Design of sphingomonad-detecting probes for a DNA array, and its application to investigate the behavior, distribution, and source of Rhizospherous sphingomonas and other sphingomonads inhabiting an acid sulfate soil paddock in Kalimantan, Indonesia

Throughout Central and South Kalimantan, Indonesia, strongly acidic soil (pH 2.1-3.7) is widely distributed, and the local acidic soil-tolerant plants, including local rice varieties, often possess sphingomonads in their rhizosphere and rhizoplane. To investigate the behavior of sphingomonads inhabiting the rhizosphere of such acid-tolerant plants, we designed 13 different DNA array probes (each of 72 mer) specific to a group of sphingomonads, using a hypervariable V6 region of the 16S rRNA gene. This DNA array system was used preliminarily for an analysis of microfloral dynamisms, particularly of sphingomonads, in acidic paddock ecosystems, and the results suggest that the acid-tolerant local rice shares rhizospherous sphingomonads with wild Juncus sp., a predominant weed that thrives in acidic paddocks during the off-season for rice farming. This tentative conclusion supports the bio-rationality of the traditional rice farming system with respect to functional rhizobacteria.     

Chemotaxonomic characterisation of Sphingomonas

Based on published results and investigations done for this study, chemotaxonomic characteristics of all validly described species of the genus Sphingomonas, as well as unnamed strains of this genus and related genera such as Rhizomonas and Blastomonas, are presented. All representatives of this group, here designated as sphingomonads, contain ubiquinone (Q-10). The two different polyamine patterns characterized either by the predominant polyamine sym-homospermidine or spermidine separate the sphingomonads into two major groups. Complex polar lipid profiles were found in sphingomonads in addition to the characteristic compound sphingoglycolipid. Identical profiles were found only in a few phylogenetically highly related species. Common to all sphingomonads is a fatty acid composition with 2-hydroxy fatty acids (14:0 2OH in all currently recognized species) and the lack of 3-hydroxy acids, which distinguishes them from taxa outside this group. Qualitative and quantitative differences in the fatty acid compositions, in several cases, were also suitable for identification at the species level. Thus, the differences in the chemotaxonomic characteristics demonstrate that the analyses of these low molecular weight cell compounds are suitable for classification of sphingomonads. Received 09 May 1999/ Accepted in revised form 10 August 1999     

The unique aromatic catabolic genes in sphingomonads degrading polycyclic aromatic hydrocarbons (PAHs)

Many members of the sphingomonad genus isolated from different geological areas can degrade a wide variety of polycyclic aromatic hydrocarbons (PAHs) and related compounds. These sphingomonads such as Sphingobium yanoikuyae strain B1, Novosphingobium aromaticivorans strain F199, and Sphingobium sp. strain P2 have been found to possess a unique group of genes for aromatic degradation, which are distantly related with those in pseudomonads and other genera reported so far both in sequence homology and gene organization. Genes for aromatics degradation in these sphingomonads are complexly arranged; the genes necessary for one degradation pathway are scattered through several clusters. These aromatic catabolic gene clusters seem to be conserved among many other sphingomonads such as Sphingobium yanoikuyae strain Q1, Sphingomonas paucimobilis strain TNE12, S. paucimobilis strain EPA505, Sphingobium agrestis strain HV3, and Sphingomonas chungbukensis strain DJ77. Furthermore, some genes for naphthalenesulfonate degradation found in Sphingomonas xenophaga strain BN6 also share a high sequence homology with their homologues found in these sphingomonads. On the other hand, protocatechuic catabolic gene clusters found in fluorene-degrading Sphingomonas sp. strain LB126 appear to be more closely related with those previously found in lignin-degrading S. paucimobilis SYK-6 than the genes in this group of sphingomonads. This review summarizes the information on the distribution of these strains and relationships among their aromatic catabolic genes.     

Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-targeted oligonucleotide probes

The increasing significance of members of the genus Sphingomonas in biotechnological applications has led to an increased interest in the diversity, abundance and ecophysiological potential of this group of Gram-negative bacteria. This general focus provides a challenge to improve means for identification of sphingomonads; eg molecular genetic methods for rapid and specific detection could facilitate screening of new isolates. Here, fluorescently labeled oligonucleotide probes targeted against 16S rRNA were used to typify strains previously assigned to the genus. All 46 sphingomonads tested including type strains of 21 Sphingomonasspecies could be detected with a probe originally designed for the genus and all but one with a probe designed for the alpha-4 subgroup of the Proteobacteria. The two probes are suitable for direct detection of sphingomonads in pure and mixed cultures as well as in environmental samples of unknown composition. The probes were used to identify sphingomonads in situ in activated sludge samples. Sphingomonads were rather abundant accounting for about 5–10% of the total cells in municipal sludges. Distinct patterns in aggregation of the cells suggest that these organisms could be involved in the formation process of sludge flocs. Received 27 May 1999/ Accepted in revised form 22 August 1999     

Use of rpsL for dominance selection and gene replacement in Streptomyces roseosporus.

We developed a gene replacement system using the rpsL gene of Streptomyces roseosporus and demonstrated its utility by constructing a deletion in the S. roseosporus glnA gene. A 1.3-kb BamHI fragment that hybridized to the Mycobacterium smegmatis rpsL gene was subcloned from an S. roseosporus cosmid library and sequenced. Plasmid pRHB514 containing the rpsL gene conferred streptomycin sensitivity (Sm(S)) to the Sm(r) S. roseosporus TH149. The temperature-sensitive plasmid pRHB543 containing rpsL and the S. roseosporus glnA gene disrupted with a hygromycin resistance (Hm(r)) gene was introduced into S. roseosporus TH149, and recombinants containing single and double crossovers were obtained after a temperature increase. Southern hybridization analysis revealed that single crossovers occurred in the glnA or rpsL genes and that double crossovers resulted in replacement of the chromosomal glnA gene with the disrupted glnA. Glutamine synthetase activity was undetectable in the recombinant containing the disrupted glnA gene.     

Nucleotide sequence analysis of the Leptospira biflexa serovar patoc rpsL and rpsG genes.

The Leptospira biflexa rpsL and rpsG genes were sequenced. Although similar in many respects, proteins encoded by these L. biflexa genes had several unusual features when compared with homologous proteins of other organisms. Unlike the rpsL genes of other eubacteria, the L. biflexa rpsL gene is adjacent to a rpoC-like gene.     

Evidence for natural horizontal transfer of the pcpB gene in the evolution of polychlorophenol-degrading sphingomonads

The chlorophenol degradation pathway in Sphingobium chlorophenolicum is initiated by the pcpB gene product, pentachlorophenol-4-monooxygenase. The distribution of the gene was studied in a phylogenetically diverse group of polychlorophenol-degrading bacteria isolated from contaminated groundwater in K?rk?l?, Finland. All the sphingomonads isolated were shown to share pcpB gene homologs with 98.9 to 100% sequence identity. The gene product was expressed when the strains were induced by 2,3,4,6-tetrachlorophenol. A comparative analysis of the 16S rDNA and pcpB gene trees suggested that a recent horizontal transfer of the pcpB gene was involved in the evolution of the catabolic pathway in the K?rk?l? sphingomonads. The full-length K?rk?l? pcpB gene allele had approximately 70% identity with the three pcpB genes previously sequenced from sphingomonads. It was very closely related to the environmental clones obtained from chlorophenol-enriched soil samples (M. Beaulieu, V. Becaert, L. Deschenes, and R. Villemur, Microbiol. Ecol. 40:345-355, 2000). The gene was not present in polychlorophenol-degrading nonsphingomonads isolated from the K?rk?l? source.     

Characterization of the rpsL and rrs genes of streptomycin-resistant clinical isolates of Mycobacterium tuberculosis in Japan

Mutations in the rpsL and rrs genes associated with streptomycin resistance in Mycobacterium tuberculosis clinically isolated in Japan were characterized. The rpsL genes of 172 clinical isolates were amplified by PCR and classified into two groups on the basis of Mbo II restriction digestion. Thirty-three out of 54 (61·1%) streptomycin-highly resistant isolates (MIC > 200 μg ml⁻¹) were not digested by Mbo II. By contrast, the remaining 21 of 54 (38·9%) streptomycin-highly resistant isolates, all of 41 isolates with streptomycin resistance at a lower level (20 μg ml⁻¹ < MIC ≤ 200 μg ml⁻¹), and all of 77 streptomycin-sensitive isolates, were restricted. Thus, all isolates resistant for Mbo II digestion showed a high level of resistance to streptomycin. Subsequently, the sequence for the rpsL and rrs genes from the 46 isolates were analysed. Eighteen out of 19 (94·7%) streptomycin-highly resistant isolates carried a mutation in any rpsL gene at position 43 or 88, or the rrs gene ; 10 out of 17 (58·8%) streptomycin-resistant isolates at a lower level were confirmed to exhibit the mutation of either the mutated rpsL gene at position 88, or the rrs gene. In the total 36 streptomycin-resistant isolates, the mutation of the rpsL or rrs gene was observed in 28 streptomycin-resistant isolates, corresponding to 77·8%, whereas none of the streptomycin-sensitive isolates had mutations in either the rpsL or rrs gene.     

Sphingomonads from marine environments

Sphingomonas species play an important role in the ecology of a range of marine habitats. Isolates and 16S-rRNA clones have been obtained from corals, natural and artificial sources of marine hydrocarbons and eutrophic and oligotrophic waters, and have been isolated as hosts for marine phages. In addition they are found in oceans spanning temperature ranges from polar to temperate waters. While less is known about marine sphingomonads in comparison to their terrestrial counterparts, their importance in microbial ecology is evident. This is illustrated by, for example, the numerical dominance of strain RB2256 in oligotrophic waters. Furthermore, the known marine sphingomonads represent a phylogenetic cross-section of the Sphingomonas genus. This review focuses on our present knowledge of cultured isolates and 16S-rDNA clones from marine environments. Received 01 May 1999/ Accepted in revised form 13 July 1999     

Use of the 16S-23S ribosomal genes spacer region for the molecular typing of sphingomonads

The ability of sphingomonads in drinking water to cause community- and hospital-acquired opportunistic infections has raised the need to establish reproducible identification assays. In this study, a total of 129 isolates recovered from drinking water with yellow- to orange-pigmented colonies were distributed among 10 biotypes on the basis of colony morphology. Polymorphisms, based on the amplification and restriction digestion of the intergenic transcribed spacer (ITS) region within the 10 assigned biotypes and 18 ATCC reference strains, were used to investigate the ability of this approach to differentiate closely related sphingomonads. ITS size, which ranged between 400 and 1100 bp, did not vary enough among the different genera. However, 16 distinct banding patterns within the ATCC reference strains and 9 within the 10 biotypes were obtained through ITS restriction digestion, and the majority of the tested biotypes produced patterns similar to those generated by the ATCC strains. To our knowledge, this study is not only the first comprehensive record of the size of the ITS region in sphingomonads, it is also the first study that describes the use of ITS restriction digestion to subtype those isolates.     

Cloning and sequence analysis of the rpsL and rpsG genes of Mycobacterium smegmatis and characterization of mutations causing resistance to streptomycin.

The Mycobacterium smegmatis rpsL and rpsG genes, encoding the ribosomal proteins S12 and S7, were cloned, and their DNA sequence was determined. The third nucleotide of the S12 termination codon overlapped the first nucleotide of the S7 translation initiation codon. A collection of 28 spontaneous streptomycin-resistant mutants of M. smegmatis were isolated. All had single-base-pair substitutions in the rpsL gene which were changed to a streptomycin-sensitive phenotype by complementation with a low-copy-number plasmid carrying the wild-type M. smegmatis rpsL gene. A total of eight different mutations were found in two specific regions of the rpsL gene. Fifty-seven percent (16 of 28) altered the Lys codon at position 43. Forty-six percent of the mutations (13 of 28) were due to a transition changing an AAG Lys codon to an AGG Arg codon, with eight changes at codon 43 and five at codon 88.     

Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium

Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE . Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium . To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium . We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL , being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL . This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity ( ram ) phenotype.     

Exchange of chromosomal and plasmid alleles in Escherichia coli by selection for loss of a dominant antibiotic sensitivity marker.

Transfer of an allele from a donor DNA to a recipient DNA molecule was selected by the loss of a dominant conditional lethal mutation previously incorporated ito the gene of interest in the recipient DNA. Both the Escherichia coli chromosome and plasmids carrying E. coli genes were used successfully as donor molecules. Recipient molecules for these exchanges were constructed in vitro by using the rpsL gene, which confers sensitivity to streptomycin, to replace segments of specific E. coli genes located either on multicopy plasmids or in the E. coli chromosome. Plasmids carrying such replacements were capable of acquiring chromosomal alleles of the gene(s) of interest, and strains carrying rpsL replacements in the chromosome were capable of acquiring plasmid-encoded alleles at the sight of the rpsL replacement. In both situations, these allele transfers resulted in loss of the rpsL gene from the recipient DNA molecule. The desired transfer events constituted a large percentage of these events, which gave rise to viable colonies when appropriate donor-recipient pairs were subjected to streptomycin selection. Thus, this is a useful approach for transferring alleles of interest from plasmids to the E. coli chromosome and vice versa.     

Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)

Certain str mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the strain carrying DeltarsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the DeltarsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the DeltarsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the DeltarsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.     

Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir

Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm(S)) to its mutant phenotype (Sm(R)). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.     

Genomic organization and genomic structural rearrangements of Sphingobium japonicum UT26, an archetypal γ-hexachlorocyclohexane-degrading bacterium

The complete genome sequencing of a γ-hexachlorocyclohexane-degrading strain, Sphingobium japonicum UT26, revealed that the genome consists of two circular chromosomes [with sizes of 3.5 Mb (Chr1) and 682kb (Chr2)], a 191-kb large plasmid (pCHQ1), and two small plasmids with sizes of 32 and 5kb. The lin genes are dispersed on Chr1, Chr2, and pCHQ1. Comparison of the UT26 genome with those of other sphingomonad strains demonstrated that the "specific"lin genes for conversion of γ-HCH to β-ketoadipate (linA, linB, linC, linRED, and linF) are located on the DNA regions unique to the UT26 genome, suggesting the acquisition of these lin genes by horizontal transfer events. On the other hand, linGHIJ and linKLMN are located on the regions conserved in the genomes of sphingomonads, suggesting that the linGHIJ-encoded β-ketoadipate pathway and the LinKLMN-type ABC transporter system are involved in core functions of sphingomonads. Based on these results, we propose a hypothesis that UT26 was created by recruiting the specific lin genes into a strain having core functions of sphingomonads. Most of the specific lin genes in UT26 are associated with IS6100. Our analysis of spontaneous linA-, linC-, and linRED-deletion mutants of UT26 revealed the involvement of IS6100 in their deduced genome rearrangements. These facts strongly suggest that IS6100 plays important roles both in the dissemination of the specific lin genes and in the genome rearrangements.     

应用4种方法检测结核分枝杆菌临床分离株对链霉素的耐受性

通过DNA测序、SSCP、RFLP和反向斑点杂交技术分析167株结核分枝杆菌临床分离株的耐药基因型,评价结核分枝杆菌rpsL或rrs基因突变与链霉素（SM）耐受性之间的关系,比较4种分子方法检测SM耐受性的临床价值。98株耐SM分离株中,78株（79．6％）rpsL 43位或88位密码子错义突变导致赖氨酸置换为精氨酸,6株（6．1％）rrs 513位碱基A突变为C或T或516位C突变为T,14株（14．3％）未发现突变;69株SM敏感的分离株未发现这两个基因突变。应用SSCP、RFLP和RDBH方法分析上述突变和野生序列的结果与DNA测序完全一致,RDBH方法可从98株耐SM分离株中正确鉴定出84株（85．7％）分离株的5种突变基因型。结果表明,应用分子技术分析rpsL和rrs基因突变可快速检测大多数结核分枝杆菌对SM的耐受性,反向斑点杂交方法是一个快速、简便和可靠地检测药物耐受性的分子方法。     

Is efficiency of suppressor tRNAs controlled at the level of ribosomal proofreading in vivo?

Ribosomal rpsD mutations did not stimulate nonsense suppressor tRNAs in a general manner according to their increased ribosomal ambiguity and decreased proofreading efficiency. Streptomycin, which stimulates error production by blocking proofreading in vitro, did not increase efficiency of suppressor tRNAs in strains with normal or streptomycin-resistant (rpsL) ribosomes. It did so only in combination with one rpsL mutation which is associated with streptomycin pseudodependence.