Spc Operon Research Articles

Synonymous rpsH variants: the common denominator in Escherichia coli adapting to ionizing radiation.

Ionizing radiation (IR) in high doses is generally lethal to most organisms. Investigating mechanisms of radiation resistance is crucial for gaining insights into the underlying cellular responses and understanding the damaging effects of IR. In this study, we conducted a comprehensive analysis of sequencing data from an evolutionary experiment aimed at understanding the genetic adaptations to ionizing radiation in Escherichia coli. By including previously neglected synonymous mutations, we identified the rpsH c.294T > G variant, which emerged in all 17 examined isolates across four subpopulations. The identified variant is a synonymous mutation affecting the 30S ribosomal protein S8, and consistently exhibited high detection and low allele frequencies in all subpopulations. This variant, along with two additional rpsH variants, potentially influences translational control of the ribosomal spc operon. The early emergence and stability of these variants suggest their role in adapting to environmental stress, possibly contributing to radiation resistance. Our findings shed light on the dynamics of ribosomal variants during the evolutionary process and their potential role in stress adaptation, providing valuable implications for understanding clinical radiation sensitivity and improving radiotherapy.

First Report of a Phytoplasma Strain in the Elm Yellows Group (16SrV) associated with Virginia Creeper in Maryland, USA.

Virginia creeper (Parthenocissus quinquefolia [L.] Planch.) is a deciduous flowering vine in the Vitaceae family. Native to eastern North America, it is often used ornamentally as a climbing vine or as ground cover due to its rapid growth and foliage color in the fall. In July of 2022, along exterior walls of a private property in Lanham, MD, two Virginia creeper (VC) vines were observed displaying symptoms of yellow mottling and premature reddening of leaves. To investigate the cause of these symptoms, two symptomatic leaf samples and one asymptomatic leaf samples from a third vine in the same vicinity were collected for further analysis. A Qiagen DNeasy Plant Mini Kit was used to extract total DNA from leaf tissues according to the manufacturer's instructions. A phytoplasma-specific real-time PCR (Hodgetts et al. 2009) was used to test the DNA extracts, which detected the presence of phytoplasmas in the two DNA samples derived from symptomatic vines. The near full-length of the 16S ribosomal RNA gene was then amplified by seminested PCR from these samples with primers P1/16S-SR followed by P1A/16S-SR (Deng, and Hiruki 1991; Lee et al. 2004) and Sanger sequenced using primers P1A and 16S-SR. Analysis of the obtained 16S rDNA sequences revealed no variation between the two plant samples, and one sequence was deposited in GenBank representing the phytoplasma strain named VC-MD1 (GenBank PP746981). A BLASTn search of the 16S rRNA gene sequence in the NCBI nucleotide database, showed 99.93% sequence identity with the phytoplasma strain AldY-WA1 (GenBank MZ557341) from red alder in Washington, a phytoplasma associated with VC plants in southern Florida (GenBank AF305198) (Harrison et al. 2001), and other strains detected in grapevines in Europe described as "flavescence dorée" phytoplasma (GenBank AF176319) (Davis, and Dally 2001). The virtual restriction fragment length polymorphism pattern derived from iPhyClassifier (Zhao et al. 2009), indicated that VC-MD1 is indeed a member of the 16SrV-C phytoplasma subgroup. To confirm the identification, the partial spc operon and the partial tuf gene were amplified as previously described (Lee et al. 2010; Makarova et al. 2012). Specifically, the spc operon region was amplified using a nested PCR approach with primer set L15F1A-a/MapR1 followed by L15F1A-b/MapR1A-b. Sequence data obtained from the two loci were deposited to GenBank with accession numbers PP746982 (spc) and PP746983 (tuf). BLAST searches querying the nucleotide sequences of the spc operon and tuf gene showed 95.39% and 99.05% identity, respectively, to the corresponding loci of 'Candidatus Phytoplasma rubi' strain RS (GenBank CP114006) and hemp dogbane yellows phytoplasma strain HD1 (GenBank FR686506). Phylogenetic analysis based on secY and tuf gene sequences suggest that the VC-MD1 strain is evolutionary closest to 16SrV-C phytoplasma strains detected in various hosts in the United States, including HD1 and AldY-WA1. These North American strains cluster together on a distinct branch within the elm yellows group phytoplasmas. For the State of Maryland, this detection represents the first report of a phytoplasma strain member of the16SrV-C subgroup infecting VC plants. A phytoplasma of the same subgroup was previously detected in Florida in asymptomatic VC vines (Harrison et al. 2001). The 16S rRNA gene sequences of the two VC phytoplasma strains are nearly identical, differing by just a single nucleotide. The disease transmission vectors of the VC-MD1 strain and the prevalence of the disease in the region remains undetermined.

Momordica charantia Is a Novel Host of 'Candidatus Phytoplasma malaysianum'-Related Strains Associated with Bitter Melon Stem Fasciation Disease in China.

Momordica charantia, also known as bitter melon, bitter gourd, and bitter squash, is a member of the Cucurbitaceae family and is widely grown in tropical and subtropical regions for its edible fruit and medicinal properties (Alves et al. 2017). In April 2022, bitter melon plants exhibiting stem fasciation and excessive tendril symptoms were observed in a 50-acre vegetable farm in Yijia Village, Weishan Yizu Huizu Autonomous County, Dali, Yunnan Province, China (Fig. 1). The farm primarily grew tomatoes, but around 400 bitter melon plants were planted in spots where tomatoes failed to establish. One plot had a 40% incidence rate, with four out of ten bitter melon plants showing symptoms. Scattered cases were observed in other plots, leading to an overall disease incidence rate of around 2% for the entire farm. Phytoplasma infection was suspected due to symptomatic plants growing in the same province as previously reported cases of phytoplasma diseases, such as happy tree (Camptotheca accuminata) witches'-broom disease, and the presence of phytoplasma-transmitting leafhoppers (Qiao et al. 2023). DNA was extracted from four symptomatic samples and two healthy controls collected from the abovementioned plot with a 40% disease incidence using Bioteke's Plant Genomic DNA Extraction Kit and then tested for phytoplasma infection. A nested PCR assay was conducted using primer pair P1/16S-SR followed by P1A/16S-SR to amplify the near full-length phytoplasma 16S rDNA (about 1.5kb) as previously described (Lee et al. 2004). None of the healthy controls tested positive for phytoplasma infection, while three out of four symptomatic plants showed positive results. The amplicons from the nested PCR were cloned into the pCRII-TOPO vector as previously described (Lee et al. 2004). The resulting clones were sequenced, and the representative sequence was deposited into GenBank (accession number PP489216). The iPhyClassifier (Zhao et al. 2009) was employed to determine the phytoplasma species and group/subgroup associated with the bitter melon stem fasciation (BMSF) disease. The results indicated that the diseased bitter melon plants were infected with a strain related to 'Candidatus Phytoplasma malaysianum' (EU371934), with a 98.07% sequence identity. The similarity coefficient was 1.00 compared to the reference strain of 16SrXXXII-D (GenBank accession: MW138004). The phytoplasma strain associated with BMSF disease was designated as BMSF1. In addition, the same DNA samples underwent further characterization of the BMSF strains. A nested PCR was conducted using primer pair rpL2F3/rpIR1A, followed by rp(III)-FN/rpIR1A to amplify a phytoplasma-specific rp gene segment (about 1.2 kb) (Martini et al. 2007; Davis et al. 2013). Three out of four samples tested positive, consistent with the 16S rRNA gene amplification results. Similarly, a primer pair L15F1/MapR1 followed by secYF1(III)/secYR1(III) was used to amplify a phytoplasma-specific partial spc operon (about 1.7 kb) that includes the complete secY gene and partial rpl15 and map genes, as previously described (Lee et al. 2010). The obtained rp and partial spc amplicons were cloned and sequenced (GenBank accession numbers PP464295 and PP464296). The rp and secY gene sequences were searched against the non-redundant nucleotide collection in the NCBI database using BLASTN. The top hit for the rp gene was 'Ca. Phytoplasma luffae' (CP054393), with 83.24% identity (1068/1283 base-matching). The top hit for the secY gene was also 'Ca. Phytoplasma luffae' (CP054393), with 72.53% identity (1294/1784 base-matching). The percent identity of the BMSF sequences compared to the top hit is low since no other group 16SrXXXII rp and secY gene sequences are available for comparison. A subgroup 16SrXXXII-D phytoplasma strain has been previously reported associated with Camptotheca acuminata witches'-broom (Qiao et al. 2023) and Trema tomentosa witches'-broom (Yu et al. 2021) in China. To our knowledge, bitter melon represents a new host of 'Candidatus Phytoplasma malaysianum'-related strains, and this is the first report of BMSF disease in China. The findings suggest that 'Candidatus Phytoplasma malaysianum'-related strains infect not only ornamental plants but also crops.

Ribosomal Protein Cluster Organization in Asgard Archaea.

It has been proposed that the superphylum of Asgard Archaea may represent a historical link between the Archaea and Eukarya. Following the discovery of the Archaea, it was soon appreciated that archaeal ribosomes were more similar to those of Eukarya rather than Bacteria. Coupled with other eukaryotic-like features, it has been suggested that the Asgard Archaea may be directly linked to eukaryotes. However, the genomes of Bacteria and non-Asgard Archaea generally organize ribosome-related genes into clusters that likely function as operons. In contrast, eukaryotes typically do not employ an operon strategy. To gain further insight into conservation of the r-protein genes, the genome order of conserved ribosomal protein (r-protein) coding genes was identified in 17 Asgard genomes (thirteen complete genomes and four genomes with less than 20 contigs) and compared with those found previously in non-Asgard archaeal and bacterial genomes. A universal core of two clusters of 14 and 4 cooccurring r-proteins, respectively, was identified in both the Asgard and non-Asgard Archaea. The equivalent genes in the E. coli version of the cluster are found in the S10 and spc operons. The large cluster of 14 r-protein genes (uS19-uL22-uS3-uL29-uS17 from the S10 operon and uL14-uL24-uL5-uS14-uS8-uL6-uL18-uS5-uL30-uL15 from the spc operon) occurs as a complete set in the genomes of thirteen Asgard genomes (five Lokiarchaeotes, three Heimdallarchaeotes, one Odinarchaeote, and four Thorarchaeotes). Four less conserved clusters with partial bacterial equivalents were found in the Asgard. These were the L30e (str operon in Bacteria) cluster, the L18e (alpha operon in Bacteria) cluster, the S24e-S27ae-rpoE1 cluster, and the L31e, L12..L1 cluster. Finally, a new cluster referred to as L7ae was identified. In many cases, r-protein gene clusters/operons are less conserved in their organization in the Asgard group than in other Archaea. If this is generally true for nonribosomal gene clusters, the results may have implications for the history of genome organization. In particular, there may have been an early transition to or from the operon approach to genome organization. Other nonribosomal cellular features may support different relationships. For this reason, it may be important to consider ribosome features separately.

Aster yellows group (16SrI), subgroups 16SrI‐A and 16SrI‐B, phytoplasmas associated with lettuce yellows in Texas

Lettuce (Lactuca sativa) is an important vegetable crop grown in the United States. Lettuce yellows (previously termed lettuce aster yellows) is a devastating disease commonly found in lettuce fields and causes significant economic loss to growers, damaging 40-100% of the crop in some seasons (Thompson, 4). The causal pathogen was not verified until a phytoplasmal aetiology was confirmed by molecular means (Lee et al., 1). In 2013, an epidemic of lettuce yellows occurred in the Winter Garden region of Texas. The infected plants were stunted with blanching and chlorosis in young heart leaves (Fig. 1). A total of thirteen samples from two different farms, including three apparently lacking symptoms from Romaine and leaf lettuce cultivars, were collected and tested for phytoplasmas. DNA was extracted from leaf veinal tissue, and nested PCRs using universal primer pair P1/16S-SR, followed by R16F2n/R16R2n and P1A/16S-SR, were performed as described previously (Lee et al., 2) to detect the presence of phytoplasmas in the samples. The taxonomic affiliation of the putative phytoplasmas detected was determined by RFLP analysis using nested PCR products (Lee et al., 1; Zhao et al., 5). A nested PCR using primer pair rpF1/rpR1 followed by rp(I)F1/rp(I)R1A as described previously (Lee et al., 2) was used to amplify a phytoplasma DNA segment (about 1.2 kb) of the ribosomal protein (rp) operon that encompassed genes rplV (rpl22) and rpsC (rps3); and a nested PCR using primer pair AYsecYF1/secYR1(I) followed by AYsecYF1/AYsecYR1 (Lee et al., 3) was used to amplify a phytoplasma DNA segment (about 1.3 kb) of the partial spc operon that includes the complete secY gene. Four PCR-amplified products from each of 16S rDNA, rp, and secY genes were cloned and sequenced as previously described (Lee et al., 2, 3), and deposited in GenBank (Accession Nos. KF573449-KF573456; rp, KF573441-KF573448; secY, KF573433-KF573440). The results indicated all 13 lettuce samples were infected with phytoplasmas. Both lettuce cultivars were infected with 16SrI-A and 16SrI-B phytoplasmas (Fig. 2). Phylogenetic analysis with 16S rRNA (Fig. 3), rp (Fig. 4) and secY gene (Fig. 5) sequences confirmed the presence of two distinct lineages (Fig. 3). This is the first report confirming that lettuce yellows in Texas is associated with two distinct phytoplasma strains. The finding implicates that multiple vectors may be involved in the disease outbreak. The information on identities of associated phytoplasmas should facilitate formulating effective strategies to combat this newly emerging lettuce yellows in Texas.

MALDI-TOF MS analysis of ribosomal proteins coded in S10 and spc operons rapidly classified the Sphingomonadaceae as alkylphenol polyethoxylate-degrading bacteria from the environment

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal subunit proteins coded in the S10-spc-alpha operon as biomarkers was applied for the classification of the Sphingomonadaceae from the environment. To construct a ribosomal protein database, S10-spc-alpha operon of type strains of the Sphingomonadaceae and their related alkylphenol polyethoxylate (APEO(n) )-degrading bacteria were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains. The observed MALDI mass spectra of intact cells were compared with the theoretical mass of the constructed ribosomal protein database. The nine selected biomarkers coded in the S10-spc-alpha operon, L18, L22, L24, L29, L30, S08, S14, S17, and S19, could successfully distinguish the Sphingopyxis terrae NBRC 15098(T) and APEO(n) -degrading bacteria strain BSN20, despite only one base difference in the 16S rRNA gene sequence. This method, named the S10-GERMS (S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum) method, is a significantly useful tool for bacterial discrimination of the Sphingomonadaceae at the strain level and can detect and monitor the main APEO(n) -degrading bacteria in the environment.

Classification of the Genus Bacillus Based on MALDI-TOF MS Analysis of Ribosomal Proteins Coded in S10 and spc Operons

A rapid bacterial identification method by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal proteins coded in S10 and spc operons as biomarkers, named the S10-GERMS (the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum) method, was applied for the genus Bacillus a Gram-positive bacterium. The S10-GERMS method could successfully distinguish the difference between B. subtilis subsp. subtilis NBRC 13719(T) and B. subtilis subsp. spizizenii NBRC 101239(T) because of the mass difference of 2 ribosomal subunit proteins, despite the difference of only 2 bases in the 16S rRNA gene between them. The 8 selected reliable and reproducible ribosomal subunit proteins without disturbance of S/N level on MALDI-TOF MS analysis, S10, S14, S19, L18, L22, L24, L29, and L30, coded in S10 and spc operons were significantly useful biomarkers for rapid bacterial classification at species and strain levels by the S10-GERMS method of genus Bacillus strains without purification of ribosomal proteins.

Phylogenetic analysis and delineation of phytoplasmas based on secY gene sequences

The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytoplasma strains, respectively, were performed to assess the efficacy of these sequences for delineating phytoplasma strains within each 16Sr group. The phylogenetic interrelatedness among phytoplasma taxa inferred by secY gene-based phylogeny was nearly congruent with that inferred by 16S rRNA gene-based phylogeny. Phylogenetic analysis based on the secY gene permitted finer differentiation of phytoplasma strains, however. The secY gene-based phylogeny not only readily resolved 16Sr subgroups within a given 16Sr group, but also delineated distinct lineages irresolvable by 16S rRNA gene-based phylogeny. Such high resolving power makes the secY gene a more useful genetic marker than the 16S rRNA gene for finer differentiation of closely related phytoplasma strains based on RFLP analysis with selected restriction enzymes. Such strains were readily identified by collective secY RFLP patterns. The genetic interrelationships among these strains were determined by pattern similarity coefficients, which coincided with delineations by phylogenetic analysis. This study also revealed two heterogeneous spc operons present in the phytoplasma clade. This latter finding may have significant implications for phytoplasma evolution.

Many nonuniversal archaeal ribosomal proteins are found in conserved gene clusters.

The genomic associations of the archaeal ribosomal proteins, (r-proteins), were examined in detail. The archaeal versions of the universal r-protein genes are typically in clusters similar or identical and to those found in bacteria. Of the 35 nonuniversal archaeal r-protein genes examined, the gene encoding L18e was found to be associated with the conserved L13 cluster, whereas the genes for S4e, L32e and L19e were found in the archaeal version of the spc operon. Eleven nonuniversal protein genes were not associated with any common genomic context. Of the remaining 19 protein genes, 17 were convincingly assigned to one of 10 previously unrecognized gene clusters. Examination of the gene content of these clusters revealed multiple associations with genes involved in the initiation of protein synthesis, transcription or other cellular processes. The lack of such associations in the universal clusters suggests that initially the ribosome evolved largely independently of other processes. More recently it likely has evolved in concert with other cellular systems. It was also verified that a second copy of the gene encoding L7ae found in some bacteria is actually a homolog of the gene encoding L30e and should be annotated as such.

Disruption ofrpmJEncoding Ribosomal Protein L36 Decreases the Expression ofsecYUpstream of thespcOperon and Inhibits Protein Translocation inEscherichia coli

The spc operon of Escherichia coli encodes 11 ribosomal proteins and SecY. The secY gene and downstream rpmJ encoding a ribosomal protein, L36, are located distal to the promoter of the spc operon. It has been suggested that the stability of SecY mRNA depends on rpmJ unless a rho-independent terminator is inserted immediately downstream of secY. Moreover, it has been suggested that RpmJ is dispensable for E. coli. We constructed rpmJ null strains, AY101 (DeltarpmJ::tetA) and AY201 (DeltarpmJ::cat), by replacing rpmJ with tetA, which encodes a membrane protein responsible for tetracycline-resistance, and cat, which encodes a cytoplasmic chloramphenicol acetyltransferase, respectively. Depletion of RpmJ did not inhibit protein synthesis, whereas the growth of AY101 was defective at high temperatures. The level of SecY mRNA decreased significantly in both disruptants even though the rho-independent terminator was inserted immediately downstream of secY. Some periplasmic proteins were missing in the disruptants with a concomitant increase in the amount of phage shock protein in the inner membrane. These phenotypes caused by the rpmJ null mutation were corrected by a plasmid carrying secY, but not by one carrying rpmJ.

Characterization of the Leptospira interrogans S10-spc-alpha operon.

A ribosomal protein gene cluster from the spirochaete Leptospira interrogans was characterized. This locus is homologous to the Escherichia coli S10, spc, and alpha operons. Analysis of L. interrogans RNA showed that the ribosomal protein genes within this cluster are co-transcribed, thus forming an operon. Two transcription initiation sites were mapped by primer extension, upstream of fus, the first gene in this cluster, and sequences from this region provided promoter activity in E. coli. Transcription terminates near a predicted stem-loop structure following rplQ, the last gene in the cluster. These data suggest that two promoters upstream of fus direct transcription of this 17.5-kb ribosomal protein gene cluster. Comparison of the L. interrogans S10-spc-alpha cluster to homologous loci from Borrelia burgdorferi and Treponema pallidum provided evidence that this region of the genome underwent several rearrangements during spirochaete evolution.

The Structure and Evolution of the Ribosomal Proteins Encoded in the spc Operon of the Archaeon (Crenarchaeota) Sulfolobus acidocaldarius

The genes for nine ribosomal proteins, L24, L5, S14, S8, L6, L18, S5, L30, and L15, have been isolated and sequenced from the spc operon in the archaeon (Crenarchaeota) Sulfolobus acidocaldarius, and the putative amino acid sequence of the proteins coded by these genes has been determined. In addition, three other genes in the spc operon, coding for ribosomal proteins S4E, L32E, and L19E (equivalent to rat ribosomal proteins S4, L32, and L19), were sequenced and the structure of the putative proteins was determined. The order of the ribosomal protein genes in the spc operon of the Crenarchaeota kingdom of Archaea is identical to that present in the Euryarchaeota kingdom of Archaea and also identical to that found in bacteria, except for the genes for r-proteins S4E, L32E, and L19E, which are absent in bacteria. Although AUG is the initiation codon in most of the spc genes, GUG (val) and UUG (leu) are also used as initiation codons in S. acidocaldarius. Over 70% of the codons in the Sulfolobus spc operon have A or U in the third position, reflecting the low GC content of Sulfolobus DNA. Phylogenetic analysis indicated that the archaeal r-proteins are a sister group of their eucaryotic counterparts but did not resolve the question of whether the Archaea is monophyletic, as suggested by the L6P, L15P, and L18P trees, or the question of whether the Crenarchaeota is separate from the Euryarchaeota and closer to the Eucarya, as suggested by the S8P, S5P, and L24P trees. In the case of the three Sulfolobus r-proteins that do not have a counterpart in the bacterial ribosome (S4E, L32E, and L19E), the archaeal r-proteins showed substantial identity to their eucaryotic equivalents, but in all cases the archaeal proteins formed a separate group from the eucaryotic proteins.

Decay of rplN and lacZ mRNA in Escherichia coli

To examine the previously proposed retroregulation model of spc mRNA degradation, two strains of Escherichia coli B/r were used; one has wild-type spc and lac operons and the other has a lac operon deletion, a wild-type spc operon, and a Pspc-rplN-lacZ fusion operon lacking the normal control sites of the spc operon (rplN is the first gene in the spc operon of ribosomal proteins). The decay of rplN mRNA and of lacZ mRNA in these strains was determined during exponential growth at different rates and after transcript initiation was inhibited by the antibiotic rifampicin. Functional decay of lacZ mRNA was monitored by measurements of β-galactosidase activity and chemical decay was monitored using probes complementary to rplN, rplX, and to the 5′ and 3′-terminal sections of lacZ. Analysis of the data was based on the assumption that the decay involves an endonucleolytic cleavage that functionally inactivates the mRNA and that this is followed by exonucleolytic degradation of the cleavage products. The major conclusions were: (1) During exponential growth, lacZ mRNA of the lac operon was translated about twice as frequently as lacZ mRNA of the spc-lac fusion, and both kinds of lacZ mRNA were translated at an elevated rate in the presence of rifampicin. (2) For lacZ mRNA from the lac operon, the endonuclease inactivation reaction was not affected by rifampicin, but the exonuclease reaction was inhibited. (3) The decay of rplN mRNA from the spc operon was accelerated in the presence of rifampicin; the average life was estimated to be six minutes during exponential growth in LB medium, and 2.8 minutes in the presence of rifampicin. (4) The decay of the rplN section of mRNA from the spc-lac operon fusion was coupled to the decay of the downstream lacZ mRNA section and was strongly inhibited (i.e. partially blocked) in the presence of rifampicin. These results show that the decay of spc mRNA differs in some important aspects from the decay of lac mRNA and support the retroregulation model. Moreover, the results indicate that rifampicin can have a significant and selective impact on the kinetics of both mRNA translation and decay.

Expression of lacZ from the Promoter of theEscherichia coli spc Operon Cloned into Vectors Carrying the W205 trp-lac Fusion

Expression of lacZ from the Promoter of theEscherichia coli spc Operon Cloned into Vectors Carrying the W205 trp-lac Fusion

Sequencing and analysis of the Thermus thermophilus ribosomal protein gene cluster equivalent to the spectinomycin operon

To assess the organization of the Thermus thermophilus ribosomal protein genes, a fragment of DNA containing the complete S10 region and ten ribosomal protein genes of the spc region was cloned, using an oligonucleotide coding for the N-terminal amino acid (aa) sequence of T. thermophilus S8 protein as hybridization probe. The nucleotide sequence of a 4290 bp region between the rps17 and rpl15 genes was determined. Comparative analysis of this gene cluster showed that the gene arrangement (S17, L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15) is identical to that of eubacteria. However, T. thermophilus ribosomal protein genes corresponding to the Escherichia coli S10 and spc operons are not resolved into two clusters: the stop codon of the rps17 gene (the last gene of the S10 operon in E. coli) and the start codon of the rpl14 gene (the first gene of the spc operon in E. coli) overlap. Most genes, except the rps14- rps8 intergenic spacer (69 bp), are separated by very short (only 3–7 bp) spacer regions or partially overlapped. The deduced aa sequences of T. thermophilus proteins share about 51–100% identities with the sequences of homologous proteins from thermophile Thermus aquaticus and Thermotoga maritima and 27–70% identities with the sequences of their mesophile counterparts.

A model for the evolution of the plastid sec apparatus inferred from secY gene phylogeny.

Plastids possess a bacteria-like sec apparatus that is involved in protein import into the thylakoid lumen. We have analyzed one of the genes essential for this process, secY. A secY gene from the unicellular red alga Cyanidium caldarium was found to be transcriptionally active, demonstrating for the first time that secY is functional in a plastid. Unlike the situation seen in bacteria the C. caldarium gene is transcribed monocistronically, despite the fact that it is part of a large ribosomal gene cluster that resembles bacterial spc operons. A molecular phylogeny is presented for 8 plastid-encoded secY genes, four of which have not been published yet. In this analysis plastid secY genes fall into two classes. One of these, comprising of genes from multicellular red algae and Cryptophyta, clusters in a neighbour-joining tree with a cyanobacterial counterpart. Separated from the aforesaid are secY genes from Chromophyta, Glaucocystophyta and a unicellular red alga. All plastid and cyanobacterial sequences are located on the same branch, separated from bacterial homologues. We postulate that the two classes of secY genes are paralogous, i.e. their gene products are involved in different protein translocation processes. Based on this assumption a model for the evolution of the plastid sec apparatus is presented.

Direct linkage of str-, S10- and spc-related gene clusters in Thermus thermophilus HB8, and sequences of ribosomal proteins L4 and S10

We have analyzed the genomic region harboring str-S10- spc-related gene clusters in the thermophilic bacterium, Thermus thermophilus (Tt) HB8. This study was initiated for the purpose of isolating the gene encoding ribosomal (r-) protein S10 which is assumed to be involved in the antitermination of transcription at the rRNA-encoding genes in Bacteria. The S10-related gene cluster encodes the same set of r-proteins as in Escherichia coli. However, the gene coding for elongation factor Tu (the last gene of the str operon in E. coli) is separated by only eight nucleotides (nt) fro the gene encoding r-protein S10 (the first gene of the S10 operon in E. coli), and the genes encoding r-protein S17 (the last gene of the S10 operon in E. coli) and L14 (the first gene of the spc operon in E. coli) overlap. This suggests that all three gene clusters are contranscribed from a single promoter preceding the str-related operon. In addition, we determined the complete nt sequences of the Tt genes encoding r-proteins L4 and S10. Tt L4 shows the lowest degree of conservation among the known L4 r-proteins from Bacteria. Tt S10 has the highest proportion of basic amino acids (aa) and the lowest number of acidic aa, as compared with its homologues from Bacteria and Archaea, which might be related to its possible role in binding to the boxA RNA of nascent rRNA transcripts at high temperatures.

Conserved gene clusters in the highly rearranged chloroplast genomes of Chlamydomonas moewusii and Chlamydomonas reinhardtii.

We have extended to about 75 the number of genes mapped on the Chlamydomonas moewusii and Chlamydomonas reinhardtii chloroplast DNAs (cpDNAs) by partial sequencing of the very closely related C. eugametos and C. moewusii cpDNAs and by hybridizations with Chlamydomonas chloroplast gene-specific sequences. Only four of these genes (tscA and three reading frames) have not been identified in any other algal cpDNAs and thus may be specific to Chlamydomonas. Although the C. moewusii and C. reinhardtii cpDNAs differ by complex sequence rearrangements, 38 genes scattered throughout the genome define 12 conserved clusters of closely linked loci. Aside from the rRNA operon, four of these gene clusters share similarity to evolutionarily primitive operons found in other cpDNAs, representing in fact remnants of these operons. Our results thus indicate that most of the ancestral bacterial operons that characterize the chloroplast genome organization of land plants and early-diverging photosynthetic eukaryotes have been disrupted before the emergence of the polyphyletic genus Chlamydomonas. All gene rearrangements between the C. moewusii and C. reinhardtii cpDNAs, with the exception of those accounting for the relocations of atpA, psbI and rbcL, occurred within corresponding regions of the genome. One of these rearrangements seems to have led to disruption of the ancestral region containing rpl23, rpl2, rps19, rpl16, rpl14, rpl5, rps8 and the psaA exon 1. This gene cluster, which bears striking similarity to the Escherichia coli S10 and spc operons, spans a continuous DNA segment in C. reinhardtii, while it maps to two separate fragments in C. moewusii.

The binding site for ribosomal protein S8 in 16S rRNA and spc mRNA from Escherichia coli: minimum structural requirements and the effects of single bulged bases on S8-RNA interaction.

Through specific interactions with rRNA and mRNA, ribosomal protein S8 of Escherichia coli plays a central role in both assembly of the 30S ribosomal subunit and translational regulation of spc operon expression. To better understand S8-RNA association, we have measured the affinity of S8 for a number of variants of its rRNA and mRNA binding sites prepared by in vitro transcription or chemical synthesis. With the aid of site-directed deletions, we demonstrate that an imperfect, 33-nucleotide helical stem encompassing nucleotides 588-603 and 635-651 possesses all of the structural information necessary for specific binding of S8 to the 16S rRNA. This segment consists of two short duplexes that enclose a conserved, asymmetric internal loop which contains features crucial for protein recognition. The S8 binding site in spc operon mRNA is very similar in both primary and secondary structure to that in 16S rRNA except for the presence of two single bulged bases in one of the duplex segments. In addition, the apparent association constant for the S8-mRNA interaction is approximately fivefold less than that for the S8-rRNA interaction. We show that the difference in affinity can be attributed to the effects of the bulged bases. Deletion of the bulged bases from the mRNA site increases its affinity for S8 to a level similar to that of the rRNA, whereas insertion of single-base bulges at equivalent positions within the rRNA site reduces its affinity for S8 to a value typical of the mRNA. Single-base bulges in the proximity of essential recognition features are therefore capable of modulating the strength of protein-RNA interactions.

Modulation of the spc operon affects growth and protein secretion in Bacillus subtilis

A proximal segment of B. subtilis secY gene was placed under the control of the inducible spac promoter/Lac repressor system. This fusion was integrated into the chromosomal spc operon of B. subtilis via Campbell-like reciprocal recombination. The growth of the resulting strain was strongly IPTG dependent. With staphylokinase and alpha-amylase as reporter proteins it was found, that the protein secretion capacity of this strain was correlated to the conditions of repression or induction of the chromosomal spac promoter.