The dgc2 gene encoding di-guanylate cyclase suppresses both motility and biofilm formation in the filamentous cyanobacterium Leptolyngbya boryana.

Colony pattern formations of bacteria with motility manifest complicated morphological self-organization phenomena. Leptolyngbya boryana is the filamentous cyanobacterial species, which has been used as a genetic model organism for studying metabolism including photosynthesis and nitrogen-fixation. Although a widely used type strain (wild type) of this species has not been reported to show any motile activity, we isolated a spontaneous mutant strain which shows active motility (gliding activity) to give rise to complicated colony patters, including comet-like wandering clusters and disk-like rotating vortices on solid media. Whole-genome resequencing identified multiple mutations on the genome in the mutant strain. We confirmed that inactivation of a candidate gene, dgc2 (LBDG_02920), in the wild type background was sufficient to give rise to motility and the morphological colony patterns. This gene encodes a protein, containing the GGDEF motif, which is conserved at the catalytic domain of diguanylate cyclase (DGC). Although DGC has been reported to be involved in biofilm formation, the mutant strain lacking dgc2 significantly facilitated biofilm formation, suggesting a role of DGC for suppressing both gliding motility and biofilm formation. Thus, L. boryana provides an excellent genetic model to study dynamic colony pattern formation, and novel insight on a role of c-di-GMP for biofilm formation.

The salivary, metal-binding peptide histatin-5 buffers extracellular copper availability

The family of human salivary histidine-rich peptides known as histatins bind zinc (Zn) and copper (Cu), but whether they contribute to nutritional immunity by influencing Zn and/or Cu availability has not been examined. We hypothesised that histatin-5 (Hst5) limits Zn availability (and promotes bacterial Zn starvation) and/or raises Cu availability (and promotes bacterial Cu poisoning). To test this hypothesis, Group A Streptococcus (GAS), which colonises the human oropharynx, was used as a model bacterium. Contrary to our hypothesis, Hst5 did not strongly influence Zn availability. This peptide did not induce expression of Zn uptake genes in GAS, nor did it suppress growth of an ΔadcAI mutant strain that is impaired in Zn uptake. Equilibrium competition measurements confirmed that Hst5 binds Zn weakly and does not compete with the high-affinity Zn uptake protein AdcAI for binding Zn. By contrast, Hst5 bound Cu with a high affinity and strongly influenced Cu availability. However, contrary to our hypothesis, Hst5 did not promote Cu toxicity. Instead, this peptide suppressed expression of Cu-inducible genes in GAS, stopped intracellular accumulation of Cu, and rescued growth of a ΔcopA mutant strain that is impaired in Cu efflux in the presence of added Cu. These findings led us to propose a new role for Hst5 and salivary histatins as major Cu buffers in saliva that reduce the potential negative effects of Cu exposure to microbes. We speculate that histatins promote oral and oropharyngeal health by contributing to microbial homeostasis in these host niches.

Prospecting the plant growth–promoting activities of endophytic bacteria Cronobacter sp. YSD YN2 isolated from Cyperus esculentus L. var. sativus leaves

Purpose Plant growth–promoting (PGP) bacteria are an environment-friendly alternative to chemical fertilizers for promoting plant growth and development. We isolated and characterized a PGP endophyte, YSD YN2, from the leaves of Cyperus esculentus L. var. sativus. Methods Specific PGP characteristics of this strain, such as phosphate solubilization ability, potassium-dissolving ability, siderophore and indole-3-acetic acid (IAA) production, and salt tolerance, were determined in vitro. In addition, positive mutants were screened using the atmospheric and room temperature plasma (ARTP) technology, with IAA level and organic phosphate solubility as indices. Furthermore, the effect of the positive mutant on seed germination, biomass production, and antioxidant abilities of greengrocery seedling was evaluated, and the genome was mined to explore the underlying mechanisms. Results The strain YSD YN2 showed a good performance of PGP characteristics, such as the production of indole acetic acid and siderophores, solubilization ability of phosphate, and potassium-dissolving ability. It was recognized through 16S rRNA sequencing together with morphological and physiological tests and confirmed as Cronobacter sp. The strain exposed to a mutation time of 125 s by ARTP had the highest IAA and organic phosphate (lecithin) concentrations of 9.25 mg/L and 16.50 mg/L, 50.41% and 30.54% higher than those of the initial strain. Inoculation of mutant strain YSD YN2 significantly increased the seed germination, plant growth attributes, and the activities of peroxidase (POD) and superoxide dismutase (SOD), respectively, but decreased the content of malondialdehyde (MDA) significantly compared with the control. Furthermore, genome annotation and functional analysis were performed through whole-genome sequencing, and PGP-related genes were identified. Conclusion Our results indicated that the mutant strain YSD YN2 with PGP characteristics is a potential candidate for the development of biofertilizers.

Screening of Cucumber Fusarium Wilt Bio-Inhibitor: High Sporulation Trichoderma harzianum Mutant Cultured on Moso Bamboo Medium

Cucumber fusarium wilt is a soil-borne disease which causes serious production decrease in cucumber cultivation world widely. Extensive using of chemical pesticides has caused serious environmental pollution and economic losses, therefore, it is particularly urgent to develop efficient, safe and pollution-free biopesticide. In this study, a mutant strain of Trichoderma harzianum cultivated in moso bamboo medium was proved to be an efficient bio-inhibitor of the disease. The mutant strain T. harzianum T334, was obtained by three microwave mutagenesis cycles with an irradiation power of 600 W and irradiation time of 40 s. In contrast to the original strain, the inhibition rate on cucumber fusarium wilt of the strain T334 increased from 63 to 78%. In this work, disk milling pretreatment of moso bamboo has shown significant beneficial effects on both biotransformation and sporulation of T334. Its sporulation reached 3.7 × 109 cfu/g in mushroom bags with 90% bamboo stem powder (pretreated by disk milli), 9.5% bamboo leaf powder and 0.5% wheat bran when the ratio of solid to liquid was 4:6, the inoculum amount was 10%, and the culture temperature was 28°C. These results provide an alternative bioinhibitor for the control of cucumber fusarium wilt, and a potential usage of moso bamboo in the production of microbial pesticide.

Enhanced Production of Citric Acid by Mutant PN12 of Aspergillus fumigatus Using Statistical Design

Distillery spent wash is an unwanted residual liquid waste generated during alcohol production. It is a potential source for production of different industrially important products. Distillery spent wash is dark colored and has many organic compounds as a waste. In this experiment, removal of color and organic compounds was carried out by anaerobic treatment. The treated spent wash was utilized for citric acid production with the help of microorganisms. The current study was performed with the treated spent wash which was applied for high level of citric acid production by a mutant strain of Aspergillus fumigatus PN12. The parent strain Aspergillus fumigatus PN12 was mutagenized by UV exposure to enhance citric acid production. After UV exposure investigation, mutant strain was selected for optimization and statistical method. The best citric acid production obtained was, 26.45 g/L at 30 ℃ with pH 6.0, 0.1 g/L of KH2PO4 and (NH4)2SO4 under OFAT. Under RSM optimization, maximum citric acid production was achieved as 30.89 g/L. Thus, the process optimization through the statistical approach resulted in a 1.16-fold enhancement in citric acid production as compared to that of the OFAT parametric conditions. Citric acid producing enzymes such as aconitase, NAD+-isocitrate dehydrogenase and NADP+ isocitrate dehydrogenase was studied. Maximum activity (U/mg) of aconitase (3.19±0.023), NAD+-isocitrate dehydrogenase (3.0±0.15) and NADP+ isocitrate dehydrogenase (2.91±0.17) was observed at 96 h. The present study can conclude that spent wash is potential source for citric acid production. Utilization of mutant strain of Aspergillus fumigatus PN12 is beneficiary for large scale industrial fermentation and citric acid production.

De novo arginine synthesis is required for full virulence of Xanthomonas arboricola pv. juglandis during Walnut Bacterial Blight Disease

Walnut blight (WB) disease caused by Xanthomonas arboricola pv. juglandis (Xaj) threatens orchards worldwide. Nitrogen metabolism in this bacterial pathogen is dependent on arginine, a nitrogen enriched amino acid that can either be synthesized or provided by the plant host. The arginine biosynthetic pathway uses argininosuccinate synthase (argG), associated with increased bacterial virulence. We examined the effects of bacterial arginine and nitrogen metabolism on the plant response during WB by proteomic analysis of the mutant strain Xaj argG-. Phenotypically, the mutant strain produced 42% fewer symptoms and survived in the plant tissue with 2.5-fold reduced growth compared to wild-type (WT) while auxotrophic for arginine in vitro. Proteomic analysis of infected tissue enabled the profiling of 676 Xaj proteins and 3,296 walnut proteins using isobaric labeling in a data-dependent acquisition approach. Comparative analysis of differentially expressed proteins revealed distinct plant responses. Xaj WT triggered processes of catabolism and oxidative stress in the host under observed disease symptoms, while most host’s biosynthetic processes triggered by Xaj WT were inhibited during Xaj argG- infection. Overall, the Xaj proteins revealed a drastic shift in carbon and energy management induced by disruption of nitrogen metabolism while the top differentially expressed proteins included a Fis transcriptional regulator and a peptidyl-prolyl isomerase. Our results show the critical role of de novo arginine biosynthesis to sustain virulence and minimal growth during WB. This study is timely and critical as current copper-based control methods are losing their effectiveness, and new sustainable methods are urgently needed in orchard environments.

Role of the Mn-Catalase in Aerobic Growth of Lactobacillus plantarum ATCC 14431

Lactobacilli are Gram-positive aerotolerant organisms that comprise the largest genus of Lactic Acid Bacteria (LAB). Most lactobacilli are devoid of the antioxidant enzymes, superoxide dismutases, and catalases, required for protection against superoxide radicals and hydrogen peroxide (H2O2), respectively. However, some lactobacilli can accumulate millimolar concentrations of intracellular manganese and spare the need for superoxide dismutase, while others possess non-heme catalases. L. plantarum is associated with plant materials and plays an important role in fermented foods and gut microbiomes. Therefore, understanding the effects of the environment on the growth and survival of this organism is essential for its success in relevant industrial applications. In this report, we investigated the physiological role of Mn-catalase (MnKat) in Lactobacillus plantarum ATCC 14431. To this end, we compared the physiological and morphological properties of a ΔMnkat mutant strain and its isogenic parental strain L. plantarum ATCC 14431. Our data showed that the MnKat is critical for the growth of L. plantarum ATCC 14431 in the presence of oxygen and resistance to H2O2. The aerobic growth of the mutant in presence or absence of H2O2 was improved in the Mn-rich medium (APT) as compared to the growth in MRS medium. Furthermore, under aerobic conditions the mutant strain possessed atypical cellular morphology (i.e., shorter, and fatter). In conclusion, the MnKat of L. plantarum ATCC 14431 is important for aerobic growth, protection against H2O2, and maintenance of the rod-shaped cell morphology under aerobic conditions.

Enhancement of membrane vesicle production by disrupting the degP gene in Meiothermus ruber H328

The phenomenon of membrane vesicle (MV) production is known to be common to all bacterial cells. Although MVs are expected to be employed in a variety of applications, improving MV productivity is essential for applications. Since the deletion of the degP gene, a periplasmic dual-function protease and chaperone, in Escherichia coli has successfully improved MV production capacity, we tried to enhance MV productivity in the thermophilic M. ruber H328 by deleting the degP gene. One gene (mrH_0331) was selected for degP gene from the H328 genome and we constructed the mutant strain ∆degP by deleting the degP gene of the H328 strain that was replaced with the htk gene showing thermophilic kanamaycin resistance by homologous recombination. The mutant strain ∆degP exhibited smooth growth but a lower level of turbidity at 60 °C although there was no difference in growth at 55 °C between the wild strain and the mutant strain. Finally, we have confirmed that incubation at 60 °C increases MV in the mutant strain ∆degP strain about fivefold by using two fluorescent dyes, DiI and FM4-64, which is followed by TEM analysis. The deletion of the degP gene presumably causes an increase in denatured proteins at 60 °C, leading to enhanced MV production. Meanwhile, the S-layer protein included in the outer membrane of the H328 strain increased in the MV fraction prepared from the mutant cells incubated at 60 °C. This indicates that this method is effective for MV production and that degP deletion enhances it in strain H328.