Purification of the bacterium-like organism associated with greening disease of citrus by immunoaffinity chromatography and monoclonal antibodies

An immunoaffinity chromatographic procedure with monoclonal antibodies (MA) has been developed for purification of the uncultivable, bacterium-like organism associated with greening disease of citrus. The greening organism (GO) was partially purified from leaf midribs of infected periwinkle plants by differential centrifugation. The GO present in such preparations was retained on an affinity matrix consisting of CNBr-activated Sepharose 4B on which GO-specific MA had been covalently linked. The unbound plant material was washed from the matrix, and the GOs were eluted with 0.1M glycine (pH 11.5). Purified GOs were compared with organisms observed in the initial plant preparation by both immunofluorescence and electron microscopic techniques. The morphology and serological characteristics of the GO were retained following purification procedures.     

Detection of several strains of the bacterium-like organism of citrus greening disease by DNA probes

Greening disease of citrus is caused by a phloem-restricted, bacterium-like organism (BLO). DNA was purified from phloem tissue of periwinkle plants infected with an indian strain of the greening BLO, restricted withHindIII endonuclease, and cloned in the replicative form of bacteriophage M13mp18.By differential hybrizations involving DNA from healthy and infected periwinkle plants, we have selected three recombinant phages containing BLO DNA. The BLO DNA inserts (In-2.6, In-1.9, and In-0.6) have been purified from the viral replicative forms and used as probes. Southern and dot hybridizations have shown that In-2.6 and In-1.9 recognized all asian strains tested (strains from India, Thailand, the Philippines, Indonesia, China, and Taiwan), but not a South African strain. In-0.6 reacted only with the indian BLO strain.     

The engL gene cluster of Clostridium cellulovorans contains a gene for cellulosomal manA

A five-gene cluster around the gene in Clostridium cellulovorans that encodes endoglucanase EngL, which is involved in plant cell wall degradation, has been cloned and sequenced. As a result, a mannanase gene, manA, has been found downstream of engL. The manA gene consists of an open reading frame with 1,275 nucleotides encoding a protein with 425 amino acids and a molecular weight of 47, 156. ManA has a signal peptide followed by a duplicated sequence (DS, or dockerin) at its N terminus and a catalytic domain which belongs to family 5 of the glycosyl hydrolases and shows high sequence similarity with fungal mannanases, such as Agaricus bisporus Cel4 (17.3% identity), Aspergillus aculeatus Man1 (23.7% identity), and Trichoderma reesei Man1 (22.7% identity). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal amino acid sequence analyses of the purified recombinant ManA (rManA) indicated that the N-terminal region of the rManA contained a DS and was truncated in Escherichia coli cells. Furthermore, Western blot analysis indicated that ManA is one of the cellulosomal subunits. ManA production is repressed by cellobiose.     

The DNA maturation domain of gpA, the DNA packaging motor protein of bacteriophage lambda, contains an ATPase site associated with endonuclease activity

Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in Escherichia coli. Biochemical characterization of gpA-DeltaN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme, DNA maturation and DNA packaging, are discussed.     

The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase

There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX₃δδ’χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-C_tag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-C_tag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III₂τ₂ γδδ’χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F’ episome.     

Nucleotide sequence of the bacteriophage T5 DNA fragment which contains the gene for tRNAAsp

The nucleotide sequence of bacteriophage T5 tRNAAsp has been determined by conventional methods using thin-layer chromatography on cellulose for oligonucleotide fractionation. It exhibits several unusual features, such as (a) the displacement of the constant residues U-8, A-14 and R-15; (b) the presence of three G X U out of four base pairs in the D-stem. The gene for T5 tRNAAsp has been cloned in pBR 322 and sequenced. The analysis of the flanking regions shows the presence of two open reading frames on both sides of this gene. It has also been shown that the cloned gene is expressed in Escherichia coli, and RNase P is involved in the T5 tRNAAsp processing.     

Direct sequencing of bacteriophage T4 DNA with a thermostable DNA polymerase

We present a simple and convenient protocol for the direct sequencing of bacteriophage T4 genomic DNA. The method utilizes the thermostable DNA polymerase from Thermus aquaticus (Taq) and 32P-end-labeled oligodeoxyribonucleotide primers to produce extension products that allow the analysis of at least 200 nucleotides (nt) on a single sequencing gel. Single-nt changes in the template were easily detectable following an overnight exposure of the autoradiograms. Comparison of sequences from fully modified T4 DNA containing glucosylated hydroxymethyldeoxycytosine or from templates containing cytosine showed little difference in sequence clarity. These techniques considerably simplify the molecular analysis of T-even bacteriophages and should be compatible with automated sequencing methods which employ 5'-end-labeled primers.     

The interleukin 1 gene cluster contains a major susceptibility locus for ankylosing spondylitis

Ankylosing spondylitis (AS) is a common and highly heritable inflammatory arthropathy. Although the gene HLA-B27 is almost essential for the inheritance of the condition, it alone is not sufficient to explain the pattern of familial recurrence of the disease. We have previously demonstrated suggestive linkage of AS to chromosome 2q13, a region containing the interleukin 1 (IL-1) family gene cluster, which includes several strong candidates for involvement in the disease. In the current study, we describe strong association and transmission of IL-1 family gene cluster single-nucleotide polymorphisms and haplotypes with AS.     

The complete genome of Bacillus amyloliquefaciens subsp. plantarum CAU B946 contains a gene cluster for nonribosomal synthesis of iturin A

The genome of the rhizobacterium Bacillus amyloliquefaciens subsp. plantarum CAU B946 was 4.02 Mb in size and harbored 3,823 genes (coding sequences [CDS]). Nine giant gene clusters were dedicated to nonribosomal synthesis of antimicrobial compounds. Remarkably, strain CAU B946 possessed a gene cluster involved in synthesis of iturin A.     

Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.     

The genome of plant growth-promoting Bacillus amyloliquefaciens subsp. plantarum strain YAU B9601-Y2 contains a gene cluster for mersacidin synthesis

The genome of rhizobacterium Bacillus amyloliquefaciens subsp. plantarum YAU B9601-Y2 was 4.24 Mb in size and harbored 3,991 coding sequences (CDS). Giant gene clusters were dedicated to nonribosomal synthesis of antimicrobial lipopeptides and polyketides. Remarkably, CAU B946 possessed a gene cluster involved in synthesis of mersacidin.     

Restriction mapping of T4 bacteriophage late gene region which contains the origins of DNA replication

DNAs of lambda T4 recombinants 596-27 (genes 50-5), 596-30 (genes 50-8), 596-29 (genes 50-12), 591-16 (genes 6-8), 591-1 (genes 9-12), 596-13 (genes 13-16), 596-17 (genes 18-20) and 596-11 (genes 25-29) were mapped with the use of EcoRI, HindIII, SmaI, SalI and BamHI restriction enzymes. T4 dcDNA was digested with HindIII restriction endonuclease and resulting fragments were cloned into HindIII lambda vector 761. The recombinants 761-7, 761-17, 761-19, 761-24, 761-44, 761-50, 761-55 contained the region of genes 25-48 and 761-42, 761-26 and 761-16 contained a single HindIII-fragment with genes 6-12 in both orientations. Data obtained with the DNA of the latter recombinants allowed to show the correctness of the map established earlier which did not contain a full set of overlapping sequences. As a result of the experiments reported, the position of EcoRI and HindIII recognition sites in the region of genes 50-20 and 25-48 was determined and in the region of genes 25-48 BglII and XhoI restriction sites were mapped. The location of a single BamHI restriction site in the region of gene 8 was also established.     

The C-terminal residues of bacteriophage T7 gene 4 helicase-primase coordinate helicase and DNA polymerase activities

The gene 4 protein of bacteriophage T7 plays a central role in DNA replication by providing both helicase and primase activities. The C-terminal helicase domain is not only responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding, but it also interacts with T7 DNA polymerase to coordinate helicase and polymerase activities. The C-terminal 17 residues of gene 4 protein are critical for its interaction with the T7 DNA polymerase/thioredoxin complex. This C terminus is highly acidic; replacement of these residues with uncharged residues leads to a loss of interaction with T7 DNA polymerase/thioredoxin and an increase in oligomerization of the gene 4 protein. Such an alteration on the C terminus results in a reduced efficiency in strand displacement DNA synthesis catalyzed by gene 4 protein and T7 DNA polymerase/thioredoxin. Replacement of the C-terminal amino acid, phenylalanine, with non-aromatic residues also leads to a loss of interaction of gene 4 protein with T7 DNA polymerase/thioredoxin. However, neither of these modifications of the C terminus affects helicase and primase activities. A chimeric gene 4 protein containing the acidic C terminus of the T7 gene 2.5 single-stranded DNA-binding protein is more active in strand displacement synthesis. Gene 4 hexamers containing even one subunit of a defective C terminus are defective in their interaction with T7 DNA polymerase.     

The pca-pob supraoperonic cluster of Acinetobacter calcoaceticus contains quiA, the structural gene for quinate-shikimate dehydrogenase.

An 18-kbp Acinetobacter calcoaceticus chromosomal segment contains the pcaIJFBDKCHG operon, which is required for catabolism of protocatechuate, and pobSRA, genes associated with conversion of p-hydroxybenzoate to protocatechuate. The genetic function of the 6.5 kbp of DNA between pcaG and pobS was unknown. Deletions in this DNA were designed by removal of fragments between restriction sites, and the deletion mutations were introduced into A. calcoaceticus by natural transformation. The mutations prevented growth with either quinate or shikimate, growth substrates that depend upon qui gene function for their catabolism to protocatechuate. The location of quiA, a gene encoding quinate-shikimate dehydrogenase, was indicated by its expression in one of the deletion mutants, and the position of the gene was confirmed by determination of its 2,427-bp nucleotide sequence. The deduced amino acid sequence of QuiA confirmed that it is a member of a family of membrane-associated, pyrrolo-quinoline quinone-dependent dehydrogenases, as had been suggested by earlier biochemical investigations. Catabolism of quinate and skikimate is initiated by NAD(+)-dependent dehydrogenases in other microorganisms, so it is evident that different gene pools were called upon to provide the ancestral enzyme for this metabolic step.     

The DNA polymerase of bacteriophage YerA41 replicates its T-modified DNA in a primer-independent manner

Yersinia phage YerA41 is morphologically similar to jumbo bacteriophages. The isolated genomic material of YerA41 could not be digested by restriction enzymes, and used as a template by conventional DNA polymerases. Nucleoside analysis of the YerA41 genomic material, carried out to find out whether this was due to modified nucleotides, revealed the presence of a ca 1 kDa substitution of thymidine with apparent oligosaccharide character. We identified and purified the phage DNA polymerase (DNAP) that could replicate the YerA41 genomic DNA even without added primers. Cryo-electron microscopy (EM) was used to characterize structural details of the phage particle. The storage capacity of the 131 nm diameter head was calculated to accommodate a significantly longer genome than that of the 145 577 bp genomic DNA of YerA41 determined here. Indeed, cryo-EM revealed, in contrast to the 25 Å in other phages, spacings of 33–36 Å between shells of the genomic material inside YerA41 heads suggesting that the heavily substituted thymidine increases significantly the spacing of the DNA packaged inside the capsid. In conclusion, YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNAP that has the ability to replicate the genome.     

The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota

Sulfolobus acidocaldarius is an aerobic thermoacidophilic crenarchaeon which grows optimally at 80 degrees C and pH 2 in terrestrial solfataric springs. Here, we describe the genome sequence of strain DSM639, which has been used for many seminal studies on archaeal and crenarchaeal biology. The circular genome carries 2,225,959 bp (37% G+C) with 2,292 predicted protein-encoding genes. Many of the smaller genes were identified for the first time on the basis of comparison of three Sulfolobus genome sequences. Of the protein-coding genes, 305 are exclusive to S. acidocaldarius and 866 are specific to the Sulfolobus genus. Moreover, 82 genes for untranslated RNAs were identified and annotated. Owing to the probable absence of active autonomous and nonautonomous mobile elements, the genome stability and organization of S. acidocaldarius differ radically from those of Sulfolobus solfataricus and Sulfolobus tokodaii. The S. acidocaldarius genome contains an integrated, and probably encaptured, pARN-type conjugative plasmid which may facilitate intercellular chromosomal gene exchange in S. acidocaldarius. Moreover, it contains genes for a characteristic restriction modification system, a UV damage excision repair system, thermopsin, and an aromatic ring dioxygenase, all of which are absent from genomes of other Sulfolobus species. However, it lacks genes for some of their sugar transporters, consistent with it growing on a more limited range of carbon sources. These results, together with the many newly identified protein-coding genes for Sulfolobus, are incorporated into a public Sulfolobus database which can be accessed at http://dac.molbio.ku.dk/dbs/Sulfolobus.