Mesophilic cellulolytic clostridia play an important role in the global carbon cycle because they are able to degrade abundantly-produced cellulosic material. Many strains may also take part in the nitrogen cycle inasmuch as they are able to satisfy their nitrogen requirement for growth by fixing dinitrogen (N$\sb2).$ The first objective of this research was to characterize nitrogenase systems of mesophilic cellulolytic clostridial species using physiological and molecular approaches. DNA probes, nifDK, vnfDGK, and anfDGK, constructed from structural genes representing three different nitrogenase systems in Azotobacter sp., were used to detect and clone potential nitrogenase genes from cellulolytic clostridia. A 7-kb genomic DNA fragment, which hybridized the nifDK probe, was cloned from Clostridium cellobioparum ATCC 18532, and a 2.1-kb subfragment was found to contain a nifH sequence. Northern analyses suggested this sequence was expressed under nitrogen-fixing conditions. Based on phylogenetic analyses involving 34 known nifH gene sequences, nifH from C. cellobioparum was most closely related to the multiple nifH genes of Clostridium pasteurianum. In addition, a 3-kb genomic DNA fragment was cloned from Clostridium hungatei B3B and found to have a high sequence identity to anfDGK from Azotobacter vinelandii. However, in studies of nitrogen fixation by C. hungatei B3B under Mo-deficient conditions, ethane was not detected during acetylene reduction assays, suggesting that the putative anfDGK genes were, if expressed in C. hungatei, part of a nitrogenase system that differed from that of A. vinelandii. The second objective of this research was to isolate plasmids indigenous to cellulolytic clostridia that may be used as vectors in strain modification. Several antibiotic resistant strains were screened for the presence of plasmids and a small plasmid (2450 bp), designated pMCF1, was isolated from strain MCF1 and ligated to the Hind III site of the vector pBluescript II KS. DNA sequence analyses identified an open reading frame encoding a peptide with homology to plasmid replication proteins. It is proposed that pMCF1 belongs to a subfamily of the single-strand DNA plasmids, which replicate via a rolling circle mechanism.

DNA replication forks are frequently detained during their progression from oriC to the replication terminus due to encounters with DNA lesions or proteins blocking the way. For survival, cells deploy a number of enzymes catalyzing replication restart at or near the damaged site. The preprimosomal protein, PriA, along with several accessory proteins, directs DnaB helicase to the lagging-strand template during assembly of the replisome. This helicase loading mechanism is analogous to that catalyzed by DnaA at oriC, except that PriA-directed fork assembly does not require oriC or any specific initiation sequence. Instead, it recognizes and binds with high specificity to various recombinational intermediates such as D loops that form during the recombination-mediated repair of replication forks. In this manner, PriA effectively links the processes of homologous recombination and DNA replication. PriA also possesses a 3′ to 5′ helicase activity that might be important for restarting stalled forks directly in the absence of homologous recombination. This lab uses a genetic approach to understand the in vivo functions of PriA and its accessory proteins, PriB, PriC, DnaT, DnaC, and Rep. Extensive phenotypic analyses of mutants of priA, priB, priC, dnaC and rep have clearly indicated that replication restart occurs by multiple pathways (Sandler 2000). The work presented in this dissertation continues to explore and test the multiple pathways model for replication restart. Three lines of investigation were pursued: (1) Can genetic evidence support a role for dnaT in replication restart? If so, then for which pathway(s) is dnaT important? (2) It has been proposed that priA mutants accumulate recombinational intermediates. Can this be detected in individual cells using a DNA stain to examine nucleoid organization? (3) PriA mutants are chronically SOS induced. Microscopy indicates that two kinds of cells are present in log phase cultures. Are SOS genes turned on in both populations of cells or only in a subpopulation?

To study polyhydroxyalkanoate (PHA) accumulation and PHA regulation in Bacillus megaterium, transposon mutagenesis with Tn917-LTV1 was carried out and seventy two mutants that produce more or less PHA than normal were isolated and partially characterized. The chromosomal regions of B. megaterium flanking the Tn917-LTV1 insertions were cloned and sequenced from two PHA over-producing and six leaky mutants. The results showed that Tn917-LTV1 was less than ideal for generating a mutant bank due to the unavoidable occurrence of sibling transposants. Furthermore, the sequence data revealed that chromosomal deletions mediated by Tn917-LTV1 insertions were common. An alternative method was developed using a direct plating procedure and it was shown to circumvent these two problems. One PHA leaky mutant and one overproducing mutant were studied in more detail. The PHA leaky mutant, T4, had significantly reduced levels of PHA accumulation in all media tested and could not form spores. Chromosomal DNA, contiguous with the transposon was cloned and sequenced. Analysis of the sequence data showed Tn917-LTV1 inserted 24-bp upstream of an operon encoding a putative RNA helicase (deaD) gene and a nuclease (nucP) gene. A chromosomal target repeat 5$\sp\prime$-TATTT-3$\sp\prime$ was found on both sides of the insertion which indicated that no deletion was involved in the Tn917-LTV1 transposition. A plasmid carrying the intact RNA helicase operon was constructed and transformed into mutant T4. The transformant recovered the ability to form spores and accumulate wild-type levels of PHA in minimal media. Deletions in either DeaD or NucP did not complement the T4 mutant to the phenotype of VT1660. The results show that both DeaD and NucP are involved in PHA accumulation and are required for spore formation. The DeaD protein of B. megaterium is the first protein in the DEAD-box helicase family that is not essential for the organism. The fact that the growth rate of mutant T4 and VT1660 were similar in both LB and minimal media indicates that this DeaD protein may target specific mRNA, such as those of pha and spo genes. B001S, a PHA over-producing mutant, unlike its parental strain VT1660, produced large quantities of PHA in rich media. The chromosomal DNA flanking the transposon was cloned from both sides of the insertion. The 8.4 kilobase pairs of chromosomal sequence from left end (IR-L side) of Tn917-LTV1 coded for sixteen open reading frames (ORFs). Ten putative products of the sixteen ORFs shared sequence homology with known gas vesicle proteins (Gvp). The 8.4-kb fragment and its deletion derivatives were cloned into pBluescriptIISK and the plasmids were transformed into E. coli. Gas vesicles were formed and observed by phase contrast microscope, differential interference contrast microscopy and electron microscopy. The deletion analysis and sequence comparison with known gas vesicle proteins suggested 14 out of the 16 ORFs formed the gvp operon of B. megaterium VT1660. The deletion analysis showed that 11 genes are the maximum required for the gas vesicle formation in E. coli. The E. coli cells containing gas vesicles showed increased buoyancy. This is the first time that a functional organelle has been transferred to E. coli.

Isolates of Burkholderia cepacia differed in their ability to utilize D-serine as a carbon and energy source. Strain 17616 is one of the strains which ordinarily fails to utilize D-serine. D-serine also inhibited the growth of this strain on alternative carbon sources such as mannitol. D-serine resistant (Dsd$\sp+$) mutants of strain 17616 were isolated which formed high constitutive levels of a D-serine deaminase not present in the wild type. The majority of such mutants also utilized D-serine as sole carbon source. A 5.5-kb fragment of B. cepacia DNA containing the dsd gene was cloned into the DsdA$\sp-$ strain E. coli AC6082. Introduction of recombinant plasmids carrying the dsd gene into strains 17616 or AC6082 resulted in high levels of constitutive D-serine deaminase activity as well as expression of a new peptide with the predicted size (45 kDa) of the the dsd gene product. Efforts were made to define the mechanism of activation of the cryptic dsd gene in strain 17616. Comparison of PCR products of the region upstream of the dsd gene in the wild type and Dsd$\sp+$ strains indicated that dsd gene expression was not a consequence of insertion of tranposable-gene-activating elements upstream of the cryptic gene as had been observed for activation of foreign bla and lac genes in this strain. Furthermore, no other alterations were detected in this upstream region that would account for dsd gene activation. Analysis the region downstream of the dsd gene revealed the presence of a marR-like repressor gene and adjacent multi-drug resistance transporter gene. The orientation of the latter two genes was opposite to that of the dsd gene. We were interested in the possibility that the marR (multiple antibiotic resistance/multiple adaptive response) protein might control dsd gene expression. To explore this possibility, I compared the nucleotide sequences of the marR gene from the representative Dsd$\sp+$ strain 249-50 with the corresponding wild type gene. The wild type and mutant sequences were identical.

Dissimilatory Fe(III) and Mn(IV) reduction is an anaerobic respiratory process common to many environments. A large number of bacteria that utilize these metals as terminal electron acceptors have been isolated and identified for the past decade. However, the molecular mechanisms of metal reduction remain unclear despite intensive research in this area. There are no genes or proteins identified, which are directly involved in metal reduction. The major goal of this work is to identify and isolate genes related to Fe(III) and Mn(IV) reduction as well as to determine the specific functions of their products. The organism used in this work is the facultatively anaerobic metal reducer S. putrefaciens MR-1. Transposon mutagenesis was used to generate mutants solely deficient in Fe(III) and Mn(IV) reduction. Analysis of the interrupted regions in two such mutants, SR-8 and SR-21, led to the identification of three genes, designated mtrC, mtrA and mtrB. The deduced amino acid sequence of mtrC and mtrA revealed that these genes encode deca-heme c-type cytochromes. The third gene, mtrB, was shown to encode an outer membrane protein of 679 amino acids. mRNA and Western blot analyses indicated that the three genes are organized in a single operon, mtrCAB, which is expressed constitutively. To elucidate the role of each protein in metal reduction, gene replacement was used to generate strains lacking either mtrC, mtrA or mtrB. Whole-cell suspensions of mtrC−A+B+, mirC+A−B+ and mtrC+A+B− mutants resulted in 4.6-fold, 31-fold and 70-fold decrease in iron reduction rates, respectively. In contrast to whole cells, the levels of iron reductase activity in the crude cell extracts of the mutants were similar to that observed in the wild type. These findings strongly suggest that the products of mtrC, mtrA and mtrB play an important role in metal reduction, however, they may not constitute the terminal metal reductase complex. Analysis of the DNA region upstream of mtrCAB revealed a presence of another multicytochrome gene cluster. Although the identified genes, mtrD, mtrE, mtrF and omcA, exhibited high degree of similarity to the mtrCAB operon, there was no evidence indicating their involvement in Fe(III) and Mn(IV) reduction in S. putrefaciens.

A newly discovered, nonoccluded baculovirus GSV has been reported to be a causative agent of sterility in adult corn ear-worms, Helicoverpa zea. Previous studies conducted by Hamm et al. (1996) and our laboratory indicated that it is an unusual insect virus with strict tissue tropism and the ability to establish persistent infections in vivo. After acquiring purified virus from infected adult insects, two permissive cell culture systems, TN-368 and Ld652Y were established for GSV replication in vitro. The cell culture-derived virus was confirmed to have the same morphology, biological activity and genetic identity as that of GSV recovered from insects. Using a cell culture system, several genetically pure GSV cloned isolates were acquired by plaque purification. The replication cycle of the virus including ultrastructural studies, viral DNA replication and virus specific protein synthesis were investigated in these two cell lines and interestingly, it was found that the exact same virus isolate had a different biology in the different permissive cell lines. Difference in the molecular biology of virus replication in these two cell lines was also observed. This suggests that host factors play an important role in determining the different host-viral interaction of the virus. In addition, biochemical properties of the GSV genome were investigated. The genome size was estimated using pulse-field gel electrophoresis to be 215-235 kb. CsCl-EtBr density gradient centrifugation indicated that GSV has a supercoiled, circular genome. Purified viral structural proteins, envelope proteins and glycoproteins were analyzed by SDS-PAGE and a total 16 of viral structural proteins were identified, three of them are glycosylated and five of these proteins are likely to be virus envelope or matrix components. Studies of GSV specific protein synthesis, DNA replication and transcription in the presence of specific inhibitors suggests that as with other most baculoviruses, GSV gene expression is temporally regulated and can be separated into early and late phases based on viral DNA replication and differential responses to the cellular RNA polymerase inhibitor, alpha-amanitin. That is, early gene expression is likely mediated by cellular RNA polymerase whereas a viral encoded or viral-modified host RNA polymerase likely mediates late viral gene expression. GSV persistent infection in vitro has been investigated using a persistently infected cell line, GSVP. GSV viral sequences and a very low level of infectious virus were detected from this normal-looking, persistently infected cell line. Co-culture of GSVP cells with another permissive cell line, Ld652Y, resulted in productive replication of GSV.

The 64.2-kilobase Bacillus subtilis (natto) plasmid pLS20 encodes functions required for conjugal transmission of plasmid DNA among a variety of Bacillus species. Localization of the transfer region on this plasmid was accomplished via the generation of transposon insertions and subsequent deletion analysis. Utilization of the temperature-sensitive transposition selection vector pTV1 allowed the isolation of a collection of pLS20::Tn917 derivatives. Insertion of Tn917 outside of the 10.8-kb BglII fragment of pLS20 did not affect the transfer abilities of the host cells harboring these plasmid mutants. Insertion of the transposon within the 10.8-kb BglII fragment led to the identification of two distinct regions of pLS20 involved in plasmid-mediated DNA exchange. Insertions into one portion of the 10.8-kb BglII fragment abolished the ability of pLS20 to transfer itself but did not affect the plasmid's ability to mobilize the tetracycline resistance plasmid pBC16. Mutants with insertions in another region of this fragment were almost completely transfer-deficient. Certain of the transposon-tagged derivatives, namely pLS20.3 and pLS20.4, incurred specific deletion following growth of host organisms for several generations in the presence of inhibitory levels of erythromycin. Deletion analyses revealed that loss of ca 15 kb of DNA encompassing of 10.8-kb BglII fragment results in total loss of conjugal transfer ability. Confirmation that this 10.8-kb BglII fragment contained all the DNA sequences necessary for conjugal DNA transfer came from cloning the fragment in the B. subtilis cloning vector pBD64. B. subtilis transformants carrying the pBD64 containing the cloned fragment were able to transfer the recombinant plasmid by conjugation at frequencies comparable to those obtained with B. subtilis donors carrying nondefective pLS20 or pLS20::Tn917 plasmids. The same region of pLS20 that is involved in transfer functions was also found to be involved in the suppression of motility of host organisms. Cells harboring pLS20 or the non-defective transposon-tagged derivatives appeared non-motile on 0.4% agar plates, and electron photomicrographs revealed the absence of flagella. However, cells cured of the plasmid or cells harboring the transfer-defective deletion derivatives pLS20.3$\Delta$1 and pLS20.4$\Delta$1 were flagellated and motile.

We exploited Pseudomonas cepacia's inability to utilize lactose to isolate strains in which transposable gene-activating elements from this bacterium activated the expression of the lac genes of Tn951 and permitted growth on lactose. Tn951 was introduced into P. cepacia on the broad-host-range plasmid pGC91.14. The lac genes of this tranposon were not expressed in P. cepacia. However, Lac$\sp{+}$ variants in which $\beta$-galactosidase was formed constitutively at high levels were isolated at a frequency of ca. 10$\sp{-6}$. RNA-DNA hybridization analysis indicated that the elevated levels of $\beta$-galactosidase in the Lac$\sp{+}$ variants were due to a marked increase in the levels of lac-specific mRNA. The plasmids of the three different Lac$\sp{+}$ strains were transferred by conjugation to Pseudomonas aeruginosa and a lac strain of Escherichia coli and the constitutive expressions of lacZ gene was observed in these strains. The plasmids were subjected to restriction enzyme analysis and found to contain additional DNA not present in pGC91.14. The insertions were due to the transposition of IS elements IS406, IS407 and IS415 from the P. cepacia chromosome to sites upstream of the lac structural genes of Tn951. Nucleotide sequence analysis of IS407 (1236 bp) confirmed the presence of features characteristics of IS-elements: terminal inverted repeat sequences and the generation of nucleotide sequence duplications at the target site. Sequence analysis of the lac promoter-operator region of pTGL67 (pGC91.14::IS407) placed the insertion site of IS407 within lacO. The insertion of IS407 separated $lacP\sb{\rm Tn951}$ from lacZ by ca. 1.3 kb. The nucleotide sequence of IS407 revealed the presence of an outwardly directed promoter proximate to the terminal inverted repeat adjacent to lacZ. Primer extension analysis of lacZ-specific mRNA isolated from E. coli determined that this promoter was operating in vivo and responsible for the constitutive expression of lacZ in this bacterium.

This study describes the nucleotide sequence analyses of three transposable elements, IS401, IS406, and IS408, from Pseudomonas cepacia. These were among a number of insertion sequences identified in this bacterium on the basis of their abilities to promote genomic rearrangements and to recruit foreign genes by replicon fusion and insertional activation. Nucleotide sequence analyses of IS401 (1316 bp), IS406 (1368 bp), and IS408 (ca. 2.8 kb), revealed features characteristic of insertion sequences: terminal inverted repeats; target site duplications; and open reading frames (ORFs) encoding putative transposases. IS406 inserted into the lac promoter of Tn951 (Tnlac) on pGC91.14, leading to the duplication 8 bp of its $-$10 region. The hybrid promoter formed may be responsible for increased lac gene expression in P. cepacia. IS406 had 41-bp terminal inverted repeats with 11 mismatches. The left terminal inverted repeat of IS406 contained a 12-bp motif also present at the ends of Tn2501, a transposon located downstream of the lacY gene in Tn951. Searches of the EMBL and GenBank databases revealed similarities, at the amino acid level, between the putative transposases of IS406 and of elements from Staphylococcus aureus, Rhizobium meliloti, and Thiobacillus ferroxidans. IS408 from pTGL68 inserted into the lacZ gene of Tn951, 1895 nucleotides upstream of the Tn951 lacY gene, generating 8-bp target sequence duplications. IS408 on pTGL68 had 49-bp terminal inverted repeats with 9 mismatches. IS408 was also present on the 170-kb cryptic plasmid, pTGL1, harbored by our laboratory strain. The bulk of the nucleotide sequence data for IS408 was obtained from analysis of the 4.1-kb HindIII fragment of pTGL5, a derivative of pTGL1 carrying a copy of IS401 next to IS408. Analysis of this fragment provided the complete nucleotide sequence of IS401 and showed that the element inserted 91 bp away from IS408, duplicating 3 bp of pTGL1 DNA. IS401 had 26-bp terminal inverted repeats with 2 mismatches. Plasmid pTGL5 carried two other copies of IS401. Their terminal inverted repeat sequences were identical to those of the copy of IS401 next to IS408. IS401 was closely related to elements from Pseudomonas syringae subsp. savastanoi and Escherichia coli.

Preparations of unrestricted DNA from Pseudomonas cepacia obtained by lysis of bacteria in agarose plugs contained small amounts of DNA with three different electrophoretic mobilities. The behavior the different species in CHEF gels corresponded to DNA fragments of 3.4, 2.5, and 0.9 Mb. Further analyses were consistent with the notion that the different species represented populations of linear DNAs derived by random double strand breakage of three distinct circular replicons. Treatment of DNA from strain 17616 with SwaI, PacI, and PmeI, restriction enzymes which recognize 8-bp sequences, generated four, six, and three fragments, respectively. Double digestion of the DNA with SwaI and PacI or with SwaI and PmeI generated fragments whose combined molecular weights was 6.8 Mb. Treatment of the DNA with AflII AseI, DraI, SpeI, and XbaI, restriction enzymes which recognize 6-bp sequences, generated about 50 fragments whose combined molecular weight also was about 6.8 Mb. The results indicate that the size of the genome of strain 17616 was 6.8 Mb. The combined molecular weights of the sets of SwaI, PacI, and PmeI fragments was 4.3, 5.9, and 5.9 Mb, respectively, reflecting the fact that the 2.5 Mb replicon contained no SwaI site and that the 0.9 Mb replicon lacked PacI and PmeI sites. The organization of SwaI, PacI, and PmeI fragments comprising each of the replicons was determined by Southern hybridization experiments using various junction fragments as probes and by further restriction analyses. Southern hybridization experiments using r-RNAs and various IS elements from P. cepacia as probes indicated that each of the replicons contained r-RNA genes and IS elements. A SwaI site was introduced into Tn5-751 and the resulting transposon was used to generate and map mutations related to amino acid biosynthesis and the degradation of various carbon sources. Derivatives of strain 17616 were identified in which large segments of the 2.5 and 0.9 Mb replicons had been deleted. The results provide a framework for further genetic analysis of P. cepacia.