Integration host factor interactions with Neisseria gene sequences: correlation between predicted binding sites and in vitro binding of Neisseria -derived IHF protein.

Putative integration host factor (IHF) binding sites are frequently being identified in Neisseria gene sequences on the basis of similarity to a degenerate Escherichia coli -derived consensus binding sequence. In this report, three different Neisseria genetic systems that contain predicted IHF binding sites were assessed for IHF binding through gel retardation analysis. The results show a positive correlation between the identification of a predicted Neisseria IHF binding site and in vitro binding of Neisseria -derived IHF protein.

Transduction by phiBB-1, a bacteriophage of Borrelia burgdorferi.

We previously described a bacteriophage of the Lyme disease agent Borrelia burgdorferi designated phiBB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus Borrelia. To demonstrate the ability of phiBB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B. burgdorferi CA-11.2A cells. The kan cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the kan cassette was packaged by phiBB-1 released from this B. burgdorferi strain. phiBB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B. burgdorferi. This is the first direct evidence of a mechanism for lateral gene transfer in B. burgdorferi.

Characterization of Borrelia burgdorferi BlyA and BlyB proteins: a prophage-encoded holin-like system.

The conserved cp32 plasmid family of Borrelia burgdorferi was recently shown to be packaged into a bacteriophage particle (C. H. Eggers and D. S. Samuels, J. Bacteriol. 181:7308-7313, 1999). This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system. Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage. An Escherichia coli mutant containing the blyAB locus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction of sheA by blyAB expression was responsible for the hemolytic activity observed previously. Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins. Consistent with holin characteristics, subcellular localization studies with E. coli and B. burgdorferi indicated that BlyA is solely membrane associated and that BlyB is a soluble protein. Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene. Finally, induction of the cp32 prophage in B. burgdorferi dramatically stimulated blyAB expression. Our results provide the first evidence of a prophage-encoded holin within Borrelia.

Bacteriophages of spirochetes.

Historically, a number of bacteriophage-like particles have been observed in association with members of the bacterial order Spirochetales, the spirochetes. In the last decade, several spirochete bacteriophages have been isolated and characterized at the molecular level. We have recently characterized a bacteriophage of the Lyme disease agent, Borrelia burgdorferi, which we have designated phiBB-1. Here we review the history of the association between the spirochetes and their bacteriophages, with a particular emphasis on phiBB-1 and its prophage, the 32-kb circular plasmid family of B. burgdorferi.

Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions.

Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall. In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella. B. burgdorferi has a bundle of 7-11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology. Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were nonmotile, but were rod-shaped. Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rod-shaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.

Disruption of the Borrelia burgdorferi gac gene, encoding the naturally synthesized GyrA C-terminal domain.

The C-terminal domain of the A subunit of DNA gyrase, which we term Gac, is naturally synthesized in Borrelia burgdorferi as an abundant DNA-binding protein. Full-length GyrA, which includes the C-terminal domain, is also synthesized by the spirochete and functions as a subunit of DNA gyrase. We have disrupted synthesis of Gac as an independent protein and demonstrated that it is not essential for growth in a coumarin-resistant background. We detected no alterations in DNA maintenance, condensation, or topology in B. burgdorferi lacking this small DNA-binding protein.

Molecular evidence for a new bacteriophage of Borrelia burgdorferi.

We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a Borrelia burgdorferi culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the B. burgdorferi genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some B. burgdorferi strains and is inducible to higher levels with 10 microg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml(-1). In addition, the prophage can be induced with MNNG from some Borrelia isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with B. burgdorferi CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of B. burgdorferi.

Natural synthesis of a DNA-binding protein from the C-terminal domain of DNA gyrase A in Borrelia burgdorferi.

We have identified a 34 kDa DNA-binding protein with an HU-like activity in the Lyme disease spirochete Borrelia burgdorferi. The 34 kDa protein is translated from an abundant transcript initiated within the gene encoding the A subunit of DNA gyrase. Translation of the 34 kDa protein starts at residue 499 of GyrA and proceeds in the same reading frame as full-length GyrA, resulting in an N-terminal-truncated protein. The 34 kDa GyrA C-terminal domain, although not homologous, substitutes for HU in the formation of the Type 1 complex in Mu transposition, and complements an HU-deficient strain of Escherichia coli. This is the first example of constitutive expression of two gene products in the same open reading frame from a single gene in a prokaryotic cellular system.

Mutations in Bartonella bacilliformis gyrB confer resistance to coumermycin A1.

This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of approximately 77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cour gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cour mutants of B. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124-->Ser), Arg184-->Gln, and Thr214-->Ala or Thr214-->Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes from Borrelia burgdorferi, E. coli, Staphylococcus aureus, and Haloferax sp. The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.

Phosphorylation of serine residues in the N-terminal domains of eukaryotic type I topoisomerases.

Eukaryotic topoisomerase I polypeptides can be partitioned into four structural domains. The function of the N-terminal domain, which is a target for serine-specific phosphorylation, has not been fully defined. The number of serine residues in the N-terminal domain of topoisomerase I from different species is inversely proportional to the number of charged amino acids in this region of the protein. The significance of this correlation is discussed in terms of a possible role for serine-specific phosphorylation in the activity of the enzyme.

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene.

The 26 to 28kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse-tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.

Cloning and expression of the Borrelia burgdorferi lon gene.

The ATP-dependent protease Lon (La) of Escherichia coli degrades abnormal proteins and is involved in the regulation of capsular polysaccharide synthesis. In addition, mutations in the E. coli lon gene suppress temperature-sensitive mutations in other genes. The lon gene of Borrelia burgdorferi, encoding a homolog of the Lon protease, has been cloned and sequenced. The gene encodes a protein of 806 amino acids. The deduced amino acid sequence of the B. burgdorferi Lon protease shares substantial sequence identity with those of other known Lon proteases. The transcription start point of the B. burgdorferi lon gene was identified by primer extension analysis and the potential promoter did not show similarities to the consensus heat-shock promoter in E. coli. The 5'-end of the B. burgdorferi lon gene appears to suppress the temperature-sensitive phenotype of an E. coli lpxA mutant.

Integration host factor is a transcriptional cofactor of pilE in Neisseria gonorrhoeae.

Integration host factor (IHF) is a small, heterodimeric DNA-binding protein with pleiotropic function. IHF was purified to apparent homogeneity from Neisseria gonorrhoeae. Gel-retardation assays demonstrated binding of IHF to the pilE promoter region. The IHF-binding site was identified by DNase I protection assays and mapped proximal to three previously defined pilE promoters. Removal of the putative IHF-binding domain from pilE promoter DNA negated retardation of the DNA fragment when assessed by gel-shift analysis. Kleinschmidt electron microscopy showed pronounced kinking of pilE promoter DNA following incubation with IHF. Isogenic N. gonorrhoeae strains were constructed that contained either a wild-type pilE locus or a deleted pilE locus where the IHF-binding domain was removed. Primer-extension analysis and Northern blotting of total gonococcal RNA showed that in the absence of IHF binding at the pilE promoter, transcription was reduced 10-fold. Together, these data indicate that IHF is a transcriptional co-activator of pilE.

Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi.

Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward facile genetic manipulation of this pathogenic spirochete, we have investigated gene inactivation by allelic exchange using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We have transformed B. burgdorferi by electroporation with a linear fragment of DNA in which this selectable marker was flanked by sequences from a native borrelial 26-kb circular plasmid. We have identified coumermycin A1-resistant transformants in which gyrB had interrupted the targeted site on the 26-kb plasmid via homologous recombination with the flanking sequences. Antibiotic resistance conferred by the mutated gyrB gene on the plasmid is dominant, and transformed spirochetes carrying this plasmid do not contain any unaltered copies of the plasmid. Coumermycin A1 resistance can be transferred to naive B. burgdorferi by transformation with borrelial plasmid DNA from the initial transformants. This work represents the first example of a directed mutation in B. burgdorferi whereby a large segment of heterologous DNA (gyrB) has been inserted via homologous recombination with flanking sequences, thus demonstrating the feasibility of specific gene inactivation by allelic exchange.

Isolation of Borrelia burgdorferi genes encoding homologues of DNA-binding protein HU and ribosomal protein S20.

Linear DNA with covalently closed ends is the predominant form of DNA in the spirochaete Borrelia burgdorferi. All bacteria examined to date have small DNA-binding proteins related to the Escherichia coli IHF and HU proteins that appear to play roles in DNA compaction and replication, but such proteins had not been isolated from bacteria with linear genomes. We found a single gene in B. burgdorferi (named hbb) whose product (named Hbb) complements the defects for gamma DNA packaging found in E. coli strains mutant in the genes for IHF and HU. The sequence of the predicted B. burgdorferi protein is similar to those of HU and IHF-like proteins in other bacteria. The gene appears to be in an operon with the order rpsT-hbb-orfH, where the rpsT gene is a homologue of the E. coli gene encoding ribosomal protein S20 and the orfH gene encodes a protein of unknown function. This operon is located upstream of the previously identified B. burgdorferi rho homologue.

Analysis of linear plasmid dimers in Borrelia burgdorferi sensu lato isolates: implications concerning the potential mechanism of linear plasmid replication.

The Borrelia genome is composed of a linear chromosome and a number of variable circular and linear plasmids. Atypically large linear plasmids of 92 to 105 kb have been identified in several Borrelia burgdorferi sensu lato isolates and characterized. These plasmids carry the p27 and ospAB genes, which in other isolates reside on a 50-kb plasmid. Here we demonstrate that these plasmids are dimers of the 50-kb ospAB plasmid (pAB50). The 94-kb plasmid from isolate VS116, pVS94, was an exception and did not hybridize with any plasmid gene probes. When this plasmid was used as a probe, homologous sequences in other isolates were not detected, suggesting that it is unique to isolate VS116. These analyses provide insight into the mechanism of linear plasmid replication and the mechanisms by which plasmid variability can arise.

Proteins binding to the promoter region of the operon encoding the major outer surface proteins OspA and OspB of Borrelia burgdorferi.

The synthesis of the major outer surface proteins OspA and OspB in Borrelia burgdorferi varies among strains and during in vitro cultivation. We examined B. burgdorferi CA-11.2A for the presence of proteins that bind to the ospAB operon promoter region. Three major DNA-protein complexes were detected using a mobility shift assay; one of these complexes was due to sequence-specific binding. These proteins may be involved in the regulation of ospAB transcription and the pathogenesis of Lyme disease.

Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB.

No useful method to genetically manipulate Borrelia burgdorferi, the causative agent of Lyme disease, has been developed previously. We have used resistance to the coumarin antibiotic coumermycin A1, an inhibitor of DNA gyrase, as a genetic marker to monitor the transformation of B. burgdorferi by electroporation. Introduction of site-directed mutations into the gyrB gene demonstrated that transformation was successful, provided evidence that homologous recombination occurs on the chromosome, and established that mutations at Arg-133 of DNA gyrase B confer coumermycin A1 resistance in B. burgdorferi. The coumermycin A1-resistant gyrB marker and genetic transformation can now be applied toward dissecting the physiology and pathogenesis of the Lyme disease agent on a molecular genetic level.

Phosphorylation of DNA topoisomerase I is increased during the response of mammalian cells to mitogenic stimuli.

DNA topoisomerase I is phosphorylated after mitogenic stimulation of 3T3-L1 mouse fibroblasts by 12-O-tetradecanoylphorbol 13-acetate (TPA), a phorbol ester tumor promoter. In vivo labeling with [32P]orthophosphate and immunoprecipitation with an anti-DNA topoisomerase I antibody has demonstrated an increase in the phosphorylation of DNA topoisomerase I in Swiss/3T3 mouse fibroblasts treated with epidermal growth factor (EGF) and H35 rat hepatoma cells treated with insulin. The only phosphorylated form of DNA topoisomerase I observed was the 100-kDa protein Digestion of DNA topoisomerase I with trypsin revealed two phosphopeptides. In addition, VT-1, a non-responsive genetic variant of 3T3-L1, and the DNA topoisomerase I inhibitor camptothecin were used to further study TPA-induced DNA topoisomerase I phosphorylation. Our results indicate that the phosphorylation of DNA topoisomerase I may be an ubiquitous response of cultured mammalian cells to mitogenic agents, even in the absence of DNA replication.