For this the application weblogo 3 (Crooks et al., 2004; http://weblogo.threeplusone.com) was used. Sequence logos have been useful in visualizing patterns in aligned sequence motifs (Schneider & Stephens, 1990) and have indeed been used to analyse Tat motifs (see e.g. Bendtsen et al., 2005). We used this to compare the Tat motifs of haloarchaeal Tat substrates with that of the consensus E. coli selleckchem motif

(S/TRRxFLK). Signal peptide-containing sequences were extracted from genomes of E. coli and three fully sequenced haloarchaea: H. marismortui, N. pharaonis, and H. salinarum. The datasets obtained (see Supporting Information, Table S1) were filtered as outlined in Materials and methods to minimize the number of false-positive hits. Current information of prokaryotic signal peptides in general and the Tat system more specifically is mostly derived from bacterial systems, and as such, our searches may have been biased towards bacterial-like signal peptides. This, and our additional filtering, has most likely led to the absence of some genuine Tat signal peptides. Indeed, some proteins that are known to be Tat substrates in E. coli are missing from our dataset, including FdnH, HyaA, and HybO, all of which have been shown experimentally selleck inhibitor to be Tat substrates (Hatzixanthis et al., 2003; Berks et al., 2005).

However, these three contain C-terminal transmembrane helices, which is the reason why our filtering steps rejected them. Nevertheless, only a fairly small proportion of Tat substrates have such additional

membrane-spanning domains, and we think that this approach has also resulted in datasets with very few or no false-positive proteins. The twin-arginine motifs obtained were aligned manually and used to generate sequence logos (Fig. 1). As can be observed from the top panel, our method used indeed led to a motif with the consensus SRRxFLK Decitabine as observed before (Berks, 1996). The twin-arginine motifs in haloarchaea were similar, but with a number of notable differences. Firstly, the dominance of Phe in position 5 is less pronounced than in E. coli; Val is found in that position in a very similar frequency. Secondly, Leu in position 6 appears to be far more frequent in haloarchaeal Tat motifs as compared with the E. coli Tat motif. Finally, the Lys in position 7 is less common in haloarchaea as compared with E. coli. Some of these differences may be attributable to the overall differences in the amino acid composition between halophilic and nonhalophilic proteins. For instance, haloarchaea contain, on average, fewer large hydrophobic residues such as Phe, as well as a relatively low percentage of lysine residues as compared with bacteria such as E. coli or Bacillus subtilis (Bolhuis et al., 2007). In this respect, the prominence of Leu in position 6 is actually interesting as this residue is, like Phe, less frequent in haloarchaeal proteins.

For this the application weblogo 3 (Crooks et al., 2004; http://weblogo.threeplusone.com) was used. Sequence logos have been useful in visualizing patterns in aligned sequence motifs (Schneider & Stephens, 1990) and have indeed been used to analyse Tat motifs (see e.g. Bendtsen et al., 2005). We used this to compare the Tat motifs of haloarchaeal Tat substrates with that of the consensus E. coli Akt inhibitor motif

(S/TRRxFLK). Signal peptide-containing sequences were extracted from genomes of E. coli and three fully sequenced haloarchaea: H. marismortui, N. pharaonis, and H. salinarum. The datasets obtained (see Supporting Information, Table S1) were filtered as outlined in Materials and methods to minimize the number of false-positive hits. Current information of prokaryotic signal peptides in general and the Tat system more specifically is mostly derived from bacterial systems, and as such, our searches may have been biased towards bacterial-like signal peptides. This, and our additional filtering, has most likely led to the absence of some genuine Tat signal peptides. Indeed, some proteins that are known to be Tat substrates in E. coli are missing from our dataset, including FdnH, HyaA, and HybO, all of which have been shown experimentally CDK inhibitor to be Tat substrates (Hatzixanthis et al., 2003; Berks et al., 2005).

However, these three contain C-terminal transmembrane helices, which is the reason why our filtering steps rejected them. Nevertheless, only a fairly small proportion of Tat substrates have such additional

membrane-spanning domains, and we think that this approach has also resulted in datasets with very few or no false-positive proteins. The twin-arginine motifs obtained were aligned manually and used to generate sequence logos (Fig. 1). As can be observed from the top panel, our method used indeed led to a motif with the consensus SRRxFLK SPTLC1 as observed before (Berks, 1996). The twin-arginine motifs in haloarchaea were similar, but with a number of notable differences. Firstly, the dominance of Phe in position 5 is less pronounced than in E. coli; Val is found in that position in a very similar frequency. Secondly, Leu in position 6 appears to be far more frequent in haloarchaeal Tat motifs as compared with the E. coli Tat motif. Finally, the Lys in position 7 is less common in haloarchaea as compared with E. coli. Some of these differences may be attributable to the overall differences in the amino acid composition between halophilic and nonhalophilic proteins. For instance, haloarchaea contain, on average, fewer large hydrophobic residues such as Phe, as well as a relatively low percentage of lysine residues as compared with bacteria such as E. coli or Bacillus subtilis (Bolhuis et al., 2007). In this respect, the prominence of Leu in position 6 is actually interesting as this residue is, like Phe, less frequent in haloarchaeal proteins.

7 kDa and the pI is 9.7. Both are predicted to have a short cytoplasmic tail adjacent to a single transmembrane region,

followed by the extracellular part containing the LCP domain, extending from aa 86 to 234 in SA0908 and from aa 90 to 236 in SA2103. The transcriptional start sites (TSS) of sa0908 and sa2103 were identified by primer extension and were 99 and 44 bp upstream of the start codons, Protein Tyrosine Kinase inhibitor respectively, and were preceded by putative promoter elements (Fig. 1a and b). Northern blots revealed that sa0908 and sa0907 were cotranscribed on a single mRNA of ∼2000 nt in wild-type MSSA1112. The deletion of sa0908 in strain RH53 resulted in a shorter, ∼800 bp, sa0907 transcript (Fig. 1c). Two transcripts hybridized to the sa2103 DIG-probe, an ∼1100 bp transcript, which initiated at the TSS, and a larger transcript of ∼2000 bp, representing a bicistronic sa2104–sa2103 transcript, which decreased in size to ∼1000 bp in the Δsa2103 mutant PS47 (Fig. 1d). Promoter–luciferase fusion constructs were used to compare the relative expression

RG7420 manufacturer levels of msrR, sa0908 and sa2103 over growth (Fig. 1D). The expression of all three genes peaked during exponential growth when cells were dividing rapidly, and then decreased as cultures entered the stationary phase. The relative expression levels of msrR were much higher than those of sa0908 and sa2103. To obtain a comprehensive overview of the functions of LCP genes, we created all possible combinations of double mutants: RH72 (Δsa0908/ΔmsrR), PS60 (Δsa2103/ΔmsrR) and PS110 (Δsa2103/Δsa0908), and a triple mutant PS111 (Δsa2103/Δsa0908/ΔmsrR). To further investigate the roles of individual LCP proteins, we complemented the triple mutant with msrR, sa0908 or sa2103 in trans. The deletion of msrR was previously shown to have no effect on

the growth rate (Hubscher et al., 2009). The deletion of sa0908 or sa2103 also had only a small, but complementable effect on growth in RH53 (Δsa0908) and PS47 (Δsa2103). The deletion of a second LCP protein had negligible further effects on the growth characteristics (data not shown). The growth of the triple mutant PS111 was severely Coproporphyrinogen III oxidase retarded, with the growth rate decreasing from 1.39 to 0.95 h−1 at 37 °C (Fig. 2a). This growth defect was further exacerbated at 42 °C (Fig. 2b). The ability of the three proteins to complement this growth defect differed, especially at the elevated temperature of 42 °C: MsrR restored growth almost to the wild-type level, followed by SA0908, which compensated growth to up to ∼70% of the wild type OD600 nm after 7 h, while SA2103 had the lowest effect (Fig. 2b). LCP mutants were analysed by TEM and the cell sizes of a minimum of 100 cells per strain were measured and expressed as the mean±SD. In single mutants, enlarged cells and irregular septa were observed in the msrR mutant (JH100 ∅1.33±0.16 μm) as reported previously (Hubscher et al., 2009). The cells of sa0908 (RH53 ∅1.04±0.07 μm) and sa2103 (PS47 ∅1.

All cases of severe malaria were due to P. falciparum, except one case attributed to P. vivax. Fifteen patients received exchange blood transfusion (10 cases) or red cell exchange (5 cases). Eleven of these patients had levels of parasitemia ≥10% (10%–40%, media 21.3%), and four patients had lower parasitemia level (1, 2, 7, and 8%, respectively), all of them with good resolution. Three women were www.selleckchem.com/products/ch5424802.html pregnant (weeks 5, 6, and 35) at the moment of the diagnosis, all of them infected

with P. falciparum. No case of congenital malaria was reported, but one of these women (week 5) suffered an abort. Other complications observed are listed in Table 4. Seven deaths were observed (mortality rate 3.8%), all due to P. falciparum: six foreign sailors and a recently arrived immigrant woman with polymyositis. Malaria in our region is imported from endemic areas and more frequent Doxorubicin chemical structure in young male travelers. This is the predominant pattern of malaria in Spain (Table 5). However, there are differences among groups of patients pertaining to their origin and travel purposes. Plasmodium falciparum was the most frequent species in our region, because a vast majority of cases are coming from the

African continent, as it is the case in Europe. However, unlike other European countries with a higher account of cases from Nigeria and Ghana,35,36 imported malaria from Equatorial Guinea, Senegal, and Mauritania is much more common in Spain.12–19,27,28 Political and geographical reasons could explain in part this fact: Equatorial Guinea was a Spanish colony until 1960s, and Senegal and Mauritania are geographically and commercially really close to the Canary Islands. next During the first period of the study, tourists and business travelers were the group with more cases, but since the year 2000, diagnosis in this group is decreasing. The last years of the study (2001–2006) showed that malaria cases are increasing among recently arrived immigrants and VFR (Figure 2). This fact reveals the importance of malaria suspicion in these individuals, considering that classic signs

and symptoms, mainly in children, are not always present; even in febrile travelers, a recent French study concludes that no single clinical or biological feature has both good sensitivity and specificity to predict malaria.37 For these reasons, we consider that a malaria diagnosis must not be ruled out in immigrant patients without fever or with levels of parasitemia so low that they could not be shown with light microscopy. In these cases, the performance of molecular biology tests such as PCR seems to be very useful. Anemia and thrombocytopenia are common laboratory findings, but it is necessary to look for other concomitant infections if high leukocyte count is observed.30 Severe malaria due to non-P. falciparum species is not frequent, but possible. We described one P.

edisan.fr). Edisan® is a commercially available software updated every 6 weeks, used to help physicians for travel advice in general Cabozantinib clinical trial and for malaria prophylaxis and vaccine prescriptions in particular. Updated specific recommendations are provided for each country, and within each country for specific areas at risk. Physicians can also use folders with updated recommendations and prefilled prescriptions for malaria prophylaxis, mosquito repellents, and mosquito nets. During the visit,

patients receive individualized travel health advice according to their medical condition, and general advice on vector-borne diseases, water-borne diseases, animal bites, as well as sexually transmitted diseases, high altitude sickness, and trauma. Patients are prescribed vaccines and malaria chemoprophylaxis when appropriate. Finally, FK506 in vitro they are encouraged to update their routine vaccination (hepatitis B, Diphtheria-Tetanus-Poliomyelitis, measles, and pertussis). Vaccinations are then performed by one of the three nurses in the center on the same day. The objective of the study was to assess the adequacy of malaria prophylaxis, yellow fever, and hepatitis A vaccination prescriptions to French recommendations. For

that purpose we used the questionnaires available in our center that were designed to assess our current practice and to ensure traceability of the advice and prescriptions

given to travelers. The questionnaire is first filled by the traveler while he/she is waiting for the physician and the ifoxetine data are then checked and completed by the physician. The questionnaires covered the following areas: age, sex, medical condition and past medical history of each traveler, ongoing treatment, pregnancy status, and vaccine status. The trip characteristics are also recorded: destinations and itineraries, duration, and type of travel (rural or urban, for tourism or visiting friends and relatives, or professional). On the same questionnaire, the physician prescribes the vaccines to be administered by the nurse and treatments recommended during the visit (chemoprophylaxis for malaria, anti-diarrheal agents or antibiotics for travelers’ diarrhea or any other specific treatment). The reasons for the choice of malaria prophylaxis prescribed are also provided by the physician. The majority of questions could be answered by “yes” or “no,” some questions provided answer choices, and a few others allowed free text entries. All physicians were informed of the study goals and time lines of implementation. At the end of the study period, all questionnaires were reviewed by two investigators who assessed the adequacy of the prescriptions to the French recommendations for malaria chemoprophylaxis and yellow fever and hepatitis A vaccines.

edisan.fr). Edisan® is a commercially available software updated every 6 weeks, used to help physicians for travel advice in general TSA HDAC in vivo and for malaria prophylaxis and vaccine prescriptions in particular. Updated specific recommendations are provided for each country, and within each country for specific areas at risk. Physicians can also use folders with updated recommendations and prefilled prescriptions for malaria prophylaxis, mosquito repellents, and mosquito nets. During the visit,

patients receive individualized travel health advice according to their medical condition, and general advice on vector-borne diseases, water-borne diseases, animal bites, as well as sexually transmitted diseases, high altitude sickness, and trauma. Patients are prescribed vaccines and malaria chemoprophylaxis when appropriate. Finally, Ponatinib cell line they are encouraged to update their routine vaccination (hepatitis B, Diphtheria-Tetanus-Poliomyelitis, measles, and pertussis). Vaccinations are then performed by one of the three nurses in the center on the same day. The objective of the study was to assess the adequacy of malaria prophylaxis, yellow fever, and hepatitis A vaccination prescriptions to French recommendations. For

that purpose we used the questionnaires available in our center that were designed to assess our current practice and to ensure traceability of the advice and prescriptions

given to travelers. The questionnaire is first filled by the traveler while he/she is waiting for the physician and the Anidulafungin (LY303366) data are then checked and completed by the physician. The questionnaires covered the following areas: age, sex, medical condition and past medical history of each traveler, ongoing treatment, pregnancy status, and vaccine status. The trip characteristics are also recorded: destinations and itineraries, duration, and type of travel (rural or urban, for tourism or visiting friends and relatives, or professional). On the same questionnaire, the physician prescribes the vaccines to be administered by the nurse and treatments recommended during the visit (chemoprophylaxis for malaria, anti-diarrheal agents or antibiotics for travelers’ diarrhea or any other specific treatment). The reasons for the choice of malaria prophylaxis prescribed are also provided by the physician. The majority of questions could be answered by “yes” or “no,” some questions provided answer choices, and a few others allowed free text entries. All physicians were informed of the study goals and time lines of implementation. At the end of the study period, all questionnaires were reviewed by two investigators who assessed the adequacy of the prescriptions to the French recommendations for malaria chemoprophylaxis and yellow fever and hepatitis A vaccines.

And while general educational efforts regarding what constitutes a ‘risky behaviour’ (e.g. unprotected anal sex) is a critical part of prevention efforts, understanding the factors that dispose people towards risky behaviours allows for targeted deployment of finite prevention resources. To that end, researchers and clinicians have been investigating a variety of predictors of sexual TRBs. Among behavioural and socio-demographic risk factors for sexual TRBs, substance abuse – especially Adriamycin order sildenafil [4–6], methamphetamine [7] and alcohol [8,9] abuse – appears to contribute to subsequent sexual TRBs separately

from the risk factors directly associated with IDU. Multiple partners [10], youth [11], sex trade GSK2126458 in vivo work [10] and limited education [12] also appear to be relevant factors. To complement these efforts, researchers and clinicians have also been examining a variety of psychological variables as potential predictors of propensity to engage in TRBs. The typical rationale is that if clinicians can, in combination with standard medical and psychiatric histories, use a relatively brief screener to identify those at greater risk of TRBs, then limited prevention resources can be directed towards those who will most benefit. Self-efficacy, treatment optimism or optimistic

attitude, perception of power within relationships [13] and supportive social norms [14,15] are all psychological variables associated with relatively low sexual TRBs. In terms of risk, depression [16], co-occurring severe mental illness and substance use [17], history of childhood sexual abuse [10], antisocial personality disorder [18] and psychological stress all have some supportive evidence as predictors of TRBs. Because providing good

medical care for cAMP persons living with HIV infection is already a challenge for time-constrained primary care providers, a lengthy sexual TRB assessment may take up time that providers do not have to spare. Following recommendations from the Centers for Disease Control and Prevention [19] and the Institute of Medicine [20], the Health Resources and Services Administration (HRSA) sponsored a 5-year initiative to develop and evaluate HIV prevention services in clinical settings. Fifteen sites received awards to tailor evidence-based prevention approaches to their settings and populations. Seattle was awarded one of the grants to conduct a 2-year, randomized controlled trial utilizing audio computer-assisted self interviews (ACASIs) to evaluate the effect of an intervention on TRBs over time. The intervention involved motivational interviewing and small group peer interventions conducted by a nurse specialist. The comparison group included patients who chose not to enrol or to delay enrolment in the intervention arm.

The second, refolding step included washing with AZD1208 cell line BB at linearly decreasing urea concentrations (from 8 to 0 M). The protein was eluted with a linear gradient of imidazole from 5 to 500 mM. Protein was collected

at 0–250 mM imidazole concentrations in a total volume of 4–5 mL. Rpf-containing fractions (30–50 μg mL−1) were dialyzed against 50 mM citric acid–sodium citrate buffer (pH 6.0). Protein samples were stored at +4 °C for 1 week without a significant loss in its activity. Myñobacterium smegmatis strains were grown under the conditions that favored the entering wild-type strain to ‘nonculturable’ (NC) state (inability to produce colonies on solid media) in stationary phase after cultivation of mycobacteria in the modified Hartman-de-Bont medium, lacking K+, at 37 °C for 120 h under aeration (Shleeva et al., 2004). In the other model, the strains under study were incubated for 4.5 months after growth in N-limited SR-1 medium to produce morphologically distinct ovoid cells (Anuchin et al., 2009). The ability of ‘NC’ cells to resuscitate in liquid medium was estimated using the most probable number (MPN) assays in triplicate repeats with inoculation of 0.1 mL cell suspensions to 0.9 mL of the modified Sauton medium in plastic 48-well microplates (Corning) as described previously (Downing et al., 2005). The Sauton medium that served for resuscitation contained (L−1): KH2PO4, 0.25 g; MgSO4·7H2O, 0.25 g; l-asparagine,

2 g; glycerol,

AUY-922 manufacturer 6 mL; ferric ammonium citrate, 0.025 g; sodium citrate, 1 g; 1% ZnSO4, 0.05 mL; ± recombinant RpfSm protein, 5 μg (pH 7.0). Cell suspensions were examined under a microscope Eclipse E4000 (Nikon, Japan) in the phase-contrast and epifluorescence modes after staining with propidium iodide (3 μM) to detect injured/dead cells or with 4′-6-diamidino-2-phenylindole (DAPI) (2 μg mL−1) bound to double-helix DNA. Excitation was at 510 and 330 nm, and emission was at >560 and >380 nm for propidium iodide and DAPI, respectively. One-milliliter aliquots were taken from stationary-phase (48 h) cultures in NB medium or from cultures stored for 4.5 months in N-limited SR-1 medium and were transferred into Petri dishes with 4 mL of liquid NB medium and then subjected to UV irradiation (BUV-30 lamp, 254 nm) as described elsewhere (Vorobjeva et al., 1995). Samples from the same Tacrolimus (FK506) cultures were also heated at 60–80 °C for 10 min. Cells after UV or heat treatment were plated onto solid NB medium for CFU assays. As already demonstrated, after cultivation for 68–70 h in the modified Hartman-de-Bont medium without K+ sources, stationary-phase wild-type M. smegmatis cells entered a dormant NC state and lost the ability to form colonies on the nutrient agar. NC cells of the wild-type strain were resuscitated in a liquid medium supplemented with Rpf. Similarly, the isogenic strain (Wt∷rpf) that harbors a plasmid containing the M.

[14] Additionally, communication between GPs and community pharmacists is currently sporadic and reactive, risking fragmentation of patient care.[15] Few studies have explored stakeholder views on pharmacist integration into general practices to date, none of which have explored the views of Australian GPs and pharmacists. The aim of this study was to elicit the views of Australian GPs and pharmacists on the integration of pharmacists Decitabine research buy into the general practice setting, the proposed roles for a general practice pharmacist, and the factors influencing integration. Advertisements and letters of invitation

were disseminated through the Victorian Divisions of General Practice (a support network for GPs in Victoria, Australia), the Australian Association of Consultant Pharmacy (AACP) (the credentialing and accreditation body for Australian consultant pharmacists) and key informants in the area. A combination of purposive, snowball and convenience sampling was used to ensure a broad sample from the two health professional groups. Participants were selected according to their role in the profession and whether they

had previous experience working with or as an on-site general practice pharmacist, or a pharmacist closely associated with a general practice. General practice staff and pharmacists were interviewed ADAMTS5 one-to-one, using a semi-structured interview guide Dactolisib clinical trial developed from the literature (Table 1). Face and content validity were established by discussion with pharmacists and the guide was

pilot tested on two interviewees. Interviews occurred over the period from December 2010 to June 2011; written consent was obtained from all participants prior to the interview. All interviews were conducted by the same interviewer (ET), either face-to-face or by telephone, according to participant preference, at a mutually convenient place and time. Recruitment and interviews continued until data saturation was reached (i.e. when no new, relevant themes were emerging). Interviews were audio-recorded and transcribed verbatim by an independent, professional transcribing service. All transcripts were verified against audio recordings by ET. Data management was facilitated using Nvivo 9.0 software (QSR, Melbourne). Interview transcripts, recordings and field notes were entered into the software. Data were analysed and coded for emergent themes using the framework approach, whereby a draft thematic framework, based on a priori issues, was applied to the data.[16] The framework was structured according to the interview guide and checked independently by all authors. This aided subsequent detailed analysis and interpretation.

, 2007; Meier et al., 2008; Pereira et al., 2009). In this study, we evaluate the inhibitory activity of PYRH-1 (sodium 3-[4-tert-butyl-3-(9H-xanthen-9-ylacetylamino)phenyl]-1-cyclohexylmethylpropoxycarbonyloxyacetate)

as a potential antimicrobial agent by targeting the bacterial UMP kinase, PyrH, which serves as a kinase in de novo pyrimidine biosynthesis pathway required for the growth of certain bacteria such as S. pneumoniae (Thanassi et al., 2002; Song et al., 2005) and H. influenzae (Akerley et al., 2002). PYRH-1 was discovered in the course of a 1536-well high throughput screening of an in-house large chemical library by the selection of chemicals directly inhibiting PyrH of S. pneumoniae. To test the inhibitory activity of PYRH-1 against PyrH, we used a luminescence-based ATP quantitative reagent. Moreover, molecular interaction analysis between PYRH-1 and S. pneumoniae Z-VAD-FMK mw PyrH by surface plasmon resonance (SPR) and susceptibility tests of PYRH-1 against some bacteria were Protein Tyrosine Kinase inhibitor performed. This is the first report that PYRH-1 inhibits PyrH. Bacterial strains used in this study are described in Table 1. Escherichia coli DH5α (competent high E. coli DH5α, Toyobo Co., Ltd.) was used for the cloning of PyrH. Escherichia

coli Rosetta-Gami B (DE3) (Novagen) was used as the host for recombinant protein expression. These were grown at 35 °C in Luria–Bertani (LB) broth or LB agar (BD Biosciences) containing 100 μg mL−1 of carbenicillin (Sigma). The culture medium used selleck inhibitor for each bacterium is as follows: S. pneumoniae, cation-adjusted Mueller–Hinton Broth (CAMHB; BD Biosciences) containing 5% of lysed horse blood (Nippon Bio-Test Laboratories Inc.) or Todd Hewitt Broth (Becton, Dickinson and Co.); S. aureus and E. coli, CAMHB; H. influenzae, Haemophilus test medium [CAMHB containing 5 mg mL−1 of Yeast Extract (BD Biosciences), 15 μg mL−1 of Hemin (Sigma) and 15 μg mL−1 of β-NAD (Sigma)]. This strain was constructed by deleting

the acrA gene and replacing it with a gene that confers resistance to chloramphenicol (cat) Streptococcus pneumoniae TIGR4 and H. influenzae Rd KW20 genomic DNA were extracted with a DNeasy Tissue Kit (Qiagen). Plasmid DNA was extracted with a QIAprep Spin Miniprep Kit (Qiagen). PCR products and plasmids digested by restriction enzyme were purified with a QIAquick PCR Purification Kit (Qiagen). PCR products digested by restriction enzyme were purified with a MinElute Reaction Cleanup Kit (Qiagen). The open reading frame of the pyrH gene was amplified from S. pneumoniae TIGR4 genomic DNA with primers SpPyrH-N-XhoI (5′- CCG CTC GAG GTG AAA ATG GCG AAT CCC AAG T -3′) and SpPyrH-C-BamHI (5′- CGC GGA TCC TTA TTC CTT TTC TTC GAT ATT ATT TGA AAC TGT TG -3′). The open reading frame of pyrH was amplified from H.