Assessment and accumulation of microplastics in sewage sludge at wastewater treatment plants located in Cádiz, Spain.

Sludge from Wastewater treatment plants (WWTPs) have been determined as a sink of microplastics (MP) removed from wastewater. The aim of this research work has been to evaluate the presence of these pollutants in the sludge of seven WWTPs (five urban and two industrial), located in southern Spain. Samples were collected in the primary, secondary and digested sludge matrixes, MPs were extracted following wet peroxide oxidation and the removal of cellulose, finally the samples were analyzed according to their abundance, size (from 100 µm to 5 mm), shape, colour, and polymer type. Subsequently, the data obtained on the WWTPs were compared, the main difference among the WWTPs and different sample points showed high heterogeneity in terms of abundance of microplastics, due to the differences in the sludge loaded, the processes and the type of sludge. The results from this study established that the most abundant shape was fibers; regarding the size, 100-355 µm fraction was the most abundant, showing that the amount of MPs increased when the size decreased. Regarding the type of polymers, 23 were identified by ATR-FTIR. Further, Acrylate, PE, EAA and PP were the most abundant found polymers. The presence of MPs in the digested sludge varied from 0.02 ± 0.006 MP g DW-1 to 57.18 ± 20.69 MP g DW-1 in the WWTP 6 (food industry) and WWTP 3 (urban city over 212,000 inhabitants), respectively; higher abundance of MPs found in the primary sludge in respect to secondary sludge; in concordance with the removal from wastewater line reported in other studies. The results obtained showed that MPs were widely present in sludge, becoming a sink of these pollutants, estimating that among 8.05 · 104 and 1.77 · 109 MPs · day-1 were loaded to sludge; therefore, these facilities act as a significant source of MPs into agriculture when sludge is used as soil amendment.

The Pathogen-annotated Tracking Resource Network (PATRN) system: a web-based resource to aid food safety, regulatory science, and investigations of foodborne pathogens and disease.

Investigation of foodborne diseases requires the capture and analysis of time-sensitive information on microbial pathogens that is derived from multiple analytical methods and sources. The web-based Pathogen-annotated Tracking Resource Network (PATRN) system (www.patrn.net) was developed to address the data aggregation, analysis, and communication needs important to the global food safety community for the investigation of foodborne disease. PATRN incorporates a standard vocabulary for describing isolate metadata and provides a representational schema for a prototypic data exchange standard using a novel data loading wizard for aggregation of assay and attribution information. PATRN currently houses expert-curated, high-quality "foundational datasets" consisting of published experimental results from conventional assays and next generation analysis platforms for isolates of Escherichia coli, Listeria monocytogenes, and Salmonella, Shigella, Vibrio and Cronobacter species. A suite of computational tools for data mining, clustering, and graphical representation is available. Within PATRN, the public curated data repository is complemented by a secure private workspace for user-driven analyses, and for sharing data among collaborators. To demonstrate the data curation, loading wizard features, and analytical capabilities of PATRN, three use-case scenarios are presented. Use-case scenario one is a comparison of the distribution and prevalence of plasmid-encoded virulence factor genes among 249 Cronobacter strains with similar attributes to that of nine Cronobacter isolates from recent cases obtained between March and October, 2010-2011. To highlight PATRN's data management and trend finding tools, analysis of datasets, stored in PATRN as part of an ongoing surveillance project to identify the predominant molecular serogroups among Cronobacter sakazakii isolates observed in the USA is shown. Use-case scenario two demonstrates the secure workspace available for private users to upload and analyze sensitive data, and for collating cross-platform datasets to identify and validate congruent datapoints. SNP datasets from WGS assemblies and pan-genome microarrays are analyzed in a combinatorial fashion to determine relatedness of 33 Salmonella enterica strains to six strains collected as part of an outbreak investigation. Use-case scenario three utilizes published surveillance results that describe the incidence and sources of O157:H7 E. coli isolates associated with a produce pre-harvest surveillance study that occurred during 2002-2006. In summary, PATRN is a web-based integrated platform containing tools for the management, analysis and visualization of data about foodborne pathogens.

Identification and characterization of Cronobacter iron acquisition systems.

Cronobacter spp. are emerging pathogens that cause severe infantile meningitis, septicemia, or necrotizing enterocolitis. Contaminated powdered infant formula has been implicated as the source of Cronobacter spp. in most cases, but questions still remain regarding the natural habitat and virulence potential for each strain. The iron acquisition systems in 231 Cronobacter strains isolated from different sources were identified and characterized. All Cronobacter spp. have both the Feo and Efe systems for acquisition of ferrous iron, and all plasmid-harboring strains (98%) have the aerobactin-like siderophore, cronobactin, for transport of ferric iron. All Cronobacter spp. have the genes encoding an enterobactin-like siderophore, although it was not functional under the conditions tested. Furthermore, all Cronobacter spp. have genes encoding five receptors for heterologous siderophores. A ferric dicitrate transport system (fec system) is encoded specifically by a subset of Cronobacter sakazakii and C. malonaticus strains, of which a high percentage were isolated from clinical samples. Phylogenetic analysis confirmed that the fec system is most closely related to orthologous genes present in human-pathogenic bacterial strains. Moreover, all strains of C. dublinensis and C. muytjensii encode two receptors, FcuA and Fct, for heterologous siderophores produced by plant pathogens. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed which genes and operons are components of the Fur regulon. Taken together, these results support the proposition that C. sakazakii and C. malonaticus may be more associated with the human host and C. dublinensis and C. muytjensii with plants.

Molecular characterization of Cronobacter lipopolysaccharide O-antigen gene clusters and development of serotype-specific PCR assays.

Cronobacter (formerly Enterobacter sakazakii) is a recently defined genus consisting of six species, C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and Cronobacter genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between galF and gnd, were used to identify serotypes in Cronobacter spp. Seven O-antigen RFLP clusters were generated, including three C. sakazakii clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two C. sakazakii strains, two C. malonaticus strains, one C. turicensis strain, and one C. muytjensii strain, revealed three O-antigen gene clusters shared among Cronobacter species. PCR assays were developed, targeting the wzx O-antigen polymerase gene, and used to screen 231 Cronobacter strains to determine the frequency of these newly identified serotypes.

Characterization of putative virulence genes on the related RepFIB plasmids harbored by Cronobacter spp.

Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA, as well as two iron acquisition systems (eitCBAD and iucABCD/iutA). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa (Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (fhaB), its specifc transporter gene (fhaC), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA-targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, the presence of cpa, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.

Cpa, the outer membrane protease of Cronobacter sakazakii, activates plasminogen and mediates resistance to serum bactericidal activity.

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ~131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii.

Molecular epidemiology of Shigella flexneri in a diarrhoea-endemic area of Lima, Peru.

A year-long community-based study of diarrhoeal diseases was conducted in Canto Grande, a periurban community in Lima, Peru. In 109 (34%) houses out of 323 that were visited, at least one individual was detected with shigellosis. The frequency of the 161 shigella isolates obtained was as follows: 117 S. flexneri (73%), 21 S. boydii (13%), 15 S. dysenteriae (9%), and 8 S. sonnei (5%). Using a non-radioactive ipaH gene probe as a molecular epidemiological tool, a total of 41 S. flexneri strains were shown to be distributed in 25 intra-family comparisons by pairs (icp). Further subdivision, based on a comparison of the serotype, plasmid profile, antibiotic resistances and ipaH hybridization patterns indicated that Group I, with 11 icp (44%), had strains that were identical. Group II with 8 icp (32%), had strains that were different and Group III with 6 icp (24%), had strains with the same serotype and identical ipaH profiles but with differences in other markers. This data indicates that a diversity of shigella clones circulated in this community resulting from both clonal spread and horizontal transfer of genetic elements. Furthermore, ipaH profiling of isolates can be used not only to differentiate between closely related shigella strains but also with other parameters, help to understand the dynamics of the generation of new clones of pathogenic bacteria.

Mutations in the extracellular protein secretion pathway genes (eps) interfere with rugose polysaccharide production in and motility of Vibrio cholerae.

Vibrio cholerae is the causal organism of the diarrheal disease cholera. The rugose variant of V. cholerae is associated with the secretion of an exopolysaccharide. The rugose polysaccharide has been shown to confer increased resistance to a variety of agents, such as chlorine, bioacids, and oxidative and osmotic stresses. It also promotes biofilm formation, thereby increasing the survival of the bacteria in the aquatic environments. Here we show that the extracellular protein secretion system (gene designated eps) is involved directly or indirectly in the production of rugose polysaccharide. A TnphoA insertion in epsD gene of the eps operon abolished the production of rugose polysaccharide, reduced the secretion of cholera toxin and hemolysin, and resulted in a nonmotile phenotype. We have constructed defined mutations of the epsD and epsE genes that affected these phenotypes and complemented these defects by plasmid clones of the respective wild-type genes. These results suggest a major role for the eps system in pathogenesis and environmental survival of V. cholerae.

Molecular evolution of the pathogenicity island of enterotoxigenic Bacteroides fragilis strains.

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.

Regulation of antioxidant enzyme gene expression in response to oxidative stress and during differentiation of mouse skeletal muscle.

Various properties of skeletal muscle, including high metabolic activity and high levels of heme-containing proteins, render it particularly susceptible to free radical injury. Indeed, cellular injury from reactive oxygen species (ROS) has been implicated in many muscle disorders. Thus muscle cell survival is critically dependent on the ability of the cell to respond to periods of oxidative stress. To investigate this important homeostatic response, we studied the effect of oxidative challenges on the expression of genes encoding the antioxidant enzymes Cu,Zn-superoxide dismutase (CuZnSOD), Mn-superoxide dismutase (MnSOD), glutathione peroxidase (GPx), and catalase (CAT) in myotube cultures. Using Northern blot analysis, we found that treatment with the pro-oxidant paraquat resulted in time- and dose-dependent increases of transcript levels that were greatest for GPx and CAT (approximately 4-5 fold). CuZnSOD and MnSOD transcripts were also increased, albeit more modestly (approximately 2-3 fold). Transcript levels were also induced by treatment of the cells with two other pro-oxidants, menadione and H2O2, and correlated with the level of oxidative injury to the cells, measured as protein carbonyl group formation. Activities of all of the enzymes increased in response to the oxidative challenges, although the magnitudes of the increases were less robust than the increases of the respective transcript levels. In studying the effect of cellular differentiation on antioxidant gene expression and susceptibility to oxidative stress, we found that pro-oxidant treatment resulted in greater oxidative injury to differentiated myotubes than to undifferentiated myoblasts. Furthermore, the increased susceptibility of myotubes correlated with decreased antioxidant defenses-as muscle cells differentiated, both transcript and activity levels of antioxidant enzymes decreased. These data suggest that muscle cells regulate antioxidant defenses in response to oxidative stress and cellular differentiation.

Identification of a third metalloprotease toxin gene in extraintestinal isolates of Bacteroides fragilis.

To further understand the epidemiology of enterotoxigenic Bacteroides fragilis (ETBF), 89 extraintestinal B. fragilis strains from Seoul, Korea, were examined for secretion of B. fragilis toxin (BFT) by the HT29/C1 biologic assay and for the B. fragilis toxin gene (bft) by colony blot hybridization and PCR. Complete agreement between the three techniques was found. Overall, 34 B. fragilis strains (38%) were identified as ETBF. Eleven of the 34 ETBF strains (32%) expressed a new isoform of BFT (Korea-BFT). This new isoform is more related to BFT-2 than to BFT-1. Like BFT-1 and BFT-2, Korea-BFT cleaves E-cadherin, the zonula adherens protein.

Evidence of oxidative stress in mdx mouse muscle: studies of the pre-necrotic state.

Considerable evidence indicates that free radical injury may underlie the pathologic changes in muscular dystrophies from mammalian and avian species. We have investigated the role of oxidative injury in muscle necrosis in mice with a muscular dystrophy due to a defect in the dystrophin gene (the mdx strain). In order to avoid secondary consequences of muscle necrosis, all experiments were done on muscle prior to the onset of the degenerative process (i.e. during the 'pre-necrotic' phase) which lasted up to 20 days of age in the muscles examined. In pre-necrotic mdx muscle, there was an induction of expression of genes encoding antioxidant enzymes, indicative of a cellular response to oxidative stress. In addition, the levels of lipid peroxidation were greater in mdx muscle than in the control. Since the free radical nitric oxide (NO*) has been shown to mediate oxidative injury in various disease states, and because dystrophin has been shown to form a complex with the enzyme nitric oxide synthase, we examined pre-necrotic mdx muscle for evidence of NO*-mediated injury by measuring cellular nitrotyrosine formation. By both immunohistochemical and electrochemical analyses, no evidence of increased nitrotyrosine levels in mdx muscle was detected. Therefore, although no relationship with NO*-mediated toxicity was found, we found evidence of increased oxidative stress preceding the onset of muscle cell death in dystrophin-deficient mice. These results lend support to the hypothesis that free radical-mediated injury may contribute to the pathogenesis of muscular dystrophies.

Cloning and characterization of the Bacteroides fragilis metalloprotease toxin gene.

Strains of Bacteroides fragilis that produce a ca. 20-kDa heat-labile protein toxin (termed B. fragilis toxin [BFT]) have been associated with diarrheal disease of animals and humans. BFT alters the morphology of intestinal epithelial cells both in vitro and in vivo and stimulates secretion in ligated intestinal segments of rats, rabbits, and lambs. Previous genetic and biochemical data indicated that BFT was a metalloprotease which hydrolyzed G (monomeric) actin, gelatin, and azocoll in vitro. In this paper, the cloning and sequencing of the entire B. fragilis toxin gene (bft) from enterotoxigenic B. fragilis (ETBF) 86-5443-2-2 is reported. The bft gene from this ETBF strain consists of one open reading frame of 1,191 nucleotides encoding a predicted 397-residue holotoxin with a calculated molecular weight of 44,493. Comparison of the predicted BFT protein sequence with the N-terminal amino acid sequence of purified BFT indicates that BFT is most probably synthesized by ETBF strains as a preproprotein. These data predict that BFT is processed to yield a biologically active toxin of 186 residues with a molecular mass of 20.7 kDa which is secreted into the culture supernatant. Analysis of the holotoxin sequence predicts a 20-residue amphipathic region at the carboxy terminus of BFT. Thus, in addition to the metalloprotease activity of BFT, the prediction of an amphipathic domain suggests that oligomerization of BFT may permit membrane insertion of the toxin with creation of a transmembrane pore. Comparison of the sequences available for the bft genes from ETBF 86-5443-2-2 and VPI 13784 revealed two regions of reduced homology. Hybridization of oligonucleotide probes specific for each bft to toxigenic B.fragilis strains revealed that 51 and 49% of toxigenic strains contained the 86-5433-2-2 and VPI 13784 bft genes, respectively. No toxigenic strain hybridized with both probes. We propose that these two subtypes of bft be termed bft-1 (VPI 13784) and bft-2 (86-5433-2-2).

Cholera in Lima, Peru, correlates with prior isolation of Vibrio cholerae from the environment.

The authors utilized a recently developed DNA probe technique to obtain quantitative data on occurrence of Vibrio cholerae in samples collected monthly from 12 environmental sites in Lima, Peru, from November 1993 through March 1995. Peak V. cholerae counts ranged from 10(2)/ml to 10(5)/ml, with the highest counts in sewage-contaminated areas and irrigation water. With our methodology, no V. cholerae cases were detected at any site during the winter months of July through October. Counts were detectable in the environment before onset of cholera in the community, with counts at "cleaner" sites upriver correlating significantly with occurrence of community disease 2 and 3 months later. In sites with heavy sewage contamination, V. cholerae could still be detected before the onset of cases in the community; however, in contrast to upriver sites, counts at these latter sites correlated most closely with the number of concurrently occurring cholera cases. These data support a model of cholera seasonality in which initial increases in number of V. cholerae in the environment (possibly triggered by temperature) are followed by onset of illness in the community, with these human cases further amplifying the organism as the epidemic cycle proceeds.

PIP: A newly developed DNA probe technique was used to analyze the occurrence of Vibrio cholerae in samples collected from 12 environmental sites in metropolitan Lima, Peru, each month from November 1993 to March 1995. Epidemic V. cholerae cases were found in 69 (34.5%) of the 200 samples collected. This represents more than twice the number of cholera-positive samples correctly identified with traditional methods. The highest V. cholerae counts were recorded in sewage-contaminated areas and irrigation water. No V. cholerae cases were detected at any site during the winter months of July through October. At the relatively cleaner upriver sites, counts were detectable 2-3 months prior to the outbreak of disease in the community. In sites with heavy sewage contamination, counts were more closely correlated with the number of concurrently occurring cholera cases in the community. These findings provide evidence for a model of cholera seasonality, in which initial increases in number of V. cholerae in the environment (possibly triggered by temperature) are followed by an onset of illness in the community, with these human cases further amplifying the organism as the epidemic cycle proceeds. Enhanced understanding of the complex web of human and environmental interactions that lead to seasonal cholera outbreaks would facilitate the design of preventive interventions.

Cloning and characterization of dnaE, encoding the catalytic subunit of replicative DNA polymerase III, from Vibrio cholerae strain C6706.

We report that Vibrio cholerae (Vc) contains a gene homologous to Escherichia coli dnaE, the structural gene for the alpha (catalytic) subunit of replicative DNA polymerase III (PolIII). Despite 24% amino acid (aa) differences in the encoded proteins, the Vc gene strongly complements an E. coli dnaE temperature sensitive (ts) mutant, indicating that all functional features essential for replication are conserved.

Stem and root nodules on the tropical wetland legume Aeschynomene fluminensis.

Aeschynomene fluminensis Veil., originally obtained from flooded areas of the Pantanal Matogrossense region of Brazil, was grown under stem-flooded or non-flooded conditions for 70 d after inoculation with isolates of photosynthetic stem nodule rhizobia obtained from native A. fluminensis. Stem nodules formed only on submerged stems of flooded plants (mean of 25 per plant), and did not form on aerial parts, although they were capable of growing and fixing N2 after drainage of the stems. Root nodules formed on both non-flooded and flooded plants but were usually decreased in number by flooding (from means of 124 to 51 per plant, respectively). Flooding (and stem-nodulation) resulted in an increase in shoot (and a decrease in root) dry weight, regardless of rhizobial isolate. Stem nodules were attached by a wide collar of aerenchymatous tissue at the base of the nodule. There were large air spaces in the stem where nodules were subtended and these were continuous with nodule aerenchyma/outer cortex. In addition, aerenchyma and spongy tissue at the base of the nodule connected both flooded and non-flooded root nodules to large intercellular spaces in the root cortex. The stem and root nodules were ovoid in shape, and essentially aeschynomenoid in type, i.e. the central infected tissue was without uninfected, interstitial cells. Root nodules had a similar structure to stem nodules (although stem nodules were generally larger), and flooded root nodules were approximately twice the size of non-flooded nodules. The infected tissue of root and stem nodules consisted of spherical, bacteroid-containing cells containing one or two rod-shaped bacteroids per peribacteroid unit and prominent organelles. Infection threads were observed in root but not in stem nodules. The cortex of stem and root nodules had an apparent oxygen diffusion barrier, consisting of concentric layers of small cells with interlocking cell walls and few intercellular spaces. Cell layers external to these consisted of larger cells and intercellular spaces, with some spaces being occluded with an electron-dense material that contained a glycoprotein recognized by the monoclonal antibodies MAC236 and MAC265. The amount of glycoprotein occlusions did not appear to differ between nodule types or treatments, although stem nodules contained intracellular glycoprotein vesicles adjacent to cell walls. The exterior of the nodules consisted of an epidermis of thin flattened cells with occasional lenticels. Amyloplasts were common in lower stem and hypocotyl nodules, but fewer in flooded or non-flooded root nodules. Upper stem nodules (i.e. those within 6 cm of the water surface) differed from more profoundly submerged stem nodules by having chloroplasts throughout the cortex. Root nodules did not contain chloroplasts, and undifferentiated plastids were found mainly in lower stem nodules.

Nitrogen-fixing stem nodules of the Legume, Discolobium pulchellum Benth.

We report on the structure of N2 -fixing nodules formed on the stem of Discolobium pulchellum Benth., an aquatic legume in the subfamily Pupilionoideae, tribe Aeschynomeneae, from the Hooded areas in the 'Pantanal Matogrossense' region of Brazil. The stern (and root) nodules were obligately aquatic, requiring permanent submergence in water or flooded soil, and receive oxygen via profuse aerenchyma covering The lower stem. Of the 69 isolates of rhizobia isolated from stem and root nodules, 70% were fast-growing acid producers and 38% were slow growers. The rhizobia were not photosynthetic. Nodules were connected to the stem, and the vascular system from the stem branched throughout the nodule, penetrating the infected, tissue within finger-like ingrowths of cortex. In both stem and root nodules, infected tissue was aeschynomenoid or desmodioid, that is, without uninfected (interstitial) cells. The infected cells in stem nodules were vacuolate, with visible infection threads. The inner cortex was rich in amyloplasts and contained the components of an oxygen diffusion barrier (a boundary cell layer without intercellular spaces and glycoprotein occlusions of intercellular spaces in other cell layers). The mid-cortex, external to the boundary layer, consisted of loosely-packed cells and these were continuous with stem aerenchyma. The outer part of the nodules was made up of phellogen-derived cells forming a periderm, or 'corky' layer of cells. The periderm formed large lenticels above cortical vascular bundles. These lenticels also connected with the stem aerenchyma. Root nodules differed only in that infected cells were not vacuolate, bacteroids were larger and contained more poly-ß-hydroxybutyrate (PHB) and there was less aerenchyma/lenticellular tissue. Stem and root nodule structure is discussed in terms of adaptations to O2 constraints in an aquatic environment.

Rhizobium tropici, a novel species nodulating Phaseolus vulgaris L. beans and Leucaena sp. trees.

A new Rhizobium species that nodulates Phaseolus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA-DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16S rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type II strains) and was recognized by its host range (which includes Leucaena spp.) and nif gene organization. In contrast to R. leguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R. tropici and describe them in this paper.