A study on the effects of some laboratory-derived genetic mutations on biofilm formation by Listeria monocytogenes

Biofilms formed by the human pathogen Listeria monocytogenes in food-processing environments can be a potential source of contamination. In this study, we investigated the ability of L. monocytogenes wild type and its laboratory-derived isogenic mutants in cwhA, prfA, agrA, flaA, degU, ami and sigB to adhere to and form biofilms on abiotic surfaces. The results suggest that inactivation of the two component regulatory system degU completely abolished biofilm formation, while inactivation of the flagellar gene flaA, two component response regulator agrA and the autolysin-adhesin gene ami lead to severe impairment of initial attachment and the subsequent development of a mature biofilm by L. monocytogenes. Mutants in the global regulator of virulence prfA and the alternative sigma factor sigB were unaffected and formed biofilms similar to wild type L. monocytogenes.     

The survival of different vibrios in association with a laboratory culture of the red-tide-causing organism Amphidinium carterae

The survival of different vibrios in association with a red-tide-causing organism Amphidinium carterae was studied in the laboratory. Vibrio alginolyticus and V. harveyi could not survive beyond 14 days in an actively growing culture of A. carterae. On the other hand, V. parahaemolyticus could be detected up to 40 days.     

Microsatellite DNA markers in the giant freshwater prawn, Macrobrachium rosenbergii: a tool for genetic analysis

Eight microsatellite loci were identified and characterized in the commercially important giant freshwater prawn, Macrobrachium rosenbergii. The microsatellite loci were detected by the random screening for dinucleotide repeat units within a partial genomic library developed for the species with biotinylated probes (CA)(15) , (AT)(15) and (GA)(15) . All the eight loci were found to be polymorphic. The number of alleles and observed heterozygosities per locus ranged between three to 16 and 0.22 to 0.71, respectively. These microsatellite markers will be useful for the conservation and management of wild and cultured stocks and population genetic studies of freshwater prawn.     

Phenotypic heterogeneity in mycobacterial stringent response

Background  

A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity.     

Meiotic diapause: how a sperm signal sets you free

Major sperm protein, a cytoskeletal molecule required for the amoeboid motility of sperm in Caenorhabditis elegans, also functions as a signaling molecule that regulates the rates of meiotic maturation and ovulation. Recent work has begun to uncover new genes required for the response to this signal in both somatic and germ line cells.     

High-temperature biotrickling filtration of hydrogen sulphide

Biofiltration of malodorous reduced sulphur compounds such as hydrogen sulphide has been confined to emissions that are at temperatures below 40°C despite the fact that there are many industrial emissions (e.g. in the pulp and paper industry) at temperatures well above 40°C. This paper describes our study on the successful treatment of hydrogen sulphide gas at temperatures of 40, 50, 60 and 70°C using a microbial community obtained from a hot spring. Three biotrickling filter (BTF) systems were set up in parallel for a continuous run of 9 months to operate at three different temperatures, one of which was always at 40°C as a mesophilic control and the other two were for exploring high-temperature operation up to 70°C. The continuous experiment and a series of batch experiments in glass bottles (250 ml) showed that addition of glucose and monosodium glutamate enhanced thermophilic biofiltration of hydrogen sulphide gas and a removal rate of 40 g m⁻³ h⁻¹ was achieved at 70°C. We suggest that the glucose is acting as a carbon source for the existing microbial community in the BTFs, whereas glutamate is acting as a compatible solute. The use of such organic compounds to enhance biodegradation of hydrogen sulphide, particularly at high temperatures, has not been demonstrated to our knowledge and, hence, has opened up a range of possibilities for applying biofiltration to hot gas effluent.     

From DNA enzymes to cone snail venom: the work of Baldomero M. Olivera

Processivity of DNA Exonucleases (Thomas, K. R., and Olivera, B. M. (1978) J. Biol. Chem. 253, 424–429)Neuronal Calcium Channel Inhibitors. Synthesis of ω-Conotoxin GVIA and Effects on ⁴⁵Ca Uptake by Synaptosomes (Rivier, J., Galyean, R., Gray, W. R., Azimi-Zonooz, A., McIntosh, J. M., Cruz, L. J., and Olivera, B. M. (1987) J. Biol. Chem. 262, 1194–1198)The two papers being recognized here as JBC Classics speak to the journeys Baldomero “Toto” Olivera at the University of Utah has made in his life. A director of a program project funded by the National Institute of General Medical Sciences and a professor at the Howard Hughes Medical Institute, Olivera''s papers highlight how doing research in two different countries ultimately influenced his focus and contributions to molecular biology and biochemistry.Open in a separate windowBaldomero “Toto” Olivera. Photo courtesy of Olivera.Olivera began his career as a DNA biophysical chemist and enzymologist. He arrived in the United States in the 1960s to do his graduate work at the California Institute of Technology after completing a bachelor''s degree in chemistry in the Philippines. He joined the laboratory of Norman Davidson to study the biophysical chemistry of DNA. When Olivera was ready to graduate with his Ph.D. degree, Davidson suggested that Olivera go to I. Robert Lehman''s laboratory at Stanford University for his postdoctoral training. “He knew it was my intention to return to the Philippines,” recalls Olivera. Davidson felt it would be easier for Olivera to study DNA enzymology, rather than biophysical chemistry, in a Philippine academic setting because the field did not necessarily demand expensive and sophisticated instrumentation.Olivera followed his thesis advisor''s suggestion and, as a result, became an expert in DNA enzymology, including exonucleases, a large class of DNA-degrading enzymes. The first JBC paper recognized here as a Classic was published in 1978 as Olivera was starting out as an independent researcher. In it, Olivera and his first graduate student, Kirk Thomas, investigated whether or not exonuclease I, first discovered in Escherichia coli by Lehman, and other exonucleases of E. coli were processive. This was at a time when little was known about nucleic acid enzymes: restriction enzymes were just starting to gain traction, and genome sequencing was far from reality. Olivera explains that no one had given much thought to how exonucleases functioned. “The significance of this paper was that it showed that the enzymes that we examined were very different using a new parameter processivity that had never been assessed for exonucleases,” he says.Olivera and Thomas designed an assay that was based on a synthetic nucleic acid chain that contained ³H on one end and ³²P on the other. Researchers knew that exonucleases selected either the 5′- or 3′-end of the DNA to start chewing. The rationale of the Thomas and Olivera assay was that if the enzyme dissociated after every single catalytic event, one label, either the ³H or ³²P, would come off the polymer. However, if the enzyme clung to the polymer and kept chewing until the whole polymer was degraded, both radioactive labels would appear simultaneously in solution.Open in a separate windowOlivera with his first graduate student, Kirk Thomas. Photo courtesy of Olivera.Thomas and Olivera demonstrated that of the eight exonucleases they tested, only the E. coli exonuclease I and λ-exonuclease were processive, meaning that once they got started, they kept on cutting the same piece of DNA before dissociating. The others, such as the spleen and T7 exonucleases, were not processive and frequently came off the DNA.Lehman explains that at the time of this JBC paper, “methods had not yet been developed to measure quantitatively the processivity of either a DNA polymerase or a DNA exonuclease. Their paper made an important contribution to the field of DNA enzymology by describing for the first time a quantitative method for doing so and applied it to eight different DNA exonucleases, an enzymological tour de force.”The second paper highlighted as a JBC Classic was published ten years later and shows a shift in Olivera''s career. The article concerns the synthesis of a peptide found in the venom of the cone snail Conus geographus, which is indigenous to the Indo-Pacific region. All 700 types of cone snails have a special tooth that they use like a harpoon. A venom gland attached to the tooth releases the poisonous peptides to paralyze or even kill prey. These snails have to be handled with great care or not handled at all. Some can sting and cause pain like bees, but C. geographus can kill humans when it stings.There is no scientific connection between DNA enzymes and snail venom. Olivera explains that when he had returned to the Philippines as an assistant professor in the College of Medicine at the University of the Philippines, his laboratory “had absolutely no equipment. It was clear I wasn''t going to be very competitive in DNA replication [research], so we decided we''d find a project that we could start without any equipment. I collected shells as a kid, so I knew about this particular snail that killed people. I had purified enzymes as a post-doc and figured I could purify toxins by injecting them into mice, which didn''t require any equipment at all.”Olivera''s group was soon isolating and characterizing peptides from the cone snail venom. The peptides are known as conotoxins. In doing so, Olivera established the field of conotoxin research, which had a significant impact on fundamental research and medicine. For example, a peptide isolated by Olivera''s group has been approved as a drug for severe pain that cannot be relieved by morphine.Olivera had part-time appointments in the United States while maintaining his full-time position in the Philippines. He first began as a visiting associate professor at Kansas State University and later at the University at Utah. “I would spend seven or eight months in the Philippines and five or four months in the U.S,” he says. Olivera became a full-time member of the faculty at the University of Utah in the 1970s after political and economic upheaval in the Philippines over Ferdinand Marcos'' rule made Olivera decide to return full-time to the United States.Open in a separate windowConus snails. Photo courtesy of Olivera.Open in a separate windowConus snail attacks a fish. Photo courtesy of Olivera.The toxins made by the Conus snails are highly specific for particular targets in the nervous system, such as ion channels. For example, the μ-conotoxins hit sodium receptor ion channels, and ω-conotoxins (one of which, ω-GVIA, is described in this JBC Classic) bind to neuronal calcium channels to inhibit calcium uptake at the presynaptic junction and shut down biochemical signaling at certain synapses.ω-Conotoxin GVIA is a 27-amino acid peptide originally called the “shaker” peptide because it made mice shake. “A number of physiological experiments were done to suggest that it acted at synapses, potentially on calcium channels,” says Olivera. “The importance of this paper is that for the first time the peptide was chemically synthesized and became available to the whole neuroscience community.”The neuroscience community desperately needed this peptide. Up to this point, neuroscientists relied on dihydropyridines to study voltage-gated calcium channels. However, these dihydropyridines had confusing effects on neuronal voltage-gated calcium channels, which made data interpretation difficult. With ω-conotoxin GVIA as a synthetic peptide, neuroscientists now had a molecular tool that clearly targeted a very specific type of neuronal voltage-gated calcium channel.The peptide was short enough to be amenable to synthesis, and Olivera is grateful to his collaborator, Jean Rivier, who was an expert in synthesizing neuropeptides, for the successful synthesis of this peptide. The peptide had only 27 amino acids but contained three disulfide bonds, “so there were fifteen possible isomers,” recalls Olivera. “You had to get the cross-linking right to end up with the biologically active isomer.”The advantage was that Olivera and colleagues had purified the native peptide, so they could compare their synthesis attempts with the native molecule. “At the beginning, we didn''t even know what the true disulfide bonding was, so we did the work qualitatively to just show the synthetic material and native material co-eluted in a column.” The investigators later established how the disulfide bonds were arranged. Rivier, Olivera, and the rest of the team went on to show that their synthetic peptide behaved just like the natural one in inhibiting calcium entry at chicken synaptosomes and was biologically active.John Exton at Vanderbilt University says “The conotoxins have proved to be extremely important molecular probes in neuroscience in defining functional roles for many receptors and ion channels.”When the paper was published, Olivera was deluged with requests for the peptide. Rivier had been able to synthesize a sizeable amount, and because it was active at subpicomolar concentrations, a little bit of it went a long way. Olivera was able to distribute the peptide, and eventually, several commercial enterprises got into the business of producing and supplying it.“I believe there is something on the order of two thousand studies in the literature using this particular peptide,” says Olivera. “It''s interesting that there are hundreds of thousands of peptides in Conus venom that we call conotoxins. But among physiologists, if you say conotoxin, this is the peptide they think of because this is the one that''s most widely used.” In fact, points out Olivera, when the neuronal calcium channel was purified eight years later, it was actually called the conotoxin receptor.