Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequence analysis

In order to determine the nearly complete 16S rRNA gene sequences of cyanobacteria originating from nonaxenic cultures, a cyanobacterium-specific oligonucleotide probe was developed to distinguish polymerase chain reaction (PCR) products of the cyanobacterial rRNA operons from those resulting from amplification of contaminating bacteria. Using this screening method the 16S rRNA genes of four nonaxenic filamentous cyanobacterial strains belonging to the generaLeptolyngbya andOscillatoria were cloned and sequenced. For the genusLeptolyngbya, the 16S rRNA sequence of the axenic strain PCC 73110 was also determined. Phylogenetic trees were constructed based on complete and partial sequences. The results show that the strainsLeptolyngbya foveolarum Komárek 1964/112,Leptolyngbya sp. VRUC 135 Albertano 1985/1, andLeptolyngbya boryanum PCC 73110 belong to the same cluster. StrainOscillatoria cf.corallinae SAG 8.92, which contains the rare photosynthetic pigment CU-phycoerythrin, is not closely related to other CU-phycoerythrin-containing cyanobacteria.Oscillatoria agardhii CYA 18, which is a representative of planktonicOscillatoria species that form toxic blooms in Norwegian inland waters, has no close relatives in the tree.     

Intraspecific variation in the magnitude and pattern of flooding-induced shoot elongation in Rumex palustris

Background and Aims

Intraspecific variation in flooding tolerance is the basic pre-condition for adaptive flooding tolerance to evolve, and flooding-induced shoot elongation is an important trait that enables plants to survive shallow, prolonged flooding. Here an investigation was conducted to determine to what extent variation in flooding-induced leaf elongation exists among and within populations of the wetland species Rumex palustris, and whether the magnitude of elongation can be linked to habitat characteristics.

Methods

Offspring of eight genotypes collected in each of 12 populations from different sites (ranging from river mudflats with dynamic flooding regimes to areas with stagnant water) were submerged, and petioles, laminas and roots were harvested separately to measure traits related to elongation and plant growth.

Key Results

We found strong elongation of petioles upon submergence, and both among- and within-population variation in this trait, not only in final length, but also in the timing of the elongation response. However, the variation in elongation responses could not be linked to habitat type.

Conclusions

Spatio-temporal variation in the duration and depth of flooding in combination with a presumably weak selection against flooding-induced elongation may have contributed to the maintenance of large genetic variation in flooding-related traits among and within populations.     

The phylogeny of the genus Nitrobacter based on comparative rep-PCR, 16S rRNA and nitrite oxidoreductase gene sequence analysis

Strains of Nitrobacter mediate the second step in the nitrification process by oxidizing nitrite to nitrate. The phylogenetic diversity of the genus is currently not well investigated. In this study, a rep-PCR profile and the nearly complete 16S rRNA gene sequence of 30 strains, comprising a wide physiological as well as ecological diversity and encompassing representatives of the four species, were determined. The sequence diversity of the 16S rRNA gene between different species was low, indicating the need for additional phylogenetic markers. Therefore, primers were developed for amplifying the complete nxrX gene and a 380bp fragment of the nxrB1 gene, which are both genes involved in the nitrite oxidation process. These genes confirmed the division into phylogenetic groups revealed by the 16S rRNA gene but showed a better discriminatory power. They can be a valuable additional tool for phylogenetic analysis within the genus Nitrobacter and can assist in the identification of new Nitrobacter isolates.     

Comparative phylogenetic analyses of Halomonas variabilis and related organisms based on 16S rRNA, gyrB and ectBC gene sequences

Halomonas variabilis and phylogenetically related organisms were isolated from various habitats such as Antarctic terrain and saline ponds, deep-sea sediment, deep-sea waters affected by hydrothermal plumes, and hydrothermal vent fluids. Ten strains were selected for physiological and phylogenetic characterization in detail. All of those strains were found to be piezotolerant and psychrotolerant, as well as euryhaline halophilic or halotolerant. Their stress tolerance may facilitate their wide occurrence, even in so-called extreme environments. The 16S rDNA-based phylogenetic relationship was complemented by analyses of the DNA gyrase subunit B gene (gyrB) and genes involved in the synthesis of the major compatible solute, ectoine: diaminobutyric acid aminotransferase gene (ectB) and ectoine synthase gene (ectC). The phylogenetic relationships of H. variabilis and related organisms were very similar in terms of 16S rDNA, gyrB, and ectB. The ectC-based tree was inconsistent with the other phylogenetic trees. For that reason, ectC was inferred to derive from horizontal transfer.     

Decolorization of sulfonated azo dyes with two photosynthetic bacterial strains and a genetically engineered Escherichia coli strain

Sulfonated azo dyes were decolorized by two wild type photosynthetic bacterial (PSB) strains (Rhodobacter sphaeroides AS1.1737 and Rhodopseudomonas palustris AS1.2352) and a recombinant strain (Escherichia coli YB). The effects of environmental factors (dissolved oxygen, pH and temperature) on decolorization were investigated. All the strains could decolorize azo dye up to 900 mg l⁻¹, and the correlations between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Repeated batch operations were performed to study the persistence and stability of bacterial decolorization. Mixed azo dyes were also decolorized by the two PSB strains. Azoreductase was overexpressed in E. coli YB; however, the two PSB strains were better decolorizers for sulfonated azo dyes.     

An essential gene of Escherichia coli that has sequence similarity to a chloroplast gene of unknown function

Summary The dedB gene of Escherichia coli has sequence similarity to the zfpA gene of the chloroplast chromosome. The functions of dedB and zfpA are unknown. We constructed derivatives of temperature-sensitive polA strains into whose chromosomes a plasmid containing the disrupted dedB gene was integrated by homologous recombination. These strains contained normal and disrupted dedB genes in their chromosomes. We then selected plasmid-segregated strains and found no cells containing the disrupted dedB gene, indicating that disruption of the dedB gene was lethal in polA strains of E. coli.     

Taxonomic re-evaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA gene sequences

The taxonomy of Aphanizomenon flos-aquae strain NH-5, a producer of cyanotoxins, was re-evaluated by comparison with six other Aphanizomenon strains using morphological characteristics and 16S rRNA gene sequences. Strain NH-5 was concluded to be improperly identified as Aph. flos-aquae based upon (1) lack of bundle formation in the trichomes, (2) location of akinetes next to heterocytes, (3) lower similarities (less than 97.5%) in the 16S rRNA gene sequences relative to Aph. flos-aquae strains, and (4) comparison within a phylogenetic tree constructed from 16S rRNA gene sequences. The Aphanizomenon strains investigated in this study are classified to four morphological groups as described by the classical taxonomy of Komárek & Kovácik (1989). This classification was supported from the phylogenetic results of 16S rRNA gene sequences. This study also discusses the generic boundaries between Aphanizomenon and Anabaena.     

rep-PCR fingerprinting and taxonomy based on the sequencing of the 16S rRNA gene of 54 elite commercial rhizobial strains

In tropical soils, diversity and biotechnological potential of symbiotic diazotrophic bacteria are high. However, the phylogenetic relationships of prominent strains are still poorly understood. In addition, in countries such as Brazil, despite the broad use of rhizobial inoculants, molecular methods are rarely used in the analysis of strains or determination of inoculant performance. In this study, both rep-PCR (BOX) fingerprintings and the DNA sequences of the 16S rRNA gene were obtained for 54 rhizobial strains officially authorized for the production of commercial inoculants in Brazil. BOX-PCR has proven to be a reliable fingerprinting tool, reinforcing the suggestion of its applicability to track rhizobial strains in culture collections and for quality control of commercial inoculants. On the other hand, the method is not adequate for grouping or defining species or even genera. Nine strains differed in more than 1.03% (15) nucleotides of the 16S rRNA gene in relation to the closest type strain, strongly indicative of new species. Those strains were distributed across the genera Burkholderia, Rhizobium, and Bradyrhizobium.     

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.     

Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12

Summary The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b ₅₆₁ and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b ₅₆₁ is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b ₅₆₁ with those of other bacterial b-type cytochromes was observed.     

Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli

Summary Efficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of M_r 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.     

Molecular phylogeny of the crab genus Brachynotus (Brachyura: Varunidae) based on the 16S rRNA gene

The crab genus Brachynotus de Haan, 1833 is restricted to the intertidal and shallow subtidal of the Mediterranean and northeastern Atlantic. It is presently recognized to consist of four species, of which three (B. foresti, B. gemmellari and B. sexdentatus) are endemic to the Mediterranean. The fourth species, B. atlanticus, is found along the Atlantic coasts of northern Africa and southern Europe, but also extends into the western Mediterranean. This high level of endemism suggests that speciation within Brachynotus is strongly correlated with the geography and geology of the Mediterranean Sea. A molecular phylogeny based on the mitochondrial large subunit (16S) rRNA gene indicates that the four species of Brachynotus form a monophyletic group within Atlantic Varunidae. The DNA sequence data also show that the genus Brachynotus can be subdivided into two species groups, one comprising B. atlanticus and B. foresti, and the other one B. gemmellari and B. sexdentatus. While B. atlanticus and B. foresti are clearly genetically distinct, B. gemmellari and B. sexdentatus are identical in the studied region of the 16S rRNA gene, suggesting a recent separation or continuing gene flow.     

Regions of Intermolecular Complementarity in Escherichia coli 16S rRNA,mRNA, and tRNA Molecules

The results of computer analysis of complementarity regions in the sequences of E. coli 16S rRNA, mRNAs and tRNAs are reported in this article. The potential regions of intermolecular RNA–RNA hybridization, or clinger fragments, in 16S rRNA, which are complementary to the sites frequently occurring in mRNAs and tRNAs, were found. Major clinger fragments on 16S rRNA are universal for genes that belong to different functional groups. Our results show there are adaptations of the structural organization of the 16S rRNA molecule to messenger and transport RNA sequences. RNA interaction with clinger fragments may contribute to upregulation of the translation process through increasing the local concentration of mRNAs and tRNAs in the vicinity of the ribosome and their proper positioning, as well as decrease the efficiency of translation through nonspecific mRNA–16SrRNA interactions.     

Discrimination of Mycobacterium avium- Mycobacterium intracellulare strains by genomic DNA fingerprinting with a 16S rRNA gene probe

Abstract Ribotyping was investigated as a means of distinguishing ten different serotyped reference strains and seven epidemiologically unrelated isolates of Mycobacterium avium - Mycobacterium intracellulare using a labelled 16S rDNA probe. Thirteen restriction enzymes were screened towards an accurate discrimination of strins. Two selected restriction enzymes ( Sac I and Cla I) enabled us to classify the 17 strains into ten ribotypes with an index of discrimination of 0.897. Typeability and reproductibility of the method reached 100%. The patterns obtained exhibited polymorphism of RE fragments within and outside the 16S rRNA gene and may be useful for epidemiological studies.     

Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE

The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.     

Identification of a nodD-like gene in Frankia by direct complementation of a Rhizobium nodD-mutant.

Summary Clones from aFrankia At4 gene bank were pooled into groups and mass conjugated into anodD mutant ofRhizobium leguminosarum bv.viciae by triparental matings. When peas were inoculated with the pooled transconjugants, nodulation was observed. A plasmid, pAt2GX containingFrankia DNA, was isolated from bacteria recovered from these nodules. This plasmid was shown to complement anodD mutant ofR. leguminosarum bv.viciae. Thus pAt2GX contains aFrankia gene that is functionally equivalent tonodD ofR. leguminosarum bv.viciae.     

Nucleotide sequence of the region encompassing the int gene of a cryptic prophage and the dna Y gene flanked by a curved DNA sequence of Escherichia coli K12

Summary The nucleotide sequence of a 2.5 kb region encompassing a curved DNA segment (BENT-9) randomly cloned from the total Escherichia coli chromosome was determined. This region was found to contain the dnaY gene encoding a transfer RNA. The curved DNA structure was demonstrated to be located just upstream of the dnaY promoter. The results of sequencing further revealed that the int gene of a cryptic prophage, qsr, which has been shown to be present in the E. coli genome, is located next to the dnaY gene.