Early Log Phase Of Growth Research Articles

EFFECTS OF MEDIA COMPOSITIONS ON AMYLASE AND D-LACTIC ACID PRODUCED BY A POTENTIAL STARCH-UTILIZING BACTERIUM

The high demand for amylase and lactic acid in various industries made enzyme and acid production from cheap substrates very attractive. This study investigated the effect of major medium constituents on amylase and D-lactic acid production by the potential starch-utilizing and optical pure D-lactic acid-producing Lactobacillus sp. SUTWR 73, to provide data for the efficient application of the lactic acid bacterium. Its amylase and D-lactic acid production was considerably affected by the addition of organic nitrogen tryptone to certain cassava starch concentrations as a carbon-source. While the other medium compositions: yeast extract and cheap nitrogen sources (soy protein, defatted rice bran, and spent brewery yeast) compared to tryptone as equal total nitrogen contents, did not have any effect on either metabolite production. The medium containing cassava starch (4.0%, w/v) and tryptone (4.5%, w/v) can be satisfactorily used for both products. The initial pH of the production medium was another factor affecting metabolite production. A suitable initial pH was 8.0 and 7.0-10.0 for amylase and D-lactic acid production, respectively. Amylase activity was detected at the early log growth phase while the D-lactic acid was produced at the late log phase. Understanding the relationship between amylase and D-lactic acid production from this study is important for designing an economical operating system for the direct production of D-lactic acid from cheap cassava starch without adding any commercial amylase as a substrate in pretreatment resulting in reduction costs of the production process.

Homologous overexpression of azoreductase (azoA) in Enterococcus sp. L2 moderated growth and azo dye decolorization while gaining an oxidative and heavy metal stress resistance: A trade-off

Heavy metals as co-pollutants significantly restrict the efficiency of bioremediation of azo dye-containing effluents wherein bacterial survival is a prerequisite. An exposure to quinones and oxidative stress via H 2 O 2 could induce the azoreductase activity in Enterococcus sp. L2 which could link the AzoA to its stress management system. Therefore, we underpinned the physiological and stress tolerance features by homologous over-expression of the azoA gene in its native strain. AzoA overexpression increased 3.4 folds azoreductase specific activity and enhanced quinone reduction profile. AzoA overexpression in native strain led to a delay in the early log phase growth and reactive violet 5R decolorization while gaining an advantageous stress resistance towards oxidative and heavy metals such as copper and chromium. This was confirmed by the 32 folds higher survival upon exposure to 20 mM H 2 O 2 supported by H 2 DCFA staining. Additionally, the genetically modified strain significantly tolerated 3 mM Cu(II) and 0.3 mM Cr(VI) supporting the development of a robust azo dye bioremediation strain. • Azo dye, quinones and H 2 O 2 enhanced azoreductase activity of Enterococcus sp. L2. • Homologous cloning in strain L2 resulted in 3.4 folds over-expression of AzoA gene. • AzoA over-expression in strain L2 moderated growth and dye decolorization. • AzoA over-expression in strain L2 increased oxidative stress (H 2 O 2 ) resistance. • AzoA over-expression in strain L2 enhanced heavy metal (Cu 2+ and Cr 6+ ) tolerance.

RsmA3 modulates RpoS through the RetS-Gac-Rsm signalling pathway in response to H2 O2 stress in the phytopathogen Pseudomonas syringae.

Reactive oxygen species are a fatal challenge to the plant pathogenic bacterium Pseudomonas syringae. In this study, we reveal that the global regulatory protein RsmA3 from the RetS-Gac/Rsm signalling pathway modulates RpoS in the early-log growth phase in the P. syringae wild-type strain MB03, thereby regulating oxidative tolerance to H2 O2 and ultimately affecting pathogenicity to the host plant. Following increased H2 O2 by external addition or endogenous induction by menadione, the resistance of the mutant strain ΔretS to H2 O2 is significantly enhanced due to rapid increases in the transcription of Rsm-related non-coding small RNAs (nc sRNAs), a sigma factor RpoS, and H2 O2 -detoxifying enzymes. Moreover, the ΔretS mutant is significantly less pathogenic in cucumber leaves. Seven Rsm-related nc sRNAs (namely, rsmZ, rsmY and rsmX1-5 ) show functional redundancy in the RetS-Gac-Rsm signalling pathway. External addition of H2 O2 stimulates increases in the transcription of both rsmY and rsmZ. Thus, we propose a regulatory model of the RetS-Gac-Rsm signalling pathway in P. syringae MB03 for the regulation of H2 O2 tolerance and phytopathogenicity in the host plant.

Improved growth and morphological plasticity of Haloferax volcanii.

Some microbes display pleomorphism, showing variable cell shapes in a single culture, whereas others differentiate to adapt to changed environmental conditions. The pleomorphic archaeon Haloferax volcanii commonly forms discoid-shaped ('plate') cells in culture, but may also be present as rods, and can develop into motile rods in soft agar, or longer filaments in certain biofilms. Here we report improvement of H. volcanii growth in both semi-defined and complex media by supplementing with eight trace element micronutrients. With these supplemented media, transient development of plate cells into uniformly shaped rods was clearly observed during the early log phase of growth; cells then reverted to plates for the late log and stationary phases. In media prepared with high-purity water and reagents, without supplemental trace elements, rods and other complex elongated morphologies ('pleomorphic rods') were observed at all growth stages of the culture; the highly elongated cells sometimes displayed a substantial tubule at one or less frequently both poles, as well as unusual tapered and highly curved forms. Polar tubules were observed forming by initial mid-cell narrowing or tubulation, causing a dumbbell-like shape, followed by cell division towards one end. Formation of the uniform early log-phase rods, as well as the pleomorphic rods and tubules were dependent on the function of the tubulin-like cytoskeletal protein, CetZ1. Our results reveal the remarkable morphological plasticity of H. volcanii cells in response to multiple culture conditions, and should facilitate the use of this species in further studies of archaeal biology.

Siderophore production by bacteria isolated from mangrove sediments: A microcosm study

Mangroves are one of the most productive ecosystems worldwide covering up to 75% of the coastline in the tropics and subtropics. They support a highly diverse community (marine and terrestrial) and serves as reservoirs of nutrients for coastal and shelf waters. Bacterial diversity in mangroves includes heterotrophs, autotrophs (nitrogen fixation) and pathogens (phytopathogens, marine, and human). All these bacterial groups require sequestration of bioavailable iron, which is largely done by the production of siderophores. In this study, microcosm experiments were conducted to test the effect of incubation conditions (temperature, iron concentration, pH, and carbon source) on growth and siderophore production in four mangrove sediment bacterial isolates- Escherichia vulneris, Enterobacter cancerogenus, Pantoea agglomerans, and Enterobacter bugandensis. Our study showed that all isolates produce more siderophores (30 to 60%) at low iron concentrations (10 nM to 1 μM) during lag-phase and early log-phase of growth. Low temperature suppressed bacterial growth without significantly altering the siderophore production, whereas low pH suppressed both growth and siderophore production in these isolates. Although all isolates could produce siderophores when using different carbon sources, glucose served as an ideal carbon source. The observed changes in growth and siderophore production may be attributed to species-specific physiological traits, changes in bioavailability of iron and/or combination of both. Our results suggest that in a changing global environment, warming of the surrounding waters may not reduce the siderophore production and hence, they will be essential in sustaining bacterial activity in sediments.

Artemisinin-resistant Leishmania parasite modulates host cell defense mechanism and exhibits altered expression of unfolded protein response genes.

Artemisinin, extracted from a medicinal herb Artemisia annua, is widely used to treat malaria and has shown potent anticancer activity. Artemisinin has been found to be effective against experimental visceral and cutaneous leishmaniasis. Despite extensive research to understand the complex mechanism of resistance to artemisinin, several questions remain unanswered. The artesunate (ART)-resistant line of Leishmania donovani was selected and cellular mechanisms associated with resistance to artemisinin were investigated. ART-resistant (AS-R) parasites showed reduced susceptibility towards ART both at promastigote and amastigote stage compared with ART sensitive (WT) parasites. WT and AS-R parasites were both more susceptible to ART at the early log phase of growth compared with late log phase. AS-R parasites were more infective to the host macrophages (p < 0.05). Evaluation of parasites' tolerance towards host microbicidal mechanisms revealed that AS-R parasites were more tolerant to complement-mediated lysis and nitrosative stress. ROS levels were modulated in presence of ART in AS-R parasites infected macrophages. Interestingly, infection of macrophages by AS-R parasites led to modulated levels of host interleukins, IL-2 and IL-10, in addition to nitric oxide. Additionally, AS-R parasites showed upregulated expression of genes of unfolded protein response pathway including methyltransferase domain-containing protein (HSP40) and flagellar attachment zone protein (prefoldin), that are reported to be associated with ART resistance in Plasmodium falciparum malaria. This study presents in vitro model of artemisinin-resistant Leishmania parasite and cellular mechanisms associated with ART resistance in Leishmania.

Effects of Anaerobic Culturing on Pathogenicity and Virulence-Related Gene Expression in Pneumococcal Pneumonia.

The pathogenicity of Streptococcus pneumoniae under anaerobic conditions remains largely unknown. We examined the pathogenicity of S. pneumoniae cultured under anaerobic conditions in a murine model of pneumococcal pneumonia. Mice were infected with S. pneumoniae grown under anaerobic or aerobic conditions. The pathogenic effects in vivo in the lower airway tract were then compared. The effect of anaerobic culture on lytA/ply transcript levels in vitro and in vivo were analyzed by quantitative real-time polymerase chain reaction. Mice inoculated with anaerobically cultured S. pneumoniae exhibited significantly lower survival rates and higher bacterial loads in the lungs and blood as compared to those infected with aerobically cultured S. pneumoniae. Aerobically cultured S. pneumoniae in the early log phase of growth was also able to induce severe pneumonia at levels equivalent to those of anaerobic S. pneumoniae. However, ply/gyrB transcript levels were significantly increased in the lungs of mice infected with anaerobically grown S. pneumoniae. In vitro, S. pneumoniae grown under anaerobic culture conditions demonstrated greater proliferation than S. pneumoniae grown under aerobic culture conditions, and bacterial concentrations were maintained for 24 hours without detectable upregulation of lytA messenger RNA. S. pneumoniae grown under anaerobic conditions had the potential to induce severe invasive bacteremic pneumococcal pneumonia in a manner different from that of S. pneumoniae grown under aerobic conditions.

Class III peroxidases in cellulose deficient cultured maize cells during cell wall remodeling.

Maize (Zea mays L.) suspension-cultured cells habituated to a cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a modified cell wall, in which the reduction in the cellulose content is compensated by a network of highly cross-linked feruloylated arabinoxylans and the deposition of lignin-like polymers. For both arabinoxylan cross-linking and lignin polymerization, class III peroxidases (POXs) have been demonstrated to have a prominent role. For the first time, a comparative study of POX activity and isoforms in control and cellulose-impaired cells has been addressed, also taking into account their cellular distribution in different compartments. Proteins from the spent medium (SM), soluble cellular (SC), ionically (ICW) and covalently bound cell wall protein fractions were assayed for total and specific peroxidase activity by using coniferyl and sinapyl alcohol and ferulic acid as substrates. The isoPOX profile was obtained by isoelectric focusing. POX activity was higher in DCB-habituated than in non-habituated cells in all protein fractions at all cell culture stages. For all substrates assayed, SC and ICW fractions showed higher activity at the early log growth phase than at the late log phase. However, the highest POX activity in the spent medium was found at the late log phase. According to the isoPOX profiles, the highest diversity of isoPOXs was detected in the ICW and SM protein fractions. The latter fraction contained isoPOXs with higher activity in DCB-habituated cells. Some of the isoPOXs detected could be involved in cross-linking of arabinoxylans and in the lignin-like polymer formation in DCB-habituated cells.

Saccharomyces cerevisiae Coq10, A Putative START Domain Protein Binds Coenzyme Q And Late‐Stage Q‐Biosynthetic Intermediates

Coenzyme Q (ubiquinone or Q) functions as an essential redox‐active lipid in respiratory electron and proton transport in cellular energy metabolism, and is an important lipid‐soluble antioxidant in cellular membranes. In Saccharomyces cerevisiae, at least 13 nuclear‐encoded genes COQ1‐COQ11, YAH1, and ARH1 have been identified as necessary for Q biosynthesis and its function in respiration and as an antioxidant. Many of the Coq polypeptides responsible for Q biosynthesis assemble into a high molecular weight, multi‐subunit complex localized to the matrix side of the mitochondrial inner membrane. This study focuses on characterizing the Coq10 polypeptide, a putative steroidogenic acute regulatory (StAR)‐related lipid transfer (StART) domain protein in S. cerevisiae. Unlike Q‐less coq1‐coq9 deletion mutants, yeast coq10 null mutants contain near wild‐type steady‐state levels of Q6, but are nonetheless respiratory deficient. Yeast coq10 null mutants show decreased de novo Q biosynthesis and accumulate high levels of the early Q‐intermediates, especially during early log phase of growth. The structure of Caulobacter crescentus CC1736, an ortholog of yeast Coq10, contains a hydrophobic tunnel characteristic of StART domain proteins. Purified CC1736 is capable of binding Q and a farnesylated analog of a late‐stage Q biosynthetic intermediate. Overexpression of CC1736 in the coq10 null mutant is able to rescue the respiratory‐deficient phenotype and partially restore efficient Q biosynthesis, suggesting a strong functional conservation. These observations suggest the Coq10 StART domain protein is required for efficient Q biosynthesis, crucial for Q function in respiratory electron transport, and for Q to function as an antioxidant. Coq10 is speculated to be a Q‐binding protein interacting with the multi‐subunit Coq polypeptide complex and/or respiratory complexes, facilitating final delivery of Q from its site of synthesis to sites of function. In order to better understand the biological function of yeast Coq10 we describe the isolation and characterization of the high molecular weight Coq10 oligomer and are characterizing lipid ligand interactions with mature Coq10. The results will provide important clues about the physiological role of Coq10 as well as the molecular interactions required for its function. This research was supported by NSF MCB‐1330803 and Ruth L. Kirschstein National Research Service Award GM007185.Support or Funding InformationThis research was supported by NSF MCB‐1330803 and Ruth L. Kirschstein National Research Service Award GM007185.

Impact of algal organic matter released from Microcystis aeruginosa and Chlorella sp. on the fouling of a ceramic microfiltration membrane

Algal blooms lead to the secretion of algal organic matter (AOM) from different algal species into water treatment systems, and there is very limited information regarding the impact of AOM from different species on the fouling of ceramic microfiltration (MF) membranes. The impact of soluble AOM released from Microcystis aeruginosa and Chlorella sp. separately and together in feedwater on the fouling of a tubular ceramic microfiltration membrane (alumina, 0.1 μm) was studied at lab scale. Multi-cycle MF tests operated in constant pressure mode showed that the AOM (3 mg DOC L−1) extracted from the cultures of the two algae in early log phase of growth (12 days) resulted in less flux decline compared with the AOM from stationary phase (35 days), due to the latter containing significantly greater amounts of high fouling potential components (protein and humic-like substances). The AOM released from Chlorella sp. at stationary phase led to considerably greater flux decline and irreversible fouling resistance compared with that from M. aeruginosa. The mixture of the AOM (1:1, 3 mg DOC L−1) from the two algal species showed more similar flux decline and irreversible fouling resistance to the AOM from M. aeruginosa than Chlorella sp. This was due to the characteristics of the AOM mixture being more similar to those for M. aeruginosa than Chlorella sp. The extent of the flux decline for the AOM mixture after conventional coagulation with aluminium chlorohydrate or alum was reduced by 70%.

A two-step method for extraction of lipopolysaccharide from Shigella dysenteriae serotype 1 and Salmonella typhimurium: An improved method for enhanced yield and purity

Bacterial lipopolysaccharide (LPS) has been widely used as an antigen and adjuvant in immunological applications. Amongst the methods developed for extraction of LPS, hot phenol extraction (HPE) method is the gold standard. However, the HPE method provides poor yield of LPS (~4.5% by weight), is associated with relatively higher impurities of proteins and nucleic acids, and the acidic hot phenol can cause a degradative effect on LPS. In this work a two-step extraction (TSE) method was developed using a non-capsulated, [Shigella dysenteriae serotype-1] (Sd1) and capsulated [Salmonella typhimurium type B (StB)] species as model pathogens. The TSE method takes advantage of growth kinetics of bacteria wherein a two-step sequential approach for LPS extraction was employed. In step-1, culture supplemented with CaCl2 during early log phase of growth was induced to release LPS by the effect of EDTA at their late exponential phase of growth. In step-II, cells with left over LPS were subjected to modified HPE method that reduced both the degradative effect of acidic hot phenol and associated impurities. The LPS produced using TSE method enabled not only enhanced yield (~2.78 and ~2.91 fold higher for Sd1 and StB respectively) requiring nearly similar duration of extraction, but also was structurally and functionally comparable with LPS produced using HPE method and commercially procured LPS. Overall, the developed TSE method is relatively more efficient (enhanced yield), clean (healthy extraction with reduced impurities), safe (reduced handling of larger pathogenic culture) and cost-effective for LPS extraction with potential for scale up.

Estimation of Candida albicans ABC Transporter Behavior in Real-Time via Fluorescence.

We present a fluorometric method for determining ABC transporter activity in the pathogenic fungus C. albicans during different growth phases and in response to glucose. The carbocyanine dye diS-C3(3) was previously used to monitor plasma membrane potentials and test the influence of surface-active compounds in membrane polarization. We used diS-C3(3) to show changes in fluorescence kinetics that reflect changes in the activity of ABC transporters in C. albicans growth. Cdr1-GFP fluorescence, revealed that Cdr1p relocates to the inside of the cell after the early-log growth phase. Addition of glucose to the cell suspension resulted in Cdr1p transporter expression in the CDR2-knockout strain. We confirmed the diS-C3(3) results by standard RT-PCR and Western blotting.

Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

BackgroundDue to its extraordinary chemical properties, the cysteine amino acid residue is often involved in protein folding, electron driving, sensing stress, and binding metals such as iron or zinc. Lactococcus lactis, a Gram-positive bacterium, houses around one hundred cysteine-rich proteins (with the CX2C motif) in the cytoplasm, but only a few in the membrane.ResultsIn order to understand the role played by this motif we focused our work on two membrane proteins of unknown function: Llmg_0524 and Llmg_0526. Each of these proteins has two CX2C motifs separated by ten amino-acid residues (CX2CX10CX2C). Together with a short intervening gene (llmg_0525), the genes of these two proteins form an operon, which is induced only during the early log growth phase. In both proteins, we found that the CX2CX10CX2C motif chelated a zinc ion via its cysteine residues, but the sphere of coordination was remarkably different in each case. In the case of Llmg_0524, two of the four cysteines were ligands of a zinc ion whereas in Llmg_0526, all four residues were involved in binding zinc. In both proteins, the cysteine-zinc complex was very stable at 37 °C or in the presence of oxidative agents, suggesting a probable role in protein stability. We found that the complete deletion of llmg_0524 increased the sensitivity of the mutant to cumene hydroperoxide whereas the deletion of the cysteine motif in Llmg_0524 resulted in a growth defect. The latter mutant was much more resistant to lysozyme than other strains.ConclusionsOur data suggest that the CX2CX10CX2C motif is used to chelate a zinc ion but we cannot predict the number of cysteine residue involved as ligand of metal. Although no other motif is present in sequence to identify roles played by these proteins, our results indicate that Llmg_0524 contributes to the cell wall integrity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0587-1) contains supplementary material, which is available to authorized users.

Inhibition of the sodium-translocating NADH-ubiquinone oxidoreductase [Na+-NQR] decreases cholera toxin production in Vibrio cholerae O1 at the late exponential growth phase

Two virulence factors produced by Vibrio cholerae, cholera toxin (CT) and toxin-corregulated pilus (TCP), are indispensable for cholera infection. ToxT is the central regulatory protein involved in activation of CT and TCP expression. We previously reported that lack of a respiration-linked sodium-translocating NADH–ubiquinone oxidoreductase (Na+-NQR) significantly increases toxT transcription. In this study, we further characterized this link and found that Na+-NQR affects toxT expression only at the early-log growth phase, whereas lack of Na+-NQR decreases CT production after the mid-log growth phase. Such decreased CT production was independent of toxT and ctxB transcription. Supplementing a respiratory substrate, l-lactate, into the growth media restored CT production in the nqrA-F mutant, suggesting that decreased CT production in the Na+-NQR mutant is dependent on electron transport chain (ETC) activity. This notion was supported by the observations that two chemical inhibitors, a Na+-NQR specific inhibitor 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO) and a succinate dehydrogenase (SDH) inhibitor, thenoyltrifluoroacetone (TTFA), strongly inhibited CT production in both classical and El Tor biotype strains of V. cholerae. Accordingly, we propose the main respiratory enzyme of V. cholerae, as a potential drug target to treat cholera because human mitochondria do not contain Na+-NQR orthologs.

Tetracycline accelerates the temporally-regulated invasion response in specific isolates of multidrug-resistant Salmonella enterica serovar Typhimurium

BackgroundMultidrug-resistant (MDR) Salmonella isolates are associated with increased morbidity compared to antibiotic-sensitive strains and are an important health and safety concern in both humans and animals. Salmonella enterica serovar Typhimurium is a prevalent cause of foodborne disease, and a considerable number of S. Typhimurium isolates from humans and livestock are resistant to three or more antibiotics. The majority of these MDR S. Typhimurium isolates are resistant to tetracycline, a commonly used and clinically and agriculturally relevant antibiotic. Because exposure of drug-resistant bacteria to antibiotics can affect cellular processes associated with virulence, such as invasion, we investigated the effect tetracycline had on the invasiveness of tetracycline-resistant MDR S. Typhimurium isolates.ResultsThe isolates selected and tested were from two common definitive phage types of S. Typhimurium, DT104 and DT193, and were resistant to tetracycline and at least three other antibiotics. Although Salmonella invasiveness is temporally regulated and normally occurs during late-log growth phase, tetracycline exposure induced the full invasive phenotype in a cell culture assay during early-log growth in several DT193 isolates. No changes in invasiveness due to tetracycline exposure occurred in the DT104 isolates during early-log growth or in any of the isolates during late-log growth. Real-time PCR was used to test expression of the virulence genes hilA, prgH, and invF, and these genes were significantly up-regulated during early-log growth in most isolates due to tetracycline exposure; however, increased virulence gene expression did not always correspond with increased invasion, and therefore was not an accurate indicator of elevated invasiveness. This is the first report to assess DT193 isolates, as well as the early-log growth phase, in response to tetracycline exposure, and it was the combination of both parameters that was necessary to observe the induced invasion phenotype.ConclusionsIn this report, we demonstrate that the invasiveness of MDR S. Typhimurium can be modulated in the presence of tetracycline, and this effect is dependent on growth phase, antibiotic concentration, and strain background. Identifying the conditions necessary to establish an invasive phenotype is important to elucidate the underlying factors associated with increased virulence of MDR Salmonella.

Stable extracellular RNA fragments of Mycobacterium tuberculosis induce early apoptosis in human monocytes via a caspase-8 dependent mechanism.

The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.

The Essential Two-Component System YhcSR Is Involved in Regulation of the Nitrate Respiratory Pathway of Staphylococcus aureus

Our previous studies revealed that a novel two-component signal transduction system, YhcSR, is essential for the survival of Staphylococcus aureus; however, the biological function of YhcSR remains unknown. In this study, we demonstrated that YhcSR plays an important role in the modulation of the nitrate respiratory pathway under anaerobic conditions. Specifically, we determined that nitrate induces yhcS transcription in the early log phase of growth under anaerobic conditions and that the downregulation of yhcSR expression eliminates the stimulatory effect of nitrate on bacterial growth. Using semiquantitative real-time reverse transcription-PCR (qPCR) and promoter-lux reporter fusions, we established that YhcSR positively modulates the transcription of the narG operon, which is involved in the nitrate respiratory pathway. Our gel shift assays revealed that YhcR binds to the promoter regions of narG and nreABC. Collectively, the above data indicate that the yhcSR system directly regulates the expression of both narG and nreABC operons, which in turn positively modulate the nitrate respiratory pathway of S. aureus under anaerobic conditions. These results provide a new insight into the biological functions of the essential two-component YhcSR system.

Optimization of electroporation conditions for Arthrobacter with plasmid PART2

A prerequisite for genetic studies of Arthrobacter is a high efficiency transformation system that allows for DNA transfer, transposon mutagenesis, and expression of specific genes. In this study, we develop a detailed electroporation method through a systematic examination of the factors involved in the entire electroporation process. Key features of this procedure, including the addition of penicillin to cells during the early log phase of growth and the presence of 0.5 M sorbitol in the electroporation and recovery media, produced the greatest increases in transformation efficiency and consistency of results. The transformation rate also varied depending on the electrical parameters, DNA concentration, and recovery time period. Using optimum conditions, we generally achieved an efficiency of 6.8 × 10 7 transformants per microgram of PART2 for Arthrobacter sp. A3. This protocol was also successfully applied to other Arthrobacter species. Therefore, we conclude that the proposed method is rapid, simple and convenient, which allows a transformation trial to be accomplished in minutes.

Assessing the toxic effect of 2,4,6-trinitrotoluene on cells of Escherichia coli K12 by flow cytofluorometry

The magnitude of transmembrane potential delta psi in cells of Escherichia coli K12 was determined by the method of flow cytofluorometry for different phases of growth. It was large in the log phase, whereas in the lag and stationary phases, the population was shown to consist of two subpopulations with low and large values of delta psi in cells. In the presence of 2,4,6-trinitrotoluene (TNT), this bimodal distribution of delta psi over the population was observed during the entire growth period until TNT was almost completely eliminated from the cultivation medium (to a concentration of 18-20 mg/l). The mean value of delta psi in cells of the population grown in the presence of TNT was substantially smaller than that in controls due to the larger size of the subpopulation with a low value of delta psi. Upon elimination of TNT, the distribution of delta psi in cells of the culture became unimodal and close to that in the control culture in the early log phase of growth. These findings are discussed from the standpoint that considers heterogeneity of the culture of Escherichia coli K12 as a mechanism of its adaptation to the presence of xenobiotics.

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

The steady-state levels of uracil residues in DNA extracted from strains of Escherichia coli were measured and the influence of defects in the genes for uracil-DNA glycosylase ( ung), double-strand uracil-DNA glycosylase ( dug), and dUTP pyrophosphatase ( dut) on uracil accumulation was determined. A sensitive method, called the Ung-ARP assay, was developed that utilized E. coli Ung, T4pdg, and the Aldehyde Reactive Probe reagent to label abasic sites resulting from uracil excision with biotin. The limit of detection was one uracil residue per million DNA nucleotides (U/10 6 nt). Uracil levels in the genomic DNA of E. coli JM105 ( ung + dug +) were at the limit of detection, as were those of an isogenic dug mutant, regardless of growth phase. Inactivation of ung in JM105 resulted in 31 ± 2.6 U/10 6 nt during early log growth and 19 ± 1.7 U/10 6 nt in saturated phase. An ung dug double mutant (CY11) accumulated 33 ± 2.9 U/10 6 nt and 23 ± 1.8 U/10 6 nt during early log and saturated phase growth, respectively. When cultures of CY11 were supplemented with 20 ng/ml of 5-fluoro-2′-deoxyuridine, uracil levels in early log phase growth DNA rose to 125 ± 1.7 U/10 6 nt. Deoxyuridine supplementation reduced the amount of uracil in CY11 DNA, but uridine did not. Levels of uracil in DNA extracted from CJ236 ( dut- 1 ung- 1) were determined to be 3000–8000 U/10 6 nt as measured by the Ung-ARP assay, two-dimensional thin-layer chromatography of metabolically-labeled 32P DNA, and LC/MS of uracil and thymine deoxynucleosides. DNA sequencing revealed that the sole molecular defect in the CJ236 dUTP pyrophosphatase gene was a C→T transition mutation that resulted in a Thr24Ile amino acid change.