Bacteriophage D3112 is a Mu-like transposable bacteriophage of Pseudomonas aeruginosa. D3112 phage has a double-stranded DNA genome of approximately 38 kb with a genetic organization similar to that of coliphages Mu and D108. Cloning and DNA sequencing of the D3112cts15 left end has revealed a unique large open reading frame spanning nucleotides 892 to 170 of the lower strand. A polypeptide of an apparent molecular weight of 19.6 kDa was visualized by SDS-PAGE when this ORF was cloned and overexpressed in Escherichia coli. / By an in vivo functional assay, it was shown that the presence of 1174 bp of the D3112 left end on an E. coli-P. aeruginosa shuttle vector in P. aeruginosa prevented lytic development of superinfecting D3112 phage, indicating that the cloned fragment encodes the D3112 (c) repressor. By an in vitro functional assay, it was shown that a crude extract, containing overexpressed repressor (c), appeared to bind specifically to a 277 bp fragment of the D3112 left end. This fragment was also bound specifically by purified E. coli IHF protein. These protein-DNA interactions, and their location, suggest a role in transcription and regulation of the repressor (c) gene for this fragment. Preliminary purification steps of the D3112 repressor (c) show no interaction with weak ion exchange and precipitation by 60% (w/v) ammonium sulfate.

In order to identify and characterize genes whose transcription is affected by arsenic and antimony, random lacZ fusions in the E. coli chromosome were constructed using the Mud I transposable phage. One clone was isolated that contained a lacZ transcriptional fusion induced by arsenic, antimony, and their associated salts. When cells were subjected to DNA-damaging agents such as mitomycin C and sodium dichromate, a low level of induction resulted. In addition, a marked increase in arsenic sensitivity was observed in the fusion clone as compared to the parent strain presumably due to the insertional inactivation of the E. coli gene(s) by the Mu prophage. By Southern Blotting analysis of the original gene fusion, as well as genetically identical transductant, only one copy of the lacZ fusion was observed. From these results, a preliminary map of the site of insertion of the reporter gene within the arsenic/antimony responding gene has been generated. This strain should prove to be useful in the rapid detection of low bioavailable concentrations of arsenic, as well as to study the molecular mechanisms of arsenic toxicity.

Termination of transcription just upstream of the late promoter on polyomavirus was studied. A mutant virus had previously been constructed. Results did not reveal any termination of transcription just upstream of the late promoter in the mutant A3-ins 5 when compared to the wild type A3 strain of polyomavirus. The effect of time of in vitro elongation on results is shown. RNA polymerases may traverse the entire genome and terminate shortly downstream of the late promoter, or termination in the region upstream of the late promoter is being masked. Alternatively, certain second termination signal(s) could be missing on the A3-ins 5 DNA leading to inefficient termination in spite of efficient cleavage and polyadenylation of the transcript. / The extent of premature termination downstream of the late promoter of polyomavirus was also studied. In vitro run-on experiments revealed that with increasing distance from the late promoter the density of RNA polymerase gradually decreased. The level of premature termination at the late promoter is not dependent on the presence of either a functional large T ag or an efficient polyadenylation signal. (Abstract shortened by UMI.)

The ferrichrome-iron receptor of Escherichia coli K-12 encoded by the fhuA gene is a multifunctional outer membrane receptor required for the binding and uptake of ferrichrome and is the receptor for the bacteriophages T5, T1 $ phi$80, and UC-1 as well as for colicin M. The complete nucleotide sequence of the fhuA gene was determined from a 2,902-base-pair fragment of DNA. A single open reading frame was found which translated into a 747-amino acid polypeptide. A signal sequence of 33 amino acids was followed by a mature protein with a molecular weight of 78,992. To identify domains of the protein which are important for FhuA activities, libraries of insertion and deletion mutants were constructed. Export of the mutant FhuA proteins to the outer membrane and receptor functions of the mutant proteins were analyzed. All of the linker insertion mutant proteins and all but three of the deletion mutant FhuA proteins co-fractionated with the outer membrane. Five linker insertion proteins and most deletion proteins were susceptible to cleavage by endogenous proteolytic activity. Some of the mutant receptors conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating as that of wild-type FhuA; killing by colicin M was also unaffected. Several mutants were altered in their sensitivities to the lethal agents. The results designated selected regions of the FhuA protein for receptor functions and demonstrate the presence of both shared and unique ligand-responsive domains.

Organotin compounds are used for a variety of applications including antifouling paints for ship hulls, wood preservatives, and UV stabilizers in plastics. When they are released into aquatic environments, they easily accumulate in various marine organisms. Organotin compounds are considered endocrine disrupting chemicals since numerous marine organisms exhibit sexual abnormalities when exposed to dibutyltin. Singe certain bacteria show resistance to these compounds, we screened for genes that are induced by organotins. 3000 Escherichia coli clones were genetically engineered to contain the luxAB reporter genes from Vibrio harveyi in single, yet different, chromosomal locations. We screened these 3000 clones in the presence and absence of organotins, and identified a uniquely responsive clone designated TBT3. Luminescence was measured in liquid media and in solid media. Upon addition of tributyltin and dibutyltin, luminescence greatly increased. The transcriptional activity was measured by RNA dot blotting, and two potential genes ygaP and b2667, were identified as induced by organotins. Also, TBT3 transcriptional activity was measured and compared with the transcriptional activity of MG1655, the parent strain of TBT3. The measured transcriptional activity of the two strains was different, although the conditions in which the measurements were taken were identical, which gives place to interesting arguments what determines this difference.

Previous work has shown that some Neisseria meningitidis strains do not express the Class 1 Outer Membrane Protein (OMP1; the porA gene product) as assessed by SDS-PAGE analysis of OMPs. Northern analysis has identified the level of control of porA expression as transcriptional. Promoter regions differed in the number of guanosine residues in a poly(G) track located between the -10 and the -35 regions. Strains that do not express the protein either had an adenosine residue within this poly(G) track or contained nine or less guanosine residues in this track. We investigated the role of the promoter region of porA in the regulation of transcription. The promoter regions of the porA gene from three strains, two of which do not express the OMP1, were analyzed. Promoters were cloned into a promoter-probe vector (pDN19/ acO) possessing a promoterless lacZ gene and expressed in Escherichia coli. Transcription activities of cloned promoter regions were measured by beta-galactosidase assays. (Abstract shortened by UMI.)

The major surface-exposed, pore-forming protein in the outer membrane (OM) of Haemophilus influenzae type b (Hib) is porin (341 amino acids; Mr 37,782 Da). Porins form water filled channels and allow the diffusion of small hydrophilic solutes up to 1400 Da including nutrients and small antibiotics across the OM. The crystal structures of porins solved to date indicate a conserved motif of a barrel composed of 16 anti-parallel beta-strands. / Molecular genetic techniques were used to introduce a hexahistidine tag into surface-exposed loops, loop 4 and loop 8, of Hib porin. Loop 4 was modified by addition of nine amino acids including six consecutive histidine residues following Val-174. Loop 8 was modified by splicing seven amino acids including six consecutive histidines following Thr-321. Plasmids harboring the mutant porins were transformed into a strain of H. influenzae deleted for the wild-type ompP2 gene. / The mutant porins, designated OmpP2.H6(L4) and OmpP2.H6(L8), were purified by metal chelate chromatography using Ni-NTA superflow and POROS 20 MC media. (Abstract shortened by UMI.)

Reactive amyloidosis characterized by the tissue deposition of insoluble AA amyloid (AA), is a potentially serious complication of certain chronic infections and recurrent inflammatory diseases. AA is derived from the amyloidogenic serum amyloid A (SAA), an acute phase protein whose plasma concentration may increase up to 1000-fold in response to tissue trauma. Chronic elevation of SAA in conjugation with inflammation-related host factors is believed to induce conversion of SAA to AA. One such host factor, called amyloid enhancing factor (AEF), has recently been identified as ubiquitin (UB). However, the molecular pathogenesis of AA amyloidosis in vivo is not well understood. / Prior studies in our laboratory using alveolar hydatid cyst infected mouse model of AA amyloidosis (AHC-mouse) has demonstrated that (a) SAA and UB co-deposit in the splenic perifollicular areas prior to AA deposition, (b) both SAA and UB co-localize to endosomes-lysosomes (ELs) inflammatory macrophages (MA) and splenic reticuloendothelial (RE) cells, and (c) UB binds to the tissue AA deposits. / My working hypothesis is that EL-mediated physiological degradation of SAA may serve as the primary clearance mechanism for exogenous SAA. As a secondary consequence, incessant overloading of ELs with ubiquitin-associated SAA during chronic inflammation may alter the rate or the extent of proteolysis leading to prolonged retention of SAA derivatives and the formation of "nascent" AA fibrils in ELs. / The objectives of the present study are the following: (a) to establish intra-macrophage topographical relationship between murine SAA3, which is synthesized by activated MA and RE cells, and two precursors of AA, SAM and SAA2, that are endocytosed by MA and RE cells, (b) to immunochemically and chemically analyze the MA-derived degradation products of SAA in order to establish whether they could be potentially amyloidogenic and (c) to seek functional clues regarding the role of ubiquitin in SAA processing and in AA formation. / Both the in vivo and in vitro experiments were carried out to pursue these objectives. I have shown that (a) murine SAA1/SAA2 localize exclusively to ELs whereas SAM occupies the cytoplasmic compartment in MAs, (b) the lysates derived from MA contain at least four AA-sized potentially amyloidogenic N-terminally intact SAM and SAA2 derivatives and (c) AA amyloid purified from AHC-mice is derived from both SAA2 and SAA1. As to the role of UB in SAA processing, I have shown that (a) UB binds avidly but non-covalently to both SAA1 and SAA2, (b) UB binds to ELs containing both immature and mature AA fibrils and (c) UB dissociated and purified from the tissue AA deposits retains its AEF activity in vivo. / Collectively, these data show for the first time a direct relationship between the localization of SAA1 and SAA2 to ELs in MAs and the intracellular generation of potentially amyloidogenic N-terminally intact SAM and SAA2 derivatives which may polymerize into "nascent" AA fibrils in ELs. We believe, as has also been proposed by others, that these "nascent" AA fibrils either after exocytosis or cell death may act as nucleation sites for extracellular AA deposition. As to the role of UB in SAA processing, we suggest that non-covalent binding of UB to SAA1 and SAA2 may (a) structurally alter theses substrates, thereby rendering them susceptible to phagocytosis by MA/RE-cells, and (b) interfere with their complete degradation culminating in the formation of UB-associated AA found in the EL. Non-covalent linkage of UB has been shown to induce structural alterations in other substrate proteins rendering them susceptible to phagocytosis.

The human T cell leukemia virus type I (HTLV-I), the etiological agent of adult T cell leukemia is the first human retrovirus linked to malignancy. The viral protein Tax, is essential for HTLV-I gene transactivation and responsible for oncogenic transformation. Tax is capable of indirectly transactivating numerous growth regulatory genes through interactions with host transcription factors. One of the targets of the Tax protein is the NF-kappaB/IkappaB family of transcriptional regulators. In HTLV-I Tax expressing cells, the maintenance of NF-kappaB activity is essential for cellular transformation. Previous studies demonstrated that Tax activated NF-kappaB through binding to the Rel homology domain of NF-kappaB. The objective of this work was to elucidate the mechanism by which Tax transactivates NF-kappaB. We addressed the specificity and function of Tax interaction with members of the NF-kappaB/IkappaBalpha family and demonstrated that: (1) Tax increased IkappaBalpha degradation, resulting in the induction of NF-kappaB DNA binding activity; (2) Tax enhanced NF-kappaB binding to DNA 40 to 100 fold by increasing NF-kappaB dimer formation which was detected in the absence of DNA; (3) Tax interacted with all NF-kappaB DNA binding subunits and with IkappaBalpha in vitro; (4) Tax physically associated with IkappaBalpha in vivo; and (5) Tax and IkappaBalpha had antagonistic effects on NF-kappaB binding and gene activity. These results suggested that Tax interaction with IkappaBalpha interferes with the formation of NF-kappaB-IkappaBalpha complexes and may play a role in targeting IkappaBalpha for degradation. To further characterize the interactions between Tax and IkappaB, co-immunoprecipitation experiments were performed that identified a complex in HTLV-I infected cells comprising Tax-IkappaBalpha and the HsN3 subunit of the 26S proteasome. Metabolic labeling and protein turnover studies indicate that Tax targets IkappaBalpha to the proteasome for degradation in a

A luxO DNA binding protein (LuxT) was purified to homogeneity from V. harveyi after five major chromatography steps including a highly effective DNA affinity chromatography step and reverse phase HPLC. The sequences of three tryptic peptides obtained on digestion of the purified protein did not match any sequences in the protein data bank indicating that LuxT is a new V. harveyi protein. Inverse PCR was conducted sequentially to obtain the complete gene (luxT) coding for a protein of 153 amino acids which shares homology with the AcrR/TetR family of transcriptional regulators. Gene disruption of luxT in V. harveyi increased luxO expression and affected the cell density dependent induction of luminescence showing that LuxT is a repressor of luxO. As LuxT also affected the survival of the V. harveyi cells at high salt concentration and close homologues were present in other bacterial species suggested that the LuxT regulatory protein appears to be a general rather than a lux-specific regulator. / The rpoS gene in V. harveyi has been cloned in this work and shown to code for a protein with high homology to the RpoS proteins in other species. The null mutant of RpoS has been constructed and the effect of rpoS deletion on stress resistance as well as the cell density dependent luminescence in V. harveyi were examined.