Weakened Cell Wall Research Articles

Efficient transformation and genome editing in a nondomesticated, biocontrol strain, Bacillus subtilis GLB191

Bacillus subtilis has been widely used as a biological control agent in agricultural production. Environmental strains of B. subtilis are an important source of biological control agents. However, due to its low genetic transformation efficiency, the genetic manipulation of the environmental and nondomesticated strains is challenging. In this study, the impact of competent cell preparation, pulse electroporation, and recovery culture on the electroporation efficiency of B. subtilis GLB191 was assessed utilizing response surface methodology. Results indicated that the concentration of glycine, DL-threonine, and Tween 80 used in a cell wall weakening solution during competent cell preparation, and the voltage applied during pulse electroporation were the primary factors affecting electroporation efficiency. Optimization of these factors led to nearly a three-fold increase (reaching 74.00 ± 5.10 CFU/µg DNA) in electroporation efficiency. The elimination of dam and dcm modifications to mitigate the influence of host restriction-modification systems was integrated to further increase the electroporation efficacy. An electroporation efficiency for replicative plasmids of 1.96 ± 0.05 × 106 CFU/µg DNA was achieved using the optimized strategy. Utilizing this improved methodology, the temperature-sensitive plasmid pJOE8899 was efficiently transformed into B. subtilis GLB191, resulting in a markerless knockout of pdeH. The optimized transformation strategy significantly enhances the efficiency of markerless genome editing of nondomesticated B. subtilis, offering the potential for future interpretation of their modes of action, which is critical for the development of the nondomesticated B. subtilis strains.

Novel triazole-nucleoside hybrids as potential antimicrobial agents: Design, synthesis, and molecular docking

Despite decoding many important genes in the formation of microbes, particularly pathogenic and mutant ones that lead to resistance to a wide range of antibiotics, this opens the door to discovering new antimicrobial compounds, whether synthetic or natural in origin. A new set of nucleoside-triazole hybrids was created to provide a more effective topoisomerase II for microbial therapy. All freshly synthesized compounds' structures were confirmed using spectral data and elemental analysis. The newly synthesized compounds were docked to the area of topoisomerase II's active site; compounds 3, 7, 15, 18, and 19 exhibited distinct hydrogen bonding formation and pi-pi interactions. The results of testing every compound against six different microbial strains were expressed as the width of the inhibitory zone.Except for Aspergillus Niger, most of the synthetic compounds inhibit all bacteria. Compounds 15 and 19 had the lowest MIC values (1.95, 3.9 µg/mL against B. subtills and 1.95, 3.9 µg/mL against S. aureus). Nucleoside 15 had MBC values between 3.9 and B. subtills. The in-vitro topoisomerase II enzyme assay revealed that compounds 3, 15, 18, and 19 had IC50 values of 0.17, 0.05, 0.13, and 0.08 µmol/mL, respectively. A transmission electron microscope (TEM) was utilized to assess morphological changes on the bacterial surface following treatment with nucleoside 15. It was discovered that nucleoside 15 induced substantial cell wall weakening, which exacerbated cell membrane rupture.

Remediation of sugarcane vinasse using Rhodotorula glutinis or Rhodotorula mucilaginosa: Biomass morphology and its potential technological applications

There is an emerging interest of use biological processes for vinasse treatment generally because they are able to produce interesting metabolites from living cells. Additionally, the production of yeasts with weakened cell wall without modifying its physiological performance would be of great technological value not only to facilitate the extraction of intracellular metabolites but also to increase the biomass digestibility. This study explored the remediation of a glycerol-fortified sugarcane vinasse using Rhodotorula glutinis or Rhodotorula mucilaginosa coupled with the production of valuable residual biomasses displaying a tailor-made weakened cell wall. Firstly, the biomass-bound ß-d-glucosidase activity was selected as a proper cell wall lytic potential marker. Secondly, in vitro-dose response assays with several metal ions showed that the presence of 6 mM of MnCl2 and FeCl3 increased the ß-d-glucosidase activity in R. glutinis and R. mucilaginosa, respectively. Thirdly, the supplementation of those salts to either YPD or vinasse-based media was evaluated. As a result, cells were bigger or even rounder than the corresponding controls (without metal ions supplementation) suggesting a weakening of the cell wall. Finally, as proof of concept, after 6 days of vinasse treatment at 30 °C under stirred conditions, the reduction of COD value for R. glutinis and R. mucilaginosa was 81.3% and 80.8%, respectively. The final biomass, lipid and protein concentrations for R. glutinis were 15.1 g/L, 31.1% and 48.12%, respectively. The final biomass, lipid and protein concentrations for R. mucilaginosa were 15.7 g/L, 31.5% and 48.12%, respectively. Potential technological applications of residual biomass were also discussed.

Dual disintegration of microalgae biomass for cost-effective biomethane production: Energy and cost assessment

The present study aims to enhance the biomethane production potential of microalgae via a dual disintegration process. During this process, the microalgae biomass was firstly subjected to cell wall weakening by thermochemical disintegration (TC) (50 to 80 °C), pH adjustment with alkali, NaOH (6 to 10) and time (0 to 10 min) and, secondly, by bacterial disintegration (BD). TC-BD disintegration was comparatively higher (33 %) than BD (24 %), TC (8.5 %), and control (7 %). A more significant VFA accumulation of 2816 mg/L was recorded for TC-BD. Similarly, a greater substrate anaerobic biodegradability was achieved in TC-BD (0.32 g COD /g COD) than BD (0.21 g COD /g COD), TC alone (0.09 gCOD/g COD) and control (0.08 g COD /g COD), respectively. The TC-BD achieves a positive net profit and an energy ratio of + 0.12 GJ/d and 1.03. The proposed dual disintegration has a promising future for commercialization.

Coordinated regulation of osmotic imbalance by c-di-AMP shapes ß-lactam tolerance in Group B Streptococcus.

Streptococcus agalactiae is among the few pathogens that have not developed resistance to ß-lactam antibiotics despite decades of clinical use. The molecular basis of this long-lasting susceptibility has not been investigated, and it is not known whether specific mechanisms constrain the emergence of resistance. In this study, we first report ß-lactam tolerance due to the inactivation of the c-di-AMP phosphodiesterase GdpP. Mechanistically, tolerance depends on antagonistic regulation by the repressor BusR, which is activated by c-di-AMP and negatively regulates ß-lactam susceptibility through the BusAB osmolyte transporter and the AmaP/Asp23/GlsB cell envelope stress complex. The BusR transcriptional response is synergistic with the simultaneous allosteric inhibition of potassium and osmolyte transporters by c-di-AMP, which individually contribute to low-level ß-lactam tolerance. Genome-wide transposon mutagenesis confirms the role of GdpP and highlights functional interactions between a lysozyme-like hydrolase, the KhpAB RNA chaperone and the protein S immunomodulator in the response of GBS to ß-lactam. Overall, we demonstrate that c-di-AMP acts as a turgor pressure rheostat, coordinating an integrated response at the transcriptional and post-translational levels to cell wall weakening caused by ß-lactam activity, and reveal additional mechanisms that could foster resistance.

Arabinogalactan-Proteins as Boron-Acting Enzymes, Cross-Linking the Rhamnogalacturonan-II Domains of Pectin

Most pectic rhamnogalacturonan-II (RG-II) domains in plant cell walls are borate-bridged dimers. However, the sub-cellular locations, pH dependence, reversibility and biocatalyst involvement in borate bridging remain uncertain. Experiments discussed here explored these questions, utilising suspension-cultured plant cells. In-vivo pulse radiolabelling showed that most RG-II domains dimerise extremely quickly (<4 min after biosynthesis, thus while still intraprotoplasmic). This tallies with the finding that boron withdrawal causes cell wall weakening within 10–20 min, and supports a previously proposed biological role for boron/RG-II complexes specifically at the wall/membrane interface. We also discuss RG-II monomer ↔ dimer interconversion as monitored in vitro using gel electrophoresis and a novel thin-layer chromatography method to resolve monomers and dimers. Physiologically relevant acidity did not monomerise dimers, thus boron bridge breaking cannot be a wall-loosening mechanism in ‘acid growth’; nevertheless, recently discovered RG-II trimers and tetramers are unstable and may thus underpin reversible wall loosening. Dimerising monomers in vitro by B(OH)3 required the simultaneous presence of RG-II-binding ‘chaperones’: co-ordinately binding metals and/or ionically binding cationic peptides. Natural chaperones of the latter type include highly basic arabinogalactan protein fragments, e.g., KHKRKHKHKRHHH, which catalyse a reaction [2 RG-II + B(OH)3 → RG-II–B–RG-II], suggesting that plants can ‘enzymically’ metabolise boron.

Dissecting the Role of Cell Wall Changes in Chilling Injury-Induced Gel Formation, Rubberiness, and Mealiness in Apricots

In apricots and other stonefruit, chilling injury (CI) symptoms like mealiness, rubberiness, and gel formation are associated with cell wall properties. Apricots were stored at 0 °C for 5 weeks and ripened at 20 °C to induce CI and compared with fruit ripened at 20 °C from harvest at similar firmness. In those apricots without CI, degradation of middle-lamella pectin during softening weakened cell-cell adhesion and intercellular junctions. Pectin was still present in middle lamella regions but pectin that filled the intercellular spaces at harvest had disappeared. Fruit with combinations of CI symptoms showed different pectin solubilities, molecular weight distribution, and differences in pectin staining compared with fruit that were severely chilling-injured, exhibiting all symptoms. The perception of mealiness correlated with the presence of pectin in the cell lumen, and rubberiness with the presence of pectin in tricellular corners. We concluded that in chilling-injured apricots, the normal softening process is not being resumed after fruit have been taken out of cold storage. Cell wall degradation is disrupted, affecting the normal weakening of cell walls during softening. Hence, cell walls were less likely to break open during chewing, and when cells did break, any juice released might be bound by pectin present in the cell walls and cell lumen, leaving a sensation of rubberiness and mealiness.

Ex-chitin-g news on drug-induced fungal epitope unmasking.

The microbial cell wall is an essential cellular organelle commonly targeted by antimicrobials. It is also a battleground of innate immune recognition where microbes can evade immune recognition by masking essential cell wall components. A recent study (A. S. Wagner, S. W. Lumsdaine, M. M. Mangrum, and T. B. Reynolds, mBio https://doi.org/10.1128/mbio.00074-23, 2023) provides insight into how echinocandin antifungals cause exposure of proinflammatory β(1,3)-glucan by driving excess chitin production in the weakened cell wall. Although many environmental and biological activities perturb cell wall integrity and regulate β(1,3)-glucan exposure, we still know little about which intracellular signaling components regulate the cell wall changes that result in disrupted cell wall architecture. Wagner et al. showed that calcineurin and the Mkc1p kinase regulate chitin deposition and β(1,3)-glucan unmasking. They further identified chitin synthesis as a key driving force in cell wall structure disruption leading to epitope exposure. Their findings highlight how fungal cell wall dynamics have important implications for antifungal immunity and future drug development.

An Update on the Therapeutic Potential of Antimicrobial Peptides against Acinetobacter baumannii Infections.

The rise in antibiotic-resistant strains of clinically important pathogens is a major threat to global health. The World Health Organization (WHO) has recognized the urgent need to develop alternative treatments to address the growing list of priority pathogens. Antimicrobial peptides (AMPs) rank among the suggested options with proven activity and high potential to be developed into effective drugs. Many AMPs are naturally produced by living organisms protecting the host against pathogens as a part of their innate immunity. Mechanisms associated with AMP actions include cell membrane disruption, cell wall weakening, protein synthesis inhibition, and interference in nucleic acid dynamics, inducing apoptosis and necrosis. Acinetobacter baumannii is a critical pathogen, as severe clinical implications have developed from isolates resistant to current antibiotic treatments and conventional control procedures, such as UV light, disinfectants, and drying. Here, we review the natural AMPs representing primary candidates for new anti-A. baumannii drugs in post-antibiotic-era and present computational tools to develop the next generation of AMPs with greater microbicidal activity and reduced toxicity.

A genetic selection for Mycobacterium smegmatis mutants tolerant to killing by sodium citrate defines a combined role for cation homeostasis and osmotic stress in cell death.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis. RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE, treZ, and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis. Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.

Endosperm cell death promoted by NAC transcription factors facilitates embryo invasion in Arabidopsis

In flowering plants, two fertilization products develop within the limited space of the seed: the embryo and the surrounding nutritive endosperm. The final size of the endosperm is modulated by the degree of embryo growth. In Arabidopsis thaliana, the endosperm expands rapidly after fertilization, but later gets invaded by the embryo that occupies most of the seed volume at maturity, surrounded by a single remaining aleurone-like endosperm layer.1,2,3,4 Embryo invasion is facilitated by the endosperm-expressed bHLH-type transcription factor ZHOUPI, which promotes weakening of endosperm cell walls.5,6 Endosperm elimination in zou mutants is delayed, and embryo growth is severely affected; the endosperm finally collapses around the dwarf embryo, causing the shriveled appearance of mature zou seeds.5,6,7 However, whether ZHOUPI facilitates mechanical endosperm destruction by the invading embryo or whether an active programmed cell death (PCD) process causes endosperm elimination has been subject to debate.2,8 Here we show that developmental PCD controlled by multiple NAC transcription factors in the embryo-adjacent endosperm promotes gradual endosperm elimination. Misexpressing the NAC transcription factor KIRA1 in the entire endosperm caused total endosperm elimination, generating aleurone-less mature seeds. Conversely, dominant and recessive higher-order NAC mutants led to delayed endosperm elimination and impaired cell corpse clearance. Promoting PCD in the zhoupi mutant partially rescued its embryo growth defects, while the endosperm in a zhoupi nac higher-order mutant persisted until seed desiccation. These data suggest that a combination of cell wall weakening and PCD jointly facilitates embryo invasion by an active auto-elimination of endosperm cells.

Cellulase mediated stress triggers the mutations of oleaginous yeast Trichosporon cutaneum with super-large spindle morphology and high lipid accumulation.

Accumulation of intracellular lipid bodies in oleaginous yeast cells is highly restricted by their natural intracellular space. Here we show a cellulase mediated adaptive evolution with ultra-centrifugation fractionation of oleaginous yeast Trichosporon cutaneum to obtain the favorable cell structure for lipid accumulation. Cellulase was added to the wheat straw hydrolysate during long-term adaptive evolution for disruption of cell wall integrity of T. cutaneum cells. The cellulase, together with ultracentrifugation force, triggered multiple mutations and transcriptional expression changes of the functional genes associated with cell wall integrity and lipid synthesis metabolism. The fractionated mutant T. cutaneum YY52 demonstrated the heavily weakened cell wall and high lipid accumulation by the super-large expanded spindle cells (two orders of magnitude greater than the parental). A record-high lipid production by T. cutaneum YY52 was achieved (55.4±0.5gL-1 from wheat straw and 58.4±0.1gL-1 from corn stover). This study not only obtained an oleaginous yeast strain with industrial application potential for lipid production but also provided a new method for generation of mutant cells with high intracellular metabolite accumulation.

Biomechanical Weakening of Paper and Plant Cell Walls by Bacterial Expansins.

Expansins are proteins that loosen plant cell walls but lack enzymatic activity. Here we describe two protocols tailored to measure the biomechanical activity of bacterial expansin. The first assay relies on the weakening of filter paper by expansin. The second assay is based on induction of creep (long-term, irreversible extension) of plant cell wall samples.

Disrupted cellulose aggregation leads to the reduced mechanical performance of wood–adhesive interphase during freeze–thaw cycles

The mechanical performance of engineered wooden products facing the freeze–thaw cycles (FTCs) arises as an attention-worthy issue since the application of timber architectures in cold climates spreads. Here, we reported an investigation to reveal the losses of the mechanical performance of the wood-phenol formaldehyde (PF) adhesive interphase after the FTCs. Results revealed that PF adhesive was barely affected by the FTCs due to the low moisture content and rigid networks, whereas the mechanical properties of the cell wall in wood-PF interphase reduced significantly (more than 30%) after 5 FTCs at – 40 °C. Cracks were observed in the cell wall and compound middle lamella after FTCs. Further investigation into the crystal structure of the cell wall in the wood-PF interphase demonstrated that the FTCs disrupt the aggregations of cellulose macromolecules. The stresses caused by the phase transition of free water and the external hydrogen bonds formed between water and cellulose disrupted hydrogen bond networks in the cell wall. A plausible mechanism for the FTCs reducing the mechanical properties of the wood-PF bonds can be concluded as the cracks and weakened cell walls crippled the structural integrity of the wood-PF interphase, making it a fragile and stress-concentrated site when subjected to load.

Comparative study on the structural and in vitro digestion properties of starch within potato parenchyma cells under different cooking methods

To study the effects of cooking methods on the structure and digestion changes of starch encapsulated by cellular structure, intact potato parenchyma cells were successfully isolated and then subjected to different domestic cooking methods, including baking, frying, boiling, and autoclaving. The morphology, crystalline structure, thermal properties, and in vitro starch digestibility of cooked cell samples were investigated. Our results indicated that potato cell walls remained intact and performed as physical barriers preventing the diffusion/absorption of α-amylase to intracellular starch substrates after baking or frying treatment. However, boiling or autoclaving treatment destroyed cell wall structure, and the disrupted cellular structure reduced the digestion rate, likely by inhibiting diffusion of amylase through a weakened cell wall barrier, but could not lower the final digestion extent when compared to the pure starch. These findings suggested that potato products with lower glycemic index can be obtained by baking or frying treatment.

The RNA-binding protein Puf5 and the HMGB protein Ixr1 contribute to cell cycle progression through the regulation of cell cycle-specific expression of CLB1 in Saccharomyces cerevisiae.

Puf5, a Puf-family RNA-binding protein, binds to 3´ untranslated region of target mRNAs and negatively regulates their expression in Saccharomyces cerevisiae. The puf5Δ mutant shows pleiotropic phenotypes including a weakened cell wall, a temperature-sensitive growth, and a shorter lifespan. To further analyze a role of Puf5 in cell growth, we searched for a multicopy suppressor of the temperature-sensitive growth of the puf5Δ mutant in this study. We found that overexpression of CLB2 encoding B-type cyclin suppressed the temperature-sensitive growth of the puf5Δ mutant. The puf5Δ clb2Δ double mutant displayed a severe growth defect, suggesting that Puf5 positively regulates the expression of a redundant factor with Clb2 in cell cycle progression. We found that expression of CLB1 encoding a redundant B-type cyclin was decreased in the puf5Δ mutant, and that this decrease of the CLB1 expression contributed to the growth defect of the puf5Δ clb2Δ double mutant. Since Puf5 is a negative regulator of the gene expression, we hypothesized that Puf5 negatively regulates the expression of a factor that represses CLB1 expression. We found such a repressor, Ixr1, which is an HMGB (High Mobility Group box B) protein. Deletion of IXR1 restored the decreased expression of CLB1 caused by the puf5Δ mutation and suppressed the growth defect of the puf5Δ clb2Δ double mutant. The expression of IXR1 was negatively regulated by Puf5 in an IXR1 3´ UTR-dependent manner. Our results suggest that IXR1 mRNA is a physiologically important target of Puf5, and that Puf5 and Ixr1 contribute to the cell cycle progression through the regulation of the cell cycle-specific expression of CLB1.

Genome-Wide Identification and Expression Analysis of XTH Gene Family during Flower-Opening Stages in Osmanthus fragrans.

Osmanthus fragrans is an aromatic plant which is widely used in landscaping and garden greening in China. However, the process of flower opening is significantly affected by ambient temperature changes. Cell expansion in petals is the primary factor responsible for flower opening. Xyloglucan endoglycolase/hydrolase (XTH) is a cell-wall-loosening protein involved in cell expansion or cell-wall weakening. Through whole-genome analysis, 38 OfXTH genes were identified in O. fragrans which belong to the four main phylogenetic groups. The gene structure, chromosomal location, synteny relationship, and cis-acting elements prediction and expression patterns were analyzed on a genome-wide scale. The expression patterns showed that most OfXTHs were closely associated with the flower-opening period of O. fragrans. At the early flower-opening stage (S1 and S2), transcriptome and qRT-PCR analysis revealed the expression of OfXTH24, 27, 32, 35, and 36 significantly increased under low ambient temperature (19 °C). It is speculated that the five genes might be involved in the regulation of flower opening by responding to ambient temperature changes. Our results provide solid foundation for the functional analysis of OfXTH genes and help to explore the mechanism of flower opening responding to ambient temperature in O. fragrans.

Effects of a novel weakened whole‐cell form of Haematococcus pluvialis on flesh pigmentation of rainbow trout ( Oncorhynchus mykiss ) when compared to synthetic astaxanthin

Fillet colour is an important quality indicator for consumers. To achieve flesh colouration in farmed salmonids, synthetic astaxanthin (Ax) pigment is added to the feed. Cost, safety and consumer preference are increasing research efforts for non-synthetic Ax alternatives. Haematococcus pluvialis (Hp) is an efficient producer of Ax but has not been effective in pigmenting salmonid flesh due to its rigid cell wall structure. A novel form of Hp has been cultivated to possess a weakened cell wall, which may increase Ax bioavailability. To assess pigmenting ability, Ax retention, tissue content and fillet colouration were measured in rainbow trout (Onchorhynchus mykiss) fed diets containing this pre-commercial Hp product. Sixty rainbow trout (992 g ± 177.24 g) were PIT (passive integrated transponder)-tagged, distributed among six tanks and fed one of three experimental diets: No Ax (N-Ax), synthetic Ax (S-Ax; 80 mg/kg Ax) or Ax from Hp whole cells (Hp-Ax; 60 mg/kg Ax). After 10 weeks, tissue Ax content and flesh colour were assessed. No significant (p > 0.05) differences were evident in either growth metrics, fillet Ax content, retention or colouration were observed between S-Ax and Hp-Ax treatments. Ax retention was 15.4 ± 0.23%, respectively, for fish fed the Hp diet. SalmoFan™ colour readings of S-Ax and Hp-Ax fillets ranged from 28 to 34 and were significantly higher than N-Ax fillets (p < 0.001). This novel whole-cell Haematococcus pluvialis was equally efficient at pigmenting the flesh of rainbow trout compared with synthetic Ax.

The C4 Atriplex halimus vs. the C3 Atriplex hortensis: Similarities and Differences in the Salinity Stress Response

Soil properties and the ability to sustain agricultural production are seriously impaired by salinity. The cultivation of halophytes is seen as a solution to cope with the problem. In this framework, a greenhouse pot experiment was set up to assess salinity response in the perennial C4 species Atriplex halimus, and in the following three cultivars of the annual C3 Atriplex hortensis: green, red, and scarlet. The four genotypes were grown for 35 days with water salinity (WS) ranging from 0 to 360 mM NaCl. Plant height and fresh weight (FW) increased at 360 vs. 0 WS. The stomatal conductance (GS) and transpiration rate (E) were more severely affected by salinity in the C4 A. halimus than in the C3 species A. hortensis. This was reflected in a lower leaf water potential indicating stronger osmotic adjustment, and a higher relative water content associated with more turgid leaves, in A. halimus than A. hortensis. In a PCA including all the studied traits, the GS and E negatively correlated to the FW, which, in turn, positively correlated with Na concentration and intrinsic water use efficiency (iWUE), indicating that reduced gas exchange associated with Na accumulation contributed to sustain iWUE under salinity. Finally, FTIR spectroscopy showed a reduced amount of pectin, lignin, and cellulose under salinity, indicating a weakened cell wall structure. Overall, both species were remarkably adapted to salinity: From an agronomic perspective, the opposite strategies of longer vs. faster soil coverage, involved by the perennial A. halimus vs. the annual A. hortensis cv. scarlet, are viable natural remedies for revegetating marginal saline soils and increasing soil organic carbon.

Efficient electrotransformation of Rhodococcus ruber YYL with abundant extracellular polymeric substances via a cell wall-weakening strategy.

Rhodococcus spp. have broad potential applications related to the degradation of organic contaminants and the transformation or synthesis of useful compounds. However, some Gram-positive bacteria are difficult to manipulate genetically due to low transformation efficiency. In this study, we investigated the effects of chemicals including glycine, isonicotinic acid hydrazide (INH), Tween 80 and penicillin G, as well as cell growth status, competent cell concentration, electroporation field strength, electroporation time and heat shock time, on the electrotransformation efficiency of the tetrahydrofuran-degrading bacterium Rhodococcus ruber YYL with low transformation efficiency. The highest electrotransformation efficiency was 1.60× 106 CFU/µg DNA after parameter optimization. GmhD (D-glycero-D-manno-heptose 1-phosphate guanosyltransferase) gene, which is important in the biosynthesis of lipopolysaccharide, was deleted via the optimized electrotransformation method. Compared with wild-type strain, YYL ΔgmhD showed extremely high electrotransformation efficiency because the surface of it had no mushroom-like extracellular polymeric substances (EPS). In addition, the results showed that cell wall-weakening reagents might cause some translucent substances like EPS, to detach from the cells, increasing the electrotransformation efficiency of strain YYL. We propose that these results could provide a new strategy for unique bacteria that are rich in EPS, for which genetic manipulation systems are difficult to establish.