Arnold Brown

Partial Sequence Analysis of the 16S-rRNA of Legionellae: Taxonomic Implications

Summary The Legionellaceae are ubiquitous Gram-negative bacilli, many of which have been associated with human disease. Controversy exists as to the number of genera needed to correctly reflect genetic differences among members of this family. DNA-DNA hybridization, 5s-rRNA sequencing, and 16s-rRNA partial digest analysis have previously been used to address this issue. Our data obtained from the partial sequencing (approximately 350–450 bases) of the 16s-rRNA of 6 legionellae supports the proposed inclusion of these organisms with the “γ-purple bacteria”. The data also indicate that the 16s-rRNA of Legionella pneumophila and Tatlockia (Legionella) micdadei strain are only 90.6 ± 1.4% (88.2–91.7%) similar, compared to 94.4% similarity observed between the 16s-rRNA of Escherichia coli and Proteus vulgaris. Additional strains must be tested, however, before a conclusion can be reached regarding the taxonomic structure of this family of microorganisms.

A general method for the extraction of DNA from bacteria

Abstract A quick and easy method was developed for the extraction of DNA from Legionella pneumophila , which can also be used to extract DNA from a variety of other Gram-negative or Gram-positive bacteria. The method may be performed in 1.5 ml polypropylene tubes, using as little as a single colony, and is suited for the extraction of many samples simultaneously. The procedure uses 6 M NaI as a chaotropic agent and isopropanol to semi-selectively precipitate the DNA. Once the cells have been lysed, the procedure takes approximately 30 min to perform. Without further clean-up, the extracted DNA can be used for restriction endonuclease digestion or polymerase chain reaction amplification.

Tatlockia, a Genetically and Chemically Distinct Group of Bacteria. Proposal to Transfer Legionella maceachernii (Brenner et al.) to the Genus Tatlockia, as Tatlockia maceachernii comb. nov.

Summary In addition to the previously reported DNA hybridization and base composition data, comparison of the nucleotide sequence of 16S-ribosomal RNA fragments and the total cellular carbohydrate content of Tatlockia micdadei and Legionella pneumophila strains strongly support their separation into different genera. In the present study Legionella maceachernii was found to be very similar to T. micdadei , and like T. micdadei , it was easily distinguished from the other legionellae. Based on the accumulated phenotypic and genotypic data Legionella maceachernii should be transferred to the genus Tatlockia , as Tatlockia maceachernii comb. nov.

Legionella pneumophila has two 60-kilodalton heat-shock proteins

Legionella pneumophila is a thermotolerant bacterium. To learn more about the thermal adaptation of this organism, we studied the properties of the Legionella 60-kDa heat-shock protein (MopA, GroEL-analog, HtpB, Lp-Hsp60) in L. pneumophila and in an Escherichia coli strain containing the cloned gene. Lp-Hsp60 was found in both cytosol and membrane fractions; however, Lp-Hsp60 in the membrane fraction of L. pneumophila was slightly larger than Lp-Hsp60 in the cytosol. In contrast, both membrane-associated and cytosolic Lp-Hsp60 in the E. coli clone were similar in size to the smaller cytosolic Lp-Hsp60 of L. pneumophila. While peptide mapping suggests there are differences between the two proteins, the larger membrane-associated Lp-Hsp60 and the smaller cytosolic LP-Hsp60 shared Legionella-specific and E. coli GroEL cross-reacting epitopes, and the sequence of their first 20 N-terminal amino acids was identical. Further, Southern blot analysis of EcoRI-digested chromosomal DNA from several strains of L. pneumophila showed two fragments reacting with an htpAB-operon probe. In summary, L. pneumophila contains two Hsp60 proteins, and possibly two hsp60 genes.

Bacterial growth and the concentrations of cyclic nucleotides inLegionella pneumophila cultures

The growth kinetics ofLegionella pneumophila, and the rates of DNA and RNA synthesis in this organism, are qualitatively similar to that of other gram negative organisms of clinical importance. After a brief stationary phase, culture viability is rapidly lost. This loss of viability is not associated with detectable cell lysis or with significant breakdown of cellular DNA; it is, however, preceded by the rapid breakdown of approximately 50% of the cumulatively labeled RNA. During logarithmic growth in buffered yeast extract broth, bacterial cells secreted into the medium large amounts of cyclic GMP at both 37°C and room temperature, while those grown in a buffered synthetic broth did not. Cyclic AMP increased moderately during stationary phase at 37°C and room temperature in both media. Neither added cyclic AMP nor added cyclic GMP influenced the rate of growth ofL. pneumophila in either broth, with or without 1-methyl-3-isobutyl-xanthine, a phosphodiestease inhibitor. Dibutyrul cyclic AMP and 8-bromo cyclic GMP individually or in combination also had no effect on growth.

Dam-like methylation in legionellae.

Abstract. The chromosomal DNA of a variety of legionellae, including clinical, environmental, and plasmid-containing strains, was examined for evidence of Dam-like methylation. It was found that Dam-like methylation was present in all the strains examined regardless of their origin or plasmid content.

A procedure for the preparation of rRNA from Legionellaceae for use in 16S-rRNA sequencing

Abstract rRNA-sequence comparison has become a commonly used method in bacterial taxonomy. To obtain intact rRNA from legioneallae for use in sequencing a simple enzymatic procedure was developed with requires no specialized equipment for release of RNA and no ultracentrifugation for its isolation. RNase inhibitors were included at each step to avoid RNA degradation. The procedure of Lane et al. was modified to decrease frequent sequence anomalies. These changes included optimization of incubation temperatures, enzyme: substratum ratios, deoxy: dideoxyribonucleotide ratios and the use of terminal deoxynucleotidyl transferase in the chase. The procedure allows the identification of 250 nucleotides from each primer reaction.

Altered rate of synthesis of specific peptides in the legionellae in response to growth temperature

The effect of a sharp temperature rise (30° to 42°C) on the rate of peptide synthesis inLegionella pneumophila (three strains),Tatlockia micdadei (Tatlock), andFluoribacter bozemanae (WIGA) was examined. Most striking was an increase in the rate of synthesis of 4–5 peptides of approximate molecular weight 85,000 daltons (85k), 78k, 70k, and either 60k (only in theL. pneumophila strains and WIGA) or 62.5k, and 40k (only in Tatlock). The response required new mRNA synthesis. In addition, the heat shock response of these organisms continued as long as the high temperature condition persisted (at least 24 h) and for at least 1 h after temperature downshift (to 30°C).