Identification of the first glyphosate transporter by genomic adaptation

The soil bacterium Bacillus subtilis can get into contact with growth-inhibiting substances, which may be of anthropogenic origin. Glyphosate is such a substance serving as a nonselective herbicide. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which generates an essential precursor for de novo synthesis of aromatic amino acids in plants, fungi, bacteria and archaea. Inhibition of the EPSP synthase by glyphosate results in depletion of the cellular levels of aromatic amino acids unless the environment provides them. Here, we have assessed the potential of B. subtilis to adapt to glyphosate at the genome level. In contrast to Escherichia coli, which evolves glyphosate resistance by elevating the production and decreasing the glyphosate sensitivity of the EPSP synthase, B. subtilis primarily inactivates the gltT gene encoding the high-affinity glutamate/aspartate symporter GltT. Further adaptation of the gltT mutants to glyphosate led to the inactivation of the gltP gene encoding the glutamate transporter GltP. Metabolome analyses confirmed that GltT is the major entryway of glyphosate into B. subtilis. GltP, the GltT homologue of E. coli also transports glyphosate into B. subtilis. Finally, we found that GltT is involved in uptake of the herbicide glufosinate, which inhibits the glutamine synthetase.     

Overproduction of, and interaction within, bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans

The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the over-produced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.     

Overproduction of,and interaction within,bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans

The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the over-produced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.     

Comparison of promoter activities of heterologous and homologous promoters in Bacillus subtilis

Summary We have constructed promoter probe vectors with the Escherichia coli galactokinase monitoring system that can be used in Bacillus subtilis. In vivo studies with these vectors demonstrated that the E. coli trp and tac(trp::lac) promoter regions could be utilized in B. subtilis. These promoter regions and the promoter region for the erythromycin resistance gene originating from Staphylococcus aureus were preferentially utilized during the stationary growth phase of B. subtilis, whereas the B. subtilis P21K and P29K promoters were utilized during the exponential growth phase and decreased rapidly during the stationary phase. The apparent strength of these promoters of E. coli in B. subtilis, in terms of galactokinase units, was comparable with those of the B. subtilis promoters.     

Across Genus Plasmid Transformation Between <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and the Effect of <Emphasis Type="Italic">Escherichia coli</Emphasis> on the Transforming Ability of Free Plasmid DNA

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.     

Folic acid and the methylation of homocysteine by Bacillus subtilis

1. Cell-free extracts of Bacillus subtilis synthesize methionine from serine and homocysteine without added folate. The endogenous folate may be replaced by tetrahydropteroyltriglutamate or an extract of heated Escherichia coli for the overall C₁ transfer, but tetrahydropteroylmonoglutamate is relatively inactive. 2. Extracts of B. subtilis contain serine transhydroxymethylase and 5,10-methylenetetrahydrofolate reductase, which are non-specific with respect to the glutamate content of the folate substrates. Methyl transfer to homocysteine requires a polyglutamate folate as methyl donor. These properties are not affected by growth of the organism with added vitamin B₁₂. 3. The synthesis of methionine from 5-methyltetrahydropteroyltriglutamate and homocysteine has the characteristics of the cobalamin-independent reaction of E. coli. No evidence for a cobalamin-dependent transmethylation was obtained. 4. S-Adenosylmethionine was not a significant precursor of the methyl group of methionine with cell-free extracts, neither was S-adenosylmethionine generated by methylation of S-adenosylhomocysteine by 5-methyltetrahydrofolate. 5. A procedure for the isolation and analysis of folic acid derivatives from natural sources is described. 6. The folates isolated from lysozyme extracts of B. subtilis are sensitive to folic acid conjugase. One has been identified as 5-formyltetrahydropteroyltriglutamate; the other is possibly a diglutamate folate. 7. A sequence is proposed for methionine biosynthesis in B. subtilis in which methyl groups are generated from serine and transferred to homocysteine by means of a cobalamin-independent pathway mediated by conjugated folate coenzymes.     

Assay-dependent effect of silver nanoparticles to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

We assess the microbial assay-dependent effect of AgNP on gram-negative Escherichia coli and gram-positive Bacillus subtilis. The experiment was conducted via three different assays: a growth inhibition assay, a colony forming unit assay, and a liquid-to-plate assay. AgNP were exposed either as liquid suspensions or in an agar state. Bacterial sensitivity to AgNP was found to be dependent on the microbial assay employed. E. coli was more sensitive than B. subtilis in the growth inhibition and CFU assays, but B. subtilis was more vulnerable than E. coli in the liquid-to-plate assay, ostensibly owing to the food stress mechanisms of B. subtilis in exposure medium. The dissolution of silver from AgNP could not explain the observed toxicity of AgNP. We detected clear evidence of AgNP uptake by cells. The results of this study showed that the microbial toxicity of AgNP and the effects of dissolved silver ions were influenced profoundly by the microbial test method employed.     

Recombinant plasmids capable to replication in B. subtilis and E. coli.

Summary The plasmid pBC16 (4.25 kbases), originally isolated from Bacillus cereus, determines tetracycline resistance and can be transformed into competent cells of B. subtilis. A miniplasmid of pBC16 (pBC16-1), 2,7 kb) which has lost an EcoRI fragment of pBC16 retains the replication functions and the tetracycline resistance. This plasmid which carries only one EcoRI site has been joined in vitro to pBS1, a cryptic plasmid previously isolated from B. subtilis and shown to carry also a single EcoRI site (Bernhard et al., 1978). The recombinant plasmid is unstable and dissociates into the plasmid pBS161 (8.2 kb) and the smaller plasmid pBS162 (2.1 kb). Plasmid pBS161 retains the tetracycline resistance. It possesses a single EcoRI site and 6 HindIII sites. The largest HindIII fragment of pBS161 carries the tetracycline resistance gene and the replication function. After circularization in vitro of this fragment a new plasmid, pBS161-1 is generated, which can be used as a HindIII and EcoRI cloning vector in Bacillus subtilis.Hybrid plasmids consisting of the E. coli plasmids pBR322, pWL7 or pAC184 and different HindIII fragments of pBS161 were constructed in vitro. Hybrids containing together with the E. coli plasmid the largest HindIII fragment of pBS161 can replicate in E. coli and B. subtilis. In E. coli only the replicon of the E. coli plasmid part is functioning whereas in B. subtilis replication of the hybrid plasmid is under the control of the Bacillus replicon. The tetracycline resistance of the B. subtilis plasmid is expressed in E. coli, but several antibiotic resistances of the E. coli plasmids (ampicillin, kanamycin and chloramphenicol) are not expressed in B. subtilis. The hybrid plasmids seem to be more unstable in B. subtilis than in E. coli.     

Interspecies Comparison of the Bacterial Response to Allicin Reveals Species‐Specific Defense Strategies

Allicin, a broad‐spectrum antimicrobial agent from garlic, disrupts thiol and redox homeostasis, proteostasis, and cell membrane integrity. Since medicine demands antimicrobials with so far unexploited mechanisms, allicin is a promising lead structure. While progress is being made in unraveling its mode of action, little is known on bacterial adaptation strategies. Some isolates of Pseudomonas aeruginosa and Escherichia coli withstand exposure to high allicin concentrations due to as yet unknown mechanisms. To elucidate resistance and sensitivity‐conferring cellular processes, the acute proteomic responses of a resistant P. aeruginosa strain and the sensitive species Bacillus subtilis are compared to the published proteomic response of E. coli to allicin treatment. The cellular defense strategies share functional features: proteins involved in translation and maintenance of protein quality, redox homeostasis, and cell envelope modification are upregulated. In both Gram‐negative species, protein synthesis of the majority of proteins is downregulated while the Gram‐positive B. subtilis responded by upregulation of multiple regulons. A comparison of the B. subtilis proteomic response to a library of responses to antibiotic treatment reveals 30 proteins specifically upregulated by allicin. Upregulated oxidative stress proteins are shared with nitrofurantoin and diamide. Microscopy‐based assays further indicate that in B. subtilis cell wall integrity is impaired.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

An active β-lactamase is a part of an orchestrated cell wall stress resistance network of Bacillus subtilis and related rhizosphere species

A hallmark of the Gram-positive bacteria, such as the soil-dwelling bacterium Bacillus subtilis, is their cell wall. Here, we report that d -leucine and flavomycin, biofilm inhibitors targeting the cell wall, activate the β-lactamase PenP. This β-lactamase contributes to ampicillin resistance in B. subtilis under all conditions tested. In contrast, both Spo0A, a master regulator of nutritional stress, and the general cell wall stress response, differentially contribute to β-lactam resistance under different conditions. To test whether β-lactam resistance and β-lactamase genes are widespread in other Bacilli, we isolated Bacillus species from undisturbed soils, and found that their genomes can encode up to five β-lactamases with differentiated activity spectra. Surprisingly, the activity of environmental β-lactamases and PenP, as well as the general stress response, resulted in a similarly reduced lag phase of the culture in the presence of β-lactam antibiotics, with little or no impact on the logarithmic growth rate. The length of the lag phase may determine the outcome of the competition between β-lactams and β-lactamases producers. Overall, our work suggests that antibiotic resistance genes in B. subtilis and related species are ancient and widespread, and could be selected by interspecies competition in undisturbed soils.     

Autolysis-mediated membrane vesicle formation in Bacillus subtilis

It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.     

Screening of Bacillus strains as potential probiotics and subsequent confirmation of the in vivo effectiveness of Bacillus subtilis MA139 in pigs

A total of 124 samples were collected from the intestine of broiler chickens, piglet faeces, fermented foods, soils and Chinese herbs. More than 750 strains of aerobic, spore-forming bacteria were isolated from these samples. The inhibitory activity of these spore-forming strains against Escherichia coli K88, E. coli K99, Salmonella typhimurium and Staphylococcus aureus was assessed using a disc plate diffusion assay. The six bacilli with the largest inhibition zones against the four indicator bacteria were chosen and assessed for their resistance to unfavorable conditions within simulated gut environments. The strain Bacillus subtilis MA139 showed full resistance to pH 2, 0.3% bile salts and exhibited the highest antimicrobial activity. Based on these results, B. subtilis MA139 was selected as a potential probiotic and fed to piglets at concentrations of 2.2 × 10⁵, 2.2 × 10⁶ or 2.2 × 10⁷ CFU/g of feed during a 28-day feeding trial. A negative control consisting of the basal diet with no additives and a positive control consisting of the basal diet supplemented with 16 g/ton flavomycin were also included. Ninety piglets between 35 and 40 days old were used in the in vivo animal trials. B. subtilis MA139 enhanced daily gain (P = 0.10) and feed conversion (P = 0.03) compared with the negative control. The performance of pigs fed B. subtilis MA139 supplemented diets did not differ from that of pigs fed the antibiotic diet. There was a significant increase in Lactobacilli cell counts (P = 0.02) and a numerical decrease in E. coli counts (P = 0.05) in the faecal samples of pigs fed B. subtilis MA139 with 2.2 × 10⁵ CFU/g of feed. The overall results of this study show that the use of initial co-culture with indicator pathogens, a disc plate diffusion assay and simulated gut environment tolerance tests is one of effective methods of screening Bacillus for probiotic use and that B. subtilis MA139 is a promising alternative to antibiotics for use as a feed additive in piglet diets.     

Distinct roles for U‐type proteins in iron–sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon

The biosynthesis of iron–sulfur (Fe–S) clusters in Bacillus subtilis is mediated by the SUF‐like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe–S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe–S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe–S cluster assembly.     

Cloning, Overexpression and Purification of Bacillus subtilis Elongation Factor Tu in Escherichia coli

To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His₆ tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.     

Iron-hydroxamate uptake systems in Bacillus subtilis: identification of a lipoprotein as part of a binding protein-dependent transport system

Bacillus subtilis was shown to utilize three types of hydroxamate siderophores, ferrichromes, ferrioxamines and shizokinen, each of which is taken up by different transport systems. Mutants deficient in the uptake of ferrichrome and/or ferrioxamine B were isolated. The gene fhuD, which was able to complement a mutant defective in ferrichrome uptake, was cloned. The deduced sequence of FhuD showed low but significant homology to the binding proteins FepB, FecB and FhuD of Escherichia coli, which are all components of binding protein-dependent, ferric siderophore transport systems. The first 23 amino acids of FhuD of B. subtilis possessed all characteristics of a lipoprotein signal sequence. The processing of FhuD in E. coli was inhibited by globomycin. Inhibition by globomycin indicated a lipid modification at the N-terminal cysteine in E. coli. It is highly likely that this step may also take place in B. subtilis. As in other binding protein-dependent transport systems of Gram-positive organisms it is proposed that the lack of a periplasm is compensated for by the lipid through which the binding protein is anchored to the cytoplasmic membrane.     

Identification and Immunochemical Location of UMP Kinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Phosphorylation of CMP and UMP is accomplished in Bacillus subtilis, as in Escherichia coli, by two different enzymes exhibiting characteristic structural and catalytic properties. UMP kinase from B. subtilis is an oligomer whose activity is strictly dependent on GTP. The B. subtilis enzyme is unstable in the absence of UTP, which acts as an allosteric inhibitor. Antibodies raised against recombinant B. subtilis UMP kinase recognized the protein both in soluble extract and in immunoelectron microscopy. UMP kinase from B. subtilis has a peripheral distribution which is related most probably to its role in the synthesis of membrane sugar components and its putative role in cell division.     

A novel class of ammonium assimilation mutants of the photosynthetic bacterium Rhodobacter capsulatus

Nitrogen assimilation in Rhodobacter capsulatus has been shown to proceed via the coupled action of glutamine synthetase (GS) and glutamate synthase (GOGAT) with no measurable glutamate dehydrogenase (GDH) present. We have recently isolated a novel class of mutants of R. capsulatus strain B100 that lacks a detectable GOGAT activity but is able to grow at wild type rates under nitrogen-fixing conditions. While NH ₄ ⁺ -supported growth in the mutants was normal under anaerobic/photosynthetic conditions, the growth rate was decreased under aerobic conditions. Ammonium and methylammonium uptake experiments indicated that there was a clear difference in the ammonium assimilatory capabilities in these mutants under aerobic versus anaerobic growth. Regulation of expression of a nifH : : lacZ fusion in these mutants was not impaired. The possible existence of alternative ammonium assimilatory pathways is discussed.     

The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration

The ftsZ gene is essential for cell division in both Escherichia coli and Bacillus subtilis. In E. coli FtsZ forms a cytokinetic ring at the division site whose formation is under cell-cycle control. In addition, the FtsZ from E. coli has a GTPase activity that shows an unusual lag in vitro. In this study we show that FtsZ in Bacillus subtilis forms a ring that is at the tip of the invaginating septum. The FtsZ ring is dynamic since it is formed as division is initiated, changes diameter during septation, and disperses upon completion of septation. In vitro the purified FtsZ from B. subtilis exhibits a GTPase activity without a demonstrable lag, but the GTPase activity is markedly dependent upon the FtsZ concentration, suggesting that the FtsZ protein must oligomerize to express the GTPase activity.