C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormone signaling intersect to promote shallow lateral root angles.

Root system architecture (RSA) influences the acquisition of heterogeneously dispersed soil nutrients. Cytokinin and C-TERMINALLY ENCODED PEPTIDE (CEP) hormones affect RSA, in part by controlling the angle of lateral root (LR) growth. Both hormone pathways converge on CEP DOWNSTREAM 1 (CEPD1) and CEPD2 to control primary root growth; however, a role for CEPDs in controlling the growth angle of LRs is unknown. Using phenotyping combined with genetic and grafting approaches, we show that CEP hormone-mediated shallower LR growth requires cytokinin biosynthesis and perception in roots via ARABIDOPSIS HISTIDINE KINASE 2 (AHK2) and AHK3. Consistently, cytokinin biosynthesis and ahk2,3 mutants phenocopied the steeper root phenotype of cep receptor 1 (cepr1) mutants on agar plates, and CEPR1 was required for trans-Zeatin (tZ)-type cytokinin-mediated shallower LR growth. In addition, the cepd1,2 mutant was less sensitive to CEP and tZ, and showed basally steeper LRs on agar plates. Cytokinin and CEP pathway mutants were grown in rhizoboxes to define the role of these pathways in controlling RSA. Only cytokinin receptor mutants and cepd1,2 partially phenocopied the steeper-rooted phenotype of cepr1 mutants. These results show that CEP and cytokinin signaling intersect to promote shallower LR growth, but additional components contribute to the cepr1 phenotype in soil.

Thalassospira aquimaris sp. nov. and Winogradskyella marincola sp. nov. two marine bacteria isolated from an agar-degrading co-culture.

Two novel Gram-stain-negative, aerobic, and non-motile strains, designated FZY0004T and YYF002T, were isolated from an agar-degrading co-culture, which was obtained from seawater of the intertidal zone of Yancheng City, the Yellow Sea of China. Strain FZY0004T optimally grew at 28 °C, pH 7.0, and 2-6% NaCl, while strain YYF002T optimally grew at 28 °C, pH 7.5, and 2-4% NaCl. Strain FZY0004T possessed Q-9 as the major respiratory quinone, and its major fatty acids (> 10%) were summed feature 8 (C18:1 ω7c), C16:0, and summed feature 3 (C16:1 ω7c/C16:1 ω6c). The polar lipids identified in strain FZY0004T were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and several unidentified phospholipids (PL) and lipids (L). On the other hand, strain YYF002T had MK-6 as the predominant respiratory quinone and its major fatty acids consisted of iso-C15:0, iso-C15:1 G, and iso-C15:0 3-OH. The polar lipids identified in strain YYF002T were aminolipid (AL), PE, and several unidentified lipids. Strain FZY0004T shared 99.5% 16S rRNA gene sequence similarity and 90.1% average nucleotide identity (ANI) with T. povalilytica Zumi 95T, and strain YYF002T shared 99.2% 16S rRNA gene sequence similarity and 88.2% ANI with W. poriferorum JCM 12885T. The genomic DNA G + C contents of strains FZY0004T and YYF002T were 54.5% and 33.5%, respectively. The phylogenetic, phenotypic, and physiological characteristics permitted the distinction of the two strains from their neighbors, and we thus propose the names Thalassospira aquimaris sp. nov. (type strain FZY0004T = JCM 35895T = MCCC 1K08380T) and Winogradskyella marincola sp. nov. (type strain YYF002T = JCM 35950T = MCCC 1K08382T).

LC-HRMS Profiling and Cytotoxic Potential of Actinomycetes Associated with the Red Sea Soft Coral Sarcophyton glaucum: In vitro and In silico Studies.

In the current study, the actinomycetes associated with the red sea-derived soft coral Sarcophyton glaucum were investigated in terms of biological and chemical diversity. Four different media, M1, ISP2, Marine Agar (MA), and Actinomycete isolation agar (AIA) were used for the isolation of three strains of actinomycetes that were identified as Streptomyces sp. UR 25, Micromonospora sp. UR32 and Saccharomonospora sp. UR 19. LC-HRMS analysis was used to investigate the chemical diversity of the isolated actinobacteria. The LC-HRMS data were statistically processed using MetaboAnalyst 5.0 viz to differentiate the extract groups and determine the optimal growth culturing conditions. Multivariate data statistical analysis revealed that the Micromonospora sp. extract cultured on (MA) medium is the most distinctive extract in terms of chemical composition. While, the Streptomyces sp. UR 25 extracts are differ significantly from Micromonospora sp. UR32 and Saccharomonospora sp. UR 19. Biological investigation using in vitro cytotoxic assay for actinobacteria extracts revealed the prominent potentiality of the Streptomyces sp. UR 25 cultured on oligotrophic medium against human hepatoma (HepG2), human breast adenocarcinoma (MCF-7) and human colon adenocarcinoma (CACO2) cell lines (IC50 =3.3, 4.2 and 6.8 µg/mL, respectively). SwissTarget Prediction speculated that among the identified compounds, 16-deethyl, indanomycin (8) could have reasonable affinity on HDM2 active site. In this respect, molecular docking study was performed for compound (8) to reveal a substantial affinity on HDM2 active site. In addition, molecular dynamics simulations were carried out at 200 ns for the most active compound (8) compared to the co-crystallized inhibitor DIZ giving deeper information regarding their thermodynamic and dynamic properties as well.

Performance evaluation of Trichoderma reseei in tolerance and biodegradation of diuron herbicide in agar plate, liquid culture and solid-state fermentation.

The present study evaluated the performance of the fungus Trichoderma reesei to tolerate and biodegrade the herbicide diuron in its agrochemical presentation in agar plates, liquid culture, and solid-state fermentation. The tolerance of T. reesei to diuron was characterized through a non-competitive inhibition model of the fungal radial growth on the PDA agar plate and growth in liquid culture with glucose and ammonium nitrate, showing a higher tolerance to diuron on the PDA agar plate (inhibition constant 98.63 mg L-1) than in liquid culture (inhibition constant 39.4 mg L-1). Diuron biodegradation by T. reesei was characterized through model inhibition by the substrate on agar plate and liquid culture. In liquid culture, the fungus biotransformed diuron into 3,4-dichloroaniline using the amide group from the diuron structure as a carbon and nitrogen source, yielding 0.154 mg of biomass per mg of diuron. A mixture of barley straw and agrolite was used as the support and substrate for solid-state fermentation. The diuron removal percentage in solid-state fermentation was fitted by non-multiple linear regression to a parabolic surface response model and reached the higher removal (97.26%) with a specific aeration rate of 1.0 vkgm and inoculum of 2.6 × 108 spores g-1. The diuron removal in solid-state fermentation by sorption on barley straw and agrolite was discarded compared to the removal magnitude of the biosorption and biodegradation mechanisms of Trichoderma reesei. The findings in this work about the tolerance and capability of Trichoderma reesei to remove diuron in liquid and solid culture media demonstrate the potential of the fungus to be implemented in bioremediation technologies of herbicide-polluted sites.

A coevolution experiment between Flavobacterium johnsoniae and Burkholderia thailandensis reveals parallel mutations that reduce antibiotic susceptibility.

One interference mechanism of bacterial competition is the production of antibiotics. Bacteria exposed to antibiotics can resist antibiotic inhibition through intrinsic or acquired mechanisms. Here, we performed a coevolution experiment to understand the long-term consequences of antibiotic production and antibiotic susceptibility for two environmental bacterial strains. We grew five independent lines of the antibiotic-producing environmental strain, Burkholderia thailandensis E264, and the antibiotic-inhibited environmental strain, Flavobacterium johnsoniae UW101, together and separately on agar plates for 7.5 months (1.5 month incubations), transferring each line five times to new agar plates. We observed that the F. johnsoniae ancestor could tolerate the B. thailandensis-produced antibiotic through efflux mechanisms, but that the coevolved lines had reduced susceptibility. We then sequenced genomes from the coevolved and monoculture F. johnsoniae lines, and uncovered mutational ramifications for the long-term antibiotic exposure. The coevolved genomes from F. johnsoniae revealed four potential mutational signatures of reduced antibiotic susceptibility that were not observed in the evolved monoculture lines. Two mutations were found in tolC: one corresponding to a 33 bp deletion and the other corresponding to a nonsynonymous mutation. A third mutation was observed as a 1 bp insertion coding for a RagB/SusD nutrient uptake protein. The last mutation was a G83R nonsynonymous mutation in acetyl-coA carboxylayse carboxyltransferase subunit alpha (AccA). Deleting the 33 bp from tolC in the F. johnsoniae ancestor reduced antibiotic susceptibility, but not to the degree observed in coevolved lines. Furthermore, the accA mutation matched a previously described mutation conferring resistance to B. thailandensis-produced thailandamide. Analysis of B. thailandensis transposon mutants for thailandamide production revealed that thailandamide was bioactive against F. johnsoniae, but also suggested that additional B. thailandensis-produced antibiotics were involved in the inhibition of F. johnsoniae. This study reveals how multi-generational interspecies interactions, mediated through chemical exchange, can result in novel interaction-specific mutations, some of which may contribute to reductions in antibiotic susceptibility.

Isolation, characterization and screening of actinomycetes associated with fijian ant-plant symbioses.

In the search for novel therapeutics to combat the ongoing antimicrobial resistance crisis, scientists are turning to underexplored environments. Defensive mutualisms between hymenopteran insects and actinomycetes represent important reservoirs for bioactive compounds. In this study, we examined the association between actinomycetes and Squamellaria ant-plants spanning three different ant and plant species combinations (Squamellaria imberbis-Philidris nagasau, Squamellaria tenuiflora- Technomyrmex vitiensis, and Squamellaria tenuiflora-Tetramorium insolens). Eight Squamellaria plants were sampled including four containing T. vitiensis, three containing P. nagasau, and a single plant containing T. insolens. A total of 47 actinomycetes were obtained from the sampled material, with 5, 16, and 26 isolates originating from cuticle, tissue, and nest samples, respectively. Cross-streaking tests showed that 12 out of 47 isolates inhibited bacterial pathogens. The most frequently inhibited pathogens in the cross-streaking tests were S. aureus and E. coli while S. enterica was the least inhibited. Among the three primary screening media used, ISP2 agar was the most suitable for secondary metabolism as more isolates exhibited antibacterial activity when grown on the medium. TFS2010 and TFS2003, which matched to Streptomyces gramineus (>99% similarity), were the most bioactive isolates in cross-streaking tests. TFS2010 displayed the strong antibacterial on Nutrient agar, Mueller Hinton agar, and ISP2 agar while TFS2003 only exhibited strong antibacterial activity on Nutrient agar. Furthermore, a difference in potency of extracts based on batch culture medium was noted in TFS2010. DNA was extracted from 19 isolates and followed by 16SrRNA gene sequencing. Analysis of sequence data revealed the presence of six genera, including Amycolatopsis, Asanoa, Jiangella, Nocardia, Nocardiopsis, and Streptomyces, with the latter being the most abundant taxon. Among these, three isolates (PNS3002, PNS3005, and TFS3001) are likely to represent new species while one (TFS2015) is likely to be a member of a novel genus. Our work represents the first attempt to study actinomycetes from Squamellaria-ant mutualisms.

Exploiting spatial dimensions to enable parallelized continuous directed evolution.

Current strategies to improve the throughput of continuous directed evolution technologies often involve complex mechanical fluid-controlling system or robotic platforms, which limits their popularization and application in general laboratories. Inspired by our previous study on bacterial range expansion, in this study, we report a system termed SPACE for rapid and extensively parallelizable evolution of biomolecules by introducing spatial dimensions into the landmark phage-assisted continuous evolution system. Specifically, M13 phages and chemotactic Escherichia coli cells were closely inoculated onto a semisolid agar. The phages came into contact with the expanding front of the bacterial range, and then comigrated with the bacteria. This system leverages competition over space, wherein evolutionary progress is closely associated with the production of spatial patterns, allowing the emergence of improved or new protein functions. In a prototypical problem, SPACE remarkably simplified the process and evolved the promoter recognition of T7 RNA polymerase (RNAP) to a library of 96 random sequences in parallel. These results establish SPACE as a simple, easy to implement, and massively parallelizable platform for continuous directed evolution in general laboratories.

High-throughput bacterial co-encapsulation in microfluidic gel beads for discovery of antibiotic-producing strains.

Bacteria with antagonistic activity inhibit the growth of other bacteria through different mechanisms, including the production of antibiotics. As a result, these microorganisms are a prolific source of such compounds. However, searching for antibiotic-producing strains requires high-throughput techniques due to the vast diversity of microorganisms. Here, we screened and isolated bacteria with antagonistic activity against Escherichia coli expressing the green fluorescent protein (E. coli-GFP). We used microfluidics to co-encapsulate and co-culture single cells from different strains within picoliter gel beads and analyzed them using fluorescence-activated cell sorting (FACS). To test the methodology, we used three bacterial isolates obtained from Mexican maize, which exhibit high, moderate, or no antagonistic activity against E. coli-GFP, as determined previously using agar plate assays. Single cells from each strain were separately co-incubated into gel beads with E. coli-GFP. We monitored the development of the maize bacteria microcolonies and tracked the growth or inhibition of E. coli-GFP using bright-field and fluorescent microscopy. We correlated these images with distinctive light scatter and fluorescence signatures of each incubated bead type using FACS. This analysis enabled us to sort gel beads filled with an antagonistic strain, starting from a mixture of the three different types of maize bacteria and E. coli-GFP. Likewise, culturing the FACS-sorted beads on agar plates confirmed the isolation and recovery of the two antagonistic strains. In addition, enrichment assays demonstrated the methodology's effectiveness in isolating rare antibiotic-producer strains (0.01% abundance) present in a mixture of microorganisms. These results show that associating light side scatter and fluorescent flow cytometry signals with microscopy images provides valuable controls to establish successful high-throughput methods for sorting beads in which microbial interaction assays are performed.

Bifunctional and monofunctional α-neoagarooligosaccharide hydrolases from Streptomyces coelicolor A3(2).

Agar is a galactan and a major component of the red algal cell wall. Agar is metabolized only by specific microorganisms. The final step of the ß-agarolytic pathway is mediated by α-neoagarooligosaccharide hydrolase (α-NAOSH), which cleaves neoagarobiose to D-galactose and 3,6-anhydro-α-L-galactose. In the present study, two α-NAOSHs, SCO3481 and SCO3479, were identified in Streptomyces coelicolor A3(2). SCO3481 (370 amino acids, 41.12 kDa) and SCO3479 (995 amino acids, 108.8 kDa) catalyzed the hydrolysis of the α-(1,3) glycosidic bonds of neoagarobiose, neoagarotetraose, and neoagarohexaose at the nonreducing ends, releasing 3,6-anhydro-α-L-galactose. Both were intracellular proteins without any signal peptides for secretion. Similar to all α-NAOSHs reported to date, SCO3481 belonged to the glycosyl hydrolase (GH) 117 family and formed dimers. On the other hand, SCO3479 was a large monomeric α-NAOSH belonging to the GH2 family with a ß-galactosidase domain. SCO3479 also clearly showed ß-galactosidase activity toward lactose and artificial substrates, but SCO3481 did not. The optimum conditions for α-NAOSH were pH 6.0 and 25 °C for SCO3481, and pH 6.0 and 30 °C for SCO3479. Enzymatic activity was enhanced by Co2+ for SCO3481 and Mg2+ for SCO3479. The ß-galactosidase activity of SCO3479 was maximum at pH 7.0 and 50 °C and was increased by Mg2+. Many differences were evident in the kinetic parameters of each enzyme. Although SCO3481 is typical of the GH117 family, SCO3479 is a novel α-NAOSH that was first reported in the GH2 family. SCO3479, a unique bifunctional enzyme with α-NAOSH and ß-galactosidase activities, has many advantages for industrial applications. KEY POINTS: • SCO3481 is a dimeric α-neoagarooligosaccharide hydrolase belonging to GH117. • SCO3479 is a monomeric α-neoagarooligosaccharide hydrolase belonging to GH2. • SCO3479 is a novel and unique bifunctional enzyme that also acts as a ß-galactosidase.

Strength in Numbers: Density-Dependent Volatile-Induced Antimicrobial Activity by Xanthomonas perforans.

For most of the 20th century, Xanthomonas euvesicatoria was the only known bacterium associated with bacterial spot of tomato in Florida. X. perforans quickly replaced X. euvesicatoria, mainly because of production of three bacteriocins (BCNs) against X. euvesicatoria; however, X. perforans outcompeted X. euvesicatoria even when the three known BCNs were deleted. Surprisingly, we observed antimicrobial activity against X. euvesicatoria in the BCN triple mutant when the triple mutant was grown in Petri plates containing multiple spots but not in Petri plates containing only one spot. We determined that changes in the headspace composition (i.e., volatiles) rather than a diffusible signal in the agar were required for induction of the antimicrobial activity. Other Xanthomonas species also produced volatile-induced antimicrobial compounds against X. euvesicatoria and elicited antimicrobial activity by X. perforans. A wide range of plant pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis, Pantoea stewartii, and Pseudomonas cichorii, also elicited antimicrobial activity by X. perforans when multiple spots of the species were present. To identify potential antimicrobial compounds, we performed liquid chromatography with high-resolution mass spectrometry of the agar surrounding the spot in the high cell density Petri plates where the antimicrobial activity was present compared with agar surrounding the spot in Petri plates with one spot where antimicrobial activity was not observed. Among the compounds identified in the zone of inhibition were N-butanoyl-L-homoserine lactone and N-(3-hydroxy-butanoyl)-homoserine lactone, which are known quorum-sensing metabolites in other bacteria.

Analysis of biofilm expansion rate of Bacillus subtilis (MTC871) on agar substrates with different stiffness.

The surface morphology of mature biofilms is heterogeneous and can be divided into concentric rings wrinkles (I), labyrinthine networks wrinkles (II), radial ridges wrinkles (III), and branches wrinkles (IV), according to surface wrinkle structure and distribution characteristics. Due to the wrinkle structures, channels are formed between the biofilm and substrate and transport nutrients, water, metabolic products, etc. We find that expansion rate variations of biofilms growing on substrates with high and low agar concentrations (1.5, 2.0, 2.5 wt.%) are not in the same phase. In the first 3 days' growth, the interaction stress between biofilm and each agar substrate increases, which makes the biofilm expansion rate decreases before wrinkle pattern IV (branches) comes up. After 3 days, in the later growth stage after wrinkle pattern IV appears, the biofilm has larger expansion rate growing on 2.0 wt.% agar concentration, which has the larger wrinkle distance in wrinkle pattern IV reducing energy consumption. Our study shows that the stiff substrate does not always inhibit the biofilm expansion, although it does in the earlier stage; after that, mature biofilms acquire larger expansion rate by adjusting the growth mode through the wrinkle evolution even in nutrient extremely depletion.

Screening and characterization of agarolytic bacteria from different sources.

According to the results of our investigation, distinct bacterial isolates capable of breaking down agar were found in various nonmarine environments. The deficiency of reducing sugar in the control media demonstrates that the agar in the experiment is broken down by the bacteria to produce various oligosaccharides because the viscosity of the medium containing the agar was found to have been extremely high before inoculation, reducing with incubation duration and attaining a maximum after 48 hours. These isolates were subsequently used in tests along with additional investigation since they could create reducing sugar. Interestingly, the deterioration of agar appears to be mainly caused by Gram-negative bacteria. In order to study the agarase properties, the relative quantity of the enzyme secreted by the bacteria that hydrolyze the agar was used. The detection of extracellular agarase surrounding the colonies and the absence of stained halos on iodine-treated agar plates show that the agarase diffusing from the bacteria impacted the characteristics of the gel. Inconclusion, these agarsase-producing bacteria can be exploited for industrial applications. Waste agar from the plant tissue culture business can be utilized for a range of applications and this degraded agar can be explored for reliable and ecologically safe alternatives.

[Inhibition of GMA-induced ubiquitination process in 16HBE malignantly transformed cells on protein CD44 and MMP14].

OBJECTIVE: To observe the effect of the ubiquitination process on the expression of CD44 antigen(CD44) and matrix metalloproteinase-14(MMP14) in human bronchial epithelial(16HBE) malignantly transformed cells induced by glycidyl methacrylate(GMA). METHODS: Successfully resuscitated 16HBE cells were cultured using a final concentration of 8 µg/mL GMA as the treatment group and 1 µg/mL dimethyl sulfoxide as the solvent control group, each time stained for 72 h, and then stained again after an interval of 24 h. After repeating the staining three times, the cells were cultured in passages respectively. The 40th generation(P40) GMA-treated group and the same-generation solvent control group were subjected to soft agar colony formation assay and concanavalin A(ConA) agglutination test to confirm that the 40th generation of GMA-induced malignant transformed 16HBE cells possessed malignant transformed cell characteristics.5, 10, 20, 40, 60 µmol/L anacardic acid were used to inhibit the ubiquitination process of GMA-induced malignant transformed 16HBE cells. The protein expression of CD44 and MMP14 were detected by western blotting, while the transcript levels of CD44, MMP14, and TFAP2A were assessed by real-time fluorescence quantitative PCR(qPCR). RESULTS: (1) In the soft agar colony formation assay, the number of clones formed by the cells in the solvent control group was 22, and the number of clones created by the malignantly transformed cells in the GMA-treated group was 208. In the ConA agglutination test, the cells in the solvent control group were uniformly dispersed in ConA solution, and no obvious agglutination occurred for 30 min, whereas the cells in the GMA-treated group were agglutinated in the 5th min, and the agglutinated cells were larger and more rapidly agglutinated. The agglomerates were more significant and faster, and the sensitivity of agglutination was increased. (2) After differential inhibition of GMA-induced ubiquitination in malignantly transformed 16HBE cells, the expression levels of CD44 and MMP14 were reduced in GMA-induced malignantly transformed 16HBE cells compared with the control group(P<0.05). The transcript levels of MMP14 and CD44 decreased with increasing inhibitor concentration(P<0.05), and the transcript levels of the upstream transcription factor TFAP2A were also simultaneously reduced(P<0.05). CONCLUSION: Inhibition of the cellular ubiquitination process mediates the down-regulation of protein expression and transcriptional expression of CD44 and MMP14 in GMA-induced malignantly transformed 16HBE cells.

1-Deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells.

Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids (SLs) that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I, resulting in the synthesis and accumulation of 1-deoxysphingolipids (deoxySLs). These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxySL synthesis, they impact numerous other metabolic pathways important for cancer cells. Here, we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxySL toxicity in cancer cells. We determined that both alanine treatment and SPTLC1C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found that spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxySL synthesis was induced via SPTLC1C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxySL synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.

Development of an Experimental Approach to Achieve Spatially Resolved Plant Root-Associated Metaproteomics Using an Agar-Plate System.

Plant-microbe interactions in the rhizosphere play a vital role in plant health and productivity. The composition and function of root-associated microbiomes is strongly influenced by their surrounding environment, which is often customized by their host. How microbiomes change with respect to space and time across plant roots remains poorly understood, and methodologies that facilitate spatiotemporal metaproteomic studies of root-associated microbiomes are yet to be realized. Here, we developed a method that provides spatially resolved metaproteome measurements along plant roots embedded in agar-plate culture systems, which have long been used to study plants. Spatially defined agar "plugs" of interest were excised and subsequently processed using a novel peptide extraction method prior to metaproteomics, which was used to infer both microbial community composition and function. As a proof-of-principle, a previously studied 10-member community constructed from a Populus root system was grown in an agar plate with a 3-week-old Populus trichocarpa plant. Metaproteomics was performed across two time points (24 and 48 h) for three distinct locations (root base, root tip, and a region distant from the root). The spatial resolution of these measurements provides evidence that microbiome composition and expression changes across the plant root interface. Interrogation of the individual microbial proteomes revealed functional profiles related to their behavioral associations with the plant root, in which chemotaxis and augmented metabolism likely supported predominance of the most abundant member. This study demonstrated a novel peptide extraction method for studying plant agar-plate culture systems, which was previously unsuitable for (meta)proteomic measurements.

AtFTCD-L, a trans-Golgi network localized protein, modulates root growth of Arabidopsis in high-concentration agar culture medium.

MAIN CONCLUSION: AtFTCD-L protein is localized on the TGN vesicles in Arabidopsis root cap cells. AtFTCD-L mutation resulted in slow root growth of Arabidopsis in high-concentration agar culture medium. Arabidopsis formiminotransferase cyclodeaminase-like protein (AtFTCD-L) in Arabidopsis is homologous to the formiminotransferase cyclodeaminase (FTCD) protein in animal cells. However, the localization and function of AtFTCD-L remain unknown in Arabidopsis. In this study, we generated and analyzed a deletion mutant of AtFTCD-L with a T-DNA insertion. We found that the growth of Arabidopsis roots with the T-DNA insertion mutation in AtFTCD-L was slower than that of wild-type roots when grown in high-concentration 1/2 MS agar culture medium. AtFTCD-L-GFP could restore the ftcd-l mutant phenotype. In addition, the AtFTCD-L protein was localized on the trans-Golgi network (TGN) vesicles in Arabidopsis root cap cells. Fluorescence recovery after photobleaching (FRAP) experiment using Arabidopsis pollen-specific receptor-like kinase-GFP (AtPRK1-GFP) stably transformed plants showed that the deficiency of AtFTCD-L protein in Arabidopsis led to slower secretion in the root cap peripheral cells. The AtFTCD-L protein deficiency also resulted in a significantly reduced monosaccharides content in the culture medium. Based on the above results, we speculate that the AtFTCD-L protein may be involved in sorting and/or transportation of TGN vesicles in root cap peripheral cells, thereby regulating the extracellular secretion of mucilage components in the root cap.

Prevalence of Type IV Pili-Mediated Twitching Motility in Streptococcus sanguinis Strains and Its Impact on Biofilm Formation and Host Adherence.

Type IV pili (Tfp) are known to mediate several biological activities, including surface-dependent twitching motility. Although a pil gene cluster for Tfp biosynthesis is found in all sequenced Streptococcus sanguinis strains, Tfp-mediated twitching motility is less commonly detected. Upon examining 81 clinical strains, 39 strains generated twitching zones on blood agar plates (BAP), while 27 strains displayed twitching on Todd-Hewitt (TH) agar. Although BAP appears to be more suitable for the development of twitching zones, 5 strains exhibited twitching motility only on TH agar, indicating that twitching motility is not only strain specific but also sensitive to growth media. Furthermore, different twitching phenotypes were observed in strains expressing comparable levels of pilT, encoding the retraction ATPase, suggesting that the twitching phenotype on agar plates is regulated by multiple factors. By using a PilT-null and a pilin protein-null derivative (CHW02) of twitching-active S. sanguinis CGMH010, we found that Tfp retraction was essential for biofilm stability. Further, biofilm growth was amplified in CHW02 in the absence of shearing force, indicating that S. sanguinis may utilize other ligands for biofilm formation in the absence of Tfp. Similar to SK36, Tfp from CGMH010 were required for colonization of host cells, but PilT only marginally affected adherence and only in the twitching-active strain. Taken together, the results suggest that Tfp participates in host cell adherence and that Tfp retraction facilitates biofilm stability. IMPORTANCE Although the gene clusters encoding Tfp are commonly present in Streptococcus sanguinis, not all strains express surface-dependent twitching motility on agar surfaces. Regardless of whether the Tfp could drive motility, Tfp can serve as a ligand for the colonization of host cells. Though many S. sanguinis strains lack twitching activity, motility can enhance biofilm stability in a twitching-active strain; thus, perhaps motility provides little or no advantage to the survival of bacteria within dental plaque. Rather, Tfp retraction could provide additional advantages for the bacteria to establish infections outside the oral cavity.

Visualization of mRNA Expression in Pseudomonas aeruginosa Aggregates Reveals Spatial Patterns of Fermentative and Denitrifying Metabolism.

Gaining insight into the behavior of bacteria at the single-cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize the expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels across heterogeneous populations such as biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the air-agar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression remained at peak levels deeper into anoxic environments than other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and they demonstrate the utility of HCR in unveiling cellular activities at the microscale level in heterogeneous populations. IMPORTANCE To understand bacteria in diverse contexts, we must understand the variations in behaviors and metabolisms they express spatiotemporally. Populations of bacteria are known to be heterogeneous, but the ways this variation manifests can be challenging to characterize due to technical limitations. By focusing on energy conservation, we demonstrate that HCR v3.0 can visualize nuances in gene expression, allowing us to understand how metabolism in Pseudomonas aeruginosa biofilms responds to microenvironmental variation at high spatial resolution. We validated probes for four catabolic genes, including a constitutively expressed oxidase, acetate kinase, nitrite reductase, and nitrous oxide reductase. We showed that the genes for different modes of metabolism are expressed in overlapping but distinct subpopulations according to oxygen concentrations in a predictable fashion. The spatial transcriptomic technique described here has the potential to be used to map microbial activities across diverse environments.

Production of selenomethionine labeled polyglycine hydrolases in Pichia pastoris.

Producing recombinant proteins with incorporated selenomethionine (SeMet) facilitates solving X-ray crystallographic structures of novel proteins. Production of SeMet labeled proteins in the yeast Pichia pastoris (syn. Komagataella phaffii) is difficult because SeMet is mildly toxic, reducing protein expression levels. To counteract this yield loss for a novel protease, Epicoccum sorghi chitinase modifying protein (Es-cmp), a novel disease promoting protease secreted by these plant pathogenic fungi, we isolated a yeast strain that secreted more protein. By comparing the expression level of 48 strains we isolated one that produced significantly more protein. This strain was found to be gene dosed, having four copies of the expression cassette. After optimization the strain produced Es-cmp in defined media with SeMet at levels nearly equal to that of the original strain in complex media. Also, we produced SeMet labeled protein for a homologous protease from the fungus Fusarium vanettenii, Fvan-cmp, by directly selecting a gene dosed strain on agar plates with increased zeocin. Linearization of plasmid with PmeI before electroporation led to high numbers of 1 mg/mL zeocin resistant clones with significantly increased expression compared to those selected on 0.1 mg/mL. The gene dosed strains expressing Es-cmp and Fvan-cmp allowed production of 8.5 and 16.8 mg of SeMet labeled protein from 500 mL shake flask cultures. The results demonstrate that selection of P. pastoris expression strains by plating after transformation on agar with 1 mg/mL zeocin rather than the standard 0.1 mg/mL directly selects gene dosed strains that can facilitate production of selenomethionine labeled proteins.

Oligosaccharides from agar extends lifespan through activation of unfolded protein response via SIR-2.1 in Caenorhabditis elegans.

PURPOSE: Agaro-oligosaccharides (AGO), hydrolysis products of agarose, is known to have antioxidant and anti-inflammatory properties. Speculating that AGO is effective for preventing aging, we investigated the longevity-supporting effects of AGO and their mechanisms using Caenorhabditis elegans. METHODS: Caenorhabditis elegans were fed AGO from young adulthood. The lifespan, locomotory activity, lipofuscin accumulation, and heat stress resistance of the worms were examined. To elucidate mechanisms of AGO-mediated longevity, we conducted comprehensive expression analysis using microarrays. Moreover, we used quantitative real-time PCR (qRT-PCR) to verify the genes showing differential expression levels. Furthermore, we measured the lifespan of loss-of-function mutants to determine the genes related to AGO-mediated longevity. RESULTS: AGO extended the lifespan of C. elegans, reduced lipofuscin accumulation, and maintained vigorous locomotion. The microarray analysis revealed that the endoplasmic reticulum-unfolded protein response (ER-UPR) and insulin/insulin-like growth factor-1-mediated signaling (IIS) pathway were activated in AGO-fed worms. The qRT-PCR analysis showed that AGO treatment suppressed sir-2.1 expression, which is a negative regulator of ER-UPR. In loss-of-function mutant of sir-2.1, AGO-induced longevity and heat stress resistance were decreased or cancelled completely. Furthermore, the pro-longevity effect of AGO was decreased in loss-of-function mutants of abnormal Dauer formation (daf) -2 and daf-16, which are IIS pathway-related genes. CONCLUSION: AGO delays the C. elegans aging process and extends their lifespan through the activations of ER-UPR and the IIS pathway.