Early Log 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.

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.

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.

Chlortetracycline and florfenicol induce expression of genes associated with pathogenicity in multidrug-resistant Salmonella enterica serovar Typhimurium

BackgroundMultidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Typhimurium) is a serious public health threat as infections caused by these strains are more difficult and expensive to treat. Livestock serve as a reservoir for MDR Salmonella, and the antibiotics chlortetracycline and florfenicol are frequently administrated to food-producing animals to treat and prevent various diseases. Therefore, we evaluated the response of MDR S. Typhimurium after exposure to these two antibiotics.ResultsWe exposed four MDR S. Typhimurium isolates to sub-inhibitory concentrations of chlortetracycline (16 and 32 µg/ml) or florfenicol (16 µg/ml) for 30 min during early-log phase. Differentially expressed genes following antibiotic treatment were identified using RNA-seq, and genes associated with attachment and those located within the Salmonella pathogenicity islands were significantly up-regulated following exposure to either antibiotic. The effect of antibiotic exposure on cellular invasion and motility was also assessed. Swimming and swarming motility were decreased due to antibiotic exposure. However, we observed chlortetracycline enhanced cellular invasion in two strains and florfenicol enhanced invasion in a third isolate.ConclusionsChlortetracycline and florfenicol exposure during early-log growth altered the expression of nearly half of the genes in the S. Typhimurium genome, including a large number of genes associated with virulence and pathogenesis; this transcriptional alteration was not due to the SOS response. The results suggest that exposure to either of these two antibiotics may lead to the expression of virulence genes that are typically only transcribed in vivo, as well as only during late-log or stationary phase in vitro.

Application of soft X-ray microscopy to environmental microbiology of hydrosphere

Microstructures of unprocessed filamentous cyanobacterium, Pseudanabaena foetida sp., producing a musty smell were observed using soft X-ray microscopy. Carbon-enriched structures and granules as well as oxygen-enriched granules which have been already reported were observed. Except for early log growth phase, the oxygen-enriched granules were observed. However, the carbon-enriched structures were observed throughout log growth phase. The result suggests there is a relationship between the oxygen-enriched granules and 2-methylisoborneol (2-MIB) productivity, since the 2-MIB productivity of each cell is increased depending on the culture period in log growth phase.

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.

Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene

The Escherichia coli gal operon has the structure Pgal-galE-galT-galK-galM. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM. However, during late-log growth, type 1 polarity is established in which galK is greater than galT, as follows: galE > galK > galT > galM. We found that type 2 polarity occurs as a result of the down-regulation of galK, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK-specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rho-mediated transcription termination at the end of galT.

Chloramphenicol and tetracycline decrease motility and increase invasion and attachment gene expression in specific isolates of multidrug-resistant Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium is one of the most common serovars isolated from humans and livestock, and over 35% of these isolates are resistant to three or more antibiotics. Multidrug-resistant (MDR) Salmonella is a public health concern as it is associated with increased morbidity in patients compared to antibiotic sensitive strains, though it is unknown how the antibiotic resistant isolates lead to a more severe infection. Cellular invasion is temporally regulated in Salmonella and normally occurs during late-log and stationary growth. However, our previous work determined that a 30 min exposure to a sub-inhibitory concentration of tetracycline can induce the full invasion phenotype during early-log growth in certain MDR S. Typhimurium isolates. The current study examined whether sub-inhibitory concentrations of other antibiotics could also induce the invasiveness in the same set of isolates. Ampicillin and streptomycin had no effect on invasion, but certain concentrations of chloramphenicol were found to induce invasion in a subset of isolates. Two of the isolates induced by chloramphenicol were also inducible by tetracycline. RNA-seq analyses demonstrated that chloramphenicol and tetracycline both down-regulated motility gene expression, while up-regulating genes associated with attachment, invasion, and intracellular survival. Eleven fimbrial operons were up-regulated, which is notable as only three fimbrial operons were thought to be inducible in culture; six of these up-regulated operons have been reported to play a role in Salmonella persistence in mice. Overall, these data show that the normal progression of the genetic pathways that regulate invasion can be expedited to occur within 30 min due to antibiotic exposure. This altered invasion process due to antibiotics may play a role in the increased intensity and duration of infection observed in patients with MDR Salmonella.

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.

Class I and class II lysyl‐tRNA synthetase mutants and the genetic encoding of pyrrolysine in Methanosarcina spp.

Methanosarcina spp. begin methanogenesis from methylamines with methyltransferases made via the translation of UAG as pyrrolysine. In vitro evidence indicates two possible routes to pyrrolysyl-tRNA(Pyl). PylS ligates pyrrolysine to tRNA(Pyl). Alternatively, class I and class II lysyl-tRNA synthetases (LysRS1 and LysRS2) together form lysyl-tRNA(Pyl), a potential intermediate to pyrrolysyl-tRNA(Pyl). The unusual possession of both LysRS1 and LysRS2 by Methanosarcina spp. may also reflect differences in catalytic properties. Here we assessed the in vivo relevance of these hypotheses. The lysK and mtmB transcripts, encoding LysRS1 and monomethylamine methyltransferase, were detectable in Methanosarcina barkeri during early log growth on trimethylamine, but not methanol. In contrast, lysS transcript encoding LysRS2 was detectable during log phase with either substrate. Methanosarcina acetivorans strains bearing deletions of lysK or lysS grew normally on methanol and methylamines with wild-type levels of monomethylamine methyltransferase and aminoacyl-tRNA(Pyl). The lysK and lysS genes could not replace pylS in a recombinant system employing tRNA(Pyl) for UAG suppression. The results support an association of LysRS1 with growth on methylamine, but not an essential role for LysRS1/LysRS2 in the genetic encoding of pyrrolysine. However, decreased lysyl-tRNA(Lys) in the lysS mutant provides a possible rationale for stable transfer of the bacterial lysS gene to methanoarchaea.

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.

An optimized Agrobacterium -mediated transformation procedure for Phaseolus acutifolius A. Gray

To improve Agrobacterium tumefaciens-mediated transformation of Phaseolus acutifolius, we examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing β-glucuronidase gene. Improved transformation frequencies were obtained with an A. tumefaciens strain carrying nopaline-type virulence genes and when calli were infected with Agrobacterium cells in the early-log growth phase. Optimized co-cultivation was performed at 22°C under a 16/8-h (day/night) photoperiod in an acidic medium (pH 5.5) in the presence of 200 µM acetosyringone. By combining the best treatments, an efficient and reproducible transformation procedure was established for the P. acutifolius genotype NI576. Southern and immunoblot analyses confirmed the stable integration and expression of the transgenes in the primary transgenic plants and their progeny.

Increased susceptibility of cells to inducible apoptosis during growth from early to late log phase: an important caveat for in vitro apoptosis research

The physiologic mode of cell death known as apoptosis has become a major focus of scientific research due to its biologic importance. Much of this research involves cells grown in culture, where induction of apoptosis is achieved through a variety of agents. We report that cell cultures in late log growth phase exhibit an increased susceptibility to apoptosis compared with cultures in early log growth phase when apoptosis is induced by sodium deoxycholate (NaDOC), anti-Fas antibody and cytosine-b- d-arabino-furanoside (Ara-C), three agents which induce apoptosis through different upstream mechanisms. We show that this phenomenon occurs in Jurkat lymphocytes, HT-29 and HCT-116 colon epithelial cells. We also present evidence that cell density alone does not affect NaDOC-induced apoptosis, but rather that the growth media plays a key role in increased susceptibility of cells in late log growth phase to NaDOC-induced apoptosis. These results indicate that growth phase is a variable that must be controlled in order to obtain reliable apoptosis data.

An optimal magnetite immobilized Pseudomonas- putida 5-x cellsystem for Cu2+ removal from industrial waste effluent

An optimal magnetite immobilized Pseudomonas putida 5-x cell system was developed to remove Cu2+ from industrial waste effluent. Cu2+ adsorption capacity of P. putida 5-x cultured in sulphate-limiting medium (SLM) was minimum in early log growth phase, and reached maximum in late stationary growth phase or early death phase. Pretreated cells by 0.6 N HCl could greatly enhance the adsorption capacity of biomass up to 85.6 mg/g and had no significant effect for the loss of P. putida 5-x cells during the pretreatment. In a semi-continuous biosorption system, the removal efficiency of Cu2+ from wastewater reached 96%, and recovery efficiency of Cu2+ was 95%, and the concentration in the recovery solution was 1.4 g/L using 0.6 N HCl as eluant. The mechanism of Cu2+ adsorption by this type of biomass was studied by using the technique of transmission electron microscopy (TEM). Degradation of a peptidoglycan layer on the cell surface was observed after acidic pretreatment, but no further degradation appeared after the adsorption-desorption cycle. TEM and X-ray analysis also showed that Cu2+ was mainly accumulated on the cell surface, so it was effectively desorpted by acidic treatment in the desorption process. The process of adsorption obeyed the Freundlich isotherm.

Salt Tolerance in Cell Suspension Cultures of the HalophyteKosteletzkya virginica

Tolerance to NaCl was studied in cell suspension cultures of Kosteletzkya virginica (L.) Presl. (Malvaceae), a dicotyledonous halophyte that grows in tidal marshes of the eastern United States. Growth of salinized cultures was significantly inhibited at high (255 mol m -3 NaCl), but not at lower external salinities. Adjustment of cell suspensions to NaCl was rapid, with the duration of the normal growth cycle unaffected by salinity. Maximum biomass was attained when cultures were exposed to NaCl during early log growth. Patterns of inorganic ion accumulation reflected the utilization of both Na + and K + as osmotica, with Na + content substantially increasing when cells were grown at an external salinity sufficient to reduce growth