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Obligate commensalism in the marine environment and its evolutionary role are still poorly understood. Although sea turtles may serve as ideal substrates for epibionts, within amphipods, only the genus Hyachelia evolved in obligate commensalism with turtles. Here, we report a new host record for Hyachelia lowryi on the hawksbill turtle and describe a larger distribution of the genus in the Atlantic Ocean on green and loggerhead turtles. Hyachelia spp. were sampled from nesting sites of Caretta caretta and feeding grounds of Eretmochelys imbricata and Chelonia mydas along the Brazilian coast. Insights regarding the coevolution of this remarkable genus with its hosts based on molecular analyses are inferred based on mitochondrial ( COI ) and nuclear (18SrRNA) genes using new and previously available sequences from the infraorder Talitrida. Divergence times for Hyachelia are around the Cretaceous (~127.66 Mya), corresponding to an ancient origin and in agreement with modern green turtle (Chelonioidea) radiation. Later, diversification of Hyachelia species is dated at about 26 Mya, suggesting a coevolutionary association between amphipods and Carettini/Chelonini sea turtles.

The genus Desmogonius (Digenea: Pronocephalidae) is known only from sea turtles and currently contains two species: D. desmogonius Stephens, 1911 and D. loossi Chattopadhyaya, 1972. The present study describes a third species, Desmogonius baldassinae n. sp. found in a juvenile green sea turtle (Chelonia mydas) from Brazil. The new species is larger than its congeners in all dimensions, with vitellinic fields ending posterior to the ovary. In addition, we present a key for species identification and an emended diagnosis of the genus Desmogonius.

Fatal infection by Cyathostoma (Cyathostoma) phenisci (Nematoda: Syngamidae), was identified in 2 of 52 brown boobies (Sula leucogaster) collected on beaches in the state of Rio de Janeiro, Brazil, and admitted to the veterinary clinic for rehabilitation. Both infected birds were in poor physical condition, with atrophied pectoral muscles, and died soon after starting treatment. The parasitological and pathological examination of the carcasses revealed the presence of C. (C.) phenisci in the trachea, resulting in tracheitis, as well as severe parasitic granulomatous bronchopneumonia caused by eggs deposited in the lungs. In our opinion, these serious pathological changes were the primary cause of chronic respiratory illness. This is the first description of fatal cyathostomiasis in a fish-eating avian host caused by infection by a member of the subgenus Cyathostoma (Cyathostoma). Therefore, it is reasonable to consider C. (C.) phenisci to be a real threat to a wide range of their definitive hosts, and cyathostomiasis should be considered in the differential diagnosis for fish-eating marine birds, even in cases without respiratory signs. This is also the first record of the genus Cyathostoma in Brazil., (© American Society of Parasitologists 2020.)

Techniques for anesthesia of green sea turtles ( Chelonia mydas ) are required for medical treatment. The use of spinal anesthesia has been reported in a few species of turtles for different purposes. The objective of this study was to evaluate the use of 2% lidocaine for spinal anesthesia of green sea turtles undergoing surgical removal of cutaneous fibropapillomas. Ten free-ranging green turtles presenting with cutaneous fibropapillomas were included in the study. Animals were accidentally captured or rescued by local fishermen and brought to the Ubatuba Research Base (Sao Paulo, Brazil) of the Brazilian Sea Turtle Conservation Program for rehabilitation. Animals were administered 2% lidocaine (0.2 ml/10 cm of carapace) in the epidural/subarachnoid space of the tail and monitored throughout surgery. The technique was effective for all animals, with fast onset of motor and sensory blockade (3 ± 1.76 min) and relatively fast recovery time (83.9 ± 16.2 min). Fibropapillomas were removed from all animals with no signs of pain (i.e., no behavioral response during surgical procedure, such as head and forelimb movement, showing discomfort) and they were all rehabilitated and successfully returned to their natural habitat. The technique was considered effective, safe, and affordable for use on green turtles undergoing surgical removal of cutaneous fibropapillomas.

The present paper reports the occurrence of the seventh species in the genus Amphiorchis (Digenea: Spirorchiidae) collected from the heart of a green turtle found in Florida, USA. A taxonomic key to the species of Amphiorchis and a literature review for the genus are presented.

The growth of human population and deficient pollution control measures pose significant challenge to the environment. Despite conservation efforts, all sea turtle species are at some risk of extinction. The present study investigated the effect of marine debris on the gastrointestinal tract of green turtles in southeastern Brazil. Of the 777 animals evaluated, 290 showed marine debris in one segment of the gastrointestinal tract. The presence of these materials in the gastrointestinal tract may be harmful, independent of the segment involved, and increases the risk of impaction. Marine debris has become a significant hazard to Chelonia mydas in the region surveyed, causing perforation, rupture, or fecal impaction that, when not treated, is potentially fatal, exposing the intestine to bacterial infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Organochlorines (OCs), such as pesticides and polychlorinated biphenyls (PCBs), are persistent, toxic and widely distributed through atmospheric transport and ocean currents. Few studies have been conducted on OCs in sea turtles, especially on the coast of Brazil. Chelonia mydas is the largest hard-shell sea turtle and is found tropical and subtropical regions in all oceans. The aim of the present study was to determine the occurrence of OCs in the green sea turtle (C. mydas). Fat, liver, kidney and muscle samples were collected from 27 juveniles found on the beach of the city of Ubatuba on the northern coast of the state of São Paulo, Brazil. OCs were extracted with organic solvents and the extract was purified with concentrated acid. Gas chromatography-mass spectrometry and electron capture detection were used for the identification and quantification of PCBs and pesticides, respectively. No organochlorine pesticides were detected in any of the samples. Concentrations of total PCBs in wet weight were <1.6 to 48.9ng/g in fat tissue, <1.6 to 17.4ng/g in liver tissue and <1.6 to 37.7ng/g in kidney tissue. The low levels found are mainly related to diet, as the green sea turtle is basically herbivorous and lower PCB contamination compared to other regions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Weight management for individuals with intellectual and developmental disabilities (IDD) has received limited attention. Studies on weight management in this population have been conducted over short time frames, in small samples with inadequate statistical power, infrequently used a randomized design, and have not evaluated the use of emerging effective dietary strategies such as pre-packaged meals (PMs). Low energy/fat PMs may be useful in individuals with IDD as they simplify meal planning, limit undesirable food choices, teach appropriate portion sizes, are convenient and easy to prepare, and when combined with fruits and vegetables provide a high volume, low energy dense meal. A randomized effectiveness trial will be conducted in 150 overweight/obese adults with mild to moderate IDD, and their study partners to compare weight loss (6 months) and weight maintenance (12 months) between 2 weight management approaches: 1. A Stop Light Diet enhanced with reduced energy/fat PMs (eSLD); and 2. A recommended care reduced energy/fat meal plan diet (RC). The primary aim is to compare weight loss (0-6 months) and weight maintenance (7-18 months) between the eSLD and RC diets. Secondarily, changes in chronic disease risk factors between the eSLD and RC diets including blood pressure, glucose, insulin, LDL-cholesterol, and HDL-cholesterol will be compared during both weight loss and weight maintenance. Finally, potential mediators of weight loss including energy intake, physical activity, data recording, adherence to the diet, study partner self-efficacy and daily stress related to dietary change will be explored., (© 2013. Published by Elsevier Inc. All rights reserved.)

Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.

Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains disrupted in this gene cluster showed that these strains grew more slowly than the wild-type strain in medium lacking iron. Additionally, the mutant strains failed to produce a chrome-azurol-S-reactive material in liquid or solid medium, and failed to produce the metabolite with iron-chelating characteristics that was identified in the wild-type strain. Addition of this purified metabolite to the growth medium of a mutant strain restored its ability to grow in iron-deficient medium. Furthermore, expression of this gene cluster was induced by growth under iron-limiting conditions, suggesting that expression of this gene cluster occurs when iron is scarce. These data are all consistent with the proposal that the proteins encoded by this gene cluster are involved in the production of a siderophore. Interestingly, these proteins show the highest level of amino acid similarity to proteins from a gene cluster found in the filamentous cyanobacterium Nostoc sp. PCC7120, rather than to known siderophore biosynthetic enzymes. Given these properties, it is proposed that the siderophore produced by A. tumefaciens C58 will have a unique chemical structure. Production of the siderophore was not required for virulence of A. tumefaciens when tested with a standard stem inoculation assay.

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

The study of microbial diversity represents a major opportunity for advances in biology and biotechnology. Recent progress in molecular microbial ecology shows that the extent of microbial diversity in nature is far greater than previously thought. Here, we discuss methods to analyse microorganisms from natural environments without culturing them and new approaches for gaining access to the genetic and chemical resources of these microorganisms.

As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.

Expression of the cobalamin biosynthetic (cob) and 1,2-propanediol utilization (cob/pdu) regulon of Salmonella typhimurium LT2 is controlled at the transcriptional level by global and specific regulatory proteins. In this paper we show that mutations in the mviA gene negatively affect cob/pdu transcription in response to 1,2-propanediol in the environment. The effects of mviA mutations were consistent with its role in the regulation of RpoS levels in the cell. Null mutations in rpoS eliminated the negative effect of mviA mutations on cob/pdu transcription, and restored growth on succinate, propionate and 1,2-propanediol. In addition, mviA mutants were deficient in the utilization of succinate, propionate and 1,2-propanediol as carbon and energy sources.

Cultured soil microorganisms have provided a rich source of natural-product chemistry. Because only a tiny fraction of soil microbes from soil are readily cultured, soil might be the greatest untapped resource for novel chemistry. The concept of cloning the metagenome to access the collective genomes and the biosynthetic machinery of soil microflora is explored here.

We show that integration host factor (IHF) is required for the activation of transcription of the cobalamin biosynthetic (cob) and 1,2-propanediol (1,2-PDL) utilization (pdu) operons in Salmonella typhimurium LT2. A lack of IHF affected transcription of the cob/pdu regulon in at least two ways. First, the level of the regulatory protein PocR was decreased in ihfB (formerly himD) mutants, as judged by Western blot analysis with polyclonal antiserum raised against PocR. Second, even when PocR was available, in the absence of IHF, PocR was unable to activate transcription of cob/pdu in response to 1,2-PDL. This result suggested an additional role for IHF in PocR-dependent transcription activation. Consistent with these findings, ihfB mutants of this bacterium were unable to use 1,2-PDL as a carbon or energy source.

The PocR protein of Salmonella typhimurium LT2 was overexpressed and used to demonstrate in vitro that it specifically binds to the cobalamin biosynthetic operon (cob) promoter region. Evidence is presented to show that PocR DNA-binding activity in vitro is regulated by the effector molecule 1,2-propanediol. Deletion analysis of the cob promoter (Pcob) suggested that two regions upstream of the promoter are needed for optimal activation of Pcob by PocR in vivo. DNase I footprinting experiments demonstrated that PocR binds to two sites within Pcob. The transcription initiation site of cob mRNA in response to 1,2-propanediol was identified and shown to be different from the one reported for transcription initiation under anoxic conditions in the absence of 1,2-propanediol.

Evidence documenting the requirement for a functional DNA polymerase I when Salmonella typhimurium LT2 uses ethanolamine (EA), 1,2-propanediol (1,2-PDL), or propionate (PRP) as the sole carbon and energy source is presented. Providing rat polymerase beta in trans demonstrated that the growth phenotypes observed were due exclusively to the lack of DNA polymerase I functions. The location of the mutation (a MudI1734 insertion) that rendered cells unable to grow on EA, 1,2-PDL, or PRP was determined by DNA sequencing to be within the polA gene. polA mutants of this bacterium may be unable to repair the damage caused by reactive aldehydes generated during the catabolism of EA, 1,2-PDL, or PRP. Consistent with this hypothesis, the inhibitory effects of acetaldehyde and propionaldehyde on the growth of this polA mutant were demonstrated. A derivative of the polA mutant unable to synthesize glutathione (GSH) was markedly more sensitive to acetaldehyde and propionaldehyde than was the polA mutant proficient in GSH synthesis. This finding was in agreement with the recently proposed role of GSH as a mechanism for quenching reactive aldehydes generated during the catabolism of these compounds (M. R. Rondon, R. Kazmierczack, and J. C. Escalante-Semerena, J. Bacteriol. 177:5434-5439, 1995).

Transcription of the cob/pdu regulon of Salmonella typhimurium is activated by the PocR regulatory protein in response to 1,2-propanediol (1,2-PDL) in the environment. Nutritional analysis and DNA sequencing confirmed that a strain defective in expression of the cob/pdu regulon in response to 1,2-PDL lacked a functional gshA gene. gshA encodes gamma-glutamylcysteine synthetase (L-glutamate:L-cysteine gamma-ligase [ADP forming]; EC 6.3.2.2), the enzyme that catalyzes the first step in the synthesis of glutathione (GSH). The DNA sequence of gshA was partially determined, and the location of gshA in the chromosome was established by two-factor crosses. P22 cotransduction of gshA with nearby markers showed 21% linkage to srl and 1% linkage to hyd; srl was 9% cotransducible with hyd. In light of these data, the gene order gshA srl hyd is suggested. The level of reduced thiols in the gshA strain was 87% lower than the levels measured in the wild-type strain in both aerobically and anaerobically grown cells. 1,2-PDL-dependent transcription of cob/pdu was studied by using M. Casadaban's Mu-lacZ fusions. In aerobically grown cells, transcription of a cbi-lacZ fusion (the cbi genes are the subset of cob genes that encode functions needed for the synthesis of the corrin ring) was 4-fold lower and transcription of a pdu-lacZ fusion was 10-fold lower in a gshA mutant than in the wild-type strain. Expression of the cob/pdu regulon in response to 1,2-PDL was restored when GSH was included in the medium. In anaerobically grown cells, cbi-lacZ transcription was only 0.4-fold lower than in the gshA+ strain; pdu-lacZ transcription was reduced only by 0.34-fold, despite the lower thiol levels in the mutant. cobA-lacZ transcription was used as negative control of gene whose transcription is not controlled by the PocR/1,2-PDL system; under both conditions, cobA transcription remained unaffected. The gshA mutant strain was unable to utilize 1,2-PDL, ethanolamine, or propionate as a carbon and energy source. The defect in ethanolamine utilization appears to be at the level of ethanolamine ammonia-lyase activity, not at the transcriptional level. Possible roles for GSH in ethanolamine, 1,2-PDL, and propionate catabolism are proposed and discussed.

The CobIII region of the cobalamin (CBL) biosynthetic (cob) operon of Salmonella typhimurium encodes functions necessary for the synthesis of the nucleotide loop of CBL and comprises three genes, designated cobU, cobS, and cobT (26). Complementation studies identified two classes of CobIII mutants: (i) 34 mutants were complemented by a plasmid carrying the cobU+ gene, and (ii) 27 mutants were complemented by a plasmid carrying the cobS+ gene; none of the mutants tested was complemented by the cobT+ clone, a result suggesting that no cobT mutations were isolated. These data were consistent with those of complementation studies done with F' cobUST plasmids, which also suggested that the CobIII region comprises two complementation groups. A plasmid carrying cobUS+ was sufficient to complement a deletion of the entire CobIII region, a result suggesting that CobT was not required for CBL biosynthesis. Nutritional studies done with synthetic putative intermediates of the CobIII pathway were performed to further classify cobIII mutants. A subset of cobU mutants were found to be responsive to exogenous dicyano-cobinamide-GDP, while cobS mutants were found to be responsive only to CBL. These results are consistent with the adenosyl-cobinamide kinase-GTP:adenosyl-cobinamide-phosphate guanylyltransferase and CBL synthase activities proposed for CobU and CobS, respectively. The cobIII genes under the control of the T7 promoter were overexpressed, and the resulting polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides with apparent molecular masses of 22, 26 and 39 kDa, consistent with the predicted masses for CobU, CobS, and CobT, respectively, were detected.

In this communication we present evidence that indicates that 1,2-propanediol (1,2-PDL) is a positive effector of the transcription of the cobalamin (vitamin B12) biosynthetic (cob) operon and of the 1,2-PDL utilization (pdu) genes. The stimulatory effects of 1,2-PDL were demonstrated using Mu d-lac transcriptional fusions to cob and pdu. Significantly increased levels of transcription of the cob and pdu operon fusions were measured in cultures grown under both anoxic and highly aerated conditions when 1,2-PDL was present in the culture medium. We have isolated mutants that carry lesions that render both pdu and cob transcription unresponsive to 1,2-PDL. These mutations were mapped to the region between cob and pdu (41 min), and they define the poc locus PDL and cobalamin). The poc locus is required for the positive regulatory effects of 1,2-PDL to be exerted. Complementation of Poc function was achieved in trans by an F' plasmid carrying the poc+ locus, suggesting that at least one diffusible product regulates the expression of cob and pdu in S. typhimurium.