Cry-like genes, in an uncommon gene configuration, produce a crystal that localizes within the exosporium when expressed in an acrystalliferous strain of Bacillus thuringiensis.

Cry proteins are pesticidal toxins produced by the bacterium Bacillus thuringiensis (Bt), which aggregate in sporulating cells to form a crystal. Except in a relatively few cases, these crystals are located outside the exosporium that surrounds the spore. Bt2-56 is a strain of Bt that has the relatively uncommon characteristic of locating its Cry protein-containing crystal within the exosporium, and in association with a long, multifiber filament. With the ultimate goal of both understanding and manipulating the localization of Cry proteins within the exosporium, we sought to identify the genes coding for the exosporium-localized Cry proteins in Bt2-56. Herein we show (i) that five cry-like genes are present in the genome of Bt2-56, (ii) that two pairs of these genes show organizational similarity to a relatively uncommon gene configuration that coexpress a cry gene along with a gene whose product aids crystal formation and (iii) that when one of these two gene pairs (cry21A-cdA) is expressed in an acrystalliferous strain of Bt, crystals are formed that localize within the exosporium. In Bt ssp. finitimus, the only other strain in which crystal localization has been studied, a Cry protein needed expression of two non-cry ORFs in order to localize within the exosporium, indicating that there are some mechanistic differences for crystal localization between Bt ssp. finitimus and Bt2-56.

Anti-cancer Parasporin Toxins are Associated with Different Environments: Discovery of Two Novel Parasporin 5-like Genes.

Cry toxins are primarily a family of insecticidal toxins produced by the bacterium Bacillus thuringiensis (Bt). However, some Cry toxins, called parasporins (PSs), are non-insecticidal and have been shown to differentially kill human cancer cells. Based on amino acid homology, there are currently six different classes of parasporins (PS1-6). It is not known what role parasporins play in nature, nor if certain PSs are associated with Bt found in particular environments. Herein, we present ten parasporin-containing isolates of Bt from the Caribbean island of Trinidad. Genes coding for PS1 and PS6 were found in isolates associated mainly with artificial aquatic environments (e.g., barrels with rain water), while Bt possessing two novel PS5-like genes (ps5-1 and ps5-2), were isolated from manure collected directly from the rectum of cattle. The amino acid sequences inferred from the two PS5-like genes were 51 % homologous to each other, while being only 41 or 45 % similar to PS5Aa1/Cry64Aa, the only reported member of the parasporin five class. The low level of amino acid homology between the two PS5-like genes and PS5Aa1 indicate that the two PS5-like genes may represent a new class of parasporins, or greatly expand the level of diversity within the current parasporin 5 class.

The Use of Polystyrene Beads to Prepare Arrayed Samples of Bacillus thuringiensis for Microscopic Examination.

A common activity in the global search for useful Cry toxins is the microscopic screening of bacterial colonies for the presence of Bacillus thuringiensis. High-throughput screens require that aliquots from large numbers of colonies be arrayed on a microscopic slide. However, precisely placing a small amount of bacteria on a slide, and at a density that is useful for microscopic examination, is both difficult to achieve and time consuming. Herein we share a simple technique that utilizes a hooked wand and small polystyrene beads to quickly collect, and uniformly apply, aliquots of bacterial colonies onto gridded microscope slides in a manner optimal for viewing. If desired, libraries of examined bacteria can simultaneously be generated by discharging the beads into indexed multiwell plates. This simple and inexpensive method is robust, suitable for both light and phase contrast microscopy, and has been also used successfully to screen randomly mutated bacteria for phenotypic changes.

A novel Bacillus thuringiensis Cry-like protein from a rare filamentous strain promotes crystal localization within the exosporium.

Mutation of a novel cry-like gene (cry256) from Bacillus thuringiensis resulted in a protein crystal, normally located within the spore's exosporium, being found predominately outside the exosporium. The cry256 gene codes for a 3-domain Cry-like protein that does not correspond to any of the known Cry protein holotypes.

An apparatus to control and automate the formation of continuous density gradients.

A common problem associated with making a continuous density gradient is the unwanted mixing of liquids as the gradient solution is being delivered from the gradient mixer to the centrifuge tube. We show that by using a Styrofoam float on a thin drawn-out glass pipette, liquids from the gradient mixer will flow down the outside surface of the glass fiber and contact the float, after which the liquid spreads across the gradient surface without unwanted mixing. As the liquid in the tube rises, the float rises up the glass fiber, thereby allowing the creation of gradients without the need to monitor the process.

Parasporins from a Caribbean Island: evidence for a globally dispersed Bacillus thuringiensis strain.

Parasporins represent a new functional class of Cry (crystal protein) toxins produced by the bacterium Bacillus thuringiensis (Bt). Unlike Cry toxins that demonstrate activity mainly against some insect cells, parasporins are characterized as being non-hemolytic, yet capable of preferentially killing some human cancer cells. Globally, six different parasporin types, PS1-PS6, based on protein sequence homology, have been identified in only four countries (Japan, Vietnam, India, and Canada). Herein we report the results of a screening study of 160 Bt isolates collected from the Caribbean island of Trinidad. One isolate (strain 64-1-94) was shown to kill human cancer cells and to contain one ps6 and two ps1 parasporin genes. The two ps1 genes were located approximately 6 kb apart from each other, sharing a similar spatial arrangement, and high sequence homology, with two plasmid-located ps1 genes, ps1Aa6 and ps1Ad1, recently isolated from a Japanese strain. Evidence is also presented that a parasporin gene reported previously for a Canadian strain, ps1Aa2, is most likely derived from a recombination event between these same two genes found in the Trinidadian and Japanese strains. Notably, all three strains share a ps6 parasporin gene, presumably located on a separate plasmid. These data suggest that the global population of ps1 genes may be have originated from a single pair of parasporin genes. Given the large geographical distance between the collection sites, which are located on both continental land masses and islands at sea, ps1 genes are able to retain a remarkable level of homology not easily explained.

Characterization of a novel Bacillus thuringiensis phenotype possessing multiple appendages attached to a parasporal body.

Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the Caribbean that produces a spore-crystal complex containing six long appendages, each comprised of numerous thinner filaments approximately 10 nm in diameter and 2.5 µm in length. Each of the multi-filament appendages was attached to a single, small parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages.

An exploratory study of methicillin-resistant Staphylococcus aureus and SCCmec elements obtained from a community setting along the Texas border with Mexico.

An exploratory study of methicillin-resistant Staphylococcus aureus (MRSA) and SCCmec elements in bacteria along the Mexican border of south Texas was performed. Between September and December of 2008, 375 swabs of anterior nares were self-collected by students attending the University of Texas-Pan American (UTPA) and cultured for MRSA. Fifty seven bacterial isolates were kept for further analysis that included suspected MRSA and other SCCmec-containing bacteria. Isolates were examined for the presence of nuc, mecA, lukS-PV, and spa genes using PCR. SCCmec and spa typing were also performed. Seven S. aureus isolates were found of which six were classified as MRSA. SCCmec typing showed five of the six MRSA strains to be type IV, while one MRSA strain, and most of the non-S. aureus strains, were untypeable, producing results that were indicative of mixed SCCmec types. Five of the six MRSA strains contained known spa types (two of which corresponded to USA300 and one to USA600), while one strain had a novel spa type. Only one isolate, a USA300 MRSA, was positive for lukS-PV. Easy access by the Texas border community to antibiotics in Mexico without a prescription, and the strong partition in SCCmec types between MRSA and non-S. aureus bacteria suggest that this border region of Texas may be uniquely suited for the study of emerging SCCmec types, their horizontal transfer, and perhaps other aspects of antibiotic resistance in bacteria.

An investigation of bacillus thuringiensis in rectal-collected fecal samples of cows.

In order to better understand the range and role of Bacillus thuringiensis (Bt) and its toxins in nature, we have undertaken a study of Bt taken directly from the rectum of 117 cows from 37 farms on the Caribbean island of Trinidad. Thirty-seven fecal samples (32%) were found to contain at least one Bt. Generally only one or two isolates with a particular crystal morphology were isolated from any one sample, however, a few samples contained more, up to 11 isolates, suggesting post-ingestion amplification. Bioassays using larvae of Musca domestica, Caenorhabditis elegans and Tetrahymena pyriformis showed no observable toxicity in gross bioassays. Very small dot-like parasporal bodies, not generally characteristic of Bt, were isolated from 44% of the samples, which in many instances appeared unstable and whose relation to Bt Cry protein-containing parasporal bodies is unknown. In conclusion, we find little evidence for a host adapted strain of Bt in the cows examined, nor toxicity to organisms that might logically be associated with either the cow or its feces. The presence of a large number of isolates containing small dot-like parasporal bodies, possibly either poly-beta-hydroxybutyrate storage bodies or Cry proteins, was unexpected and merits further investigation.

Rapid in vivo exploration of a 5S rRNA neutral network.

A partial knockout compensation method to screen 5S ribosomal RNA sequence variants in vivo is described. The system utilizes an Escherichia coli strain in which five of eight genomic 5S rRNA genes were deleted in conjunction with a plasmid which is compensatory when carrying a functionally active 5S rRNA. The partial knockout strain is transformed with a population of potentially compensatory plasmids each carrying a randomly generated 5S rRNA gene variant. a The ability to compensate the slow growth rate of the knockout strain is used in conjunction with sequencing to rapidly identify variant 5S rRNAs that are functional as well as those that likely are not. The assay is validated by showing that the growth rate of 15 variants separately expressed in the partial knockout strain can be accurately correlated with in vivo assessments of the potential validity of the same variants. A region of 5S rRNA was mutagenized with this approach and nine novel variants were recovered and characterized. Unlike a complete knockout system, the method allows recovery of both deleterious and functional variants.. The method can be used to study variants of any 5S rRNA in the E. coli context including those of E. coli.

Comparison of polymerase chain reaction and bacterial culture for Salmonella detection in the Muscovy duck in Trinidad and Tobago.

OBJECTIVES: The purpose of this study was to investigate the presence and serovar identity of Salmonella, at the national level, in farmed Muscovy ducks (Cairina moschata) in Trinidad and Tobago, and to compare the relative benefits of bacterial culture to those of polymerase chain reaction (PCR) for use in the routine detection and surveillance of Salmonella in these ducks. METHODS: From March-September 2003, 110 fecal samples were collected from 82 farms across the islands of Trinidad and Tobago. Salmonella was isolated from fresh and frozen samples and the serotype of each was determined through bacterial culture. An in-house, nested PCR that detects all pathogenic Salmonella species was utilized in analyzing the samples. RESULTS: Five samples were positive for Salmonella by bacterial culture, whereas 44 were positive by the nested PCR. Serovars isolated were Kiambu, Orion, Uganda, and two isolates from Group E1 whose H antigens could not be fully characterized. Of the samples, 87 (79%) gave equivalent PCR results for both enrichment broths-28 were positive for both and 59 were negative for both). However, 16 samples were positive for one broth, but not for the other, with the majority (14 of the 16) resulting positive for Selenite broth. PCR results for seven samples were inconclusive due to ambiguous band size or multiple bands near the expected band size. CONCLUSIONS: In Trinidad and Tobago, the Muscovy duck does not appear to be a significant source of S. typhimurium or S. enteritidis, but it does harbor other Salmonella species. In-house, nested PCR represents a simple, relatively inexpensive and potentially more sensitive method than bacterial culture for the routine surveillance of pathogenic Salmonella in the Muscovy duck.

Comparison of polymerase chain reaction and bacterial culture for Salmonella detection in the Muscovy duck in Trinidad and Tobago / Comparación de la reacción en cadena de la polimerasa y el cultivo bacteriano como métodos de detección de Salmonella en patos criollos en Trinidad y Tobago

OBJECTIVES: The purpose of this study was to investigate the presence and serovar identity of Salmonella, at the national level, in farmed Muscovy ducks (Cairina moschata) in Trinidad and Tobago, and to compare the relative benefits of bacterial culture to those of polymerase chain reaction (PCR) for use in the routine detection and surveillance of Salmonella in these ducks. METHODS: From March-September 2003, 110 fecal samples were collected from 82 farms across the islands of Trinidad and Tobago. Salmonella was isolated from fresh and frozen samples and the serotype of each was determined through bacterial culture. An in-house, nested PCR that detects all pathogenic Salmonella species was utilized in analyzing the samples. RESULTS: Five samples were positive for Salmonella by bacterial culture, whereas 44 were positive by the nested PCR. Serovars isolated were Kiambu, Orion, Uganda, and two isolates from Group E1 whose H antigens could not be fully characterized. Of the samples, 87 (79 percent) gave equivalent PCR results for both enrichment broths-28 were positive for both and 59 were negative for both). However, 16 samples were positive for one broth, but not for the other, with the majority (14 of the 16) resulting positive for Selenite broth. PCR results for seven samples were inconclusive due to ambiguous band size or multiple bands near the expected band size. CONCLUSIONS: In Trinidad and Tobago, the Muscovy duck does not appear to be a significant source of S. typhimurium or S. enteritidis, but it does harbor other Salmonella species. In-house, nested PCR represents a simple, relatively inexpensive and potentially more sensitive method than bacterial culture for the routine surveillance of pathogenic Salmonella in the Muscovy duck.

OBJETIVOS: Investigar la presencia de Salmonella en patos criollos (Cairina moschata) criados en Trinidad y Tobago e identificar los serotipos circulantes en el país, así como comparar los beneficios relativos del cultivo bacteriano con respecto a la reacción en cadena de la polimerasa (RCP) en la detección y la vigilancia cotidianas de la Salmonella en estos patos. MÉTODOS: Entre marzo y septiembre de 2003 se tomaron 110 muestras de heces fecales de 82 granjas distribuidas por las islas de Trinidad y Tobago. Se aisló Salmonella de muestras frescas y congeladas y se determinaron los serotipos mediante el cultivo bacteriano. Se utilizó un sistema autóctono de RCP anidada que detecta todas las especies patógenas de Salmonella en las muestras. RESULTADOS: Cinco muestras resultaron positivas para Salmonella mediante el cultivo bacteriano, mientras que 44 fueron positivas mediante la RCP anidada. Se asilaron los serotipos Kiambu, Orion, Uganda y dos aislamientos del grupo E1, cuyos antígenos H no se pudieron caracterizar totalmente. Hubo coincidencia en 87 (79 por ciento) de las muestras analizadas por RCP en ambos caldos de enriquecimiento (28 positivas y 59 negativas). Sin embargo, 16 muestras positivas en un caldo resultaron negativas en el otro; la mayoría de ellas (14 de 16) resultaron positivas en caldo selenito. Siete muestras resultaron indefinidas mediante la RCP debido a tallas ambiguas de las bandas o a múltiples bandas cerca de la talla esperada. CONCLUSIONES: El pato criollo no parece ser una fuente importante de infección por S. typhimurium y S. enteritidis en Trinidad y Tobago, aunque hospeda otras especies de Salmonella. El sistema autóctono de RCP anidada constituye un método simple, relativamente económico y posiblemente más sensible que el cultivo bacteriano en la vigilancia cotidiana de especies patógenas de Salmonella en el pato criollo.

In-house polymerase chain reaction for affordable and sustainable Chlamydia trachomatis detection in Trinidad and Tobago.

OBJECTIVES: To provide a preliminary assessment of in-house polymerase chain reaction (PCR) as an alternative to the more costly commercial test for detection of asymptomatic infection by Chlamydia trachomatis and to provide much needed demographic data on infection indicators within the Trinidad and Tobago public health care system. METHODS: An inexpensive in-house nested-PCR with an Internal Amplification Control was used to detect C. trachomatis and Neisseria gonorrhoeae in urine samples collected from 273 apparently healthy, pregnant women from March-September 2004 in Trinidad, West Indies. Demographic information on participants was collected and subjected to statistical analyses. RESULTS: C. trachomatis was detected in 57/273 (21 per cent) samples, of which 5 (2 per cent) were also positive for N. gonorrhoeae. Infection correlated well with certain demographic parameters, with the highest incidence of C. trachomatis infection found among pregnant women that were single or of African descent. CONCLUSIONS: Given the lack of commercial tests in Trinidad, in-house PCR is an inexpensive alternative that can be used to detect asymptomatic infections of C. trachomatis and to provide demographic information needed for interventions by the public health care system.

In-house polymerase chain reaction for affordable and sustainable Chlamydia trachomatis detection in Trinidad and Tobago

OBJECTIVES: To provide a preliminary assessment of in-house polymerase chain reaction (PCR) as an alternative to the more costly commercial test for detection of asymptomatic infection by Chlamydia trachomatis and to provide much needed demographic data on infection indicators within the Trinidad and Tobago public health care system. METHODS: An inexpensive in-house nested-PCR with an Internal Amplification Control was used to detect C. trachomatis and Neisseria gonorrhoeae in urine samples collected from 273 apparently healthy, pregnant women from March-September 2004 in Trinidad, West Indies. Demographic information on participants was collected and subjected to statistical analyses. RESULTS: C. trachomatis was detected in 57/273 (21 percent) samples, of which 5 (2 percent) were also positive for N. gonorrhoeae. Infection correlated well with certain demographic parameters, with the highest incidence of C. trachomatis infection found among pregnant women that were single or of African descent. CONCLUSIONS: Given the lack of commercial tests in Trinidad, in-house PCR is an inexpensive alternative that can be used to detect asymptomatic infections of C. trachomatis and to provide demographic information needed for interventions by the public health care system.

OBJETIVOS: Hacer una evaluación preliminar de un sistema autóctono para la detección de la infección asintomática por Chlamydia trachomatis mediante la reacción en cadena de la polimerasa (RCP), como alternativa a los costosos sistemas comerciales, y ofrecer datos demográficos muy necesarios relacionados con los indicadores de esta infección en el sistema de salud pública de Trinidad y Tobago. MÉTODOS: Se empleó un sistema autóctono y económico de RCP anidada con control interno de la amplificación para la detección de C. trachomatis y Neisseria gonorrhoeae en muestras de orina de 273 mujeres embarazadas asintomáticas, entre marzo y septiembre de 2004 en Trinidad y Tobago, Indias Occidentales. Se obtuvo la información demográfica de las participantes y se sometió a análisis estadístico. RESULTADOS: Se detectó C. trachomatis en 57/273 (21 por ciento) muestras, de las cuales 5 (2 por ciento) fueron también positivas para N. gonorrhoeae. La infección se correlacionó bien con algunos parámetros demográficos; la mayor incidencia de la infección por C. trachomatis se observó en las mujeres embarazadas solteras o de ascendencia africana. CONCLUSIONES: Debido al déficit de sistemas de diagnóstico comerciales en Trinidad, la RCP autóctona es una alternativa económica que puede emplearse para detectar la infección asintomática por C. trachomatis y obtener la información demográfica necesaria para que el sistema de salud pública implemente intervenciones.

Versatile blood bags for laboratory feeding of mosquitoes.

Research programs in mosquito-borne diseases have necessitated the bloodfeeding of mosquitoes in captivity. Parafilm has been shown to be an effective membrane through which mosquitoes can access blood, and numerous feeding devices using parafilm membranes have been reported. However, these devices can be incompatible with experimental conditions, which may require small blood volumes, exact and constant blood temperatures, and inexpensive or disposable materials. We report herein on 2 methodologies that can be used to make parafilm blood bags with a support side and a feeding side made up of a thinner sheet of parafilm. Blood bags were easily and cheaply made to handle volumes as little as 150 microl or as large as 5 ml, to be disposable, and to be easily fastened to a heat source to provide constant and reproducible blood temperatures. Blood bags represent perhaps the most versatile and inexpensive approach to parafilm membrane feeding yet reported.

In-house polymerase chain reaction for affordable and sustainable Chlamydia trachomatis detection in Trinidad and Tobago.

OBJECTIVES: To provide a preliminary assessment of in-house polymerase chain reaction (PCR) as an alternative to the more costly commercial test for detection of asymptomatic infection by Chlamydia trachomatis and to provide much needed demographic data on infection indicators within the Trinidad and Tobago public health care system. METHODS: An inexpensive in-house nested-PCR with an Internal Amplification Control was used to detect C. trachomatis and Neisseria gonorrhoeae in urine samples collected from 273 apparently healthy, pregnant women from March-September 2004 in Trinidad, West Indies. Demographic information on participants was collected and subjected to statistical analyses. RESULTS: C. trachomatis was detected in 57/273 (21%) samples, of which 5 (2%) were also positive for N. gonorrhoeae. Infection correlated well with certain demographic parameters, with the highest incidence of C. trachomatis infection found among pregnant women that were single or of African descent. CONCLUSIONS: Given the lack of commercial tests in Trinidad, in-house PCR is an inexpensive alternative that can be used to detect asymptomatic infections of C. trachomatis and to provide demographic information needed for interventions by the public health care system.

A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results.

BACKGROUND: Bacillus thuringiensis is a bacterium known for producing protein crystals with insecticidal properties. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. There is great interest in isolating strains with improved or novel toxin characteristics, however isolating B. thuringiensis from the environment is time consuming and yields relatively few isolates of interest. New approaches to B. thuringiensis isolation have been, and continue to be sought. In this report, candidate B. thuringiensis isolates were recovered from environmental samples using a combination of a novel stain, high throughput and reduced selection. Isolates were further characterized by SDS-PAGE, light microscopy, PCR, probe hybridization, and with selected isolates, DNA sequencing, bioassay or Electron Microscopy. RESULTS: Based on SDS-PAGE patterns and the presence of cry genes or a crystal, 79 candidate, non-clonal isolates of B. thuringiensis were identified from 84 samples and over 10,000 colonies. Although only 16/79 (20%) of the isolates showed DNA homology by Probe Hybridization or PCR to common cry genes, initial characterization revealed a surprisingly rich library that included a putative nematocidal gene, a novel filamentous structure associated with a crystal, a spore with spikes originating from a very small parasporal body and isolates with unusually small crystals. When compared to reports of other screens, this screen was also atypical in that only 3/79 isolates (3.8%) produced a bipyramidal crystal and 24/79 (30%) of the isolates' spores possessed an attached, dark-staining body. CONCLUSION: Results suggest that the screening methodology adopted in this study might deliver a vastly richer and potentially more useful library of B. thuringiensis isolates as compared to that obtained with commonly reported methodologies, and that by extension, methodologies fundamentally different from current methods should also be explored.