Paths of lateral gene transfer of lysyl-aminoacyl-tRNA synthetases with a unique evolutionary transition stage of prokaryotes coding for class I and II varieties by the same organisms

Background  

While the premise that lateral gene transfer (LGT) is a dominant evolutionary force is still in considerable dispute, the case for widespread LGT in the family of aminoacyl-tRNA synthetases (aaRS) is no longer contentious. aaRSs are ancient enzymes, guarding the fidelity of the genetic code. They are clustered in two structurally unrelated classes. Only lysine aminoacyl-tRNA synthetase (LysRS) is found both as a class 1 and a class 2 enzyme (LysRS1-2). Remarkably, in several extant prokaryotes both classes of the enzyme coexist, a unique phenomenon that has yet to receive its due attention.     

Synthesis of <Emphasis Type="Italic">scl</Emphasis>-poly (3-hydroxyalkanoates) by <Emphasis Type="Italic">Bacillus cereus</Emphasis> found in freshwater,from monosaccharides and disaccharides

Background

Polyhydroxyalkanoates are a good substitute for synthetic plastic because they are highly biocompatible, ecofriendly, and biodegradable. Bacteria in freshwater bodies such as rivers, tube wells, and canals are exposed to alternating high and low concentrations of substrates that induce PHA production.

Methods

Fresh water samples were collected for isolation of bacterial strains. Screening of PHA in bacterial cells was performed with Sudan and Nile Red staining. Extracted PHA was characterized by FTIR.

Results

In this study, nine bacterial isolates were selected for PHA production on the basis of phenotypic screening. Their ability to accumulate PHAs was determined using different monosaccharides and disaccharides. Two bacterial isolates Bacillus cereus T1 (KY746353) and Bacillus cereus R3 (KY746354) produced PHAs. Optimal growth of the bacterial strain (T1) was observed in the presence of glucose, followed by maximum production of PHAs (63% PHAs) during the logarithmic phase of growth. B. cereus R3 (KY746354) accumulated 60% PHAs by dry cell weight.

Conclusion

PHA accumulation was relatively less with fructose, but both strains showed increased production (up to 50%) with sucrose. The polymer produced was characterized by Fourier-transform infrared spectroscopy (FTIR), which showed that the compound contains short-chain PHAs.

     

Genetic diversity of clinical isolates of <Emphasis Type="Italic">Bacillus cereus</Emphasis>using multilocus sequence typing

Background  

Bacillus cereus is most commonly associated with foodborne illness (diarrheal and emetic) but is also an opportunistic pathogen that can cause severe and fatal infections. Several multilocus sequence typing (MLST) schemes have recently been developed to genotype B. cereus and analysis has suggested a clonal or weakly clonal population structure for B. cereus and its close relatives B. anthracis and B. thuringiensis. In this study we used MLST to determine if B. cereus isolates associated with illnesses of varying severity (e.g., severe, systemic vs. gastrointestinal (GI) illness) were clonal or formed clonal complexes.     

Toxin production in a rare and genetically remote cluster of strains of the <Emphasis Type="Italic">Bacillus cereus</Emphasis> group

Background  

Three enterotoxins are implicated in diarrhoeal food poisoning due to Bacillus cereus: Haemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe), and Cytotoxin K (CytK). Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of B. cereus group strains. B. cereus strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other B. cereus group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of cytK expression. To date, only three strains containing cytK-1 have been identified; B. cereus strains NVH 391/98, NVH 883/00, and INRA AF2.     

Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

Background  

Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown.     

<Emphasis Type="Italic">Bacillus cereus</Emphasis> cytotoxins Hbl,Nhe and CytK are secreted via the Sec translocation pathway

Background  

Bacillus cereus and the closely related Bacillus thuringiensis are Gram positive opportunistic pathogens that may cause food poisoning, and the three secreted pore-forming cytotoxins Hbl, Nhe and CytK have been implicated as the causative agents of diarrhoeal disease. It has been proposed that the Hbl toxin is secreted using the flagellar export apparatus (FEA) despite the presence of Sec-type signal peptides. As protein secretion is of key importance in virulence of a microorganism, the mechanisms by which these toxins are secreted were further investigated.     

Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization

Background  

Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH) was used to identify specific chromosomal sequences unique to B. anthracis.     

Crystal structures of <Emphasis Type="Italic">Leptospira interrogans</Emphasis> FAD-containing ferredoxin-NADP<Superscript>+</Superscript> reductase and its complex with NADP<Superscript>+</Superscript>

Background  

Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs.     

Discrimination of cognate and noncognate substrates at the active site of class I lysyl-tRNA synthetase

The aminoacyl-tRNA synthetases are divided into two unrelated structural classes, with lysyl-tRNA synthetase (LysRS) being the only enzyme represented in both classes. On the basis of the structure of l-lysine complexed with Pyrococcus horikoshii class I LysRS (LysRS1) and homology to glutamyl-tRNA synthetase (GluRS), residues implicated in amino acid recognition and noncognate substrate discrimination were systematically replaced in Borrelia burgdorferi LysRS1. The catalytic efficiency of steady-state aminoacylation (k(cat)/K(M)) with lysine by LysRS1 variants fell by 1-4 orders of magnitude compared to that of the wild type. Disruption of putative hydrogen bonding interactions through replacement of G29, T31, and Y269 caused up to 1500-fold reductions in k(cat)/K(M), similar to changes previously observed for comparable variants of class II LysRS (LysRS2). Replacements of W220 and H242, both of which are implicated in hydrophobic interactions with the side chain of lysine, resulted in more dramatic changes with up to 40000-fold reductions in k(cat)/K(M) observed. This indicates that the more compact LysRS1 active site employs both electrostatic and hydrophobic interactions during lysine discrimination, explaining the ability of LysRS1 to discriminate against noncognate substrates accepted by LysRS2. Several of the LysRS1 variants were found to be more specific than the wild type with respect to noncognate amino acid recognition but less efficient in cognate aminoacylation. This indicates that LysRS1 compromises between efficient catalysis and substrate discrimination, in contrast to LysRS2 which is considerably more effective in catalysis but is less specific than its class I counterpart.     

Identification of three Zwittermicin A biosynthesis-related genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> strain YBT-1520

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.     

Characterization and genomic analysis of chromate resistant and reducing <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain SJ1

Background  

Chromium is a toxic heavy metal, which primarily exists in two inorganic forms, Cr(VI) and Cr(III). Chromate [Cr(VI)] is carcinogenic, mutational, and teratogenic due to its strong oxidizing nature. Biotransformation of Cr(VI) to less-toxic Cr(III) by chromate-resistant and reducing bacteria has offered an ecological and economical option for chromate detoxification and bioremediation. However, knowledge of the genetic determinants for chromate resistance and reduction has been limited so far. Our main aim was to investigate chromate resistance and reduction by Bacillus cereus SJ1, and to further study the underlying mechanisms at the molecular level using the obtained genome sequence.     

Optimization of Inactivation of Endospores of <Emphasis Type="Italic">Bacillus cereus</Emphasis> in Milk by Surfactin and Fengycin Using a Response Surface Method

In this paper, the sterilization of surfactin and fengycin to Bacillus cereus was observed, and the optimization of the inactivation of surfactin and fengycin to spores of B. cereus by a response surface methodology was studied. Results showed that surfactin and fengycin had high sterilization to B. cereus, whose minimal inhibitory concentration was 31.25 μM and 62.5 μM respectively. The optimization result indicated that spores of B. cereus could be inactivated by two orders of magnitude when the temperature was 20.41°C, the action time was 21.13 h, and the concentration (surfactin/fengycin molar ratio 1:1) was 54.20 μM.     

An antibiotic,heavy metal resistant and halotolerant <Emphasis Type="Italic">Bacillus cereus</Emphasis> SIU1 and its thermoalkaline protease

Background  

Many workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min.     

Extracellular ATP reduces HIV-1 transfer from immature dendritic cells to CD4<Superscript>+</Superscript>T lymphocytes

Background

Dendritic cells (DCs) are considered as key mediators of the early events in human immunodeficiency virus type 1 (HIV-1) infection at mucosal sites. Previous studies have shown that surface-bound virions and/or internalized viruses found in endocytic vacuoles of DCs are efficiently transferred to CD4⁺ T cells. Extracellular adenosine triphosphate (ATP) either secreted or released from necrotic cells induces a distorted maturation of DCs, transiently increases their endocytic capacity and affects their migratory capacity. Knowing that high extracellular ATP concentrations are present in situations of tissue injury and inflammation, we investigated the effect of ATP on HIV-1 transmission from DCs to CD4⁺ T lymphocytes.

Results

In this study, we show that extracellular ATP reduces HIV-1 transfer from immature monocyte-derived DCs (iDCs) to autologous CD4⁺ T cells. This observed decrease in viral replication was related to a lower proportion of infected CD4⁺ T cells following transfer, and was seen with both X4- and R5-tropic isolates of HIV-1. Extracellular ATP had no effect on direct CD4⁺ T cell infection as well as on productive HIV-1 infection of iDCs. These observations indicate that extracellular ATP affects HIV-1 infection of CD4⁺ T cells in trans with no effect on de novo virus production by iDCs. Additional experiments suggest that extracellular ATP might modulate the trafficking pathway of internalized virions within iDCs leading to an increased lysosomal degradation, which could be partly responsible for the decreased HIV-1 transmission.

Conclusion

These results suggest that extracellular ATP can act as a factor controlling HIV-1 propagation.

     

Specificity of translation of spore polypeptides in bacillus in vitro systems

Cell free extracts prepared from exponentially growing Escherichia coli and Bacillus cereus as well as from Bacillus cereus at the end of exponential growth were optimized for various factors required for amino acid incorporation when programmed with Bacillus ribonucleic acid. All three preparations synthesized glutamine synthetase antigen when ribonucleic acid from a Bacillus subtilis strain that overproduces glutamine synthetase was added. The post exponential Bacillus cereus extract, however, was most active for the synthesis of Bacillus cereus spore coat antigen when supplemented with the appropriate ribonucleic acid. There appears to be some specificity in the translation of at least this sporulation messenger RNA.Non-Standard Abbreviations PMSF phenyl methyl sulfonylfluoride - GS glutamine synthetase - UDS 8 M urea, 1% (W/V) sodium dodecyl sulfate, 50 mM dithioerythritol, 2 mM PMSF, 5 mM cyclohexylaminoethane sulfonic acid, pH 9.6     

DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria

Background  

Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs). Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP) analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs.     

The control of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript>regulatory T cell survival

   

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that T_reg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of T_reg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25⁺ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in T_reg cells. Our study reveals that the survival of T_reg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate T_reg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate T_reg cells that could be utilized for their therapeutic applications.     

Arg<Superscript>235</Superscript> is an essential catalytic residue of <Emphasis Type="Italic">Bacillus pumilus</Emphasis> DKS1 pectate lyase to degum ramie fibre

After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of 35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5–9.0. Both Ca²⁺ and Mn²⁺ ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn²⁺ and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1 pectate lyase.