<p> The genetic code is highly conserved across all domains of life, enabling horizontal gene transfer (HGT) between organisms and across ecosystems via horizontally-transferred genetic elements such as viruses and plasmids. While HGT increases genetic diversity, it poses a risk to engineered biological systems by introducing new genes that destabilize engineered functions or allowing the expression of engineered genes in wild organisms with unknown effects. A model organism engineered with an alternative genetic code may provide new insight into the origins of the genetic code while also providing a stable chassis for engineered biological systems.</p><p> The Isaacs Lab recently developed an <i>Escherichia coli</i> strain lacking both UAG stop codons and Release Factor 1, resulting in the first genomically recoded organism (GRO) with an unassigned codon in its genetic code. Here, we demonstrate that this alternative genetic code lacking UAG codon assignment confers resistance to multiple viruses (&lambda;, M13, PI, MS2) at titers up to 10¹¹ PFU/mL and impairs conjugative plasmid function (F and RK2) up to 10⁵-fold. Propagating viruses on a mixed microbial community containing standard and alternative genetic codes also reduced viral population fitness and prompted viral adaptation to the alternative genetic code. In investigating the molecular mechanism underlying the resistance to viruses and conjugative plasmids, we found that UAG-ending genes elicit ribosomal stalling and the tmRNAmediated ribosomal rescue response, resulting in degradation of UAG-ending proteins and suggesting that genomic recoding may be a broadly applicable strategy to impair horizontal gene transfer into other organisms.</p><p> To prevent the expression of engineered genes in wild organisms, we reassigned the UAG codon in the GRO to a sense codon incorporating the non-standard amino acid <i>4</i>-acetylphenylalanine (pAcF) through the introduction of an orthogonal translation system (OTS). We then created a library of UAG-containing variants and assessed escape of UAG-containing genes from the GRO into wild-type organisms for both a non-selective green fluorescent protein (GFP) and selective chloramphenicol acetyltransferase (CAT) gene. While 1 UAG codon impaired the expression of GFP in wild-type organisms, at least 2 UAG codons were required in CAT to consistently prevent escaped expression in wild-type organisms with a standard genetic code. Additionally, sequencing revealed that wild-type organisms enabled expression of CAT by mutating UAG codons to UGG coding for tryptophan or CAG coding for glutamine. By placing UAG at sites in proteins that cannot tolerate a tryptophan or glutamine substitution, we can create UAG-containing genes further isolated from expression in wild organisms.</p><p> As biotechnology increasingly targets open-environment applications such as bioremediation or disease treatment in humans, we require methods to stabilize and control the genetic information that we encode in engineered biological systems. Because alternative genetic codes can both confer resistance to horizontal gene transfer into an engineered system and restrict expression of engineered genes in wild-type organisms, genomic recoding of organisms to contain alternative genetic codes is a promising path towards increasing the stability and safety of engineered biological systems. However, open-environment applications will expose engineered biological systems to new stresses not represented in the laboratory environment, and further work is required to validate these methods will be robust in conditions of limiting nutrients or other cellular stresses. Additionally, while we have demonstrated genetic isolation of the GRO with respect to genes both entering and leaving the cell, we cannot currently have both properties simultaneously because UAG is the sole open codon. We envision that current research into further codon reassignments, including the reassignment of sense codons, will pave the way for alternate genetic codes with multiple codon reassignments. By expanding recoding efforts to multiple species, we envision the development of synthetic microbial communities with alternate genetic codes that are genetically isolated and robust to perturbation by HGT.</p>

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.

Actinomycetes have been used with enormous success in industrial processes; however, little is known about biofilm development by these filamentous microbes, and the presence of community development on insoluble cellulosic substrates such as cellulose. Cellulose is the most abundant biopolymer and renewable energy source on Earth, and its decomposition, which is carried out almost exclusively by microorganisms, is a key step in the cycling of carbon in the biosphere. It has long been known that cellulolytic bacteria may adhere to their insoluble substrate as it is degraded, although surprisingly little is known about microbial growth, colonization and community development on insoluble cellulosic substrates and non-nutritive surfaces. Previous investigations indicated that two Gram-positive cellulolytic soil bacteria, Cellulomonas uda, a facultative aerobe, and Clostridium phytofermentans , an obligate anaerobe, specifically adhered to nutritive surfaces forming a biofilm, but cells did not colonize non-nutritive surfaces. In this study is hypothesized that biofilm formation is a general strategy used by microbes in the degradation of insoluble substrates, and that it may serve as a means for microbes to secure a nutrient and persist in their environments. The objective of this study was to characterize biofilms produced by Thermobifida fusca, a Gram-positive cellulolytic actinomycete isolated from compost that rapidly degrades cellulose by means of a well-characterized extracellular cellulase system, and is a causative agent of Farmers Lung, the most common type of hypersensitivity pneumonitis. T. fusca was cultured with dialysis tubing as a nutritive surface for biofilm formation, and by using non-nutritive surfaces such as glass, plastic, metal and Teflon. Dialysis tubing was colonized by T. fusca aleuriospores but not by mycelial pellets. Surface-attached growth, examined by confocal scanning laser and scanning electron microscopy revealed structures resembling biofilms with cells embedded in fibrous material suggestive of an exopolymeric (EPS) matrix. T. fusca cells possessed higher hydrophobicity than C. uda and C. phytofermentans cells implicating higher capacity to bind to surfaces. DNase1 inhibited biofilm formation when assayed on microtiter plates suggesting a role for extracellular DNA in T. fusca biofilm formation. Concanavalin-A bound to the EPS material of biofilms and mycelial pellets, indicating alpha-linked D-mannosyl and/or alpha-linked D-glucosyl residues. The carbohydrate content of biofilms and mycelial pellets increased during growth. T. fusca biofilm formation is reduced when lack or excess of nutrients such as; iron, nitrogen and salt. Robust biofilms were developed between pHs 7 and 9, whereas minimum biofilms were produced at pH 3 and 11. Cellulose degradation rate and celE (endoglucanase E5) expression was similar for T. fusca biofilms and mycelial pellets. Also, results of this study indicate that in the life cycle of this actinomycete, cellulose is specifically colonized by aleuriospores, which germinate and degrade cellulose, ultimately developing into biofilms encased in a carbohydrate-containing EPS matrix, a hallmark of biofilm production.

Signal transduction in bacterial chemotaxis system is mediated by a family of transmembrane proteins that are clustered in the membrane and form noncovalent complexes with cytoplasmic signaling proteins. Recent evidence suggests that receptor clusters play an important role in mediating the signaling in bacterial chemotaxis. To understand the functional role of receptor clusters in kinase regulation and the mechanism of cluster formation, we employed a strategy to engineer an intrasubunit disulfide bond between the two methylation regions in the cytoplasmic domain of E. coli aspartate receptor (Tar CF). We assumed the potential stabilizing effect of an internal disulfide bond might change receptor's propensity to cluster. Furthermore, the intrasubunit disulfide bond effectively prevents domain swapping, thus providing a tool to test the recently proposed "domain swapping model" as a mechanism that generates cluster formation and CheA activation. Our results showed one disulfide-bonded CF, termed CFSP3, exhibited a slight increase in protein stability together with a dramatic increase in CheA activation, high cooperativity and a strong propensity to form large receptor clusters in solution. Conversely, we did not observe notable cluster formation in any CFs in solution that failed to activate CheA. Yet when these assembled into clusters by templating methods, some measure of CheA activation was achieved. We also found that addition of CheW significantly decreased the cluster size, suggesting CheW might bind to the same region as where Us make contact to oligomerize. The results demonstrate that domain swapping is not required for the formation of active receptor clusters. We also propose a clustering model in which a receptor's propensity to form large clusters correlate with the receptor-coupled kinase activity in solution. Tar CF is highly dynamic, and the regulation of dynamics has been proposed to be involved in signaling. Therefore, HDX properties of the engineered CFs were studied to gauge dynamics. Overall Us showed near complete exchange, although a detectable difference in HDX protection was observed between the reduced and oxidized forms of CFSP3, which may be functionally significant. Future experiments conducted with ternary complexes should provide invaluable information to clarify the functional role of receptor dynamics in signaling.

Cystic Fibrosis (CF) is a common autosomal genetic disorder in Caucasian populations. CF is caused by mutations in the cftr gene, which encodes the CF transmembrane conductance regulator (CFTR). CFTR regulates chloride and sodium ion transport across the epithelial cells lining the exocrine organs. Mutations in the cftr result in a failure to mediate chloride transport, which leads to dehydration of the mucus layer surrounding the epithelial cells. The mucus coating in the lung epithelia provides a favorable environment for invasion and growth of several opportunistic bacterial pathogens resulting in life threatening respiratory infections in CF patients. Pseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) are associated with chronic lung infections and are responsible for much of the mortality in CF. Little is known about interactions between these two, often co-infecting, species. When in competition, it is not known whether Bcc replaces the resident Pa or if the two species co-exist in the CF lung. Bacteriocins are potent toxins produced by bacteria. They have a quite narrow killing range in comparison to antibiotics and have been implicated in intra-specific and inter-specific bacterial competition brought on by limited nutrients or niche space. Both Pa and Bcc produce bacteriocins known as pyocins and cepaciacins, respectively. More than 90% of Pa strains examined to date produce one or more of three pyocin types: R, F, and S. A limited number of phenotypic surveys suggest that approximately 30% of Bcc also produce bacteriocins. The goals of my thesis study were to determine if clinical strains of Pa and Bcc produce bacteriocins and to determine whether these toxins play a role in mediating intra- and inter-specific bacterial interactions in the CF lung. The final goal was to identify novel bacteriocins from clinical Pa and Bcc strains. First, I designed a phenotypic bacteriocin survey to evaluate bacteriocin production in 66 clinical Pa (38) and Bcc (28) strains procured from CF patients. This study revealed that 97% of Pa strains and 68% of Bcc strains produce bacteriocin-like inhibitory activity. Further phenotypic and molecular based assays showed that the source of inhibition is different for Pa and Bcc. In Pa, much of the inhibitory activity is due to the well known S- and RF-type pyocins. S- and RF pyocins were the source of within species inhibitory activity while RF pyocins were primarily implicated in the between species inhibitory activity of Pa strains. In contrast, Bcc inhibition appeared to be due to novel inhibitory agents. Finally, I constructed genome libraries of B. multivorans, B. dolosa, and B. cenocepacia to screen for genes responsible for the inhibitory activity previously described in Bcc. ∼10,000 clones/genome were screened, resulting in fifteen clones with the anticipated inhibition phenotype. Of these fifteen, only five clones had stable inhibitory activity. These clones encoded proteins involved in various metabolic pathways including bacterial apoptosis, amino acid biosynthesis, sugar metabolism, and degradation of aromatic compounds. Surprisingly, none of Bcc clones possessed typical bacteriocin-like genes. These data suggest that, in contrast to all bacterial species examined in a similar fashion to date, Bcc may not produce bacteriocins. Instead, Bcc may be using novel molecular strategies to mediate intra- and inter-specific bacterial interactions.

Staphylococcal Enterotoxin B (SEB) is one of the potent exotoxins synthesized by Staphylococcus aureus that causes toxic shock, is a primary cause of food poisoning and is a Class B bioterrorism agent. SEB, a superantigen, mediates antigen-independent activation of a major subset of the T-cell population by crosslinking TCRs of T-cells with MHC class II molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. This excessive secretion of cytokines, some of which are inflammatory, causes immune dysregulation, systemic inflammation and disease. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin’s interaction with the TCR or MHC class II and provide protection against the debilitating effects of this superantigen. In a series of experiments, we derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of noncrossreactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically-acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human PBMCs, or splenocytes derived from BALB/c or HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T cell activation and cytokine production. When tested in vivo in HLA-DR3 transgenic mouse TSS model, the two chimeric antibodies acted synergistically and provided full protection against SEB-mediated TSS symptoms and lethality of SEB. Furthermore, combination of chimeric anti-SEB, an extracellular inhibitor of SEB, and pharmacological agents (γ-secretase inhibitors, rapamycin, or lovastatin), an inhibitor of intracellular pathways recruited by SEB, provided significant reduction of SEB-induced T cell activation in cultures of mouse splenocytes and human PBMCs. Combination of chimeric anti-SEB antibody and lovastatin also provided in vivo protection against lethal doses of SEB in HLA-DR3 transgenic TSS model. In this study, we have developed a pair of chimeric anti-SEB antibodies (for the first time) that neutralize SEB efficiently in vitro as well as in vivo. In addition, we demonstrated that in vivo protection against lethal doses of SEB can be achieved by a statin of proven safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts. Both these findings have provided potential treatment options for diseases mediated by SEB, as there is no prophylaxis, or therapy against accidental or malicious exposure.

Chlamydia trachomatis is an obligate intracellular parasite which has a trophism for columnar epithelial cells. It is recognized as a major cause of endemic trachoma and conjunctivitis, as well as being the most prevalent sexually transmitted disease in the United States. The species can be subdivided into two biovars according to their host cell specificity and disease epidemiology. The trachoma biovar is associated with infections of ocular origin, or with the genital epithelia. The lymphogranuloma venereum (LGV) biovar is a sexually transmitted biovar causing a more invasive disease involving the lymphoid tissues. Inguinal and femoral lymph nodes become infected, producing swelling, necrosis and lymphocutaneous fistulae. The differences between biovar infectivity may in part be due to specific membrane antigens of the infectious elementary body and soluble antigens. Soluble antigens released during the infective cycle are likely to be crucial for the organism's survival. The identification of antigenic epitopes present on these molecules can aid in both serodiagnosis and selective immunization procedures which combat this pathogen. A soluble Chlamydia trachomatis esterase has been identified within the supernatant of 48 hour infected McCoy cell tissue cultures. The extraction procedure utilized a combination of ammonium sulfate precipitation, gel filtration, and immunoprecipitation techniques. The isolated protein has an approximate molecular weight of 26,000 to 30,000 M$\sb{\rm r}$ as demonstrated by gel filtration and SDS-PAGE size estimation. The esterase co-isolates with a chlamydial protein, and also contains serovar-specific epitopes. Use of rabbit anti-B serovar antibodies to deplete the esterase activity from the protein fraction confirms this molecule as being a chlamydial protein. Primates infected with B serovar C. trachomatis produce antibodies directed towards this antigen. This indicates that this molecule is immunogenic during infection, and perhaps plays a biological role in the pathogenesis of infection. Isoelectric focusing techniques has enabled the partial purification of a 26,000 M$\sb{\rm r}$ molecule, with a pI value of approximately 6.6, released during chlamydial infection. This molecule co-isolates with fractions containing esterase activity. This molecule is absent in mock-infected McCoy cell supernatants. Guinea pig antisera directed towards this protein is able to recognize specific epitopes present on the surface of infectious chlamydial elementary bodies.

Anabaena 7120 is a filamentous cyanobacterium that fixes nitrogen in heterocysts, specialized cells that develop at intervals along the filament in response to combined nitrogen deprivation. This study began as an investigation of nifH*, a gene 83% identical to the sequence encoding nitrogenase reductase, part of the nitrogen fixation complex. Although mutational analysis was not successful, transcriptional analysis demonstrated that nifH* is not expressed along with nifH in heterocysts of filaments deprived of combined nitrogen under aerobic conditions. Rather, expression of nifH* was detected only during anaerobic induction, at which time nifH is also highly expressed. Sequencing of the region adjacent to nifH* revealed four open reading frames. One of these was identified in a database search by homology to other sequences and encodes an enzyme which appears to act in the catabolism of endogenous glycogen reserves. The cyanobacterial gene is transcribed in exponentially growing cultures but not during anaerobic induction in which metabolic processes have been severely curtailed. There is no evidence that the gene is specifically involved in nitrogen fixation although it is expressed in heterocysts. The other three open reading frames were not identifiable by database comparison. ORF2 is expressed at low levels under the same condition as nifH* (anaerobic induction). ORF3 is probably expressed at very low levels under the same conditions as nifH (nitrogen deficiency and anaerobic induction) and the transcript may be large enough to include unsequenced regions upstream. Transcription of ORF4 was not detected under the conditions investigated.

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.

The 3$\sp\prime$ minor domain of the 16S rRNA is associated with ribosomal decoding. Host cells expressing C1395U or C1395U/G1505U mutant rRNAs are inviable or slow-growing, respectively; those accumulating G1505U mutant rRNA show growth similar to wild-type. At the third doubling after expression of the mutant rRNA, host cells containing C1395U or C1395U/G1505U accumulated larger amounts of free 50S and 30S subunits than those containing the wild-type or G1505U mutant rRNA. The proportion of mutant rRNA reached 50% in 30S subunit populations but was marginal in 70S subunits and polysomes in all three mutant-containing strains. 30S subunits containing C1395U or C1395U/G1505U rRNA exhibited a marginal tRNA binding capacity and no response to IF2 stimulation while those containing mutant G1505U rRNA possessed an affinity for tRNA similar to their wild-type counterparts but failed to respond to IF2. These observations imply that mutations in decoding domain of 16S rRNA provoke changes related to translational initiation, and that the accumulation of the mutant 30S subunits impedes the function of the wild-type 30S subunits in the host cell. The binding site for ribosomal protein Ll lies within a sequence spanning nucleotides 2067-2235 of the E. coli 23S rRNA. A DNA fragment encoding the Ll-specific rRNA was inserted into a T3 transcription vector and subjected to random and site-directed mutagenesis. Wild-type and mutant transcripts were then prepared, and their affinities for Ll were determined by a quantitative filter assay. The results were as follows. (1) Mutations that disrupted base pairing within helix 77 significantly reduced the affinity of Ll for the rRNA. (2) The replacement of conserved purines at positions 2126 and 2168-2173 in the loop between helices 77 and 78 also severely impaired Ll-rRNA interaction. (3) Deletion of 1-4 non-conserved bases from the same loop, however, led to less than a 50% reduction in Ll association. Nondenaturing polyacrylamide gel electrophoresis revealed that most of the mutant rRNA transcripts exhibiting defects in Ll binding also migrated more slowly than their wild-type counterparts, indicating that they were improperly folded. Conformational differences in several of these transcripts were pinpointed through an analysis of their susceptibility to chemical modification. Finally, a smaller RNA transcript, encompassing helices 77 and 78 and loop 77/78, was synthesized and found to be active in binding protein Ll.