Progress 10/01/04 to 09/30/09
Outputs
OUTPUTS: Activities All objectives were addressed, except 1.2 which was abandoned early on due to publications on the topic by competitors. Functional genomics of the model soil bacteria Bacillus cereus and B. subtilis were conducted using a suite of biochemical, genetic and microscopic approaches. Post-doc S. Vilain worked on biofilm formation of B. cereus, and published several papers. One 2006 paper was highlighted by the Journal of Proteome Research as news item. Our 2009 paper on eDNA of B. cereus biofilm was linked to the front cover on Appl. Environ. Microbiol. Dr Vilain and MS student Y. Luo worked on ecophysiology of B. cereus, producing strong evidence that this food-associated pathogen is able to grow and translocate in soil. Their seminal paper on the life cycle of B. cereus has been highlighted in several recent reviews by key persons in the Bacillus field. In collaboration with Prof Heckers group at Greifswald we conducted a systems approach to growth of B. subtilis in soil, and published a paper on the spectrum of bacterial nutrients in forest and field soil. We were also invited to write a chapter in a new book on Bacillus in soil. Work on ecophysiology of human pathogens in soil was conducted with MS student A. Christie, visiting scientist C Leonard, and also collaboration with Dr Fanus Venter. The initial choice on Salmonella was expanded to include Escherichia coli O157:H7 and Listeria monocytogenes. Work on the effect of landspreading of manure or biosolids harbouring growth promotant antibiotics was completed, leading to the MS degree of K Lehnert. A related project on effects of growth promotant antibiotic on ileal microbiota was conducted with various on campus collaborators, and published in Appl. Environ. Microbiol. Events Findings were reported at various professional meetings, including the American Society of Microbiology, American Society of Microbiology North Central Branch Meetings, The American Society of Agronomy and South Dakota Academy of Science. Products: A number of students conducted their research in areas of the project. This includes three students who have all graduated, and about eight undergraduate students. The project PI is part of a regional (South and North Dakota) team that has developed a training series on soil health. The team, funded by NC-SARE, has developed a series of workshops directed at training extension officers and interested farmers in aspects of soil biology and soil health. Several collaborative projects have emanated from the project during the funding period. These include work on functional genomic analysis of Bacillus subtilis with Prof Hecker and team members in Greifswald, Germany. This group is an international leader in the area of bacterial systems biology. Additional collaborations are with Dr Theron, University of Pretoria, on eDNA, and Dr Venter on pathogen reservoirs in the environment. We have also enjoyed strong interaction with the group of Drs Sharon and Davis Clay at SDSU, working on soil bacterial community analysis for their various studies on management regimens. PARTICIPANTS: Post-doc Sebastien Vilain worked with MS student Yun Luo on the ecophysiology of B. cereus in soil. He also conducted the proteomic analyses of B. cereus biofilm. The discovery of eDNA as obligate part of the extracellular matrix of B. cereus biofilm was conducted by Dr Vilain in collaboration with Dr Theron at the University of Pretoria. Various undergraduate students (Laura Weyrich, Kelsey Wick, Brittany Brown, Jeff Vahrenkamp, Andrea Lucas and Jarred Sutton) worked over the years on further screening of transposon mutant libraries for genes involved in multicellularity of B. cereus. Research on the effect of manure-associated growth promotant antibiotics was performed in my laboratory by graduate student Kelly Lehnert in collaboration with Dr S. Clay (SDSU Plant Science) and Dr S. Gibson (SDSU Biology and Microbiology). PhD student Elizabeth Rettedal and undergraduate student Justin Luetgers did the work to determine soil bacterial diversity as influenced by various agronomic practices. This project is in collaboration with Dr D. Clay at SDSU. The study on effect of growth promotant CTC on porcine ileal microbiota was conducted in collaboration with others on campus, primarily David Francis and Radhey Kaushik. Analysis of 16SrRNA gene sequence pools was conducted by PhD student Elizabeth Rettedal. TARGET AUDIENCES: Work on ecophysiology of Bacillus, and on thediversity of bacterial meta-communities in soils is directed at understanding soil biological processes, and therefore at audiences concerned with soil fertility and use of biomass as feedstock for biofuels. Research into B. cereus biofilms is of relevance to food safety, specifically under processing conditions. It should be of interest to food processors and packagers, food microbiologists and also regulatory agencies. Work on growth of human pathogenic bacteria in soil is of relevance to the broad public health arena, and specifically to producers and processors of fresh produce grown in contact with soil. Research into the effect of landspreading of manure harbouring growth promotant antibiotics is of primary interest to farmers, regulators, and environmentalists who seek to understand the possible impact of such practices on soil processes. Work on effect of growth promotant antibiotic on ileal bacterial community of pigs is relevant to the swine industry, regulators and those interested in seeking alternatives to using growth promotant antibiotics to develop alternative approaches to improved feed intake and feed - to mass conversion ratio. PROJECT MODIFICATIONS: All objectives were addressed, except 1.2 on biofilm of Pseudomonas aeruginosa, which was abandoned early on due to publications on the topic by competitors.
Impacts
Changes in knowledge A key finding of our work was that the food- and soil-associated human pathogen B. cereus is able to germinate and grow on the organic matter present in various soils. This is also true the related B. subtitles and B. licheniformis. Previously it was held that B. cereus occurs in soil only as endospores, growing in association with insects or mammals. Analysis of the organic composition of the water soluble components in a forest soil revealed a wide spectrum of mono- and disaccharides, organic acids, fatty acids and amino acids, with glucose present at 700 nM/kg soil. Functional genomic (transcriptomic, proteomic and metabolomic) analysis of B. subtilus revealed that, under soil conditions, the species is not subject to carbon catabolite repression, a phenomenon where catabolic operons are controlled by a complex regulatory circuit which has been studied extensively. Rather, all substrates were utilized simultaneously and leaving residual glucose in the environment. As a true soil autochthon, B. subtilis should have evolved its regulatory circuits under soil conditions. This begs further investigation into the nature of the regulatory circuits involved in the control of catabolic pathways of soil bacteria. We had previously established that the food and soil-associated pathogen B. cereus is able to form biofilms. During this project period we determined that the species displays two interface-associated phenotypes - the biofilm and biofilm-associated free swimming populations which we termed PGW. Through analysis of a transposon mutant screen we identified biofilm-required genes in the purine biosynthesis pathway. A subsequent suite of biochemical and microscopic analyses supplied evidence for eDNA as a required component of the extracellular matrix. A number of human bacterial pathogens may have reservoirs in the environment. We obtained data to indicate that Salmonella Typhimurium, E. coli O157:H7 and L. monocytogenes are able to grow on soil organic matter under controlled laboratory conditions. These species may be able to maintain populations in the environment, despite predation and competition, as increasingly suggested by in situ studies. Bacterial community analysis of experimental plots land spread with manure from pigs fed the growth-promotant antibiotic CTC versus various controls was carried out by DGGE of the V3 region of the 16SrRNA gene pool. DGGE band patterns were analyzed by Raup-Crick multivariate analysis. The CTC did not exert a significant effect on the soil microbiota when compared to manure alone. In a parallel study on the effect of CTC as growth promotant on the ileal microbiota of weanling pigs, we employed partial sequencing of 16SrRNA gene amplicon pools. Sequences analysis of amplicon pools from twelve pigs using LibSHUFF, SONS and DOTUR showed that CTC did cause shifts in the ileal microbiota. Change in conditions Three graduate student completed their MS thesis research on aspects of the project, and a PhD student is about half-way done. Eight SDSU students conducted undergraduate research on the various projects, acquiring a series of molecular microbiology and microscopy skills.
Publications
- Vilain, S. Pretorius, J.M., Theron, J. and Brozel, V.S. 2009 DNA as an adhesin: Bacillus cereus requires extracellular DNA to form biofilm. Appl. Environ. Microbiol. 75: 2861 - 2868
- Liebeke, M., Brozel, V.S., Hecker, M. and Lalk, M. 2009 Chemical characterisation of soil extract as bacterial growth media for environmental systems biology approaches. Appl. Microbiol. Biotechnol. 83: 161 - 173
- Elizabeth Rettedal, Sebastien Vilain, Stacy Lindblom, Kelly Lehnert, Clay Scofield, Sajan George, Sharon Clay, Radhey S. Kaushik, Artur J. M. Rosa, David Francis and Volker S. Brozel 2009 Alteration of the Ileal Microbiota of Weanling Pigs by the Growth-Promoting Antibiotic Chlortetracycline. Appl. Environ. Microbiol. 75: 5489 - 5495
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Activities during 2008 were spread between field and laboratory work, and data analysis. A number of new techniques were established in the lab, including methods for analyzing large sequence data sets, and genetic tools. Work on the effect of landspreading of manure or biosolids harbouring growth promotant antibiotics was completed, and written up as a masters thesis. Work during 2008 included completion of gel image analysis and sequence analysis of the 100 bands of interest, previously chosen. Preparation of a manuscript is underway. As we have the techniques for DNA extraction, PCR and DGGE of bacterial communities established in our laboratory, two new projects were initiated as part of multidisciplinary studies headed out of the SDSU Plant Science Department. The first study addresses the effects of complete removal of plant residue from corn fields in order to ascertain the long-term effect of using the entire corn plant as biomass for bio fuel production. The first of three growing seasons has been completed at the experimental farm at Aurora, SD, and the soil DNA extracted, 16SrRNA gene pool amplified and resolved by DGGE. The second study aims to study whether proximal growth of weeds, or merely the shading caused, affects growth of corn. Our lab conducted the soil microbial analyses to look at any possible effects of weed growth on soil bacterial meta community. Laboratory work on the effect of the growth promotant antibiotic CTC on the porcine ileal microbiota had been completed during 2006, generating 2,050 sequences of about 700 bp in length, but the raw data was not analyzed at the time. Extensive efforts were expended to familiarize ourselves with the current approach to analyzing large 16SrRNA gene datasets, establishing the respective software and analyzing the data. Sequences were aligned with ARB, LIBSHUFF, DOTUR and SONS. SONS analysis showed that about half the number of OTU observed were unique to either treated or control pigs, and that the treated and control pigs had different community structures. Collectively these analyses indicate that the growth promotant antibiotic CTC is associated with significant shifts in the meta community in the porcine ileum. A manuscript was written and submitted to Appl. Environ. Microbiol. It was accepted subject to modifications which were made and the manuscript re-submitted. We had previously indicated an interest in studying the physiology of Turicibacter sanguis - like bacteria, a group found so prevalent in the ileum of weanling pigs. None of our attempts to isolate representatives of this apparently strict anaerobic Gram positive rod were successful to date. Work on multicellularity and translocation through soil of Bacillus cereus continued as a peripheral project during 2008. Extensive efforts we applied to establish some more current genetic tools. These included generation of markerless deletion mutants using a system developed by Dr Stibitz of the Food and Drug Administration in Bethesda, Maryland for B. anthracis, and establishment of two new transposon delivery systems developed for B. anthracis at the Scripps Institute. PARTICIPANTS: Research on the effect of manure-associated growth promotant antibiotics was performed in my laboratory by graduate student Kelly Lehnert in collaboration with Dr S. Clay (SDSU Plant Science) and Dr S. Gibson (SDSU Biology and Microbiology). Masters student Elizabeth Rettedal and undergraduate student Justin Luetgers did the work to determine soil bacterial diversity as influenced by various agronomic practices. Analysis of 16SrRNA gene sequence pools was conducted in my laboratory by masters student Elizabeth Rettedal. Undergraduate student Kelsey Wick worked with the PI on establishing genetic tools for studying multicellularity of Bacillus cereus. TARGET AUDIENCES: Research into the effect of landspreading of manure harbouring growth promotant antibiotics is of primary interest to farmers, regulators, and environmentalists who seek to understand the possible impact of such practices on soil processes. Work on effect of growth promotant antibiotic on ileal meta community of pigs is relevant to the swine industry, regulators and those interested in seeking alternatives to using growth promotant antibiotics to develop alternative approaches to improved feed intake and feed - to mass conversion ratio. Work on diversity of bacterial meta-communities in soils is directed at understanding soil biological processes, and therefore at audiences concerned with soil fertility and use of biomass as feedstock for biofuels. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Changes in knowledge results obtained during 2008 have shown that landspreading of manure or biosolids harbouring growth promotant antibiotics does shift the soil bacterial meta-community, but has little effect on soil bacterial activity as measured by degradation of the herbicide 2,4-D. Analysis of meta community from porcine ileum has shown that CTC as growth promotant antibiotic is associated with a significant shift in the bacterial community. One graduate student completed her masters thesis research on the effect of landspreading manure with growth promotant antibiotic. A second masters student conducted thesis research on soil bacterial diversity as affected by agronomic practices, acquiring a series of molecular microbiology and analytical skills. Two SDSU students conducted undergraduate research on the various projects during 2008, acquiring a series of molecular microbiology skills.
Publications
- Koh, S., George, S., Brozel, V.S., Moxley,R., Francis, D. & Kaushik, R.S. 2008 Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli. Vet. Microbiol. 130: 191 - 197
- Lehnert, K. 2008 The Effects of Swine Manure or Biosolids Containing Antibiotics on Soil Microbial Communities in Eastern South Dakota. Masters thesis, South Dakota State University.
- Burke, L.M., Brozel, V.S., and Venter, S.N. 2008 Construction and Evaluation of a gfp-tagged Salmonella Typhimurium for Environmental Applications. Water SA 34: 19 - 24
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities: Research into the effect of landspreading of manure harbouring growth promotant antibiotics continued during 2007 along two lines. The field study performed during 2006 was repeated using biodegradation of the herbicide attrazine in place of 2,4D. This entailed a randomized plot design (n=4), amending ring-plots with manure, manure with chlortetracycline, biosolids and biosolids with tetracycline, all with and without the herbicide added. Samples were analyzed on d1, 7, 28, 42, 56 and 84 for aerobic culturable count, attrazine degrader count, attrazine and antibiotic content in collaboration with Dr S. Clay. The second line of investigation entailed analysis of the microbial communities of 2006 samples by denaturing gradient gel electrophoresis (DGGE) of the V3 region of the 16SrRNA gene pool. Gels were analyzed by densitometry and principle component analysis to determine the respective degrees of community shift. Bands of interest were cloned and sequenced to
obtain information on the respective bacterial phylotypes. Work on functional genomics of soil bacteria was conducted largely during faculty improvement leave in the group of Prof M. Hecker, Ernst Moritz Arndt University, Greifswald, Germany. The soil extracted soluble organic matter (SESOM) was analyzed for sugar, amino acid, organic acid, fatty acid content by both 600 MHz 1-H NMR and GC-MS of derivatized fractions. A manuscript reporting for the first time a detailed analysis of the organic composition of the water soluble components in a soil was written and submitted. Having refined this analytical approach, we were then able to quantify consumption of organic matter by the soil bacterium Bacillus subtilis 168 over time. B. subtilis was analyzed by both proteomics and transcriptomics (Eurogentec slide for the 168 strain) at various stages during growth in order to quantify the expression of genes involved in specific metabolic pathways, and therefore in consumption and excretion
of specific products. This data could then be compared with the metabolomic data. Analysis of the data will continue into 2008. Additional projects included continuation of work on multicellularity of Bacillus cereus, and on the effect of growth promotant antibiotics on porcine intestinal flora. Both projects are ongoing. Events Findings of work on manure-associated growth promotant antibiotics on soil microbial activity were reported at professional meetings including South Dakota Academy of Science meeting (04/07), American Society of Microbiology North Central Branch Meeting (10/07), and American Society of Agronomy meeting (11/07). Findings on functional genomics of soil bacteria were communicated at the South Dakota Academy of Science meeting (04/07), and American Society of Microbiology North Central Branch Meeting (10/07). Products: Work on functional genomic analysis of soil bacteria led to strong and ongoing collaborative ties with Prof Hecker and team members working in the
areas of proteomics, transcriptomics and metabolomics. This group is an international leader in the area of bacterial systems biology. Our collaboration with the group of Dr S. Clay has also grown during 2007.
PARTICIPANTS: Functional genomic analysis of the model soil bacterium Bacillus subtilis was performed during faculty improvement leave together with various researchers working under Prof M. Hecker, Ernst Moritz Arndt University, Greifswald, Germany. Prof Hecker's group is an international leader in the area of bacterial systems biology. Group leaders worked with included Dr B. Voigt (proteomics), Dr U. Mader (transcriptomics), and Dr M. Lalk (metabolomics). I was able to acquire a great deal of detail skills in this broad, yet high-end analytical area of research. The daily interactions during this six-month period strengthened our collaborative ties and one proposal has been submitted to fund future collaborative research. Work on genes involved in multicellularity of Bacillus cereus was continued with undergraduate student Laura Weyrich, now a PhD student at Penn State. Research on the effect of manure-associated growth promotant antibiotics was performed in my laboratory by
graduate student Kelly Lehnert and undergraduate student Kelsey Wick in collaboration with Dr S. Clay (SDSU Plant Science) and Dr S. Gibson (SDSU Biology and Microbiology). Undergraduate students Justin Luetgers and Josh Steinbronn continued work on effect of the growth promotant antibiotic CTC on the porcine digestive tract, specifically determining community shifts of ileal microflora by DGGE.
TARGET AUDIENCES: Research into the effect of landspreading of manure harbouring growth promotant antibiotics is of primary interest to farmers, regulators, and environmentalists who seek to understand the possible impact of such practices on soil processes. Work on functional genomics of soil bacteria is directed at understanding soil biological processes, and therefore at audiences concerned with soil fertility.
Impacts
Changes in knowledge: Results obtained during 2007 have contributed to our understanding of the effect of landspreading of manure harbouring growth promotant antibiotics. The antibiotics chlortetracycline and Tetracycline introduced to soil through manure or biosolid land spreading did not affect soil microbial numbers, or herbicide degrader numbers, supporting data from the 2006 season. Yet these antibiotics were the cause of a significant shift in the soil microbial community as determined by DGGE of the V3 region of the 16SrRNA gene pool. Analysis of the organic composition of the water soluble components in a model soil (Oak Lake forest) revealed a wide spectrum of mono- and disaccharides, organic acids, fatty acids and amino acids. Surprisingly, glucose was present at 700 nM/kg soil. This residual level was surprising to many as a wide variety of microbes use glucose in preference to other substrates. Functional genomic analysis revealed a novel phenomenon. Under
laboratory conditions bacteria display preference for a specific nutrient, the so-called preferred substrate as described by Monod. This phenomenon is regulated by a complex regulatory circuit which has been studied extensively in several species, and called carbon catabolite repression.. Yet under soil conditions, B. subtilis does not appear to display carbon catabolite repression as shown by metabolomic, transriptomic and proteomic data. It may be that carbon catabolite repression only functions where a limited number of substrates are present in high concentrations. When a wide range of substrates is present at low concentrations, the system may be subject to override. As a true soil autochthon, B. subtilis should have evolved its regulatory circuits under soil conditions. This begs further investigation into the nature of the regulatory circuits involved in the control of catabolic pathways of soil bacteria. Change in conditions: One graduate student conducted her thesis research
in the project, acquiring a series of molecular microbiology and analytical skills. Three SDSU students conducted undergraduate research on the various projects during 2007, acquiring a series of molecular microbiology skills.
Publications
- Luo, Y., Vilain, S. Voigt, B., Albrecht, D., Hecker, M. and Brozel, V.S. 2007 Proteomic analysis of Bacillus cereus growing in liquid soil organic matter. FEMS. Microbiol. Letts.271: 40 - 47
- September, S.M., Els, F.A., Venter, S.N. and Brozel, V.S. 2007 Prevalence of bacterial pathogens in biofilms of drinking water distribution systems. J. Wat. Health. 5: 219 - 227
- George, S., Oh, Y., Lindblom, S., Vilain, S., Rosa, A., Francis, D., Brozel, V. and Kaushik, R. 2007 Lectin binding profile on the small intestine of 5-week old pigs in response to the use of chlortetracycline as a growth promotant, and in the absence of intestinal microbiota. J. Anim. Sci. 85, 1640 - 1650.
- Hawumba, J.F., Brozel, V.S. and Theron, J. 2007 Cloning and characterization of a 4-hydroxyphenylacetate 3-hydroxylase from the thermophile Geobacillus sp. PA-9. Curr. Microbiol. 55: 480 - 484
- Christie, A. 2007 Fate of Escherichia coli O157:H7 in soil. Masters thesis, South Dakota State University.
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Progress 01/01/06 to 12/31/06
Outputs
The second year of this project allowed us to begin capitalizing on established techniques and collaborations, both in the area of bacterial response to growth promotant antibiotics and fate of bacteria in soils. Field trials to study the effect of manure from antibiotic-fed pigs on soil microbial activity were conducted during the summer and fall seasons in collaboration with Dr Sharon Clay (Plant Science, SDSU). Pigs were fed a diet with and without the growth promotant antibiotic chlortetracycline (CTC) and the manure was spread at agronomic field rates. Manure-associated CTC did not exert a significant effect on the bacterial culturable count over time, an indicator of number but not of diversity or activity. Degradation of the herbicide 2,4D in soil was not retarded significantly by the manure-associated CTC. Microbial diversity analysis through denaturing gradient gel electrophoresis (DGGE) of 16SrRNA gene amplicon pools is still under way, but preliminary
results indicate an antibiotic-induced shift. A parallel project in collaboration with Drs R. Kaushik, A. Rosa and D. Francis addressed the effect of the growth promotant antibiotic CTC on the porcine digestive tract. While beneficial for feed intake and feed to weight gain ratio, use of these antibiotics has been banned in Europe and no alternatives are available, due largely to a lack of understanding of the underlying mechanisms. Our lab studied the effect of CTC on the ileal bacterial community structure using 16SrRNA gene sequence as marker. DNA samples were extracted from the lumen and mucus of six control and six antibiotic-fed pigs (half-sib pigs obtained by cesarean section and raised using surrogate mothers). The 16SrRNA gene pool was amplified by PCR, ligated into pGEM T-Easy, and 96 clones of each sample were sequenced. The results show a significant shift in phylotype diversity in both the lumen and mucus of CTC-fed pigs. The major shifts were decreases in Lactobacillus
johnsonii, Clostridium glycolicum and Turicibacter sanguinis, with an increase in L. amylovorus. Future work will focus on the physiology of T. sanguinis, a bacterium with no known function in the porcine intestinal tract. Previous results showed that the bacterial pathogens Bacillus cereus and Escherichia coli O157:H7 are able to grow on soil organic matter. Work during 2006 was directed at proteomic analysis to improve our understanding of the underlying mechanisms. The proteome of both pathogens shifted significantly in populations growing in soil extracted soil organic matter (SESOM). The identities of differentially expressed spots pointed to broad-ranging differences in several key metabolic pathways when growing in SESOM. Shifts in abundance of specific proteins in SESOM-grown populations pointed to catabolism of fatty acids, sugars and possibly amino acids, biosynthesis of amino acids and nucleotides and changes in overall cell physiology and structure. Future work will be
directed at understanding the relationship between soil organic matter components and induction of metabolic processes.
Impacts
Pig manure containing the growth promotant antibiotic chlortetracycline did not slow down the rate of biodegradation of the herbicide 2,4D in soil. This indicates that manure from antibiotic-fed pigs can be land-spread without negatively impacting soil processes such as biodegradation of the herbicide 2,4D. The changes in porcine intestinal bacteria when fed with chlortetracycline may be exploited to develop alternative strategies to feed antibiotics. The bacterial pathogen Bacillus cereus and Escherichia coli O157:H7 are able to grow on soil organic matter, challenging the current paradigm that their occurrence in soil and water is indicative of pollution by animal or human activity. This should influence the way that presence/absence data in soil and water are interpreted by epidemiologists.
Publications
- Lindsay, D., Brozel, V.S. and Von Holy, A. 2006 Biofilm / spore response in Bacillus cereus and Bacillus subtilis during nutrient limitation. J. Food Prot.69, 1168 - 1172
- Vilain, S., Luo, Y., Hildreth, M. and Brozel, V.S 2006 Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil. Appl. Environ. Microbiol. 72: 4970- 4977
- Vilain, S. and Brozel, V.S. 2006 Multivariate approach to comparing whole-cell proteomes of Bacillus cereus indicates a biofilm-specific proteome. J. Prot. Res. 5: 1924 - 1930
- Luo, Y. 2006 Multicellular behaviour of Bacillus cereus growing as a biofilm in soil. Masters thesis, South Dakota State University.
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Progress 01/01/05 to 12/31/05
Outputs
At the end of the first year of this project our laboratory has gained significant insights into how bacteria behave in soil. By employing two simple model systems developed in the laboratory, we were able to demonstrate that, once exposed to soil organic matter the model soil bacterium Bacillus cereus switches to a filamentous mode of growth. The parallel bundles of filaments serve to translocate the species through the soil matrix, facilitating colonization at a rate of up to 1.2 mm/day. This work relies heavily on our initial establishment of two simple models for studying bacterial growth. Growth-kinetic and proteomic studies are performed in Soil Extracted Soluble Organic Matter (SESOM), and translocation studies are performed using artificial soil microcosms (ASM). While the current paradigm holds that the large numbers of Bacillus found in soils occur solely as resting spores, multiplying only in insect and mammalian hosts, our data supports the hypothesis of
growth in bulk soil, achieving densities up to 20,000,000 cells per g of soil. B. cereus is a close relative of both the insect control agent B. thuringiensis, and the mammalian pathogen B. anthracis, suggesting that the latter may well be able to proliferate in soils under specific conditions. A survey of 20 Bacillus isolated from soils of South Dakota Experiment Station fields revealed that this soil-associated multicellular growth mode is common to soil Bacillus. Translocation was not due to swimming or swarming as shown when supplementing ASM with the flagellar inhibitor L-ethionine. In collaboration with a group from the University of Pretoria, we have been able to show that the food and water associated bacterial pathogens Salmonella Typhimurium, E. coli O157:H7 and Listeria monocytogenes are also able to grow in SESOM. These pathogens are widely held to occur in soils only when introduced from animal sources. Survival studies in soils are underway. A related project has
addressed the development of biofilms of the food and dairy-associated pathogen B. cereus at solid-liquid interfaces. A transposon mutant screen revealed the obligate requirement of three genes involved in purine biosynthesis (purA, purC & purL). While the mutants were able to grow in liquid medium containing nucleotides, they were unable to form biofilms. Enzymatic and confocal scanning laser microscopic investigations using nucleic acid-excited fluorophores confirmed the presence and structural role of DNA in the extracellular matrix, from the early stages of conditioning film formation through to mature biofilm. The matrix further contains three DNA-associated proteins identified by MALDI-TOF-MS, oligopeptide-binding protein OppA, ATP synthase beta chain and enolase. Preliminary data was obtained to demonstrate that the manure-associated antibiotic chlortetracycline, but not tylosin, suppresses herbicide-degrading activity of bacterial flora in soil. Chlortetracycline at 2.5 ppm
suppressed both the growth and attrazine-degrading activity of Pseudomonas ADP, a degrader isolated from soil. Funding for this work was subsequently obtained from the EPA with Dr S. Clay as PI, and further work is underway.
Impacts
A model system to study the growth and functional genomics of bacteria in soil has been developed. The soil-associated Bacillus cereus is able to grow in soil and to move to colonize new zones by switching to a multicellular filamentous mode. While most bacteria produce extracellular polysaccharides in order to stick to surfaces, B. cereus requires self-produced extracellular DNA to attach to surfaces and form biofilms, both at man-made interfaces such as food-contact surfaces, and in soil. This information can lead to new strategies to sanitize food contact surfaces. Various pathogenic bacteria, E. coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes are able to grow in soils, challenging the current paradigm that their occurence in soil and water is indicative of pollution by animal or human activity. This will influence the way that presence/absence data in soil and water are interpreted.
Publications
- Lindsay, D., Brozel, V.S., and Von Holy, A. 2005 Spore formation in Bacillus subtilis biofilms J. Food Protection 68, 860 - 865.
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