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PubMed2004

Evaluation of the rrn operon copy number in Bifidobacterium using real-time PCR.

AIMS: A real-time PCR-based method was developed to evaluate the Bifidobacterium rRNA operon copy number. As a result of their repetitive nature, rRNA operons are very suitable targets for chromosomal integration of heterologous genes. METHODS AND RESULTS: The rrn operon multiplicity per chromosome was determined by real-time PCR quantification of the 16S rRNA amplicons obtained from genomic DNA. The values obtained in several bifidobacterial strains of human origin ranged from 1 to 5. The reliability of the method developed was confirmed by Southern hybridization technique. CONCLUSIONS: In the Bifidobacterium genus the rrn operon copies showed variability at species and strain level. The identification of Bifidobacterium strains with high rRNA multiplicity allowed the selection of potential hosts for chromosomal integration. SIGNIFICANCE AND IMPACT OF THE STUDY: The methodology here proposed represents a rapid, reliable and sensitive new tool for the quantification of rrn operon copy number in bacteria.

Bifidobacterium↗

PubMed1996

Variable numbers of rRNA gene operons in Bacillus cereus strains.

Ribosomal RNA operon organisation was analysed in two Bacillus cereus strains of different chromosome size, ATCC 10987 (5.4 Mb) and F0837/76 (2.4 Mb). We estimated that there were twelve and nine copies of the rRNA operons in these two strains, respectively. In B. cereus ATCC 10987 six rRNA operons were less than 10 kb apart, while in B. cereus F0837/76 four rRNA operons were similarly clustered. The origin of replication was located in the vicinity of a rRNA operon in both strains.

Bacillus cereus↗

PubMed2002

Analysis of the zwf-pgl-eda-operon in Pseudomonas putida strains H and KT2440.

A 3.9-kb fragment of the genome of Pseudomonas putida H, containing the complete zwf-pgl-eda-operon, encoding glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase and 2-keto-3-deoxy-6-phosphogluconate-aldolase, respectively, and part of the divergently transcribed regulatory gene, hexR, was cloned and analyzed. The nucleotide sequences of these genes showed high similarities to the corresponding DNA sequences of P. putida KT2440 and also to sequences of Pseudomonas aeruginosa PAO1. Derivatives of strains H and KT2440, containing transcriptional lacZ fusions to P(zwf) were generated and used to study the expression of these operons. In both strains, this operon was induced by carbohydrates such as glucose, gluconate, fructose and glycerol. The transcription rate of the zwf-pgl-eda-operon was found to be about three times higher in the KT2440 background than in strain H. In both strains the induction of the zwf-pgl-eda-operon by carbohydrates during growth on carboxylic acids was not affected by carbon catabolite repression.

Aldehyde-Lyases↗

PubMed2006

Operon prediction for sequenced bacterial genomes without experimental information.

Various computational approaches have been proposed for operon prediction, but most algorithms rely on experimental or functional data that are only available for a small subset of sequenced genomes. In this study, we explored the possibility of using phylogenetic information to aid in operon prediction, and we constructed a Bayesian hidden Markov model that incorporates comparative genomic data with traditional predictors, such as intergenic distances. The prediction algorithm performs as well as the best previously reported method, with several significant advantages. It uses fewer data sources and so it is easier to implement, and the method is more broadly applicable than previous methods--it can be applied to essentially every gene in any sequenced bacterial genome. Furthermore, we show that near-optimal performance is easily reached with a generic set of comparative genomes and does not depend on a specific relationship between the subject genome and the comparative set. We applied the algorithm to the Bacillus anthracis genome and found that it successfully predicted all previously verified B. anthracis operons. To further test its performance, we chose a predicted operon (BA1489-92) containing several genes with little apparent functional relatedness and tested their cotranscriptional nature. Experimental evidence shows that these genes are cotranscribed, and the data have interesting implications for B. anthracis biology. Overall, our findings show that this algorithm is capable of highly sensitive and accurate operon prediction in a wide range of bacterial genomes and that these predictions can lead to the rapid discovery of new functional relationships among genes.

Algorithms↗

PubMed1999

The groESL chaperone operon of Lactobacillus johnsonii.

The Lactobacillus johnsonii VPI 11088 groESL operon was localized on the chromosome near the insertion element IS1223. The operon was initially cloned as a series of three overlapping PCR fragments, which were sequenced and used to design primers to amplify the entire operon. The amplified fragment was used as a probe to recover the chromosomal copy of the groESL operon from a partial library of L. johnsonii VPI 11088 (NCK88) DNA, cloned in the shuttle vector pTRKH2. The 2,253-bp groESL fragment contained three putative open reading frames, two of which encoded the ubiquitous GroES and GroEL chaperone proteins. Analysis of the groESL promoter region revealed three transcription initiation sites, as well as three sets of inverted repeats (IR) positioned between the transcription and translation start sites. Two of the three IR sets bore significant homology to the CIRCE elements, implicated in negative regulation of the heat shock response in many bacteria. Northern analysis and primer extension revealed that multiple temperature-sensitive promoters preceded the groESL chaperone operon, suggesting that stress protein production in L. johnsonii is strongly regulated. Maximum groESL transcription activity was observed following a shift to 55 degrees C, and a 15 to 30-min exposure of log-phase cells to this temperature increased the recovery of freeze-thawed L. johnsonii VPI 11088. These results suggest that a brief, preconditioning heat shock can be used to trigger increased chaperone production and provide significant cross-protection from the stresses imposed during the production of frozen culture concentrates.

Amino Acid Sequence↗

PubMed2000

Identification and characterization of a bile acid 7alpha-dehydroxylation operon in Clostridium sp. strain TO-931, a highly active 7alpha-dehydroxylating strain isolated from human feces.

Clostridium sp. strain TO-931 can rapidly convert the primary bile acid cholic acid to a potentially toxic compound, deoxycholic acid. Mixed oligonucleotide probes were used to isolate a gene fragment encoding a putative bile acid transporter from Clostridium sp. strain TO-931. This DNA fragment had 60% nucleotide sequence identity to a known bile acid transporter gene from Eubacterium sp. strain VPI 12708, another bile acid-7alpha-dehydroxylating intestinal bacterium. The DNA (9.15 kb) surrounding the transporter gene was cloned from Clostridium sp. strain TO-931 and sequenced. Within this larger DNA fragment was a 7.9-kb region, containing six successive open reading frames (ORFs), that was encoded by a single 8.1-kb transcript, as determined by Northern blot analysis. The gene arrangement and DNA sequence of the Clostridium sp. strain TO-931 operon are similar to those of a Eubacterium sp. strain VPI 12708 bile acid-inducible operon containing nine ORFs. Several genes in the Eubacterium sp. strain VPI 12708 operon have been shown to encode products required for bile acid 7alpha-dehydroxylation. In Clostridium sp. strain TO-931, genes potentially encoding bile acid-coenzyme A (CoA) ligase, 3alpha-hydroxysteroid dehydrogenase, bile acid 7alpha-dehydratase, bile acid-CoA hydrolase, and a bile acid transporter were similar in size and exhibited amino acid homology to similar gene products from Eubacterium sp. strain VPI 12708 (encoded by baiB, baiA, baiE, baiF, and baiG, respectively). However, no genes similar to Eubacterium sp. strain VPI 12708 biaH or baiI were found in the Clostridium sp. strain TO-931 bai operon, and the two putative Eubacterium sp. strain VPI 12708 genes, baiC and baiD, were arranged in one continuous ORF in Clostridium sp. strain TO-931. Intergene regions showed no significant DNA sequence similarity, but primer extension analysis identified a region 115 bp upstream from the first ORF that exhibited 58% identity to a bai operator/promoter region identified in Eubacterium sp. strain VPI 12708. These results indicate that the gene organization, gene product amino acid sequences, and promoters of the bile acid-inducible operons of Clostridium sp. strain TO-931 and Eubacterium sp. strain VPI 12708 are highly conserved.

3-Hydroxysteroid Dehydrogenases↗

PubMed2005

Identification of a positive transcription regulatory element within the coding region of the nifLA operon in Azotobacter vinelandii.

Nitrogen fixation in Azotobacter vinelandii is regulated by the nifLA operon. NifA activates the transcription of nif genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels. However, transcriptional regulation of the nifLA operon of A. vinelandii itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the nifLA operon, showing it to be similar to the sigma54-dependent promoters. We also identified a positive cis-acting regulatory element (+134 to +790) of the nifLA operon within the coding region of the nifL gene of A. vinelandii. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2 (+745 to +765), are involved in regulating the nifLA promoter activity. The absence of NtrC-like binding sites in the upstream region of the nifLA operon in A. vinelandii makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within the BglII-BglII fragment may play, in part; an important role in nifLA regulation in A. vinelandii.

Azotobacter vinelandii↗

PubMed2005

Differential expression of the two kdp operons in the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31.

In several types of bacteria, the Kdp ATPase (comprising of the KdpABC complex) is an inducible, high-affinity potassium transporter that scavenges K+ from the environment. The cyanobacterium Anabaena sp. strain L-31 showed the presence of not one but two distinct kdp operons in its genome. The kdp1 consisted of kdpA1B1G1C1D genes, whereas the kdp2 contained the kdpA2B2G2C2 genes. Among the regulatory genes, the kdpD open reading frame of Anabaena sp. strain L-31 was truncated compared to the kdpD of other bacteria, whereas a kdpE-like gene was absent in the vicinity of the two kdp operons. In response to K+ limitation (<0.05 mM external K+), only kdp2 (and not kdp1) expression could be detected as a 5.3-kb transcript on Northern blots, indicating that kdpA2B2G2C2 genes constitute a polycystronic operon. Unlike E. coli, addition of osmolytes like NaCl, or a change in pH of the medium did not enhance the kdp expression in Anabaena sp. strain L-31. Interestingly, the Anabaena sp. strain L-31 kdp2 operon was strongly induced in response to desiccation stress. The addition of K+ to K+ -starved cultures resulted in repression and degradation of kdp2 transcripts. Our results clearly show that kdp2 is the major kdp operon expressed in Anabaena sp. strain L-31 and may play an important role in adaptation to K+ limitation and desiccation stress.

Adenosine Triphosphatases↗

PubMed1988

Distribution and degree of heterogeneity of the afimbrial-adhesin-encoding operon (afa) among uropathogenic Escherichia coli isolates.

The afimbrial adhesin (AFA-I) from a pyelonephritic Escherichia coli isolate (KS52) is a mannose-resistant, P-independent, X-binding adhesin, expressed by the afa-1 operon. It is distinct from the E. coli X-binding adhesins with M and S specificity. A total of 138 E. coli isolates belonging to various serotypes, mostly from urinary tract infections, were screened for the presence of DNA sequences related to the afa operon and for the expression of an X-adhesin able to mediate mannose-resistant hemagglutination (MRHA) and adhesion to uroepithelial cells. Fifteen strains were shown to harbor DNA sequences related to the AFA-I-encoding operon, and 13 of them expressed an X-adhesin. Using as probes different DNA segments of the AFA-I-encoding operon in Southern experiments, we demonstrated that only three of these clinical isolates contained genetic determinants closely related to those identified in the original afa prototype strain (KS52): presence of the afaA, afaB, afaC, afaD, and afaE genes associated with the expression of a 16,000-dalton hemagglutinin-adhesin which strongly cross-reacted with AFA-I-specific antibodies. The other E. coli isolates harbored DNA sequences homologous to the afaA, afaB, afaC, and afaD genes, but lacked the sequence corresponding to the adhesin-producing gene afaE; Western blots allowed the detection of polypeptides (15,000, 15,500, or 16,000 daltons) in these strains which cross-reacted with variable intensity with antibodies raised against the denatured AFA-I protein, but did not cross-react with native AFA-I-specific antibodies. Following DNA cloning experiments from chromosomal DNA of two of those strains (A22 and A30), we demonstrated that although the AFA-related operon in A22 and A30 strains lacked the AFA-I adhesin-encoding gene, they synthesized a functional X-adhesin. Thus, strains A22 and A30 encode adhesins designated AFA-II and AFA-III, which were cloned on recombinant plasmids pILL72 and pILL61, respectively. Southern hybridization experiments and Western blot analyses of the 15 AFA-related strains demonstrate the heterogeneity of the genetic sequences encoding the structural adhesin and suggest the bases for the serological diversity of the AFA adhesins.

Adhesins, Escherichia coli↗

PubMed1999

The virulence plasmid-encoded impCAB operon enhances survival and induced mutagenesis in Shigella flexneri after exposure to UV radiation.

Upon exposure to UV radiation, Shigella flexneri SA100 displayed survival and mutation frequencies comparable to those of Escherichia coli AB1157, which contains a functional UmuDC error-prone DNA repair system. Survival of SA100 after UV irradiation was associated with the presence of the 220-kb virulence plasmid, pVP. This plasmid encodes homologues of ImpA and ImpB, which comprise an error-prone DNA repair system encoded on plasmid TP110 that was initially identified in Salmonella typhimurium, and ImpC, encoded upstream of ImpA and ImpB. Although the impB gene was present in representatives of all four species of Shigella, not all isolates tested contained the gene. Shigella isolates that lacked impB were more sensitive to UV radiation than isolates that contained impB. The nucleotide sequence of a 2.4-kb DNA fragment containing the imp operon from S. flexneri SA100 pVP was 96% identical to the imp operon from the plasmid TP110. An SA100 derivative with a mutation in the impB gene had reduced survival following UV irradiation and less UV-induced mutagenesis relative to the parental strain. We also found that S. flexneri contained a chromosomally encoded umuDC operon; however, the umuDC promoter was not induced by exposure to UV radiation. This suggests that the imp operon but not the umuDC operon contributes to survival and induced mutagenesis in S. flexneri following exposure to UV radiation.

Bacterial Proteins↗

PubMed2000

Genetic characterization of a Streptococcus mutans LraI family operon and role in virulence.

Proteins belonging to the LraI (for "lipoprotein receptor antigen") family function as adhesins in several streptococci, as a virulence factor for endocarditis in at least one of these species, and potentially as metal transporters in many bacteria. We have identified and characterized the chromosomal locus containing the LraI family gene (designated sloC) from Streptococcus mutans, an agent of dental caries and endocarditis in humans. Northern blot analysis indicated that sloC is cotranscribed with three other genes. As with other LraI operons, the sloA and sloB genes apparently encode components of an ATP-binding cassette transport system. The product of the fourth gene, sloR, has homology to the metal-dependent regulator from Corynebacterium diphtheriae, DtxR. A potential binding site for SloR was identified upstream from the sloABCR operon and was conserved upstream from LraI operons in several other streptococci. Potential SloR homologs were identified in the unfinished genomic sequences from two of these, S. pneumoniae and S. pyogenes. Mutagenesis of sloC in S. mutans resulted in apparent loss of expression of the entire operon as assessed by Northern blot analysis. The sloC mutant was indistinguishable from its wild-type parent in a gnotobiotic rat model of caries but was significantly less virulent in a rat model of endocarditis. Virulence for endocarditis was restored by correction of the sloC mutation but not by provision of the sloC gene in trans, suggesting that virulence requires the expression of other genes in the sloC operon.

ATP-Binding Cassette Transporters↗

PubMed2006

Characterization of the primary starch utilization operon in the obligate anaerobe Bacteroides fragilis: Regulation by carbon source and oxygen.

The opportunistic pathogen Bacteroides fragilis is a commensal organism in the large intestine, where it utilizes both dietary and host-derived polysaccharides as a source of carbon and energy. In this study, a four-gene operon required for starch utilization was identified. The operon also was found to be oxygen responsive and thus was designated osu for oxygen-induced starch utilization. The first three genes in the operon were predicted to encode outer membrane proteins involved in starch binding, and a fourth gene, osuD, encoded an amylase involved in starch hydrolysis. Insertional mutation of the osuA gene (Omega osuA) resulted in the inability to utilize starch or glycogen and an insertional mutation into the osuD gene (Omega osuD) was severely impaired for growth on starch media. Transcriptional studies indicated that maltose, maltooligosaccharides, and starch were inducers of osu expression and that maltose was the strongest inducer. A transcriptional activator of osuABCD, OsuR, was identified and found to mediate maltose induction. The Omega osuA and Omega osuD mutants were able to grow on maltose but not starch, whereas a mutation in osuR abolished growth on both substrates, indicating that additional genes under the control of OsuR are needed for maltose utilization. The osuABCD operon also was induced by exposure to oxygen and was shown to be part of the oxidative stress response important for aerotolerance of B. fragilis. Transcriptional analyses showed that osuA was induced 20-fold by oxygen, but OsuR was not required for this activation. Analysis of osu mutants suggested that expression of the operon was important for survival during oxygen exposure but not to hydrogen peroxide stress.

Amylases↗

PubMed2006

The pio operon is essential for phototrophic Fe(II) oxidation in Rhodopseudomonas palustris TIE-1.

Phototrophic Fe(II)-oxidizing bacteria couple the oxidation of ferrous iron [Fe(II)] to reductive CO(2) fixation by using light energy, but until recently, little has been understood about the molecular basis for this process. Here we report the discovery, with Rhodopseudomonas palustris TIE-1 as a model organism, of a three-gene operon, designated the pio operon (for phototrophic iron oxidation), that is necessary for phototrophic Fe(II) oxidation. The first gene in the operon, pioA, encodes a c-type cytochrome that is upregulated under Fe(II)-grown conditions. PioA contains a signal sequence and shares homology with MtrA, a decaheme c-type cytochrome from Shewanella oneidensis MR-1. The second gene, pioB, encodes a putative outer membrane beta-barrel protein. PioB is a homologue of MtrB from S. oneidensis MR-1. The third gene, pioC, encodes a putative high potential iron sulfur protein (HiPIP) with a twin-arginine translocation (Tat) signal sequence and is similar to the putative Fe(II) oxidoreductase (Iro) from Acidithiobacillus ferrooxidans. Like PioA, PioB and PioC appear to be secreted proteins. Deletion of the pio operon results in loss of Fe(II) oxidation activity and growth on Fe(II). Complementation studies confirm that the phenotype of this mutant is due to loss of the pio genes. Deletion of pioA alone results in loss of almost all Fe(II) oxidation activity; however, deletion of either pioB or pioC alone results in only partial loss of Fe(II) oxidation activity. Together, these results suggest that proteins encoded by the pio operon are essential and specific for phototrophic Fe(II) oxidation in R. palustris TIE-1.

Base Sequence↗

PubMed2006

Chlamydial type III secretion system is encoded on ten operons preceded by sigma 70-like promoter elements.

Many gram-negative bacterial pathogens employ type III secretion systems for infectious processes. Chlamydiae are obligate intracellular bacteria that encode a conserved type III secretion system that is likely requisite for growth. Typically, genes encoding type III secretion systems are located in a single locus; however, for chlamydiae these genes are scattered throughout the genome. Little is known regarding the gene regulatory mechanisms for this essential virulence determinant. To facilitate identification of cis-acting transcriptional regulatory elements, the operon structure was determined. This analysis revealed 10 operons that contained 37 genes associated with the type III secretion system. Linkage within these operons suggests a role in type III secretion for each of these genes, including 13 genes encoding proteins with unknown function. The transcriptional start site for each operon was determined. In conjunction with promoter activity assays, this analysis revealed that the type III secretion system operons encode sigma(70)-like promoter elements. Transcriptional initiation by a sigma factor responsible for constitutive gene expression indicates that undefined activators or repressors regulate developmental stage-specific expression of chlamydial type III secretion system genes.

Bacterial Proteins↗

PubMed1973

Mapping of asporogenous mutations of Bacillus subtilis: a minimum estimate of the number of sporeulation operons.

Sporulation-specific mutations in Bacillus subtilis have been mapped by transduction and transformation. The mutations caused blocks at stages 0, II, III, and IV of sporulation; more than one phenotype was found for each of these stages. On the basis of the criteria used to define a sporulation operon, a minimum estimate could be made of the number of operons activated during sporulation. Nine operons were identified for stage 0, eight for stage II, five for stage III, and six for stage IV. It is probable that several of these 28 operons are activated in groups so that the number of steps in the dependent sequence of sporulation events should turn out to be less than the number of operons.

Alkaline Phosphatase↗

PubMed1974

Mutations in the L-arabinose operon of Escherichia coli B-r that result in hypersensitivity to catabolite repression.

Two independent mutants resistant to l-arabinose inhibition only in the presence of d-glucose were isolated from an l-arabinose-sensitive strain containing the araD139 mutation. Preliminary mapping studies indicate that these mutations are closely linked to the araIOC region. Addition of d-glucose to growing cultures of these mutants results in a 95 to 98% repression of ara operon expression, as compared to a 50% repression of the parental control. Since cultures of both mutant and parental strains undergo a 50% repression of lac operon expression upon addition of glucose, the hypersensitivity to catabolite repression exhibited by these mutants is specific for the ara operon. Addition of cyclic adenosine monophosphate reverses the catabolite repression of the ara operon in both mutant and parent strains to 70 to 80% of the control. It is suggested that in these mutants the affinity of the ara operon initiator region for the cAMP-catabolite-activator protein complex may have been altered.

Arabinose↗

PubMed1974

Statistical estimate of the total number of operons specific for Bacillus subtilis sporulation.

As an alternative to exhaustive mapping, an attempt has been made to obtain a rough estimate of the total number of sporulation operons by statistical treatment. Sixteen sporulation mutants taken at random were characterized biochemically and morphologically. The mutations they contained were mapped to determine whether they fell into any one of 23 known operons. From the proportion that do so (10/16), it is calculated that the most probable number of sporulation operons is 37 (68% confidence limits of 31 and 46). If allowance is made for the fact that two of the operons apparently contain mutagenic "hot spots" and the calculation is amended accordingly, the most probable numbers of operons becomes 42 (limits 33 and 59).

Bacillus subtilis↗

PubMed1977

Threonyl-transfer ribonucleic acid synthetase and the regulation of the threonine operon in Escherichia coli.

Two threonine-requiring mutants with derepressed expression of the threonine operon were isolated from an Escherichia coli K-12 strain containing two copies of the thr operon. One of them carries a leaky mutation in ilvA (the structural gene for threonine deaminase), which creates an isoleucine limitation and therefore derepression of the thr operon. In the second mutant, the enzymes of the thr operon were not repressed by threonine plus isoleucine; the threonyl-transfer ribonucleic acid(tRNA) synthetase from this mutant shows an apparent Km for threonine 200-fold higher than that of the parental strain. The gene, called thrS, coding for threonyl-tRNA synthetase was located around 30 min on the E. coli map. The regulatory properties of this mutant imply the involvement of charged threonyl-tRNA or threonyl-tRNA synthetase in the regulation of the thr operon.

Amino Acyl-tRNA Synthetases↗