Streptomyces lividans possesses a GroEL-like chaperonin

Streptomyces lividans grown at 45 degrees C produces a GroEL-like chaperonin. This protein is specifically synthesized in bacterial cell cultures upon heat shock induction. It has a similar size (62 kDa) to the GroEL-like proteins from Escherichia coli and Bacillus subtilus and shows immunological cross-reaction with serum raised against GroEL from E. coli. The S. lividans 62-kDa protein assembles into oligomers around 20S that show a morphology consistent with a barrel showing six-fold and seven-fold symmetries as previously described in E. coli and B. subtilis.     

Cell size and the initiation of DNA replication in bacteria

In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ~30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication) has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA.     

Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and Bacillus subtilis observed by fluorescence ratio imaging microscopy

The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The E. coli cytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an E. coli biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells. B. subtilis cells were acid shifted from pH 7.5 to pH 6.0. In B. subtilis, unlike the case with E. coli, cytoplasmic pH showed no "overshoot" but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual B. subtilis cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.     

Impact of Cell Wall Structure on the Behavior of Bacterial Cells as Sorbents of Cadmium and Lead

Batch metal sorption studies were conducted to compare the behavior of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli as sorbents of Cd 2+ and Pb 2+ . A pH range from 3.0 to 6.5 was investigated at total metal concentrations of 1 2 10 -4.0 and 3.2 2 10 -5 M. Concentration apparent equilibrium sorption constants (K s n M ) and sorption capacity (S max n ) values were determined for the bacteria by fitting experimental data to one- ( n = 1) and two-site ( n = 2) Langmuir sorption isotherms. The sorption data for each of the bacteria were described well by a one-site model (r 2 > 0.9), Cd 2+ exhibited somewhat lower sorption affinities (log K s M =- 1.5 for B. subtilis , and -0.7 for E. coli ) than Pb 2+ (log K s M =-0.6 for B. subtilis and -0.8 for E. coli ). Corresponding S max values for Cd 2+ and Pb 2+ on B. subtilis were 0.36 mmole/g and 0.27 mmole/g, respectively. For E. coli Cd 2+ and Pb 2+ S max values were lower at 0.10 mmole/g and 0.21 mmole/g. A two-site sorption model yielded an improved fit for only the E. coli data with several orders of magnitude difference evident between high (Cd 2+ log K s1 M = 0.9; Pb 2+ log K s1 M = 1.5) and low (Cd 2+ log K s2 M =- 1.1; Pb 2+ log K s2 M = -1.6) affinity sorption sites. In addition, allowing for the presence of low affinity sorption (i.e., S max2 ) sites further increased the total E. coli metal sorption capacity closer to that of B. subtilis . As expected, the sorption of Cd 2+ and Pb 2+ by the bacteria exhibited a strong dependence on pH with sorption edges in the range of pH 4.2 to 5.6. The results of this study show that, despite differences in cell wall structure and composition, B. subtilis and E. coli exhibit remarkably similar sorption behavior toward Cd 2+ and Pb 2+ , respectively. These similarities can be attributed to the specific chemical reactivity of acidic functional groups (e.g., carboxyl, phosphoryl) that occur in the cell walls of both bacteria.     

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.     

The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes

Ribosomes from Gram-negative bacteria such as Escherichia coli exhibit non-specific translation of bacterial mRNAs. That is, they are able to translate mRNAs from a variety of sources in a manner independent of the "strength" of the Shine-Dalgarno region, in contrast to ribosomes from many Gram-positive bacteria, such as Bacillus subtilis, which show specific translation in only being able to translate other Gram-positive mRNA, or mRNAs that have "strong" Shine-Dalgarno regions. There is an evolutionary correlation between the translational specificity and the absence of a protein analogous to E. coli ribosomal protein S1. The specificity observed with B. subtilis ribosomes is a function of their 30 S subunit which lacks S1; translation of Gram-negative mRNA can occur with heterologous ribosomes containing the 30 S subunit of E. coli ribosomes and the 50 S subunit of B. subtilis ribosomes. However, the addition of E. coli S1 alone to B. subtilis ribosome does not overcome their characteristic inability to translate mRNA from Gram-negative organisms. By contrast, the removal of S1 from E. coli ribosomes results in translational behavior similar to that shown by B. subtilis ribosomes in that the S1-depleted E. coli ribosomes can translate mRNA from Gram-positive sources in the absence of added S1, although addition of S1 stimulates further translation of such mRNAs by the E. coli ribosomes.     

微囊藻毒素对典型微生物生长及生理生化特性的影响

研究了不同浓度微囊藻毒素对典型微生物大肠杆菌和枯草芽孢杆菌生长及生理生化特性的影响。微囊藻毒素对大肠杆菌和枯草芽孢杆菌的生长和细胞活性具有一定的剂量效应,较高浓度微囊藻毒素对其生长和活性有短时间的抑制作用,随着处理时间的延长,细胞的生长和活性逐渐恢复。细胞内可溶性糖和可溶性蛋白的含量,处理组和对照组相比均有先上升后下降的趋势。结果表明,微囊藻毒素的处理对大肠杆菌和枯草芽孢杆菌具有一定的胁迫作用,细胞通过调节细胞内可溶性蛋白和可溶性糖的含量来抵抗外界胁迫,但随着处理时间的延长,细菌逐渐适应了这种胁迫,恢复正常的生长。     

Expression of Bacillus circulans Teri-42 xylanase gene in Bacillus subtilis

The xylanase gene of Bacillus circulans Teri-42 was cloned in both B. subtilis and Escherichia coli. The enzyme activity was almost 87% higher in B. subtilis (pBA7) than in E. coli (pAQ4). No cellulase activity was detected in the clones, B. subtilis (pBA7) and E. coli (pAQ4). Approximately 1120 U (80%) of the xylanase was secreted extracellularly by the clone B. subtilis (pBA7) as compared to 79 U (88%) excreted in E. coli (pAQ4). In B. subtilis (pBA7) the optimal xylanase activity was at pH 7.0 and 50 degrees C, which was the same as that of the parent B. circulans Teri-42. The recombinant xylanase in B. subtilis was more stable at higher temperatures than the parent B. circulans Teri-42. Purification of xylanase from the clone B. subtilis (pBA7) showed a 71 kDa polypeptide similar to that observed in B. circulans Teri-42.     

Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase.

The Bacillus subtilis genes encoding threonine synthase (thrC) and homoserine kinase (thrB) have been cloned via complementation of Escherichia coli thr mutants. Determination of their nucleotide sequences indicates that the thrC stop codon overlaps the thrB start codon; this genetic organization suggests that the two genes belong to the same operon, as in E. coli. However, the gene order is thrC-thrB in B. subtilis whereas it is thrB-thrC in the thr operon of E. coli. This inversion of the thrC and thrB genes between E. coli and B. subtilis is indicative of a possible independent construction of the thr operon in these two organisms. In other respects, comparison of the predicted amino acid sequences of the B. subtilis and E. coli threonine synthases with that of Saccharomyces cerevisiae threonine dehydratase and that of E. coli D-serine dehydratase revealed extensive homologies between these pyridoxal phosphate-dependent enzymes. This sequence homology, which correlates with similarities in the catalytic mechanisms of these enzymes, indicates that these proteins, catalyzing different reactions in different metabolic pathways, may have evolved from a common ancestor.     

Molecular cloning of promoter-containing fragments from Bacillus stearothermophilus and their expression in Escherichia coli and Bacillus subtilis

Abstract Bacillus stearothermophilus DNA fragments containing a promoter were isolated in Escherichia coli using a shuttle promoter-probe vector. The molecular sizes of the isolated fragments ranged from 0.78 to 10 kb. The 0.78 and 1.1 kb fragments were selected and examined in some detail for promoter activity in both E. coli and Bacillus subtilis by analysis of expression of erythromycin-resistance (Em^r) and β-galactosidase. The results showed that the two fragments exhibit a high promoter activity in both bacteria. In vitro promoter activity of the 1.1 kb fragment was also shown by RNA syntheses catalyzed by RNA polymerases prepared from E. coli, B. subtilis and B. stearothermophilus .     

Functional implementation of the posttranslational SecB-SecA protein-targeting pathway in Bacillus subtilis

Bacillus subtilis and its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria, B. subtilis does not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway into B. subtilis by coexpressing SecB from Escherichia coli together with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of the B. subtilis SecA were replaced by the corresponding part of SecA from E. coli. In vitro pulldown experiments showed that, in contrast to B. subtilis SecA, the hybrid SecA protein gained the ability to efficiently bind to E. coli SecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins by B. subtilis was significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.     

The rpsD gene, encoding ribosomal protein S4, is autogenously regulated in Bacillus subtilis.

Although the mechanisms for regulation of ribosomal protein gene expression have been established for gram-negative bacteria such as Escherichia coli, the regulation of these genes in gram-positive bacteria such as Bacillus subtilis has not yet been characterized. In this study, the B. subtilis rpsD gene, encoding ribosomal protein S4, was found to be subject to autogenous control. In E. coli, rpsD is located in the alpha operon, and S4 acts as the translational regulator for alpha operon expression, binding to a target site in the alpha operon mRNA. The target site for repression of B. subtilis rpsD by protein S4 was localized by deletion and oligonucleotide-directed mutagenesis to the leader region of the monocistronic rpsD gene. The B. subtilis rpsD leader exhibits little sequence homology to the E. coli alpha operon leader but may be able to form a pseudoknotlike structure similar to that found in E. coli.     

Extra cytoplasmic function σ factor activation

The bacterial cell envelope is essential for cell viability and is a target for numerous antibiotics and host immune defenses. Thus bacteria must sense and respond to damage to the cell envelope. Many bacteria utilize alternative σ factors such as extracytoplasmic function (ECF) σ factors to respond to cell envelope stress. Although ECF σ factors are utilized by both Gram negative and Gram positive bacteria to respond to cell envelope stress, the mechanisms of sensing differ. In this review, we examine the events and proteins that are required for activation of two model extracytoplasmic function σ factors, σ(E) in E. coli and σ(W) in B. subtilis.     

PO-independent termination of transcription of catabolite operons in Escherichia coli and Bacillus subtilis]

The review discusses the Po-independent antitermination in E. coli and B. subtilis. The functional role of antiterminators BglG (in E. coli), SacY, SacT, BglG, LicT, and GlcT (in B. subtilis) is described. Special attention is paid to the role of antiterminators in connection with the phosphoenolpyruvate-dependent phosphotransferase system involved in the carbohydrate transport in bacteria.     

Peptide mapping of Bacillus subtilis RNA polymerase alpha factors and core-associated polypeptides

An analysis of the peptide maps of the sigma factors and core-associated subunits of Bacillus subtilis RNA polymerase has revealed that all the sigma factors ad core-associated polypeptides are derived from separate genes and are not proteolytically modified products of the major 55,000-dalton sigma factor. A comparison of the peptide pattern of the major B. subtilis and Escherichia coli sigma factors revealed limited homology between them. Furthermore, antibody prepared against the 55,000-dalton B. subtilis sigma factor cross-reacted against the E. coli sigma factor, but not against any of the other B. subtilis sigma factors and core-associated polypeptides. These results unambiguously demonstrate the independently derived nature of the B. subtilis RNA polymerase core-associated subunits and the partial relationship between the major sigma factors of B. subtilis and E. coli.     

Sequence comparison of new prokaryotic and mitochondrial members of the polypeptide chain release factor family predicts a five-domain model for release factor structure.

We have recently reported the cloning and sequencing of the gene for the mitochondrial release factor mRF-1. mRF-1 displays high sequence similarity to the bacterial release factors RF-1 and RF-2. A database search for proteins resembling these three factors revealed high similarities to two amino acid sequences deduced from unassigned genomic reading frames in Escherichia coli and Bacillus subtilis. The amino acid sequence derived from the Bacillus reading frame is 47% identical to E.coli and Salmonella typhimurium RF-2, strongly suggesting that it represents B.subtilis RF-2. Our comparison suggests that the expression of the B.subtilis gene is, like that of the E.coli and S. typhimurium RF-2 genes, autoregulated by a stop codon dependent +1 frameshift. A comparison of prokaryotic and mitochondrial release factor sequences, including the putative B.subtilis RF-2, leads us to propose a five-domain model for release factor structure. Possible functions of the various domains are discussed.