Point mutations that lock Salmonella typhimurium flagellar filaments in the straight right-handed and left-handed forms and their relation to filament superhelicity.

We have determined the nucleotide sequence of two mutant and the parent fliC genes, encoding the protein flagellin (serotype i), of Salmonella typhimurium. The flagellar filaments of the two mutants, SJW1655 and SJW1660, are locked in the straight-right-handed (R) and straight-left-handed (L) conformations, respectively. Their normal, wild-type, parent strain is SJW1103. These mutant strains differ from the wild-type by only one base-pair: the mutation of SJW1655 occurs at nucleotide 1346 in the flagellin gene, changing a C.G pair to T.A (alanine 449 to valine). The mutation of SJW1660 occurs at nucleotide 1277, changing a G.C pair to C.G (glycine 426 to alanine). The resulting amino acid substitutions are near the C terminus predicted to form an alpha-helical coiled coil. The region contains six heptad repeats. Similar alpha-helical segments (three and four repeats long) are present near the N terminus. Alignment of the 17 flagellin sequences available to date confirms the generality of these segments. The mutations are within that portion of the sequence assigned, by proteolytic cleavage, to the middle flagellin domain whose length corresponds to the six heptad repeats found in the sequence (approximately 50 A). We have shown that these mutations are the sole cause of the straight phenotype by replacing the mutated segments with a wild-type one and restoring both superhelicity and motility.

DNA probes for Mycoplasma gallisepticum and Mycoplasma synoviae: application in experimentally infected chickens.

DNA probes specific for Mycoplasma gallisepticum and M. synoviae were selected from genomic libraries prepared in the pUC13 vector. The probes hybridized with the DNA of a wide spectrum of strains within each homologous species, but did not react with the heterologous species or with DNA from any other avian mycoplasma or bacteria tested. Experimental infection and contact exposure of chickens to M. gallisepticum served as models to test the effectiveness of the DNA probe in diagnosis as compared with serological and culture detection methods carried out in parallel. A correlation was generally found between the level of M. gallisepticum in tracheal swabs and the effectiveness of the probe, although a predictably reactive level of mycoplasmas was not always detected. Treatment of clinical specimens with acetylcysteine to disrupt mucus improved the detection rate. Dot-blot hybridization with probe pMG4 enabled positive identification of M. gallisepticum at an early stage of infection, prior to the development of a serological response in the infected chicken. Results are obtainable within 4 days of sampling, much more rapidly than culture, and also in clinical specimens from which mycoplasma isolation is impossible, such as carcasses. The results indicate that the use of DNA probes for the early and rapid detection of M. gallisepticum infection is feasible; a development which can replace laborious culture techniques and less effective serological methods, and thus reduce the time required for diagnosis.

Chemiprobe, a nonradioactive system for labeling nucleic acid. Principles and applications.

The Chemiprobe Kit provides a complete system for nonradioactive labeling of DNA probes and their detection in hybridization studies. The system is highly sensitive, permitting the detection of 0.2-0.4 pg DNA which allows detection of a single gene sequence in 0.5-1 microgram of bacterial DNA or in 3-5 micrograms of mammalian DNA. In this paper the authors show that the rRNA genes of M. capricolum can be detected by using only 50 ng/ml of sulfonated probe cloned from another mycoplasma, M. pneumoniae. The Chemiprobe system has been successfully used in the detection of the single copy human gene for glucocerobrosidase from total embryonic DNA by hybridization to a specific sulfonated cDNA. 5 x 10(4) M. pneumoniae cells can be detected either free or mixed with sputum using a standard dot blot technique: mycoplasma cells were lysed by a mucolytic agent, denaturated by NaOH, immobilized on a nylon membrane filter, and then hybridized with pPN4, a plasmid DNA probe specific for M. pneumoniae. The resulting hybrids were then detected by the standard Chemiprobe procedure. A new kit based on the Chemiprobe system has been designed especially for the detection of mycoplasmas in tissue culture. This kit has been tested on 70 random samples collected from tissue culture fluids from 11 different sources. Of these, 42 were found to be contaminated by the Chemiprobe procedure, whereas 41 were found to be contaminated by classical microbiological methods. No false negatives were found.

Promoter of the Mycoplasma pneumoniae rRNA operon.

RNA transcripts starting from the 5' end of the single Mycoplasma pneumoniae rRNA operon were analyzed by several methods. By primer extension analysis a start site was found 62 nucleotides upstream from the start site of the 16S rRNA. This site was preceded by a putative Pribnow box; however, a defined -35 recognition region was absent. The cloned rRNA operon was transcribed in vitro by using purified RNA polymerase of Escherichia coli. A single start site could be demonstrated within a few nucleotides of the start site found by primer extension analysis of M. pneumoniae transcripts. When fragments from the cloned operon were used as hybridization probes, S1 nuclease mapping yielded a single transcript extending approximately 193 nucleotides upstream from the 16S rRNA start site. The region surrounding this endpoint did not resemble any known promoter sequence. Dot blot hybridization of M. pneumoniae RNA to three oligonucleotides consisting of nucleotides -5 to -21, -38 to -54, and -112 to -132 (from the start of the 16S rRNA gene) indicated that most rRNA transcripts were processed at the stem site preceding the 16S rRNA gene. The majority of the longer precursor transcripts, extending beyond this point, did not extend further upstream to an oligonucleotide consisting of nucleotides -112 to -132. It was concluded that transcription of the rRNA operon of M. pneumoniae is initiated by a single promoter. The nucleotide sequence of the region is presented.

Promoters of Mycoplasma capricolum ribosomal RNA operons: identical activities but different regulation in homologous and heterologous cells.

The 5' region of the rRNA operon, rrnA, of M. capricolum was cloned. Sequence analysis revealed two tRNA genes, tRNA(leu) and tRNA(lys), upstream to the promoter of the rRNA operon. The in vivo transcription start sites of the rRNA operon and of the tRNA genes were mapped. The same promoters used by M. capricolum RNA polymerase are also recognized by E. coli RNA polymerase both in vivo and in vitro. We find that high levels of ppGpp in E. coli, resulting from amino acid starvation or from spoT mutation, activate rather than repress the transcription of the mycoplasma rrnA operon.

DNA probes for detection and identification of mycoplasmas (Mollicutes).

DNA probes are expected to prove a specific, sensitive, rapid and inexpensive means for diagnosis of mycoplasma infections, replacing procedures that depend on cultivation of the fastidious organisms. Probes made up of conserved genes, such as rRNA genes, do offer the advantage of identifying and distinguishing multiple species with a single labeled reagent. The mycoplasmal rRNA gene probe pMC5 was effective in detection and identification of mycoplasmas infecting cell cultures. However, use of pMC5 for detection of spiroplasmas and mycoplasma-like organisms (MLOs) in infected plants was hindered by hybridization of this probe with chloroplast rRNA genes. Moreover, for identifying species and strains by pMC5, a complex hybridization procedure--involving DNA purification, digestion, electrophoresis, and Southern blot hybridization--is required. More specific DNA probes, on the other hand, can identify specific Mollicutes by the much simpler, faster and more sensitive dot blot technique. Thus, a probe made of a cloned Spiroplasma citri plasmid could detect by this technique as little as 10 pg of S. citri DNA (equivalent to about 10(3) organisms) in infected plants and insects. DNA probes specific for Mycoplasma pneumoniae and M. genitalium were selected from genomic libraries and prepared in pUC13 by screening the libraries for inserts hybridizing only with DNA of the specific mycoplasma. The probes, labeled by nick translation with 32P-nucleotides, could detect as little as approximately 100 pg of the specific mycoplasmal DNA by dot blot hybridization. To eliminate radioactivity, the above DNA probes were labeled by biotinylation of sulfonation systems. Dot blot hybridization with these probes showed decreased sensitivity of detection by about one order of magnitude, and some nonspecific background reaction with large quantities of nonhomologous DNAs.

Transcription control elements of the Mycoplasma pneumoniae rRNA operon.

The single RNA operon of Mycoplasma pneumoniae was cloned into a lambda vector and subcloned into pBR322. This was carried out in order to enable the analysis of the transcription control regions of this operon. S1 nuclease mapping was used to locate the 5' ends of RNA transcripts synthesized from the operon. The 5' ends of the 23S, 16S, and a precursor RNA synthesized in vivo in M. pneumoniae were mapped on the DNA template. Preliminary in vitro transcription experiments using RNA polymerase of Escherichia coli led to the conclusion that E. coli recognizes one promoter in the 5' region of the M. pneumoniae rRNA operon. The startsite of the in vitro transcript seems to lie downstream from the 5' end of the M. pneumoniae precursor transcript. Preliminary sequencing of the 5' regions of the M. pneumoniae rRNA operon and of the M. capricolum rRNA B operon enabled their comparison to each other and to known sequences from other organisms.

DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium.

DNA probes specific for Mycoplasma pneumoniae and Mycoplasma genitalium were selected from genomic libraries prepared in pUC13. The 32P-labeled probes could detect, by dot blot hybridization, down to about 0.1 ng of the specific mycoplasma DNA or 10(5) CFU. Biotinylation of probe decreased the sensitivity of detection and produced nonspecific background reactions with nonhomologous DNAs. Sulfonation of probe yielded a similar level of sensitivity with less background.

Transcription termination and processing sites in the bacteriophage lambda pL operon.

S1 nuclease mapping was performed on transcripts from the major leftward operon of the bacteriophage lambda in order to locate the 3' ends of stable RNA species produced in vivo. The analysis was carried out on RNA purified from either an induced lambda prophage or bacteria carrying a plasmid containing a large segment of lambda including the intact PL operon through the bet gene. The S1 nuclease mapping was performed on transcripts produced in the presence and the absence of the N antitermination function, and in the presence and the absence of either the RNase III processing enzyme or the Rho factor. The results of this work indicate that the intercistronic region between the N and ral genes of lambda contains three sites at which transcripts end under N-Rho+ conditions (positions on the lambda sequence: 34,826, 34,558 and 34,393). The distal two correspond to the two sites previously described in this region as tL1 (on both sides of the BamHI site). In the region between ral and Ea10, we mapped the 3' ends of three species of RNA. The 3' end of one species was found to be located 90 nucleotides proximal to tL2a, at 34,000 in the lambda sequence. The terminator at this site may be partially N-resistant. In an RNase III deficient host, an additional RNA species is formed. The 3' end of this RNA species is located at tL2a (33,910 on the lambda sequence). In the presence of the antitermination N gene product, the readthrough transcripts are processed to form a 3' end at position 33,980 on the lambda sequence. These results suggest that elongation of transcription of the lambda PL operon is reduced gradually by clusters of termination located between genes and that the expression of the terminated products is further controlled by processing of the mRNA.

The use of the plasmid pHA10 in the isolation of lambda PL promoter mutations.

The plasmid pHA10 provides a good positive selection system for PL promoter mutations. It contains the lambda EcoR1-D fragment cloned into the pBR322 plasmid. Inactivation of the temperature sensitive lambda repressor at 42 degrees C leads to transcription of the kil gene and host death. One group of survivors at 42 degrees C is saved due to mutations in the PL promoter. After several screening procedures one such mutant was isolated. Restriction enzyme analysis of the plasmid DNA followed by sequence determination showed it to be a 24 base pair deletion beginning half-way through the "Pribnow box" continuing through part of the -35 region. It removes the OL1 section of the OL operator plus one base pair of OL2. This mutation (called dpl) may be useful to the study of the lambda OLPL regulation system. The effect of base changes within the PL promoter on the promoter activity are discussed.