Preferential Inhibition of Protein Synthesis by Ketolide Antibiotics in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> Cells

The ketolide antibiotics are semi-synthetic derivatives of erythromycin A with enhanced inhibitory activity in a wide variety of microorganisms. They have significantly lower MICs than the macrolide antibiotics for many Gram-positive organisms. Two ketolides, telithromycin and ABT-773, were tested for growth-inhibitory effects in Haemophilus influenzae. Both antibiotics increased the growth rate and reduced the viable cell number with IC₅₀ values of 1.5 μg/ml. Protein synthesis was inhibited in cells with a similar IC₅₀ concentration (1.25 μg/ml). Macrolide and ketolide antibiotics have been shown to have a second equivalent target for inhibition in cells, which is blocking the assembly of the 50S ribosomal subunit. Pulse and chase labeling assays were conducted to examine the effect of the ketolides on subunit formation in H. influenzae. Surprisingly, both antibiotics inhibited 50S and 30S subunit assembly to the same extent, with no specific effect of the compounds on 50S assembly. Over a range of antibiotic concentrations, 30S particle synthesis was diminished to the same extent as 50S formation. H. influenzae cells seem to have only one significant target for these antibiotics, and this may help to explain why these drugs are not more effective than the macrolides in preventing the growth of this microorganism. Received: 21 February 2002 / Accepted: 30 April 2002     

Telithromycin Inhibition of Protein Synthesis and 50S Ribosomal Subunit Formation in <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> Cells

The new ketolide antibiotic telithromycin (HMR3647) has been examined for inhibitory effects in cells of Streptococcus pneumoniae. The antibiotic caused a proportional decline in cell growth rate and viability with an IC₅₀ of 15 ng/ml. At a concentration of 7.5 ng/ml, protein synthesis in these cells was reduced by 50%. As seen in other organisms, this compound was also a very effective inhibitor of the formation of the 50S ribosomal subunit in growing cells. Pulse and chase labeling assays defined the reduced rate of 50S synthesis in antibiotic treated cells. At 7.5 ng/ml the rate was reduced to 50% of the control synthesis rate. An IC₅₀ of 15 ng/ml was found for the effect on this process. 30S ribosomal subunit formation was unaffected by the antibiotic. Inhibition of translation and 50S particle formation are equivalent targets for this antibiotic. The effects of telithromycin in S. pneumoniae are compared with those found in Staphylococcus aureus cells. Received: 29 October 2001 / Accepted: 1 February 2002     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.     

Biological characterization of two <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains toxic against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Two Bacillus thuringiensis strains isolated from diseased Spodoptera frugiperda larvae collected in the northwest of Argentina were molecularly and phenotypically characterized. Insecticidal activity against Spodoptera frugiperda larvae was also determined. Both strains were highly toxic against first instar larvae. One strain (Bacillus thuringiensis LSM) was found to be even more toxic than the reference strain Bacillus thuringiensis var. kurstaki 4D1. This strong biological effect was represented by both a higher mortality which reached 90%, and a shorter LT₅₀. Molecular characterization showed that Bacillus thuringiensis LSM carried a cry gene profile identical to that of Bacillus thuringiensis var. kurstaki 4D1. Evaluation of length polymorphism of the intergenic transcribed spacers between the 16S and 23S rDNA genes revealed an identical pattern between native strains and Bacillus thuringiensis var. kurstaki 4D1. In contrast, phenotypic characterization allowed differentiation among the isolates by means of their extracellular esterase profiles. Lytic activity that would contribute to Bacillus thuringiensis effectiveness was also studied in both strains. Analyses like those presented in the current study are essential to identify the most toxic strains and to allow the exploitation of local biodiversity for its application in biological control programmes.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected. An erratum to this article can be found at     

Neomycin and Paromomycin Inhibit 30S Ribosomal Subunit Assembly in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

A number of different antibiotics that prevent translation by binding to the 50S ribosomal subunit of bacterial cells have recently been shown to also prevent assembly of this subunit. Antibacterial agents affecting 30S particle activities have not been examined extensively for effects on small subunit formation. The aminoglycoside antibiotics paromomycin and neomycin bind specifically to the 30S ribosomal subunit and inhibit translation. These drugs were examined in Staphylococcus aureus cells to see whether they had a second inhibitory effect on 30S particle assembly. A ³H-uridine pulse and chase assay was used to examine the kinetics of subunit synthesis in the presence and absence of each antibiotic. 30S subunit formation was inhibited by both compounds. At 3 µg/mL each antibiotic reduced the rate of 30S formation by 80% compared with control cells. Both antibiotics showed a concentration-dependent inhibition of particle formation, with a lesser effect on 50S particle formation. For neomycin, the IC₅₀ for 30S particle formation was equal to the IC₅₀ for inhibition of translation. Both antibiotics reduced the viable cell number with an IC₅₀ of 2 µg/mL. They also inhibited protein synthesis in the cells with different IC₅₀ values (2.5 and 1.25 µg/mL). This is the second demonstration of 30S ribosomal subunit-specific antibiotics that prevent assembly of the small subunit.Received: 13 August 2002 / Accepted: 4 November 2002     

Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> virulence genes in <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis> from Vellore,India

Streptococcus dysgalactiae subsp. equisimilis (SDSE), belonging to the group C and G streptococci, are human pathogens reported to cause clinical manifestations similar to infections caused by Streptococcus pyogenes. To scrutinize the distribution of gene coding for S. pyogenes virulence factors in SDSE, 255 isolates were collected from humans infected with SDSE in Vellore, a region in southern India, with high incidence of SDSE infections. Initial evaluation indicated SDSE isolates comprising of 82.35% group G and 17.64% group C. A multiplex PCR system was used to detect 21 gene encoding virulence-associated factors of S. pyogenes, like superantigens, DNases, proteinases, and other immune modulatory toxins. As validated by DNA sequencing of the PCR products, sequences homologous to speC, speG, speH, speI, speL, ssa and smeZ of the family of superantigen coding genes and for DNases like sdaD and sdc were detected in the SDSE collection. Furthermore, there was high abundance (48.12% in group G and 86.6% in group C SDSE) of scpA, the gene coding for C5a peptidase in these isolates. Higher abundance of S. pyogenes virulence factor genes was observed in SDSE of Lancefield group C as compared to group G, even though the incidence rates in former were lower. This study not only substantiates detection of S. pyogenes virulence factor genes in whole genome sequenced SDSE but also makes significant contribution towards the understanding of SDSE and its increasing virulence potential.     

The Role of ClpP in Protein Expression of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Previous reports suggest that ClpP proteolytic activity is important not only for cell physiology but also for regulation of virulence properties of Streptococcus pneumoniae (S. pneumoniae). In order to get a more comprehensive picture of the role of ClpP protease on protein expression in S. pneumoniae D39 and how it relates to physiology and virulence, a clpP mutant strain was constructed in S. pneumoniae D39, and global proteome expression was studied by 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. We report here that clpP deletion affects the expression of proteins which are involved in the general stress response, nucleotide metabolism, energy metabolism, and proteins metabolism. These provide clues for understanding the role of ClpP in the physiology and pathogenesis of pneumococcus.     

Antimicrobial activity of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains

A bacterial strain with a high level of antimicrobial activity was isolated from soil and identified as Bacillus megaterium. Production of antibiotics by nine strains of this species from the collection of the State Research Institute for Genetics and Selection of Industrial Microorganisms was investigated. In submerged cultures, nine out of ten B. megaterium strains were found to produce antibacterial antibiotics differing in their spectra of action. Physicochemical characteristics of five compounds were described. Three of them belonged to peptide antibiotics. All five compounds were active against the methicillin-resistant strain Staphylococcus aureus INA 00761. Three of them were shown to be the previously undescribed compounds. Antibiotics produced by various B. megaterium strains were also active against the Leuconostoc mesenteroides VKPM B-4177 strain resistant to glycopeptide antibiotics and against gram-negative bacteria Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922.     

Gene Sequence Phylogenies of the Family <Emphasis Type="Italic">Microbacteriaceae</Emphasis>

The type strains of 32 species of 13 genera of the family Microbacteriaceae were analysed with respect to gene-coding phylogeny for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA), and polyphosphate kinase (ppk). The resulting gene trees were compared with the 16S rRNA gene phylogeny of the same strains. The topology of neighbour-joining and maximum parsimony phylogenetic trees, based on nucleic-acid sequences and protein sequences of housekeeping genes, differed from one another, and no gene tree was identical to that of the 16S rRNA gene tree. Most genera analysed containing >1 strain formed phylogenetically coherent taxa. The three pathovars of Curtobacterium flaccumfaciens clustered together to the exclusion of the type strains of other Curtobacterium species in all DNA - and protein-based analyses. In no tree did the distribution of a major taxonomic marker, i.e., diaminobutyric acid versus lysine and/or ornithine in the peptidoglycan, or acyl type of peptidoglycan, correlate with the phylogenetic position of the organisms. The changing phylogenetic position of Agrococcus jenensis was unexpected: This strain defined individual lineages in the trees based on 16S rRNA and gyrB and showed identity with Microbacterium saperdae in the other three gene trees.     

Improving Toxicity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Contains the <Emphasis Type="Italic">cry8Ca</Emphasis> Gene Specific to <Emphasis Type="Italic">Anomala corpulenta</Emphasis> Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73⁻), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC₅₀) of the two mutants were 0.2334 × 10⁸ and 0.2591 × 10⁸ colony-forming units g⁻¹, considerably lower than the LC₅₀ of the wild-type strain HBF-1 (0.9583 × 10⁸ CFU g⁻¹) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 10⁸ CFU g⁻¹).     

Inhibition of 50S Ribosomal Subunit Assembly in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> Cells by Azithromycin and Erythromycin

Azithromycin is an important antibiotic for the treatment of several different Gram-positive and Gram-negative bacterial infections. Erythromycin and clarithromycin are less useful antibiotics against Gram-negative infections. This difference in inhibitory activity was explored by comparing the effects of azithromycin and erythromycin on cellular functions in Haemophilus influenzae cells. Effects of both antibiotics on translation, cell viability, and growth rates have been measured. An IC₅₀ of 0.4 μg/ml was found for the effects of azithromycin on each of these processes. For erythromycin, an IC₅₀ of 1.5 μg/ml was observed, indicating a fourfold lower sensitivity of the organisms to this compound. The features of a second target for macrolide antibiotic inhibition in H. influenzae cells have also been examined. Inhibition of the synthesis of the large 50S ribosomal subunit was measured. Subunit formation was prevented in a concentration dependent fashion, with azithromycin showing a ninefold greater effect on this process compared with erythromycin. Synthesis of the 30S ribosomal subunit was not effected. Pulse and chase labeling kinetics confirmed the slower synthesis rate of the 50S particle in the presence of each antibiotic. The results are discussed in terms of the stronger effect of azithromycin on ribosome biosynthesis in this organism. Received: 24 July 2001 / Accepted: 25 September 2001     

Increase in Insecticidal Toxicity by Fusion of the <Emphasis Type="Italic">cry1Ac</Emphasis> Gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> with the Neurotoxin Gene <Emphasis Type="Italic">hwtx-I</Emphasis>

A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC₅₀ (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis cry gene and other foreign toxin genes and recombinant strains with high toxicity. LiQiu Xia and XiaoShan Long contributed equally to this work.     

Enhanced 1,3-propanediol production in recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> carrying the gene <Emphasis Type="Italic">yqh</Emphasis>D encoding 1,3-propanediol oxidoreductase isoenzyme

The yqhD gene from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme (PDORI) and the tetracycline resistant gene (tetR) from plasmid pHY300PLK were amplified by PCR. They were inserted into vector pUC18, yielding the recombinant expression vector pUC18-yqhD-tetR. The recombinant vector was then cloned into Klebsiella pneumoniae ME-308. The overexpression of PDORI in K. pneumoniae surprisingly led to higher 1,3-propanediol production. The final 1,3-propanediol concentration of recombinant K. pneumoniae reached 67.6 g/l, which was 125.33% of that of the original strain. The maximum activity of recombinant PDORI converting 3-HPA to 1,3-PD reached 110 IU/mg after induction by IPTG at 31°C during the fermentation, while it was only 11 IU/mg under the same conditions for the wild type strain. The K _m values of the purified PDORI for 1,3-propanediol and NADP were 12.1 mM and 0.15 mM, respectively. Compared with the original strains, the concentration of the toxic intermediate 3-hydroxypropionaldehyde during the fermentation was also reduced by 22.4%. Both the increased production of 1,3-propanediol and the reduction of toxic intermediate confirmed the significant role of 1,3-propanediol oxidoreductase isoenzyme from E. coli in converting 3-hydroxypropionaldehyde to 1,3-propanediol for 1,3-PD production.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.     

Probiotic properties of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains isolated from porcine gastrointestinal tract

One strain of Lactobacillus salivarius, two strains of Lactobacillus reuteri and Lactobacillus amylovorus, and two strains of Bifidobacterium thermacidophilum with antagonistic effect against Clostridium perfringens were isolated from porcine gastrointestinal tract. Isolates were assayed for their ability to survive in synthetic gastric juice at pH 2.5 and were examined for their ability to grow on agar plate containing porcine bile extract. There was a large variation in the survival of the isolates in gastric juice and growth in the medium containing 0.3% (w/v) bile. L. salivarius G11 and L. amylovorus S6 adhered to the HT-29 epithelial cell line. Cell-free supernatant of L. amylovorus S6 showed higher antagonistic activity as effective as the antibiotics such as neomycin, chlortetracycline, and oxytetracycline against bacterial pathogens including C. perfringens, Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, Edwardsiella tarda, and Aeromonas salmonicida subsp. salmonicida.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.