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Characterization of Novel Marine Oligotrophic Bacteria Isolated from the Pacific Ocean: Description Ofmarinivirgulalluito Gen

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Characterization of Novel Marine Oligotrophic Bacteria Isolated from the Pacific Ocean: Description Ofmarinivirgulalluito Gen AN ABSTRACT OF THE THESIS OF Eun Jung Shin for the degree of Master of Science in Microbiology presented on August 25. 2003. Title: Characterization of Novel Marine Oligotrophic Bacteria Isolated from the Pacific Ocean: Description ofMarinivirgulalluito gen. nov., sp. nov., Marinivirgula obesa gen. nov.. sp. nov. and Litincola parvulus gen. nov.. sp. nov. Abstract approved: Giovannoni A new high-throughput culturing (HTC) method using a low nutrient heterotrophic medium (LNHM) has led to the isolation of many novel strains of oligotrophic bacteria from marine ecosystems. Four strains belonging to a single dade, HTCC2151, HTCC218OT,HTCC2178T and HTCC2188T,were isolated from the coast of Oregon by the HTC method. Phylogenetic analysis based on their 1 6S rDNA sequences showed that they fell into the OM1 82 dade in the oligotrophic marine Gammaproteobacteria (0MG) group, which is distantly related to previously cultivated genera in Gammaproteobacteria. This analysis, along with DNA-DNA hybridization results, indicated that strains HTCC2151 and HTCC218OTwere the same species, showing 89.6% genomic DNA relatedness between these strains. Strain HTCC2 was revealed to fall into the same genus with strains HTCC2 151 and HTCC2180T,but a different species, and this was supported by 96.5% 96.7% of 16S rRNA gene sequence similarity with the strains and 27.4% of genomic DNA relatedness between strains HTCC2178Tand HTCC218OT. Strain HTCC2188T differed significantly from HTCC2151, HTCC218OT and HTCC2178T, with less than 12.1% of the DNA-DNA hybridization result with any of the strains, and thus it was proposed that strain HTCC2188Tbe placed in a separate genus in the 0M182 dade. G+C mol% of strains HTCC2151, HTCC218OT,HTCC2178TandHTCC2188Twere 36.2%, 41%, 54.2% and 49.3%, respectively (HPLC method). All the HTCC strains in 0M182 dade were Gram-negative, and microscopic observations revealed that they were short rods of 0.06xm3to 0.13 m3in size on average and divided by binary fission. Growth curves and specific growth rates at six different temperatures and six different mixed carbon concentrations were determined. Their growth characteristics showed that these bacteria were slowly growing, obligate or facultative oligotrophic and psychro-to- mesophilic bacteria. Two of the strains,HTCC2178Tand HTCC2188T, started forming tiny colonies on l/1OR2A at 16°C after a 10-day incubation, but not HTCC2151 and HTCC2180TFive different liquid artificial seawater (A SW) media were tested for their ability to support growth of the strains, and strainsHTCC218OT, HTCC2 178T and HTCC2188T grew in one or two different ASW media, but not HTCC215O. pH ranges for growth of strains HTCC2151,HTCC218OT,HTCC2178Tand HTCC2188Twere pH 7-8.5, pH 7.5-10, pH 7-11, andpll 7.5 10. Ranges of NaCI concentrations for growth of strains HTCC2180T,HTCC2178Tand HTCC2188Twere 1.5-3.3%, 1.0-3.3%, and2.0-3.3%. Thirty four different carbon sources were utilized by strain HTCC2180T, fourteen by HTCC2178T and fifteen by strainHTCC21S8Tas sole carbon source among forty seven different carbon sources. Strain HTCC2180Tutilized all kinds of hexoses and sugar alcohols tested, most of pentoses, oligosaccharides and organic acids, some of amino acids, and one of amino sugars as sole carbon sources. Most of pentoses and hexoses, some of oligosaccharides and organic acids, and only one kind of sugar alcohols and amino acids were utilized by strain HTCC2178T, however, none of amino sugars. Strain HTCC2188T utilized most of hexoses, some of pentoses, oligosaccharides and sugar alcohols, and one of amino acids and amino sugars, but none of organic acids was utilized by this strain as sole carbon source. All the strains in the OM 182 dade showed positive reactions for alkaline phosphatase, esterase, esterase lipase and naphthol-AS-BI-phosphohydrolase. Presence of acid phosphatase and naphthol-AS-BI-phosphohydrolase was detected from strains HTCC2151 andHTCC218OT. Onlyone strain,HTCC2188T, exhibited positive results for leucine arylamidase, a-chymotrypsin and N-acetyl-f3- glucosaminidase. HTCC strains in the 0M182 dade contained the fatty acids, 16:0, 18:0 and 16:1, commonly found in previously cultivated genera in Gammaproteobacteria. They also showed the presence of 17:1 o7c or 17:1 w8c as one of the major fatty acids, which was not usually found as a dominant fatty acid in other genera in Gammaproteobacteria. From the genotypic and polyphasic evidences, it is proposed that strains HTCC2151, HTCC218OT andHTCC2178T should be placed intoa new genus named Marinivirgula gen. nov. including Marinivirgulafluito gen. nov., sp. nov. with the type strain HTCC2180T,and Marinivirgula obesa gen. nov., sp. nov. with the type strain HTCC2178T. The name Litincolaparvulus gen. nov., sp. nov. is suggested for the type strain HTCC2 188T ©Copyright by Eun Jung Shin August 25, 2003 All Rights Reserved Characterization of Novel Marine Oligotrophic Bacteria Isolated from the Pacific Ocean: Description ofMarinivirgulafluito gen. nov., sp. nov., Marinivirgula obesa gen. nov., sp. nov. and Litincola parvulus gen. nov., sp. nov. by Eun Jung Shin A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented August 25, 2003 Commencement June 2004 Master of Science thesis of Eun Jung Shin presented on August 25, 2003. APPROVED: Major Professor, representing Microbiology Chair of the Department of Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Eun Jung Shin, Author ACKNOWLEDGMENTS I would like to express my gratitude to my major professor, Dr. Stephen Giovannoni, for the opportunity to pursue a graduate degree, and his guidance and patience.Special thanks to Dr. Jang-Cheon Cho for his assistance and helpful discussion throughout this entire thesis project.I also wish to acknowledge my committee members, Dr. Peter Bottomley, Dr. Daniel Rockey, and Dr. Julia Jones for their comments on this thesis.Finally, I would like to thank my friends and family for their endless love and encouragement. This research was supported by Oregon Sea Grant. TABLE OF CONTENTS INTRODUCTION. 1 MATERIALS AND METHODS.................................................... 5 Experimental Design.......................................................... 5 Isolation............................................................................ 5 Microscopy.....................................................................7 Cells stained with 4', 6-diamidino-2-phenylindole..............7 Transmission electron microscopy.................................7 Growth Conditions............................................................ 8 MediumTests..................................................................8 pH and Salinity Tests........................................................... 10 Antibiotic Susceptibility....................................................... 11 Oxygen Requirement.......................................................... 11 Carbon Source Tests............................................................ 11 Enzymatic Assay...............................................................12 DNAPreparation................................................................13 Phylogenetic Analysis Based on 1 6S rRNA Gene Sequences.......... 13 Sequencing of 16S rRNA genes.................................... 13 Phylogenetic analysis................................................ 14 DNA-DNA Hybridization.................................................... 14 G + C mol% Measurements................................................. 15 Analysis of Cellular Fatty Acids............................................ 15 RESULTS AND DISCUSSION..................................................... 16 Phenotypic Characterizations...................................................16 Morphology and biovolume........................................ 16 TABLE OF CONTENTS (Continued) Growth characteristics at different temperatures and carbon concentrations.........................................................23 Medium tests.........................................................29 Ranges and optima of pH and salinity for growth...............31 Antiobiotic susceptibility...........................................33 Oxygen requirement.................................................35 Sole carbon source utilization......................................35 Enzymatic activity...................................................37 Phylogeny, DNA Composition and DNA Relatedness........................40 Phylogenetic analysis based on 16S rRNA gene sequences 40 G + C contents of DNA.............................................43 DNA-DNA hybridization...........................................44 Cellular Fatty Acid Profiles..................................................45 Taxonomic Conclusions......................................................50 Descriptions of the HTCC Strains in the 0M182 dade..................51 Description of Marinivirgula gen. nov........................... 51 Description of Marinivirgulafluito sp. nov.......................52 Description of Marinivirgula obesa sp. nov......................52 Description of Litincola gen. nov.................................. 53 Description of Litincola parvulus sp. nov........................54 REFERENCES.........................................................................55
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