Halobacterium sp. sustains a short Period of phototrophic growth by synthesizing ATP using a light-driven proton pump, bacteriorhodopsin. The expression of bop, which encodes the protein component of bacteriorhodopsin, bacterioopsin, is induced in mid- to late-log phase with maximal expression in the stationary phase. Three cis-acting elements within the minimal 53 bp bop promoter were predicted to be responsible in mediating expression and regulation of this gene, viz. UAS, TATA box, and RY box. Finally, computational analysis of the complete genome sequence was projected as a means of identifying putative transcription factors and regulators. I have used a saturation mutagenesis approach to derive a consensus for 35 bp of the 53 bp minimal bop promoter. In the process, we identified two cis-acting elements required for transcription of bop (TATA box and UAS). Based on the presence of an alternate TATA box consensus sequence and lack of a TFB responsive element (BRE) we predicted presence of multiple TBPs and TFBs in Halobacterium NRC-1. Complete genome sequence analysis identified a total of six TBPs and seven TF13s. The UAS was highly immutable at its 3′ and 5 ′ extremities. Three nucleotides (−27A, −23 A and −19A), within the RY box, were identified to be the major sites for DNA supercoiling mediated regulation of bop. We identified the UAS upstream to three other genes, viz. brp, blp and crtB1. Expression profiles for these genes indicated that (a) they are coordinately regulated in Halobacterium NRC-1 and S9, (b) Bat, the putative bacterio-opsin activator, is required for expression of bop, blp and crtB1 and (c) like the bop promoter, the crtB1 and brp promoters contain alternating purine-pyrimidine sequences (10 and 11 bp long respectively), and are also regulated by changes in DNA supercoiling. A detailed analysis of Bat revealed the presence of a redox-sensing PAS/PAC domain, a cGMP responsive GAF domain and a DNA-binding helix-turn-helix motif. These results lead us to conclude that carotenoid and bacterio-opsin biosyntheses are coordinately regulated by Bat in Halobacterium NRC-1. Also, Bat probably functions as a repressor and activator in high- and low-oxygen tension respectively; and the repressor state is transdominant. A model has been proposed to explain bop gene regulation by DNA supercoiling, multiple transcription factors, and Bat. (Abstract shortened by UMI.)

The genome of Halobacterium NRC-1 is 2,571,010 bp and encodes 2,630 proteins. The genome is arranged into 3 replicons, a large chromosome and two smaller mini-chromosomes, pNRC100 and pNRC200. The mini-chromosomes share approximately 145 Kb of sequence similarity and harbor the majority of the 91 IS elements. This large amount of repeated DNA required mapping pNRC200 for final assembly of the genome sequence. The proteome of NRC-1 is extremely acidic, with an average pI of 4.9, and represents the major adaptation to the high internal salt concentration. Homology modeled structures showed the majority of additional charge is at the surface of the protein where it compensates for lower water activity and depressed dielectric constant to maintain solubility at saturating salt concentrations. GC and amino acid compositions reflect the extremely acid proteome. An overrepresentation of codons GAC and GAG, coding for aspartic acid and glutamic acid respectively, are found in the genome. NRC-1 showed extensive evidence of lateral gene transfer (LGT). Although 16S rDNA analysis shows halophiles to be a monophyletic group branching from the methanogens in the Euryarchaeota, several features important to environmental adaptation appear to be acquired laterally from bacteria. The nuo and men genes corresponding to electron transport were identified as laterally transferred, giving evidence of the evolutionary past of this halophile. Further analysis of local databases and the Clusters of Orthologous Genes (COG) databases revealed, among the 2411 non-redundant protein coding genes from NRC-1, 783 proteins with bacterial character by BLAST score. Phylogenetic analysis showed several biosynthetic, transport, and energy systems, including histidine utilization, purine metabolism, and glycerol utilization, were likely acquired by LGT. However, no link between the 91 IS elements and the laterally transferred genes was found. While the structure of the genome, with its three replicons, mini-chromosomes, IS elements, different GC-fractions, and observed plasticity, have been the driving force towards sequencing and subsequent analysis, the ultimate role of these various features remains mostly in the realm of the unknown. I discuss several scenarios to explain the presence of IS elements, duplication of genes, and overall genomic organization.

Halobacterium halobium strain NRC-1 harbors a 200-kilobase pair (kb) plasmid, pNRC100, which contains a cluster of genes that code for synthesis of buoyant gas-filled vesicles. A restriction map of pNRC100 was constructed using AflII, DraI, HindIII, and SfiI with the aid of pulsed-field agarose gel electrophoresis. The identity and approximate positions of seventeen insertion sequences (IS) in pNRC100 were determined by Southern hybridization and limited nucleotide sequence analysis across the IS element-target site junctions: ISH2, a 0.5-kb element, was found in four copies; ISH3, a 1.4-kb heterogeneous family of elements, was present in seven copies; ISH8, a 1.4-kb element, was found in five copies; and ISH50, a 1.0-kb element, was present in a single copy. Additionally, a large (35-38 kb) inverted repeat (IR) sequence was also found. Inversion isomers of pNRC100 were demonstrated by Southern hybridization analysis using two restriction enzymes, AflII and SfiI, that cut asymmetrically within the intervening small single copy (SSC) region and the large single copy (LSC) region, respectively, but not within the large IRs. No inversion isomers were observed for a deletion derivative of pNRC100 lacking one IR, which suggests that both copies are required for inversion to occur. One end of the large IRs terminated at an ISH2 element while the other end terminated at an ISH3 element. The pNRC100 derivatives had suffered deletions which removed the gas vesicle genes and flanking DNA sequences. An autonomous replicating sequence of pNRC100 was mapped to the 19-kb HindIII-C fragment by cloning it into an E. coli plasmid containing the Haloferax volcanii mevinolin-resistance (mev$\sp{R})$ gene, transformation of H. halobium with the resulting plasmid pNGHCMEV1, and recovery of pNGHCMEV1 DNA from mevinolin-resistance transformants (Rep$\sp+).$ The nucleotide sequence of the replication origin was determined and found to contain a 550 bp highly A+T-rich segment and a 3,027 nucleotide open reading frame, named repH. The repH transcription start site was shown by primer extension analysis to be 17-18 nucleotides upstream of the putative start codon. The predicted RepH protein, an acidic protein of 113,442 molecular weight, showed 24.2 to 27.1% identity with the putative gene products of Hf. volcanii plasmid pHV2 and H. halobium halophage ($\phi$H)-derived plasmid p$\phi$HL. The replication origin was subjected to linker scanning mutagenesis and the results showed that repH is required for replication. (Abstract shortened by UMI.)

Two novel antinematodal strains of bacteria, Streptomyces sp. (CR-43) and Bacillus thuringiensis is (Bt) (CR-371) originating in Costa Rica were differentiated from other closely related strains and compared to rifampicin-resistant derivatives in greenhouse and field studies. The name Streptomyces costaricus is proposed for strain CR-43. The generic attribution was based on its typical morphology, production of LL-diaminopimelic acid and fatty acid composition. To clarify its taxonomic position, the isolate was compared with type strains of similar Streptomyces spp. The results of biochemical tests and profile analysis of hydrolyzable fatty acids indicated that CR-43 differed from previously described species and represents a new species. CR-43 (= ATCC 55274 = NRRL B-16897) is the type strain. A genotaxonomic approach to the differentiation of the streptomycetes was also taken. A modified RAPD analysis, conserved mini-sequence primed PCR (CMSP-PCR), was designed to target short conserved sequences dispersed throughout the streptomycete genome. All species had distinct DNA profiles when DNA was amplified with either 15-mer primer 5SSU3 (5$\sp{\prime}$-TGCGGCCGTACTCCC) or SSU5 (5$\sp{\prime}$-CGGCAGGCCTAACAC). The DNA profile of CR-43 resembled most that of S. h. decoyicus using primer SSU5. Species delineation was easily achieved with CMSP-PCR DNA amplification without further enzymatic processing. The differentiation of the novel Bt strain from five patented antinematodal Bt strains was required for patent prosecution. This was achieved by analysis of hydrolyzable fatty acid compositions of the strains by GC-MS and by DNA profile analysis. Thirty compounds in the total ion chromatograms were identified and analyzed using non-parametric statistics, the major fatty acids being i-15:0 (13.7-23.2%), i-13:0 (6.8-10.7%), i-14:0 (5.0-7.7%), 14:0 (3.0-4.1%), a-15:0 (3.9-9.9%), i-16:0 (3.6-7.2%), 16:0 (3.2-11.6%), i-17:0 (3.2-9.8%), 18:0 (tr-13.5%), a monounsaturated C16 (4.5-9.9%), a monounsaturated branched C17 (2.8-5.8%), a diunsaturated C18 (0-5.8%), and a monounsaturated C18 acid (0.3-4.8%). Molecular amplification of the DNA using a random nanomeric primer 5$\sp{\prime}$-CCGAGTCCA (that could discriminate serovars) showed that RAPD profiles of the Bt's were different. Spontaneous rifampicin-resistance (rif$\sp+$) was selected in CR-371 and CR-43. Rif$\sp+$ had a positively pleiotropic effect on the biocontrol activity of the Bt strain and a negatively pleiotropic effect on the novel streptomycete. Rif$\sp+$ was a useful marker for monitoring survival of CR-371 and CR-43 in soil.

Phytophthora capsici is a destructive pathogen of tomato, pepper and cucurbits worldwide. Its genetic variation has a significant impact on disease management. Morphological analysis, fungicide resistance, isozyme analysis, internal transcribed spacers (ITS) and random amplified polymorphic DNA (RAPD) analysis were used to investigate the genetic variation in the genome of Phytophthora capsici from Western Massachusetts, Long Island, New York and Quebec, Canada. Isolates from the first two locations contained both mating types: Al and A2. Only one mating type, A2, was present in Quebec, Canada. The morphological characters used in this study included length and width of sporangia, length of pedicel, length and width of antheridia, and diameters of oospore and oogonia of P. capsici. All morphological characters had significant variation among the three collections. Within each collection, there were significant differences between isolates and between characters. There were no significant differences in resistance to the fungicide metalaxyl among the three collections, but more fungicide resistance was observed in the collection from Massachusetts. Crosses between the most resistant and sensitive isolates were performed. The banding patterns of the isozymes: glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphate isomerase (GPI), Malate dehydrogenase (MDH) and Malate dehydrogenase NADP (ME) were examined. The results from isozyme analysis showed no difference among isolates. ITS analysis revealed no difference among isolates, and suggested that the isolates may have a very close genotype. In RAPD analysis, twenty 10-mer random primers were tested and the banding patterns were analyzed with MacClade 3.5.1. The results suggested that (1) isolates of the same mating type tended to cluster together; (2) Massachusetts isolates were spread throughout in tree, suggesting more genetic variation; (3) New York and Canadian isolates were relatively close, implying less variation; (4) progenies of metalaxyl resistant and susceptible isolates tended to cluster with one parental isolate, although some were widely divergent indicating that genetic variation arose from sexual reproduction. Canadian isolates, which lacked sexual reproduction, had less genetic variation, and less metalaxyl resistance. This research demonstrated that DNA recombination during sexual reproduction can generate genetic variation that can lead to increased opportunity of occurrence of fungicide resistance. The RAPD technique was used to develop a Phytophthora capsici specific probe and primers for pathogen identification and detection.

The budding yeast (S. cerevisiae) centromere component, Cse4p is an evolutionarily conserved histone H3-like protein, with homologues identified in fission yeast, worm, fly and human. All histone H3-like proteins have C-terminal histone fold domains (HFD) that are highly similar to the HFD of H3, but carry very different N-termini with unknown functions. The Cse4p N-terminus contains 135 residues, with a large portion of charged amino acids and a high concentration of serines within the first 22 residues. Based on the current model that suggests that Cse4p replaces H3 in a specialized centromeric nucleosome, the Cse4p N-terminus would extend out from the putative Cse4p-nucleosome and may play a variety of roles in centromere function. To elucidate the function of the Cse4p N-terminus, we conducted two comprehensive and systematic mutagenesis studies involving alanine scanning and sequence deletions, and we defined a 33-amino acid domain that is essential for cell viability and chromosome segregation. This essential N-terminal domain (END) has functions distinct from that of the HFD as demonstrated by interallelic complementation between cse4 END and HFD mutant alleles and heterodimer formation of END-HFD mutant proteins. Mutating all the potential posttranslational sites in the END indicates that the END function does not require posttranslational phosphorylation or acetylation. Genetic studies involving dosage suppression, synthetic lethality and two-hybrid analysis reveal that the END interacts with the Ctf19p/Mcm21p/Okp1p kinetochore complex. These results are consistent with the current Cse4p-nucleosome model. Although Cse4p has an HFD resembling that of H3, unlike H3, Cse4p exclusively localizes at the centromere. An important question is whether the N-terminus of Cse4p is responsible for the specific centromere targeting of the protein. Lethal Cse4p proteins lacking regions of the N-terminus can localize to the centromere in the presence or absence of wildtype Cse4p as determined by chromatin immunoprecipitation. In contrast, some lethal Cse4p HFD mutant proteins as well as chimeric proteins consisting of the Cse4p N-terminus fused to the HFD of either H3 or the Cse4p human homologue, CENP-A, fail to localize to the centromere. We conclude that the N-terminus of Cse4p is not required for centromere targeting of the protein and that the Cse4p HFD is necessary and sufficient to confer centromere localization.

Bulk soil is a stressful environment low in readily available nutrients and characterized by a low oxygen tension. Additionally, many soils undergo conditions of drought. Strategies that common soil bacteria take to cope with adverse conditions may be crucial to the ability of soil microorganisms to persist in soil. Therefore, studying genetic loci inducible by stress conditions commonly applied to soil microorganisms may shed light on the strategies used, and a thorough understanding of molecular responses to these stresses is critical. This dissertation explores in detail the responses of S. meliloti 1021 to experimentally induced salinity and desiccation. We have identified a number of factors central to S. meliloti 1021's ability to survive desiccation, including temperature, stationary growth phase, chloride and sulfate availability. We were also successful in identifying two genetic loci that are important in the adaptation of S. meliloti 1021 to increased levels of NaCl. One such locus ( asnO) was shown to contain a hypothetical glutamine-dependent asparagin synthetase. We showed that asnO::tn5LuxAB (a transcriptional fusion with luciferase on a Tn5 derivative) was induced by chloride and osmotic stress, and during the stationary phase. The main function of AsnO is not asparagine synthesis since addition of aminoacids to cells at elevated NaCl concentrations did not complement growth. However, AsnO is not involved in NaCl mediated survival during desiccation. We also tested a second locus for its involvement in NaCl response and desiccation. Open reading frame smb20482, contains a putative dAla-dala ligase domain and is induced by osmotic stress. Disruption of this locus caused NaCl mediated desiccation sensitivity. Penicillin sensitivity indicates a function for this gene in crosslinking of peptidoglycan. Interestingly, homologs of this gene are only found among the proteobacteria. In conclusion, (1) chloride availability enhances survival during desiccation of S. meliloti 1021, (2) we identified two NaCl responsive loci also involved in adaptation to NaCl and, (3) one locus (smb20482) is also involved in survival during desiccation. Thus, our strategy to identify genetic loci involved in the survival during desiccation has been successful.

Mesophilic cellulolytic Clostridium papyrosolvens C7 potentially could be used by industry for ethanol production from cellulose fermentation. The first goal of this study was to determine the pathways utilized by C. papyrosolvens C7 for the fermentation of cellulose and cellobiose, the soluble disaccharide product of cellulose hydrolysis. High-performance-liquid chromatography and gas chromatography analyses showed that acetate, ethanol, formate, lactate, malate, CO2, and H 2 were the end products of cellobiose fermentation by C. papyrosolvens C7. These products were quantified and a fermentation balance was calculated. Based on these analyses and the results of enzyme assays, a biochemical model for the fermentation pathways of C. papyrosolvens C7 is presented. It is suggested that formation of malate, a very uncommon fermentation product, served as an electron consuming reaction used to regenerate electron carriers (e.g., NAD+) during cell growth. In order to facilitate the metabolic engineering of C. papyrosolvens C7 for increased ethanol production and other properties desirable for industrial applications, a genetic exchange system involving the conjugative transposon Tn916 was developed. Tn916 was transferred from Enterococcus faecalis to C. papyrosolvens C7 in filter mating experiments. The tetM marker, conferring resistance to tetracycline, was used in the primary selection of transconjugants. Tn916 transfer frequencies ranged from 10 −7 to 10−5 per recipient. Highest transfer frequencies were obtained when recipient cells received a heat shock treatment, and both the donor and recipient cells were in the logarithmic phase of growth. A tetM gene probe hybridized to chromosomal DNA extracted from randomly selected C. papyrosolvens C7 transconjugants. These experiments indicated that Tn916 inserted into different sites on the chromosome of C. papyrosolvens C7. Tn 916 transposon mutagenesis was used to select low-acid-producing mutants. Fourteen mutant strains were selected by using the proton suicide method. Most of the mutant strains selected showed higher ethanol production. The third goal of this project was to determine the genome size of C. papyrosolvens C7. The restriction enzyme NotI was used to cleave intact C. papyrosolvens C7 DNA in agarose plugs. The resulting five DNA fragments were resolved by using pulse-field gel electrophoresis. The minimum size of the C. papyrosolvens C7 genome was determined to be 4.88 Mb.

The purpose of this dissertation is to evaluate the proposition that plasmids can be maintained in bacteria populations as parasites, i.e., by infectious transfer while conferring a selective disadvantage to their hosts (relative to plasmid-free cells). Towards this end, I have extended existing theoretical and experimental studies of the population dynamics of plasmids to consider (1) surface growth, (2) the effects of environmental factors (antibiotics) on rates of plasmid transfer, and (3) conjugative R-plasmids of eight different incompatibility groups. I have developed (1) an experimental method for the quantitative study of population growth and plasmid transfer rates in surface cultures of bacteria, the Surface Slide System, (2) a protocol to estimate the plasmid transfer rate parameter in liquid batch culture, the "end-point method," and (3) a mathematical model of bacterial growth and plasmid transfer in surface populations. The highest rate of plasmid transfer in surface culture obtained when the bacterial population was a confluent layer of micro-colonies. Under these conditions, the kinetics of plasmid transfer fit a simple mass-action model developed for bacteria in liquid culture. When the surface population forms fewer dispersed colonies, less transconjugants are produced per donor and recipient cell. The cost (measured as the decrease in the population growth rate) on the E. coli K12 host strain of carrying each plasmid, was similar in liquid and surface seasonal habitats and substantially less than previously reported for IncFII plasmids in chemostats. The surface model provided a reasonably good fit with the data observed in SSS culture, despite the fact that this model is only a simple first approximation of the complex process of growth and plasmid transfer on surfaces. This study provided evidence in support of the longstanding hypothesis that plasmid fertility is augmented by the presence of antibiotics. In the absence of antibiotic-mediated selection, the two antibiotics tested, ampicillin and kanamycin, augmented the transfer rate parameter of IncFII plasmids by as much as ten-fold. Several of the plasmids studied in serial transfer experiments could become established as parasites in populations with stationary phase densities on the order of 10$\sp8$ cells ml$\sp{-1}$. For most plasmids, the invasion conditions were broader in surface than in liquid culture. Despite this, I argue that in natural populations, a parasitic lifestyle is unlikely.

An investigation of the mechanism responsible for genetic hypervariability in Halobacterium halobium gas vesicle synthesis was conducted. Four partially vacuolated mutants (Vac$\sp{\delta-})$ H. halobium mutants were analyzed by Southern hybridization, cloning, and DNA sequence analysis. In each mutant a different halobacterial insertion element was responsible for the observed phenotype. The insertions mapped upstream of the H. halobium gvpA gene. DNA sequence analysis of the 5$\sp\prime$ and 3$\sp\prime$ regions of gvpA revealed 10 open reading frames; gvpD, E, F, G, H, I, J, K, L, and M; 5$\sp\prime$ to the gvpA gene in the opposite strand and two open reading frames, gvpC and N, in the region 3$\sp\prime$ to gvpA with the same transcriptional orientation as gvpA. A study was conducted to determine if the products of the gvpA, gvpC, gvpD, gvpE, gvpF, gvpJ and gvpM genes could be detected in purified H. halobium gas vesicles or whole cell lysates using immunological techniques. To do so, LacZ-Gvp fusion proteins were produced in E. coli and used to immunize rabbits. The antisera and protein-A column purified antibodies were used in immunoblot analysis of purified gas vesicles and cell lysates. The antiserum directed against the LacZ-GvpC fusion protein was successful in identifying a protein present in both purified gas vesicles and whole cell lysates, and this indicates that the gvpC gene product is a structural gas vesicle protein. Techniques were developed to allow for genetic analysis of gas vesicle synthesis in H. halobium. An H. halobium/E. coli shuttle vector, pJHGV3, which contains the gvpA gene cluster was constructed. Transformation of Vac$\sp-$ H. halobium strains, in which the gvpA gene cluster is deleted, with pJHGV3 resulted in complementation of gas vesicle synthesis. Methods were developed to allow non-polar mutations to be introduced into gvp genes present on pJHGV3. A plasmid containing a disruption of the region 3$\sp\prime$ to the gvpN gene, pJHGV33$\sp\prime$::$\kappa,$ was constructed and used to transform Vac$\sp-$ deletion mutants. Resulting transformants were Vac$\sp+$ indicating that there are not additional contiguous gvp genes downstream from gvpN. Together these techniques will provide useful tools in further analysis of the gas vesicle structure and its assembly.