In fact, the response to a certain stress is often accompanied by

In fact, the response to a certain stress is often accompanied by seemingly unrelated responses. For example, glucose- or nitrogen-starved cultures of Escherichia coli exhibit enhanced resistance to heat, H2O2, or osmotic challenge (Jenkins et al., 1988; Jenkins et al., 1990); furthermore, when bacteria are challenged with high osmolarity, they acquire increased resistance to high temperature and oxidative stresses (Tesone et al., 1981; Hengge-Aronis et al., 1993; Canovas et al., 2001; Gunasekera et al., 2008). Elucidation of bacterial stress responses

will facilitate understanding of bacterial physiology. The stationary phase-dependent regulatory protein (SdrP) is a CRP/FNR family transcriptional regulator from Thermus thermophilus Selleckchem PI3K inhibitor HB8 (Agari et al., 2008), which is an extremely thermophilic bacterium isolated from the water at a Japanese hot spring. Thermus thermophilus HB8 can grow at 47–85 °C, and its optimum

temperature range is from 65 to 72 °C (Oshima & Imahori, 1974). Previously, we demonstrated that sdrP mRNA increases upon entry into the stationary phase, and SdrP positively regulates the expression of several kinds of genes, which are possibly involved in nutrient and energy supply, redox control, and polyadenylation of mRNA (Agari et al., 2008). Transcriptional activation occurs independently of any added effector GDC-0980 molecule, which is supported by the observation that the three-dimensional structure of apo-SdrP is similar to that of almost the DNA-binding form of E. coli CRP (Agari et al., 2008). In this study, to gain further insight into the cellular function of SdrP, we developed a new approach to identify novel genes whose expression was regulated by SdrP. The T. thermophilus wild-type and csoR gene-deficient (ΔcsoR) strains (Sakamoto et al., 2010) were cultured at 70 °C in a rich or synthetic medium (Supporting Information, Table S1). The details of the culture conditions

are given in the NCBI Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/projects/geo/), the accession numbers being GSE21433 [for N,N,N′,N′-tetramethylazodicarboxamide (diamide) treatment], GSE21430 (for H2O2 treatment), GSE20900 (for ZnSO4 treatment), GSE21432 (for tetracycline treatment), GSE21289 (for NaCl treatment), GSE21435 (for ethanol treatment), GSE19508 (for CuSO4 treatment of the wild-type strain), and GSE19509 (for CuSO4 treatment of the ΔcsoR strain). Total RNA was isolated from each strain, as described previously (Shinkai et al., 2007). Using the RNA (1 μg) as a template, RT-PCR was performed in 20 μL reaction mixtures with a PrimeScript RT-PCR kit (Takara Bio. Inc.) according to the manufacturer’s instructions. The reverse transcription reaction was performed at 42 °C for 20 min. Using 1 μL of the reaction mixture as a template, PCR was performed in the presence of 0.

brasilense Sp245 (Pothier et al, 2008) Azospirillum brasilense

brasilense Sp245 (Pothier et al., 2008). Azospirillum brasilense is able to produce considerable quantities of NO under aerobic conditions, and as stated before, NO production is required for Azospirillum-induced lateral root formation (Creus et al., 2005). Interestingly,

the mutant Faj164 that produces 5% of NO compared to the Sp245 wt strain in supplemented media was unable to induce the promoting effect on the tomato root growth system (Molina-Favero et al., 2008). Consequently, NO production might be another beneficial trait for plants inoculated with Azospirillum (Molina-Favero et al., 2008; Bashan & de-Bashan, 2010; Fibach-Paldi et al., 2012). To produce beneficial effects, Azospirillum has to interact with the plant surface to form complex

multicellular assemblies such as aggregates and biofilms that are initiated by an attachment process (Burdman et al., 2000). Biofilms Dasatinib mouse are defined as surface-attached multicellular aggregates, typically encased in a self-produced extracellular polymeric matrix (Ramey et al., 2004). Several factors like mechanical and nutritional stress, and inorganic and quorum-sensing molecules among others, regulate biofilms assembly and disassembly (Karatan & Watnick, 2009). In response to these factors, secondary messengers like cyclic diguanosine monophosphate (c-di-GMP) are activated (Hengge, 2009) leading to biofilm formation or modification (Karatan & Watnick, 2009). PAK6 Recently, it was shown that NO learn more stimulates biofilm formation by controlling the levels of

c-di-GMP (Plate & Marletta, 2012). On the other hand, Barraud et al. (2006, 2009) showed that NO triggered the disassembly of Pseudomonas aeruginosa biofilms acting upstream of c-di-GMP signaling pathway. More evidences of this complex picture are the results reported by Schmidt et al. (2004) who showed that cultures of Nitrosomonas europaea treated with exogenous NO gas enhanced biofilm formation. Considering that A. brasilense produces high amounts of NO in supplemented medium (Molina-Favero et al., 2008), it was interesting to test the effect of endogenous NO production on the ability of this beneficial bacterium to form biofilms. Hence, we proposed that NO could be involved in the signaling process for biofilm formation in A. brasilense. To determine this, we tested cultures of A. brasilense Sp245 and its isogenic Nap mutant Faj164 under static growth conditions for their ability to form biofilm on abiotic surfaces. We also evaluated the effects of the addition of a NO donor on biofilm formation. Azospirillum brasilense Sp245 wt, isolated from surface-sterilized wheat roots (Baldani et al., 1986), and A. brasilense Faj164, a knockout mutant of Sp245 with a Tn5 insertion in the napA gene of the operon (Steenhoudt et al., 2001a), were used.

Single cross-over recombinants were selected on VMM with streptom

Single cross-over recombinants were selected on VMM with streptomycin and gentamicin. There was no statistically significant difference

in the gusA activity of the chromosomal fusions and the plasmid fusions. The enzyme assays for β-glucuronidase activity were carried out based on the β-galactosidase activity method of Miller (1972), with modifications described by Yost et al. (2004). To measure the gusA activity of the pEV65, pEV60, pEV58, and pDF4 fusions from bacteroids, 5–10 nodules were placed into 500 μL of sterile 0.25 M mannitol, 0.05 M Tris-HCl, pH 8.0, and crushed with a sterile inoculating stick. The nodule debris was allowed to settle for several minutes, and the supernatant was used in a standard GusA assay as described previously. It was recently shown in S. meliloti that the acpXL gene, located upstream of fabZXL, is part of an operon with fabZXL, fabF2XL, and fabF1XL, buy Roxadustat while the adh2XL and lpxXL genes comprise a second operon (Haag et al., 2011). We used RT-PCR to determine the operon structure of the acpXL gene in R. leguminosarum. Primers binding within the acpXL and

fabF2XL genes did not amplify a PCR product following RT of R. leguminosarum 3841 mRNA, indicating that these genes are not cotranscribed (Fig. 1). Primers binding within the acpXL gene confirmed expression of the acpXL gene, and primers binding to the 16S rRNA gene were used to confirm the quality of the mRNA and the success of the RT reactions. The GusA activity observed from the gusA transcriptional fusion with the upstream DNA from fabZXL (pEV60) further confirms the CP868596 presence of a promoter between the acpXL and fabZXL genes. The DNA sequences immediately upstream of the fabZXL gene from a number of different species

within the Rhizobiaceae were aligned to investigate the difference in operon structure between the rhizobial strains. While the acpXL and fabZXL gene sequences are ≥ 88% and ≥ 90% identical, Ixazomib molecular weight respectively, the intergenic region between acpXL and fabZXL is heterogeneous among the different species. There is also variability in the length of the intergenic sequence between the acpXL and fabZ genes. In R. leguminosarum bv. viciae 3841 and R. leguminosarum bv. trifolii WSM1325, the sequence is 205 and 204 bp, respectively. In S. meliloti, the sequence is 172 bp, and in Agrobacterium tumefaciens str C58, the intergenic sequence is further reduced to 90 bp. These differences in length of the intergenic region can be partially explained by a unique 72-bp insertion found in R. leguminosarum bv. viciae 3841 and R. leguminosarum bv. trifolii WSM1325 (Fig. 1). These results demonstrate that while the individual genes for biosynthesis of the VLCFA are homologous between different rhizobial species, the arrangement of those genes into operons has not been as stringently conserved.

Every man who uses BCN Checkpoint services is tested for and coun

Every man who uses BCN Checkpoint services is tested for and counselled regarding HIV infection and syphilis. Peer counselling is offered by an openly gay staff, and some of the

counsellors are PLWHIV themselves. VCT lasts 1 h on the first visit and 30 min on subsequent visits (although it can take longer depending on the client’s needs) where men are able to talk openly about sexuality, their perceptions of the risk of HIV transmission, and sexual safety without fearing prejudice or stigma. Education is also provided on post-exposure prophylaxis (PEP) and other STIs. Men with an HIV-positive result receive immediate emotional support from a peer, have the result confirmed by a Western blot test, and are offered

an appointment at one of Barcelona’s HIV units. Men with IWR-1 cost an HIV-negative result receive counselling encouraging them to maintain sexual safety for risk reduction, and are invited to repeat PD-0332991 purchase the test at least every 6 or 12 months. Only data regarding HIV were included in this study. We determined (1) the number of tests performed and the number of persons tested, (2) the global HIV prevalence and the HIV prevalence for first visits to the centre, (3) the proportion of reported HIV cases in MSM in Catalonia detected at BCN Checkpoint, (4) the proportion of HIV-positive individuals with a previous negative test result within the last 18 months, (5) the linkage to care rate: the proportion of newly diagnosed individuals successfully linked to medical care (a successful linkage was considered an HIV unit referral within 4 weeks). Table 1 shows the HIV positivity rates from 2007 to 2012. The numbers of tests (row 1), persons tested (row 2) and HIV-positive cases (row 3) increased progressively. BCN Checkpoint achieved a maximum of 5051 tests offered to a population of 4049 different men in 2012. As a result of the promotion of regular testing for MSM, the proportion of people returning to

the centre increased over the years. Nevertheless, the number of persons who visited BCN Checkpoint for the first time (row 5) Aprepitant remained steady and the average prevalence of HIV positivity for these individuals (row 7) was 5.4% (range: 4.1−5.8%). Regarding the detection of HIV in MSM in Catalonia, BCN Checkpoint detected a substantial proportion of all new cases of HIV infection in MSM between 2007 and 2011 (row 9), according to the Catalan National HIV Surveillance System (row 8; no data from 2012 yet available). During 2009–2011 the average proportion was 36.6% (range: 35.0−40.4%). The proportion of individuals newly diagnosed at BCN Checkpoint between 2009 and 2012 who had had at least one previous negative test result within the last 18 months was 62.1% (284 out of 457). Some of these detections were recent, acute infections.

However, intracellular M bovis CFU decreases drastically after 2

However, intracellular M. bovis CFU decreases drastically after 24 h, which could be attributed to the massive cellular death observed. The CFU assessment shows no significant difference in the intracellular bacterial load of M. bovis between MDMs from tuberculosis and healthy control cattle. BTB is a chronic infectious disease caused by the pathogen M. bovis and continues to pose a threat to livestock

worldwide. Mycobacterium bovis is the causative agent of most cases of tuberculosis in cattle and M. bovis Beijing strains cause a substantial proportion of tuberculosis cases worldwide (Chen et al., 2009; Kremer et al., 2009). Understanding the specific immune response to BTB will aid in developing improved control and diagnostic strategies. Studies on tuberculosis in humans indicate that innate immunity, Apitolisib in vivo TLR signaling and the Th1/Th2 bias of the immune response are essential for host defense against tuberculosis (Doherty & Arditi, 2004; Winek et al., 2009; Ahmad, 2011). However, these specific cell signal pathways and immune responses are poorly defined in cattle. Meade et learn more al. (2006)

examined the gene expression profiles of PBMCs from BTB-infected and healthy cattle and demonstrated the differential expression of innate immunity-related genes. In this study, gene expressions of MDMs cells from tuberculosis and healthy groups stimulated with M. bovis were detected. Seven genes (IL1β, IL1R1, IL1A, TNF-α, IL10, TLR2 and TLR4) implicated in immune responses were examined. In MDMs, the expression of the seven examined genes was increased in both stimulated tuberculosis and stimulated healthy cattle. The expression of the proinflammatory cytokine TNF-α, IL1β and its receptor IL1R1 markedly increased, indicating that these genes may play a key role in the early interaction of host cells and M. bovis. The expression of these three genes, although elevated in response to M. bovis stimulation,

showed no significant difference between the two groups. This finding may indicate that the macrophages from tuberculosis cattle have a capability similar to healthy cattle in generating proinflammatory cytokine (IL1β and TNF-α) during early immune response to M. bovis stimulation. In agreement, why it is frequently reported that the tuberculosis infection could induce a burst of inflammatory cytokines IL1β and TNF-α in the infected location (Arcila et al., 2007; Qiu et al., 2008; Winek et al., 2009). Two Toll-like receptor genes (TLR2 and TLR4) were examined. The two genes have been studied widely, because they are very important in innate immunity and TLR signaling aids the activation of antigen-specific T cells (Cooper, 2009). Previous studies demonstrated that M. tuberculosis products can be recognized by TLR2 or TLR4 (Aliprantis et al., 1999; Underhill et al., 1999; Abel et al., 2002).

However, intracellular M bovis CFU decreases drastically after 2

However, intracellular M. bovis CFU decreases drastically after 24 h, which could be attributed to the massive cellular death observed. The CFU assessment shows no significant difference in the intracellular bacterial load of M. bovis between MDMs from tuberculosis and healthy control cattle. BTB is a chronic infectious disease caused by the pathogen M. bovis and continues to pose a threat to livestock

worldwide. Mycobacterium bovis is the causative agent of most cases of tuberculosis in cattle and M. bovis Beijing strains cause a substantial proportion of tuberculosis cases worldwide (Chen et al., 2009; Kremer et al., 2009). Understanding the specific immune response to BTB will aid in developing improved control and diagnostic strategies. Studies on tuberculosis in humans indicate that innate immunity, GSK2126458 chemical structure TLR signaling and the Th1/Th2 bias of the immune response are essential for host defense against tuberculosis (Doherty & Arditi, 2004; Winek et al., 2009; Ahmad, 2011). However, these specific cell signal pathways and immune responses are poorly defined in cattle. Meade et see more al. (2006)

examined the gene expression profiles of PBMCs from BTB-infected and healthy cattle and demonstrated the differential expression of innate immunity-related genes. In this study, gene expressions of MDMs cells from tuberculosis and healthy groups stimulated with M. bovis were detected. Seven genes (IL1β, IL1R1, IL1A, TNF-α, IL10, TLR2 and TLR4) implicated in immune responses were examined. In MDMs, the expression of the seven examined genes was increased in both stimulated tuberculosis and stimulated healthy cattle. The expression of the proinflammatory cytokine TNF-α, IL1β and its receptor IL1R1 markedly increased, indicating that these genes may play a key role in the early interaction of host cells and M. bovis. The expression of these three genes, although elevated in response to M. bovis stimulation,

showed no significant difference between the two groups. This finding may indicate that the macrophages from tuberculosis cattle have a capability similar to healthy cattle in generating proinflammatory cytokine (IL1β and TNF-α) during early immune response to M. bovis stimulation. In agreement, Obatoclax Mesylate (GX15-070) it is frequently reported that the tuberculosis infection could induce a burst of inflammatory cytokines IL1β and TNF-α in the infected location (Arcila et al., 2007; Qiu et al., 2008; Winek et al., 2009). Two Toll-like receptor genes (TLR2 and TLR4) were examined. The two genes have been studied widely, because they are very important in innate immunity and TLR signaling aids the activation of antigen-specific T cells (Cooper, 2009). Previous studies demonstrated that M. tuberculosis products can be recognized by TLR2 or TLR4 (Aliprantis et al., 1999; Underhill et al., 1999; Abel et al., 2002).

, 2008) between examined Sodalis isolates, C melbae, and C colu

, 2008) between examined Sodalis isolates, C. melbae, and C. columbae symbionts. The ompA, ompC, and rcsF loci (Fig. 2) appear to be more informative toward the phylogenetic resolution of the Sodalis-like symbiont clade. With CX-5461 nmr rcsF, sufficient phylogenetic signal was provided to enable clustering of the Glossina symbionts, with strong support, separate from the C. melbae symbiont (Fig. 2b). Interestingly, rcsF in E. coli has been shown to be involved in signaling transduction of perturbations and/or environmental cues from the cell surface (Majdalani et al., 2005). Diversification between Sodalis and C. melbae isolates may indicate functional

adaptations, such as differences in the type of signaling encountered within the host species background. The Sodalis symbionts also formed a distinct clade with the ompC phylogeny, with most mutations noted outside of the seven putative extracellular loops (Basle et al., 2006) of the different Glossina isolates. The one exception occurred in extracellular loop 4, where host interspecies diversity was observed with Sodalis isolates. Relative to the other surface encoding genes analyzed in this study, the ompA gene exhibited the greatest diversity among symbionts due to a combination of point mutations

and indels. The best-studied ompA gene variant, that of E. coli K-12, encodes a 325 amino acid polypeptide Dimethyl sulfoxide (Chen et al., 1980). The N-terminal domain forms an eight-stranded β-barrel in the outer membrane, creating four surface-exposed loops (Pautsch & Schulz, 1998), while the C-terminus is Selleck Y 27632 periplasmic (Klose et al., 1988). Amino acid variations within outer membrane proteins mainly occur in the

domains located in the extracellular regions, while interspaced residues making up the β-strands tend to be conserved. In our analyses, relative to Glossina symbionts, a total of nine nonsynonymous mutations were observed among C. melbae, C. columbae, and Sitophilus (i.e. Sitophilus oryzae primary symbiont, SOPE) symbionts occurring in loops 1–4 of the OmpA protein. Differences noted in the ompA sequence between the Glossina symbionts were localized outside of the extracellular regions, similar to our observations with ompC. In relation to ompA, the C. columbae symbiont exhibited the greatest nucleotide divergence resulting in its sister taxon placement relative to the other symbionts of interest with strong MP bootstrap support. MP, Bayesian, and NJ analyses all grouped Glossina symbionts within their own clade indicative of diversification potentially arising from host adaptation processes. The Sodalis ompA gene demonstrated a wide nucleotide variation (π) within tsetse species (Table 1), with the highest π exhibited within G. morsitans (π=0.11) and the lowest within G. brevipalpis (π=0.001).

For example, pyocyanin is the blue/green pigmented toxin that giv

For example, pyocyanin is the blue/green pigmented toxin that gives P. aeruginosa cultures their characteristic color and acts as an antimicrobial that can kill competing microorganisms. However, it also disrupts

eukaryotic cellular processes, which can have a detrimental effect on human cells (Rada & Leto, 2013). The qualities which make pseudomonads evolutionarily fit have been both beneficial and detrimental to humans. On the one hand, we have harnessed the power of pseudomonads for bioremediation and biocontrol. For example, P. fluorescens Omipalisib price and P. protegens have proved particularly successful in pest control and crop protection, where they are thought to outcompete and/or antagonize plant pathogens (Kupferschmied et al., 2013). The catabolic power of pseudomonads has also been wielded for biodegradation and/or detoxification of pesticides, heavy metals, and hydrocarbons (e.g. oil spills), as see more well as many other pollutants (Wasi et al., 2013). On the other hand, some species of Pseudomonas are pathogenic to plants and animals, causing infections that can be extremely difficult to eradicate. For example, P. aeruginosa is one of the most frequent causes of hospital-acquired infections worldwide, mainly owing to its abilities to thrive in water-related hospital reservoirs and survive killing by disinfectants and antibiotics. Once

again demonstrating its ability to occupy diverse niches,

it can cause infections at many anatomical sites, including the skin, brain, eyes, ears, urinary tract, and lungs. Immunosuppressed individuals, particularly those with excessive burn wounds, cystic fibrosis, or neutropenia, are particularly at risk. The exceptional ability of P. aeruginosa and other Pseudomonas species to cause such a diverse array of infections is their capacity this website to produce a veritable arsenal of virulence factors, including toxins, proteases, and hemolysins. Considering the medical importance of P. aeruginosa, it is not surprising that much of the research effort in the Pseudomonas field has been devoted to trying to understand the regulation, biosynthesis, and environmental cues influencing the release of these virulence factors. Prof. Gerd Döring is an example of one such researcher who devoted his career to investigating the pathogenic mechanisms of P. aeruginosa in the lungs of patients with cystic fibrosis. In their touching obituary, Burkhard Tümmler and Dieter Haas detail the contributions Prof. Doring made to the field. The many new treatment strategies that have helped dramatically increase the average life span of patients with cystic fibrosis is due, in no small part, to the research of Prof. Döring and others in his field. The first Pseudomonas genome was sequenced in 2000, and at 6.

For example, pyocyanin is the blue/green pigmented toxin that giv

For example, pyocyanin is the blue/green pigmented toxin that gives P. aeruginosa cultures their characteristic color and acts as an antimicrobial that can kill competing microorganisms. However, it also disrupts

eukaryotic cellular processes, which can have a detrimental effect on human cells (Rada & Leto, 2013). The qualities which make pseudomonads evolutionarily fit have been both beneficial and detrimental to humans. On the one hand, we have harnessed the power of pseudomonads for bioremediation and biocontrol. For example, P. fluorescens Palbociclib datasheet and P. protegens have proved particularly successful in pest control and crop protection, where they are thought to outcompete and/or antagonize plant pathogens (Kupferschmied et al., 2013). The catabolic power of pseudomonads has also been wielded for biodegradation and/or detoxification of pesticides, heavy metals, and hydrocarbons (e.g. oil spills), as MDV3100 manufacturer well as many other pollutants (Wasi et al., 2013). On the other hand, some species of Pseudomonas are pathogenic to plants and animals, causing infections that can be extremely difficult to eradicate. For example, P. aeruginosa is one of the most frequent causes of hospital-acquired infections worldwide, mainly owing to its abilities to thrive in water-related hospital reservoirs and survive killing by disinfectants and antibiotics. Once

again demonstrating its ability to occupy diverse niches,

it can cause infections at many anatomical sites, including the skin, brain, eyes, ears, urinary tract, and lungs. Immunosuppressed individuals, particularly those with excessive burn wounds, cystic fibrosis, or neutropenia, are particularly at risk. The exceptional ability of P. aeruginosa and other Pseudomonas species to cause such a diverse array of infections is their capacity PtdIns(3,4)P2 to produce a veritable arsenal of virulence factors, including toxins, proteases, and hemolysins. Considering the medical importance of P. aeruginosa, it is not surprising that much of the research effort in the Pseudomonas field has been devoted to trying to understand the regulation, biosynthesis, and environmental cues influencing the release of these virulence factors. Prof. Gerd Döring is an example of one such researcher who devoted his career to investigating the pathogenic mechanisms of P. aeruginosa in the lungs of patients with cystic fibrosis. In their touching obituary, Burkhard Tümmler and Dieter Haas detail the contributions Prof. Doring made to the field. The many new treatment strategies that have helped dramatically increase the average life span of patients with cystic fibrosis is due, in no small part, to the research of Prof. Döring and others in his field. The first Pseudomonas genome was sequenced in 2000, and at 6.

, 2001) All components of both systems were heterologously produ

, 2001). All components of both systems were heterologously produced in Escherichia coli (Schilhabel et al., 2009). Both MT I are zinc-containing enzymes (Schilhabel et al., 2009). Zinc may have structural or catalytic functions in proteins (Vallee & Auld, 1990a, b). The metal is generally bound to the side chains of histidine, cysteine, aspartate or glutamate (Vallee & Auld,

1990a). In most cases, zinc is bound to three amino acid side chains and one water molecule when the metal has a catalytic function in enzymes (Auld, 2001). These zinc-binding motifs usually exhibit common characteristics with regard to the distances between the zinc-binding amino acids in the primary structure of the proteins (Auld, 2001). Two of these amino acids are separated by a short distance of one to three amino acids; the third ligand

is located at a distance of 20–120 amino acids to the other ligands Venetoclax clinical trial (Vallee & Auld, 1990a). Exceptions to this rule are the cofactor-dependent alcohol dehydrogenase (Vallee & Auld, 1990a) and the cobalamin-dependent methanol methyltransferase of Methanosarcina barkeri (Hagemeier et al., 2006). In this study, we report on the identification of the zinc-binding motifs of MT Ivan of the vanillate-O-demethylase and MT Iver of the veratrol-O-demethylase of A. dehalogenans using site-directed mutagenesis. Acetobacterium dehalogenans was cultivated anaerobically as described PF-562271 earlier (Traunecker et al., 1991). Syringate (20 mM) or fructose (20 mM) was used as a growth substrate. The production of the recombinant proteins and the purification of the methyltransferases and of CP were performed as described earlier (Schilhabel et al., 2009). For the activation reaction, crude extracts of E. coli containing the recombinant AE were used. These crude extracts did not exhibit methyltransferase MTMR9 activity. Cells of A. dehalogenans (0.2 g wet weight) were

suspended in 1 mL 10 mM Tris-HCl, pH 8.0, containing 10 mM EDTA. The genomic DNA was isolated according to Bollet et al. (1991). After incubation with 0.01% RNase (w/v) for 15 min at 37 °C, the DNA was stored at 4 °C. Expression cassettes of the mutated genes of MT Ivan and MT Iver (GenBank accession no. AF087018 and AY318856) as fusion proteins with a C-terminal Strep-tag and with restriction sites for the cloning in pET11a (Agilent Technologies, Böblingen, Germany) were constructed from PCR products. Point mutations of both enzymes were generated using overlap extension PCR essentially using the method described by An et al. (2005). The mutations were inserted using multistep PCR. In the first PCR step, two fragments were amplified: one by the combination of primer 1 (MT Ivan) or 3 (MT Iver) (Table 1) with the mutated reverse primer and the other fragment by the combination of the mutated forward primer with primer 2 (MT Ivan) or 4 (MT Iver) (Table 1).