Strain MT1 Research Articles

Lactobacillus johnsonii is a dominant Lactobacillus in the murine oral mucosa and has chitinase activity that compromises fungal cell wall integrity.

The interactions between the opportunistic pathogen Candida albicans and resident oral bacteria are particularly crucial in maintaining oral health. Emerging antifungal drug-resistant strains, slow-paced drug discovery, and the risk of side effects can compromise the effectiveness of current treatments available for oropharyngeal candidiasis. This study advances the search for alternative microbiome-targeted therapies in oral fungal infections. We report that Lactobacillus johnsonii strain MT4 prevents the Candida-induced bloom of dysbiotic oral enterococci and reduces oral mucosal lesions in an oropharyngeal candidiasis murine model. We also show that this strain directly compromises the cell wall and reduces fungal metabolic activity, partly due to its chitinase activity.

Expression of Concern: Bacillus rubiinfantis sp. nov. strain mt2(T), a new bacterial species isolated from human gut

Expression of Concern: Bacillus rubiinfantis sp. nov. strain mt2(T), a new bacterial species isolated from human gut

Microbial Decolorization of Crystal Violet Dye by a Native Multi-Metal Tolerant <i>Aeromonas caviae</i> MT-1 Isolate from Dye-Contaminated Soil: Optimization and Phytotoxicity Study

In the recent past, one of the main environmental issues is the contamination of textile dye wastes. The toxicity of dyes poses adverse effects on the flora and fauna of the ecosystem. The present study aimed to isolate bacteria that decolourize crystal violet dye, optimization of various environmental factors for effective decolourization, and phytotoxicity analysis. Out of 13 isolated bacteria, a single isolate was able to grow at 250 mg/L crystal violet dye concentration in a synthetic medium and identified as Aeromonas caviae MT-1 strain (accession number; LC720408) using morphological, biochemical and molecular analyses. Presumably, this is the first report of crystal violet dye decolourization by a native Aeromonas caviae isolate. In this study, after a 72-hour incubation period, a maximum of 98.0% dye decolourization was observed at neutral pH and 35°C with 5% v/v bacterial culture under static culture conditions. Dye decolourization was inhibited to a significant degree by the rising of its concentration. UV-Vis spectra analysis of samples before and after decolourization showed the possible degradation of crystal violet dye by A. caviae. The strain MT-1 was also tolerant to toxic heavy metals like arsenic, lead, and chromium. Phytotoxicity tests revealed that decolourized dye products inhibited Vigna radiata growth less than the un-decolourized dye solution. The findings revealed that a native multi-metal tolerant A. caviae MT-1 isolate could decolourize crystal violet dye rapidly, and possibly have the ability for extensive treatment of dyecontaminated waste.

Subcellular Architecture of the xyl Gene Expression Flow of the TOL Catabolic Plasmid of Pseudomonas putida mt-2.

ABSTRACTDespite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida strain mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid, and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they colocalize with ribosome-rich domains of the intracellular milieu. This arrangement was maintained even when the xyl genes were artificially relocated to different chromosomal locations. The same held true when genes were expressed through a heterologous T7 polymerase-based system, which likewise led to mRNA foci outside the DNA. In contrast, rifampin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs exit from their initiation sites to move to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest the distinct subcellular motion of xyl mRNAs results from both innate properties of the sequences and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed within the background of current knowledge on the three-dimensional organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism.

Phytohormones producing rhizobacterium alleviates chromium toxicity in Helianthus annuus L. by reducing chromate uptake and strengthening antioxidant system

Contamination of agricultural land with heavy metal is a serious biological and environmental issue. Such threat can be challenged by exploring the plant symbiotic microbes that can improve plant growth through phyto-hormones secretion and chromate chelation. In the current study, chromate resistant rhizospheric Staphylococcus arlettae strain MT4 was isolated from the rhizosphere of Malvestrum tricuspadatum L. The strain showed potential to secrete phytohormones and plant growth promoting secondary metabolites under induced chromate stress, making it a best suitable candidate in chromate stress alleviation. Moreover, the rhizobacterium MT4 significantly promoted the net assimilation and relative growth rate of sunflower grown in the presence of chromate (100 ppm). Chromate stress alleviation strategy of MT4 strain was three-fold. MT4 alleviated chromate stress and promoted the sunflower growth by suppressing the chromate intake by the host, modulating phytohormones and strengthening of the host’s antioxidant system. The improved antioxidant system was confirmed by noticing lower ROS accumulation and improved ROS scavenging, lower peroxidase activity and higher accumulation of phenols and flavonoids.

Biochemical characterisation and application of keratinase from Bacillus thuringiensis MT1 to enable valorisation of hair wastes through biosynthesis of vitamin B-complex

This study reports on the exploitation of keratinous hydrolysate by keratinase enzymes to produce vitamin B-complex. Toward this end, keratinase enzyme was produced by Bacillus thuringiensis strain MT1, newly isolated from cattle-yard utilising donkey hairs. Scanning electron microscope (SEM) and Fourier transform infrared spectrophotometer (FTIR) analyses demonstrated hairs disintegration and the disruption of the disulphide bonds of the keratin structure, respectively. The biochemical characterisation of the produced enzyme exhibited optimal activity of 422 U/ml at 50 °C and pH 9 with a molecular mass of 80 kDa. The enzyme activity was entirely deactivated by Ethylenediaminetetraacetic acid (EDTA), implying the existence of a metallokeratinase group. Donkey hairs were thus treated with metallokeratinase, emancipating eight essential and eight more non-essential amino acids, which were identified employing amino acid analyser. These amino acids were subsequently utilised by Saccharomyces cerevisiae strain ATCC 64712, at different concentrations, to produce vitamin B-complex. High-performance liquid chromatography (HPLC) analysis revealed the synthesis of vitamins B1, B2, and B12 at various levels associated with concentrations of supplemented amino acids. This report thus highlights the feasible application of keratinase enzyme as an eco-friendly approach to managing hair waste, and concurrently promotes the implementation of hair-based hydrolysate in vitamin B-complex biosynthesis.

Whole Genome Sequence of Dermacoccus abyssi MT1.1 Isolated from the Challenger Deep of the Mariana Trench Reveals Phenazine Biosynthesis Locus and Environmental Adaptation Factors.

Dermacoccus abyssi strain MT1.1T is a piezotolerant actinobacterium that was isolated from Mariana Trench sediment collected at a depth of 10898 m. The organism was found to produce ten dermacozines (A‒J) that belonged to a new phenazine family and which displayed various biological activities such as radical scavenging and cytotoxicity. Here, we report on the isolation and identification of a new dermacozine compound, dermacozine M, the chemical structure of which was determined using 1D and 2D-NMR, and high resolution MS. A whole genome sequence of the strain contained six secondary metabolite-biosynthetic gene clusters (BGCs), including one responsible for the biosynthesis of a family of phenazine compounds. A pathway leading to the biosynthesis of dermacozines is proposed. Bioinformatic analyses of key stress-related genes provide an insight into how the organism adapted to the environmental conditions that prevail in the deep-sea.

Isolation of marine xylene-utilizing bacteria and characterization of Halioxenophilus aromaticivorans gen. nov., sp. nov. and its xylene degradation gene cluster.

Seven xylene-utilizing bacterial strains were isolated from seawater collected off the coast of Japan. Analysis of 16S rRNA gene sequences indicated that six isolates were most closely related to the marine bacterial genera Alteromonas, Marinobacter or Aestuariibacter. The sequence of the remaining strain, KU68FT, showed low similarity to the 16S rRNA gene sequences of known bacteria with validly published names, the most similar species being Maricurvus nonylphenolicus strain KU41ET (92.6% identity). On the basis of physiological, chemotaxonomic and phylogenetic data, strain KU68FT is suggested to represent a novel species of a new genus in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria, for which the name Halioxenophilus aromaticivorans gen. nov., sp. nov. is proposed. The type strain of Halioxenophilus aromaticivorans is KU68FT (=JCM 19134T=KCTC 32387T). PCR and sequence analysis revealed that strain KU68FT possesses an entire set of genes encoding the enzymes for the upper xylene methyl-monooxygenase pathway, xylCMABN, resembling the gene set of the terrestrial Pseudomonas putida strain mt-2.

The Dichotomy of the Poly(ADP-Ribose) Polymerase-Like Thermozyme from Sulfolobus solfataricus

The first evidence of an ADP-ribosylating activity in Archaea was obtained in Sulfolobus solfataricus(strain MT-4) where a poly(ADP-ribose) polymerase (PARP)-like thermoprotein, defined with the acronymous PARPSso, was found. Similarly to the eukaryotic counterparts PARPSso cleaves beta-nicotinamide adenine dinucleotide to synthesize oligomers of ADP-ribose; cross-reacts with polyclonal anti-PARP-1 catalytic site antibodies; binds DNA. The main differences rely on the molecular mass (46.5 kDa) and the thermophily of PARPSso which works at 80 °C. Despite the biochemical properties that allow correlating it to PARP enzymes, the N-terminal and partial amino acid sequences available suggest that PARPSso belongs to a different group of enzymes, the DING proteins, an item discussed in detail in this review.This finding makes PARPSso the first example of a DING protein in Archaea and extends the existence of DING proteins into all the biological kingdoms. PARPSsohas a cell peripheral localization, along with the edge of the cell membrane. The ADP-ribosylation reaction is reverted by a poly(ADP-ribose) glycohydrolase-like activity, able to use the eukaryotic poly(ADP-ribose) as a substrate too. Here we overview the research of (ADP-ribosyl)ation in Sulfolobus solfataricus in the past thirty years and discuss the features of PARPSso common with the canonical poly(ADP-ribose) polymerases, and the structure fitting with that of DING proteins.

Regulation of general amino acid permeases Gap1p, GATA transcription factors Gln3p and Gat1p on 2-phenylethanol biosynthesis via Ehrlich pathway

In Saccharomyces cerevisiae, when l-phenylalanine (l-Phe) is used as the sole nitrogen source, 2-phenylethanol (PE) is mainly synthesized via the Ehrlich pathway. General amino acid permease Gap1p is response of aromatic amino acids transportation, and GATA transcription factors Gln3p and Gat1p regulate the transcription of permease gene and catabolic enzyme genes for nitrogen sources and aromatic amino acids utilization. In this study, it was demonstrated that over-expressing GLN3 gene from industrial yeast strain MT2 or S. cerevisiae haploid strain YS58, 2-PE synthesis levels of recombinant strains increased 54% or 40% than that of the control strain, which suggested that higher Gln3p activity in yeast has positive regulation effect on 2-PE biosynthesis via Ehrlich pathway. The recombinant strains with over-expression of GAT1 gene from MT2 or YS58 also up-regulated Ehrlich pathway for 2-PE biosynthesis and increased 2-PE production. Similarly, when GAP1 gene respectively from MT2 or YS58 was over-expressed, 2-PE yield was improved obviously, suggesting that GAP1 over-expressing in yeast also promoted Ehrlich pathway to produce 2-PE. The synergistic regulation of GLN3/GAT1 or GLN3/GAP1 over-expression was similar to that of single factor over-expression. Among these regulatory factors, Gln3p of industrial yeast strain MT2 caused stronger regulation on target genes than that of haploid strain YS58, which might be due to the differences in translational efficiency or nuclear localization of each Gln3p, or due to their different spatial structures and binding domains. Further results showed that efficient Gln3p expression in MT2 brought about higher 2-PE, 3.59gL−1, which was of potential significant for commercial exploitation.

A Glycosyltransferase from Sulfolobus solfataricus MT-4 Exhibits Poly(ADP-ribose) Glycohydrolase Activity

Anti-poly(ADP-ribose) glycohydrolase immunoblotting of a lysate from Sulfolobus solfataricus (strain MT-4) cells showed a main intense signal close to the 37 kDa protein marker. The immunoreactive protein was purified by electroelution and showed a hydrolysing activity towards oligomers (1- 6 residues) of ADP-ribose similar to eukaryotc poly(ADP-ribose) glycohydrolase. This protein was characterized as it regards enzymatic inhibition by adenosine diphosphate- (hydroxymethyl)pyrrolidine-3,4-diol, a known inhibitor of eukaryotic poly(ADP-ribose) glycohydrolase, and by analysis of reaction products. ADP-ribose polymer electrophoresis and thin layer chromatography clearly showed that the enzyme was able to monomerize Sulfolobus solfataricus MT-4 (ADP-ribose)1-6, an oligomer recognized also by eukaryotic poly (ADP-ribose) glycohydrolases. Edman degradation of the purified protein allowed to determine a short N-terminal sequence: Met-Ile-Ser-Val-Ala. This pentapeptide was used for a blast search towards Sulfolobus solfataricus genomes. It gave evidence of a 40 kDa-protein present only in two strains (P2 and 98/2) of Sulfolobus solfataricus. Oligonucleotide primers drawn on the cDNA of human poly(ADP-ribose) glycohydrolase gave a fragment of the corresponding Sulfolobus solfataricus MT-4 gene overlapping the sequences from the genomes of Sulfolobus solfataricus P2 and 98/2. Translation of the sequence confirmed the occurrence of a region with some amino acids matching the human poly(ADP-ribose) glycohydrolase “signature”. Keywords: Crenarchaeon, hyperthermophile, poly(ADP-ribose), poly(ADP-ribose) glycohydrolase, Sulfolobus; thermoprotein.

Designing a Response Surface Model for Removing Phosphate and Organic Compound from Wastewater by Pseudomonas Strain MT1

Improvement in wastewater bioremediation in water treatment refineries has attracted attention recently. For this, a strain of Pseudomonas aeruginosa MT1 with phosphate accumulating capacity was isolated from the topsoil of an agricultural land through screening in an enrichment medium based on decolorizing Toluidine blue-O dye. Two experimental design approaches were adopted to find the optimum condition for phosphate and organic carbon removal using the strain. In the first approach, one factor at a time method was employed with different carbon sources (glycerol, sodium citrate, sodium acetate and frying oil), pH and temperature range as well as KH2PO4 and NH4Cl concentrations. Maximum bacterial growth (250 mg/l) and phosphate sequestration (10.5 % of total phosphate) were achieved at pH 7.5, 30–35 °C and in frying oil as carbon source within 48 h incubation, while KH2PO4 in the range of 5–15 mg/l was regarded as ineffective. KH2PO4, sodium acetate concentrations, pH and agitation speed ranges were re-used in the second experimental approach using central composite design from response surface methodology. All experimental runs were conducted at lab-scale in a liquid synthetic wastewater with the variation of the factors whose supernatant chemical oxygen demand and phosphate contents were measured at the end of the batch incubation. In comparison, the response surface method resulted in more promising yield (50 and 38 % improvement in organic carbon and phosphate removal, respectively) and was more informative than one factor at a time approach in understanding the distinctive and interactive effects on the objective responses.

Thermus anatoliensis sp. nov., a thermophilic bacterium from geothermal waters of Buharkent, Turkey.

A Gram-stain-negative, lack of motility, catalase- and oxidase- positive bacterium (strain MT1(T)) was isolated from Buharkent hot spring in Aydin, Turkey. Its taxonomy was investigated using a polyphasic approach. The strain was able to grow at 45-80 °C, pH 5.5-10.5 and with a NaCI tolerance up to 2.0% (w/v). Strain MT1(T) was able to utilize d-mannitol and l-arabinose, not able to utilize d-cellobiose as sole carbon source. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Thermus; strain MT1(T) detected low-level similarities of 16S rRNA gene sequences (below 97%) compared with all other species in this genus. The predominant fatty acids of strain MT1(T) were iso-C(15:0) (43.0%) and iso-C(17:0) (27.4%). Polar lipid analysis revealed a major phospholipid, one major glycolipid, one major aminophospholipid, two minor aminolipids, one minor phospholipid, and several minor glycolipids. The major isoprenoid quinone was MK-8. The DNA G+C content of MT1(T) was 69.6 mol%. On the basis of a taxonomic study using a polyphasic approach, strain MT1(T) is considered to represent a novel species of the genus Thermus, for which the name Thermus anatoliensis sp. nov. is proposed. The type strain is MT1(T) (=NCCB 100425(T) =LMG 26880(T)).

Engineering of global regulator cAMP receptor protein (CRP) in Escherichia coli for improved lycopene production

Transcriptional engineering has received significant attention for improving strains by modulating the behavior of transcription factors, which could be used to reprogram a series of gene transcriptions and enable multiple simultaneous modifications at the genomic level. In this study, engineering of the cAMP receptor protein (CRP) was explored with the aim of subtly balancing entire pathway networks and potentially improving lycopene production without significant genetic intervention in other pathways. Amino acid mutations were introduced to CRP by error-prone PCR, and three variants (mcrp26, mcrp159 and mcrp424) with increased lycopene productivity were screened. Combinations of three point mutations were then created via site-directed mutagenesis. The best mutant gene (mcrp26) was integrated into the genome of E. coli BW25113-BIE to replace the wild-type crp gene (MT-1), which resulted in a higher lycopene production (18.49mg/g DCW) compared to the original strain (WT). The mutant strain MT-1 was further investigated in a 10-L bench-top fermentor with a lycopene yield of 128mg/l at 20h, approximately 25% higher than WT. DNA microarray analyses showed that 396 genes (229 up-regulated and 167 down-regulated) were differentially expressed in the mutant MT-1 compared to WT. Finally, the introduction of the mutant crp gene (mcrp26) increased β-carotene production in E. coli. This is the first report of improving the phenotype for metabolite overproduction in E. coli using a CRP engineering strategy.

Genome Sequence of the Deep-Sea Denitrifier Pseudomonas sp. Strain MT-1, Isolated from the Mariana Trench.

Pseudomonas sp. strain MT-1 was the first deep-sea denitrifier isolated and characterized from mud recovered from a depth of 11,000 m in the Mariana Trench. We report here the genome sequence of this bacterium, which contributes to our understanding of denitrification and bioenergetics in the deep sea.

Dermacozines H–J Isolated from a Deep-Sea Strain of Dermacoccus abyssi from Mariana Trench Sediments

Dermacoccus abyssi sp. nov. strains MT1.1 and MT1.2 are actinomycetes isolated from a Mariana Trench sediment at a depth of 10 898 m. The fermentation process using complex media led to the production of three new pigmented heteroaromatic (oxidized and reduced) phenazine compounds, dermacozines H-J (1-3). Extensive use was made of 1D and 2D NMR experiments and high-resolution MS to determine the structures of the compounds. The new dermacozines showed radical scavenging activity, and the highest activity was observed for dermacozine H (1), with an IC50 value of 18.8 μM.

Correlation of thermostability and conformational changes of catechol 2, 3-dioxygenases from two disparate micro-organisms

We have investigated the structure of recombinant catechol 2, 3-dioxygenase (C23O) purified from two species in which the enzyme has evolved to function at different temperature. The two species are mesophilic bacterium Pseudomonas putida strain mt-2 and thermophilic archaea Sulfolobus acidocaldariusDSM639. Using the primary sequence analysis, we show that both C23Os have only 30% identity and 48% similarity but contain conserved amino acid residues forming an active site area around the iron ion. The corresponding differences in homology, but structural similarity in active area residues, appear to provide completely different responses to heating the two enzymes. We confirm this by small angle X-ray scattering and demonstrate that the overall structure of C23O from P. putida is slightly different from its crystalline form whereas the solution scattering of C23O from S. acidocaldarius at temperatures between 4 and 85°C ideally fits the calculated scattering from the single crystal structure. The thermostability of C23O from S. acidocaldarius correlates well with conformation in solution during thermal treatment. The similarity of the two enzymes in primary and tertiary structure may be taken as a confirmation that two enzymes have evolved from a common ancestor.

As(V)-reduction to As(III) by arsenic-resistant Bacillus spp. bacterial strains isolated from low-contaminated sediments of the Oliveri-Tindari Lagoon, Italy

Five arsenic-resistant bacterial strains designated MT1, MT2, MT3, V1 and V2 were isolated from sediments of the Oliveri-Tindari Lagoon (Italy), which comprises six small lakes whose sediments contain low arsenic concentrations. Phylogenetic analysis of the 16S rRNA gene sequences assigned them to the genus Bacillus. Bacillus sp. strain MT3 showed higher tolerance to As(III) and As(V), as indicated by minimum inhibitory concentrations of 14 and 135 mmol−1, respectively. Bacillus sp. strain V1 showed growth inhibition at 14 mmol−1 in the presence of As(III) and at 68 mmol−1 in the presence of As(V), whereas the arsenic resistance of Bacillus sp. strain MT1 was 10 and 27 mmol−1 for As(III) and As(V), respectively. The strains Bacillus spp. MT2 and V2 showed low levels of As(III) and As(V) resistance, as it was unable to grow at concentrations>7 and 14 mmol−1, respectively. The isolated arsenic-resistant Bacillus spp. strains were able to reduce As(V) to As(III), especially Bacillus spp. strain MT3. This study suggests that the isolated bacterial strains play a role in the arsenic biogeochemical cycle of arsenic-poor sediments in the Oliveri-Tindari Lagoon.

Kinetics of carbon sharing in a bacterial consortium revealed by combining stable isotope probing with fluorescence-activated cell sorting

To determine the kinetics of substrate fluxes in a microbial community in order to elucidate the roles of the community members. The kinetics of substrate sharing in a bacterial consortium were measured by a new analytical approach combining immunostaining, stable isotope probing and fluorescence-activated cell sorting (FACS). The bacterial consortium, consisting of four strains and growing on 4-chlorosalicylate (4-CS), was pulse-dosed with the degradation intermediate [U-(13) C]-4-chlorocatechol (4-CC). Cells were stained with strain-specific antibodies sorted by FACS and the (13) C-incorporation into fatty acids of the two most abundant members of the community was determined by isotope ratio mass spectrometry. From the two most abundant strains, the primary degrader Pseudomonas reinekei MT1 incorporated the labelled substrate faster than strain Achromobacter spanius MT3 but the maximal incorporation in strain MT3 was almost three times higher than in MT1. It has been reported that strain MT1 produces 4-CC as an intermediate but has a lower LD50 for it than strain MT3; therefore, MT3 still degrades 4-CC when the concentrations of 4-CC are already too toxic, even lethal, for MT1. By degrading 4-CC, produced by MT1, MT3 protects the entire community against this toxin. The higher affinity but lower tolerance of strain MT1 for 4-chlorocatechol compared to strain MT3 explains the complementary function these two strains have in the consortium adding exceptional stability to the entire community. The novel approach can reveal carbon fluxes in microbial communities generating quantitative data for systems biology of the microbial community.

Consortia modulation of the stress response: proteomic analysis of single strain versus mixed culture

The high complexity of naturally occurring microbial communities is the major drawback limiting the study of these important biological systems. In this study, a comparison between pure cultures of Pseudomonas reinekei sp. strain MT1 and stable community cultures composed of MT1 plus the addition of Achromobacter xylosoxidans strain MT3 (in a steady-state proportion 9:1) was used as a model system to study bacterial interactions that take place under simultaneous chemical and oxidative stress. Both are members of a real community isolated from a polluted sediment by enrichment in 4-chlorosalicylate (4CS). The analysis of dynamic states was carried out at the proteome, metabolic profile and population dynamic level. Differential protein expression was evaluated under exposure to 4CS and high concentrations of toxic intermediates (4-chlorocatechol and protoanemonin), including proteins from several functional groups and particularly enzymes of aromatic degradation pathways and outer membrane proteins. Remarkably, 4CS addition generated a strong oxidative stress response in pure strain MT1 culture led by alkyl hydroperoxide reductase, while the community showed an enhanced central metabolism response, where A. xylosoxidans MT3 helped to prevent toxic intermediate accumulation. A significant change in the outer membrane composition of P. reinekei MT1 was observed during the chemical stress caused by 4CS and in the presence of A. xylosoxidans MT3, highlighting the expression of the major outer membrane protein OprF, tightly correlated to 4CC concentration profile and its potential detoxification role.