Early Exponential Growth Phase Research Articles

σB-dependent protein induction in Listeria monocytogenes during vancomycin stress

Listeria monocytogenes is a food-borne pathogen that can survive under a wide range of environmental and energy stress conditions. The general stress response controlled by sigma(B) largely contributes to stress resistance in L. monocytogenes. Moreover, the bacterial cell wall is the first defense against cellular stress and as such is the target of numerous antibiotics. We therefore hypothesize that sigma(B) contributes to monitoring the integrity of cell walls. We evaluated sigma(B) activity in wild type and DeltasigB mutant L. monocytogenes containing reporter fusions (sigma(B)-dependent opuCA promoter and a lacZ reporter gene) during the early exponential growth phase by measuring the specific activity of beta-galactosidase after vancomycin (2 microg mL(-1) final concentration) stress. sigma(B) activity is significantly induced only in the wild-type strain by addition of vancomycin. In addition, we identified sigma(B)-dependent vancomycin-inducible proteins using LC-ESI-MS/MS analysis. Two independent proteomic analyses confirmed the minimum twofold upregulation of 18 vancomycin-inducible sigma(B)-dependent stress response proteins in the wild-type strain compared with the DeltasigB mutant. The functions of these proteins are associated with cell wall biogenesis, intracellular transport, general stress response, cell metabolism and virulence. These results suggest that the sigma(B) protein may contribute to the monitoring of cell wall integrity.

Effects of growth conditions and NAPL presence on transport of Pseudomonas saccharophilia P15 through porous media

Extensive research has been done to characterize transport of bacteria in porous media; however, little is understood on how the presence of non-aqueous phase liquids (NAPLs) coupled with the growth state and carbon source of bacteria affect bacterial transport. The objective of this research is to quantify the bacterial adhesion of Pseudomonas saccharophilia P15 (P15), which is known to biodegrade polycyclic aromatic hydrocarbons (PAH) and to interact with coal tars, within a NAPL–water–mineral system. Through a series of short-pulse column experiments, the transport and deposition of P15 in porous media (quartz sand) as a function of growth state and carbon sources (peptone and naphthalene), and in the presence and absence of residual NAPL (hexadecane), is measured and evaluated. Coating 20% of the quartz grain with hexadecane as a model NAPL increased the retention of P15 by as much as a factor of 26 as compared to the retention exhibited in quartz sand with no NAPL present. P15 grown on peptone and in the late exponential growth state exhibited a greater amount of deposition within the hexadecane column than when it was grown on naphthalene or was in early exponential growth phase. During early growth stage P15 grown on naphthalene adhered stronger to the porous media compared to when grown on peptone. Results were compared with results of MATH assays, where P15 partitioning to hexadecane was evaluated as a function of carbon source and growth state.

Two widely expressed plasma membrane H+-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine

The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

Bacterial responses to Cu-doped TiO 2 nanoparticles

The toxicity of Cu-doped TiO 2 nanoparticles (NPs, 20 nm), synthesized by a flame aerosol reactor, to Mycobacterium smegmatis and Shewanella oneidensis MR-1, is the primary focus of this study. Both doped and non-doped TiO 2 NPs (20 nm) tended to agglomerate in the medium solution, and therefore did not penetrate into the cell and damage cellular structures. TiO 2 particles (< 100 mg/L) did not apparently interfere with the growth of the two species in aqueous cultures. Cu-doped TiO 2 NPs (20 mg/L) significantly reduced the M. smegmatis growth rate by three fold, but did not affect S. oneidensis MR-1 growth. The toxicity of Cu-doped TiO 2 NPs was driven by the release of Cu 2+ from the parent NPs. Compared to equivalent amounts of Cu 2+, Cu-doped TiO 2 NPs exhibited higher levels of toxicity to M. smegmatis ( P-value < 0.1). Addition of EDTA in the culture appeared to significantly decrease the anti-mycobacterium activity of Cu-doped TiO 2 NPs. S. oneidensis MR-1 produced a large amount of extracellular polymeric substances (EPS) under NP stress, especially extracellular protein. Therefore, S. oneidensis MR-1 was able to tolerate a much higher concentration of Cu 2+ or Cu-doped TiO 2 NPs. S. oneidensis MR-1 also adsorbed NPs on cell surface and enzymatically reduced ionic copper in culture medium with a remediating rate of 61 µg/(liter∙OD 600∙ hour) during its early exponential growth phase. Since the metal reducing Shewanella species can efficiently “clean” metal-oxide NPs, the activities of such environmentally relevant bacteria may be an important consideration for evaluating the ecological risk of metal-oxide NPs.

Functional ingredients produced by culture of Koliella antarctica

Unicellular algae represent an interesting source of bioactive food ingredients. In this study, fatty acids and carotenoids of Koliella antarctica, a psychrophylic Antarctic unicellular alga, were investigated. K. antarctica was cultivated at different temperatures and harvested at the early exponential growth phase and at the late exponential growth phase. After 240 h, at the end of the late exponential growth phase, the cell biomass concentration was 1.1 g L − 1 at 10 °C while it was 2.2 g L − 1 at 15 °C. The lipid profile of K. antarctica was rich in polyunsaturated fatty acids (PUFA) particularly at the early exponential phase (50.8% of total fatty acids). The comparison of data obtained from algae cultivated at 10 °C and 15 °C, showed at 15 °C a strong increase in saturated lipids and a decrease in PUFA. At the same time the presence of EPA and DHA was marginally affected, with a percentage decrease around 15–20%. The productivity of EPA and DHA was similar at the two growth temperatures with maximum values of 0.6 mg L − 1 day − 1 and 1.3 mg L − 1 day − 1 , at 10 and 15 °C, respectively. In K. antarctica cultures, the concentration of lutein and astaxanthin increased during growth up to 1.4 g per 100 g dry weight for both compounds. This is one of the highest concentrations reported in the literature for carotenoid production by unicellular algae, with a total carotenoid productivity of 3.1 mg L − 1 day − 1 . Although optimization studies to increase K. antarctica biomass production were not yet carried out, data suggested that this alga is a promising source for low cost production of valuable bioactive compounds. The performance recorded at 15 °C suggested that it could be particularly suitable for outdoor cultivation during the winter season in the Mediterranean region.

Aggregatibacter actinomycetemcomitansarcB influences hydrophobic properties, biofilm formation and adhesion to hydroxyapatite

The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly.This study investigated in A. actinomycetemcomitans the role of ArcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitans arcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis.The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase ArcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces.

AI-2 signalling is induced by acidic shock in probiotic strains of Lactobacillus spp.

Survival and ability to respond to various environmental stresses such as low pH are important factors for lactobacilli for their function as probiotics. LuxS-mediated quorum sensing mechanism, which is based on the production of universal signal molecule called autoinducer-2 (AI-2), regulates important physiological traits and a variety of adaptive processes in different bacteria. The aim of this study was to investigate the effect of acidic stress on LuxS-mediated quorum sensing (AI-2 signalling) in four probiotic strains of different Lactobacillus species. Initially, the production of AI-2-like molecule was investigated in four strains of Lactobacillus spp. at standard growth conditions using Vibrio harveyi bioluminescence assay. Species variation in AI-2 activity was observed. AI-2 activity started at early-exponential growth phase and increased during the mid-exponential phase concomitant with the reduction of pH, reaching maximum at late exponential phase ( L. rhamnosus GG) or at stationary phase ( L. salivarius UCC118, L. acidophilus NCFM and L. johnsonii NCC533). Acidic shock experiments were conducted on L. rhamnosus GG and L. acidophilus NCFM after exposure to different acidic shocks (pH 5.0, 4.0 and 3.0) and to pH 6.5 as control, measuring AI-2 activity and transcription of the luxS gene. AI-2 activity increased by lowering the pH in a dose dependent manner and was negatively influenced by acid adaptation. In both species, the luxS gene was repressed after exposure to pH 6.5 as control. However, after acidic shock (pH 4.0) a transient response of luxS gene was observed and the transcription augmented over time, reaching a maximum level and decreased subsequently. Acid adaptation of cells attenuated the transcription of this gene. Based on the observations done in the present study, the luxS gene appears to have a clear role in acidic stress response in probiotic lactobacilli. This might be important in the survival of these bacteria during the passage through the gastrointestinal tract, and further influence the cell-to-cell communication among bacteria in the intestinal microbiota.

Study of kinetic parameters for the production of recombinant rabies virus glycoprotein

Gene expression in insect cells is an advantageous system for recombinant protein production, mainly because of its capacity to produce complex proteins with correct post-translational modifications. Recently, we identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of rabies virus glycoprotein, expressed in Drosophila melanogaster cells, by about 60%. In this work, the kinetic parameters for cell growth and recombinant rabies virus glycoprotein production were determined in cultures of transfected Drosophila melanogaster Schneider 2 (S2) cells expressing recombinant rabies virus glycoprotein (rRVGP), enriched and non-enriched with the hemolymph of Lonomia obliqua (Hb). The highest concentration of rRVGP was achieved at the beginning of the culture enriched with Hb, indicating that the cells produce greater amounts of rRVGP per cell (specific rRVGP concentration) at the early exponential growth phase. After day 8, a decrease in the concentration of rRVGP (ng/mL) was observed, probably due to protein decomposition. The average specific rRVGP production rate (mu(rRVGP)) was 30 ng rRVGP/10(7)cell.day, higher than that observed in the non-enriched culture.

Phosphate levels and expression of phosphoribosylpyrophosphate synthetase isozymes in suspension-cultured Arabidopsis thaliana cells

In plants, as well as the generally distributed phosphate-dependent phosphoribosylpyrophosphate (PRPP) synthetase (class I), phosphate-independent PRPP synthetase (class II) is also present. To investigate the effect of the inorganic phosphate (Pi) level on the two classes of PRPP synthetase, we first estimated the cellular phosphate level using 33Pi in suspension-cultured Arabidopsis thaliana cells. 33Pi in the culture medium was taken up by the cells, and the concentration of Pi in cells increased up to 5.5 μmol/g fresh weight within 24 h once the cells were transferred to the fresh medium; its concentration then fell because of the conversion of Pi to organic compounds. In vitro activity of PRPP synthetase increased after inoculation and maintained a high activity until the early exponential growth stage. The transcript levels of PRS1 and PRS2 encoding class I and PRS3 encoding class II enzymes increased rapidly after the cells were transferred to the fresh medium, then remained almost constant during the early exponential growth phase. In contrast, constitutive expression of PRS4 encoding cytosolic class II enzyme was observed during culture. During long-term Pi-starvation the transcript levels of PRS1 and PRS2 were reduced, but PRS3 and PRS4 were expressed continually during the Pi-starvation. Pi-dependent PRPP synthetase activity was simultaneously reduced, but Pi-independent activity did not change. Expression of PRS1 and PRS2 and the activity of Pi-dependent enzyme grew to normal rates by 24 h after supply of Pi.

Structure-Function, Stability, and Chemical Modification of the Cyanobacterial Cytochrome b6f Complex from Nostoc sp. PCC 7120

The crystal structure of the cyanobacterial cytochrome b(6)f complex has previously been solved to 3.0-A resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b(6)f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical to those in the M. laminosus b(6)f complex. Purified b(6)f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b(6)f complex, determined to a resolution of 3.0A (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme b(p) that is rotated 180 degrees about the alpha- and gamma-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme c(n) is similar to that previously found in the b(6)f complex from other sources.

Specific activity and viability of Nitrosomonas europaea during discontinuous and continuous fermentation

The energy conservation and number of viable cells of Nitrosomonas europaea fluctuate dramatically during cultivation. In discontinuous culture the specific activity (SA) reaches its maximum after 9 h with about 2700 nmol O 2 (mg protein) −1 min −1, where the highest number of viable N. europaea cells is detectable after 21 h with 2 × 10 8 cell ml −1. Afterwards, both SA and viable cell number immediately start to decrease. Accordingly, the exponential growth turns into a linear growth, whereby the number of viable cells permanently decreases. The exponential growth phase can be extended from about 21 to 38 h by increasing the concentration of CO 2 or trace elements. In continuous fermentation of N. europaea, SA of about 2500 nmol O 2 (mg protein) −1 min −1 and viable cell number of 2.5 × 10 8 cell ml −1 is detectable at dilution rates between 1 and 1.8 day −1. At dilution rates below 1 day −1, SA and number of viable cells are reduced. The minimal doubling time is 13 and 15 h during continuous and discontinuous fermentation, respectively. Consequently, cell production of N. europaea should be performed in continuous fermentation. When bacteria are grown in discontinuous systems, they should be harvested in the early exponential growth phase.

Transcriptomic and metabolomic profiling of Zymomonas mobilis during aerobic and anaerobic fermentations

BackgroundZymomonas mobilis ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. Z. mobilis performs best under anaerobic conditions, but is an aerotolerant organism. However, the genetic and physiological basis of ZM4's response to various stresses is understood poorly.ResultsIn this study, transcriptomic and metabolomic profiles for ZM4 aerobic and anaerobic fermentations were elucidated by microarray analysis and by high-performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Greater amounts of other end-products such as acetate, lactate, and acetoin were detected under aerobic conditions and at 26 h there was only 1.7% of the amount of ethanol present aerobically as there was anaerobically. In the early exponential growth phase, significant differences in gene expression were not observed between aerobic and anaerobic conditions via microarray analysis. HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point.Differences in extracellular metabolite profiles between conditions became greater as the fermentations progressed. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 contained lower levels of amino acids such as alanine, valine and lysine, and other metabolites like lactate, ribitol, and 4-hydroxybutanoate under anaerobic conditions relative to aerobic conditions. Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold. Transcripts for Entner-Doudoroff (ED) pathway genes (glk, zwf, pgl, pgk, and eno) and gene pdc, encoding a key enzyme leading to ethanol production, were at least 30-fold more abundant under anaerobic conditions in the stationary phase based on quantitative-PCR results. We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation.ConclusionHigh oxygen concentrations present during Z. mobilis fermentations negatively influence fermentation performance. The maximum specific growth rates were not dramatically different between aerobic and anaerobic conditions, yet oxygen did affect the physiology of the cells leading to the buildup of metabolic byproducts that ultimately led to greater differences in transcriptomic profiles in stationary phase.

Aggregatibacter actinomycetemcomitans arcB influences hydrophobic properties, biofilm formation and adhesion to hydroxyapatite

The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly.This study investigated in A. actinomycetemcomitans the role of ArcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitans arcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis.The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase ArcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces.

SarZ , a sarA Family Gene, Is Transcriptionally Activated by MgrA and Is Involved in the Regulation of Genes Encoding Exoproteins in Staphylococcus aureus

The expression of genes involved in the pathogenesis of Staphylococcus aureus is controlled by global regulatory loci, including two-component regulatory systems and transcriptional regulators (e.g., sar family genes). Most members of the SarA family have been partially characterized and shown to regulate a large numbers of target genes. Here, we describe the characterization of sarZ, a sarA paralog from S. aureus, and its regulatory relationship with other members of its family. Expression of sarZ was growth phase dependent with maximal expression in the early exponential phase of growth. Transcription of sarZ was reduced in an mgrA mutant and returned to a normal level in a complemented mgrA mutant strain, which suggests that mgrA acts as an activator of sarZ transcription. Purified MgrA protein bound to the sarZ promoter region, as determined by gel shift assays. Among the sarA family of genes analyzed, inactivation of sarZ increased sarS transcription, while it decreased agr transcription. The expression of potential target genes involved in virulence was evaluated in single and double mutants of sarZ with mgrA, sarX, and agr. Northern and zymogram analyses indicated that the sarZ gene product played a role in regulating several virulence genes, particularly those encoding exoproteins. Gel shift assays demonstrated nonspecific binding of purified SarZ protein to the promoter regions of the sarZ-regulated target genes. These results demonstrate the important role played by SarZ in controlling regulatory and virulence gene expression in S. aureus.

Effects of monoamine neuromediators on the growth-related variables of Escherichia coli K-12

The monoamine neuromediators serotonin (5-HT), histamine, dopamine (DA), and norepinephrine (NE), added to an Escherichia coli K-12 strain MC 4100 culture upon inoculation, stimulate cell proliferation (determined from CFU formation) and biomass accumulation (monitored nephelometrically) during the late lag phase and the early exponential growth phase. These effects are less significant in the late exponential and stationary phase cultures. According to the concentration dependence of the stimulatory effects, the neuromediators can be classified into two groups: (i) the catecholamines DA and NE, whose effects increase almost linearly with increasing concentrations within the range of 0.1-100 microM, and (ii) histamine and 5-HT, which are characterized by bell-shaped concentration dependence curves with maxima at 0.1 (histamine) and 1 microM (5-HT). On an agar-containing medium, the growing E. coli population includes solitary cells and compact cell groups (microcolonies). In this system, both tested catecholamines exert a relatively weak stimulatory influence that manifests itself as an increase in the number of both solitary cells and cell groups, and occurs at concentrations of 10 microM and higher. In analogy to the culture grown on the liquid medium, 5-HT and histamine are distinguished by nonlinear concentration dependence curves: their effects peak at 0.1 microM (histamine) or 1 microM (5-HT); an increase in the neuromediator concentrations results in a decrease in effects that are enhanced by further increasing the concentrations to the submillimolar range. DA increases the percentage of solitary cells, whereas the other tested amines promote cell group formation. The results are interpreted in terms of specific (probably receptor-dependent) mechanisms of action of the neuromediators involved.

Analysis of Dibenzothiophene Desulfurization in a Recombinant Pseudomonas putida Strain

Biodesulfurization was monitored in a recombinant Pseudomonas putida CECT5279 strain. DszB desulfinase activity reached a sharp maximum at the early exponential phase, but it rapidly decreased at later growth phases. A model two-step resting-cell process combining sequentially P. putida cells from the late and early exponential growth phases was designed to significantly increase biodesulfurization.

Prophage induction in Lactococcus lactis by the bacteriocin Lactococcin 972

Lactococcin 972 (Lcn972) is a non-pore forming bacteriocin with a narrow spectrum of activity restricted to Lactococcus. Lcn972 inhibits the incorporation of cell wall precursors in the septum area, thereby inhibiting cell division. In this work, an additional inhibitory effect is described, namely, the induction of the lytic cycle of resident prophages in the lysogenic strain L. lactis IPLA 513. Lcn972 triggered the release of prophages in a concentration-dependent fashion. The extent of prophage induction was influenced by the physiological status of the cultures, being maximal at the early exponential growth phase. A microtiter based protocol was designed and the induction ability of several antimicrobials was compared. Prophages were activated by all cell wall biosynthesis inhibitors tested, although the levels of induction were lower than those obtained after activation of the SOS response. As far as we know, this is the first report of prophage induction by an antimicrobial peptide. Since Lcn972 is active against L. lactis strains currently used in commercial starters, promising applications for dairy fermentations are discussed.

Regulation of Hemin Binding Proteins by a Novel Transcriptional Activator in Porphyromonas gingivalis

One of the features of the periodontal pathogen Porphyromonas gingivalis is the presence of complex iron acquisition systems that include an hmuYRSTUV locus. HmuY and HmuR are hemin binding proteins required for P. gingivalis growth. Previous studies have demonstrated that expression of the hmu locus is regulated in response to environmental changes, such as growth phases. However, the mechanisms involved in hmu gene regulation are poorly understood. Here we report that a novel transcriptional activator, PG1237, is required for the expression of humY and humR, but not other iron acquisition-related genes, such as fetB and tlr, which also encode hemin binding proteins. Real-time reverse transcription-PCR analysis revealed that a mutation in the pg1237 gene decreased expression of hmuY and hmuR 149- and 25-fold, respectively, compared to that observed in the wild-type strain. In addition, differential expression of hmuY, hmuR, and the pg1237 gene was found to be quorum-sensing dependent, such that higher expression levels of these genes were observed when P. gingivalis was grown at a lower cell density, such as that seen during the early exponential growth phase. This work demonstrates the involvement of a novel transcriptional activator, PG1237, in expression of the hmu operon in a cell density-dependent fashion.

Global transcriptional analysis of Mycoplasma hyopneumoniae following exposure to norepinephrine

Mycoplasma hyopneumoniae, a component of the porcine respiratory disease complex, colonizes the respiratory tract of swine by binding to the cilia of the bronchial epithelial cells. Mechanisms of pathogenesis are poorly understood for M. hyopneumoniae, but previous work has indicated that it responds to the environmental stressors heat shock, iron deprivation and oxidative compounds. For successful infection, M. hyopneumoniae must effectively resist host responses to the colonization of the respiratory tract. Among these are changes in hormonal levels in the mucosal secretions. Recent work in the stress responses of other bacteria has included the response to the catecholamine norepinephrine. The idea that M. hyopneumoniae can respond to a host hormone, however, is novel and has not previously been demonstrated. To test this, organisms in the early exponential phase of growth were exposed to 100 muM norepinephrine for 4 h, and RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. The M. hyopneumoniae response included slowed growth and changes in mRNA transcript levels of 84 genes, 53 of which were upregulated in response to norepinephrine. A larger proportion of the genes upregulated than those downregulated were involved with transcription and translation. The downregulated genes were mostly involved with metabolism, which correlated with the reduction in growth of the mycoplasma. Approximately 51 % of the genes were hypothetical with no known function. Thus, in response to norepinephrine, M. hyopneumoniae appears to upregulate protein expression while downregulating general metabolism.

Production of Anti-Methicillin-Resistant Staphylococcus Activity from Bacillus subtilis sp. Strain B38 Newly Isolated from Soil

B38 bacterial strain, isolated from Tunisian soil showed a strong antimicrobial activity. Based on biochemical characterization and 16S rDNA sequence analysis, B38 strain was identified as Bacillus subtilis. Cell culture supernatant showed antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus species and several Gram-positive and Gram-negative bacteria. Antifungal activity against phytopathogenic fungi was also observed. Antibacterial activity production started at early exponential growth phase, and maximum activity was reached at the stationary phase. This antibacterial activity was neither affected by proteases, lipase, and organic solvents, nor by surfactants. It was stable over a wide pH range and still active after autoclaving at 121 degrees C during 20 min. Thin layer chromatography followed by bioautography assay allowed the detection of four active spots with R(f) values of 0.30, 0.47, 0.70, and 0.82. The single spot with R (f) 0.30 showed antifungal activity, whereas the spots with R(f) values of 0.47, 0.70, and 0.82 exhibited antibacterial activity.