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“H2 energy carrier Microalgae have gained relevance recently as versatile organisms that are able to harvest solar energy and convert it into a variety of products of commercial

significance, from nutraceuticals to fuels. One of the useful products of algal metabolism is the energy carrier hydrogen (H2). Besides being the third most abundant element on the earth, H2 can be produced by a variety of sustainable Meloxicam technologies and can be easily interconverted into electricity for storage selleck chemical and transport. One of the major advantages of H2 as an energy carrier is the fact that its combustion does not release toxic products. Available technologies for production of H2 gas mostly involve reforming methanol. However, sustainable methods to extract H2 from water through photocatalytic, nuclear, photobiological, or photohybrid water electrolysis are being explored and offer the potential for a totally carbon-neutral process. Moreover, the use of wind turbines to drive water electrolysis and generate H2 is being tested

as a feasible technology to store energy during off-peak hours. Many microalgae have a H2-centered metabolism in which H2 serves as a source of reductant, and protons act as a sink for intracellular reductant under different environmental conditions. Of major interest, though, is the fact that microalgae are able to directly link photosynthetic water oxidation to H2 production by hydrogenases, thus holding the promise of plentiful energy from essentially inexhaustible sources—water and sunlight. Microalgae H2 pathways As many other chlorophytes, the green unicellular alga Chlamydomonas reinhardtii is capable of producing H2 following a period of anaerobic induction (Gaffron and Rubin 1942; Healey 1970). Its genome is sequenced (Merchant et al. 2007), and many genetic and genomic tools to manipulate this organism are available.

Cells were gated on forward scatter and side scatter to exclude clumps and debris. DCs were CD14- and DC-SIGN+ (constituting approximately 90%

of gated cells). Results were analysed using Summit software version 4.3 (Dako/Beckman Coulter). Cytokine Analysis Dendritic cells were infected with live H37Ra or streptomycin-killed H37Ra at MOI 1 for 24 or 48 h. LPS was applied for 24 h (Sigma; 1 μg/ml) as a positive control for DC maturation and cytokine secretion. Cytokine secretion was measured in cell-free supernatants by ELISA using the Meso Scale Discovery SECTOR Imager 2400 and the following assays: human IL-6 assay, and human Th1/Th2 10-cytokine multiplex assay, capable of detecting IFN-γ, IL-1β, IL-10, IL-12p70, IL-13, IL-2, IL-4, IL-5, IL-8 and TNF-α (Meso Scale Discovery, Gaithersburg, MD). IL-4 measurements were disregarded, as DCs were maintained in culture with exogenous IL-4, rendering it impossible to distinguish levels

check details secreted by the cells themselves. Colony forming units and BacT/ALERT 3D Dendritic cells were harvested 24 h or 72 h after infection with M. tuberculosis. VEGFR inhibitor Cells were centrifuged and washed 3 times at 800 rpm to remove extracellular bacteria. The cells were lysed in 0.1% Triton X-100 (Sigma) for 10 min. The resultant bacterial suspension was then passed through a 25 gauge needle eight times to disperse clumps. The bacilli were serially diluted x10-1 – x10-5 in Middlebrook 7H9 medium and plated on Middlebrook 7H10 agar (Difco) supplemented with oleic acid-albumin-dextrose-catalase (Becton Dickinson) and cycloheximide (Sigma), or inoculated into BacT/ALERT MP bottles (bioMérieux, Durham, NC). Agar plates were incubated at 37°C for 14 – 21 days and colony forming units were counted. BacT/ALERT MP bottles were incubated in a BacT/ALERT 3D automated microbial detection system (bioMérieux) and time to reach positivity was recorded, and a growth index was calculated, using the equation ((TTPDay1-TTPDay3)/TTPDay1)x100 as we have already published for the BD BACTEC

liquid culture platform [69]. 4��8C In this equation TTP Day 1 is the time to culture positivity for infected DC lysates at Day 1, and TTP Day 3 is the time to positivity for infected DC lysates at Day 3. Statistical analysis Results are expressed as means ± the standard errors of the mean (SEM). The data were analyzed with GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA) statistical software using by repeated measures ANOVA with Tukey’s post test, or (where stated) by the Friedman test followed by Dunns multiple comparison test. A P value of < 0.05 was considered statistically significant. Graphs were compiled using GraphPad Prism 5 software. Acknowledgements The authors wish to thank Dr Timothy Grant, Centre for Support and Analysis in Research (CSTAR) for providing advise on statistical analysis of the data.

0005 0.0030 0.0114 Impeller tip speed (ITS): (1) where π = 3.142 N = Agitation speed DI = Impeller diameter Agitation speed (N): (2) Inoculum volume (Vx): (3) Analytical methods The 1,3-PD, glycerol and organic acids were assayed by high-performance liquid chromatography. Samples for chemical analysis were first centrifuged at 10,000 g for 10 min at 4°C (Multifuge 3SR, Germany), filtered through a 0.22 μm membrane filter (Millex-GS, Millipore, USA), and then analyzed on an HPLC system (Agilent Technologies 1200 series). An Agilent Technolgies 1200 series system equipped with a refractive index detector was used. Analyses were performed isocratically GSK2118436 price at a flow rate of 0.6 mL/min on an Aminex HPX-87H 300 × 7.8 column (Bio-Rad,

CA, USA) at a constant temperature of 65°C. H2SO4 (0.5 mN) was the mobile phase. External standards were applied for identification and quantification of peak areas. Retention times (Rt) determined for the target compounds were as follows: 1,3-PD – 17.17 min; glycerol – 13.03 min; butyric acid – 20.57 min; acetic acid – 14.4 min and lactic acid – 11.19 min. Protein analyses Proteins Palbociclib molecular weight were reduced (10 mM DTT, 30 min, 56°C) and alkylated with iodoacetamide in darkness (45 min, 20°C) and digested overnight with 10 ng/μL trypsin. The resulting peptide mixtures were applied to the RP-18 pre-column of a UPLC system (Waters) using water containing 0.1% FA as a mobile phase and then transferred to a nano-HPLC

RP-18 column (internal diameter 75 μm, Waters) using ACN gradient (0 – 35% ACN in 160 min) in the presence of 0.1% FA at a flow rate of 250 μL/min. The column outlet was coupled directly to the ion source of an Orbitrap Velos mass spectrometer (Thermo). Each sample was measured in duplicate – once for protein sequencing (data-dependent MS to MS/MS switch) and once for quantitative information (MS only, sequencing disabled). The acquired MS/MS data

were pre-processed with Mascot Distiller software ADAMTS5 (v. 2.3, MatrixScience) and a search was performed with the Mascot Search Engine MatrixScience, Mascot Server 2.4) against the set of Clostridium protein sequences derived from Uniprot, merged with its randomized version (16294 sequences; 5095802 residues). The proteins that exactly matched the same set of peptides were combined into a single cluster. The mass calibration and data filtering were carried out with MScan software. The lists of peptides that matched the acceptance criteria from the LC-MS/MS runs were merged into one common list. This common list was overlaid onto 2-D heat maps generated from the LCMS profile datasets by tagging the peptide-related isotopic envelopes with corresponding peptide sequence tags on the basis of the measured/theoretical mass difference, the deviation from the predicted elution time, and the match between the theoretical and observed isotopic envelopes. The abundance of each peptide was determined as the height of a 2-D fit to the monoisotopic peak of the tagged isotopic envelope.

Richardson [18] summarized the results of aggressive surgical management for oesophageal perforation. All were treated by operative repairs, buttressed with muscle Proteasome assay or pleura. Sternocleidomastoid muscle was used to buttress or primarily close the defects in the neck, and a flap of diaphragm was often used for thoracic perforation. Patients with perforated cancer or severe underlying disease had an oesophagectomy. With these techniques, 50 of 64 patients underwent preservation of the oesophagus after closure of the perforation and 14 underwent resection. The leak rate was 17%, but all

healed. One patient treated with primary closure died (1.5% mortality) and only 1 patient required subsequent oesophagectomy. Vallböhmer [19] described an institutional experience of 44 patients over a period BMN 673 concentration of 12 years. Iatrogenic injury was the most frequent cause of oesophageal perforation. Eight patients (18%) underwent conservative treatment with cessation of oral intake,

antibiotics, and parenteral nutrition. Twelve (27%) patients received an endoscopic stent implantation. Surgical therapy was performed in 24 (55%) patients with suturing of the lesion in nine patients, oesophagectomy with delayed reconstruction in 14 patients, and resection of the distal oesophagus and gastrectomy in one patient. The hospital mortality rate was 6.8% (3 of 44 patients): one patient with an iatrogenic perforation after conservative treatment, and two patients after surgery (one with Boerhaave syndrome, one with iatrogenic rupture). No death

occurred in the 25 patients when the diagnosis was made in less than 24 hours. When it was delayed, 19% of 16 patients died (P = 0.05). Keeling et al. [20] in 2010 retrospectively reviewed all cases of oesophageal perforation from 1997 Tobramycin through 2008 at Emory University. Among 91 patients, the perforation was iatrogenic in 50 (52%), spontaneous in 23 (24%), and idiopathic in 22 (23%). The authors concluded that the overall mortality from oesophageal perforation can be less than 10%. Primary repair should be considered as first-line treatment when appropriate even in patients who present more than 24 hours after perforation. Non- operative management, in appropriate patients, can be used in selected patients. Similar results were recorded by the Houston group [21] and two recent meta-analyses [22, 23]. Results and prognostic considerations In the multi-institutional series reported by Asensio [4], a logistic regression of 346 patients reaching the O.R. after penetrating trauma established that a delay in preoperative evaluation, AAST organ injury score > 2 and resection and diversion were independent factors for increased oesophagus-related complications.

Moreover, polycrystalline hydroxyapatite is reported to exhibit plasticity at higher temperature [19, 20], but no plasticity has been reported at room temperature for nanostructured transparent ceramics. Furthermore, for ceramic materials, the

plasticity is limited at low loads, and the influence of dislocation can be important [21, 22]. GW-572016 supplier Thus, the faceted pile-up region suggests that dislocations generated during the indentation are attributed to the residual strain of nanostructured transparent ceramics. Figure 2 SPM image and corresponding cross-sectional profile. SPM image of an indented area (A) and the corresponding cross-sectional profile (B) along the bluish grey line in (A). In order to further investigate the mechanical properties of nanostructured transparent ceramics, we used HRTEM to examine the microstructures of the sample indented at 9,000 μN. The HRTEM image is shown in Figure 3.

The inset in this figure is a selected area electron diffraction pattern of the indented sample, indicative of a magnesia-alumina spinel crystal structure. The left part of the HRTEM image reveals well-ordered atomic structures. However, there are dislocations close to the triangular grain boundary, suggesting that the generation, movement, and interaction of dislocations check details during the indentation play an important role in the plastic deformation as well as the resulting mechanical properties. Figure 3 HRTEM image of the nanostructured transparent MgAl ADP ribosylation factor 2 O 4 ceramic. Inset shows the selected area

diffraction pattern. Hardness and Young’s modulus of the nanostructured transparent MgAl2O4 ceramics are shown in Figure 4 as a function of the applied load. Both hardness and Young’s modulus decrease with increasing loads. Furthermore, it also indicates that there appears to be a larger decrease in the hardness than in the Young’s modulus with increasing load. These phenomena have been attributed to the well-known indentation size effect. Gong et al. [14] studied an alumina ceramic by nanoindentation testing and found that more cracks were generated at higher loads. However, the absence of cracks in the vicinity of the indented zone (Figure 2) suggests that it should not be reasonable to explain the load-dependent mechanical properties of our nanostructured transparent ceramics only by the indentation size effect. Dislocation activity, as evidenced in Figure 3, compared to HRTEM images of the sample at atmospheric pressure [11] should be considered as an important factor that can influence the mechanical properties of nanostructured transparent ceramics. A more detailed study is clearly needed to understand how the dislocation activity influences the mechanical properties. Figure 4 Hardness (A) and Young’s modulus (B) as a function of applied load. Inset shows TEM image of the sample.

PCR was performed

in a 50-μl reaction mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 0.5 μM of each primer, 50 ng of DNA template, and 2.5 U of Taq DNA polymerase (Promega, USA). Sorafenib price The PCR conditions consisted of an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 56-60°C for 1 min and extension at 72°C for 1-2 min depending on the PCR product size (Table 2), and a final extension at 72°C for 7 min. The PCR products were analyzed by agarose gel electrophoresis and purified using the QIAquick PCR Purification Kit (Qiagen, Germany) prior to submission for DNA sequencing. Table 2 Primers used for amplification and sequencing of M.tuberculosis clinical strains Gene Primer name (position*)

Primer sequence (5′→3′) Annealing temp (°C) PCR product size (bp) Purpose Reference rrs F-rrs (-44) 5′-TTCTAAATACCTTTGGCTCCCT-3′ 51 1,680 PCR/Seq [42] R-rrs (1,636) 5′-TGGCCAACTTTGTTGTCATGCA-3′ 53   PCR/Seq [42] F-rrs1 (554) 5′-CTGGGCGTAAAGAGCTCGTA-3′ 54   Seq This study F-rrs2 (1,114) 5′-GTTGCCAGCACGTAATGGTG-3′ ITF2357 purchase 54   Seq This study R-rrs1 (483) 5′-TCCACCTACCGTCAATCCGA-3′ 54   Seq This study R-rrs2 (1,073) 5′-ATCTCACGACACGAGCTGAC-3′ 54   Seq This study eis (Rv2416c) F-Rv2417c (-316) 5′-GCGGTGCATCACGTCGCCGA-3′ 60 1,661 PCR/Seq This study R-eis-Rv2415c (1,345) 5′-GCAACGCGATCCGCGAGTGC-3′ 60   PCR/Seq This study F-eis1 (247) 5′-AGTTTCGTCGCGGTGGCGCC-3′ 60   Seq This study F-eis2 (816) 5′-GGACCCGTTACCCCACCTGC-3′ 60   Seq This study R-eis1 (240) Cyclic nucleotide phosphodiesterase 5′-GGCGGTCGGGAGCACCACTT-3′ 60   Seq This study R-eis2 (769) 5′-TCAGGGCCCGCCACAACGCA-3′ 60   Seq This study tap (Rv1258c) F-Rv1259 (-496) 5′-CAGGCCGGCCCTATGCAGTG-3′ 60 1,847 PCR/Seq This study R-Rv1257c (1,351) 5′-CGGTCTTGCCGGTAGCCGTC-3′ 60   PCR/Seq This study F-tap1 (41) 5′-TCGCAACGCTGATGGCGGCC-3′ 60   Seq This study F-tap2 (641) 5′-AGGGGCTGCGCTTCGTCTGG-3′ 60   Seq This study R-tap1 (210) 5′-CCCGAAGTAGTCGACCGCGG-3′ 60   Seq This study R-tap2 (862) 5′-GACGGGGAACGCGGATAGCC-3′

60   Seq This study whiB7 (Rv3197A) F URT-whiB7 (-451) 5′-GCTGGTTCGCGGTCGGACCT-3′ 60 550 PCR/Seq This study R whiB7 (99) 5′-CGGGGTATCGGCGAACCACA-3′ 58   PCR/Seq This study tlyA (Rv1694) F-tlyA (1) 5′-GTGGCACGACGTGCCCGCGT-3′ 62 807 PCR/Seq This study R-tlyA (807) 5′-CTACGGGCCCTCGCTAATCG-3′ 58   PCR/Seq This study *The first 5′nucleotide position of each primer was counted from the translation start codon of each gene. DNA sequencing analysis Nucleotide sequencing was performed with the Big-Dye™ Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, USA) using an ABI PRISMR 3700 DNA analyzer at First BASE Laboratories (Malaysia). The PCR products were sequenced in both directions. The obtained nucleotide sequences were compared with those of M. tuberculosis H37Rv (Accession no. NC_000962) by pairwise alignment using the ClustalW program [43].

These compound concentrations were established according to the purpose of each experiment. Experimental procedure Spore germination and inoculum preparation consisted of two pre-cultures with 24-hour cultivation each in shake flasks. Inoculum volume comprised 10% of suspension cell volume per culture medium volume throughout this study. Submerged cultures for cephamycin C production were performed 5-Fluoracil concentration in 500 ml Erlenmeyer

shake flasks at 28°C and 260 rpm (5 cm eccentricity). To prevent problems of oxygen limitation during the shake-flask procedure, the broth volume was kept under 10% of the Erlenmeyer flask nominal volume. Samples were collected at 24-hour intervals. Experiments in the bench-scale bioreactor (New Brunswick Bioflo 2000; 5 l working volume) were performed at 1.0 vvm aeration rate, 6.8 ± 0.1 pH, 28°C temperature, and 50% dissolved oxygen saturation level automatically

controlled by varying the agitation speed. Analytical methods The supernatant was obtained after centrifugation of the culture medium at 15,550 x g for 10 min, 4°C, for further analyses. The cell density was quantified as grams Opaganib of dry weight per liter of sample (gDWC l-1). Cephamycin C was determined by means of the agar-diffusion assay method using cephalosporin C zinc salt (Sigma) as standard. Penase® (BD Difco) was employed at 20 μL per ml of sample, reacting at 25°C for 20 min to degrade penicillin N. In this method, the measure of cephamycin C represents the total amount of cephalosporins in the sample (in mg l-1) [36]. A calibration curve was performed using ten cephalosporin C concentration values from 5 to 120 mg l-1 and 24 replicates for each concentration. Antibiotic analyses were also carried out via high-performance liquid chromatography as described in Baptista

Neto et DCLK1 al. [37]. Lysine and alpha-aminoadipic acid analyses were conducted by means of the post-column derivatization method with orthophtalaldehyde and quantified in a fluorescence detector [38]. The starch concentration was determined after acid hydrolysis, by quantifying the total reducing sugars by the dinitrosalicylic acid method [39]. Experimental design CCF experimental designs, including four replicates of an experiment under the same conditions, were employed to investigate individual and combined effects of lysine and compounds, one at a time, putrescine, 1,3-diaminopropane, cadaverine, and alpha-aminoadipic acid, on cephamycin C production. The response surface methodology was used to investigate the relationship between cephamycin C production (response variable) and the compounds that enhance beta-lactam antibiotic production (independent variables) [40, 41].

Among them, the pCS20 real-time PCR TaqMan probe assay provides the best sensitivity with a detection limit of one gene copy per reaction, which is 100 times higher than that of conventional pCS20 PCR [20]. However, this assay was reported to cross-react with both E. chaffeensis and E. canis [20]. Moreover, although this assay performs well in the sensitive detection and quantification of E. ruminantium, it is not readily transferable

to low-technology settings where there is limited access to expensive fluorescence detector based thermocyclers. Loop-mediated isothermal amplification (LAMP) assay is a rapid DNA amplification method originally developed by Notomi et al. [21], and it has been applied for the detection of viral [22, 23], bacterial [24, 25], fungal [26], and parasitic agents [27,

selleck products 28], but it has never previously been applied to rickettsial agents. The method requires a specially designed primer set that recognizes at least six independent regions of the target gene, which increases the specificity as well as the rapidity of the reaction. LAMP results are visualized by turbidity that can be seen by the naked eye [29], and optionally by agarose gel electrophoresis or by addition of fluorescent dyes visualized under UV light [30, 31]. Since the Bst DNA polymerase used in LAMP allows strand displacement-DNA synthesis, LAMP reactions are performed under isothermal conditions using a simple incubator, such as a water bath or heating block. Furthermore, LAMP reagents are relatively stable for a month, even when stored at 37°C, which is a warmer temperature than recommended by the manufacturer [32]. With these advantages, LAMP MG-132 clinical trial has the potential to be used even in clinical laboratories often poorly equipped, facing problems of constant electricity supply in tropical and sub-tropical countries where heartwater is endemic. The purpose of the present study was to develop LAMP assays for the detection science of E. ruminantium and to evaluate the diagnostic sensitivity

and specificity of these assays using a panel of bacterial DNA samples, quantitated plasmid standards, and field samples derived from both animal blood and ticks. The newly developed LAMP assays successfully detected E. ruminantium with rapidity, specificity, and high sensitivity. Results Optimization of LAMP The reactions for both pCS20 and sodB LAMP were performed under isothermal conditions at a range of 58 to 66°C using plasmid DNA (106 copies per reaction) for 120 min, with monitoring of the turbidity. Although amplifications with the LAMP assays were observed at all temperatures tested, the reactions reached the threshold value (0.1) with the shortest incubation times at 61°C for pCS20 and 63°C for sodB (data not shown). No nonspecific amplification was detected for the negative cell culture until after at least 120 min incubation. Thus, subsequent LAMP reactions were conducted at these temperatures for 60 min.

As control, mice were administered with lip + LAg vaccine

intraperitoneally, whereas negative control mice received PBS or adjuvant alone (subcutaneously). Mice were then challenged with L. donovani promastigotes 10 days after vaccination. Inoculation of BALB/c mice with L. donovani strain AG83 leads to progressive infection in the liver and spleen, corresponding with hepato- and splenomegaly [4, 18]. We therefore evaluated the kinetics of increasing parasitic burden at 2 and 4 months after challenge, and the parasite loads in liver and spleen see more were quantitated as Leishman Donovan Units (Figure 1). Figure 1 Parasite burdens in vaccinated mice after L. donovani challenge infection. BALB/c mice were vaccinated subcutaneously with PBS, LAg, alum, alum + LAg, saponin and saponin + LAg, or intraperitoneally with Lip and Lip + LAg. Ten days post-immunization, mice were challenged intravenously

with 2.5 × 107 promastigotes of L. donovani. Liver (A) and spleen (B) parasite burden was measured buy Ku-0059436 2 and 4 months after challenge, and expressed as Leishman Donovan Units. Bars represent the mean ± SE of five individual mice per group, representative of two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to PBS as well as free adjuvant immunized groups as assessed by a one-way ANOVA and Tukey’s multiple comparison

test. In the liver, we observed a trend of decreased Avelestat (AZD9668) parasitic load in both alum + LAg and saponin + LAg immunized mice as compared to PBS immunized control group, reaching statistical significance at 2 months postinfection (p < 0.05, Figure 1A). However, this effect was minor, and notably neither vaccine statistically improved the protective efficacy over immunization with adjuvant alone. Mice immunized with LAg alone also did not exhibit significantly reduced parasite load compared to controls, consistent with our earlier observation that free LAg administered subcutaneously did not influence parasite growth in the liver [6]. In contrast, significantly reduced parasite burden was seen following intraperitoneal immunization with lip + LAg as compared to both PBS and empty liposome immunized mice (p < 0.001) [4, 6]. At 4 months postinfection both alum + LAg and saponin + LAg immunized mice failed to maintain the slight reduction in the parasite levels seen at the 2 month time point, instead demonstrating infection levels comparable to PBS and free adjuvant-immunized controls. In contrast, lip + LAg immunized animals maintained lower levels of parasite burden versus controls (p < 0.001). Immunization with alum + LAg fails to reduce splenic L.

2   LSA1771 comC DNA uptake machinery 0 4.10E-06 3.2 ± 0.2 608 ± 199 DNA metabolism: replication, repair, recombination, RM LSA0008 ssb Single-stranded DNA binding protein > threshold 3.88E-02 1.4 ± 0.1 1.2 ± 0.3 LSA0146   Putative DNA methyltransferase (apparently stand-alone) 1.55E-04 > threshold 1.6 ± 0.4   LSA1299   Putative DNA methyltransferase (apparently stand-alone) 2.48E-08 > threshold 1.9 ± 0.4   LSA1338 exoA Exodeoxyribonuclease III 1.36E-07 > threshold 1.8 ± 0.3   Purines, pyrimidines, nucleosides and nucleotides LSA0533 iunh2 Inosine-uridine preferring GSK2118436 nucleoside hydrolase

1.14E-05 > threshold 1.7 ± 0.4   Energy metabolism LSA1298 ack2 Acetate kinase 4.27E-09 > threshold 1.9 ± 0.4   Translation LSA0009 rpsR Ribosomal protein 1.67E-02 > threshold 1.5 ± 0.4   Regulatory function LSA0421   Putative transcriptional regulator, MerR

family 0 3.56E-03 2.5 ± 0.5   Hypothetical protein LSA0040   Hypothetical protein, conserved in some lactobacilli 0 3.56E-03 2.5 ± 0.5   LSA0409   Hypothetical Palbociclib integral membrane protein 3.02E-05 7.25E-03 0.61 ± 0.01   LSA0536   Hypothetical protein with putative NAD-binding domain, NmrA structural superfamily 6.28E-06 3.32E-02 1.6 ± 0.4   LSA0779   Hypothetical protein, peptidase S66 superfamily 4.77E-05 > threshold 0.6 ± 0.1   LSA0991   Hypothetical protein with putative NAD-binding domain, NmrA structural superfamily 1.02E-04 > threshold 1.6 ± 0.2   LSA1475   Hypothetical protein, conserved in bacteria 1.62-12 > threshold 2.1 ± 0.5   CDS £ Gene Name Product       qPCR LSA0487 recA DNA recombinase A       2.7 ± 0.7 LSA0992 dprA DNA protecting protein, ADAMTS5 involved in DNA transformation       2163 ± 1242 $ Expression ratios represent the fold change in amounts of transcripts in the strain overexpressing SigH relative to the WT control strain. For the microarray experiment they were calculated from log2ratio; for the qPCR they were calculated by the 2-ΔΔCt

method described in Methods. Genes underexpressed in the context of SigH overexpression have a ratio < 1. Standard deviation is indicated (weak accuracy for qPCR experiments may be due to Ct at the detection limit for basal level). § see additional file 3: Competence DNA uptake machinery of B. subtilis and comparison with L. sakei. £ not found statistically differentially expressed in the microarray transcriptome experiment, checked by qPCR. Two genes coding for hypothetical proteins, LSA0409 and LSA0779, were down-regulated in the sigH Lsa overexpression strain. As sigma factors are usually positive regulators, we consider it likely that down-regulation of these genes is an indirect effect of sigH Lsa overexpression, e.g., this effect could correspond to σH-mediated activation of an unidentified repressor. The sole transcriptional regulator (LSA0421) listed as σH-activated in Table 2 is probably not responsible for this effect, since MerR-type regulators reportedly act as activators [34].