FliH and fliI of Borrelia burgdorferi are similar to flagellar and virulence factor export proteins of other bacteria.

Two motility genes (fliH and fliI) of the Lyme disease spirochete Borrelia burgdorferi were cloned, physically mapped and sequenced, FliH and FliI showed extensive homology to the proteins involved in the export of flagellar components and to virulence factors found in both animal and plant bacterial pathogens. The results suggest that the flagellar apparatus and associated protein export pathway are well conserved in evolution.

Cloning and analysis of the leuB gene of Leptospira interrogans serovar pomona.

The leuB gene of Leptospira interrogans serovar pomona strain kenniwicki has been cloned on a 9.5 kb plasmid, pWVL1, by complementation of Escherichia coli leuB mutants. Subcloning and Tn5 mutagenesis showed that the region required for complementation was approximately 1.2 kb in length. Enzyme assays showed that the product of the cloned gene was a beta-isopropylmalate dehydrogenase. Defects in the leuA, leuC and leuD genes of E. coli were not complemented by pWVL1. The nucleotide sequence of the leuB-complementing region and surrounding DNA has been determined. Three open reading frames were found which encode proteins of 40.9, 38.8 and 15 kDa. Analysis of subclones containing nucleotide deletions of varying sizes showed that only the 38.8 kDa protein was necessary to obtain complementation of E. coli leuB mutations. The PIR data base was searched and the enzyme 3-isopropylmalate dehydrogenase from six different micro-organisms was found to share significant amino acid sequence similarity (43-57%) with the 38.8 kDa L. interrogans leuB gene product. The organization of the leucine biosynthetic genes in L. interrogans differs from that found in E. coli, Salmonella typhimurium and Bacillus subtilis.

Molecular genetic analysis of a class B periplasmic-flagellum gene of Treponema phagedenis.

Treponema phagedenis is a host-associated spirochete with multiple polypeptides making up its periplasmic flagella (PFs). Each PF has a 39-kDa polypeptide making up the sheath (class A PF polypeptide) and two to four antigenically similar 33- to 34-kDa polypeptide species making up the core (class B PF polypeptides). A genetic analysis of the PF-deficient mutants T-40 and T-55 has shown that the PFs are involved in motility. To better understand the synthesis and assembly of these complex organelles and to compare the PF genes with those of other spirochetes, we cloned and characterized the T. phagedenis flaB2 gene, which encodes one class B polypeptide. The flaB2 gene consists of an open reading frame of 858 nucleotides capable of encoding a protein of 31.5 kDa. The predicted amino acid sequence of the FlaB2 polypeptide was 92% identical to that of T. pallidum FlaB2, with a 76% identity at the nucleotide level. These results confirm previous immunological and N-terminal-sequence analyses which suggested that the PF genes are well conserved in the spirochetes. Primer extension analysis of T. phagedenis flaB2 indicated that the start site of transcription was 127 nucleotides upstream from the ATG initiation codon. Preceding the start site is a DNA sequence similar to the sigma 28 consensus promoter sequence commonly found associated with motility genes. Northern (RNA) blots probed with a segment of flaB2 DNA revealed a 1,000-nucleotide monocistronic transcript in the wild type and in PF-deficient mutants T-40 and T-55. DNA sequencing of most of the flaB2 gene of the mutants revealed no differences from the wild-type gene. Because the mutants fail to synthesize detectable class B PF polypeptides yet synthesize extensive amounts of flaB2 mRNA, PF synthesis in T. phagedenis is likely to involve regulation at the translational level.

Genetic approaches to cell biology and metabolism of spirochetes.

Genetic analysis and methodology have only comparatively recently been applied to the study of spirochetes. Although genetic transfer procedures for spirochetes are not widely available, there are several examples of progress in genetic analysis of spirochetes by other approaches. Some examples of these approaches are the following. 1) Genes for synthetic pathways in Treponema and Leptospira have been cloned by complementation of Escherichia coli serving as plasmid hosts. 2) The OspA protein of Borrelia burgdorferi has been overexpressed in E. coli without the signal peptide; the recombinant product has been suitable for circular dichroism as well as other biochemical analyses. 3) The heat shock proteins of B. burgdorferi are homologous to heat shock proteins of E. coli. 4) Enzyme activity profiles of B. burgdorferi and other spirochetes show strain heterogeneity and also indicate which biosynthetic and enzymatic activities are conserved within different spirochetes. 5) The gene organization of rRNA genes have revealed differences between spirochetes and other types of bacteria.

Treponema phagedenis encodes and expresses homologs of the Treponema pallidum TmpA and TmpB proteins.

We cloned and sequenced the genes from Treponema phagedenis Kazan 5 encoding proteins homologous to the TmpA and TmpB proteins of Treponema pallidum subsp. pallidum Nichols (hereafter referred to as T. pallidum). Although previous reports suggested that the TmpA and TmpB proteins were specific for T. pallidum, we found that homologs for both were expressed in T. phagedenis Kazan 5 and Reiter. The TmpA protein from T. phagedenis contained the consensus sequence that bacterial lipoproteins require for posttranslational modification and subsequent proteolytic cleavage by signal peptidase II and showed 42% amino acid sequence identity with the TmpA protein from T. pallidum. The TmpB proteins of T. phagedenis and T. pallidum had similar amino acid sequences at their amino- and carboxy-terminal ends. The central portions of both of these proteins contained four repeats of the amino acid sequence EAARKAAE. The TmpB protein from T. phagedenis had an additional amino acid sequence repeat (consensus sequence KAAKE/D) that was not found in the TmpB protein from T. pallidum; this repeat was most remarkable, as it occurred 17 times in succession. These repeated amino acid sequences probably created an extensive alpha-helix region within the TmpB proteins. As with T. pallidum, the stop codon of the T. phagedenis tmpA gene overlapped the start codon of its tmpB gene. Northern blot analysis showed that the T. phagedenis tmpA and tmpB genes were probably transcribed into a single 2.5-kb mRNA molecule. Western blot (immunoblot) analysis demonstrated that both proteins were expressed by T. phagedenis. The high degree of amino acid sequence conservation seen with the TmpA and TmpB proteins from two different Treponema species suggests that they may play crucial roles in the biology of these organisms.

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.

Analysis of cloned DNA from Leptospira biflexa serovar patoc which complements a deletion of the Escherichia coli trpE gene.

To analyze the cloned region of the chromosome of the spirochete Leptospira biflexa serovar patoc which complemented a defect in the trpE gene of Escherichia coli, we performed a series of experiments involving subcloning, transposon mutagenesis, and maxicells. By subcloning into pBR322 we were able to isolate the Leptospira genes on a 9.7-kilobase pair plasmid (pYC6). Transposon mutagenesis with Tn5 identified a 2.8-kilobase pair region of this plasmid as being necessary to complement a trpE deletion mutation in E. coli. Transformation of plasmid pYC6 into E. coli cells deleted for trpE and the proximal end of trpD showed that the Leptospira DNA complemented both defects. A maxicell analysis of various transposon-induced mutations of the plasmid revealed that three proteins (53.5, 23.6, and 22 kilodaltons) were encoded by the 2.8-kilobase pair region of the Leptospira genome. Two different promoters controlled the production of these three proteins.

Cloning of a gene required for tryptophan biosynthesis from Leptospira biflexa serovar patoc into Escherichia coli.

A clone bank, consisting of approx. 8100 colonies, has been created for the spirochete Leptospira biflexa serovar patoc in Escherichia coli using pBR322 as the vector. One of these clones contains the genetic information needed to complement a defect in the trpE gene of E. coli. The information resides on a 20.5-kb plasmid designated pYC1, which carries a 16-kb insert consisting of three HindIII fragments. It does not complement defects in other genes needed for the biosynthesis of tryptophan in E. coli.

Phenotypic transformation of the host cell enhances polyoma pseudovirion formation.

Phenotypic transformation of the host cell affected the formation of polyoma pseuodovirions. Polyoma virus infection of various transformed derivatives of mouse 3T3 cells resulted in the formation of predominantly pseudovirions, whereas infection of mouse 3T3 cells produced mainly polyoma virus. The effect that transformation of the host cell had on polyoma pseudovirus formation was further demonstrated by using phenotypic revertants isolated from some of the transformed cell lines. The revertants were characterized by their morphology, saturation densities, and colony-forming ability in methylcellulose suspension. By these criteria they were distinct from their transformed parents and similar to 3T3 cells. After infection, the revertants produced predominantly polyoma virus and few pseudovirus. Thus, for the cell lines used in this study, phenotypic transformation enhanced the formationof polyoma pseudovirions.

Biochemical and biophysical properties of hyphomicrobium bacteriophage hyphi30.

Hyphomicrobium bacteriophage Hyphi30 and its nucleic acid were studied to determine some of their biochemical and biophysical properties. The molecular weight of the phage is 55.4 x 10(6), and its buoyant density is 1.508 g/ml. The nucleic acid of Hyphi30 is linear, double-stranded DNA with a molecular weight of 29.7 x 10(6). The guanine-plus-cytosine content of the DNA was 62% as determined from its melting temperature and buoyant density.

Polyoma pseudovirions. II. Influence of host cell on pseudovirus production.

The type of host cell influenced the relative amounts of pseudovirions and polyoma virions produced. The infection of primary mouse embryo cells resulted in the production of particles that were predominantly pseudovirions. Infection of baby mouse kidney or 3T3D cells yielded mainly infectious polyoma virus. The length of time that infection was allowed to continue also affected the amount of pseudovirions relative to polyoma virions. The longer the viral infection was allowed to proceed, the greater the quantity of pseudovirions produced. Pseudovirion production could be correlated with the fragmentation of host cell DNA to a size of approximately 3 x 10(6) daltons. The fragmentation of host cell DNA was much more extensive in primary mouse embryo cells than in the other cell types.

Polyoma pseudovirions. I. Sequence of events in primary mouse embryo cells leading to pseudovirus production.

The relationship of the intracellular events leading to the production of polyoma pseudovirions in primary mouse embryo cells has been investigated. Replication of polyoma deoxyribonucleic acid (DNA) began 18 hr after infection. Assembly of viral capsid protein occurred 12 hr later. Intracellular fragments of host cell DNA, of the size found in pseudovirions, were first detected 36 hr after infection. The amount of intracellular 14S host DNA that was produced during infection was seven times greater than the amount of polyoma DNA synthesized. The relative pool sizes of polyoma DNA and 14S DNA at the time of virus assembly may dictate the amounts of polyoma virus and pseudovirus produced.

Comparison of Bacillus cereus bacteriophages CP-51 and CP-53.

Transducing bacteriophages CP-51 and CP-53 were compared. Unlike CP-51, CP-53 appeared to be a lysogenizing phage. CP-51 gave greater frequencies of co-transduction for linked markers than did CP-53. CP-51 was found to be a larger phage which carried more deoxyribonucleic acid (DNA) than CP-53. CP-51 DNA contained about 43% guanine plus cytosine and in addition contained 5-hydroxymethyluracil in place of thymine. CP-53 DNA contained no unusual bases; its guanine plus cytosine content was 37%.

Transduction in Bacillus cereus by each of two bacteriophages.

The ability of phage CP-51 to mediate transduction both homologously and heterologously in some of its hosts was investigated. CP-51 was shown to transduce Bacillus cereus strains 6464, 9139, and T in addition to 569 which was reported earlier from this laboratory. Furthermore, CP-51 grown on B. thuringiensis was shown to transduce some mutants of B. cereus. During this investigation, a second transducing phage for B. cereus 569 was isolated from lysates of phage CP-51 grown on B. cereus 6464. This phage, designated CP-53, is carried by wild-type strain 6464 possibly as prophage. All auxotrophic mutants of B. cereus 569 tested, those requiring tryptophan, histidine, methionine, and leucine, were transduced to prototrophy by CP-53. Electron micrographs of the two phages revealed that CP-51 has a tail core surrounded by a contractile sheath and CP-53 has a long flexible tail without a contractile sheath. CP-53 is stable in the cold, whereas CP-51 is rapidly inactivated at 4 C.