Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: Evidence for a novel site of streptomycin resistance in the small subunit rRNA

Summary A major obstacle to out understanding of the mechanisms governing the inheritance, recombination and segregation of chloroplast genes in Chlamydomonas is that the majority of antibiotic resistance mutations that have been used to gain insights into such mechanisms have not been physically localized on the chloroplast genome. We report here the physical mapping of two chloroplast antibiotic resistance mutations: one conferring cross-resistance to erythromycin and spiramycin in Chlamydomonas moewusii (er-nM1) and the other conferring resistance to streptomycin in the interfertile species C. eugametos (sr-2). The er-nM1 mutation results from a C to G transversion at a well-known site of macrolide resistance within the peptidyl transferase loop region of the large subunit rRNA gene. This locus, designated rib-2 in yeast mitochondrial DNA, corresponds to residue C-2611 in the 23 S rRNA of Escherichia coli. The sr-2 locus maps within the small subunit (SSU) rRNA gene at a site that has not been described previously. The mutation results from an A to C transversion at a position equivalent to residue A-523 in the E. coli 16 S rRNA. Although this region of the E. coli SSU rRNA has no binding affinity for streptomycin, it binds to ribosomal protein S4, a protein that has long been associated with the response of bacterial cells to this antibiotic. We propose that the sr-2 mutation indirectly affects the nearest streptomycin binding site through an altered interaction between a ribosomal protein and the SSU rRNA.     

Variety of Hybrid Characters among Recombinants Obtained by Interspecific Protoplast Fusion in Streptomycetes

To discover whether the protoplast fusion method is useful or not for interspecific breeding, some methods were devised, and the appearance of various hybrids with different characters and the change of antibiotic activities in the recombinants obtained by the protoplast fusion were investigated. The purification of protoplasts, the choice of parental natural characters as selection markers, and the adoption of a replica method for selecting all types of recombinants were devised and used for these experiments. Protoplast fusion was done between S. griseus KCC S-0644 and each strain of 5 species that were clearly different species from S. griseus, in addition to being streptomycin sensitive (SM^s) and capable of L-arabinose utilization for growth (Ara⁺). Recombinants (SM^r, Ara⁺) obtained by protoplast fusion displayed a great variety of hybrids in their taxonomic characters, e.g., 21 recombinant strains obtained by the cross between S. griseus and S. griseoruber consisted of 14 types of hybrids. Antibiotic productivity was examined in all recombinants obtained. Although both parental species produced their respective antibiotics, 60% of the recombinants did not produce any antibiotic and 24% produced different antibiotics from those of their parents. Among those recombinants, it was also found that the distribution of the productivity of each antibiotic among the recombinants was entirely different from that of the allelo-character in each taxonomic feature.     

Coevolution of antibiotic production and counter‐resistance in soil bacteria

We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance‐only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.     

Selection of the Internal Control Gene for Real-Time Quantitative RT-PCR Assays in Temperature Treated <Emphasis Type="Italic">Leptospira</Emphasis>

Analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. To avoid bias, real-time RT-PCR is referred to one or several internal control genes. Here, we sought to identify a gene to be used as normalizer by analyzing three functional distinct housekeeping genes (lipL41, flaB, and 16S rRNA) for their expression level and stability in temperature treated Leptospira cultures. Leptospira interrogans serovar Hardjo subtype Hardjoprajitno was cultured at 30°C for 7 days until a density of 10⁶ cells/ml was reached and then shifted to physiological temperatures (37°C and 42°C) and to environmental temperatures (25°C and 30°C) during a 24 h period. cDNA was amplified by quantitative PCR using SYBR Green I technology and gene-specific primers for lipL41, flaB, and 16S rRNA. Expression stability (M) was assessed by geNorm and Normfinder v.18. 16S rRNA registered an average expression stability of M = 1.1816, followed by flaB (M = 1.682) and lipL41 (M = 1.763). 16S rRNA was identified as the most stable gene and can be used as a normalizer, as it showed greater expression stability than lipL41 and flaB in the four temperature treatments. Hence, comparisons of gene expression will have a higher sensitivity and specificity.     

Evidence that in higher plants the 25S and 18S rRNA genes are not interspersed with genes for 5S rRNA

DNA samples from various higher plants (Phaseolus aureus, Glycine max, Matthiola incana, Brassica pekinensis, Cucumis melo) were centrifuged in actinomycin-caesium chloride gradients and the genes coding for the ribosomal RNAs were detected by hybridisation with tritium labelled 5S and 25S+18S rRNA, respectively. With DNA of low molecular weight (< 5×10⁶ daltons) the 5S and 25S+18S rRNA genes are often fractionated together. A good separation of the genes for 25S+18S rRNA from the 5S rRNA genes occurred only with high molecular weight DNA (> 10×10⁶ daltons) indicating that at least most of the 5S rRNA genes are not linked to, or interspersed with, the genes coding for 25S and 18S rRNA. This result is in agreement with the situation in animal cells and in contrast to that reported for bacteria, lower eukaryotes and chloroplasts.     

Ribosomal protein S12 as a site for streptomycin resistance in Nicotiana chloroplasts

Summary A streptomycin resistant Nicotiana plastome mutant, X/str ^R6, was subjected to molecular analysis. In this mutant, a single nucleotide transition, C » T, in the chloroplast gene for ribosomal protein S12 alters codon 90 from proline to serine while the nucleotide sequence of the chloroplast 16 S rRNA gene is identical to that of the wild type. Mutant X/str ^R6 thus differs from several previously reported streptomycin resistant chloroplast mutants which are altered in the gene for 16 S rRNA.     

Cytological heterozygosity and the hybrid origin of sweet orange [Citrus sinensis (L.) Osbeck]

Citrus sinensis chromosomes, although small in size, present a remarkable differentiation of bands with the fluorochromes CMA and DAPI. These bands suggest that some heteromorphisms are fixed in this species. To investigate the extension of these heteromorphisms, ten cultivars of C. sinenesis were analysed with CMA/DAPI staining and, in some of them, the 18S–5.8S–25S rRNA and 5S rRNA genes were located by in situ hybridization. CMA/DAPI staining showed exactly the same CMA⁺/DAPI⁻ banding pattern for all cultivars. In situ hybridization revealed three 18S–5.8S–25S rRNA gene sites, two proximally located on two similar chromosomes and one terminally located on a third non-related chromosome. Two 5S rRNA gene sites were observed in this species, with one located proximal to the telomeric 18S–5.8S–25S rDNA site. Both cytological approaches revealed an invariable, heterozygotic karyotype among sweet orange cultivars. Based on these data, the putative hybrid origin of the species is discussed. Received: 9 April 1999 / Accepted: 22 June 1999     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Mutations conferring lincomycin, spectinomycin, and streptomycin resistance in Solanum nigrum are located in three different chloroplast genes

A number of Solanum nigrum mutants resistant to the antibiotics spectinomycin, streptomycin and lincomycin have been isolated from regenerating leaf strips after mutagenesis with nitroso-methylurea. Selection of streptomycin- and spectinomycin-resistant mutants has been described earlier. Lincomycin-resistant mutants show resistance to higher levels of the antibiotic than used in the initial selection, and in the most resistant mutant (Ll7A1) maternal inheritance of the trait was demonstrated. The lincomycin-resistant mutant L17A1 and a streptomycin plus spectinomycin resistant double mutant (StSpl) were chosen for detailed molecular characterisation. Regions of the plastid DNA, within the genes encoding 16S and 23S rRNA and rps12 (3) were sequenced. For spectinomycin and lincomycin resistance, base changes identical to those in similar Nicotiana mutants were identified. Streptomycin resistance is associated with an A C change at codon 87 of rps 12 (converting a lysine into a glutamine), three codons upstream from a mutation earlier reported for Nicotiana. This site has not previously been implicated in streptomycin resistance mutations of higher plants, but has been found in Escherichia coli. The value of these mutants for studies on plastid genetics is discussed.     

Molecular cloning and characterization of ribosomal RNA genes from a blue-green alga, Anacystis nidulans

Summary Two PstI fragments (5.3x10⁶ and 4.3x10⁶ daltons) coding for Anacystis nidulans rRNA genes were cloned. The cloned rDNAs were characterized by restriction endonuclease mapping, DNA-RNA hybridization analysis and the R-loop technique. The results indicated that both fragments contained 16S, 23S and 5S rRNA genes in this order. A tRNA gene(s) was detected in the spacer region between 16S and 23S rRNA genes. The organization of A. nidulans rRNA genes resembles those of E. coli and of Euglena chloroplasts rather than those of higher plant chloroplasts.     

On the interaction between heparin and some aminoglycoside antibiotics

An interaction between the aminoglycoside antibiotics and heparin wherein charge transfer complexes are formed has been investigated to determine the degree of inhibition of antibacterial function of the antibiotic in the complexed form.Minimum inhibitory concentration (MIC) values have been obtained for the action of the aminoclycoside antibiotics tobramycin, gentamicin, amikacin, kanamycin, and streptomycin, on a sensitive strain ofE. coli. Growth curves ofE. coli determined at concentrations of these antibiotics just below the MIC demonstrated significant lengthening of the lag phase relative to control growth curves generated in the absence of antibiotic. Heparin (1 U ml^–1 and 10 U ml^–1) had no effect on control growth curves; however, particularly at the higher concentration, it reduced the effect on the lag phase produced by the aminoglycoside antibiotics. Thus kanamycin, gentamicin, and tobramycin were most affected, while amikacin and streptomycin were least affected. The rank order of inhibition of antibiotic activity by interaction with heparin was in qualitative agreement with previously published figures for the degree of complexation between antibiotics and heparin.     

Physical mapping of genes on yeast mitochondrial DNA: Localization of antibiotic resistance loci, and rRNA and tRNA genes

Summary We have physically mapped the loci conferring resistance to antibiotics that inhibit mitochondrial protein synthesis (erythromycin, chloramphenicol and paromomycin) or respiration (oligomycin I and II), as well as the 21s and 14s rRNA and tRNA genes on the restriction map of the mitochondrial genome of the yeast Saccharomyces cerevisiae. The mitochondrial genes were localized by hybridization of labeled RNA probes to restriction fragments of grande (strain MH41-7B) mitochondrial DNA (mtDNA)¹ generated by endonucleases EcoRI, HpaI, BamHI, HindIII, SalI, PstI and HhaI. We have derived the HhaI restriction fragment map of MH41-7B mit DNA, to be added to our previously reported maps for the six other endonucleases.The antibiotic resistance loci (ant ^R) were mapped by hybridization of ³H-cRNA transcribed from single marker petite mtDNA's of low kinetic complexity to grande restriction fragments. We have chosen the single Sal I site as the origin of the circular physical map and have positioned the antibiotic loci as follows: C (99.5-1.Ou)-P(27-36.Ou)-O_II (58.3-62u)-O_I (80-84u)-E (94.4-98.4u). The 21s rRNA is localized at 94.4-99.2u, and the 14s rRNA is positioned between 36.2-39.8u. The two rRNA species are separated by 36% of the genome. Total mitochondrial tRNA labeled with ¹²⁵I hybridized primarily to two regions of the genome, at 99.5-11.5u and 34-44u. A third region of hybridization was occasionally detected at 70-76u, which probably corresponds to seryl and glutamyl tRNA genes, previously located to this region by petite deletion mapping.Supported by USPHS Training Grant T32-GM-07197.Supported by USPHS Training Grant 5-T01-GM-0090-19.The Franklin McLean Memorial Research Institute is operated by the University of Chicago for the U. S. Energy Research and Development Administration under Contract EY-76-C-02-0069.     

Phylogenetic relationship of the green alga Nanochlorum eukaryotum deduced from its chloroplast rRNA sequences

The marine green coccoidal alga Nanochlorum eukaryotum (N.e.) is of small size with an average diameter of 1.5 m. It is characterized by primitive-appearing biochemical and morphological properties, which are considerably different from those of other green algae. Thus, it has been proposed that N.e. may be an early developed algal form. To prove this hypothesis, DNA of N.e. was isolated by a phenol extraction procedure, and the chloroplast DNA separated by preparative CsCl density-gradient centrifugation. The kinetic complexity of the nuclear and of the chloroplast DNA was evaluated by reassociation kinetics to 3 × 10⁷ by and 9 × 10⁴ bp, respectively. Several chloroplast genes, including the rRNA genes, were cloned on distinct fragments. The order of the rRNA genes corresponds to the common prokaryotic pattern. The 16S rRNA gene comprises 1,548 bases and is separated from the 23S rRNA gene with its 2,920 bases by a short spacer of 460 bases, which also includes the tRNA^Ile and tRNA^Ala genes. The 5S rRNA gene has not been found; it must start further than 500 bases downstream from the 3-end of the 23S rRNA gene. From the chloroplast rRNA sequences, we have deduced secondary structures of the 16S and 23S rRNAs, which are in agreement with standard models. The rRNA sequences were aligned with corresponding chloroplast sequences; phylogenetic relationships were calculated by several methods. From these calculations, we conclude that N.e. is most closely related to Chlorella vulgaris. Therefore, N.e. does not represent an early developed algal species; the primitive-appearing morphological and biochemical characteristics of N.e. must rather be explained by secondary losses. Correspondence to: D. Weinblum     

Pseudopropionibacterium rubrum sp. nov., a novel red‐pigmented species isolated from human gingival sulcus

In this study, sStrain SK‐1^T, a novel gram‐positive, pleomorphic, rod‐shaped, non‐spore forming, non‐motile organism, designated SK‐1^T, was isolated from human gingival sulcus and found to produce acetic acid, propionic acid, lactic acid, and succinic acid as end products of glucose fermentation. Strain SK‐1^T is most closely related to Pseudopropionibacterium (Propionibacterium) propionicum with sequence homologies of the 16S rRNA and RNA polymerase β subunit (rpoB) genes of 96.6% and 93.1%, respectively. The genomic DNA G + C content of the isolate was 61.8 mol%. On the basis of the sequence data of the 16S rRNA and housekeeping (rpoB) genes, a novel taxon is here proposed, Pseudopropionibacterium rubrum sp. nov. (type strain SK‐1^T = JCM 31317^T = DSM 100122^T). The 16S rRNA and rpoB gene sequences of strain SK‐1^T have been deposited in the DDBJ under the accession numbers LC002971 and LC102236, respectively.

     

Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

Intraspecific nucleotide divergence in Saccharomycodes ludwigii,and proposal of Saccharomycodes pseudoludwigii sp. nov,a new apiculate yeast isolated from China

The six synonyms currently accepted under Saccharomycodes ludwigii were investigated for by phenotypic properties, however, the sequence diversity of the rRNA and protein coding genes have not yet been determined. Nine strains including the type strains of synonyms of S. ludwigii deposited in the CBS yeast collection, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, were analyzed using a multi-locus sequence analysis (MLSA) approach that included sequences of 18S ribosomal RNA (rRNA), the D1/D2 domains of the 26S rRNA, the ITS region (including the 5.8S rRNA) and fragments of genes encoding the largest subunit of the RNA polymerase II (RPB1 and RPB2) and translation elongation factor 1-α (TEF1). Our results showed that the nine strains have identical D1/D2, 18S and RPB2 sequences and similar ITS, RPB1 and TEF1 sequences, which indicated that they are conspecific. In addition, a novel species of Saccharomycodes, S. pseudoludwigii sp. nov. (type CGMCC 2.4526 ^T) that was isolated from fruit and tree bark in China, is proposed. The MycoBank number of this new species is MB 811,650.

     

Molecular identification of filterable bacteria and archaea in the water of acidic lakes of northern Russia

Wetland ecosystems are the natural centers of freshwater formation in northern Russia lowland landscapes. The humic acidic waters formed in bogs feed the numerous lakes of the northern regions. One milliliter of the water in these lakes contains up to 10⁴ ultrasmall microbial cells that pass through “bacterial” filters with a pore size of 0.22 μm. The vast majority of these cells do not grow on nutrient media and cannot be identified by routine cultivation-based approaches. Their identification was performed by analysis of clone libraries obtained by PCR amplification of archaeal and bacterial 16S rRNA genes from the fraction of cells collected from water filtrates of acidic lakes. Most of the obtained bacterial 16S rRNA gene sequences represented the class Betaproteobacteria and exhibited the highest homology of (94–99%) with 16S rRNA genes of representatives of the genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia. The archaeal 16S rRNA gene clone library comprised genes of Euryarchaeota representatives. One-third of these genes exhibited 97–99% homology to the 16S rRNA genes of taxonomically described organisms of the orders Methanobacteriales and Methanosarcinales. The rest of the cloned archaeal 16S rRNA genes were only distantly related (71–74% homology) to those in all earlier characterized archaea.     

Diversity and abundance of anammox bacterial community in the deep-ocean surface sediment from equatorial Pacific

The community structure and diversity of anaerobic ammonium oxidation (anammox) bacteria in the surface sediments of equatorial Pacific were investigated by phylogenic analysis of 16S rRNA and hydrazine oxidoreductase (hzo) genes and PCoA (principal coordinates analysis) statistical analysis. Results indicated that 16S rRNA and hzo sequences in the P2 (off the center of western Pacific warm pool) and P3 (in the eastern equatorial Pacific) sites all belong to the Candidatus “Scalindua”, the dominate anammox bacteria in the low-temperature marine environment proved by previous studies. However, in the P1 site (in center of warm pool of western Pacific), large part of 16S rRNA gene sequences formed a separated cluster. Meanwhile, hzo gene sequences from P1 sediment also grouped into a single cluster. PCoA analysis demonstrated that the anammox community structure in the P1 has significant geographical distributional difference from that of P2, P3, and other marine environments based on 16S rRNA and hzo genes. The abundances of anammox bacteria in surface sediments of equatorial Pacific were quantified by q-PCR analysis of hzo genes, which ranged from 3.98 × 10³ to 1.17 × 10⁴ copies g⁻¹ dry sediments. These results suggested that a special anammox bacteria phylotypes exist in the surface sediment of the western Pacific warm pool, which adapted to the specific habitat and maybe involved in the nitrogen loss process from the fixed inventory in the habitat.