在兽疫链球菌中表达vgb基因和HA合成基因提高透明质酸产量

透明质酸(HA)是一种在医药及化妆品领域具有广泛应用的天然粘多糖。兽疫链球菌(Streptococcuszooepidemicus)是工业上生产透明质酸的菌种之一。透明颤菌血红蛋白(VHb)具有增强细胞摄氧的作用。对生产透明质酸的兽疫链球菌进行了基因改造:将兽疫链球菌HA的合成基因hasABC以及合成透明颤菌血红蛋白的vgb基因(Vitreoscillahemoglobingene,vgb)分别或同时插入阳性菌表达质粒pEU308中,通过电转化导入兽疫链球菌中。通过一氧化碳(CO)差光谱检测到了VHb的表达。在摇瓶实验中,同时带有hasABC和vgb基因的重组菌比野生菌的透明质酸产量提高了30％。而在发酵罐中,带有这2个基因的重组菌的透明质酸产量达到了6．9g／L,高于重组菌5．5g／L的产量。实验结果表明,vgb基因的存在促进了细胞的生长,hasABC操纵子的过表达增强了透明质酸的合成。首次将VHb导入兽疫链球菌中,获得了表达,并证明其对菌体生长及透明质酸合成有促进作用。通过研究,VHb将可以在阳性菌中获得更广泛的应用。     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

Distribution of transposons Tn<Emphasis Type="Italic">5044</Emphasis> and Tn<Emphasis Type="Italic">5070</Emphasis> with noncanonical <Emphasis Type="Italic">mer</Emphasis> operons in environmental bacterial populations

The distribution of noncanonical mercury resistance transposons, Tn5044 and Tn5070 , was examined. A characteristic feature of Tn5044 is temperature sensitivity of its mercury operon and the presence in the mer operon of the gene homologous to RNA polymerase subunit. Structural organization of mercury operon Tn5070 , containing minimum gene set (merRTPA), differs from mer operons of both Gram-negative and Gram-positive bacteria. None of more than two thousand environmental bacterial strains displaying mercury resistance and isolated from the samples selected from different geographical regions hybridized to Tn5044- and Tn5070-specific probes. A concept on the existence of cosmopolite, endemic, and rare transposons in environmental bacterial populations was formulated.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1717–1721.Original Russian Text Copyright © 2004 by Gorlenko, Kalyaeva, Bass, Petrova, Mindlin.     

Functional analysis of an intergenic non-coding sequence within <Emphasis Type="Italic">mce1</Emphasis> operon of <Emphasis Type="Italic">M.tuberculosis</Emphasis>

Background  

The mce operons play an important role in the entry of M. tuberculosis into macrophages and non-phagocytic cells. Their non-redundant function as well as complex regulation is implied by the phenotype of mce mutants. Recently, mce1 operon was found to extend over 13 genes, fadD5 (Rv0166) being the first gene of the operon. The presence of a non-coding sequence of 200 base pairs between Rv0166 and Rv0167 is peculiar to mce1 among the four mce operons of M.tuberculosis. We have examined the function of this region.     

The role of <Emphasis Type="Italic">stj</Emphasis> fimbrial operon in the intestinal persistence of <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium in mice

Salmonella Typhimurium contains 13 operons coding for fimbriae with unique binding specificities to host epithelial surfaces. stj operon is only detected in S. Typhimurium genome suggesting that Stj fimbria may effect serovarspecific virulence characteristics. In this study, the role of stj fimbrial operon in the long-term persistence of S. Typhimurium was identified by competitive infection experiment in genetically resistant mouse (CBA) model system. Knock-out mutation of stjA (major subunit of the Stj fimbria) gene reduced recovery of S. Typhimurium from fecal samples and its colonization to spleen, cecum and mesenteric lymph nodes over a 34-day time period (p < 0.05). This data indicate that stj fimbrial operon has a role in long-term intestinal persistence of S. Typhimurium in CBA mice.     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

Diverse bacteria isolated from root nodules of <Emphasis Type="Italic">Trifolium</Emphasis>, <Emphasis Type="Italic">Crotalaria</Emphasis> and <Emphasis Type="Italic">Mimosa</Emphasis> grown in the subtropical regions of China

We investigated the regulation of the two of the three groE operons (cpn.1 and cpn.2) of the root-nodulating bacterium R. leguminosarum strain A34. Both are heat inducible, and both have a CIRCE sequence in their upstream regions, suggesting regulation by an HrcA repressor. Mutagenesis of the CIRCE sequence upstream of cpn.1 led to an increase in the levels of cpn.1 mRNA, and knock-out of the hrcA gene increased the level of Cpn60.1 protein (the GroEL homologue encoded by the cpn.1 operon). Inactivation of the hrcA gene also caused increased expression of a 29 kDa protein that was identified as RhiA, a component of a quorum-sensing system. However, neither loss of the upstream CIRCE sequence, nor loss of HrcA function, had any effect on expression from the cpn.2 promoter. Further analysis of the cpn.2 upstream region suggested regulation could be mediated by an RpoH system, and this was confirmed by deleting the rpoH gene from the chromosome, which led to a decreased level of Cpn60.2 expression. Inactivation of RpoH led to a reduction in growth rate which could be partly compensated for by inactivation of HrcA, indicating an overlap in the in vivo function of the proteins regulated by these two systems. Accession numbers: DQ173160 (hrcA operon); DQ173161 (rpoH gene).     

Sulfolipid accumulation in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> disrupted in the <Emphasis Type="Italic">mce2</Emphasis> operon

Mycobacterium tuberculosis, the causative agent of tuberculosis, has a lipid-rich cell wall that serves as an effective barrier against drugs and toxic host cell products, which may contribute to the organism’s persistence in a host. M. tuberculosis contains four homologous operons called nice (mce1–4) that encode putative ABC transporters involved in lipid importation across the cell wall. Here, we analyzed the lipid composition of M. tuberculosis disrupted in the mce2 operon. High resolution mass spectrometric and thin layer chromatographic analyses of the mutant’s cell wall lipid extracts showed accumulation of SL-1 and SL₁₂₇₈ molecules. Radiographic quantitative analysis and densitometry revealed 2.9, 3.9 and 9.8-fold greater amount of [³⁵S] SL-1 in the mce2 operon mutant compared to the wild type M. tuberculosis during the early/mid logarithmic, late logarithmic and stationary phase of growth in liquid broth, respectively. The amount of [³⁵S] SL₁₂₇₈ in the mutant also increased progressively over the same growth phases. The expression of the mce2 operon genes in the wild type strain progressively increased from the logarithmic to the stationary phase of bacterial growth in vitro, which inversely correlated with the proportion of radiolabel incorporation into SL-1 and SL₁₂₇₈ at these phases. Since the mce2 operon is regulated in wild type M. tuberculosis, its cell wall may undergo changes in SL-1 and SL₁₂₇₈ contents during a natural course of infection and this may serve as an important adaptive strategy for M. tuberculosis to maintain persistence in a host.     

Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> virulence genes in <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis> from Vellore,India

Streptococcus dysgalactiae subsp. equisimilis (SDSE), belonging to the group C and G streptococci, are human pathogens reported to cause clinical manifestations similar to infections caused by Streptococcus pyogenes. To scrutinize the distribution of gene coding for S. pyogenes virulence factors in SDSE, 255 isolates were collected from humans infected with SDSE in Vellore, a region in southern India, with high incidence of SDSE infections. Initial evaluation indicated SDSE isolates comprising of 82.35% group G and 17.64% group C. A multiplex PCR system was used to detect 21 gene encoding virulence-associated factors of S. pyogenes, like superantigens, DNases, proteinases, and other immune modulatory toxins. As validated by DNA sequencing of the PCR products, sequences homologous to speC, speG, speH, speI, speL, ssa and smeZ of the family of superantigen coding genes and for DNases like sdaD and sdc were detected in the SDSE collection. Furthermore, there was high abundance (48.12% in group G and 86.6% in group C SDSE) of scpA, the gene coding for C5a peptidase in these isolates. Higher abundance of S. pyogenes virulence factor genes was observed in SDSE of Lancefield group C as compared to group G, even though the incidence rates in former were lower. This study not only substantiates detection of S. pyogenes virulence factor genes in whole genome sequenced SDSE but also makes significant contribution towards the understanding of SDSE and its increasing virulence potential.     

Primary Structure of NM.<Emphasis Type="Italic">Bst</Emphasis>SEI Operon from <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis>, the Producer of N.<Emphasis Type="Italic">Bst</Emphasis>SEI Site-Specific Nicking Endonuclease

The nucleotide sequence of Bacillus stearothermophilus SE-589 DNA fragment including an operon for the site-specific nicking-modification (NM) system with a gene for BstSEI nicking endonuclease (nickase) has been determined. An analysis of the regions adjacent to the nickase gene has revealed two genes encoding DNA methyltransferases belonging to different classes. Three genes that form the system operon are separated by short open reading frames (ORFs). An analysis of these ORFs has shown that the polypeptides they encode are homologous to different parts of BstSEI nickase, NatB protein, and arginase. A difference in the GC content of the beginning and ending regions of the cloned DNA fragment and the presence of short ORFs similar to genes for known proteins indicate that the NM.BstSEI system operon has probably evolved by horizontal DNA transfer.     

Molecular cloning and analysis of the UDP-Glucose Pyrophosphorylase in <Emphasis Type="Italic">Streptococcus equi</Emphasis> subsp<Emphasis Type="Italic">. zooepidemicus</Emphasis>

UDP-Glucose Pyrophosphorylase (EC 2.7.7.9, UGPase) plays an important role in Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) cell envelope Hyaluronic acid (HA) biosynthesis and it is also recognized as a virulence determinant in several bacterial species. HA is valuable biopolymer used in the pharmaceutical and cosmetic industry. In addition, encapsulation by HA is considered an important virulence factor in other streptococci. Research UGPase will contribute to the vaccine development of S. zooepidemicus and the production of HA. In this study, The UGPase gene fragment (789 bp) obtained from previous research was amplified using PCR, and located by Genome walking technology (Genebank No.GQ423507). The UGPase was expressed, purified and identified using UGPase antibody. The enzyme kinetic parameters were determined, the temperature and pH of the highest activity for the cloned UGPase were 37°C, pH 7.5. The K _m and K _cat value against UTP and G-1-P was 8.5 μM, 69.05 s⁻¹ and 36.41 μM, 48.81 s⁻¹, respectively. The homology-modeling was operated. Overexpression of the UGPase in S. zooepidemicus, its virulence was slightly affected, and HA yield reduced. Real-time PCR was carried out to determine the UGPase expression levels of both SEZp and SEZugp in different grow period, the level is high in logarithmic phase and low in Decline phase.     

Phenotypical Assays and Partial Sequencing of the <Emphasis Type="Italic">hsp60</Emphasis> Gene for Identification of <Emphasis Type="Italic">Streptococcus equi</Emphasis>

Strangles is an acute and contagious disease characterized by inflammation of the upper respiratory tract of horses. The etiological agent of strangles is the bacteria S. equi subsp. equi, which belongs to the Lancefield group C. Opportunistic agents from the same group are frequently isolated from horses with strangles and may induce mistaken diagnoses. Among the subspecies of S. equi, the phenotypic features are almost undistinguishable; however, the pathogenic potential is widely differentiated. The aim of this study was to characterize S. equi isolates obtained from clinical samples of strangles by phenotypic tests and to analyze the partial sequences obtained from fragments of the hsp60 gene. In this work, 26 strains of Streptococcus spp. isolated from horse clinical samples were analyzed. By phenotypical assays, 18 were characterized as S. equi subsp. equi, five as S. equi subsp. zooepidemicus, two as S. dysgalactiae subsp. equisimilis, and one as Streptococcus sp. However 21 isolates were identified as S. equi subsp. equi and five as S. equi subsp. zooepidemicus by DNA sequencing. The sequencing of the partial hsp60 gene was demonstrated to be an alternative method to analyze and differentiate strains of Streptococcus spp. In addition, this method can be useful as a discriminatory tool for characterization of atypical isolates.     

Expression of the entire polyhydroxybutyrate operon of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> in plants

Background

Previously we demonstrated that an entire bacterial operon (the PRN operon) is expressible in plants when driven by the Tomato -yellow-leaf-curl-virus (TYLCV) -derived universal vector IL-60.Petroleum-derived plastics are not degradable, and are therefore harmful to the environment. Fermentation of bacteria carrying operons for polyhydroxyalkanoates (PHAs) produces degradable bioplastics which are environmentally friendly. However, bacterial production of bioplastics is not cost-effective, and attention is turning to their production in plants. Such “green” plastics would be less expensive and environmentally friendly. Hence, attempts are being made to substitute petroleum-derived plastics with “green” plastics. However, transformation of plants with genes of operons producing bioplastics has deleterious effects. Transformation of plastids does not cause deleterious effects, however it is a complicated procedures.

Results

We have developed another TYLCV-based vector (SE100) and show that yet another bacterial operon (the phaCAB operon) when driven by SE100 is also expressed in plants. We employed the combination of SE100 and the phaCAB operon to drive the operon to the plastids and produce in plants a biodegradable plastic [polyhydroxybutyrate (PHB)].Here we indicate that the bacterial operon (phaCAB), when driven by the newly developed universal plant vector SE100 is directed to chloroplasts and produces in plants PHB, a leading PHA. The PHB-producing plants circumvent the need for complicated technical procedures.

Conclusion

The viral vector system SE100 facilitated the production of the bio-plastic poly-3-hydroxybutyrate. This was achieved by using the full pha-CAB operon indicating that TYLCV based system can transcribe and translate genes from bacterial operons controlled by a single cis element. Our data hints to the participation of the chloroplasts in these processes.

     

Assembly of a complete genome sequence for <Emphasis Type="Italic">Gemmata obscuriglobus</Emphasis> reveals a novel prokaryotic rRNA operon gene architecture

Gemmata obscuriglobus is a Gram-negative bacterium with several intriguing biological features. Here, we present a complete, de novo whole genome assembly for G. obscuriglobus which consists of a single, circular 9 Mb chromosome, with no plasmids detected. The genome was annotated using the NCBI Prokaryotic Genome Annotation pipeline to generate common gene annotations. Analysis of the rRNA genes revealed three interesting features for a bacterium. First, linked G. obscuriglobus rrn operons have a unique gene order, 23S–5S–16S, compared to typical prokaryotic rrn operons (16S–23S–5S). Second, G. obscuriglobus rrn operons can either be linked or unlinked (a 16S gene is in a separate genomic location from a 23S and 5S gene pair). Third, all of the 23S genes (5 in total) have unique polymorphisms. Genome analysis of a different Gemmata species (SH-PL17), revealed a similar 23S–5S–16S gene order in all of its linked rrn operons and the presence of an unlinked operon. Together, our findings show that unique and rare features in Gemmata rrn operons among prokaryotes provide a means to better define the evolutionary relatedness of Gemmata species and the divergence time for different Gemmata species. Additionally, these rrn operon differences provide important insights into the rrn operon architecture of common ancestors of the planctomycetes.