Genomic and physiological analyses of an indigenous strain, <Emphasis Type="Italic">Enterococcus faecium</Emphasis> 17OM39

The human gut microbiome plays a crucial role in human health and efforts need to be done for cultivation and characterisation of bacteria with potential health benefits. Here, we isolated a bacterium from a healthy Indian adult faeces and investigated its potential as probiotic. The cultured bacterial strain 17OM39 was identified as Enterococcus faecium by 16S rRNA gene sequencing. The strain 17OM39 exhibited tolerance to acidic pH, showed antimicrobial activity and displayed strong cell surface traits such as hydrophobicity and autoaggregation capacity. The strain was able to tolerate bile salts and showed bile salt hydrolytic (BSH) activity, exopolysaccharide production and adherence to human HT-29 cell line. Importantly, partial haemolytic activity was detected and the strain was susceptible to the human serum. Genomics investigation of strain 17OM39 revealed the presence of diverse genes encoding for proteolytic enzymes, stress response systems and the ability to produce essential amino acids, vitamins and antimicrobial compound Bacteriocin-A. No virulence factors and plasmids were found in this genome of the strain 17OM39. Collectively, these physiological and genomic features of 17OM39 confirm the potential of this strain as a candidate probiotic.     

Genome sequence of strain IMCC3088, a proteorhodopsin-containing marine bacterium belonging to the OM60/NOR5 clade

Strain IMCC3088, cultivated from the Yellow Sea, is a novel isolate belonging to the OM60/NOR5 clade and is closely related to clone OM241, Congregibacter litoralis, and strain HTCC2080. Here, the genome sequence of strain IMCC3088 is presented, showing the absence of photosynthetic gene clusters and the presence of proteorhodopsin.     

The structural genes encoding CO dehydrogenase subunits (cox L,M and S) in Pseudomonas carboxydovorans OM5 reside on plasmid pHCG3 and are,with the exception of Streptomyces thermoautotrophicus,conserved in carboxydotrophic bacteria

Employing deoxyoligonucleotide probes and Southern hybridizations, we have examined in carboxydotrophic bacteria the localization on the genome of genes encoding the large, medium and small subunits of CO dehydrogenase (coxL, M and S, respectively). In Pseudomonas carboxydovorans OM5 coxL, M and S were identified on the plasmid pHCG3; they were absent on the chromosome. This was evident from positive hybridizations with plasmid DNA of the wild-type strain OM5 and the absence of hybridizations with chromosomal DNA from the plasmid cured mutant strain OM5–12. The genes coxL, M and S were found on plasmids in all other plasmid-containing carboxydotrophic bacteria e.g. Alcaligenes carboxydus, Azomonas B1, Pseudomonas carboxydoflava, Pseudomonas carboxydovorans OM2 and OM4. Cox L, M and S could be identified on the chromosome of the plasmid-free bacteria Arthrobacter 11/x, Bacillus schlegelii, Pseudomonas carboxydohydrogena, and Pseudomonas carboxydovorans OM3. These results essentially confirm and extend former reports that cox genes are rather conserved among carboxydotrophic bacteria of distinct taxonomic position. However, Streptomyces thermoautotrophicus is an noteworthy exception since none of the three cox genes could be detected. This refers to a new type of CO dehydrogenase and is in accord with results indicating that the S. thermoautotrophicus CO dehydrogenase has an unusual electron acceptor specificity and some other properties setting it apart from the classical CO dehydrogenases.Abbreviations CODH carbon monoxide dehydrogenase - H₂ase hydrogenase - kb kilobase - PRK phosphoribulokinase - Rubisco ribulosebisphosphate carboxylase - SDS sodium dodecylsulfate     

Genome sequence of strain HIMB55, a novel marine gammaproteobacterium of the OM60/NOR5 clade

Strain HIMB55 is a phylogenetically unique member of the OM60/NOR5 clade of the Gammaproteobacteria isolated from coastal seawater of Kaneohe Bay on the northeastern shore of Oahu, Hawaii, by extinction culturing in seawater-based oligotrophic medium. Here we present the genome sequence of strain HIMB55, including genes for bacteriochlorophyll-based phototrophy.     

Biogeography and phylogeny of the NOR5/OM60 clade of Gammaproteobacteria

The phylogeny, abundance, and biogeography of the NOR5/OM60 clade was investigated. This clade includes “Congregibacter litoralis” strain KT71, the first cultured representative of marine aerobic anoxygenic phototrophic Gammaproteobacteria. More than 500 16S rRNA sequences affiliated with this clade were retrieved from public databases. By comparative sequence analysis, 13 subclades could be identified, some of which are currently restricted to discrete habitat types. Almost all sequences in the largest subclade NOR5-1 and related subclade NOR5-4 originated from marine surface water samples. Overall, most of the NOR5/OM60 sequences were retrieved from marine coastal settings, whereas there were fewer from open-ocean surface waters, deep-sea sediment, freshwater, saline lakes and soil.     

The 5'-terminal sequence of TMV RNA. Question on the polymorphism found in vulgare strain

The complete nucleotide sequence of TMV RNA (common strain) reported in [Proc. Natl. Acad. Sci. USA (1982) 79, 5818] its 5'-end to be represented by two variants which differed in length. We have tested that result and sequenced the 5'-terminal regions of two strains of TMV RNA (common strain OM and tomato strain L) using cloned cDNA copies. The results showed that the 5'-terminal region of the TMV genome is not polymorphic and that one of the two variants cited above represents a tomato strain but not the common strain.     

Genome sequence of the chemolithoautotrophic bacterium Oligotropha carboxidovorans OM5T

Oligotropha carboxidovorans OM5(T) (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium with the capability to utilize carbon monoxide, carbon dioxide, and hydrogen. It is also capable of heterotrophic growth under appropriate environmental conditions. Here we report the annotated genome sequence of the circular chromosome of this organism.     

Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by virulence plasmid pJM1.

Vibrio anguillarum 775 harboring the virulence plasmid pJM1 synthesized an outer membrane protein of 86 kilodaltons, OM2, that was inducible under conditions of iron limitation. pJM1 DNA fragments obtained by digestion with restriction endonucleases were cloned into cosmid vectors and transferred into Escherichia coli. The OM2 protein was synthesized in E. coli, demonstrating that it is actually encoded by the pJM1 plasmid. Mobilization of the recombinant plasmids to V. anguillarum was accomplished by using the transfer factor pRK2013. A V. anguillarum exconjugant harboring the recombinant derivative pJHC-T7 and synthesizing the OM2 protein took up 55Fe3+ and grew under iron-limiting conditions, only in presence of the pJM1-mediated siderophore. Exconjugants harboring recombinant plasmids, such as pJHC-T2 which did not encode the OM2 protein, were transport negative. Membrane protein iodination experiments, together with protease treatment of whole cells, indicated that the OM2 protein is exposed to the outside environment of the V. anguillarum cells.     

Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18

We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.     

陆地棉栽培品种转复合启动子控制下的cry1Ac3基因获得高效抗虫植株(英文)

以根癌土壤杆菌 (Agrobacteriumtumefaciens)介导法分别将植物表达载体pBinMoBc和pBinoBc导入陆地棉(GossypiumhirsutumL .)栽培品种“新陆早 1号”、“晋棉 7号”、“晋棉 12号”和“冀合 32 1”。pBinMoBc携带有高效启动子复合OM启动子控制下的cry1Ac3基因 ,pBinoBc携带有 35S启动子控制下的cry1Ac3基因。经过共培养、卡那霉素筛选抗性愈伤组织及体细胞胚的诱导 ,得到了再生植株。对T2 代的PCR、Southernblotting、ELISA检测及Westernblotting证明cry1Ac3基因已整合入受体棉花基因组并得到表达。抗虫性检测表明转基因后代对棉铃虫 (Heliothisarmigera )具有良好的抗性 ,转pBinMoBcT2 代与转pBinoBcT2 代相比 ,对棉铃虫具有更快的致死速度。本研究建立了一套高效的陆地棉栽培品种转化体系 ;进一步的检测结果表明 ,复合OM启动子可以提高外源基因的表达量从而增强转基因棉的抗虫性。     

酵母菌色氨酸合成酶基因的克隆与表达

用RemHI酶切酿酒酵母(Saceharomyces cercuisiae) 1412-4D染色体DNA,通过蔗糖梯度分离2-4kb DNA片段并插入穿棱质粒pCN60,构成1412-4D基因文库。从基因文库中提取重组质粒,转化受体菌C9(a,trp5,adcl,ade6),用直接功能互补法,分离到9株重组质粒,它们都含有3．2kb的TRP5 DNA片段,分别命名为pCN60(trps)1-90转化体中色氨酸合成酶的酶活水平比原始菌株1412-4D高3倍。     

Homology and distribution of CO dehydrogenase structural genes in carboxydotrophic bacteria

The 17 (S), 30 (M) and 87 kDa (L) subunits of CO dehydrogenases from the CO-oxidizing bacteria Pseudomonas carboxydoflava, Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans OM5 were isolated and purified. The N-terminal sequences of same subunits from different bacteria showed distinct homologies. Dot blot hybridization employing oligonucleotide probes derived from the sequences of the S-subunit of P. carboxydovorans OM5 and the M-subunit of P. carboxydohydrogena and DNA of the plasmid-containing CO-oxidizing bacteria Alcaligenes carboxydus, Azomonas B1, P. carboxydoflava, P. carboxydovorans OM2, OM4 and OM5 indicated that all genes encoding these subunits reside on plasmids. That in P. carboxydovorans OM5 CO dehydrogenase structural genes are located entirely on plasmid pHCG3 was evident from the absence of hybridization employing DNA from the cured mutant strain OM5-12. CO dehydrogenase structural genes could be identified on the chromosome of the plasmid-free bacteria Arthrobacter 11/x, Bacillus schlegelii, P. carboxydohydrogena and P. carboxydovorans OM3. There was no example of a plasmid-harboring carboxydotrophic bacterium that did not carry CO dehydrogenase structural genes on the plasmid. The N-terminal sequences of CO dehydrogenase structural genes were found to be conserved among carboxydotrophic bacteria of distinct taxonomic position, independent of the presence of plasmids. It is discussed whether this might be the consequence of horizontal gene transfer.     

Identification and characteristics of plasmids of the strains of erythromycin-producing Streptomyces erythreus

Presence of plasmid DNA was investigated in laboratory strains 2 and 4 (NRRL 2338) of S. erythreus, as well as in strains 1 and 3 of S. erythreus subjected to improvement with respect to erythromycin production. Families of plasmids close by their molecular weights were identified in S. erythreus strains 3 and 4 (NRRL 2338). A plasmid DNA fraction of S. erythreus strain 3 was studied with electron microscopy. It enabled to identify 5 plasmids: pSE11, pSE12, pSE13, pSE14 and pSE15 with length of 5.3, 12.4, 16.3, 29.6 and 86.9 kb respectively. Using of various procedures for isolation of extrachromosomal DNA did not provide its detection in S. erythreus strains 1 and 2. At least a part of the plasmids detected in S. erythreus strains 3 and 4 (NRRL 2338) was conjugative. 32R-Labeled plasmid DNA of S. erythreus strain 3 was subjected to hydridization according to Sauthern with total DNA of the 4 strains treated with restrictases BamHI, PstI and BgIII. The studies showed that the genome of S. erythreus strain 2 was not homologous with the probe while S. erythreus strain 1 contained one of the plasmids or its part in chromosome-integrated state. In strains 3 and 4 (NRRL 2338) of S. erythreus certain plasmid DNAs were present in both autonomous and chromosome-inserted states. 32P-Labeled gene of erythromycin resistance (ermE) was subjected to hybridization according to Southern with total DNA of the 4 strains and with DNA plasmid fraction of S. erythreus strain 3. The signal was positive only in hydridization of the probe with total DNA of S. erythreus strains 1, 3, and 4 (NRRL 2338).(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic and physiological characterization of 23S rRNA and ftsJ mutants of Borrelia burgdorferi isolated by mariner transposition

Borrelia burgdorferi contains one 16S rRNA gene and two tandem sets of 23S and 5S rRNA genes located in a single chromosomal region. This unusual rRNA gene organization has been speculated to be involved in the slow growth of this organism. Because we were repeatedly unable to isolate a 23S ribosomal mutant in B. burgdorferi by allelic exchange, we developed a transposition mutagenesis system for this bacterium. To this end, Himar1 transposase is expressed in B. burgdorferi from a resident plasmid containing an erythromycin resistance marker, and this strain is then electroporated with suicide plasmids containing mariner transposons and kanamycin resistance genes expressible in B. burgdorferi. This system permitted us to generate hundreds of erythromycin/kanamycin-resistant B. burgdorferi clones with each of three suicide plasmids. DNA sequencing of several kanamycin-resistant clones generated with one of the suicide plasmids showed stable and random insertion of the transposon into the B. burgdorferi chromosomal and plasmid genome. One mutant was inactivated in rrlA (23S rRNA), another in ftsJ (rrmJ). rrlA disruption had no effect on growth rate under a wide range of culture conditions, but disruption of ftsJ interfered significantly with growth rate and bacterial morphology. These data show it is possible to isolate random and stable B. burgdorferi transposition mutants for physiological analysis of this pathogenic spirochete.     

Draft genome sequence of Streptomyces clavuligerus NRRL 3585, a producer of diverse secondary metabolites

Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.     

Plasmid-encoded gamma-hexachlorocyclohexane degradation genes and insertion sequences in Sphingobium francense (ex-Sphingomonas paucimobilis Sp+)

The lin genes encode the gamma-hexachlorocyclohexane (gamma-HCH or lindane) catabolic pathway in lindane-degrading strains. The location and stability of these genes have been explored in the lindane-degrading Sphingobium francense strain Sp+, and in two non-lindane-degrading mutants (Sp1- and Sp2-). The lin genes, linA, linB, linE and linX were localized by hybridization on three of the six plasmids of the S. francense strain Sp+ showing dispersal within the genome. The linC gene was detected by PCR, but was not detected by hybridization on any of the plasmids. The hybridization of the linA and linX genes was negative with the two non-lindane-degrading mutants S. francense strains, Sp1- and Sp2-. The dynamic of this genome associated with gene loss and acquisition, and plasmid rearrangement was explored by a search for associated insertion sequences. A new insertion sequence, ISSppa4, belonging to the IS21 family was detected and compared with IS6100 and ISsp1. Insertion sequence localization was explored on different hybridization patterns (plasmid, total genome) with the lindane-degrading Sp+ strain and the two non-degrading derivatives (Sp1-, Sp2-). Insertion sequence movement and plasmid rearrangement could explain the emergence of the non-lindane-degrading mutants.     

Complete genome sequence of the type strain Cupriavidus necator N-1

Here we announce the complete genome sequence of the copper-resistant bacterium Cupriavidus necator N-1, the type strain of the genus Cupriavidus. The genome consists of two chromosomes and two circular plasmids. Based on genome comparison, the chromosomes of C. necator N-1 share a high degree of similarity with the two chromosomal replicons of the bioplastic-producing hydrogen bacterium Ralstonia eutropha H16. The two strains differ in their plasmids and the presence of hydrogenase genes, which are absent in strain N-1.     

Characterization of Three Plasmid Deoxyribonucleic Acid Molecules in a Strain of Streptococcus faecalis: Identification of a Plasmid Determining Erythromycin Resistance

Three plasmids designated alpha, beta, and gamma, distinguishable by their molecular weights (6, 17, and 34 million, respectively) were isolated from Streptococcus faecalis strain DS-5 (ATCC 14508). Derivatives of this strain "cured" for erythromycin resistance lacked the beta-plasmid. In the parent strain the beta-plasmid was estimated to be present to the extent of one to two copies per chromosomal genome equivalent whereas the alpha- and gamma-plasmids were about nine and five copies, respectively.     

Identification and characterization of Streptomyces ghanaensis ATCC14672 integration sites for three actinophage-based plasmids

Streptomyces ghanaensis produces the antibiotic moenomycin A, which is the only known direct inhibitor of bacterial peptidoglycan glycosyltransferases (transglycosylases). Recent progress in understanding moenomycin biosynthesis opens the door to the generation of novel moenomycins via biocombinatorial approaches. To realize the promise of such an approach, one needs better knowledge of the S. ghanaensis genome and diverse genetic tools for stable expression of recombinant constructs in this strain. In this respect, we report the intergeneric Escherichia coli–S. ghanaensis conjugal transfer of plasmids pRT801 and pSOK804 based on the actinophage BT1 and VWB integrase systems, respectively. The attB sites for these two plasmids and for pSET152 were characterized. In particular, sequencing revealed that a putative Arg-tRNA gene serves as an integration site for both phage VWB and pSAM2-like actinomycete integrative and conjugative element recently suggested to be widespread and functional in actinomycetes. The stability of the studied plasmids and their neutrality with respect to antibiotic production warrant their use for manipulations of S. ghanaensis genome.     

Symbiosis Island Shuffling with Abundant Insertion Sequences in the Genomes of Extra-Slow-Growing Strains of Soybean Bradyrhizobia

Extra-slow-growing bradyrhizobia from root nodules of field-grown soybeans harbor abundant insertion sequences (ISs) and are termed highly reiterated sequence-possessing (HRS) strains. We analyzed the genome organization of HRS strains with the focus on IS distribution and symbiosis island structure. Using pulsed-field gel electrophoresis, we consistently detected several plasmids (0.07 to 0.4 Mb) in the HRS strains (NK5, NK6, USDA135, 2281, USDA123, and T2), whereas no plasmids were detected in the non-HRS strain USDA110. The chromosomes of the six HRS strains (9.7 to 10.7 Mb) were larger than that of USDA110 (9.1 Mb). Using MiSeq sequences of 6 HRS and 17 non-HRS strains mapped to the USDA110 genome, we found that the copy numbers of ISRj1, ISRj2, ISFK1, IS1632, ISB27, ISBj8, and IS1631 were markedly higher in HRS strains. Whole-genome sequencing showed that the HRS strain NK6 had four small plasmids (136 to 212 kb) and a large chromosome (9,780 kb). Strong colinearity was found between 7.4-Mb core regions of the NK6 and USDA110 chromosomes. USDA110 symbiosis islands corresponded mainly to five small regions (S1 to S5) within two variable regions, V1 (0.8 Mb) and V2 (1.6 Mb), of the NK6 chromosome. The USDA110 nif gene cluster (nifDKENXSBZHQW-fixBCX) was split into two regions, S2 and S3, where ISRj1-mediated rearrangement occurred between nifS and nifB. ISs were also scattered in NK6 core regions, and ISRj1 insertion often disrupted some genes important for survival and environmental responses. These results suggest that HRS strains of soybean bradyrhizobia were subjected to IS-mediated symbiosis island shuffling and core genome degradation.