and ΔotsA (compatible solutes synthesis), and ΔdegP (quality control of nascent extracytoplasmic proteins). ΔdegP presented the highest sensitivity to salt stress, Δtbdr, andΔmtlK also showed increased sensitivity, but ΔotsA was not affected. This is the first demonstration that DegP protein, a protease with minor chaperone activity, is essential for tolerance to salt stress in G. diazotrophicus. Our data contribute to a better understanding of the molecular bases that control the bacterial response/tolerance to salt stress, shedding light on quality control of nascent extracytoplasmic proteins., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

In addition to encoding the tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determine the speed of folding. How these kinetic barriers are encoded is not well understood. Here, we use evolutionary sequence variation in the α-lytic protease (αLP) protein family to probe the relationship between sequence and energy landscape. αLP has an unusual energy landscape: the native state of αLP is not the most thermodynamically favored conformation and, instead, remains folded due to a large kinetic barrier preventing unfolding. To fold, αLP utilizes an N-terminal pro region similar in size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant time scale. Without the pro region, αLP folds on the order of millennia. A phylogenetic search uncovers αLP homologs with a wide range of pro region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro region size. By studying homologs naturally lacking a pro region, we demonstrate they can be thermodynamically stable, fold much faster than αLP, yet retain the same fold as αLP. Key amino acids thought to contribute to αLP's extreme kinetic stability are lost in these homologs, supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on the protein sequence.

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx by forming multicellular biofilms. Due to the high level of asymptomatic carriage, transition to infections, such as otitis media, pneumonia, sepsis, and meningitis, occurs often enough that the pneumococcus remains a major cause of disease and death globally. Virus infection and virus-induced responses, such as increased temperature (fever), trigger release of virulent bacteria from colonizing biofilms. The exact mechanisms involved in pneumococcal egress during biofilm dispersal remain unknown, although we hypothesize that disruption of the biofilm matrix encasing the bacteria is necessary. Here, we utilized established in vitro biofilm dispersal models to investigate the involvement of proteases in bacterial egress from pneumococcal biofilms. We demonstrate the importance of protease activity, both through increased bacterial release following addition of proteases and reduced heat-induced biofilm dispersal in the presence of protease inhibitors. We identify a key role for the surface-exposed serine protease HtrA, but not PrtA, in heat-induced biofilm dispersal. Bacterial release from htrA-negative biofilms was significantly reduced compared to wild-type isogenic strains but was restored and increased above wild-type levels following addition of recombinant HtrA. Understanding the specific mechanisms involved in bacterial egress may provide novel targets for future strategies aimed to specifically interfere with disease progression without disturbing nasopharyngeal biofilm colonization.

The membrane protease SppA of Bacillus subtilis was first described as a signal peptide peptidase and later shown to confer resistance to lantibiotics. Here, we report that SppA forms octameric complexes with YteJ, a membrane protein of thus-far-unknown function. Interestingly, sppA and yteJ deletion mutants exhibited no protein secretion defects. However, these mutant strains differed significantly in their resistance to antimicrobial peptides. In particular, sppA mutant cells displayed increased sensitivity to the lantibiotics nisin and subtilin and the human lysozyme-derived cationic antimicrobial peptide LP9. Importantly, YteJ was shown to antagonize SppA activity both in vivo and in vitro , and this SppA-inhibitory activity involved the C-terminal domain of YteJ, which was therefore renamed SppI. Most likely, SppI-mediated control is needed to protect B. subtilis against the potentially detrimental protease activity of SppA since a mutant overexpressing sppA by itself displayed defects in cell division. Altogether, we conclude that the SppA-SppI complex of B. subtilis has a major role in protection against antimicrobial peptides. IMPORTANCE Our study presents new insights into the molecular mechanism that regulates the activity of SppA, a widely conserved bacterial membrane protease. We show that the membrane proteins SppA and SppI form a complex in the Gram-positive model bacterium B. subtilis and that SppI inhibits SppA protease activity in vitro and in vivo Furthermore, we demonstrate that the C-terminal domain of SppI is involved in SppA inhibition. Since SppA, through its protease activity, contributes directly to resistance to lantibiotic peptides and cationic antibacterial peptides, we propose that the conserved SppA-SppI complex could play a major role in the evasion of bactericidal peptides, including those produced as part of human innate immune defenses., (Copyright © 2020 Henriques et al.)

Chlamydia trachomatis is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, the elementary body (EB) and reticulate body (RB), each of which expresses its own specialized repertoire of proteins. Both primary (EB to RB) and secondary (RB to EB) differentiations require protein turnover, and we hypothesize that proteases are critical for mediating differentiation. The Clp protease system is well conserved in bacteria and important for protein turnover. Minimally, the system relies on a serine protease subunit, ClpP, and an AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia , ClpX is encoded within an operon 3' to clpP2 We present evidence that the chlamydial ClpX and ClpP2 orthologs are essential to organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability with observable defects in EB morphology noted. Conversely, overexpression of a catalytically inactive ClpP2 mutant significantly impacted developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in trans by a plasmid copy of clpP2 Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation. IMPORTANCE Chlamydia trachomatis is the leading cause of infectious blindness globally and the most reported bacterial sexually transmitted infection both domestically and internationally. Given the economic burden, the lack of an approved vaccine, and the use of broad-spectrum antibiotics for treatment of infections, an understanding of chlamydial growth and development is critical for the advancement of novel targeted antibiotics. The Clp proteins comprise an important and conserved protease system in bacteria. Our work highlights the importance of the chlamydial Clp proteins to this clinically important bacterium. Additionally, our study implicates the Clp system playing an integral role in chlamydial developmental cycle progression, which may help establish models of how Chlamydia spp. and other bacteria progress through their respective developmental cycles. Our work also contributes to a growing body of Clp-specific research that underscores the importance and versatility of this system throughout bacterial evolution and further validates Clp proteins as drug targets., (Copyright © 2020 Wood et al.)

Feedback mechanisms are fundamental to the control of physiological responses. One important example in gene regulation, termed negative autoregulation (NAR), occurs when a transcription factor (TF) inhibits its own production through transcriptional repression. This enables more-rapid homeostatic control of gene expression. NAR circuits presumably evolve to limit the fitness costs of gratuitous gene expression. The key biochemical reactions of NAR can be parameterized using a mathematical model of promoter activity; however, this model of NAR has been studied mostly in the context of synthetic NAR circuits that are disconnected from the target genes of the TFs. Thus, it remains unclear how constrained NAR parameters are in a native circuit context, where the TF target genes can have fitness effects on the cell. To quantify these constraints, we created a panel of Escherichia coli strains with different lexA- NAR circuit parameters and analyzed the effect on SOS response function and bacterial fitness. Using a mathematical model for NAR, these experimental data were used to calculate NAR parameter values and derive a parameter-fitness landscape. Without feedback, survival of DNA damage was decreased due to high LexA concentrations and slower SOS "turn-on" kinetics. However, we show that, even in the absence of DNA damage, the lexA promoter is strong enough that, without feedback, high levels of lexA expression result in a fitness cost to the cell. Conversely, hyperfeedback can mimic lexA deletion, which is also costly. This work elucidates the lexA -NAR parameter values capable of balancing the cell's requirement for rapid SOS response activation with limiting its toxicity. IMPORTANCE Feedback mechanisms are critical to control physiological responses. In gene regulation, one important example, termed negative autoregulation (NAR), occurs when a transcription factor (TF) inhibits its own production. NAR is common across the tree of life, enabling rapid homeostatic control of gene expression. NAR behavior can be described in accordance with its core biochemical parameters, but how constrained these parameters are by evolution is unclear. Here, we describe a model genetic network controlled by an NAR circuit within the bacterium Escherichia coli and elucidate these constraints by experimentally changing a key parameter and measuring its effect on circuit response and fitness. This analysis yielded a parameter-fitness landscape representing the genetic network, providing a window into what gene-environment conditions favor evolution of this regulatory strategy., (Copyright © 2020 Kozuch et al.)

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro , and that the Δ fakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla Δ fakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the Δ fakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the Δ fakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the Δ fakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in Δ fakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δ aur and Δ sspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the Δ fakA mutant during skin infection., (Copyright © 2020 American Society for Microbiology.)

The human pathogen Mycobacterium tuberculosis (Mtb) harbors a well-orchestrated Clp (caseinolytic protease) proteolytic machinery consisting of two oligomeric segments, a barrel-shaped heterotetradecameric protease core comprising the ClpP1 and ClpP2 subunits, and hexameric ring-like ATP-dependent unfoldases composed of ClpX or ClpC1. The roles of the ClpP1P2 protease subunits are well-established in Mtb, but the potential roles of the associated unfoldases, such as ClpC1, remain elusive. Using a CRISPR interference-mediated gene silencing approach, here we demonstrate that clpC1 is indispensable for the extracellular growth of Mtb and for its survival in macrophages. The results from isobaric tags for relative and absolute quantitation-based quantitative proteomic experiments with clpC1 - and clpP2 -depleted Mtb cells suggested that the ClpC1P1P2 complex critically maintains the homeostasis of various growth-essential proteins in Mtb, several of which contain intrinsically disordered regions at their termini. We show that the Clp machinery regulates dosage-sensitive proteins such as the small heat shock protein Hsp20, which exists in a dodecameric conformation. Further, we observed that Hsp20 is poorly expressed in WT Mtb and that its expression is greatly induced upon depletion of clpC1 or clpP2 Remarkably, high Hsp20 protein levels were detected in the clpC1( - ) or clpP2( - ) knockdown strains but not in the parental bacteria, despite significant induction of hsp20 transcripts. In summary, the cellular levels of oligomeric proteins such as Hsp20 are maintained post-translationally through their recognition, disassembly, and degradation by ClpC1, which requires disordered ends in its protein substrates., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Lunge et al.)

Mycobacterium smegmatis MSMEG_0129 and Rv0164, its homologue in Mycobacterium tuberculosis, are single START-domain proteins essential for bacterial growth and survival, but their biochemical activities and biological roles remain undetermined. Here, we probed the possible functions of MSMEG_0129 and its underlying mechanisms by determining its cellular location, searching for its interaction partners and monitoring its transcription profile. MSMEG_0129, and Rv0164 by extension, were found to be cytosolic proteins rather than secreted components as previously understood. Increases in MSMEG_0129 expression at physiological levels accelerated bacterial growth in a proportional manner, but additional growth acceleration was not observed when MSMEG_0129 was overexpressed up to 20 fold. MSMEG_0129 is a short-lived protein, unstable at both the mRNA and protein levels. Co-IP and GST pull-down assays showed that MSMEG_0129 interacts with the ClpP2 protease and a global transcription factor, CarD, their expression being correlated with that of MSMEG_0129. Nutrient deficiency led to the downregulation of MSMEG_0129 but upregulation of CarD. However, in the context of constitutive MSMEG_0129 overexpression under nutrient-rich or starvation conditions, the mRNA level of CarD was reduced 3 fold. Conversely, expression of ClpP2 decreased with MSMEG_0129 downregulation under starvation conditions, but increased 4-8 fold when MSMEG_0129 was overexpressed. Our data suggest that MSMEG_0129, and Rv0164 by analogy, are likely to be nutrition sensing factors that regulate mycobacterial growth and may be involved in signal transfer under nutrient deficiency, possibly via physical and regulatory interactions with CarD and ClpP2., Competing Interests: Declaration of Competing Interest The authors declare there are no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The bacterial DNA damage response (the SOS response) is a key pathway involved in antibiotic evasion and a promising target for combating acquired antibiotic resistance. Activation of the SOS response is controlled by two proteins: the repressor LexA and the DNA damage sensor RecA. Following DNA damage, direct interaction between RecA and LexA leads to derepression of the SOS response. However, the exact molecular details of this interaction remain unknown. Here, we employ the fluorescent unnatural amino acid acridonylalanine (Acd) as a minimally perturbing probe of the E. coli RecA:LexA complex. Using LexA labeled with Acd, we report the first kinetic model for the reversible binding of LexA to activated RecA. We also characterize the effects that specific amino acid truncations or substitutions in LexA have on RecA:LexA binding strength and demonstrate that a mobile loop encoding LexA residues 75-84 comprises a key recognition interface for RecA. Beyond insights into SOS activation, our approach also further establishes Acd as a sensitive fluorescent probe for investigating the dynamics of protein-protein interactions in other complex systems.

The aim of this study was to investigate whether HtrA is responsible for differences in adherence and invasion of Campylobacter jejuni towards human and chicken cell lines. Gentamicin protection assays were performed with either human Caco-2 or chicken 2G4 cells using C. jejuni strain NCTC11168 to compare the adhesion and invasion rates towards these two cell types. The results revealed significant differences in the adhesion and invasion rates between the human and avian cells. Deletion of the Campylobacter htrA gene, coding for the dual function of serine protease and chaperonin with a role in pathogenesis, led to a reduction of the rates in both cell lines. Using a single-amino acid substitution mutant (ΔhtrA/htrA S197A ) that lacked protease activity, but retained chaperonin activity, we show that the first is involved in the invasion of human Caco-2 and chicken 2G4 cells, whereas the latter mutant invaded at lower levels. Adherence towards the chicken cells is higher than towards Caco-2 cells and this is also dependent on HtrA. Together, these data suggest that the proteolytic activity of HtrA is involved in the difference in host response of C. jejuni towards human and chicken-derived cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter jejuni is the main cause for bacterial foodborne enterocolitis worldwide. While colonization of the human intestine can lead to severe problems, avian hosts - as the major source of infection - remain unaffected by the bacteria. We showed that the bacterial serine protease and chaperonin HtrA are involved in adhesion and invasion in both species and not responsible for the discrepancy of virulence between the different hosts. In future, HtrA might act as a target for inhibitors to avoid or eradicate colonization in chickens as a less problematic alternative to antibiotics in commercial livestock breeding., (© 2020 The Society for Applied Microbiology.)

The DNA damage response of the multidrug-resistant pathogen Acinetobacter baumannii , which induces mutagenic UmuD' 2 C error-prone polymerases, differs from that of many bacteria. Acinetobacter species lack a LexA repressor, but induce gene transcription after DNA damage. One regulator, UmuDAb, binds to and represses the promoters of the multiple A. baumannii ATCC 17978 umuDC alleles and the divergently transcribed umuDAb and ddrR genes. ddrR is unique to the genus Acinetobacter and of unknown function. 5' RACE (rapid amplification of cDNA ends) PCR mapping of the umuDAb and ddrR transcriptional start sites revealed that their -35 promoter elements overlapped the UmuDAb binding site, suggesting that UmuDAb simultaneously repressed expression of both genes by blocking polymerase access. This coordinated control of ddrR and umuDAb suggested that ddrR might also regulate DNA damage-inducible gene transcription. RNA-sequencing experiments in 17 978 ddrR - cells showed that ddrR regulated approximately 25 % ( n =39) of the mitomycin C-induced regulon, with umuDAb coregulating 17 of these ddrR -regulated genes. Eight genes (the umuDC polymerases, umuDAb and ddrR ) were de-repressed in the absence of DNA damage, and nine genes were uninduced in the presence of DNA damage, in both ddrR and umuDAb mutant strains. These data suggest ddrR has multiple roles, both as a co-repressor and as a positive regulator of DNA damage-inducible gene transcription. Additionally, 57 genes were induced by mitomycin C in the ddrR mutant but not in wild-type cells. This regulon contained multiple genes for DNA replication, recombination and repair, transcriptional regulators, RND efflux, and transport. This study uncovered another regulator of the atypical DNA damage response of this genus, to help describe how this pathogen acquires drug resistance through its expression of the error-prone polymerases under DdrR and UmuDAb control.

Aims: LexA protein is a transcriptional repressor which regulates the expression of more than 60 genes belonging to the SOS global regulatory network activated by damages to bacterial DNA. Considering its role in bacteria, LexA represents a key target to counteract bacterial resistance: the possibility to modulate SOS response through the inhibition of LexA autoproteolysis may lead to reduced drug susceptibility and acquisition of resistance in bacteria. In our study we investigated boron-containing compounds as potential inhibitors of LexA self-cleavage., Main Methods: The inhibition of LexA self-cleavage was evaluated by following the variation of the first-order rate constant by LC-MS at several concentrations of inhibitors. In silico analysis was applied to predict the binding orientations assumed by the inhibitors in the protein active site, upon covalent binding to the catalytic Ser-119. Bacterial filamentation assay was used to confirm the ability of (3-aminophenyl)boronic acid to interfere with SOS induced activation., Key Findings: Boron-containing compounds act as inhibitors of LexA self-cleavage, as also confirmed by molecular modelling where the compounds interact with the catalytic Ser-119, via the formation of an acyl-enzyme intermediate. A new equation for the description of the inhibition potency in an autoproteolytic enzyme is also disclosed. Bacterial filamentation assays strongly support the interference of our compounds with the SOS response activation through inhibition of septum formation., Significance: The obtained results demonstrated that phenylboronic compounds could be exploited in a hit-to-lead optimization process toward effective LexA self-cleavage inhibitors. They would sustain the rehabilitation in therapy of several dismissed antibiotics., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fat separation is a limiting factor for the shelf life of UHT milk. It may be promoted by the proteolysis of fat surface-adsorbed proteins (FSAP) by proteases that remain active after UHT treatment. The aim of this research was to explore the relationship between the proteolysis of FSAP and fat destabilization. In this study, we developed a full-fat UHT milk-based model system and added either the major bacterial protease AprX from Pseudomonas fluorescens or the major native milk protease plasmin at high levels to induce fast destabilization of the milk fat globules. We monitored changes in physical properties and FSAP composition, and structural changes in fat globules, over 24 h. Our results showed that AprX-induced sedimentation as a result of the flocculation of fat globules, and plasmin induced cream to float as a result of the coalescence of fat globules. This study confirmed that AprX and plasmin can both lead to fat destabilization in full-fat UHT milk, and it provides insights in the underlying mechanisms., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Filamentous hemagglutinin (FHA) is a critically important virulence factor produced by Bordetella species that cause respiratory infections in humans and other animals. It is also a prototypical member of the widespread two partner secretion (TPS) pathway family of proteins. First synthesized as a ~370 kDa protein called FhaB, its C-terminal ~1,200 amino acid 'prodomain' is removed during translocation to the cell surface via the outer membrane channel FhaC. Here, we identify CtpA as a periplasmic protease that is responsible for the regulated degradation of the prodomain and for creation of an intermediate polypeptide that is cleaved by the autotransporter protease SphB1 to generate FHA. We show that the central prodomain region is required to initiate degradation of the prodomain and that CtpA degrades the prodomain after a third, unidentified protease (P3) first removes the extreme C-terminus of the prodomain. Stepwise proteolysis by P3, CtpA and SphB1 is required for maturation of FhaB, release of FHA into the extracellular milieu, and full function in vivo. These data support a substantially updated model for the mechanism of secretion, maturation and function of this model TPS protein., (© 2019 John Wiley & Sons Ltd.)

Objective: Dichelobacter nodosus is the primary aetiological agent of footrot in sheep. Ovine footrot causes considerable economic losses and substantial animal welfare issues in the Australian sheep industry. Current methods for detecting D. nodosus are difficult, laborious and time-consuming. Recently, we developed a robust LAMP assay (VDN LAMP) that was able to identify aprV2 positive D. nodosus in-field. A major advantage of LAMP technology is the ability of the assay to be performed by non-specialists with minimal training. We aimed to assess the performance of the VDN LAMP in-field in comparison to a laboratory-based aprV2/aprB2 rtPCR when used by secondary users after training by the authors., Results: Two animal health officers (termed secondary users) from Department of Primary Industries and Regions, South Australia (PIRSA) were trained in the use of VDN LAMP, before carrying out in-field testing on several locations in South Australia. The performance of VDN LAMP assay by secondary user 1 was shown to successfully detect 73.91% (n = 53) aprV2 positive samples, while secondary user 2 detected 37.93% (n = 30) aprV2 positive samples. Overall, the ability to identify virulent D. nodosus by VDN LAMP by secondary users was mixed for various reasons, however, this could be rectified by additional training and commercial production of the LAMP kits to increase stability. We envisaged in the future VDN LAMP will able to be used by non-specialists to aid control programs.

Tick-borne relapsing fever (TBRF), characterized by recurring febrile episodes, is globally distributed and among the most common bacterial infections in some African countries. Despite the public health concern that this disease represents, little is known regarding the virulence determinants required by TBRF Borrelia during infection. Because the chromosomes of TBRF Borrelia show extensive colinearity with those of Lyme disease (LD) Borrelia , the exceptions represent unique genes encoding proteins that are potentially essential to the disparate enzootic cycles of these two groups of spirochetes. One such exception is a gene encoding an HtrA family protease, BtpA, that is present in TBRF Borrelia , but not in LD spirochetes. Previous work suggested that btpA orthologs may be important for resistance to stresses faced during mammalian infection. Herein, proteomic analyses of the TBRF spirochete, Borrelia turicatae , demonstrated that BtpA, as well as proteins encoded by adjacent genes in the B. turicatae genome, were produced in response to culture at mammalian body temperature, suggesting a role in mammalian infection. Further, transcriptional analyses revealed that btpA was expressed with the genes immediately upstream and downstream as part of an operon. To directly assess if btpA is involved in resistance to environmental stresses, btpA deletion mutants were generated. btpA mutants demonstrated no growth defect in response to heat shock, but were more sensitive to oxidative stress produced by t- butyl peroxide compared to wild-type B. turicatae . Finally, btpA mutants were fully infectious in a murine relapsing fever (RF) infection model. These results indicate that BtpA is either not required for mammalian infection, or that compensatory mechanisms exist in TBRF spirochetes to combat environmental stresses encountered during mammalian infection in the absence of BtpA.

Binary expression systems like the LexA-LexAop system provide a powerful experimental tool kit to study gene and tissue function in developmental biology, neurobiology, and physiology. However, the number of well-defined LexA enhancer trap insertions remains limited. In this study, we present the molecular characterization and initial tissue expression analysis of nearly 100 novel StanEx LexA enhancer traps, derived from the StanEx 1 index line. This includes 76 insertions into novel, distinct gene loci not previously associated with enhancer traps or targeted LexA constructs. Additionally, our studies revealed evidence for selective transposase-dependent replacement of a previously-undetected KP element on chromosome III within the StanEx 1 genetic background during hybrid dysgenesis, suggesting a molecular basis for the over-representation of LexA insertions at the NK7.1 locus in our screen. Production and characterization of novel fly lines were performed by students and teachers in experiment-based genetics classes within a geographically diverse network of public and independent high schools. Thus, unique partnerships between secondary schools and university-based programs have produced and characterized novel genetic and molecular resources in Drosophila for open-source distribution, and provide paradigms for development of science education through experience-based pedagogy., (Copyright © 2019 Kockel et al.)

Bacteria identify and respond to DNA damage using the SOS response. LexA, a central repressor in the response, has been implicated in the regulation of lysogeny in various temperate bacteriophages. During infection of Bacillus thuringiensis with GIL01 bacteriophage, LexA represses the SOS response and the phage lytic cycle by binding DNA, an interaction further stabilized upon binding of a viral protein, gp7. Here we report the crystallographic structure of phage-borne gp7 at 1.7-Å resolution, and characterize the 4:2 stoichiometry and potential interaction with LexA using surface plasmon resonance, static light scattering, and small-angle X-ray scattering. These data suggest that gp7 stabilizes LexA binding to operator DNA via coordination of the N- and C-terminal domains of LexA. Furthermore, we have found that gp7 can interact with LexA from Staphylococcus aureus, a significant human pathogen. Our results provide structural evidence as to how phage factors can directly associate with LexA to modulate the SOS response., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Plant innate immunity restricts growth of bacterial pathogens that threaten global food security. However, the mechanisms by which plant immunity suppresses bacterial growth remain enigmatic. Here we show that Arabidopsis thaliana secreted aspartic protease 1 and 2 (SAP1 and SAP2) cleave the evolutionarily conserved bacterial protein MucD to redundantly inhibit the growth of the bacterial pathogen Pseudomonas syringae. Antibacterial activity of SAP1 requires its protease activity in planta and in vitro. Plants overexpressing SAP1 exhibit enhanced MucD cleavage and resistance but incur no penalties in growth and reproduction, while sap1 sap2 double mutant plants exhibit compromised MucD cleavage and resistance against P. syringae. P. syringae lacking mucD shows compromised growth in planta and in vitro. Notably, growth of ΔmucD complemented with the non-cleavable MucD F106Y is not affected by SAP activity in planta and in vitro. Our findings identify the genetic factors and biochemical process underlying an antibacterial mechanism in plants.

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook-associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non-motile mutant cells that are unable to assemble flagellar filaments and pentagon-shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella-deficient mutants (i.e., in the hook, rod, or MS-ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm., (© 2019 John Wiley & Sons Ltd.)

Mycobacterium tuberculosis, the causative agent of tuberculosis, withstands diverse environmental stresses in the host. The periplasmic protease HtrA is required only to survive extreme conditions in most bacteria but is predicted to be essential for normal growth in mycobacteria. We confirm that HtrA is indeed essential in Mycobacterium smegmatis and interacts with another essential protein of unknown function, LppZ. However, the loss of any of three unlinked genes, including those encoding Ami3, a peptidoglycan muramidase, and Pmt, a mannosyltransferase, suppresses the essentiality of both HtrA and LppZ, indicating the functional relevance of these genes' protein products. Our data indicate that HtrA-LppZ is required to counteract the accumulation of active Ami3, which is toxic under the stabilizing influence of Pmt-based mannosylation. This suggests that HtrA-LppZ blocks the toxicity of a cell wall enzyme to maintain mycobacterial homeostasis., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Knowledge of the molecular mechanisms of specific bacterial virulence factors can significantly contribute to antibacterial drug discovery. Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects almost half of the world's population, leading to gastric disorders and even gastric cancer. H. pylori expresses a series of virulence factors in the host, among which high-temperature requirement A ( Hp HtrA) is a newly identified serine protease secreted by H. pylori. Hp HtrA cleaves the extracellular domain of the epithelial cell surface adhesion protein E-cadherin and disrupts gastric epithelial cell junctions, allowing H. pylori to access the intercellular space. Here we report the first crystal structure of Hp HtrA at 3.0 Å resolution. The structure revealed a new type of HtrA protease trimer stabilized by unique N-terminal domain swapping distinct from other known HtrA homologs. We further observed that truncation of the N terminus completely abrogates Hp HtrA trimer formation as well as protease activity. In the presence of unfolded substrate, Hp HtrA assembled into cage-like 12-mers or 24-mers. Combining crystallographic, biochemical, and mutagenic data, we propose a mechanistic model of how Hp HtrA recognizes and cleaves the well-folded E-cadherin substrate. Our study provides a fundamental basis for the development of anti- H. pylori agents by using a previously uncharacterized HtrA protease as a target., (© 2019 Zhang et al.)

Integrons recruit resistance genes through integrase-driven recombination events that are regulated by the bacterial SOS response and require the repressor LexA. Class 1 integrons genes are expressed from a common promoter, Pc, of which at least 5 predominant variants, classified from weak to strong, have been described. In Escherichia coli, there is an intertwined regulation between gene cassette expression and integrase activity: the stronger the promoter, the weaker the integrase. Class 1 integrons have been frequently described in Acinetobacter baumannii. However, Acinetobacter spp. lack the LexA repressor, suggesting that the integrase is constitutively expressed. We characterized the integron content of 83 clinical and environmental A. baumannii strains. We found a predominance of Pc variants described as strong in E. coli. The Pc expression level was 2- to 4-fold lower in A. baumannii than in E. coli, and the diversity of the gene cassette array was low. In A. baumannii, integrons with a PcS promoter might have been selected to enable sufficient resistance while avoiding the toxicity of a highly active integrase. Furthermore, a transcriptional interference between PcS and PintI1 (as shown in E. coli) may limit the expression of the integrase and thus counterbalance the lack of LexA-driven integrase repression to prevent the cost of the integrase., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The sapRH gene, which encodes the serine alkaline protease SAPRH, from Bacillus safensis RH12, was isolated and its DNA sequence was determined. The deduced amino-acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with SAPB from B. pumilus CBS, with only 9 amino-acids of difference. The region, encoding SAPRH was heterologously expressed in E. coli BL21-AI™ cells using GATEWAY™ pDEST™17 expression-vector. The recombinant (His) 6 -tag enzyme (His 6 -rSAPRH) was purified in a single affinity chromatography step and its biochemical properties were determined and compared to those of SAPRH and rSAPB. Interestingly, His 6 -rSAPRH showed improved thermostability compared to SAPRH and rSAPB. The molecular dynamics of SAPRH compared to SAPB revealed a more thermostable structure, thus confirming the in vitro results showing that His 6 -rSAPRH has a t 1/2 of 120 min against 90 and 30 min for SAPRH and rSAPB, respectively, at 70 °C and different kinetic parameters to synthetic peptides. The docking simulations data allow in getting an insight into the involvement of some key amino-acids in substrate binding and account for the selectivity. Overall, this is the first report of a sapRH gene cloned from B. safensis which can be a promising potential candidate for future applications in detergent formulations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Quorum sensing (QS) system is an important bacterial cell-to-cell signaling system controlling expression of various genes in response to cell densities. In vibrios, LuxR/AphA are two established master QS regulators (MQSRs), and VqsA is recently identified to be the third putative MQSR. As a novel LysR-type regulator, the regulon and the underlying regulation mechanisms of VqsA remains to be elucidated. Here our investigation indicated that the yields of alkaline serine protease (Asp), the exotoxin in Vibrio alginolyticus was dependent on both LuxR and VqsA in growth phase dependent manner. Various in vivo and in vitro analyses including electrophoretic mobility shift assays (EMSA) along with DNase I footprinting investigations demonstrated that VqsA positively controls asp expression through directly binding to the partially palindromic 29 bp binding motif in the promoter region of asp. Moreover, RNA-seq analysis validated the regulatory roles of VqsA in various processes in the organism. Collectively, our data showed that VqsA positively regulates the expression of exotoxin and other virulence-associated genes and is essential for the QS regulation in V. alginolyticus., (© FEMS 2019.)