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September 22, 2019

Transcriptional regulation of cysteine and methionine metabolism in Lactobacillus paracasei FAM18149.

Lactobacillus paracasei is common in the non-starter lactic acid bacteria (LAB) community of raw milk cheeses. This species can significantly contribute to flavor formation through amino acid metabolism. In this study, the DNA and RNA of L. paracasei FAM18149 were sequenced using next-generation sequencing technologies to reconstruct the metabolism of the sulfur-containing amino acids cysteine and methionine. Twenty-three genes were found to be involved in cysteine biosynthesis, the conversion of cysteine to methionine and vice versa, the S-adenosylmethionine recycling pathway, and the transport of sulfur-containing amino acids. Additionally, six methionine-specific T-boxes and one cysteine-specific T-box were found. Five of these were located upstream of genes encoding transporter functions. RNA-seq analysis and reverse-transcription quantitative polymerase reaction assays showed that expression of genes located downstream of these T-boxes was affected by the absence of either cysteine or methionine. Remarkably, the cysK2-ctl1-cysE2 operon, which is associated with te methionine-to-cysteine conversion and is upregulated in the absence of cysteine, showed high read coverage in the 5′-untranslated region and an antisense-RNA in the 3′-untranslated region. This indicates that this operon is regulated by the combination of cis- and antisense-mediated regulation mechanisms. The results of this study may help in the selection of L. paracasei strains to control sulfuric flavor formation in cheese.

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September 22, 2019

Co-culture of soil biofilm isolates enables the discovery of novel antibiotics

Bacterial natural products (NPs) are considered to be a promising source of drug discovery. However, the biosynthesis gene clusters (BGCs) of NP are not often expressed, making it difficult to identify them. Recently, the study of biofilm community showed bacteria may gain competitive advantages by the secretion of antibiotics, implying a possible way to screen antibiotic by evaluating the social behavior of bacteria. In this study, we have described an efficient workflow for novel antibiotic discovery by employing the bacterial social interaction strategy with biofilm cultivation, co-culture, transcriptomic and genomic methods. We showed that a biofilm dominant species, i.e. Pseudomonas sp. G7, which was isolated from cultivated soil biofilm community, was highly competitive in four-species biofilm communities, as the synergistic combinations preferred to exclude this strain while the antagonistic combinations did not. Through the analysis of transcriptomic changes in four-species co-culture and the complete genome of Pseudomonas sp. G7, we finally discovered two novel non-ribosomal polypeptide synthetic (NRPS) BGCs, whose products were predicted to have seven and six amino acid components, respectively. Furthermore, we provide evidence showing that only when Pseudomonas sp. G7 was co-cultivated with at least two or three other bacterial species can these BGC genes be induced, suggesting that the co-culture of the soil biofilm isolates is critical to the discovery of novel antibiotics. As a conclusion, we set a model of applying microbial interaction to the discovery of new antibiotics.

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September 22, 2019

Analysis of the complete genome sequence of Bacillus atrophaeus GQJK17 reveals its biocontrol characteristics as a plant growth-promoting rhizobacterium

Bacillus atrophaeus GQJK17 was isolated from the rhizosphere of Lycium barbarum L. in China, which was shown to be a plant growth-promoting rhizobacterium as a new biological agent against pathogenic fungi and gram-positive bacteria. We present its biological characteristics and complete genome sequence, which contains a 4,325,818 bp circular chromosome with 4,181 coding DNA sequences and a G+C content of 43.3%. A genome analysis revealed a total of 8 candidate gene clusters for producing antimicrobial secondary metabolites, including surfactin, bacillaene, fengycin, and bacillibactin. Some other antimicrobial and plant growth-promoting genes were also discovered. Our results provide insights into the genetic and biological basis of B. atrophaeus strains as a biocontrol agent for application in agriculture.

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September 22, 2019

Characterization and high-quality draft genome sequence of Herbivorax saccincola A7, an anaerobic, alkaliphilic, thermophilic, cellulolytic, and xylanolytic bacterium.

An anaerobic, cellulolytic-xylanolytic bacterium, designated strain A7, was isolated from a cellulose-degrading bacterial community inhabiting bovine manure compost on Ishigaki Island, Japan, by enrichment culture using unpretreated corn stover as the sole carbon source. The strain was Gram-positive, non-endospore forming, non-motile, and formed orange colonies on solid medium. Strain A7 was identified as Herbivorax saccincola by DNA-DNA hybridization, and phylogenetic analysis based on 16S rRNA gene sequences showed that it was closely related to H. saccincola GGR1 (= DSM 101079T). H. saccincola A7 (= JCM 31827=DSM 104321) had quite similar phenotypic characteristics to those of strain GGR1. However, the optimum growth of A7 was at alkaline pH (9.0) and 55°C, compared to pH 7.0 at 60°C for GGR1, and the fatty acid profile of A7 contained 1.7-times more C17:0 iso than GGR1. The draft genome sequence revealed that H. saccincola A7 possessed a cellulosome-like extracellular macromolecular complex, which has also been found for Clostridium thermocellum and C. clariflavum. H. saccincola A7 contained more glycoside hydrolases (GHs) belonging to GH families-11 and -2, and more diversity of xylanolytic enzymes, than C. thermocellum and C. clariflavum. H. saccincola A7 could grow on xylan because it encoded essential genes for xylose metabolism, such as a xylose transporter, xylose isomerase, xylulokinase, and ribulose-phosphate 3-epimerase, which are absent from C. thermocellum. These results indicated that H. saccincola A7 has great potential as a microorganism that can effectively degrade lignocellulosic biomass. Copyright © 2018 Elsevier GmbH. All rights reserved.

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September 22, 2019

Complete genome sequence provides insights into the biodrying-related microbial function of Bacillus thermoamylovorans isolated from sewage sludge biodrying material.

To enable the development of microbial agents and identify suitable candidate used for biodrying, the existence and function of Bacillus thermoamylovorans during sewage sludge biodrying merits investigation. This study isolated a strain of B. thermoamylovorans during sludge biodrying, submitted it for complete genome sequencing and analyzed its potential microbial functions. After biodrying, the moisture content of the biodrying material decreased from 66.33% to 50.18%, and B. thermoamylovorans was the ecologically dominant Bacillus, with the primary annotations associated with amino acid transport and metabolism (9.53%) and carbohydrate transport and metabolism (8.14%). It contains 96 carbohydrate-active- enzyme-encoding gene counts, mainly distributed in glycoside hydrolases (33.3%) and glycosyl transferases (27.1%). The virulence factors are mainly associated with biosynthesis of capsule and polysaccharide capsule. This work indicates that among the biodrying microorganisms, B. thermoamylovorans has good potential for degrading recalcitrant and readily degradable components, thus being a potential microbial agent used to improve biodrying. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis.

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

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September 22, 2019

Biosynthesis of abscisic acid in fungi: identification of a sesquiterpene cyclase as the key enzyme in Botrytis cinerea.

While abscisic acid (ABA) is known as a hormone produced by plants through the carotenoid pathway, a small number of phytopathogenic fungi are also able to produce this sesquiterpene but they use a distinct pathway that starts with the cyclization of farnesyl diphosphate (FPP) into 2Z,4E-a-ionylideneethane which is then subjected to several oxidation steps. To identify the sesquiterpene cyclase (STC) responsible for the biosynthesis of ABA in fungi, we conducted a genomic approach in Botrytis cinerea. The genome of the ABA-overproducing strain ATCC58025 was fully sequenced and five STC-coding genes were identified. Among them, Bcstc5 exhibits an expression profile concomitant with ABA production. Gene inactivation, complementation and chemical analysis demonstrated that BcStc5/BcAba5 is the key enzyme responsible for the key step of ABA biosynthesis in fungi. Unlike what is observed for most of the fungal secondary metabolism genes, the key enzyme-coding gene Bcstc5/Bcaba5 is not clustered with the other biosynthetic genes, i.e., Bcaba1 to Bcaba4 that are responsible for the oxidative transformation of 2Z,4E-a-ionylideneethane. Finally, our study revealed that the presence of the Bcaba genes among Botrytis species is rare and that the majority of them do not possess the ability to produce ABA.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Comparative analysis reveals unexpected genome features of newly isolated Thraustochytrids strains: on ecological function and PUFAs biosynthesis.

Thraustochytrids are unicellular fungal-like marine protists with ubiquitous existence in marine environments. They are well-known for their ability to produce high-valued omega-3 polyunsaturated fatty acids (?-3-PUFAs) (e.g., docosahexaenoic acid (DHA)) and hydrolytic enzymes. Thraustochytrid biomass has been estimated to surpass that of bacterioplankton in both coastal and oceanic waters indicating they have an important role in microbial food-web. Nevertheless, the molecular pathway and regulatory network for PUFAs production and the molecular mechanisms underlying ecological functions of thraustochytrids remain largely unknown.The genomes of two thraustochytrids strains (Mn4 and SW8) with ability to produce DHA were sequenced and assembled with a hybrid sequencing approach utilizing Illumina short paired-end reads and Pacific Biosciences long reads to generate a highly accurate genome assembly. Phylogenomic and comparative genomic analyses found that DHA-producing thraustochytrid strains were highly similar and possessed similar gene content. Analysis of the conventional fatty acid synthesis (FAS) and the polyketide synthase (PKS) systems for PUFAs production only detected incomplete and fragmentary pathways in the genome of these two strains. Surprisingly, secreted carbohydrate active enzymes (CAZymes) were found to be significantly depleted in the genomes of these 2 strains as compared to other sequenced relatives. Furthermore, these two strains possess an expanded gene repertoire for signal transduction and self-propelled movement, which could be important for their adaptations to dynamic marine environments.Our results demonstrate the possibility of a third PUFAs synthesis pathway besides previously described FAS and PKS pathways encoded in the genome of these two thraustochytrid strains. Moreover, lack of a complete set of hydrolytic enzymatic machinery for degrading plant-derived organic materials suggests that these two DHA-producing strains play an important role as a nutritional source rather than a nutrient-producer in marine microbial-food web. Results of this study suggest the existence of two types of saprobic thraustochytrids in the world’s ocean. The first group, which does not produce cellulosic enzymes and live as ‘left-over’ scavenger of bacterioplankton, serves as a dietary source for the plankton of higher trophic levels and the other possesses capacity to live on detrital organic matters in the marine ecosystems.

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September 22, 2019

Genome mining-mediated discovery of a new avermipeptin analogue in Streptomyces actuosus ATCC 25421.

Streptomyces actuosus ATCC 25421 was famous for producing thiopeptide nosiheptide, which has widely been used as a feed additive for the promotion of animal growth. Herein, we report the complete genome sequence of S. actuosus ATCC 25421, which consists of an 8,145,579-bp circular chromosome with a G+C content of 72.53?% containing 7?536 protein-coding genes. The antiSMASH 3.0 program was used to identify 49 biosynthetic gene clusters for putative secondary metabolites, including a putative lantipeptide gene cluster that showed 85?% similarity to the reported informatipeptin biosynthetic gene cluster, indicating that the putative lantipeptide gene cluster has the ability to generate the informatipeptin analogue. Compared with avermipeptin, the lantipeptide precursor peptide (termed avermipeptin B) from S. actuosus ATCC 25421 contains a 14-aa leader peptide and a 24-aa core peptide, in which Ile15 was different from Val15 in avermipeptin. We also deduced the structure and the biosynthetic mechanism of avermipeptin B. Heterologous expression of the avermipeptin B biosynthetic gene cluster in S. lividans TK24 was characterized by high-resolution mass spectrometry (ESI-MS/MS). Finally, we found that avermipeptin B displayed strong activity against Gram-positive strains. The genome sequence reported here can encourage us to mine novel secondary metabolites and investigate their biosynthetic mechanism in the future.

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September 22, 2019

Biosynthetic Baeyer-Villiger chemistry enables access to two anthracene scaffolds from a single gene cluster in Deep-Sea-derived Streptomyces olivaceus SCSIO T05.

Four known compounds, rishirilide B (1), rishirilide C (2), lupinacidin A (3), and galvaquinone B (4), representing two anthracene scaffolds typical of aromatic polyketides, were isolated from a culture of the deep-sea-derived Streptomyces olivaceus SCSIO T05. From the S. olivaceus producer was cloned and sequenced the rsd biosynthetic gene cluster (BGC) that drives rishirilide biosynthesis. The structural gene rsdK2 inactivation and heterologous expression of the rsd BGC confirmed the single rsd BGC encodes construction of 1-4 and, thus, accounts for two anthracene scaffolds. Precursor incubation experiments with 13C-labeled acetate revealed that a Baeyer-Villiger-type rearrangement plays a central role in construction of 1-4. Two luciferase monooxygenase components, along with a reductase component, are presumably involved in the Baeyer-Villiger-type rearrangement reaction enabling access to the two anthracene scaffold variants. Engineering of the rsd BGC unveiled three SARP family transcriptional regulators, enhancing anthracene production. Inactivation of rsdR4, a MarR family transcriptional regulator, failed to impact production of 1-4, although production of 3 was slightly improved; most importantly rsdR4 inactivation led to the new adduct 6 in high titer. Notably, inactivation of rsdH, a putative amidohydrolase, substantially improved the overall titers of 1-4 by more than 4-fold.

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September 22, 2019

Nine draft genome sequences of Claviceps purpurea s.lat., including C. arundinis, C. humidiphila, and C. cf. spartinae, pseudomolecules for the pitch canker pathogen Fusarium circinatum, draft genome of Davidsoniella eucalypti, Grosmannia galeiformis, Quambalaria eucalypti, and Teratosphaeria destructans.

This genome announcement includes draft genomes from Claviceps purpurea s.lat., including C. arundinis, C. humidiphila and C. cf. spartinae. The draft genomes of Davidsoniella eucalypti, Quambalaria eucalypti and Teratosphaeria destructans, all three important eucalyptus pathogens, are presented. The insect associate Grosmannia galeiformis is also described. The pine pathogen genome of Fusarium circinatum has been assembled into pseudomolecules, based on additional sequence data and by harnessing the known synteny within the Fusarium fujikuroi species complex. This new assembly of the F. circinatum genome provides 12 pseudomolecules that correspond to the haploid chromosome number of F. circinatum. These are comparable to other chromosomal assemblies within the FFSC and will enable more robust genomic comparisons within this species complex.

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September 22, 2019

Quorum-quenching bacteria isolated from Red Sea sediments reduce biofilm formation by Pseudomonas aeruginosa.

Quorum sensing (QS) is the process by which bacteria communicate with each other through small signaling molecules such as N-acylhomoserine lactones (AHLs). Certain bacteria can degrade AHL molecules by a process called quorum quenching (QQ); therefore, QQ can be used to control bacterial infections and biofilm formation. In this study, we aimed to identify new species of bacteria with QQ activity. Red Sea sediments were collected either from the close vicinity of seagrass or from areas with no vegetation. We isolated 72 bacterial strains, which were tested for their ability to degrade/inactivate AHL molecules. Chromobacterium violaceum CV026-based bioassay was used for the initial screening of isolates with QQ activity. QQ activity was further quantified using high-performance liquid chromatography-tandem mass spectrometry. We found that these isolates could degrade AHL molecules of different acyl chain lengths as well as modifications. 16S-rRNA sequencing of positive QQ isolates showed that they belonged to three different genera. Specifically, two isolates belonged to the genus Erythrobacter; four, Labrenzia; and one, Bacterioplanes. The genome of one representative isolate from each genus was sequenced, and potential QQ enzymes, namely, lactonases and acylases, were identified. The ability of these isolates to degrade the 3OXOC12-AHLs produced by Pseudomonas aeruginosa PAO1 and hence inhibit biofilm formation was investigated. Our results showed that the isolate VG12 (genus Labrenzia) is better than other isolates at controlling biofilm formation by PAO1 and degradation of different AHL molecules. Time-course experiments to study AHL degradation showed that VG1 (genus Erythrobacter) could degrade AHLs faster than other isolates. Thus, QQ bacteria or enzymes can be used in combination with an antibacterial to overcome antibiotic resistance.

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September 22, 2019

Fusarium species complex causing Pokkah Boeng in China

Sugarcane is one of the most important crops for sugar production in sugarcane-growing areas. Many biotic and abiotic stresses affected the sugarcane production which leads to severe losses. Pokkah boeng is now playing a very important role due to its economic threats. Currently, the occurrence and rigorousness of pokkah boeng disease have been spread like wildfire from major sugarcane-growing countries. Pokkah boeng is a fungal disease that can cause serious yield losses in susceptible varieties. Infection of the disease is caused either by spores or ascospores. It may cause serious yield losses in commercial plantings. However, there have been many reported outbreaks of the disease which have looked spectacular but have caused trade and industry loss. Fusarium species complex is the major causal agent of this disease around the world, but some researchers have documented the increased importance of Fusarium. Three Fusarium species have been identified to cause the sugarcane pokkah boeng disease in China. Moreover, Fusarium may be accompanied of its mycotoxin production, genomic sequencing, and association with nitrogen application in China. Many studies on disease investigations, breeding of disease-resistant varieties, and strategy of disease control have also been carried out in China.

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September 22, 2019

Genetic and biochemical characterization of 5-hydroxypicolinic acid metabolism in Alcaligenes faecalis JQ135.

5-Hydroxypicolinic acid (5HPA), a natural pyridine derivative, is microbially degraded in the environment. However, the physiological, biochemical, and genetic foundations of the 5HPA metabolism remain unknown. In this study, an operon (hpa), responsible for 5HPA degradation, was cloned from Alcaligenes faecalis JQ135. HpaM was a monocomponent FAD-dependent monooxygenase and shared low identity (only 28-31%) with reported monooxygenases. HpaM catalyzed the ortho decarboxylative hydroxylation of 5HPA, generating 2,5-dihydroxypyridine (2,5DHP). The monooxygenase activity of HpaM was FAD and NADH-dependent. The apparent Km values of HpaM for 5HPA and NADH were 45.4 µM and 37.8 µM, respectively. The genes hpaX, hpaD, and hpaF were found to encode 2,5DHP dioxygenase, N-formylmaleamic acid deformylase, and maleamate amidohydrolase, respectively; however, the three genes were not essential for 5HPA degradation in A. faecalis JQ135. Furthermore, the gene maiA, which encodes a maleic acid cis-trans isomerase, was essential for the metabolism of 5HPA, nicotinic acid, and picolinic acid in A. faecalis JQ135, indicating that it might be a key gene in the metabolism of pyridine derivatives. The genes and proteins identified in this study showed a novel degradation mechanism of pyridine derivatives.Importance Unlike the benzene ring, the uneven distribution of the electron density of pyridine ring influences the positional reactivity and the interaction with enzymes, e.g., the ortho and para oxidation are more difficult than the meta oxidations. Hydroxylation is an important oxidation process for the pyridine derivative metabolism. In previous reports, the ortho hydroxylation of pyridine derivatives were catalyzed by multicomponent molybdenum-containing monooxygenases, while the meta hydroxylations were catalyzed by monocomponent FAD-dependent monooxygenases. This study identified the new monocomponent FAD-dependent monooxygenase HpaM that catalyzed the ortho decarboxylative hydroxylation of 5HPA. In addition, we found that the maiA coding for maleic acid cis-trans isomerase was pivotal for the metabolism of 5HPA, nicotinic acid, and picolinic acid in A. faecalis JQ135. This study provides novel insights into the microbial metabolism of pyridine derivatives. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

The plant growth-promoting rhizobacterium Variovorax boronicumulans CGMCC 4969 regulates the level of indole-3-acetic acid synthesized from indole-3-acetonitrile.

Variovorax is a metabolically diverse genus of plant growth-promoting rhizobacteria (PGPR) that engages in mutually beneficial interactions between plants and microbes. Unlike most PGPR, Variovorax cannot synthesize the phytohormone indole-3-acetic acid (IAA) via tryptophan. However, we found that V. boronicumulans strain CGMCC 4969 could produce IAA using indole-3-acetonitrile (IAN) as the precursor. Thus, in the present study, the IAA synthesis mechanism of V. boronicumulans CGMCC 4969 was investigated. V. boronicumulans CGMCC 4969 metabolized IAN to IAA through both a nitrilase-dependent pathway and a nitrile hydratase (NHase) and amidase-dependent pathway. Cobalt enhanced the metabolic flux via the NHase/amidase, by which IAN was rapidly converted to indole-3-acetamide (IAM) and in turn to IAA. IAN stimulated the metabolic flux via the nitrilase, by which IAN was rapidly converted to IAA. Subsequently, the IAA was degraded. V. boronicumulans CGMCC 4969 could use IAN as the sole carbon and nitrogen source for growth. Genome sequencing confirmed the IAA synthesis pathways. Gene cloning and overexpression in Escherichia coli indicated that NitA has the nitrilase activity, and IamA has the amidase activity to respectively transform IAN and IAM to IAA. Interestingly, NitA showed a close genetic relationship with the nitrilase of the phytopathogen Pseudomonas syringae Quantitative PCR analysis indicated that the NHase/amidase system is constitutively expressed, whereas the nitrilase is inducible. The present study helps our understanding of the versatile functions of Variovorax nitrile-converting enzymes that mediate IAA synthesis and the interactions between plants and these bacteria.IMPORTANCE We demonstrated that Variovorax boronicumulans CGMCC 4969 has two enzymatic systems-nitrilase and nitrile hydratase/amidase-that convert indole-3-acetonitrile (IAN) to the important plant hormone indole-3-acetic acid (IAA). The two IAA synthesis systems have very different regulatory mechanisms, affecting the IAA synthesis rate and duration. The nitrilase was induced by IAN, which was rapidly converted to IAA; subsequently IAA was rapidly consumed for cell growth. The NHase and amidase system was constitutively expressed and slowly but continuously synthesized IAA. In addition to synthesizing IAA from IAN, CGMCC 4969 has a rapid IAA degradation system, which would be helpful for a host plant to eliminate redundant IAA. This study indicates that the plant growth-promoting rhizobacterium V. boronicumulans CGMCC 4969 has the potential to be used by host plants to regulate the IAA level. Copyright © 2018 American Society for Microbiology.

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