A preliminary study of the mechanism of nitrate-stimulated remarkable increase of rifamycin production in Amycolatopsis mediterranei U32 by RNA-seq.

BACKGROUND: Rifamycin is an important antibiotic for the treatment of infectious disease caused by Mycobacteria tuberculosis. It was found that in Amycolatopsis mediterranei U32, an industrial producer for rifamycin SV, supplementation of nitrate into the medium remarkably stimulated the yield of rifamycin SV. However, the molecular mechanism of this nitrate-mediated stimulation remains unknown. RESULTS: In this study, RNA-sequencing (RNA-seq) technology was employed for investigation of the genome-wide differential gene expression in U32 cultured with or without nitrate supplementation. In the presence of nitrate, U32 maintained a high transcriptional level of genes both located in the rifamycin biosynthetic cluster and involved in the biosynthesis of rifamycin precursors, including 3-amino-5-dihydroxybenzoic acid, malonyl-CoA and (S)-methylmalonyl-CoA. However, when nitrate was omitted from the medium, the transcription of these genes declined sharply during the transition from the mid-logarithmic phase to the early stationary phase. With these understandings, one may easily propose that nitrate stimulates the rifamycin SV production through increasing both the precursors supply and the enzymes for rifamycin biosynthesis. CONCLUSION: It is the first time to thoroughly illustrate the mechanism of the nitrate-mediated stimulation of rifamycin production at the transcriptional level, which may facilitate improvement of the industrial production of rifamycin SV, e.g. through optimizing the global rifamycin biosynthetic pathways on the basis of RNA-seq data.

Comparative analysis of rapamycin biosynthesis clusters between Actinoplanes sp. N902-109 and Streptomyces hygroscopicus ATCC29253.

The present study was designed to identify the difference between two rapamycin biosynthetic gene clusters from Streptomyces hygroscopicus ATCC29253 and Actinoplanes sp. N902-109 by comparing the sequence and organization of the gene clusters. The biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus ATCC29253 was reported in 1995. The second rapamycin producer, Actinoplanes sp. N902-109, which was isolated in 1995, could produce more rapamycin than Streptomyces hygroscopicus ATCC29253. The genomic map of Actinoplanes sp. N902-109 has been elucidated in our laboratory. Two gene clusters were compared using the online software anti-SMASH, Glimmer 3.02 and Subsystem Technology (RAST). Comparative analysis revealed that the organization of the multifunctional polyketide synthases (PKS) genes: RapA, RapB, RapC, and NRPS-like RapP were identical in the two clusters. The genes responsible for precursor synthesis and macrolactone modification flanked the PKS core region in N902-109, while the homologs of those genes located downstream of the PKS core region in ATCC29253. Besides, no homolog of the gene encoding a putative type II thioesterase that may serve as a PKS "editing" enzyme accounted for over-production of rapamycin in N902-109, was found in ATCC29253. Furthermore, no homologs of genes rapQ (encoding a methyltransferase) and rapG in N902-109 were found in ATCC29253, however, an extra rapM gene encoding methyltransferase was discovered in ATCC29253. Two rapamycin biosynthetic gene clusters displayed overall high homology as well as some differences in gene organization and functions.

iBrick: a new standard for iterative assembly of biological parts with homing endonucleases.

The BioBricks standard has made the construction of DNA modules easier, quicker and cheaper. So far, over 100 BioBricks assembly schemes have been developed and many of them, including the original standard of BBF RFC 10, are now widely used. However, because the restriction endonucleases employed by these standards usually recognize short DNA sequences that are widely spread among natural DNA sequences, and these recognition sites must be removed before the parts construction, there is much inconvenience in dealing with large-size DNA parts (e.g., more than couple kilobases in length) with the present standards. Here, we introduce a new standard, namely iBrick, which uses two homing endonucleases of I-SceI and PI-PspI. Because both enzymes recognize long DNA sequences (>18 bps), their sites are extremely rare in natural DNA sources, thus providing additional convenience, especially in handling large pieces of DNA fragments. Using the iBrick standard, the carotenoid biosynthetic cluster (>4 kb) was successfully assembled and the actinorhodin biosynthetic cluster (>20 kb) was easily cloned and heterologously expressed. In addition, a corresponding nomenclature system has been established for the iBrick standard.

Synthesis, antifungal activities and qualitative structure activity relationship of carabrone hydrazone derivatives as potential antifungal agents.

Aimed at developing novel fungicides for relieving the ever-increasing pressure of agricultural production caused by phytopathogenic fungi, 28 new hydrazone derivatives of carabrone, a natural bioactive sesquisterpene, in three types were designed, synthesized and their antifungal activities against Botrytis cinerea and Colletotrichum lagenarium were evaluated. The result revealed that all the derivatives synthesized exhibited considerable antifungal activities in vitro and in vivo, which led to the improved activities for carabrone and its analogues and further confirmed their potential as antifungal agents.

Synthesis and antifungal activities of carabrol ester derivatives.

Thirty-eight new ester derivatives of carabrol were designed, synthesized, and characterized by (1)H and (13)C NMR and HR-ESI-MS. Their antifungal activities against the fungal pathogen Colletotrichum lagenarium were evaluated using a spore germination assay. Of these 38 ester derivatives, 16 showed higher antifungal activity than that of carabrol and 7 showed higher antifungal activity than that of carabrone. It was found that the C-4 position of carabrol was a key position involving its antifungal activity, which showed the variation of 50% inhibition concentration (IC(50)) from 2.70 to 52.33 µg/mL. When substituted by the phenyl ring, the ester derivatives with electron-attracting groups showed higher activity than those with electron-donating ones. Two ester derivatives, carabryl 4-cynaobenzoate (II-17, IC(50) 2.70 µg/mL) and carabryl 4-isopropylbenzoate (II-27, IC(50) 2.82 µg/mL), showed only slightly lower antifungal activity than that of the positive control chlorothalonil (IC(50) 0.87 µg/mL) and have been identified as promising leads for development of new environmentally friendly fungicides.

[Cloning and structural analysis of mouse genomic nucleophosmin gene].

Nucleophosmin (NPM) is an abundant nucleolar phosphoprotein. NPM gene involved chromosomal translocations were found in the patients with anaplastic large cell lymphomas (ALCL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL). To generate NPM gene knockout mice and study its biological function in vivo, we screened the lambda phage genomic library derived from 129S1 mice with mouse NPM cDNA probe. A positive phage clone which contained the full-length NPM genomic DNA was obtained and the insert of 15.3 kb genomic DNA in this clone was sequenced with shotgun method. BLAST analysis showed that the sequence of insert are 99.8% identity to that of NPM gene of C57BL/6 mouse strain. Based on the sequence, bioinformatics analysis on genomic structure of NPM and the transcription factor binding sites in the NPM 5' flanking region were performed.

Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris.

Xanthomonas campestris pathovar campestris (Xcc) is the causative agent of crucifer black rot disease, which causes severe losses in agricultural yield world-wide. This bacterium is a model organism for studying plant-bacteria interactions. We sequenced the complete genome of Xcc 8004 (5,148,708 bp), which is highly conserved relative to that of Xcc ATCC 33913. Comparative genomics analysis indicated that, in addition to a significant genomic-scale rearrangement cross the replication axis between two IS1478 elements, loss and acquisition of blocks of genes, rather than point mutations, constitute the main genetic variation between the two Xcc strains. Screening of a high-density transposon insertional mutant library (16,512 clones) of Xcc 8004 against a host plant (Brassica oleraceae) identified 75 nonredundant, single-copy insertions in protein-coding sequences (CDSs) and intergenic regions. In addition to known virulence factors, full virulence was found to require several additional metabolic pathways and regulatory systems, such as fatty acid degradation, type IV secretion system, cell signaling, and amino acids and nucleotide metabolism. Among the identified pathogenicity-related genes, three of unknown function were found in Xcc 8004-specific chromosomal segments, revealing a direct correlation between genomic dynamics and Xcc virulence. The present combination of comparative and functional genomic analyses provides valuable information about the genetic basis of Xcc pathogenicity, which may offer novel insight toward the development of efficient methods for prevention of this important plant disease.

Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome.

Comparative genomic analysis of the coding sequences (CDSs) of Leptospira interrogans revealed a pair of closely linked genes homologous to the vapBC loci of many other bacteria with respect to both deduced amino acid sequences and operon organizations. Expression of single vapC gene in Escherichia coli resulted in inhibition of bacterial growth, whereas co-expression of vapBC restored the growth effectively. This phenotype is typical for three other characterized toxin-antitoxin systems of bacteria, i.e., mazEF, relBE and chpIK. The VapC proteins of bacteria and a thermophilic archeae, Solfolobus tokodaii, form a structurally distinguished group of toxin different from the other known toxins of bacteria. Phylogenetic analysis of both toxins and antitoxins of all categories indicated that although toxins were evolved from divergent sources and may or may not follow their speciation paths (as indicated by their 16s RNA sequences), co-evolution with their antitoxins was obvious.

[Mutation analysis of hMSH2 and hMLH1 genes in Chinese hereditary nonpolyposis colorectal cancer families].

OBJECTIVES: To determine the germ-line mutations of hMSH2 and hMLH1 genes in Chinese hereditary nonpolyposis colorectal cancer (HNPCC) families' probands or in patients fulfilling different clinical criteria or guidelines; to clarify the nature and distribution of the mutations; to evaluate the sensitivity of different clinical criteria in mutation prediction. METHODS: The entire coding regions (35 exons including exon-intron boundaries) of hMSH2 and hMLH1 genes were directly sequenced in 24 Amsterdam criteria (AC) probands, 15 Japanese criteria (JC) probands (except AC kindreds) and 19 Bethesda guidelines (BG) patients (except two former groups). All available affected and unaffected members from families of those with mutations were screened for mutation. RESULTS: In 16 unrelated families selected by the different clinical criteria, 17 germ-line mutations were found with 11 (64.7%) of hMLH1 and 6 (35.3%) of hMSH2. Two mutations were identified in one of the families. Among the 17 germ-line mutations, 12 had not been reported previously. A diversified mutation spectrum was found, but 6 hMLH1 mutations were found to be concentrated in the region encompassing exon 14, 15 and 16. There was a wide spectrum of mutation type including frame shift, nonsense, splice site mutation, in frame insertion or deletion and missense mutations. The mutation detection rate of hMSH2 and hMLH1 in the AC group was significantly higher than that in the JC group (12/24 vs. 3/15). On the other hand, a low mutation rate (1/19) was detected in 19 BG patients. The mutation cosegregated with disease. Besides, three different genotypes in tumors from probands of mutation-positive families were found. CONCLUSIONS: hMSH2 and hMLH1 mutations in Chinese HNPCC families show a wide spectrum. It seems that hMLH1 gene is involved more frequently than hMSH2 gene in Chinese HNPCC families. Different clinical criteria predict mutations with different sensitivities. The Amsterdam Criteria are most sensitive, while Japanese Criteria are highly practical and the Bethesda Guidelines are also practical to some extent. Gene mutations cosegregate with the disease phenotype. Carriers with no symptom in HNPCC families are most vulnerable groups, follow-ups are required for this group to get early diagnosis and to prevent the development of CRCs.

Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228).

Staphylococcus epidermidis strains are diverse in their pathogenicity; some are invasive and cause serious nosocomial infections, whereas others are non-pathogenic commensal organisms. To analyse the implications of different virulence factors in Staphylococcus epidermidis infections, the complete genome of Staphylococcus epidermidis strain ATCC 12228, a non-biofilm forming, non-infection associated strain used for detection of residual antibiotics in food products, was sequenced. This strain showed low virulence by mouse and rat experimental infections. The genome consists of a single 2499 279 bp chromosome and six plasmids. The chromosomal G + C content is 32.1% and 2419 protein coding sequences (CDS) are predicted, among which 230 are putative novel genes. Compared to the virulence factors in Staphylococcus aureus, aside from delta-haemolysin and beta-haemolysin, other toxin genes were not found. In contrast, the majority of adhesin genes are intact in ATCC 12228. Most strikingly, the ica operon coding for the enzymes synthesizing interbacterial cellular polysaccharide is missing in ATCC 12228 and rearrangements of adjacent genes are shown. No mec genes, IS256, IS257, were found in ATCC 12228. It is suggested that the absence of the ica operon is a genetic marker in commensal Staphylococcus epidermidis strains which are less likely to become invasive.

Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing.

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum and Borrelia burgdorferi, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.

Variant-type PML-RAR(alpha) fusion transcript in acute promyelocytic leukemia: use of a cryptic coding sequence from intron 2 of the RAR(alpha) gene and identification of a new clinical subtype resistant to retinoic acid therapy.

The physiologic actions of retinoic acids (RAs) are mediated through RA receptors (RARs) and retinoid X receptors (RXRs). The RAR(alpha) gene has drawn particular attention because it is the common target in all chromosomal translocations in acute promyelocytic leukemia (APL), a unique model in cancer research that responds to the effect of RA. In the great majority of patients with APL, RAR(alpha) is fused to the PML gene as a result of the t(15;17) translocation. Three distinct types of PML-RAR(alpha) transcripts, long (L), short (S), and variant (V), were identified. The V-type is characterized by truncation of exon 6 of PML and in some cases by the insertion of a variable "spacer" sequence between the truncated PML and RAR(alpha) mRNA fusion partners, although the precise mechanisms underlying formation of the V-type transcript remain unclear. To get further insights into the molecular basis of the t(15;17), we sequenced the entire genomic DNA region of RAR(alpha). Of note, all previously reported "spacer" sequences in V-type transcripts were found in intron 2 of the RAR(alpha) gene and most of these sequences were flanked by gt splice donor sites. In most cases, these "cryptic" coding sequences maintained the ORF of the chimeric transcript. Interestingly, two cases with a relatively long spacer sequence showed APL cellular and clinical resistance to RA treatment. In these cases, the aberrant V-type PML-RAR(alpha) protein displayed increased affinity to the nuclear corepressor protein SMRT, providing further evidence that RA exerts the therapeutic effect on APL through modulation of the RAR-corepressor interaction. Finally, among patients with the L- or S-type PML-RAR(alpha) fusion transcript, some consensus motifs were identified at the hotspots of the chromosome 17q breakpoints within intron 2 of RAR(alpha), strengthening the importance of this intron in the molecular pathogenesis of APL.