UC San Diego

Chapter 1 introduces the deep sea environment. Recent studies of sequence-independent diversity of deep-sea microbial communities are reviewed and re-analyzed. These are compared to sequence-dependent studies evidencing the need for new culturing and isolation methods for deep-sea bacteria. The chapter ends by reviewing the current state- of-the-art of deep-sea genetics and genomics and describes the general features of the genomes of piezophiles. Chapter 2 describes the development of three new vectors for use in Photobacterium profundum and other Gram- negative bacteria. The first one (pEE3) is a suicide vector for insertional inactivation of genes. The second one is a general purpose cloning vector (pFL122) based on the broad-host-range replicon RSF1010. The third one is a broad-host-range vector carrying an arabinose-inducible promoter to use for gene expression studies. Chapter 3 explores the diversity evolution of cultured piezophiles. Three novel strains of piezophilic bacteria were isolated, one of them being the first Gram-positive piezophile ever isolated. Most of the cultured piezophiles, belong only to a narrow range of phylogenetic lineages which cluster within clades of known psychrophiles. In the ensuing discussion the hypothesis that cold-adapted piezophiles might evolve from polar water psychrophiles and disseminated through deep-sea oceanic currents is presented. During the study specific ribosomal RNA features of the 16S of piezophiles were identified. These features are correlated with the pressure adaptation of the strains. Chapter 4 describes a high-throughput transposon mutagenesis approach to identify genes important for growth at low temperature and high pressure. These genes belong to a wide variety of categories which are discussed in detail. A large fraction of the mutants impaired in growth at low temperature had gene disruptions in genes for the synthesis of extracellular polysaccharides, providing the first direct evidence of the role of the cell envelope in adaptation to low temperature. Chapter 5 presents the results of the sequencing and the analysis of the genome of the shallow water strain P. profundum 3TCK. The genome is compared to that of the deep-water strain P. profundum SS9 identifying unique regions that might be conferring fitness in different habitats. Among these is a cluster of genes for motility important for swimming in high viscosity found only in the deep-water strain, a set of genes for UV resistance found only in the shallow water strain, and the specific rRNA features described in chapter 3 found only in the deep-water strain. The motility gene clusters are further characterized evidencing the role for particular flagellins and motor proteins for swimming at high pressure. The UV and ribosomal features are exchanged between the shallow- and the deep- water strains and the phenotypes of the resulting strains are characterized