Laboratory Growth Conditions Research Articles

Chitosan-strigolactone mimics with synergistic effect: A new concept for plant biostimulants

The paper reports new multifunctional plant biostimulant formulations obtained via in situ hydrogelation of chitosan with salicylaldehyde in the presence of a mimetic naphthalimide-based strigolactone, in specific conditions. Various analytical techniques (FTIR, 1H NMR, SEM, POM, TGA, WRXD) were employed to understand the particularities of the hydrogelation mechanism and its consequences on the formulations' properties. Further, in order to evaluate their potential for the targeted application, the swelling in media of pH characteristic for different soils, water holding capacity, soil biodegradability, in vitro release of the strigolactone mimic and impact on tomatoes plant growth in laboratory conditions were investigated and discussed. It was found that the strigolactone mimic has the ability to bond to the chitosan matrix via physical forces, favoring a prolonged release. Moreover, the combination of chitosan with the strigolactone mimic in an optimal mass ratio triggered a synergistic effect on the plant growth, up to 4 times higher compared to the neat control soil.

In vitro and in vivo Effect of Weeds (Root) Extracts on Soil Borne Fungal Phytopathogens and Fungal Infected Legume Crop Bengal Gram (Cicer arietinum)

Background: Weed management during growing season has been a serious problem for many years. Worldwide, a 10% loss of agricultural products can be attributed to the competitive effect of weeds, despite their intensive control. Dactyloctenium aegyptium and Chenopodium album L. is an annual weed of cultivated fields. Methods: This investigation was done in 2020-2021. Root parts of both weeds Dactyloctenium aegyptium and Chenopodium album were collected from a nearby area, Sardar Vallabh Bhai Patel University of Agriculture and Technology, Meerut. To determine the antifungal potentials of both weeds Dactyloctenium aegyptium and Chenopodium album root extracts were determined by the food poisoning method against fungal phytopathogens. Methane, Ethyl acetate, Butyl alcohol, Benzene and Water-soluble root extracts of both weeds at different concentrations (5%, 10% and 15%) were used against fungal phytopathogens growth in laboratory conditions and also used on fungal infected related attributes of Bengal gram in a pot culture experiment in 1:10 ratio. Antioxidants of both weed extracts were done by DPPH, FRAP and ABTS methods. Result: Findings showed that application of 15% concentrated extracts has maximum antifungal effects against Fusarium oxysporum and Sclerotium rolfsi (70-80%). In the pot experiment, we find that at increased concentrations of methanolic extract (15%) infected Bengal gram crops show high effectiveness against fungal phytopathogens and improve different parameters such as germination, plant physiology and morphological characteristics. However, higher concentrations of methanol extract promoted these parameters and reduced fungal growth in both in vivo and vitro conditions Results of this study reported highly significant suppressive effects of higher concentrations (10% and 15%).

Multifactorial genetic control and magnesium levels govern the production of a Streptomyces antibiotic with unusual cell density dependence.

Streptomyces bacteria are renowned both for their antibiotic production capabilities and for their cryptic metabolic potential. Their metabolic repertoire is subject to stringent genetic control, with many of the associated biosynthetic gene clusters being repressed by the conserved nucleoid-associated protein Lsr2. In an effort to stimulate new antibiotic production in wild Streptomyces isolates, we leveraged the activity of an Lsr2 knockdown construct and successfully enhanced antibiotic production in the wild Streptomyces isolate WAC07094. We determined that this new activity stemmed from increased levels of the angucycline-like family member saquayamycin. Saquayamycin has both antibiotic and anti-cancer activities, and intriguingly, beyond Lsr2-mediated repression, we found saquayamycin production was also suppressed at high density on solid or in liquid growth media; its levels were greatest in low-density cultures. This density-dependent control was exerted at the level of the cluster-situated regulatory gene sqnR and was mediated in part through the activity of the PhoRP two-component regulatory system, where deleting phoRP led to both constitutive antibiotic production and sqnR expression. This suggests that PhoP functions to repress the expression of sqnR at high cell density. We further discovered that magnesium supplementation could alleviate this density dependence, although its action was independent of PhoP. Finally, we revealed that the nitrogen-responsive regulators GlnR and AfsQ1 could relieve the repression exerted by Lsr2 and PhoP. Intriguingly, we found that this low density-dependent production of saquayamycin was not unique to WAC07094; saquayamycin production by another wild isolate also exhibited low-density activation, suggesting that this spatial control may serve an important ecological function in their native environments.IMPORTANCEStreptomyces specialized metabolic gene clusters are subject to complex regulation, and their products are frequently not observed under standard laboratory growth conditions. For the wild Streptomyces isolate WAC07094, production of the angucycline-family compound saquayamycin is subject to a unique constellation of control factors. Notably, it is produced primarily at low cell density, in contrast to the high cell density production typical of most antibiotics. This unusual density dependence is conserved in other saquayamycin producers and is driven by the pathway-specific regulator SqnR, whose expression is influenced by both nutritional and genetic elements. Collectively, this work provides new insights into an intricate regulatory system governing antibiotic production and indicates there may be benefits to including low-density cultures in antibiotic screening platforms.

The antagonistic transcription factors, EspM and EspN, regulate the ESX-1 secretion system in M. marinum.

Bacterial pathogens use protein secretion systems to transport virulence factors and regulate gene expression. Among pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum, the ESAT-6 system 1 (ESX-1) secretion is crucial for host interaction. Secretion of protein substrates by the ESX-1 secretion system disrupts phagosomes, allowing mycobacteria cytoplasmic access during macrophage infections. Deletion or mutation of the ESX-1 system attenuates mycobacterial pathogens. Pathogenic mycobacteria respond to the presence or absence of the ESX-1 system in the cytoplasmic membrane by altering transcription. Under laboratory conditions, the EspM repressor and WhiB6 activator control transcription of specific ESX-1-responsive genes, including the ESX-1 substrate genes. However, deleting the espM or whiB6 gene does not phenocopy the deletion of the ESX-1 substrate genes during macrophage infection by M. marinum. In this study, we identified EspN, a critical transcription factor whose activity is masked by the EspM repressor under laboratory conditions. In the absence of EspM, EspN activates transcription of whiB6 and ESX-1 genes during both laboratory growth and macrophage infection. EspN is also independently required for M. marinum growth within and cytolysis of macrophages, similar to the ESX-1 genes, and for disease burden in a zebrafish larval model of infection. These findings suggest that EspN and EspM coordinate to counterbalance the regulation of the ESX-1 system and support mycobacterial pathogenesis.IMPORTANCEPathogenic mycobacteria, which are responsible for tuberculosis and other long-term diseases, use the ESX-1 system to transport proteins that control the host response to infection and promote bacterial survival. In this study, we identify an undescribed transcription factor that controls the expression of ESX-1 genes and is required for both macrophage and animal infection. However, this transcription factor is not the primary regulator of ESX-1 genes under standard laboratory conditions. These findings identify a critical transcription factor that likely controls expression of a major virulence pathway during infection, but whose effect is not detectable with standard laboratory strains and growth conditions.

ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis.

Salmonella enterica is a major pathogen responsible for foodborne gastroenteritis, and a leading model organism for genetic and molecular studies of bacterial virulence mechanisms. One key trait of this pathogen is the ability to survive within infected host cells. During infection, the bacteria employ a type three secretion system that deliver effector proteins to target and manipulate host cell processes. The transcriptional regulation of this virulence program is well understood. By contrast, the factors and mechanisms operating at the post-transcriptional level to control virulence gene expression are less clear. In this study, we have charted the global RNA ligand repertoire of the RNA-binding protein ProQ during in vitro conditions mimicking the host cell environment. This identified hundreds of binding sites and revealed ProQ-dependent stabilization of intracellular-specific small RNAs. Importantly, we show that ProQ post-transcriptionally activates the expression of PhoP, a master transcriptional activator of intracellular virulence in Salmonella.

The role of GpsB in Staphylococcus aureus cell morphogenesis.

Staphylococcus aureus is a Gram-positive clinical pathogen, which is currently the second cause of death by antibiotic-resistant infections worldwide. For decades, S. aureus cells were thought to be spherical and lack the ability to undergo elongation. However, super-resolution microscopy techniques allowed us to observe the minor morphological changes that occur during the cell cycle of this pathogen, including cell elongation. S. aureus elongation is not required for normal growth in laboratory conditions. However, it seems to be essential in the context of some infections, such as osteomyelitis, during which S. aureus cells apparently elongate to invade small channels in the bones. In this work, we uncovered new determinants required for S. aureus cell elongation. In particular, we show that GpsB has an important role in the spatio-temporal regulation of PBP2 and PBP4, two proteins involved in peptidoglycan synthesis, contributing to the maintenance of the correct cell morphology in S. aureus.

Short communication: Investigating optimal laboratory growth conditions of Gracilibacillus halotolerans in media supplemented with salt

Gracilibacillus halotolerans, a new and relatively unstudied extremophile, extracted from the Great Salt Lake USA, survives in an extreme saline environment. Uncovering optimal laboratory growth conditions can be useful to improve treatment strategies against antibiotic resistance and biofilm formation. In the current study, G. halotolerans growth optimization was tested to determine the ideal saline concentration. In addition, a variety of G. halotolerans'-derived survival strategies were reviewed. The major findings of the current study includes the optimal laboratory growth condition for G. halotolerans that requires the supplement of 5% NaCl. In addition, optimal growth was observed up to 72 h in Luria Bertani (LB) broth. Identifying the optimal laboratory growth conditions for G. halotolerans will standardize growth methods, reduce laboratory cost, and can improve future investigations of extremophile bacteria as model organisms to combat antibiotic resistance, biofilm, and other persister cell characteristics that negatively affect research and clinical settings.

Harnessing regulatory networks in Actinobacteria for natural product discovery.

This review provides insights into advances in experimental and computational methods aimed at predicting transcription factor binding sites and their applications to guide natural product discovery.

Generation and characterization of temperature-sensitive alleles of the glucanosyltransferase Gas1 in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe Gas family of β-1,3-glucanosyltransferases modify the cell wall by elongating β-1,3-glucan chains. While gas1Δ cells are inviable under standard laboratory growth conditions, they are viable in the presence of an osmotic stabilizer. Even under these conditions however, gas1Δ cells are slow-growing and display cell separation and morphology defects. Here, we isolated and characterized two gas1 temperature-sensitive alleles. Our data support that Gas1 is the primary S. pombe β-1,3-glucanosyltransferase important for cell separation and cell viability and provide useful tools for further analysis of S. pombe cell wall formation.

Isolation and characterization of the novel phage BP14 for lysing Vibrio parahaemolyticus and reducing virulence proteins

Accumulating evidence has indicated that Vibrio parahaemolyticus stains resistant to multiple drugs may pose a serious threat to public health and could lead to global economic. Phage therapy is an alternative and eco-friendly biocontrol strategy for the prevention and control of diseases in the aquatic systems. Therefore, this study was oriented to isolate and characterize the applicability of phages for V. parahaemolyticus (VP) lysis in aquaculture. Here, twenty-two lytic bacteriophages (BP1–22) were isolated from sewage collected in a local shrimp farming area and from the tissue of AHPND-infected shrimp and oysters collected in Taiwan. Among these, phage BP14 could effectively lyse V. parahaemolyticus, this bacterium causes acute hepatopancreatic necrosis disease (AHPND), resulting in severe mortality in Litopenaeus vannamei and Penaeus monodon, two marine shrimp species that are commonly grown in aquaculture farms. Characterization of the morphology of the virion particle via electron microscopy revealed that the virus belonged to the family Inoviridae, as suggested by its filamentous shape. The genome of phage BP14 was composed of 9071 bp with a 43.85% GC content and was inserted into a chromosome depending on the occurrence of Xer site-specific recombination. Furthermore, phage BP14 established a chronic productive infection, thus continuously releasing virions from infected cells. A viral load of approximately 12 plaque-forming units (PFUs) per cell was determined for phage BP14 under laboratory growth conditions. Western blotting and ELISA analyses revealed that phage BP14 decreased the levels of toxin-PirB and PirA protein by approximately 40%–60% compared to the wild-type VP. Collectively, our findings demonstrated the potential applicability of phage BP14 for the biological control of VPAHPND.

Anti-persister efficacy of colistin and meropenem against uropathogenic Escherichia coli is dependent on environmental conditions.

Antibiotic persistence is a phenomenon observed when genetically susceptible cells survive long-term exposure to antibiotics. These 'persisters' are an intrinsic component of bacterial populations and stem from phenotypic heterogeneity. Persistence to antibiotics is a concern for public health globally, as it increases treatment duration and can contribute to treatment failure. Furthermore, there is a growing array of evidence that persistence is a 'stepping-stone' for the development of genetic antimicrobial resistance. Urinary tract infections (UTIs) are a major contributor to antibiotic consumption worldwide, and are known to be both persistent (i.e. affecting the host for a prolonged period) and recurring. Currently, in clinical settings, routine laboratory screening of pathogenic isolates does not determine the presence or the frequency of persister cells. Furthermore, the majority of research undertaken on antibiotic persistence has been done on lab-adapted bacterial strains. In the study presented here, we characterized antibiotic persisters in a panel of clinical uropathogenic Escherichia coli isolates collected from hospitals in the UK and Australia. We found that a urine-pH mimicking environment not only induces higher levels of antibiotic persistence to meropenem and colistin than standard laboratory growth conditions, but also results in rapid development of transient colistin resistance, regardless of the genetic resistance profile of the isolate. Furthermore, we provide evidence for the presence of multiple virulence factors involved in stress resistance and biofilm formation in the genomes of these isolates, whose activities have been previously shown to contribute to the formation of persister cells.

Analysis of programmed frameshifting during translation of prfB in Flavobacterium johnsoniae.

Ribosomes of Bacteroidia fail to recognize Shine-Dalgarno (SD) sequences due to sequestration of the 3' tail of the 16S rRNA on the 30S platform. Yet in these organisms, the prfB gene typically contains the programmed +1 frameshift site with its characteristic SD sequence. Here, we investigate prfB autoregulation in Flavobacterium johnsoniae, a member of the Bacteroidia. We find that the efficiency of prfB frameshifting in F. johnsoniae is low (∼7%) relative to that in Escherichia coli (∼50%). Mutation or truncation of bS21 in F. johnsoniae increases frameshifting substantially, suggesting that anti-SD (ASD) sequestration is responsible for the reduced efficiency. The frameshift site of certain Flavobacteriales, such as Winogradskyella psychrotolerans, has no SD. In F. johnsoniae, this W. psychrotolerans sequence supports frameshifting as well as the native sequence, and mutation of bS21 causes no enhancement. These data suggest that prfB frameshifting normally occurs without SD-ASD pairing, at least under optimal laboratory growth conditions. Chromosomal mutations that remove the frameshift or ablate the SD confer subtle growth defects in the presence of paraquat or streptomycin, respectively, indicating that both the autoregulatory mechanism and the SD element contribute to F. johnsoniae cell fitness. Analysis of prfB frameshift sites across 2686 representative bacteria shows loss of the SD sequence in many clades, with no obvious relationship to genome-wide SD usage. These data reveal unexpected variation in the mechanism of frameshifting and identify another group of organisms, the Verrucomicrobiales, that globally lack SD sequences.

Adaptation mechanisms of Listeria monocytogenes to quaternary ammonium compounds.

Listeria monocytogenes is ubiquitously found in nature and can easily enter food-processing facilities due to contaminations of raw materials. Several countermeasures are used to combat contamination of food products, for instance, the use of disinfectants that contain quaternary ammonium compounds, such as benzalkonium chloride (BAC) and cetyltrimethylammonium bromide (CTAB). In this study, we assessed the potential of the commonly used wild-type strain EGD-e to adapt to BAC and CTAB under laboratory growth conditions. All BAC-tolerant suppressors exclusively carried mutations in fepR, encoding a TetR-like transcriptional regulator, or its promoter region, likely resulting in the overproduction of the efflux pump FepA. In contrast, CTAB tolerance was associated with mutations in sugR, which regulates the expression of the efflux pumps SugE1 and SugE2. L. monocytogenes strains lacking either FepA or SugE1/2 could still acquire tolerance toward BAC and CTAB. Genomic analysis revealed that the overproduction of the remaining efflux system could compensate for the deleted one, and even in the absence of both efflux systems, tolerant strains could be isolated, which all carried mutations in the diacylglycerol kinase-encoding gene lmo1753 (dgkB). DgkB converts diacylglycerol to phosphatidic acid, which is subsequently reused for the synthesis of phospholipids, suggesting that alterations in membrane composition could be the third adaptation mechanism. IMPORTANCE Survival and proliferation of Listeria monocytogenes in the food industry are ongoing concerns, and while there are various countermeasures to combat contamination of food products, the pathogen still successfully manages to withstand the harsh conditions present in food-processing facilities, resulting in reoccurring outbreaks, subsequent infection, and disease. To counteract the spread of L. monocytogenes, it is crucial to understand and elucidate the underlying mechanism that permits their successful evasion. We present various adaptation mechanisms of L. monocytogenes to withstand two important quaternary ammonium compounds.

Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M. tuberculosis.

Cyclic AMP (cAMP) is a ubiquitous second messenger that transduces signals from cellular receptors to downstream effectors. Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, devotes a considerable amount of coding capacity to produce, sense, and degrade cAMP. Despite this fact, our understanding of how cAMP regulates Mtb physiology remains limited. Here, we took a genetic approach to investigate the function of the sole essential adenylate cyclase in Mtb H37Rv, Rv3645. We found that a lack of rv3645 resulted in increased sensitivity to numerous antibiotics by a mechanism independent of substantial increases in envelope permeability. We made the unexpected observation that rv3645 is conditionally essential for Mtb growth only in the presence of long-chain fatty acids, a host-relevant carbon source. A suppressor screen further identified mutations in the atypical cAMP phosphodiesterase rv1339 that suppress both fatty acid and drug sensitivity phenotypes in strains lacking rv3645. Using mass spectrometry, we found that Rv3645 is the dominant source of cAMP under standard laboratory growth conditions, that cAMP production is the essential function of Rv3645 in the presence of long-chain fatty acids, and that reduced cAMP levels result in increased long-chain fatty acid uptake and metabolism and increased antibiotic susceptibility. Our work defines rv3645 and cAMP as central mediators of intrinsic multidrug resistance and fatty acid metabolism in Mtb and highlights the potential utility of small molecule modulators of cAMP signaling.

The Pseudomonas aeruginosa RpoH (σ32) Regulon and Its Role in Essential Cellular Functions, Starvation Survival, and Antibiotic Tolerance.

The bacterial heat-shock response is regulated by the alternative sigma factor, σ32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose-inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH (σ32) regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden change in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurred without arabinose induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased five-fold within 15 min of arabinose addition. Transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many genes essential for P. aeruginosa growth are induced by RpoH. Other stress response genes induced by RpoH are involved in damaged nucleic acid repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. Phenotypic analysis of P. aeruginosa ∆rpoH (PBAD-rpoH) showed that it is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also had increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside sensitivity of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes, such as aminoglycoside acetyl transferases, that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient-limiting conditions.

Expression of virulence and antimicrobial related proteins in Burkholderia mallei and Burkholderia pseudomallei.

Burkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics. The expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared. Proteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, PenA as well as several other efflux pump related proteins and putative β-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB-OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in β-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified. Analyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei.

Auxin and cytokinin control fate determination of cotyledons in the one-leaf plant Monophyllaea glabra.

One-leaf plants in the Gesneriaceae family initially have two cotyledons of identical size; one cotyledon stops growing shortly after germination, whereas the other continues indeterminate growth. Factors involved in the unequal growth have been investigated, and a competitive relationship between the two cotyledons was previously proposed. However, questions regarding the fate determination of the two cotyledons remain: Why does only one cotyledon grow indeterminately while the other stops; is the fate of the cotyledons reversible; and what role does light quality play in the fate determination of the cotyledons? In this study, physiological experiments using the one-leaf plant species Monophyllaea glabra suggest that a biased auxin concentration between the two cotyledons and subsequent cytokinin levels may determine the fate of the cotyledons. In addition, observation of relatively mature individuals without hormone treatment and younger individuals with cytokinin treatment under laboratory growth conditions revealed that the fate determination of the microcotyledon is reversible. Although light quality has been suggested to be important for the determination of cotyledon fate in Streptocarpus rexii, an anisocotylous species, we conclude that light quality is not important in M. glabra.

Multiple Stochastic Parameters Influence Genome Dynamics in a Heterozygous Diploid Eukaryotic Model.

The heterozygous diploid genome of Candida albicans displays frequent genomic rearrangements, in particular loss-of-heterozygosity (LOH) events, which can be seen on all eight chromosomes and affect both laboratory and clinical strains. LOHs, which are often the consequence of DNA damage repair, can be observed upon stresses reminiscent of the host environment, and result in homozygous regions of various sizes depending on the molecular mechanisms at their origins. Recent studies have shed light on the biological importance of these frequent and ubiquitous LOH events in C. albicans. In diploid Saccharomyces cerevisiae, LOH facilitates the passage of recessive beneficial mutations through Haldane’s sieve, allowing rapid evolutionary adaptation. This also appears to be true in C. albicans, where the full potential of an adaptive mutation is often only observed upon LOH, as illustrated in the case of antifungal resistance and niche adaptation. To understand the genome-wide dynamics of LOH events in C. albicans, we constructed a collection of 15 strains, each one carrying a LOH reporter system on a different chromosome arm. This system involves the insertion of two fluorescent marker genes in a neutral genomic region on both homologs, allowing spontaneous LOH events to be detected by monitoring the loss of one of the fluorescent markers using flow cytometry. Using this collection, we observed significant LOH frequency differences between genomic loci in standard laboratory growth conditions; however, we further demonstrated that comparable heterogeneity was also observed for a given genomic locus between independent strains. Additionally, upon exposure to stress, three outcomes could be observed in C. albicans, where individual strains displayed increases, decreases, or no effect of stress in terms of LOH frequency. Our results argue against a general stress response triggering overall genome instability. Indeed, we showed that the heterogeneity of LOH frequency in C. albicans is present at various levels, inter-strain, intra-strain, and inter-chromosomes, suggesting that LOH events may occur stochastically within a cell, though the genetic background potentially impacts genome stability in terms of LOH throughout the genome in both basal and stress conditions. This heterogeneity in terms of genome stability may serve as an important adaptive strategy for the predominantly clonal human opportunistic pathogen C. albicans, by quickly generating a wide spectrum of genetic variation combinations potentially permitting subsistence in a rapidly evolving environment.

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling.

Gram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions. An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the ~20 other known PrkA targets to PG biosynthesis regulation is unknown. We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to activate PG biosynthesis and further enhanced the intrinsic cephalosporin resistance of L. monocytogenes. This latter effect required the RodA3/PBP B3 transglycosylase/transpeptidase pair. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during standard laboratory growth conditions and intracellular growth in macrophages. These findings have important implications for the understanding of PG biosynthesis regulation and β-lactam resistance of L. monocytogenes and related Gram-positive bacteria.

Inducing Global Expression of Actinobacterial Biosynthetic Gene Clusters.

Bacteria produce an impressive array of bioactive specialized metabolites, with Streptomyces (and the actinobacteria more generally) being unusually diverse and prolific producers. However, the biosynthetic potential of these organisms has yet to be fully explored, as many of the biosynthetic gene clusters that direct the synthesis of these natural products are transcriptionally silent under laboratory growth conditions. Here, we describe strategies that can be employed to broadly stimulate the expression of biosynthetic gene clusters in Streptomyces and their relatives, follow the transcription of these genes, and assess the antimicrobial activity of the resulting molecules.