Exponential Phase Cells Research Articles

Protocol for the transformation of Bacillus subtilis through inducing natural competence

Transformation is the key step in genetic engineering as it governs the facile introduction of plasmid DNA or genome editing tools into the microbial cells. More importantly, approach taken for transformation differs between different microbial chassis depending on the structure of the cell envelope and other barriers. In this aspect, Bacillus subtilis requires a different transformation technique compared to the heat shock approach commonly used for Escherichia coli. Given that B. subtilis enters competence state during stationary phase, DNA could theoretically be introduced into the Gram-positive bacterium during this differentiation program. This aspect of B. subtilis cell biology has been exploited in biotechnology through the use of xylose for inducing the comK master competence regulator. With xylose as inducer, comK gene is transcribed, which would set off a transcriptional program that guides B. subtilis stationary phase cells to enter the natural competence state, where the cell envelope of the bacterium is permeabilized to afford facile entry of plasmid DNA. This protocol details the experimental approach and procedure for inducing natural competence in B. subtilis cells for enabling plasmid DNA to enter the bacterial cell during transformation. One methodological innovation in the described protocol is the use of stationary phase B. subtilis cells as compared to exponential phase cells in earlier incarnations of the approach.

Magnetically driven active topography for long-term biofilm control

Microbial biofilm formation on indwelling medical devices causes persistent infections that cannot be cured with conventional antibiotics. To address this unmet challenge, we engineer tunable active surface topographies with micron-sized pillars that can beat at a programmable frequency and force level in an electromagnetic field. Compared to the flat and static controls, active topographies with the optimized design prevent biofilm formation and remove established biofilms of uropathogenic Escherichia coli (UPEC), Pseudomonas aeruginosa, and Staphylococcus aureus, with up to 3.7 logs of biomass reduction. In addition, the detached biofilm cells are found sensitized to bactericidal antibiotics to the level comparable to exponential-phase planktonic cells. Based on these findings, a prototype catheter is engineered and found to remain clean for at least 30 days under the flow of artificial urine medium, while the control catheters are blocked by UPEC biofilms within 5 days.

Inorganic Cadmium Detection Using a Fluorescent Whole-Cell Bacterial Bioreporter

Cadmium pollution has become a serious environmental issue due to its toxicity and frequent entrance into environment components such as soil, water, and air via many anthropogenic sources. Over the last two decades, whole-cell bacterial bioreporters have been acknowledged as bio-sentinels for the determination of toxic heavy metals. Herein a sensitive and quite specific bacterial bioreporter was developed to cope with the need for the rapid and simple determination of cadmium. The construction and characterization of a fluorescence-based whole-cell cadmium bioreporter strain, Escherichia coli MG1655 (pBR-PzntA), was described which is based on the expression of green fluorescent protein under the control of the zntA gene promoter of heavy metal resistance determinant. The developed bioreporter was able determine cadmium at 5 µg/L after 3.5 hours of induction in a defined medium while the cadmium detection limit was improved to 2 µg/L after 1.5 hours by the use of an inorganic phosphate-limiting defined medium. Drastic changes in cadmium sensitivity were obtained between bioreporter cells induced at different growth phases. The maximum fluorescence performance was obtained for early exponential growth phase cells. This cadmium bioreporter was found to be more sensitive and specific to cadmium ions than to a wide range of heavy metals and was sensitive to only cadmium at drinking water quality standard concentrations. These findings will lead to future studies including integration of the bioreporter cells into a portable device to assess bioavailable cadmium levels in environmental samples which will provide a rapid and practical field detection technique.

Dps Protects Enterohemorrhagic Escherichia coli against Acid-Induced Antimicrobial Peptide Killing.

Dps, a DNA-binding protein from starved cells in Escherichia coli, is part of the bacterial defense system that protects DNA against various cellular stresses. Our lab previously demonstrated that a novel antimicrobial peptide, WRWYCR, enhances acid-induced killing of enterohemorrhagic Escherichia coli (EHEC) and ameliorates infection in a Citrobacter rodentium mouse model of EHEC infection. WRWYCR has previously been shown to compromise DNA damage repair and to increase chelatable iron within the cell. These findings, combined with the effects of peptide and acid stress on DNA damage, suggest a key defense role for Dps in peptide-induced killing of EHEC. The goal of this study is to evaluate the role of Dps in peptide-induced killing of EHEC through survival assays and flow cytometric analyses of DNA damage and hydroxyl radical formation. Our results demonstrate that disruption of the dps gene in stationary-phase EHEC O157:H7 cells, but not in exponential-phase cells, enhances acid-, peptide-, and peptide-acid-induced killing relative to that of wild-type (WT) EHEC. Using flow cytometric analysis, we have also demonstrated increased levels of hydroxyl radicals in peptide-treated wild-type EHEC relative to those in the untreated control. Disruption of the dps gene further increases this. These findings indicate that peptide treatment of EHEC enhances the formation of hydroxyl radicals, likely through the Fenton reaction, thereby contributing to the killing action of the peptide, and that dps protects against peptide killing of EHEC. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials.IMPORTANCE The research presented in this paper explores the role of the DNA-binding protein Dps as a key defense mechanism of enterohemorrhagic Escherichia coli (EHEC) strains in protecting against killing by the novel antimicrobial peptide WRWYCR. Our results demonstrate that Dps protects against peptide-induced killing of EHEC through direct protection against acid stress and hydroxyl radical formation, both of which are mechanisms targeted by the antimicrobial peptide. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials through specific targeting of Dps in order to allow a more potent response to the antimicrobial WRWYCR.

A novel mechanism of ribonuclease regulation: GcvB and Hfq stabilize the mRNA that encodes RNase BN/Z during exponential phase

RNase BN, the Escherichia coli RNase Z family member, plays a limited role in tRNA metabolism, in contrast to most other organisms. However, RNase BN does act on 6S RNA, the global transcription regulator, degrading it in exponential-phase cells and maintaining it at low levels during this phase of growth. RNase BN levels decrease in stationary-phase cells, leading to elevation of 6S RNA and subsequent regulation of RNA polymerase. These findings were the first indication that RNase BN itself is growth phase-regulated. Here, we analyze the mechanism of this regulation of RNase BN. We find that RNase BN decreases in stationary phase because its mRNA becomes unstable, due primarily to its degradation by RNase E. However, in exponential-phase cells rbn mRNA is stabilized due to binding by the sRNA, GcvB, and the protein, Hfq, which reduce cleavage by RNase E. Because the amount of GcvB decreases in stationary phase, rbn mRNA is less protected and becomes increasingly unstable resulting in reduction in the amount of RNase BN. The small RNA-dependent, positive regulation of RNase BN in exponential-phase cells is the first example of this novel mechanism for RNase regulation.

Blue Light Sensing in Listeria monocytogenes Is Temperature-Dependent and the Transcriptional Response to It Is Predominantly SigB-Dependent

Listeria monocytogenes is an important food-borne pathogen that is tolerant to many of the stresses commonly used during food preservation. Outside the host, the bacterium has a saprophytic lifestyle that includes periodic exposure to solar irradiance. The blue component of this light is known to influence the activity of the stress-inducible sigma factor Sigma B (σB). In this study, the influence of temperature and growth phase on the response of L. monocytogenes to blue light was investigated and the global transcriptional response to blue light was elucidated using an RNAseq-based approach. Stationary phase cells were found to be significantly more resistant to killing by blue light (470 nm) than exponential phase cells. Temperature also had a marked effect on blue light resistance with cells cultured at 37°C being much more sensitive than cells grown at 30°C. The role of σB in light tolerance was confirmed but this effect was observed only at 30°C. σB activation by blue light was assessed by measuring the transcriptional response of known σB-dependent genes (sigB, lmo2230, and opuCA) to light. The transcripts were induced by blue light only at 30°C suggesting that blue light fails to activate σB at 37°C. The light-induced transcription at 30°C was dependent on a functional blue light sensor, Lmo0799 (which we rename herein as RsbL). A transcriptomic analysis of the response to sub-lethal levels of blue light found that the changes in transcription were almost entirely σB-dependent. A mutant where the light sensing mechanism of RsbL was inactivated through an amino acid substitution (Cys56Ala) was found to have an attenuated response to blue light, but residual activation of σB-dependent genes suggested that alternative routes for activation of σB by light are likely to exist. Overall, the study highlights the central role of σB in the response of this pathogen to visible light and further shows that light sensing is absent at temperatures that exist within the mammalian host.

Matrices (re)loaded: Durability, viability, and fermentative capacity of yeast encapsulated in beads of different composition during long-term fed-batch culture.

Encapsulated microbes have been used for decades to produce commodities ranging from methyl ketone to beer. Encapsulated cells undergo limited replication, which enables them to more efficiently convert substrate to product than planktonic cells and which contributes to their stress resistance. To determine how encapsulated yeast supports long‐term, repeated fed‐batch ethanologenic fermentation, and whether different matrices influence that process, fermentation and indicators of matrix durability and cell viability were monitored in high‐dextrose, fed‐batch culture over 7 weeks. At most timepoints, ethanol yield (g/g) in encapsulated cultures exceeded that in planktonic cultures. And frequently, ethanol yield differed among the four matrices tested: sodium alginate crosslinked with Ca2+ and chitosan, sodium alginate crosslinked with Ca2+, Protanal alginate crosslinked with Ca2+ and chitosan, Protanal alginate crosslinked with Ca2+, with the last of these consistently demonstrating the highest values. Young's modulus and viscosity were higher for matrices crosslinked with chitosan over the first week; thereafter values for both parameters declined and were indistinguishable among treatments. Encapsulated cells exhibited greater heat shock tolerance at 50°C than planktonic cells in either stationary or exponential phase, with similar thermotolerance observed across all four matrix types. Altogether, these data demonstrate the feasibility of re‐using encapsulated yeast to convert dextrose to ethanol over at least 7 weeks.

De novo transcriptome assembly and analysis of the freshwater araphid diatom Fragilaria radians, Lake Baikal

Diatoms are a group of eukaryotic microalgae populating almost all aquatic and wet environments. Their abundance and species diversity make these organisms significant contributors to biogeochemical cycles and important components of aquatic ecosystems. Although significant progress has been made in studies of Diatoms (Bacillariophyta) over the last two decades, since the spread of “omics” technologies, our current knowledge of the molecular processes and gene regulatory networks that facilitate environmental adaptation remain incomplete. Here, we present a transcriptome analysis of Fragilaria radians isolated from Lake Baikal. The resulting assembly contains 27,446 transcripts encoding 21,996 putative proteins. The transcriptome assembly and annotation were coupled with quantitative experiments to search for differentially expressed transcripts between (i) exponential growth phase and dark-acclimated cell cultures, and (ii) those changing expression level during the early response to light treatment in dark-acclimated cells. The availability of F. radians genome and transcriptome data provides the basis for future targeted studies of this species. Furthermore, our results extend taxonomic and environmental sampling of Bacillariophyta, opening new opportunities for comparative omics-driven surveys.

Electrotransformation of Saccharomyces cerevisiae.

Intact yeast cell transformation is easily achieved by gene electrotransfer (GET). The procedure is fast and efficient in terms of transformants/μg DNA. Yeast cells in exponential growth phase are washed, treated for a short period with dithiothreitol (DTT) and then mixed with the plasmid DNA in a buffer with a low conductivity. A single well defined electric pulsed is delivered. After a 1h incubation in the growth medium without selection, transformants are obtained on a selective plate medium. After a short description of the present knowledge on the events affecting the yeast cell as a consequence of the pulsed electric field, a step-by-step protocol is reported for Saccharomyces cerevisiae.

Modified semi-continuous fermentation for resuscitating nongrowing cells during high-temperature gluconic acid production by Acetobacter senegalensis.

The formation of metabolically inactive and nongrowing cells is an inevitable by-product of intensive fermentation. This study investigated whether co-feeding can be used to resuscitate nongrowing Acetobacter senegalensis cells to enable them to produce gluconic acid in successive fermentation runs at 38°C. In the first fermentation cycle, 75gl-1 of glucose were converted to gluconic acid. Subsequently, however, stationary-phase cells were unable to initiate a new fermentation cycle. The majority of stationary-phase cells (97%) were nonculturable on glucose at 38°C. In addition, 54 and 41% of cells contained non-active cellular dehydrogenases and a compromised cell envelope respectively. Co-feeding stationary-phase cells with a mixture of ethanol, glucose and acetic acid for 7h enabled these cells to grow on 75gl-1 of glucose and produce gluconic acid. Additionally, 74% of cells contained active forms of cellular dehydrogenases after 7h of co-feeding. However, co-feeding did not improve cell envelope integrity. Quantification of cellular NAD content showed that stationary-phase cells contained moderately reduced levels of total NAD (NADt) as compared with exponential-phase cells. Interestingly, the analysis of stationary-phase cells showed that co-feeding resulted in higher levels of NADt and NADH, suggesting that the regeneration of NADH is one of the limiting factors of glucose consumption. Expression of catalase and superoxide dismutase was increased in stationary-phase cells, but analysis of protein carbonylation and lipid peroxidation did not confirm an extensive oxidative stress. Co-feeding with favourable nutrients may enable resuscitation of cells and utilization of less-favourable carbon sources in successive cycles. This study proposed a unique method for resuscitation of nongrowing cells during high-temperature fermentation. By applying this method, cells can be used for consecutive fermentation cycles.

Citric acid can force Staphylococcus aureus into viable but nonculturable state and its characteristics

Pathogens in viable but nonculturable (VBNC) state can escape traditional detection methods based on culturable ability, thus bringing risks to food safety and human health. Considering Staphylococcus aureus as a kind of primary foodborne pathogen, this study attempted to investigate whether citric acid, a food additive commonly used, can force S. aureus into VBNC state along with low temperature. Treated with citric acid solution (pH 4.0) at 4 °C, S. aureus was confirmed to enter into VBNC state after induction for 18 days. Meanwhile, resuscitation was achieved in culture medium rather than in nutrition-free saline solution. In VBNC cells, ATP concentration still maintained at a high level, as about two-thirds of exponential-phase cells. For survival, intracellular structure of VBNC cells changed remarkably, including irregular cell shape, denser cytoplasm, space between cell wall and cell membrane, and decreased density of nuclear region. Notably, resistance of VBNC cells to simulated gastric fluid improved when compared with exponential-phase cells. What are noted above suggests that VBNC state adopted by S. aureus might be a survival strategy to the adverse environment (acidity stress and low temperature). In conclusion, our study sounds an alarm for the safety of citric acid-containing foods.

Global Proteomic Analysis of the Resuscitation State of Vibrio parahaemolyticus Compared With the Normal and Viable but Non-culturable State.

Vibrio parahaemolyticus is a common pathogen which has become a major concern of seafood products. The bacteria in the viable but non-culturable (VBNC) state are unable to form colonies on growth media, but under appropriate conditions they can regain culturability. In this study, V. parahaemolyticus was induced into VBNC state at low temperature and oligotrophic condition, and was resuscitated to culturable state. The aim of this study is to explore the comparative proteomic profiles of the resuscitation state compared with the VBNC state and the exponential phase of V. parahaemolyticus using isobaric tags for relative and absolute quantitation (iTRAQ) technique. The differentially expressed proteins (DEPs) were subjected to GO functional annotations and KEGG pathway analysis. The results indicated that a total of 429 proteins were identified as the significant DEPs in the resuscitation cells compared with the VBNC cells, including 330 up-regulated and 99 down-regulated DEPs. Meanwhile, the resuscitation cells displayed 25 up-regulated and 36 down-regulated DEPs (total of 61 DEPs) in comparison with the exponential phase cells. The remarkable DEPs including ribosomal proteins, ABC transporters, outer membrane proteins and flagellar proteins. GO annotation showed that the 429 DEPs were classified into 37 GO terms, of which 17 biological process (BP) terms, 9 cellular component (CC) terms and 11 molecular function (MF) terms. The up-regulated proteins presented in all GO terms except two terms of developmental process and reproduction. The 61 DEPs were assigned to 23 GO terms, the up- and down-regulated DEPs were both mainly involved in cellular process, establishment of localization, metabolic process and so on. KEGG pathway analysis revealed that the 429 DEPs were assigned to 35 KEGG pathways, and the pathways of ribosome, glyoxylate and dicarboxylate metabolism were significantly enriched. Moreover, the 61 DEPs located in 26 KEGG pathways, including the significantly enriched KEGG pathways of ABC transporters and two-component system. This study would contribute to a better understanding of the molecular mechanism underlying the resuscitation of the VBNC state of V. parahaemolyticus.

Induction of viable but putatively non-culturable Lactobacillus acetotolerans by thermosonication and its characteristics

This study aimed at investigating for the first time whether thermosonication (TS), a non-thermal processing technique, could induce the beer-spoilage bacterium, Lactobacillus acetotolerans CGMCC 7.150, into the viable but putatively non-culturable (VPNC) state. After determining culturable, viable and total cell counts, we found that L. acetotolerans in beer could enter the VPNC state by TS at a constant frequency of 24 kHz at 50 °C at three relatively low volumetric powers (0.6, 1.2 and 1.8 W/mL, but not 2.4 W/mL). Furthermore, with the increase of volumetric power, the time required for L. acetotolerans cells to enter the VPNC state shortened. The addition of Tween 80 at a concentration of 0.1% v/v into de Man Rogosa Sharpe agar enabled the VPNC cells to regain culturability with a recovery time of approximately 3 days. However, their recovery capability decreased with the increasing volumetric power. Scanning electron microscopy analysis showed that cells decreased in size and gradually changed morphology from short rods to coccoids upon entering the VPNC state. An insignificant difference in beer-spoilage ability was found between the exponential-phase and VPNC cells. The resistance of these VPNC cells to 0.025 M NaOH and 0.025 M KOH was enhanced compared with the exponential-phase cells.

DNA susceptibility of Saccharomyces cerevisiae to Zeocin depends on the growth phase.

The aim of this study was to evaluate the level of Zeocin-induced double-strand breaks (DSBs) in Saccharomyces cerevisiae cells in a different growth phase, using constant-field gel electrophoresis (CFGE). Saccharomyces cerevisiae diploid strain D7ts1 with enhanced cellular permeability was used. The effects of growth phase and treatment time were evaluated based on Zeocin-induced DSBs, measured by CFGE. Survival assay was also applied. No protoplast isolation was necessary for the detection of DSBs in strain D7ts1. Differences in the response of cells depending on the growth phase were obtained. Cells in exponential growth phase had increased DSB levels only after Zeocin treatment with concentrations equal or higher than 200μgml-1. Increasing treatment time did not result in higher DSB levels. Oppositely, treatment of cells at the beginning of stationary phase with Zeocin concentrations resulted in more than 1.5-fold increase in DSB levels in comparison with those in untreated cells. Increased DSB levels were measured for all the treatment times. A dose-dependent decrease in cell survival was observed after Zeocin treatment with concentrations in the range of lethality LD20-LD50. A strong negative correlation was calculated between the levels of DSBs and cell survival. New information is provided concerning DNA susceptibility depending on the growth phase. DNA susceptibility is higher in cells at the beginning of stationary phase than those in exponential phase. Data presented here illustrate that the optimized by us CFGE protocol is sensitive and could be used successfully for DSB measurement in Saccharomyces cerevisiae strains with enhanced cellular permeability.

Changes in nanomechanical properties and adhesion dynamics of algal cells during their growth

Nanomechanical and structural characterisations of algal cells are of key importance for understanding their adhesion behaviour at interfaces in the aquatic environment. We examine here the nanomechanical properties and adhesion dynamics of the algal cells during two phases of their growth using complementary surface methods and the mathematical modelling. Mechanical properties of motile cells are hard to assess while keeping cells viable, and studies to date have been limited. Immobilisation of negatively charged cells to a positively charged substrate enables high-resolution AFM imaging and nanomechanical measurements. Cells were stiffer and more hydrophobic in the exponential than in the stationary phase, suggesting molecular modification of the cell envelope during aging. The corresponding properties of algal cells were in agreement with the increase of critical interfacial tensions of adhesion, determined amperometrically. Cells in exponential phase possessed a larger cell volume, in agreement with the large amount of amperometrically measured displaced charge at the interface. Differences in the kinetics of adhesion and spreading of cells at the interface were attributed to their various volumes and nanomechanical properties that varied during cell aging. Our findings contribute to the present body of knowledge on the biophysics of algal cells on a fundamental level.

Toxic effect and bioaccumulation of selenium in green alga Chlorella pyrenoidosa

Se is required for the growth and metabolism of many species of microalgae. However, high concentrations of Se show toxicity to microalgal cells. In the study, the biomass, growth rate, total Se content, organic Se content, SOD, CAT, and LPO of Chlorella pyrenoidosa in an environment containing Se were determined to explore the effects of selenite on the growth of C. pyrenoidosa in different growth phases (exponential phase, late exponential phase, and early stationary phase). Bioaccumulation and conversion of selenium in C. pyrenoidosa and toxic effect of selenium were further explored. A low dosage of selenite could promote the growth of C. pyrenoidosa, but the higher Se concentration strongly inhibited growth and led to the low biomass. Algae cells in exponential phase could be exposed to high doses of selenium (≤ 5 mg L−1), which was higher than that for the cells in late exponential phase (≤ 2 mg L−1) and the cells in early stationary phase (≤ 2 mg L−1). The optimal growth conditions for enriching selenium in C. pyrenoidosa in exponential phase were determined as 2 mg L−1 selenite and 5-day cultivation. The highest transformation mass was 78 ± 0.5% and corresponding intracellular accumulation of organic Se was 72 ± 0.1 μg g−1 dry weight. The lower Se concentration induced the activities of oxidases, which was conducive to the growth of microalgal cells. The higher concentration of Se significantly induced the activities of oxidases, indicating that Se accumulation had a significant toxic effect on C. pyrenoidosa. In this way, a great quantity of Se-enriched biomass can be produced for nutritional purposes.

The effects of resveratrol on mitochondrial biogenesis dysfunction induced by fluoride in human neuroblastoma SH-SY5Y cells

Objective: To explore the role of mitochondrial biogenesis and the neuroprotective mechanism of resveratrol in fluoride neurotoxicity. Methods: SH-SY5Y cells in exponential phase were treated with different concentrations (20, 40, 60 mg/L) of sodium fluoride (NaF) for 24 h. Co-treatment with 60 mg/L NaF, 20 μmol/L resveratrol (RSV) was administrated in the resveratrol intervene trial. Western blotting was used to determine the expression levels of mitochondrial biogenesis key regulating factor of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) , nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) in SH-SY5Y cells. The mRNA levels of PGC-1α, NRF1 and TFAM were determined by Quantitative Real-time PCR in SH-SY5Y cells, as well as the relative mitochondrial DNA (mtDNA) contents and mRNA expression of mitochondrial respiratory chain complexes subunit CO1 and ATP8. Flow cytometry was used to determine mitochondrial membrane potential in SH-SY5Y cells. Results: Both the protein and mRNA levels of PGC-1α, NRF1 and TFAM were decresed after 60 mg/L NaF treatment in SH-SY5Y cells (P<0.05) . The relative mtDNA contents and mRNA expression of complexes subunit CO1 and ATP8 were also significantly decreased compared with control (P<0.05) . Mitochondrial membrane potential were also significantly decreased after 60 mg/L NaF treatment in SH-SY5Y cells (P<0.05) . Compared with 60 mg/L NaF group, the protein and mRNA levels of PGC-1α, NRF1 and TFAM in 20 μmol/L RSV+60 mg/L NaF group were significantly increased (P<0.05) . The relative mtDNA contents, mitochondrial membrane potential and mRNA levels of complexes subunit CO1 and ATP8 in 20 μmol/L RSV+60 mg/L NaF group were also significantly higher than that in 60 mg/L NaF group (P<0.05) . Conclusion: Resveratrol may alleviate the fluoride-induced mitochondrial biogenesis dysfunction in SH-SY5Y cells.

Heat resistance, membrane fluidity and sublethal damage in Staphylococcus aureus cells grown at different temperatures

In this work the influence of growth temperature (10–42 °C) on Staphylococcus aureus heat resistance was studied, and its relationship with the ability of cells to repair sublethal damages and with membrane fluidity was evaluated. Non-linear, convex from above survival curves were obtained, and therefore a special case of the Baranyi model was used to fit them. For exponential phase cells, heat resistance did not change with growth temperature in the range between 10 and 37 °C, but cells grown at 42 °C were significantly more resistant, showing D58 and shoulder length (sl58) values 2.5 and 4 times greater than the others, respectively. For stationary growth phase cells, an increase in growth temperature above 20 °C resulted in an increase in D58 values, and cells grown at 42 °C also displayed the highest D58 and sl58 values. The increased heat resistance at 58 °C of stationary growth phase cells grown at higher temperatures was coincident with the appearance of a higher proportion of sublethally damaged cells capable of recovery and outgrowth in non-selective medium. Membrane fluidity was measured at treatment temperatures, and it was observed that those cells with more rigid membranes displayed greater heat resistance (Pearson coefficient = 0.969***). Additionally, S. aureus cells whose membrane was fluidized through exposure to benzyl alcohol were notably sensitized against the action of heat, in a concentration-dependent manner. Results obtained in this research indicate that membrane physical state could be an important factor determining the survival capacity of bacterial cells to a heat treatment.

L-Proline catabolism by the high G + C Gram-positive bacterium Paenarthrobacter aurescens strain TC1.

Paenarthrobacter aurescens (formerly called Arthrobacter aurescens) strain TC1 is a high G + C Gram-positive aerobic bacterium that can degrade the herbicide atrazine. Analysis of its genome indicated strain TC1 has the potential to form a bifunctional PutA protein containing L-proline dehydrogenase and L-glutamate-γ-semialdehyde dehydrogenase (L-Δ1-pyrroline-5-carboxylate dehydrogenase) activities. P. aurescens strain TC1 grew well in minimal media with L-Proline as a supplemental nutrient, the nitrogen source, or the sole carbon and nitrogen source. Multicellular myceloids induced by NaCl or citrate also grew on L-proline. The specific activity of L-proline dehydrogenase in whole cells was higher whenever L-proline was added to the medium. Both L-proline dehydrogenase and L-glutamate-γ-semialdehyde dehydrogenase (L-Δ1-pyrroline-5-carboxylate dehydrogenase) activities were found primarily in a membrane fraction from exponential-phase cells. The two activities co-eluted from a Bio-Gel P-60 column after precipitation of proteins with ammonium sulfate and solubilization with 0.1% Tween 20. The PutA protein in the active fraction also oxidized 3,4-dehydro-DL-proline, but there was no activity with other L-proline analogues. When P. aurescens strain TC1 was grown in minimal media containing increasing concentrations of NaCl, there was a progressive decrease in the specific activity of L-proline dehydrogenase and a concomitant increase in the intracellular concentration of L-proline. These results indicate that P. aurescens strain TC1 can use L-proline as a nutrient in a regulated fashion. Because this bacterium also showed the ability to degrade most of the other common amino acids, it can serve as a useful model for the control of amino acid catabolism in the high G + C Actinobacteria.

Assessment of Sep1virus interaction with stationary cultures by transcriptional and flow cytometry studies.

The majority of phage infection studies are performed in bacteria that are growing exponentially, although in nature, phages usually interact also with non-replicating cells. These stationary-phase cells differ from exponential cells morphologically, physiologically and metabolically. The interaction of a Sep1virus with Staphylococcus epidermidis stationary and exponential phase cells was explored. Phage SEP1 efficiently infected both cell culture states, without the addition of any fresh nutrients to stationary cultures. Phage-host interactions, analysed by flow cytometry, showed stationary-phase cells response to phage immediately after SEP1 addition. Quantitative PCR experiments corroborate that phage genes are expressed within 5 min of contact with stationary phase cells. The increase of host RNA polymerase transcripts in stationary cells suggests that SEP1 infection leads to the upregulation of host machinery fundamental for completion of its lytic life cycle. SEP1 infection and replication process highlights its potential clinical interest targeting stationary phase cells.