Effect of colony morphology variation of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro

Background  

Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays.     

The structure of pyogenecin immunity protein, a novel bacteriocin-like immunity protein from Streptococcus pyogenes

Background  

Many Gram-positive lactic acid bacteria (LAB) produce anti-bacterial peptides and small proteins called bacteriocins, which enable them to compete against other bacteria in the environment. These peptides fall structurally into three different classes, I, II, III, with class IIa being pediocin-like single entities and class IIb being two-peptide bacteriocins. Self-protective cognate immunity proteins are usually co-transcribed with these toxins. Several examples of cognates for IIa have already been solved structurally. Streptococcus pyogenes, closely related to LAB, is one of the most common human pathogens, so knowledge of how it competes against other LAB species is likely to prove invaluable.     

Purification and characterization of endonucleases DraII and III from Deinococcus radiophilus

Deinococcus radiophilus strain ATCC 27603 contains, apart from endonuclease DraI [1], two additional sequence-specific endonucleases. These enzymes, designated DraII and DraIII, recognize nucleotide sequences with novel specificities, PuG↓GNCCPy and CACNN↓GTG, respectively.     

Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

Background  

The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.     

Ribonucleotide reduction - horizontal transfer of a required function spans all three domains

Background  

Ribonucleotide reduction is the only de novo pathway for synthesis of deoxyribonucleotides, the building blocks of DNA. The reaction is catalysed by ribonucleotide reductases (RNRs), an ancient enzyme family comprised of three classes. Each class has distinct operational constraints, and are broadly distributed across organisms from all three domains, though few class I RNRs have been identified in archaeal genomes, and classes II and III likewise appear rare across eukaryotes. In this study, we examine whether this distribution is best explained by presence of all three classes in the Last Universal Common Ancestor (LUCA), or by horizontal gene transfer (HGT) of RNR genes. We also examine to what extent environmental factors may have impacted the distribution of RNR classes.     

Pulsed-field gel electrophoresis profile homogeneity of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> isolates from cattle and heterogeneity of those from sheep and goats

Background  

Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Characterization of strains led to classify paratuberculosis isolates in two main types, cattle type strains, found affecting all host species, and sheep type strains, reported affecting mainly sheep. In order to get a better understanding of the epidemiology of paratuberculosis a large set of Map isolates obtained from different species over the last 25 years have been characterized. Five-hundred and twenty isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) and origins had been cultured and typed by IS1311 restriction-endonuclease-analysis. Two-hundred and sixty-nine isolates were further characterized by pulsed-field gel electrophoresis (PFGE) using SnaBI and SpeI endonucleases. Differences in strain isolation upon various media conditions were also studied.     

Evaluation of multicomponent recombinant vaccines against <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> in mice

Background  

Porcine contagious pleuropneumonia (PCP) is a highly contagious disease that is caused by Actinobacillus pleuropneumoniae (APP) and characterized by severe fibrinous necrotizing hemorrhagic pleuropneumonia, which is a severe threat to the swine industry. In addition to APP RTX-toxins I (ApxI), APP RTX-toxin II (ApxII), APP RTX-toxin III (ApxIII) and Outer membrane protein (OMP), there may be other useful antigens that can contribute to protection. In the development of an efficacious vaccine against APP, the immunogenicities of multicomponent recombinant subunit vaccines were evaluated.     

Variation of cytosine methylation in 57 sweet orange cultivars

Sweet orange is an important group of citrus cultivars, which includes a number of bud sport cultivars. Little is known about the CpG methylation status of the CCGG sequences in the orange genome. In this study, methylation-sensitive amplification polymorphism (MSAP), based on the application of isoschizomers (Hpa II and Msp I), was first used to analyze cytosine methylation patterns in 57 orange cultivars that were not fully differentiated by regular DNA molecular markers. Three types of bands were generated from ten primer pairs. Type I bands were present following restriction with Eco RI + Hpa II and Eco RI + Msp I; type II or type III were present only following restriction with either Eco RI + Hpa II or with Eco RI + Msp I. The total number of these three types of bands was 802, 72, and 157, respectively. Among these, the number of polymorphic bands were 244 (30.2%), 23 (31.9%), and 32 (20.4%), in type I, II and III, respectively. The methylation patterns of these 57 cultivars are discussed and assessed by dendrograms derived from the analysis of polymorphic MSAP bands. The distribution of polymorphic bands of the above three types demonstrate the methylation patterns and frequency at the cytosine loci. We suggest that methylation events could be more frequent than demethylation events, and that the methylation patterns maybe associated with phenotypic traits.     

A simple and rapid method for screening bacteria for type II restriction endonucleases: enzymes in Aphanothece halophytica

A method is described which allows a large number of bacterial strains to be rapidly and easily screened for the presence of site-specific endonucleases. The method involves selective permeabilization of the bacterial cell and analysis of the exuded material. Type II restriction endonucleases from cyanobacteria and Gram-negative eubacteria have been detected and new enzymes have been found. The method should be widely applicable and easy to modify for use in genera other than those tested. Three-site-specific endonuclease activities, detected by this method in Aphanothece halophytica PCC 7412, were purified and their recognition and cleavage specificities were determined AhaI and AhaII recognise and cleave the same DNA sequences as CauII and AcyI respectively; the specificity of AhaIII (TTTAAA) has been reported previously (Whitehead and Brown, 1982, FEBS Letters 143:296–300).Abbreviations Brij-58 20 cetyl ether - Pu purine nucleoside - Py pyrimidine nucleoside     

Chromosomal aberrations in a fish, Channa punctata after in vivo exposure to three heavy metals

The studies were designed to assess the extent of chromosomal aberrations (CA) under the exposure of three common heavy metalic compounds, viz. mercuric chloride, arsenic trioxide and copper sulphate pentahydrate, in vivo using fish, Channa punctata (2n = 32), as a test model. Prior acclimatized fishes were divided into five groups. Group I and II served as negative and positive control, respectively. An intramuscular injection of Mitomycin-C (@ 1 mg/kg body wt.) was administered to group II only. Fishes of groups III, IV and V were subjected to sublethal concentrations (10% of 96 h LC₅₀), of HgCl₂ (0.081 mg/L), As₂O₃ (6.936 mg/L) and CuSO₄·5H₂O (0.407 mg/L). Fishes of all the groups were exposed uninterrupted for 24, 48, 72, 96 and 168 h. Observations of kidney cells of exposed fishes revealed chromatid and chromosome breaks, chromatid and chromosome gaps along with ring and di-centric chromosomes. A significant increase over negative control in the frequency of chromosomal aberrations (CA) was observed in fish exposed to Mitomycin-C, Hg(II), As(III) and Cu(II). As the average ± SE total number of CA, average number of CA per metaphase and %incidence of aberrant cells in Hg(II) was 104.40 ± 8.189, 0.347 ± 0.027 and 10.220 ± 0.842, respectively; in As(III) 109.20 ± 8.309, 0.363 ± 0.027 and 10.820 ± 2.347, respectively and in Cu(II) 89.00 ± 19.066, 0.297 ± 0.028 and 8.900 ± 0.853, respectively. Hence, it reveals that the order of induction of frequency of CA was Cu < Hg < As. The findings depict genotoxic potential of these metals even in sublethal concentrations.     

Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities

Background

We previously defined a family of restriction endonucleases (REases) from Thermus sp., which share common biochemical and biophysical features, such as the fusion of both the nuclease and methyltransferase (MTase) activities in a single polypeptide, cleavage at a distance from the recognition site, large molecular size, modulation of activity by S-adenosylmethionine (SAM), and incomplete cleavage of the substrate DNA. Members include related thermophilic REases with five distinct specificities: TspGWI, TaqII, Tth111II/TthHB27I, TspDTI and TsoI.

Results

TspDTI, TsoI and isoschizomers Tth111II/TthHB27I recognize different, but related sequences: 5'-ATGAA-3', 5'-TARCCA-3' and 5'-CAARCA-3' respectively. Their amino acid sequences are similar, which is unusual among REases of different specificity. To gain insight into this group of REases, TspDTI, the prototype member of the Thermus sp. enzyme family, was cloned and characterized using a recently developed method for partially cleaving REases.

Conclusions

TspDTI, TsoI and isoschizomers Tth111II/TthHB27I are closely related bifunctional enzymes. They comprise a tandem arrangement of Type I-like domains, like other Type IIC enzymes (those with a fusion of a REase and MTase domains), e.g. TspGWI, TaqII and MmeI, but their sequences are only remotely similar to these previously characterized enzymes. The characterization of TspDTI, a prototype member of this group, extends our understanding of sequence-function relationships among multifunctional restriction-modification enzymes.     

A physical map of the permuted genome of bacteriophage T1

Summary A restriction map has been constructed for the DNA of coliphage T1 which locates the cleavage sites of the restriction endonucleases, BglI (6 cuts), BglII (16 cuts), EcoRI (2 cuts), HindIII (2 cuts) and PstI (2 cuts). Digestions with BglI and BglII reveal fragments which are present in sub-molar quantities. Two methods, one using the selective removal of molecular ends with exonuclease III and the other involving the comparison of digestion patterns of concatemeric and virion DNA, have shown that the submolar fragments are at or close to the ends of the molecules. Digestions with BglI show that one terminal fragment has a very precise molecular weight whereas all the others are of heterogenous molecular weight. These results are consistent with the model for DNA packaging in which maturation is initiated at a precise site on a concatemeric precursor and proceeds by the encapsidation of up to four successive headfuls of 1.065 genome equivalents (MacHattie and Gill 1977).     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

Cloning and heterologous expression of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> uroporphyrinogen III synthase/methyltransferase gene (cobA/hemD): preliminary characterization

Purpose of work  

To clone, express and characterize uroporphyrinogen III synthase/methyltransferase gene (cobA/hemD) from Lactobacillus reuteri.     

The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNA<Superscript>Asn</Superscript>

Background  

Lysyl-tRNA synthetase (LysRS) is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1) and class II (LysRS2) enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820) and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1) of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression.     

Sequence variation of non-coding regions of chloroplast DNA of soybean and related wild species and its implications for the evolution of different chloroplast haplotypes

Soybean chloroplast DNAs (cpDNAs) are classified into three types (I, II and III) based on RFLP profiles. Type I is mainly observed in cultivated soybean (Glycine max), while type II and type III are frequently found in both cultivated and wild soybean (Glycine soja), although type III is predominant in wild soybean. In order to evaluate the diversity of cpDNA and to determine the phylogenetic relationship among different chloroplast types, we sequenced nine non-coding regions of cpDNA for seven cultivated and 12 wild soybean accessions with different cpDNA types. Eleven single-base substitutions and a deletion of five bases were detected in a total of 3849 bases identified. Five mutations distinguished the accessions with types I and II from those with type III, and seven were found in the accessions with type III, independently of their taxa. Four species of the subgenus Glycine shared bases that were identical to those with types I and II at two of the five mutation sites and shared bases that were identical to those with type III at the remaining three sites. Therefore, the different cpDNA types may not have originated monophyletically, but rather may have differentiated from a common ancestor in different evolutionary directions. A neighbor-joining tree resulting from the sequence data revealed that the subgenus Soja connected with Glycine microphylla which formed a distinct clade from Clycine clandestina and the tetraploid cytotypes of Glycine tabacina and Glycine tomentella. Several informative length mutations of 54 to 202 bases, due to insertions or deletions, were also detected among the species of the genus Glycine. Received: 16 December 1999 / Accepted: 12 February 2000     

Sequence diversity of the intergenic spacer region of the rRNA gene of Cryptococcus albidus isolated from the skin of patients with atopic dermatitis and healthy individuals

The yeast species Cryptococcus albidus var. albidus was found to more often colonize the skin surface of patients with atopic dermatitis (77.0%, 47/61) than that of  healthy subjects (37.5%, 15/40). The intergenic spacer 1 region of the rRNA gene of this species consists of four sequence types: I, II, III and IV. Types I and II were predominant among healthy subjects and atopic dermatitis patients, respectively.     

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata: I. Sequence evolution and transcription of the maxicircle

Kinetoplast DNA from the insect trypanosome Crithidia luciliae contains a maxicircle of 22 × 10⁶ D. We have cleaved this DNA with endonucleases PstI, XbaI, XhoI, SstI, HaeIII, SalII, HindIII, EcoRI, and HapII and constructed a physical map of the 31 cleavage sites. The maxicircle segments hybridizing with total cellular RNA are clustered on one-half of the maxicircle; the genes for the 9 and 12 S mitochondrial (r)RNAs are located on a 1.7-kb segment. Restriction enzyme analysis indicates a sequence homology of more than 96% between the maxicircles of C. luciliae and C. fasciculata, which is not lower than that found between maxicircles of individual Trypanosoma brucei stocks. We conclude therefore that C. luciliae and C. fasciculata are one species and propose to name this species C. fasciculata and to rename C. luciliae as C. fasciculata, var. luciliae. Furthermore we show that the overall maxicircle genome organization of Crithidia resembles that of Trypanosoma.     

Cloning and sequence comparison ofAvaI andBsoBI restriction-modification systems

AvaI andBsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5CYCGRG3 and cleave between the first C and second Y to generate a four-base 5 extension. TheAvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned intoEscherichia coli by the methylase selection method. TheBsoBI restriction endonuclease gene (bsoBIR) and part of theBsoBI methylase gene (bsoBIM) were cloned by the endo-blue method (SOS induction assay), and the remainder ofbsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated thatAvaI andBsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. TheavaIM gene precedesavaIR in theAvaI RM system, while thebsoBIR gene is located upstream ofbsoBIM in theBsoBI RM system. BothAvaI andBsoBI methylases contain motifs conserved among the N⁴ cytosine methylases.     

Mapping of additional restriction enzyme cleavage sites on bacteriophage MB78 genome

A physical map of bacteriophage MB78 DNA indicating the cleavage sites for the enzymeBglII,ClaI,EcoRI,PvuII,SalI andSmaI comprising of a total of 34 cleavage sites have been constructed earlier. The cleavage sites for a few more restriction endonucleases likeApaI,AvaI,BglI,HindIII,KpnI andXhoI have now been mapped. A total of 72 cleavage sites on MB78 DNA are known by now. Relative positions ofEcoRI I and J fragments which could not be decided earlier has now been determined.