Macromolecular Synthesis Operon Research Articles

Open Questions about the Roles of DnaA, Related Proteins, and Hyperstructure Dynamics in the Cell Cycle.

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function. In these dynamics, hyperstructures can (1) emit and receive signals or (2) fuse and separate from one another. We ask whether the DnaA-based initiation hyperstructure acts as a logic gate receiving information from the membrane, the chromosome, and metabolism to trigger replication; we try to phrase some of these questions in terms of DNA supercoiling, strand opening, glycolytic enzymes, SeqA, ribonucleotide reductase, the macromolecular synthesis operon, post-translational modifications, and metabolic pools. Finally, we ask whether, underpinning the regulation of the cell cycle, there is a physico-chemical clock inherited from the first protocells, and whether this clock emits a single signal that triggers both chromosome replication and cell division.

Simultaneous enumeration of Cronobacter sakazakii and Staphylococcus aureus in powdered infant foods through duplex TaqMan real-time PCR

Cronobacter sakazakii and Staphylococcus aureus are important pathogens. Targeting the species-specific macromolecular synthesis (MMS) operon and thermostable nuclease gene (nuc), a duplex TaqMan real-time PCR (dRT-PCR) assay was developed for their simultaneous enumeration within two kinds of powdered infant foods (PIFs). The assay was highly specific, producing fluorescent signals exclusively from target bacteria. The quantification limits were 29.10 and 8.97 copies μL−1 for standard plasmids containing MMS and nuc fragments. Without enrichment, the dRT-PCR could quantify as low as 102 and 101 cfu mL−1C. sakazakii and S. aureus in both pure culture and spiked PIFs. Quantitative linearity ranged from 102 to 107 cfu mL−1 for C. sakazakii and from 101 to 107 cfu mL−1 for S. aureus. Bacterial counts derived from dRT-PCR were consistent with those calculated from plating for randomly spiked PIFs. The dRT-PCR assay provides a promising culture-independent alternative enabling direct, simultaneous and robust pathogen quantification.

Novel duplex real-time Taqman PCR assay for rapid detection of Enterobacter sakazakii in powdered infant formula

To develop a dulplex real-time Taqman polymerase chain reaction(PCR)assay for the detection of Enterobacter sakazakii. Primers and probes were designed based on the sequences of the macromolecular synthesis(MMS)operon and outer member protein A(ompA). Sensitivity and specificity of the assay were evaluated. Detection limit of the assay in pure culture was 4.3×103 CFU/mL and as few as 2 CFU/100 g of Enterobacter sakazakii could be detected in artificially contaminated infant formula through 24 h of enrichment. All of 15 Enterobacter sakazakii strains were successfully identified,no specific amplification were presented when 24 non-Enterobacter sakazakii were tested. The duplex real time Taqman PCR assay developed in study was sensitivity,specific and rapid. It could be used for detection of Enterobacter sakazakii from infant milk powder for food safety.

Real-time PCR with internal amplification control for the detection of Cronobacter spp. (Enterobacter sakazakii) in food samples

Cronobacter spp. (Enterobacter sakazakii) is an opportunistic pathogen and is linked with life-threatening infections in neonates. The organism has been isolated from a wide variety of foods and environments. In this study, a Taqman real-time PCR assay incorporating an internal amplification control (IAC) was developed and evaluated for specific detection of Cronobacter spp. in foods. Previously reported macromolecular synthesis (MMS) operon sequence was selected for specificity, and 67 bacterial strains, including four strains of Cronobacter spp., were evaluated. All Cronobacter strains were successfully identified, however no cross-reactivity was observed with non-Cronobacter strains. Detection limit of the assay in pure culture and formula infant without enrichment was 1.2 × 103 CFU/ml (1.2 × 101 CFU/assay). After 24 h enrichment in broth, as few as 100 CFU/ml or g of Cronobacter could be detected in artificially contaminated food samples (infant formula, sterilized milk and chicken meat). The detection limit of the real-time PCR assay however remained unaffected in the presence of 108 CFU/ml Salmonella typhimurium in another analysis. A total of 92 food samples were analyzed for the presence of Cronobacter, out of which two pork samples were found as positive by real-time PCR, whereas only one was detected by the ISO standard method. The adjusted real-time PCR assay can be adopted to rapidly detect Cronobacter spp. in food samples with high specificity and sensitivity, and can prevent false negative results by using the IAC.

Genetic analysis of the Staphylococcus epidermidis Macromolecular Synthesis Operon: Serp1129 is an ATP binding protein and sigA transcription is regulated by both σA- and σB-dependent promoters

BackgroundThe highly conserved macromolecular synthesis operon (MMSO) contains both dnaG (primase) and sigA (primary sigma factor). However, in previously evaluated gram-positive species, the MMSO is divergent upstream of dnaG. The MMSO of Bacillus subtilis contains three open reading frames (ORFs) that are differentially regulated by multiple promoters. In conjunction with studies to determine the expression profile of dnaG, the MMSO of Staphylococus epidermidis was characterized.ResultsThe ORFs of S. epidermidis were compared to the previously described MMSO of B. subtilis and two additional ORFs in S. epidermidis, serp1129 and serp1130, were identified. The largest transcript, 4.8 kb in length, was expressed only in exponential growth and encompassed all four ORFs (serp1130, serp1129, dnaG, and sigA). A separate transcript (1.5 kb) comprising serp1130 and serp1129 was expressed in early exponential growth. Two smaller transcripts 1.3 and 1.2 kb in size were detected with a sigA probe in both exponential and post-exponential phases of growth. Western blot analysis correlated with the transcriptional profile and demonstrated that Serp1129 was detected only in the exponential phase of growth. Computational analysis identified that Serp1130 contained a CBS motif whereas Serp1129 contained an ATP/GTP binding motif. Functional studies of Serp1129 demonstrated that it was capable of binding both ATP and GTP. Comparisons with a sigB:dhfr mutant revealed that the 1.3 kb sigA transcript was regulated by a σB-dependent promoter.ConclusionsThese studies demonstrated that the S. epidermidis 1457 MMSO contains two ORFs (serp1129 and serp1130) not described within the B. subtilis MMSO and at least three promoters, one of which is σβ-dependent. The transcriptional regulation of sigA by σB provides evidence that the staphylococcal σB-dependent response is controlled at both the transcriptional and post-transcriptional level. The conservation of serp1129 across multiple gram-positive organisms and its capability to bind ATP and GTP support the need for further investigation of its role in bacterial growth.

Identification of the Macromolecular Synthesis Operon ofOenococcus oeni

The macromolecular synthesis (MMS) operon and two adjacent genes have been sequenced in the wine bacterium <i>Oenococcus oeni</i>. This bacterium is significant for carrying out malolactic fermentation, which is important for flavor maturation of wine. To grow in wine, <i>O. oeni</i> must adapt to low pH (3.0 to 4.0) and ethanol concentrations as high as 16% (v/v). Understanding the regulation of this key operon will enable improvements in starter culture preparation to ensure successful malolactic fermentation. Preliminary analysis has identified several characteristics emblematic of the typical gram-positive MMS operon and has revealed significant differences from bacteria within closely related genera. The <i>O. oeni</i> MMS operon has two genes, <i>dnaG</i> and <i>sigA</i>, located ~6 Kbp downstream from <i>rpsU</i>, the third gene of the MMS operon. They are encoded by a 3.3 kb genome segment ending with a putative stem-loop transcriptional termination structure most similar to that of <i>Lactococcus lactis</i>. A conserved open-reading-frame with identity to genes in other gram-positive bacteria is located downstream of the primary sigma factor and is here named <i>orfCE1</i>. The recently annotated <i>O. oeni</i> genome sequence incompletely describes the organization and sequence of the MMS operon. We describe the architecture of the <i>O. oeni</i> MMS operon and its location relative to adjacent genes. Further, we describe and compare the organization, identity, and phylogenetic relationship of the <i>O. oeni</i> MMS to other gram-positive bacteria.

Rapid, Specific Detection of Enterobacter sakazakii in Infant Formula Using a Real-Time PCR Assay

Enterobacter sakazakii is a rare cause of invasive infection with high mortality rates in neonates. Powdered milk–based infant formulas have been associated with the E. sakazakii–related outbreaks in premature or other immunocompromised infants. In this study, an assay was developed for the specific detection of E. sakazakii in infant formula using an application of the fluorogenic 5′ nuclease assay (TaqMan). A set of primers and probe was designed using the E. sakazakii partial macromolecular synthesis operon: the rpsU gene 3′ end and the primase (dnaG) gene 5′ end. The specificity of the assay was evaluated using 68 Enterobacter and 55 non-Enterobacter strains. The newly developed assay enables us to detect 100 CFU/ml in pure culture and in reconstituted infant formula in 50 cycles of PCR without enrichment. The assay was specific enough to discriminate E. sakazakii from all other Enterobacter and non-Enterobacter strains tested. The developed real-time PCR assay could save up to 5 days and eliminate the need for plating samples on selective or diagnostic agars and for biochemical confirmation steps. The real-time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.

Identification and characterization of a stress-responsive promoter in the macromolecular synthesis operon of Bacillus subtilis.

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant. Induction of sigmaA has been observed exclusively in this mutant harbouring extra copies of the plasmid-borne Ts sigA gene transcriptionally controlled by the P1P2 promoters of the B. subtilis macromolecular synthesis (MMS; rpoD or sigA) operon. Investigation of the mechanisms leading to the induction has allowed us to identify a sigmaB-type promoter, P7, in the MMS operon for the first time. Therefore, at least seven promoters in total are responsible for the regulation of the B. subtilis MMS operon, including the four known sigmaA- and sigmaH-type promoters, as well as two incompletely defined promoters. The P7 promoter was activated in B. subtilis after the imposition of heat, ethanol and salt stresses, indicating that the MMS operon of B. subtilis is subjected to the control of general stress. The significant heat induction of P7 in B. subtilis DB1005 harbouring a plasmid-borne Ts sigA gene can be explained by a model of competition between sigmaA and sigmaB for core binding; very probably, the sigmaB factor binds more efficiently to core RNA polymerase under heat shock. This mechanism may provide a means for the expression of the B. subtilis MMS operon when sigmaA becomes defective in core binding.

A Carboxyl-terminal Cys2/His2-type Zinc-finger Motif in DNA Primase Influences DNA Content inSynechococcus PCC 7942

The DNA primase gene, dnaG, has been isolated from the cyanobacterium Synechococcus PCC 7942. It is not part of a macromolecular synthesis operon but is co-transcribed with pheT and located adjacent to the metallothionein divergon, smt. At the carboxyl terminus of this DnaG is a Cys2/His2 zinc-finger motif. The carboxyl-terminal 91 residues bound 65Zn and 0.95 g atom of Zn2+ mol-1 were detected with 4-(2-pyridylazo)resorcinol. Following exposure to Cd2+, 0.95 g atom of Cd2+ was displaced by 2 equivalents of p-(hydroxymercuri) phenylsulfonate mol-1, while only 0.03 g atom of Cd2+ was displaced mol-1 polypeptide missing the carboxyl-terminal (residue 592 onward) zinc-finger motif. Zn2+ caused an increase in intensity, and a reduction in wavelength, of Trp fluorescence at the tip of the predicted zinc-finger, while EDTA caused the converse. Cells containing a single chromosomal codon substitution (C597S), altering the zinc-finger, were generated by exploiting Zn2+-sensitive smt mutants and the proximity of dnaG to smt. Cells in which smt and dnaG(C597S) had integrated into the chromosome were selected via restored Zn2+ tolerance. Synechococcus PCC 7942 and its dnaG(C597S) mutant grew at equivalent rates, but the latter had a reduced number of chromosomes.

Characterization of the dnaG locus in Mycobacterium smegmatis reveals linkage of DNA replication and cell division.

We have isolated a UV-induced temperature-sensitive mutant of Mycobacterium smegmatis that fails to grow at 42 degrees C and exhibits a filamentous phenotype following incubation at the nonpermissive temperature, reminiscent of a defect in cell division. Complementation of this mutant with an M. smegmatis genomic library and subsequent subcloning reveal that the defect lies within the M. smegmatis dnaG gene encoding DNA primase. Sequence analysis of the mutant dnaG allele reveals a substitution of proline for alanine at position 496. Thus, dnaG is an essential gene in M. smegmatis, and DNA replication and cell division are coupled processes in this species. Characterization of the sequences flanking the M. smegmatis dnaG gene shows that it is not part of the highly conserved macromolecular synthesis operon present in other eubacterial species but is part of an operon with a dgt gene encoding dGTPase. The organization of this operon is conserved in Mycobacterium tuberculosis and Mycobacterium leprae, suggesting that regulation of DNA replication, transcription, and translation may be coordinated differently in the mycobacteria than in other bacteria.

Temperature-dependent regulation of the ribosomal small-subunit protein S21 in the cyanobacterium Anabaena variabilis M3.

The rpsU gene, which encodes the ribosomal small-subunit protein S21 in Anabaena, is not a part of the macromolecular-synthesis operon as in most enterobacteria but rather is located downstream of the rbpA1 gene, which encodes an RNA-binding protein. Two types of transcripts were detected for this gene cluster. The level of the major rbpA1-rpsU transcript was about 10 times higher at 22 degrees C than at 38 degrees C, whereas the minor monocistronic rpsU transcript was more abundant at the higher temperature. The level of the S21 protein in relation to total protein was three times lower at 38 degrees C than at 22 degrees C. Analysis of isolated ribosomes indicated that S21 was present at an equimolar ratio with regard to other ribosomal proteins at 22 degrees C but that its level decreased with temperature. Conversely, the relative abundance of S5 increased with temperature. A decrease in the level of S21 at high temperature was also found in Synechocystis, in which rpsU is located downstream of the rrn operon. These results suggest that S21 is involved in the adaptation to changes in temperature in cyanobacteria.

Transcriptional characterization of the Rickettsia prowazekii major macromolecular synthesis operon.

Recent studies have demonstrated that Rickettsia prowazekii can regulate transcription of selected genes at the level of initiation. However, little information concerning the existence of operons and coordinate gene regulation in this obligate intracellular parasitic bacterium is available. To address these issues, we have focused on the rpoD gene linkage group (greA-open reading frame 23 [ORF23]-dnaG-rpoD), which includes the rickettsial analog (ORF23-dnaG-rpoD) of the major macromolecular synthesis operon (MMSO). The rickettsial MMSO consists of an ORF coding for a protein of unknown function the structural genes for DNA primase (dnaG) and the major sigma factor of RNA polymerase (rpoD). RNase protection assays (RPA) were used to determine if these genes are organized into an operon controlled by multiple promoters and the quantities of transcripts produced by these genes relative to each other. RPA with a probe spanning the 270-base greA-ORF23 intervening region identified a putative transcriptional promoter within the intervening sequence. Multiple RPA probes spanning the next 4,041 bases of the linkage group demonstrated the presence of a continuous transcript and thus the existence of an operon. A probe spanning the dnaG-rpoD region revealed that two additional mRNA fragments were also protected, which enabled us to identify additional putative promoters for rpoD within dnaG. Primer extension determined that the 5' ends of the three transcripts consist separately of adenine (located 227 bases upstream of ORF23) and uracil and adenine (located 336 and 250 bases upstream of rpoD, respectively). Quantitation of transcripts produced by the three ORFs determined the relative amounts of transcripts (ORF23 to dnaG to rpoD) to be 1:2.7:5.1.

A missense mutation in rpoD results in an A-signalling defect in Myxococcus xanthus.

The Myxococcus xanthus asg genes (asgA, asgB, and asgC) are necessary for production of extracellular A-signal, which is thought to function as a cell-density signal. Previous analyses of the asgA and asgB genes suggest that they perform regulatory functions. In this work, we localized asgC to a region that contains genes homologous to rpsU, dnaG, and rpoD of the Escherichia coli macromolecular synthesis (MMS) operon. Surprisingly, asgC767 was found to be a mutant allele of rpoD, the gene encoding the major sigma factor of M. xanthus. The mutation in asgC767 results in a glutamate to lysine substitution at amino acid 598, which lies within conserved region 3.1 of the major sigma factors. Previous studies have shown that the asg mutants share a number of growth and developmental phenotypes. We found that A-signal restores developmental expression of an A-signal-dependent gene (omega 4521) in the asgC767 (rpoDEK598) mutant background in a manner similar to that seen in the asgA and asgB mutants. Because the asg mutants have very similar phenotypes and the asg genes encode proteins that appear to have regulatory functions, we hypothesize that the asg gene products function together in a regulatory pathway that is required for extracellular A-signal production.

Cloning and characterization of the sigA gene encoding the major sigma subunit of Rhizobium meliloti.

Using PCR to create a probe based on conserved region 2 of sigma factors, we have cloned the sigA gene coding for the major sigma factor of Rhizobium meliloti. The 684-residue protein encoded by the sigA gene was expressed in vitro in coupled transcription-translation experiments with R. meliloti extracts and migrated aberrantly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its deduced amino acid sequence is similar to that of RpoD of Escherichia coli and is nearly identical to that of SigA of the closely related bacterium Agrobacterium tumefaciens. Through Southern analysis, we located the gene on the R. meliloti main chromosome rather than on one of the megaplasmids. The sigA locus does not appear to be part of a macromolecular synthesis operon (MMS), as in many other bacterial species, but rather lies downstream of a partial open reading frame showing similarity to the threonine dehydrogenase gene (tdh) of E. coli.

Functional analysis of mutations in the transcription terminator T1 that suppress two dnaG alleles in Escherichia coli.

The mutations dnaG2903 and parB are both temperature-sensitive conditional lethal alleles of the Escherichia coli dnaG gene, which encodes the protein primase. The lesions are located in the 3' end of the gene, 9 basepairs apart, and both cause Glu-to-Lys substitutions in the carboxy terminus of primase. Previously, it was shown that dnaG2903 can be suppressed by point mutations in the rho-independent transcription terminator T1, which is located just upstream of dnaG in the rpsU-dnaG-rpoD macromolecular synthesis operon. We report here that parB can also be suppressed by point mutations in T1, demonstrating that parB can be suppressed in the same manner as dnaG2903. We also identified additional suppressors of dnaG2903 that are point mutations in T1, suggesting that defective transcription termination leading to overexpression of dnaG2903 and parB suppresses the temperature-sensitive phenotype of strains harboring these mutations. Utilizing two mutant rpoB alleles whose transcription termination phenotypes at rho-independent terminators have been previously characterized, we demonstrate that defective transcription termination leading to the overexpression of dnaG does indeed suppress dnaG2903 and parB. The point mutations in T1 identified in this study were analyzed for their effects on termination efficiency at T1. Our results indicate that the thermodynamic stability of the hairpin structures may not be the sole determinant of termination efficiency in vivo.

Characterization of the macromolecular synthesis (MMS) operon from Listeria monocytogenes

The macromolecular synthesis (MMS) operon consists of three genes: rpsU, which encodes the S21 ribosomal protein (in Bacillus subtilis (Bs), rpsU is replaced by orfP23 which encodes a protein of unknown function), dnaG, encoding the DNA primase involved in the initiation of chromosome replication, and rpoD, which encodes the principal sigma subunit of RNA polymerase. The operon was cloned in three segments from Listeria monocytogenes (Lm), initially using a probe designed from a highly conserved region of RpoD. Analysis of the nucleotide sequence revealed three genes: orfP17 (whose product, P17, is homologous to Bs P23), dnaG and rpoD. The Lm DnaG resembles the primase from Escherichia coli through the first two-thirds of the sequence. C-terminal similarity was observed between DnaG from Lm and Bs. Lm RpoD is similar to Bs SigA, shares identical DNA-binding domains with SigA, and is a member of the σ4 subgroup of the σ70 family.

Conservation and evolution of the rpsU‐dnaG‐rpoD macromolecular synthesis operon in bacteria

The macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD) whose products (S21, primase, sigma-70) are necessary for the initiation of protein, DNA, and RNA synthesis respectively. PCR amplifications with primers complementary to conserved regions within these three genes, and subsequent DNA sequencing of rpsU-dnaG PCR products, demonstrate that the three genes appear to be contiguous in 11 different Gram-negative species. Within the Gram-negative enteric bacterial lineage, the S21 amino acid sequence is absolutely conserved in 10 species examined. The putative nuteq antiterminator sequence in rpsU consists of two motifs, boxA and boxB, conserved in primary sequence and secondary structure. The terminator sequence, T1, located between rpsU and dnaG is conserved at 31 positions in nine enterobacterial species, suggesting the importance of primary sequence in addition to secondary structure for transcription termination. The intergenic region between rpsU and dnaG varies in size owing to the presence or absence of the Enterobacterial Repetitive Intergenic Consensus (ERIC) DNA element. The rpoD gene contains rearrangements involving a divergent sequence, although two carboxy-terminal regions which encode functional domains are conserved in primary sequence and spacing. Our data suggest that primary sequence divergence and DNA rearrangements in both coding and non-coding sequences account for the interspecies variation in operon structure. However, MMS operon gene organization and cis-acting regulatory sequences appear to be conserved in diverse bacteria.

Molecular and Enzymological Evidence for Two Classes of Fumarase in Bacillus Stearothermophilus (var. Non-Diastaticus)

The gene (fumABst) encoding an oxygen-labile fumarase of Bacillus stearothermophilus has been cloned and sequenced. The structural gene (1542 bp) encodes a product (FumABst) of M(r) 56,788 containing 514 amino acid residues. The amino acid sequence is 23% identical (37% similar) to FumA and FumB, the labile [4Fe-4S]-containing fumarases (Class I enzymes) of Escherichia coli. It exhibits no significant similarity to FumC and CitG, the stable fumarases (Class II enzymes) of E. coli and Bacillus subtilis (respectively). Enzymological studies indicated that FumABst resembles the iron-sulphur-containing fumarases in being dimeric (M(r) 2 x 58,500), oxygen labile and partially reactivated by Fe2+ plus DTT. The fumABst gene is the first gene encoding a Class I fumarase to be characterized in any organism other than E. coli. Enzymological and DNA-hybridization studies further indicated that B. stearothermophilus resembles E. coli in containing an oxygen-stable fumarase (Class II enzyme). Sequence comparisons revealed significant similarities between the Class I fumarases and the products of adjacent open-reading frames (orfZ1 and orfZ2) located upstream of the macromolecular synthesis operon (rpsU-dnaG-rpoD) at 67 min in the E.coli linkage map. Located downstream of fumABst, there is an unidentified gene (orf2), which is homologous to the rhizobial nodB genes involved in the initiation of root nodule formation.

Role of the 5' upstream sequence and tandem promoters in regulation of the rpsU-dnaG-rpoD macromolecular synthesis operon.

Bal31 exonuclease deletion analysis and transposon Tn5 mutagenesis of the 5' regulatory region of the rpsU-dnaG-rpoD macromolecular synthesis operon fused to the chloramphenicol acetyltransferase gene (pGLR301) demonstrated that sequences 5' to the operon promoters were not involved in operon transcriptional regulation and that the three tandem promoters P1, P2, and P3 were functionally independent. P2 was the strongest promoter, and P3 was the weakest. P1, P2, and P3 acting in combination appeared to be stronger than the individual promoters.

Possible new genes as revealed by molecular analysis of a 5-kb Escherichia coli chromosomal region 5′ to the rpsU-dnaG-rpoD macromolecular-synthesis operon

The complete nucleotide sequence of 5-kb DNA fragment immediately 5′ to the rpsU-dnaG-rpoD macro-molecular-synthesis operon in Escherichia coli has been determined. It encodes for six open reading frames. Transcriptional and translational analysis have shown that three of them ( orf x, orf z1 and orf z2) are expressed in exponentially growing E. coli cells. The orf x, directly 5′ to the rpsU gene but transcribed in the opposite direction, may be part of the rpsU-dnaG-rpoD macromolecular-synthesis operon.