Genomics of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon mobility within Gram-negative bacteria

Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of Gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway that converts tryptophan to PRN is composed of four genes, prnA through D, whose diversity, genomic context and spread over bacterial genomes are poorly understood. Therefore, we launched an endeavour aimed at retrieving, by in vitro and in silico means, diverse bacteria carrying the prnABCD biosynthetic loci in their genomes. Analysis of polymorphisms of the prnD gene sequences revealed a high level of conservation between Burkholderia, Pseudomonas and Serratia spp. derived sequences. Whole-operon- and prnD-based phylogeny resulted in tree topologies that are incongruent with the taxonomic status of the evaluated strains as predicted by 16S rRNA gene phylogeny. The genomic composition of c. 20 kb DNA fragments containg the PRN operon varied in different strains. Highly conserved and distinct transposase-encoding genes surrounding the PRN biosynthetic operons of Burkholderia pseudomallei strains were found. A prnABCD-deprived genomic region in B. pseudomallei strain K96243 contained the same gene composition as, and shared high homology with, the flanking regions of the PRN operon in B. pseudomallei strains 668, 1106a and 1710b. Our results strongly suggest that the PRN biosynthetic operon is mobile. The extent, frequency and promiscuity of this mobility remain to be understood.

Bacterial communities and biogeochemical transformations of iron and sulfur in a high saltmarsh soil profile

Microbial community structure in saltmarsh soils is stratified by depth and availability of electron acceptors for respiration. However, the majority of the microbial species that are involved in the biogeochemical transformations of iron (Fe) and sulfur (S) in such environments are not known. Here we examined the structure of bacterial communities in a high saltmarsh soil profile and discuss their potential relationship with the geochemistry of Fe and S. Our data showed that the soil horizons Ag (oxic-suboxic), Bg (suboxic), Cri (anoxic with low concentration of pyrite Fe) and Cr-2 (anoxic with high concentrations of pyrite Fe) have distinct geochemical and microbiological characteristics. In general, total S concentration increased with depth and was correlated with the presence of pyrite Fe. Soluble + exchangable-Fe, pyrite Fe and acid volatile sulfide Fe concentrations also increased with depth, whereas ascorbate extractable-Fe concentrations decreased. The occurrence of reduced forms of Fe in the horizon Ag and oxidized Fe in horizon Cr-2 suggests that the typical redox zonation, common to several marine sediments, does not occur in the saltmarsh soil profile studied. Overall, the bacterial community structure in the horizon Ag and Cr-2 shared low levels of similarity, as compared to their adjacent horizons, Bg and Cr-1, respectively. The phylogenetic analyses of bacterial 16S rRNA gene sequences from clone libraries showed that the predominant phylotypes in horizon Ag were related to Alphaproteobacteria and Bacteroidetes. In contrast, the most abundant phylotypes in horizon Cr-2 were related to Deltaproteo-bacteria, Chloroflexi, Deferribacteres and Nitrospira. The high frequency of sequences with low levels of similarity to known bacterial species in horizons Ag and Cr-2 indicates that the bacterial communities in both horizons are dominated by novel bacterial species. (c) 2008 Elsevier Ltd. All rights reserved.

Diversity of endophytic bacteria from Eucalyptus species seeds and colonization of seedlings by Pantoea agglomerans

The diversity and beneficial characteristics of endophytic microorganisms have been studied in several host plants. However, information regal-ding naturally, occurring seed-associated endophytes and vertical transmission among different life-history stages of hosts is limited. Endophytic bacteria were isolated from seeds and seedlings of 10 Eucalyptus species and two hybrids. The results showed that endophytic bacteria, Such as Bacillus, Enterococcus, Paenibacillus and Methylobacterium, are vertically transferred from seeds to seedlings. In addition, the endophytic bacterium Pantoea agglomerans was tagged with the gfp gene, inoculated into seeds and further reisolated from seedlings. These results suggested it novel approach to change the profile of the plants, where the bacterium is a delivery vehicle for desired traits. This is the first report of an endophytic bacterial community residing in Eucalyptus seeds and the transmission of these bacteria from seeds to seedlings. The bacterial species reported ill this work have been described as providing benefits to host plants. Therefore, we Suggest that endophytic bacteria can be transmitted vertically from seeds to seedlings, assuring the support of the bacterial community in the host plant.

Analysis of vaginal lactobacilli from healthy and infected Brazilian women

Culture-dependent PCR-amplified rRNA gene restriction analysis and culture-independent (PCR-denaturing gradient gel electrophoresis) methodologies were used to examine vaginal lactobacilli from Brazilian women who were healthy or had been diagnosed with vulvovaginal candidiasis (VVC) or bacterial vaginosis. Only Lactobacillus crispatus was detected accordingly by both methods, and H2O2-producing lactobacilli were not associated with protection against VVC.

wsp Gene sequences from the Wolbachia of filarial nematodes

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences.

Bellerophon: a program to detect chimeric sequences in multiple sequence alignments

Bellerophon is a program for detecting chimeric sequences in multiple sequence datasets by an adaption of partial treeing analysis. Bellerophon was specifically developed to detect 16S rRNA gene chimeras in PCR-clone libraries of environmental samples but can be applied to other nucleotide sequence alignments.

Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus

The nifH gene sequence of the nitrogen-fixing bacterium Acetobacter diazotrophicus was determined with the use of the polymerase chain reaction and universal degenerate oligonucleotide primers. The gene shows highest pair-wise similarity to the nifH gene of Azospirillum brasilense. The phylogenetic relationships of the nifH gene sequences were compared with those inferred from 16S rRNA gene sequences. Knowledge of the sequence of the nifH gene contributes to the growing database of nifH gene sequences, and will allow the detection of Acet. diazotrophicus from environmental samples with nifH gene-based primers.

Molecules and morphology in phylogenetic studies of the Hemiuroidea (Digenea : Trematoda : Platyhelminthes)

Phylogenies of trematodes based on characters derived from morphology and life cycles have been controversial. Here, we add molecular data to the phylogenetic study of a group of trematodes, members of the superfamily Hemiuroidea Looss, 1899. DNA sequences from the V4 domain of the nuclear small subunit (18S) rRNA gene and a matrix of morphological characters modified from a previous study were used. There was no significant incongruence between the molecular and the morphological data. However, this was probably due largely to the limited resolving power of the morphological data. Analyses support a monophyletic Hemiuroidea containing at least the families Accacoeliidae, Derogenidae, Didymozoidae, Hirudinellidae, Sclerodistomidae, Syncoeliidae, Isoparorchiidae, Lecithasteridae, and Hemiuridae. These families fall into two principal clades. One contains the first six families and the other the Hemiuridae and lecithasterine lecithasterids. The positions of the hysterolecithine lecithasterids and the Isoparorchiidae were poorly resolved. The Ptychogonimidae may be the sister group of the remaining Hemiuroidea, but there was no support from the molecular data for the placement of the Azygiidae within the superfamily. (C) 1998 Academic Press.

Sensitive and specific detection of Clavibacter xyli subsp. xyli, causal agent of ratoon stunting disease of sugarcane, with a polymerase chain reaction-based assay

A sensitive, specific polymerase chain reaction-based assay was developed for the detection of the causal agent of ratoon stunting disease of sugarcane, Clavibacter xyli subsp. xyli. This assay uses oligonucleotide primers derived from the internal transcribed spacer region between the 16S and 23S rRNA genes of the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplification product from the template of the closely related bacterium C. xyli subsp. cynodontis, nor from other bacterial species. The assay was successfully applied to the detection of C. xyli subsp. xyli in fibrovascular fluid extracted from sugarcane and was sensitive to approximately 22 cells per PCR assay. A multiplex PCR test was also developed which identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.

Distinguishing species and populations of Rhipicephaline ticks with its 2 ribosomal RNA

Most populations and some species of ticks of the genera Boophilus (5 spp.) and Rhipicephalus (ca. 75 spp.) cannot be distinguished phenotypically. Moreover, there is doubt about the validity of species in these genera. I studied the entire second internal transcribed spacer (ITS 2) rRNA of 16 populations of rhipicephaline ticks to address these problems: Boophilus,microplus from Australia, Kenya, South Africa and Brazil (4 populations); Boophilus decoloratus from Kenya; Rhipicephalus appendiculatus from Kenya, Zimbabwe and Zambia (7 populations); Rhipicephalus zambesiensis from Zimbabwe (3 populations); and Rhipicephalus evertsi from Kenya. Each of the 16 populations had a unique ITS 2, but most of the nucleotide variation occurred among species and genera. ITS 2 rRNA can be used to distinguish the populations and species of Boophilus and Rhipicephalus studied here. Little support was found for the hypothesis that B. microplus from Australia and South Africa are different species. ITS 2 appears useful for phylogenetic inference in the Rhipicephalinae because in genetic distance, maximum likelihood, and maximum parsimony analyses, most branches leading to species had >95% bootstrap support. Rhipicephalus appendiculatus and R, zambeziensis are closely related, yet their ITS 2 sequences could be distinguished unambiguously. This lends weight to a previous proposal that Rhipicephalus sanguineus and Rhipicephalus turanicus, and Rhipicephalus pumlilio and Rhipicephalus camicasi, respectively, are conspecific, because each of these pairs of species had identical sequences for ca. 250 bp of ITS 2 rRNA.

Increased expression of mitochondrial genes in human alcoholic brain revealed by differential display

Polymerase chain reaction (PCR)-based differential display was used to screen for alterations in gene expression in the mesolimbic system of the human alcoholic brain. Total RNA was extracted from the nucleus accumbens of five alcoholic and five control brains. A selected subpopulation of mRNA was reverse-transcribed to cDNA and amplified by PCR. A differentially expressed cDNA fragment was recovered, cloned, and sequenced. Full sequence analysis of this 467 bp fragment revealed 98.2% homology with the human mitochondrial 12S rRNA gene. Dot-blot analysis showed increased expression of this gem in nucleus accumbens and hippocampus, but not in the superior frontal cortex, primary motor cortex, caudate, and pallidus/putamen In a total of eight human alcoholic brains, compared with seven control brains. A similar increased expression was observed by dot-blot analysis, using RNA from the cerebral cortex of rats chronically treated with alcohol vapor. Hybridization of a 16S rRNA oligonucleotide probe indicated that the expression of both rRNAs genes was significantly increased in nucleus accumbens. These results indicate that chronic alcohol consumption induces alteration in expression of mitochondrial genes in selected brain regions. The altered gene expression may reflect mitochondrial dysfunction In the alcohol-affected brain.

Phylogenetic position of Chitinophaga pinensis in the Flexibacter-Bacteroides-Cytophaga phylum

Comparison of the 16S rRNA gene sequence determined for Chitinophaga pinensis showed that this species is most closely related to Flexibacter filiformis in the Flexibacter-Bacteroides-Cytophaga phylum, These two chitinolytic bacteria, which are characterized by transformation into spherical bodies on ageing, belong to a strongly supported lineage that also includes Cytophaga arvensicola, Flavobacterium ferrugineum and Flexibacter sancti, The lineage is distinct from the microcyst-forming species Sporocytophaga myxococcoides.

The phylogenetic relationships of Caulobacter, Asticcacaulis and Brevundimonas species and their taxonomic implications

The phylogenetic relationships among the species of Caulobacter, Asticcacaulis and Brevundimonas were studied by comparison of their 16S rDNA sequences. The analysis of almost complete sequences confirmed the early evolutionary divergence of the freshwater and marine species of Caulobacter reported previously [Stahl, D. A., Key, R,, Flesher, B, & Smit, J. (1992), J Bacteriol 174, 2193-2198]. The freshwater species formed two distinct clusters. One cluster contained the species Caulobacter bacteroides, Caulobacter crescentus, Caulobacter fusiformis and Caulobacter henricii. C, bacteroides and C, fusiformis are very closely related (sequence identity 99.8%). The second cluster was not exclusive and contained the species Caulobacter intermedius, Caulobacter subvibrioides and Caulobacter variabilis, as well as Brevundimonas diminuta and Brevundimonas vesicularis, The marine species Caulobacter halobacteroides and Caulobacter maris were very closely related, with a sequence identity of 99.7%, These two species were most closely but distantly related to the marine hyphal/budding bacteria Hyphomonas jannaschiana and Hirschia baltica, which formed a deep phylogenetic line with Rhodobacter sphaeroides and Rhodobacter capsulatus, Caulobacter leidyia is unrelated to the other species of Caulobacter and belongs to the alpha-4 subclass of the Proteobacteria, forming a distinct cluster with Asticcacaulis excentricus and Asticcacaulis biprosthecium, The taxonomic implications of the polyphyletic nature of the genus Caulobacter and the absence of a type culture for the type species of the genus, Caulobacter vibrioides, are discussed.

Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al. 1987) gen. nov., comb. nov.

The phylogenetic relationships among members of the family Comamonadaceae and several unclassified strains were studied by direct sequencing of their PCR-amplified 16S rRNA genes. Based on the 16S rRNA gene sequence analysis, members of the family formed a coherent group. The closest relatives are species of the Rubrivivax sub-group: Leptothrix discophora, Ideonella dechloratans and Rubrivivax gelatinosus. The genus Hydrogenophaga formed two subclusters, as did the species of Acidovorax, whereas the five species of the genus [Aquaspirillum] were polyphyletic. Comamonas acidovorans was phylogenetically distant from the type species of Comamonas, Comamonas terrigena. On the basis of this work and previous studies, Comamonas acidovorans is removed from the genus Comamonas and renamed as Delftia acidovorans gen. nov., comb, nov. Descriptions of the new genus Delftia and of the type species Delftia acidovorans, for which the type strain is ATCC 15668(T), are presented.