Chloramphenicol-erythromycin resistance mutations in a 23S rRNA gene of Escherichia coli.

Two chloramphenicol resistance mutations were isolated in an Escherichia coli rRNA operon (rrnH) located on a multicopy plasmid. Both mutations also confer resistance to 14-atom lactone ring macrolide antibiotics, but they do not confer resistance to 16-atom lactone ring macrolide antibiotics or other inhibitors of the large ribosomal subunit. Classic genetic and recombinant DNA methods were used to map the two mutations to 154-base-pair regions of the 23S RNA genes. DNA sequencing of these regions revealed that chloramphenicol-erythromycin resistance results from a guanine-to-adenine transition at position 2057 of the 23S RNA genes of both independently isolated mutants. These mutations affect a region of 23S RNA strongly implicated in peptidyl transfer and known to interact with a variety of peptidyl transferase inhibitors.     

The nucleotide sequence of the gene coding for the 16S rRNA from the archaebacterium Halobacterium halobium

The complete 1473-bp sequence of the 16S rRNA gene from the archaebacterium Halobacterium halobium has been determined. Alignment with the sequences of the 16S rRNA gene from the archaebacteria Halobacterium volcanii and Halococcus morrhua reveals similar degrees of homology, about 88%. Differences in the primary structures of H. halobium and eubacterial (Escherichia coli) 16S rRNA or eukaryotic (Dictyostelium discoideum) 18S rRNA are much higher, corresponding to 63% and 56% homology, respectively. A comparison of the nucleotide sequence of the H. halobium 16S rRNA with those of its archaebacterial counterparts generally confirms a secondary structure model of the RNA contained in the small subunit of the archaebacterial ribosome.     

Molecular cloning of the cytochrome aa3 gene from the archaeon (Archaebacterium) Halobacterium halobium.

A novel aa3-type cytochrome oxidase from the extremely halophilic archaeon, Halobacterium halobium, differs significantly from those of other prokaryotic and eukaryotic cytochrome oxidases (Fujiwara, T., Fukumori, Y., and Yamanaka, T. (1989) J. Biochem. 105, 287-292). In the present study, we cloned and sequenced the gene which encodes the cytochrome aa3 by using the polymerase chain reaction methods. The deduced amino acid sequence of subunit I of H. halobium cytochrome aa3 was more similar to that of subunit I of the eukaryotic cytochrome (44%, maize mitochondria) than that of the cytochrome from other bacteria (36%, Paracoccus denitrificans). The consensus sequence in putative metal binding residues is well-conserved also in H. halobium cytochrome aa3.     

Putative promoter region of rRNA operon from archaebacterium Halobacterium halobium.

The 100 bp sequence from the beginning of the 16S rRNA gene of archaebacterium Halobacterium halobium and the adjacent 800 bp upstream sequence were determined. Four long (80 bp) direct repeats were found in the region preceeding the structural gene of the 16S rRNA. These repeats are proposed to constitute the promoter region of the rRNA operon of H. halobium.     

A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance.

Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archael vector.     

Genetic variability in Halobacterium halobium.

Halobacterium halobium exhibits an extraordinary degree of spontaneous variability. Mutants which are defective in the formation of gas vacuoles (vac) arise at a frequency of 10(-2). Other easily detectable phenotypes, like the synthesis of bacterioruberin (Rub) or the synthesis of retinal (Ret) and bacterio-opsin (Ops), the two components which form the purple membrane (Pum) of H. halobium, are lost at a frequency of about 10(-4). With the same frequency a mutant type appears which exhibits an extremely high variability in these phenotypes. With the exception of the ret mutants, all spontaneously arising mutants show alterations, i.e., insertions, rearrangements, or deletions, in the plasmid pHH1. It appears that the introduction of one insertion into pHH1 triggers further insertions, which makes the identification of relationships between phenotypic and genotypic alterations rather difficult. From the analysis of a large number of spontaneous vac mutants and their vac+ revertants it can be concluded that the formation of the gas vacuoles is determined or controlled by plasmid genes. No such conclusion is yet possible for the rub mutants, although all mutants of this type so far analyzed exhibit a defined insertion. pum mutants which have lost the capability of forming bacterio-opsin carry insertions in the plasmid which are distributed over a rather large region of the plasmid. No strains of H. halobium could be obtained which had lost plasmid pHH1 completely.     

Bacteriorhodopsin-mediated photophosphorylation in Halobacterium halobium.

The rate of halobacterial photophosphorylation was found to be a linear function of light intensity over a wide range (between 1 and 20 mW/cm2). At higher light intensities (above 25 mW/cm2) the ATP-synthesizing system itself limits the maximal rate of photophosphorylation. The optimal external pH range for this type of photophosphorylation is between pH 6.2 and 7.2 external. The photophosphorylation rate is directly proportional to the bacteriorhodopsin content of the cells. The quantum requirement for photophosphorylation was found to be 22 +/- 5 photons per ATP molecule synthesized. According to Mitchell's chemiosmotic hypothesis of energy coupling phosphorylation can be driven by a membrane potential or a pH gradient or a combination of both. From the results of experiments with drugs which abolish or reduce either one of the two components we conclude that the major driving force for photophosphorylation above an external pH value of 6.5 is the membrane potential, while at more acidic pH value the pH gradient becomes dominating. We did not observe a correlation between a transient alkalinization of the medium and ATP-synthesis upon illumination under certain conditions.     

Chemosensory responses of Halobacterium halobium.

Responses of Halobacterium halobium cells to chemical stimuli have been shown by a capillary technique. Cells were attacted by D-glucose and several amino acids and repelled by phenol. Certain chemicals, such as acetate, benzoate, indole, and NiSO4, that are known to act as repellents of Escherichia coli cells served as attractants for Halobacterium. In the presence of ethionine, sensitivity to attractants was reduced. Arsenate prevented the attraction by glucose without lowering the cellular adenosine 5'-triphosphate level. The ability for chemo-accumulation toward glucose and histidine was interfered with by the formation of photosensory systems. Light-induced motor responses and chemosensory behavior toward glucose and histidine became detectable in the late stationary growth phase only. The behavior toward acetate and indole was not connected to photobehavior in that way: both substances acted as attractants already in the late log phase. Inhibition of bacteriorhodopsin synthesis by L-nicotine allowed chemo-accumulation toward glucose and histidine already in the late logarithmic phase.     

Characterization of insertions affecting the expression of the bacterio-opsin gene in Halobacterium halobium.

We have determined the sequence of the inverted repeats and duplicated target DNA of the halobacterial insertion elements ISH2 (520 bp), ISH23 (900 bp) and ISH24 (3000 bp) associated with bacterio-opsin (bop) mutants. ISH2 has a perfect 19 bp inverted repeat (3,5), while both ISH23 and ISH24 have imperfect inverted repeats of 29 bp and 14 bp respectively. ISH23 was shown to be highly homologous to ISH50 (6). Variable lengths of duplicated target DNA are found when ISH2 and ISH23 (ISH50) transpose into different sites. A 550 bp DNA insert ("ISH25") reverts the Bop mutation caused by ISH24. "ISH25" lacks typical structural features of a transposable element. "ISH25" and ISH24 are found adjacent to each other upstream of the bop gene. An identical arrangement of "ISH25" and ISH24 is found in the cccDNA of H. halobium NRC817. Comparative sequence analysis of both areas suggests that the translocation of "ISH25" to the bop gene region occurred by a recombination event.     

Efficient transfection of the archaebacterium Halobacterium halobium.

We developed an efficient polyethylene glycol-mediated spheroplast transfection method for the extremely halophilic archaebacterium Halobacterium halobium. The 59-kilobase-pair linear phage phi H DNA molecule routinely produced between 5 X 10(6) and 2 X 10(7) transfectants per microgram of DNA. Between 0.5 and 1% of spheroplasts were transfected per microgram of luminal diameter H DNA. Under our conditions, survival and regeneration of H. halobium spheroplasts were also quite efficient, suggesting that this method will be useful for introducing other DNAs into these bacteria.     

Dynamic plasmid populations in Halobacterium halobium.

Deletion events occurring in the major 150-kilobase-pair (kb) plasmid pHH1 of the archaebacterium Halobacterium halobium were investigated. We found four deletion derivatives of pHH1 in gas-vacuole-negative mutants, two of which (pHH23) [65 kb] and pHH4 [36 kb]) we analyzed. Both plasmids incurred more than one deletion, leading to the fusion of noncontiguous pHH1 sequences. pHH23 and pHH4 overlapped by only 4 kb of DNA sequence. A DNA fragment derived from this region was used to monitor the production of further deletion variants of pHH4. A total of 25 single colonies were characterized, 23 of which contained various smaller pHH4 derivatives. Of the 25 colonies investigated, 2 had lost pHH4 entirely and contained only large (greater than or equal to 100-kb) minor covalently closed circular DNAs. One colony contained the 17-kb deletion derivative pHH6 without any residual pHH4. The sizes of the pHH4 deletion derivatives, produced during the development of a single colony, ranged from 5 to 20 kb. In five colonies, pHH4 was altered by the integration of an additional insertion element. These insertions, as well as copies of the various insertion elements already present in pHH4, presumably serve as hot spots for recombination events which result in deletions. A second enrichment procedure led to the identification of colonies containing either a 16-kb (pHH7) or a 5-kb (pHH8) deletion derivative of pHH4 as the major plasmid. pHH8, the smallest plasmid found, contained the 4 kb of unique DNA sequence shared by pHH23 and pHH4, as well as some flanking pHH4 sequences. This result indicates that the 4-kb region contains the necessary sequences for plasmid maintenance and replication.     

Light-driven proton translocations in Halobacterium halobium.

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.     

Methyl-accepting taxis proteins in Halobacterium halobium.

Methyl-accepting taxis proteins were identified and characterized in Halobacterium halobium, an archaebacterial species that is both chemotactic and phototactic. The data suggest direct involvement of methylation and demethylation in mechanisms of both chemotaxis and phototaxis and identify adaptation as the sensory process in which those reactions are likely to be involved. Analysis by electrophoresis and fluorography revealed methyl-accepting species, of apparent Mr between 90,000 and 135,000, that exhibited characteristics of sensory components. Those methyl-3H-labeled species were absent in a mutant blocked in taxis. Methylation of specific bands increased after positive chemostimuli and decreased after negative stimuli. Other methyl-3H-labeled bands, from 17 to 29 kd, exhibited features of biosynthetic intermediates, not of sensory components. Assay of rates of demethylation by measuring release of volatile forms of radiolabeled methyl groups revealed transient changes following chemo- or photostimuli that persisted for periods roughly equivalent to adaptation times. Negative chemostimuli induced increased rates of demethylation, as expected from fluorographic analysis, but positive chemostimuli also resulted in an increase. Photostimuli of either sign were followed by increases in rates of demethylation of shorter duration and lesser magnitude than chemostimuli-induced increases, a relationship that corresponded to differences in adaptation time.     

Characterization of the 23S and 5S rRNA genes of Coxiella burnetii and identification of an intervening sequence within the 23S rRNA gene.

Characterization of the rRNA operon from the obligate intracellular bacterium Coxiella burnetii has determined the order of the rRNA genes to be 16S-23S-5S. A 444-bp intervening sequence (IVS) was identified to interrupt the 23S rRNA gene beginning at position 1176. The IVS is predicted to form a stem-loop structure formed by flanking inverted repeats, and the absence of intact 23S rRNA molecules suggests that the loop is removed. An open reading frame in the IVS has been identified that shows 70% similarity at the amino acid level to IVS open reading frames characterized from four species of Leptospira.     

16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences analysis of the genus Myxococcus.

Phylogenetic relationships of the species belonging to the genus Myxococcus were elucidated based on the sequences of 16S rRNA genes and 16S-23S rRNA gene internal transcribed spacer (ITS) regions. The Myxococcus species were consequently classified into four distinct groups. The type strain of Myxococcus coralloides occupied an independent position (Group 1); it has been recently reclassified as Corallococcus coralloides. Group 2 comprised the type strains of both Myxococcus virescens and Myxococcus xanthus, and some strains assigned to Myxococcus flavescens. The type strain of M. flavescens was contained in Group 3 along with the strains of Myxococcus fulvus. Group 4 included the strains belonging to C. coralloides, M. fulvus, and M. stipitatus. The type strain of M. fulvus that was allocated outside Group 4 in the 16S rRNA gene tree belonged to Group 3 in the ITS tree. These results strongly suggest that the morphological characteristics of Myxococcus species are not consistent with the phylogenetic relationships. The Myxococcus species must therefore be redefined according to the phylogenetic relationships revealed in this study.