Molecular cloning and characterization of Borrelia burgdorferi rpoB

Borrelia burgdorferi rpoB, the gene encoding the β-subunit of RNA polymerase, has been cloned and sequenced. The full-length gene encodes a protein of 1154 amino acids with a calculated molecular mass of 129.8 kDa. The amino-acid sequence is 49% identical to the corresponding protein from Escherichia coli. B. burgdorferi rpoB is a component of a gene cluster, which includes rplJ, rplL and rpoC. A temperature-sensitive E. coli rpoB mutant could be complemented by introduction of the B. burgdorferi gene, indicating that the B. burgdorferi rpoB is expressed in E. coli and the β-subunit can be assembled into functional holoenzyme. The wild-type amino-acid sequence of the B. burgdorferi β-subunit is consistent with those of spontaneously arising rifampicin-resistant mutants of E. coli and Mycobacterium tuberculosis at certain critical residues. This suggests that the natural resistance of B. burgdorferi to rifampicin may be due to the primary amino-acid sequence of its β-subunit.     

Molecular serotyping of Salmonella enterica by complete rpoB gene sequencing

Serotyping has been the gold standard for identifying Salmonella, but it requires large amounts of standard antisera. Multilocus sequence typing (MLST) has been applied to identify Salmonella serovars, but the recombination of 4–7 housekeeping genes and multiple analytic steps diminish its applicability. In the present study, we determined the complete sequences of the RNA polymerase beta subunit gene (rpoB) and 7 housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA) for 76 strains of 33 Salmonella enterica serovars and conducted phylogenetic analyses together with the corresponding gene sequences of 24 reference strains registered in the GenBank database. Based on the phylogenetic analyses, 100 strains from 40 serovars and 91 strains from 37 serovars were classified into 60 rpoB (RST) and 49 multilocus sequence types (ST), respectively. The nucleotide similarities were 98.8–100% and 96.9–100% for the complete rpoB gene and the seven concatenated housekeeping genes, respectively. The strains of 35 and 30 serovars formed serovar-specific branches or clusters in the rpoB and housekeeping gene phylogenetic trees, respectively. Therefore, complete rpoB gene sequencing and phylogenetic analysis may be a useful method for identifying Salmonella serovars that is a simpler, more cost-effective, and less time-consuming alternative or complementary method to MLST and conventional serotyping.     

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.     

rpoB gene as a novel molecular marker to infer phylogeny in Planctomycetales

The 16S rRNA gene has been used in the last decades as a gold standard for determining the phylogenetic position of bacteria and their taxonomy. It is a well conserved gene, with some variations, present in all bacteria and allows the reconstruction of genealogies of microorganisms. Nevertheless, this gene has its limitations when inferring phylogenetic relationships between closely related isolates. To overcome this problem, DNA–DNA hybridization appeared as a solution to clarify interspecies relationships when the sequence similarity of the 16S rRNA gene is above 97 %. However, this technique is time consuming, expensive and laborious and so, researchers developed other molecular markers such as sequencing of housekeeping or functional genes for accurate determination of bacterial phylogeny. One of these genes that have been used successfully, particularly in clinical microbiology, codes for the beta subunit of the RNA polymerase (rpoB). The rpoB gene is sufficiently conserved to be used as a molecular clock, it is present in all bacteria and it is a mono-copy gene. In this study, rpoB gene sequencing was applied to the phylum Planctomycetes. Based on the genomes of 19 planctomycetes it was possible to determine the correlation between the rpoB gene sequence and the phylogenetic position of the organisms at a 95–96 % sequence similarity threshold for a novel species. A 1200-bp fragment of the rpoB gene was amplified from several new planctomycetal isolates and their intra and inter-species relationships to other members of this group were determined based on a 96.3 % species border and 98.2 % for intraspecies resolution.     

RifR mutations in the beginning of the Escherichia coli rpoB gene

In Escherichia coli, mutations conferring rifampicin (Rif) resistance map to the rpoB gene, which encodes the 1342-amino acid β subunit of RNA polymerase. Almost all sequenced RifR mutations occur within the Rif region, encompassing rpoB codons 500–575. A strong Rif^R mutation lying outside the Rif region, which changed Val¹⁴⁶ to Phe was previously reported, but was not recovered in subsequent studies. Here, we used site-directed mutagenesis followed by selection on Rif to search for Rif^R mutations in the evolutionarily conserved segment of rpoB around codon 146. Strong Rif^R mutations were obtained when Val¹⁴⁶ was mutated, and several weak Rif^R mutations were also isolated near position 146. The results define a new, N-terminal cluster of Rif^R mutations, in addition to the classical central Rif region.     

Utilization of the rpoB Gene as a Specific Chromosomal Marker for Real-Time PCR Detection of Bacillus anthracis

The potential use of Bacillus anthracis as a weapon of mass destruction poses a threat to humans, domesticated animals, and wildlife and necessitates the need for a rapid and highly specific detection assay. We have developed a real-time PCR-based assay for the specific detection of B. anthracis by taking advantage of the unique nucleotide sequence of the B. anthracis rpoB gene. Variable region 1 of the rpoB gene was sequenced from 36 Bacillus strains, including 16 B. anthracis strains and 20 other related bacilli, and four nucleotides specific for B. anthracis were identified. PCR primers were selected so that two B. anthracis-specific nucleotides were at their 3′ ends, whereas the remaining bases were specific to the probe region. This format permitted the PCR reactions to be performed on a LightCycler via fluorescence resonance energy transfer (FRET). The assay was found to be specific for 144 B. anthracis strains from different geographical locations and did not cross-react with other related bacilli (175 strains), with the exception of one strain. The PCR assay can be performed on isolated DNA as well as crude vegetative cell lysates in less than 1 h. Therefore, the rpoB-FRET assay could be used as a new chromosomal marker for rapid detection of B. anthracis.     

Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types

The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I–III). To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C. butyricum strain BL6340. The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C. Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C. butyricum strain BL6340. However, the genes of ORF-X₁ (435 bp) and ORF-X₂ (partially sequenced) were present just upstream of that of P47. The orientation of these genes was in inverted direction to that of p47. The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex.     

Developing a Novel Approach of rpoB Gene as a Powerful Biomarker for the Environmental Microbial Diversity

Use of the rpoB gene (the encoding the β-subunit of RNA Polymerase gene), a potential alternative biomarker to the 16S rRNA gene, has been limited to environmental microbial investigation for a long time because of the lack of effective primers. Here we developed a novel rpoB gene-based approach using a newly designed primer pair and tested it in three different environmental water samples with the traditional library method. The results showed that our novel approach presented different microbial diversity patterns from the different environmental samples. Compared to previous rpoB gene based reports, the first retrieved groups in our approach included mainly α-Proteobacteria, δ-Proteobacteria, Planctomycetes, Verrucomicrobia, Firmicutes, Chlorofexi and Actinobacteria, which greatly expanded the potential ability of the rpoB gene approach for environmental surveys. Most importantly, the use of the traditional clone library approach with the novel rpoB gene primers greatly supplement the microbial diversity based on the 16S rRNA gene approach with the universal primer pair (27f and 1492r), at all phylum, class, order, family and genus levels, indicating a powerful complementary method and a potential alternative biomarker of the current popular NGS (next-generation sequences) technologies for the environmental microbial investigation.     

A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity

Background

The 16S rRNA gene is the gold standard in molecular surveys of bacterial and archaeal diversity, but it has the disadvantages that it is often multiple-copy, has little resolution below the species level and cannot be readily interpreted in an evolutionary framework. We compared the 16S rRNA marker with the single-copy, protein-coding rpoB marker by amplifying and sequencing both from a single soil sample. Because the higher genetic resolution of the rpoB gene prohibits its use as a universal marker, we employed consensus-degenerate primers targeting the Proteobacteria.

Methodology/Principal Findings

Pyrosequencing can be problematic because of the poor resolution of homopolymer runs. As these erroneous runs disrupt the reading frame of protein-coding sequences, removal of sequences containing nonsense mutations was found to be a valuable filter in addition to flowgram-based denoising. Although both markers gave similar estimates of total diversity, the rpoB marker revealed more species, requiring an order of magnitude fewer reads to obtain 90% of the true diversity. The application of population genetic methods was demonstrated on a particularly abundant sequence cluster.

Conclusions/Significance

The rpoB marker can be a complement to the 16S rRNA marker for high throughput microbial diversity studies focusing on specific taxonomic groups. Additional error filtering is possible and tests for recombination or selection can be employed.     

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

Aim:  To investigate the applicability of rpoB gene, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP‐ and BOX‐polymerase chain reaction) were also used. Methods and Results:  rpoB DNA (458 bp) were amplified from 21 Geobacillus‐ and Bacillus type strains, producing different BOX‐ and REP‐PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. Conclusion:  BOX‐ and REP‐PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. Significance and Impact of the Study:  The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.     

Transformation of Rickettsia prowazekii to Rifampin Resistance

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism’s unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rif^s) Madrid E strain and a rifampin-resistant (Rif^r) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of the R. prowazekii RNA polymerase β subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rif^s Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rif^r region of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.