Extraordinary long-stem confers resistance of intrinsic terminators to processive antitermination.

Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. This is the first P-AT resistance mechanism to be reported.

Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by monomeric Soj/ParA during growth and sporulation.

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.

A novel bipartite antitermination system widespread in conjugative elements of Gram-positive bacteria.

Transcriptional regulation allows adaptive and coordinated gene expression, and is essential for life. Processive antitermination systems alter the transcription elongation complex to allow the RNA polymerase to read through multiple terminators in an operon. Here, we describe the discovery of a novel bipartite antitermination system that is widespread among conjugative elements from Gram-positive bacteria, which we named conAn. This system is composed of a large RNA element that exerts antitermination, and a protein that functions as a processivity factor. Besides allowing coordinated expression of very long operons, we show that these systems allow differential expression of genes within an operon, and probably contribute to strict regulation of the conjugation genes by minimizing the effects of spurious transcription. Mechanistic features of the conAn system are likely to decisively influence its host range, with important implications for the spread of antibiotic resistance and virulence genes.

Reversible regulation of conjugation of Bacillus subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor RappLS20.

Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, Pc, is by default repressed by a regulator RcopLS20 involving DNA looping. A plasmid-encoded signalling peptide, Phr*pLS20, inactivates the anti-repressor of RcopLS20, named RappLS20, which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two RcopLS20 tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that RappLS20 is sufficient for activating conjugation in vivo. We also show that RappLS20 actively detaches RcopLS20 from DNA by preferentially acting on the RcopLS20 molecules involved in DNA looping, resulting in sequestration but not inactivation of RcopLS20. Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.

Cohesion of Sister Chromosome Termini during the Early Stages of Sporulation in Bacillus subtilis.

During sporulation of Bacillus subtilis, the cell cycle is reorganized to generate separated prespore and mother cell compartments, each containing a single fully replicated chromosome. The process begins with reorganization of the nucleoid to form an elongated structure, the axial filament, in which the two chromosome origins are attached to opposite cell poles, with the remainder of the DNA stretched between these sites. When the cell then divides asymmetrically, the division septum closes around the chromosome destined for the smaller prespore, trapping the origin-proximal third of the chromosome in the prespore. A translocation pore is assembled through which a DNA transporter, SpoIIIE/FtsK, transfers the bulk of the chromosome to complete the segregation process. Although the mechanisms involved in attaching origin regions to the cell poles are quite well understood, little is known about other aspects of axial filament morphology. We have studied the behavior of the terminus region of the chromosome during sporulation using time-lapse imaging of wild-type and mutant cells. The results suggest that the elongated structure involves cohesion of the terminus regions of the sister chromosomes and that this cohesion is resolved when the termini reach the asymmetric septum or translocation pore. Possible mechanisms and roles of cohesion and resolution are discussed.IMPORTANCE Endospore formation in Firmicutes bacteria provides one of the most highly resistant life forms on earth. During the early stages of endospore formation, the cell cycle is reorganized so that exactly two fully replicated chromosomes are generated, before the cell divides asymmetrically to generate the prespore and mother cell compartments that are critical for the developmental process. Decades ago, it was discovered that just prior to asymmetrical division the two chromosomes enter an unusual elongated configuration called the axial filament. This paper provides new insights into the nature of the axial filament structure and suggests that cohesion of the normally separated sister chromosome termini plays an important role in axial filament formation.

Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane.

To proliferate efficiently, cells must co-ordinate division with chromosome segregation. In Bacillus subtilis, the nucleoid occlusion protein Noc binds to specific DNA sequences (NBSs) scattered around the chromosome and helps to protect genomic integrity by coupling the initiation of division to the progression of chromosome replication and segregation. However, how it inhibits division has remained unclear. Here, we demonstrate that Noc associates with the cell membrane via an N-terminal amphipathic helix, which is necessary for function. Importantly, the membrane-binding affinity of this helix is weak and requires the assembly of nucleoprotein complexes, thus establishing a mechanism for DNA-dependent activation of Noc. Furthermore, division inhibition by Noc requires recruitment of NBS DNA to the cell membrane and is dependent on its ability to bind DNA and membrane simultaneously. Indeed, Noc production in a heterologous system is sufficient for recruitment of chromosomal DNA to the membrane. Our results suggest a simple model in which the formation of large membrane-associated nucleoprotein complexes physically occludes assembly of the division machinery.

Synergy of N-(3-oxohexanoyl)-L-homoserine lactone and tryptophan-like outer extracellular substances in granular sludge dominated by aerobic ammonia-oxidizing bacteria.

Nitrogen removal via nitrite is an energy-saving method for high-strength ammonia wastewater treatment. A better understanding of the formation of granular sludge dominated by aerobic ammonia-oxidizing bacteria (AerAOB) could facilitate the improved use of rapid sludge granulation for nitritation. In this study, AerAOB-dominated activated sludge (NAS) and granular sludge (NGS) produced different N-scyl-homoserine lactones (AHLs). N-(3-oxohexanoyl)-L-homoserinelactone (OHHL), only released from NGS, was shown to accelerate sludge aggregation by increasing the biomass growth rate, microbial activity, extracellular protein, and AerAOB biomass. For both NAS and NGS, sludge cells were glued together by inner extracellular polymeric substances (EPSs) with similar components to form microcolony. Different from the characterized negative effect of NAS's outer-EPS on cell adhesion, the outer-EPS of NGS played a positive role in the attached growth of AerAOB-dominated sludge and contained more tryptophan-like substances. More interesting, OHHL enhanced the yields of tryptophan-like substances after mixing with the outer-EPS of NGS, enhancing cell adhesion. In a word, OHHL and more tryptophan-like substances were produced in the process of granulation under the selective sludge discharge condition, which was proved to be able to accelerate NAS granulation. Therefore, the sludge granulation process for nitritation can be improved by increasing the levels of OHHL and tryptophan in the initial startup stage. The appropriate engineering strategy should be further studied to facilitate the actual application of granular sludge for nitrogen removal on a large scale.

Cell Cycle Machinery in Bacillus subtilis.

Bacillus subtilis is the best described member of the Gram positive bacteria. It is a typical rod shaped bacterium and grows by elongation in its long axis, before dividing at mid cell to generate two similar daughter cells. B. subtilis is a particularly interesting model for cell cycle studies because it also carries out a modified, asymmetrical division during endospore formation, which can be simply induced by starvation. Cell growth occurs strictly by elongation of the rod, which maintains a constant diameter at all growth rates. This process involves expansion of the cell wall, requiring intercalation of new peptidoglycan and teichoic acid material, as well as controlled hydrolysis of existing wall material. Actin-like MreB proteins are the key spatial regulators that orchestrate the plethora of enzymes needed for cell elongation, many of which are thought to assemble into functional complexes called elongasomes. Cell division requires a switch in the orientation of cell wall synthesis and is organised by a tubulin-like protein FtsZ. FtsZ forms a ring-like structure at the site of impending division, which is specified by a range of mainly negative regulators. There it recruits a set of dedicated division proteins to form a structure called the divisome, which brings about the process of division. During sporulation, both the positioning and fine structure of the division septum are altered, and again, several dedicated proteins that contribute specifically to this process have been identified. This chapter summarises our current understanding of elongation and division in B. subtilis, with particular emphasis on the cytoskeletal proteins MreB and FtsZ, and highlights where the major gaps in our understanding remain.

A benzamide-dependent ftsZ mutant reveals residues crucial for Z-ring assembly.

In almost all bacteria, cell division is co-ordinated by the essential tubulin homologue FtsZ and represents an attractive but as yet unexploited target for new antibiotics. The benzamides, e.g. PC190723, are potent FtsZ inhibitors that have the potential to yield an important new class of antibiotic. However, the evolution of resistance poses a challenge to their development. Here we show that a collection of PC190723-resistant and -dependent strains of Staphylococcus aureus exhibit severe growth and morphological defects, questioning whether these ftsZ mutations would be clinically relevant. Importantly, we show that the most commonly isolated substitution remains sensitive to the simplest benzamide 3-MBA and likely works by occluding compound binding. Extending this analysis to Bacillus subtilis, we isolated a novel benzamide-dependent strain that divides using unusual helical division events. The ftsZ mutation responsible encodes the substitution of a highly conserved residue, which lies outside the benzamide-binding site and forms part of an interface between the N- and C-terminal domains that we show is necessary for normal FtsZ function. Together with an intragenic suppressor mutation that mimics benzamide binding, the results provide genetic evidence that benzamides restrict conformational changes in FtsZ and also highlights their utility as tools to probe bacterial division.

Postpartum depression and partner support during the period of lactation: Correlation research and its influencing factors.

BACKGROUND: Postpartum depression (PPD) not only affects the psychological and physiological aspects of maternal health but can also affect neonatal growth and development. Partners who are in close contact with parturient women play a key role in communication and emotional support. This study explores the PPD support relationship with partners and its influencing factors, which is believed to establish psychological well-being and improve maternal partner support. AIM: To explore the correlation between PPD and partner support during breastfeeding and its influencing factors. METHODS: Convenience sampling was used to select lactating women (200 women) who underwent postpartum examinations at the Huzhou Maternity and Child Health Care Hospital from July 2022 to December 2022. A cross-sectional survey was conducted on the basic information (general information questionnaire), depression level [edinburgh postnatal depression scale (EPDS)], and partner support score [dyadic coping inventory (DCI)] of the selected subjects. Pearson's correlation analysis was used to analyze the correlation between PPD and DCI in lactating women. Factors affecting PPD levels during lactation were analyzed using multiple linear regression. RESULTS: The total average score of EPDS in 200 lactating women was (9.52 ± 1.53), and the total average score of DCI was (115.78 ± 14.90). Dividing the EPDS, the dimension scores were: emotional loss (1.91 ± 0.52), anxiety (3.84 ± 1.05), and depression (3.76 ± 0.96). Each dimension of the DCI was subdivided into: Pressure communication (26.79±6.71), mutual support (39.76 ± 9.63), negative support (24.97 ± 6.68), agent support (6.87 ± 1.92), and joint support (17.39 ± 4.19). Pearson's correlation analysis demonstrated that the total mean score and individual dimension scores of EPDS during breastfeeding were inversely correlated with the total score of partner support, stress communication, mutual support, and co-support (P < 0.05). The total mean score of the EPDS and its dimensions were positively correlated with negative support (P < 0.05). Multiple linear regression analysis showed that the main factors affecting PPD during breastfeeding were marital harmony, newborn health, stress communication, mutual support, negative support, co-support, and the total score of partner support (P < 0.05). CONCLUSION: PPD during breastfeeding was associated with marital harmony, newborn health, stress communication, mutual support, negative support, joint support, and the total DCI score.

Noc protein binds to specific DNA sequences to coordinate cell division with chromosome segregation.

Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis, the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS array recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division, but that its activity is strictly localized by the interaction with NBS sites in vivo. We propose that Noc serves not only as a spatial regulator of cell division to protect the nucleoid, but also as a timing device with an important role in the coordination of chromosome segregation and cell division.

Localisation of DivIVA by targeting to negatively curved membranes.

DivIVA is a conserved protein in Gram-positive bacteria and involved in various processes related to cell growth, cell division and spore formation. DivIVA is specifically targeted to cell division sites and cell poles. In Bacillus subtilis, DivIVA helps to localise other proteins, such as the conserved cell division inhibitor proteins, MinC/MinD, and the chromosome segregation protein, RacA. Little is known about the mechanism that localises DivIVA. Here we show that DivIVA binds to liposomes, and that the N terminus harbours the membrane targeting sequence. The purified protein can stimulate binding of RacA to membranes. In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved. On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers. This model may explain why DivIVA localises at cell division sites. A Monte-Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.

Repurposing drugs with specific activity against L-form bacteria.

Cell wall deficient "L- form" bacteria are of growing medical interest as a possible source of recurrent or persistent infection, largely because of their complete resistance to cell wall active antibiotics such as ß-lactams. Antibiotics that specifically kill L-forms would be of potential interest as therapeutics, but also as reagents with which to explore the role of L-forms in models of recurrent infection. To look for specific anti-L-form antibiotics, we screened a library of several hundred FDA-approved drugs and identified compounds highly selective for L-form killing. Among the compounds identified were representatives of two different classes of calcium channel blockers: dihydropyridines, e.g., manidipine; and diphenylmethylpiperazine, e.g., flunarizine. Mode of action studies suggested that both classes of compound work by decreasing membrane fluidity. This leads to a previously recognized phenotype of L-forms in which the cells can continue to enlarge but fail to divide. We identified a considerable degree of variation in the activity of different representatives of the two classes of compounds, suggesting that it may be possible to modify them for use as drugs for L-form-dependent infections.

Structural evolution and functional diversification analyses of argonaute protein.

Argonaute (AGO) proteins are highly specialized small-RNA-binding modules and small RNAs are anchored to their specific binding pockets guiding AGO proteins to target mRNA molecules for silencing or destruction. The 135 full-length AGO protein sequences derived from 36 species covering prokaryote, archaea, and eukaryote are chosen for structural and functional analyses. The results show that bacteria and archaeal AGO proteins are clustered in the same clade and there exist multiple AGO proteins in most eukaryotic species, demonstrating that the increase of AGO gene copy number and horizontal gene transfer (HGT) have been the main evolutionary driving forces for adaptability and biodiversity. And the emergence of PAZ domain in AGO proteins is the unique evolutionary event. The analysis of middle domain (MID)-nucleotide contaction shows that either the position of sulfate I bond in Nc_QDE2 or the site of phosphate I bond in Hs_AGO2 represents the 5'-nucleotide binding site of miRNA. Also, H334, T335, and Y336 of Hs_AGO1 can form hydrogen bonds with 3'-overhanging ends of miRNAs and the same situation exists in Hs_AGO2, Hs_AGO3, Hs_AGO4, Dm_AGO1, and Ce_Alg1. Some PIWI domains containing conserved DDH motif have no slicer activity, and post-translational modifications may be associated with the endonucleolytic activities of AGOs. With the numbers of AGO genes increasing and fewer crystal structures available, the evolutionary and functional analyses of AGO proteins can help clarify the molecular mechanism of function diversification in response to environmental changes, and solve major issues including host defense mechanism against virus infection and molecular basis of disease.

Multiple effects of benzamide antibiotics on FtsZ function.

Cell division in almost all bacteria is orchestrated by the essential tubulin homologue FtsZ, which assembles into a ring-like structure and acts as a scaffold for the division machinery. Division was recently validated as an important target for antibiotics by the demonstration that low-molecular-weight inhibitors of FtsZ, called benzamides, can cure mice infected with Staphylococcus aureus. In treated cells of Bacillus subtilis we show that FtsZ assembles into foci throughout the cell, including abnormal locations at the cell poles and over the nucleoid. These foci are not inactive aggregates because they remain dynamic, turning over almost as rapidly as untreated polymers. Remarkably, although division is completely blocked, the foci efficiently recruit division proteins that normally co-assemble with FtsZ. However, they show no affinity for components of the Min or Nucleoid occlusion systems. In vitro, the benzamides strongly promote the polymerization of FtsZ, into hyperstable polymers, which are highly curved. Importantly, even at low concentrations, benzamides transform the structure of the Z ring, resulting in abnormal helical cell division events. We propose that benzamides act principally by promoting an FtsZ protomer conformation that is incompatible with a higher-order level of assembly needed to make a division ring.

Inhibition of Bacillus subtilis natural competence by a native, conjugative plasmid-encoded comK repressor protein.

Under certain growth conditions, Bacillus subtilis can develop natural competence, the state in which it is able to bind, adsorb and incorporate exogenous DNA. Development of competence is a bistable process and is subject to complex regulation. Rok is a repressor of the key transcriptional activator of competence genes, comK, and limits the size of the subpopulation that develops competence. Here we report the finding that the large conjugative B. subtilis plasmid pLS20 harbours a rok homologue rok(LS20). Although the deduced product of rok(LS20) is considerably shorter than the chromosomally encoded Rok protein, we show that ectopic expression of the plasmid-encoded Rok(LS20) leads to inhibition of competence by repressing comK, and that the effects of the plasmid and chromosomally encoded Rok proteins are additive. We also show that pLS20 inhibits competence in a rok(LS20) -dependent manner and that purified Rok(LS20) preferentially binds to the comK promoter. By analysing the available databases we identified several additional rok-like genes. These putative rok genes can be divided into two groups and we propose that rok(LS20) is the prototype of a newly identified subgroup of nine rok genes. Finally, we discuss the possible role of the plasmid-located rok and its relatedness with other rok genes.

[Molecular mechanism for dynamic regulation of endogenous ABA signal level].

The process from stress signal perception and the trigger of ABA biosynthesis to dynamic regulation of ABA level is an important stress signaling pathway in cells. Compared to the downstream events in ABA signal transduction, the researches in this field are relatively lagged. Expression of synthase genes, such as ZEP in roots and rate-limiting enzyme genes NCED, AtRGS1 and ABA2, can be activated in response to stresses. However, the expression of genes encoding degradative enzymes, including 7'-, 8'-, 9'-hydroxylase and glucosyltransferase, negatively regulates ABA accumulation. Meanwhile, the expressions of the synthases, such as ZEP and NCED3, are induced by increasing endogenous ABA contents. Additionally, the analyses of gene expression and source-sink dynamics indicates that sustained supply from root-sourced ABA is required for the maintenance of leaf ABA dynamic pool. It is notable that miRNAs should be involved in ABA signal origin and ABA level dynamic adjustment. Further dynamic analysis of ABA metabolism revealed that endogenous ABA signal levels are synergistically controlled by the expressions of synthases and degradative enzymes.

Discovery of Demurilactone A: A Specific Growth Inhibitor of L-Form Bacillus subtilis.

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.

Conjugation Operons in Gram-Positive Bacteria with and without Antitermination Systems.

Genes involved in the same cellular process are often clustered together in an operon whose expression is controlled by an upstream promoter. Generally, the activity of the promoter is strictly controlled. However, spurious transcription undermines this strict regulation, particularly affecting large operons. The negative effects of spurious transcription can be mitigated by the presence of multiple terminators inside the operon, in combination with an antitermination system. Antitermination systems modify the transcription elongation complexes and enable them to bypass terminators. Bacterial conjugation is the process by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugation involves many genes that are mostly organized in one or a few large operons. It has recently been shown that many conjugation operons present on plasmids replicating in Gram-positive bacteria possess a bipartite antitermination system that allows not only many terminators inside the conjugation operon to be bypassed, but also the differential expression of a subset of genes. Here, we show that some conjugation operons on plasmids belonging to the Inc18 family of Gram-positive broad host-range plasmids do not possess an antitermination system, suggesting that the absence of an antitermination system may have advantages. The possible (dis)advantages of conjugation operons possessing (or not) an antitermination system are discussed.

Mirubactin C rescues the lethal effect of cell wall biosynthesis mutations in Bacillus subtilis.

Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a protein machine called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg2+ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening > 2,000 extracts from a diverse collection of actinobacteria, we identified a compound, mirubactin C, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed using synthetic mirubactin C. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin C rescuing activity is not due simply to iron sequestration. The results support an emerging view that the functions of bacterial siderophores extend well beyond simply iron binding and uptake.