Mid-log Phase Research Articles

Non-photochemical quenching-dependent acclimation and thylakoid organization of Chlamydomonas reinhardtii to high light stress.

Light is essential for all photosynthetic organisms while an excess of it can lead to damage mainly the photosystems of the thylakoid membrane. In this study, we have grown Chlamydomonas reinhardtii cells in different intensities of high light to understand the photosynthetic process with reference to thylakoid membrane organization during its acclimation process. We observed, the cells acclimatized to long-term response to high light intensities of 500 and 1000µmolm-2s-1 with faster growth and more biomass production when compared to cells at 50µmolm-2s-1 light intensity. The ratio of Chl a/b was marginally decreased from the mid-log phase of growth at the high light intensity. Increased level of zeaxanthin and LHCSR3 expression was also found which is known to play a key role in non-photochemical quenching (NPQ) mechanism for photoprotection. Changes in photosynthetic parameters were observed such as increased levels of NPQ, marginal change in electron transport rate, and many other changes which demonstrate that cells were acclimatized to high light which isan adaptive mechanism. Surprisingly, PSII core protein contents have marginally reduced when compared to peripherally arranged LHCII in high light-grown cells. Further, we also observed alterations in stromal subunits of PSI and low levels of PsaG, probably due to disruption of PSI assembly and also its association with LHCI. During the process of acclimation, changes in thylakoid organization occurred in high light intensities with reduction of PSII supercomplex formation. This change may be attributed to alteration of protein-pigment complexes which are in agreement with circular dichoismspectra of high light-acclimatized cells, where decrease in the magnitude of psi-type bands indicates changes in ordered arrays of PSII-LHCII supercomplexes. These results specify that acclimation to high light stress throughNPQ mechanism by expression of LHCSR3 and also observedchanges in thylakoid protein profile/supercomplex formation lead to low photochemical yield and more biomass production in high light condition.

Antioxidative responses of Nostoc ellipsosporum and Nostoc piscinale to salt stress

The responses of the cyanobacteria Nostoc ellipsosporum and Nostoc piscinale to salt stress during various growth stages were studied. Nostoc ellipsosporum was more NaCl tolerant and attained more biomass under salinity than N. piscinale. Accumulation of proline was detected in N. ellipsosporum under salinity stress. Malondialdehyde content decreased in both species on day 9 (mid log phase) under salt stress. A similar trend was detected in hydrogen peroxide (H2O2) content in N. ellipsosporum. Strong induction in catalase (CAT) activity was observed in N. ellipsosporum on day 9 in the presence of salt. The increase in CAT activity of N. piscinale was observed only at higher concentrations of NaCl. In contrast to N. ellipsosporum, induction in peroxidase and polyphenol oxidase (PPO) activities on day 9 was stronger in N. piscinale. Salinity enhanced superoxide dismutase (SOD) and PPO activity in N. ellipsosporum at all growth stages. Moreover, different isoforms of CAT and SOD were detected in these cyanobacteria. Apparently, selection pressure in these cyanobacteria has led to the evolution of SODs and CATs as the main antioxidant enzymes against reactive oxygen species. Phycobiliprotein content in N. ellipsosporum under all conditions was significantly higher than that in N. piscinale. NaCl at moderate concentrations significantly increased phycobiliprotein content in the middle of the log phase in both species. Moreover, contrary to N. piscinale, the size of phycobilisomes [phycoerythrin + phycocyanin / allophycocyanin] in N. ellipsosporum increased significantly under salt stress at the mid of log phase and later. The increase in size of phycobilisomes in N. ellipsosporum could help this species to withstand salt stress by enhancing energy transfer capacity. These results suggest that N. ellipsosporum cells could be better protected against salinity-induced oxidative damage by maintaining higher levels of antioxidative enzymes, proline, and phycobiliproteins than the cells of N. piscinale.

Coordinated regulation of core and accessory genes in the multipartite genome of Sinorhizobium fredii.

Prokaryotes benefit from having accessory genes, but it is unclear how accessory genes can be linked with the core regulatory network when developing adaptations to new niches. Here we determined hierarchical core/accessory subsets in the multipartite pangenome (composed of genes from the chromosome, chromid and plasmids) of the soybean microsymbiont Sinorhizobium fredii by comparing twelve Sinorhizobium genomes. Transcriptomes of two S. fredii strains at mid-log and stationary growth phases and in symbiotic conditions were obtained. The average level of gene expression, variation of expression between different conditions, and gene connectivity within the co-expression network were positively correlated with the gene conservation level from strain-specific accessory genes to genus core. Condition-dependent transcriptomes exhibited adaptive transcriptional changes in pangenome subsets shared by the two strains, while strain-dependent transcriptomes were enriched with accessory genes on the chromid. Proportionally more chromid genes than plasmid genes were co-expressed with chromosomal genes, while plasmid genes had a higher within-replicon connectivity in expression than chromid ones. However, key nitrogen fixation genes on the symbiosis plasmid were characterized by high connectivity in both within- and between-replicon analyses. Among those genes with host-specific upregulation patterns, chromosomal znu and mdt operons, encoding a conserved high-affinity zinc transporter and an accessory multi-drug efflux system, respectively, were experimentally demonstrated to be involved in host-specific symbiotic adaptation. These findings highlight the importance of integrative regulation of hierarchical core/accessory components in the multipartite genome of bacteria during niche adaptation and in shaping the prokaryotic pangenome in the long run.

Effects of increasing hydrophobicity by N-terminal myristoylation on the antibacterial and hemolytic activities of the C-terminal cationic segments of human-β-defensins 1-3.

Analogs of the cationic C-terminal segments of human-β-defensins HBD1-3, Phd1-3 with a single disulfide bond, exhibited comparable antimicrobial activity that was salt sensitive. They did not show hemolytic activity. In this study, N-terminal myristoylation was carried out on Phd1-3 to examine whether increasing hydrophobicity would result in improved antibacterial activity. The antibacterial activity of the oxidized myristoylated peptides MPhd1-3 and their reduced forms MPhd1r-3r was determined. These peptides showed enhanced antibacterial activity as compared to Phd1-3, on mid-log phase and stationary phase of Staphylococcus aureus and Escherichia coli, except MPhd1r-3r that were inactive on stationary-phase E.coli. In the presence of 150mm NaCl, MPhd1-3 showed activity against S. aureus. MPhd1and two exhibited activity against E.coli but MPhd3 was inactive. Zeta potential measurements indicated that MPhd1-3 were more effective in surface charge neutralization of bacteria as compared to Phd1-3. MPhd1-3 exhibited hemolytic activity to varying extents with MPhd1 being most hemolytic. The data indicate that myristoylation enhances antibacterial activity and modulates hemolytic activity to different extents. Apart from hydrophobicity, distribution of cationic residues in MPhd1-3 plays important roles for these activities.

Roles of cell cycle phases and DNA end structures in determining requirements for the Yku, Mrx and Ligase IV complexes in nonhomologous end‐joining repair of plasmid DNA

Deoxyribonucleic acid (DNA) can be damaged through a variety of different damaging agents such as free radicals, radiation and environmental chemicals. Types of damage include single‐strand lesions such as base or sugar modifications and more deleterious lesions such as interstrand crosslinks and DNA double‐stranded breaks (DSBs). Unrepaired or misrepaired DSBs in a cell can lead to mutations and chromosome rearrangements, potentially leading to loss of cell function and even cell death. There are two independent pathways used to repair DSBs in DNA; these pathways are homologous recombination (HR) and nonhomologous end‐joining (NHEJ). NHEJ involves the functions of three major protein complexes. In the budding yeast Saccharomyces cerevisiae, these assemblages include the Yku, Mrx and DNA ligase IV complexes. Each complex has a specific function. Yku (Yku70 and Yku80) protects the broken ends from degradation and recruits other repair proteins. Mrx, composed of Mre11, Rad50 and Xrs2, may act to tether the ends together and perform nuclease processing of the ends if necessary. The ligase IV complex, containing Dnl4 and Lif1 and possibly Nej1, performs the final step, ligating the two broken ends of DNA thus ending the NHEJ process.Repair of DSBs by NHEJ is frequently analyzed using plasmid‐based assays. In these experiments, a plasmid with a single DSB is transformed into cells and the efficiency of repair/recircularization inside cells is monitored. The region around the DSB site is designed so that it lacks homology with the host cell's chromosomal DNA to prevent repair by HR. An uncut plasmid is usually also transformed into the cells to normalize repair efficiencies to simple cell transformation efficiencies. Many past studies involving NHEJ repair using plasmid assays have observed that repair in yku, mrx, and dnl4 mutants is reduced similarly, typically 10–30 fold, suggesting equal importance of the three major complexes in the pathway. Using a high efficiency yeast transformation method involving treatment with dithiothreitol (DTT) and inclusion of a rich broth recovery step, we observed that repair of broken plasmids with 5′ single‐stranded DNA overhangs (4 nt long) was strongly reduced in yku mutants (yku70 or yku80) and DNA ligase IV mutants (dnl4, lif1, or nej1), but not in mrx strains (mre11, rad50 or xrs2). The accuracy of repair was also strikingly different among mrx cells vs. yku and dnl4 mutants. By contrast, efficiency and accuracy of repair of plasmids containing 3′ overhangs was reduced to similar levels in yku70, mre11 and dnl4 mutants. Since NHEJ repair efficiency is most active in G1 phase, we have also assessed the differential repair of broken plasmids in yku, mrx and dnl4 mutants in cultures with different proportions of G1 cells. These experiments have included mid‐log phase, early stationary phase, stationary phase and alpha factor‐arrested G1 phase cells. Work utilizing linear plasmids containing different end sequences and structures will be described.Support or Funding InformationNational institute of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in Clostridium perfringens

Clostridium perfringens produces various exotoxins and enzymes that cause food poisoning and gas gangrene. The genes involved in virulence are regulated by the agr-like quorum sensing (QS) system, which consists of a QS signal synthesis system and a VirSR two-component regulatory system (VirSR TCS) which is a global regulatory system composed of signal sensor kinase (VirS) and response regulator (VirR). We found that the perfringolysin O gene (pfoA) was transiently expressed during mid-log phase of bacterial growth; its expression was rapidly shut down thereafter, suggesting the existence of a self-quorum quenching (sQQ) system. The sQQ system was induced by the addition of stationary phase culture supernatant (SPCS). Activity of the sQQ system was heat stable, and was present following filtration through the ultrafiltration membrane, suggesting that small molecules acted as sQQ agents. In addition, sQQ was also induced by pure acetic and butyric acids at concentrations equivalent to those in the stationary phase culture, suggesting that organic acids produced by C.perfringens were involved in sQQ. In pH-controlled batch culture, sQQ was greatly diminished; expression level of pfoA extended to late-log growth phase, and was eventually increased by one order of magnitude. Furthermore, hydrochloric acid induced sQQ at the same pH as was used in organic acids. SPCS also suppressed the expression of genes regulated by VirSR TCS. Overall, the expression of virulence factors of C.perfringens was downregulated by the sQQ system, which was mediated by primary acidic metabolites and acidic environments. This suggested the possibility of pH-controlled anti-virulence strategies.

SpeG Is Required for Intracellular Replication of Salmonella in Various Human Cells and Affects Its Polyamine Metabolism and Global Transcriptomes.

The speG gene has been reported to regulate polyamine metabolism in Escherichia coli and Shigella, but its role in Salmonella remains unknown. Our preliminary studies have revealed that speG widely affects the transcriptomes of infected in vitro M and Caco-2 cells and that it is required for the intracellular replication of Salmonella enterica serovar Typhimurium (S. Typhimurium) in HeLa cells. In this study, we demonstrated that speG plays a time-dependent and cell type-independent role in the intracellular replication of S. Typhimurium. Moreover, high-performance liquid chromatography (HPLC) of four major polyamines demonstrated putrescine, spermine, and cadaverine as the leading polyamines in S. Typhimurium. The deletion of speG significantly increased the levels of the three polyamines in intracellular S. Typhimurium, suggesting the inhibitory effect of speG on the biosynthesis of these polyamines. The deletion of speG was associated with elevated levels of these polyamines in the attenuated intracellular replication of S. Typhimurium in host cells. This result was subsequently validated by the dose-dependent suppression of intracellular proliferation after the addition of the polyamines. Furthermore, our RNA transcriptome analysis of S. Typhimurium SL1344 and its speG mutant outside and inside Caco-2 cells revealed that speG regulates the genes associated with flagellar biosynthesis, fimbrial expression, and functions of types III and I secretion systems. speG also affects the expression of genes that have been rarely reported to correlate with polyamine metabolism in Salmonella, including those associated with the periplasmic nitrate reductase system, glucarate metabolism, the phosphotransferase system, cytochromes, and the succinate reductase complex in S. Typhimurium in the mid-log growth phase, as well as those in the ilv–leu and histidine biosynthesis operons of intracellular S. Typhimurium after invasion in Caco-2 cells. In the present study, we characterized the phenotypes and transcriptome effects of speG in S. Typhimurium and reviewed the relevant literature to facilitate a more comprehensive understanding of the potential role of speG in the polyamine metabolism and virulence regulation of Salmonella.

A comprehensive assessment of the biosynthetic pathways of ascorbate, α-tocopherol and free amino acids in Euglena gracilis var. saccharophila

Euglena gracilis produces several important health-enhancing metabolites including ascorbate, α-tocopherol and free amino acids (faa). The yield of metabolites is dependent on the strain of E. gracilis and the metabolic growth condition. Here we investigated the effects of photoautotrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on the synthesis of ascorbate, α-tocopherol and faa in E. gracilis var. saccharophila, using label-free shotgun proteomics, and metabolite analysis using colourimetric assay, high-performance and ultra-performance liquid chromatography (HPLC/UPLC). PT cultivation resulted in the production of more antioxidants (up to 4.13mgg−1 ascorbate and 2.52mgg−1 α-tocopherol) than the MT and HT growth conditions (up to 0.97 and 0.50mgg−1 ascorbate, and 1.40 and 0.21mgg−1 α-tocopherol, respectively). The relative abundance of several faa varied between mid-log and initial stationary growth phases, but the total amount of faa remained about the same, with arginine as the most abundant amino acid. Proteomic analysis revealed a total of 3843 non-redundant proteins in E. gracilis var. saccharophila, of which 1890 were common among all cultivations. Gene ontology annotations suggested derivatisation of metabolic pathways from different organisms, such as lysine biosynthesis from fungi and serine biosynthesis from plants, while a few pathways were unique to Euglena, such as those of ascorbate and arginine. Some enzymes exhibited several isoforms that were influenced by the metabolic growth condition. For example, one of the isozymes of threonine aldolase was expressed in HT/MT cultures only, and one of the isozymes of phosphoglycerate dehydrogenase was expressed in PT cultures only. This is the first proteomic study of E. gracilis var. saccharophila, which provides a mechanistic insight into the biosynthetic pathway dynamics of primary metabolites (antioxidants and faa). This new information can serve as a framework for further development of Euglena as a producer of nutraceuticals.

Temporal regulation of σB by partner-switching mechanism at a distinct growth stage in Bacillus cereus

The alternative transcription factor σB in Bacillus cereus governs the transcription of a number of genes that confer protection against general stress. This transcription factor is regulated by protein-protein interactions among RsbV, RsbW, σB, RsbY, RsbM and RsbK, all encoded in the sigB cluster. Among these regulatory proteins, RsbV, RsbW and σB comprise a partner-switching mechanism. Under normal conditions, σB remains inactive by associating with anti-sigma factor RsbW, which prevents σB from binding to the core RNA polymerase. During environmental stress, RsbK activates RsbY to hydrolyze phosphorylated RsbV, and the dephosphorylated RsbV then sequesters RsbW to liberate σB from RsbW. Although the σB partner-switching module is thought to be the core mechanism for σB regulation, the actual protein-protein interactions among these three proteins in the cell remain to be investigated. In the current study, we show that RsbW and RsbV form a long-lived complex under transient stress treatment, resulting in high persistent expression of RsbV, RsbW and σB from mid-log phase to stationary phase. Full sequestration of RsbW by excess RsbV and increased RsbW:RsbV complex stability afforded by cellular ADP contribute to the prolonged activation of σB. Interestingly, the high expression levels of RsbV, RsbW and σB were dramatically decreased beginning from the transition stage to the stationary phase. Thus, protein interactions among σB partner-switching components are required for the continued induction of σB during environmental stress in the log phase and significant down-regulation of σB is observed in the stationary phase. Our data show that σB is temporally regulated in B. cereus.

Long and branched polyamines are required for maintenance of the ribosome, tRNAHis and tRNATyr in Thermus thermophilus cells at high temperatures.

Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T.thermophilus are synthesized from spermidine, the speB and speD1 gene-deleted strains (ΔspeB and ΔspeD1, respectively) cannot synthesize long and branched polyamines. Although neither strain grew at high temperatures (>75°C) in minimal medium, both strains survived at 80°C when they were cultured at 70°C until the mid-log phase and then shifted to 80°C. We therefore prepared the ΔspeB and ΔspeD1 cells using this culture method. Microscopic analysis showed that both strains can survive for 10h after the temperature shift. Although the modification levels of 2'-O-methylguanosine at position 18, N7 -methylguanosine at position 46, 5-methyluridine at position 54 and N1 -methyladenosine at position 58 in the class I tRNA from both strains were normal, amounts of tRNATyr , tRNAHis , rRNAs and 70S ribosomes were decreased after the temperature shift. Furthermore, invivo protein synthesis in both strains was completely lost 10h after the temperature shift. Thus, long and branched polyamines are required for at least the maintenance of 70S ribosome and some tRNA species at high temperatures.

Determination of volumetric gas–liquid mass transfer coefficient of carbon monoxide in a batch cultivation system using kinetic simulations

A mathematical model of microbial kinetics was introduced to predict the overall volumetric gas–liquid mass transfer coefficient (kLa) of carbon monoxide (CO) in a batch cultivation system. The cell concentration (X), acetate concentration (Cace), headspace gas (Nco and Nco2), dissolved CO concentration in the fermentation medium (Cco), and mass transfer rate (R) were simulated using a variety of kLa values. The simulated results showed excellent agreement with the experimental data for a kLa of 13/hr. The Cco values decreased with increase in cultivation times, whereas the maximum mass transfer rate was achieved at the mid-log phase due to vigorous microbial CO consumption rate higher than R. The model suggested in this study may be applied to a variety of microbial systems involving gaseous substrates.

Bacterial Associates Modify Growth Dynamics of the Dinoflagellate Gymnodinium catenatum.

Marine phytoplankton cells grow in close association with a complex microbial associate community known to affect the growth, behavior, and physiology of the algal host. The relative scale and importance these effects compared to other major factors governing algal cell growth remain unclear. Using algal-bacteria co-culture models based on the toxic dinoflagellate Gymnodinium catenatum, we tested the hypothesis that associate bacteria exert an independent effect on host algal cell growth. Batch co-cultures of G. catenatum were grown under identical environmental conditions with simplified bacterial communities composed of one-, two-, or three-bacterial associates. Modification of the associate community membership and complexity induced up to four-fold changes in dinoflagellate growth rate, equivalent to the effect of a 5°C change in temperature or an almost six-fold change in light intensity (20–115 moles photons PAR m-2 s-1). Almost three-fold changes in both stationary phase cell concentration and death rate were also observed. Co-culture with Roseobacter sp. DG874 reduced dinoflagellate exponential growth rate and led to a more rapid death rate compared with mixed associate community controls or co-culture with either Marinobacter sp. DG879, Alcanivorax sp. DG881. In contrast, associate bacteria concentration was positively correlated with dinoflagellate cell concentration during the exponential growth phase, indicating growth was limited by supply of dinoflagellate-derived carbon. Bacterial growth increased rapidly at the onset of declining and stationary phases due to either increasing availability of algal-derived carbon induced by nutrient stress and autolysis, or at mid-log phase in Roseobacter co-cultures potentially due to the onset of bacterial-mediated cell lysis. Co-cultures with the three bacterial associates resulted in dinoflagellate and bacterial growth dynamics very similar to more complex mixed bacterial community controls, suggesting that three-way co-cultures are sufficient to model interaction and growth dynamics of more complex communities. This study demonstrates that algal associate bacteria independently modify the growth of the host cell under non-limiting growth conditions and supports the concept that algal–bacterial interactions are an important structuring mechanism in phytoplankton communities.

Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress.

The present study shows the existence of two specific sub-populations of Mycobacterium smegmatis and Mycobacterium tuberculosis cells differing in size and density, in the mid-log phase (MLP) cultures, with significant differential susceptibility to antibiotic, oxidative, and nitrite stress. One of these sub-populations (~10% of the total population), contained short-sized cells (SCs) generated through highly-deviated asymmetric cell division (ACD) of normal/long-sized mother cells and symmetric cell divisions (SCD) of short-sized mother cells. The other sub-population (~90% of the total population) contained normal/long-sized cells (NCs). The SCs were acid-fast stainable and heat-susceptible, and contained high density of membrane vesicles (MVs, known to be lipid-rich) on their surface, while the NCs possessed negligible density of MVs on the surface, as revealed by scanning and transmission electron microscopy. Percoll density gradient fractionation of MLP cultures showed the SCs-enriched fraction (SCF) at lower density (probably indicating lipid-richness) and the NCs-enriched fraction (NCF) at higher density of percoll fractions. While live cell imaging showed that the SCs and the NCs could grow and divide to form colony on agarose pads, the SCF, and NCF cells could independently regenerate MLP populations in liquid and solid media, indicating their full genomic content and population regeneration potential. CFU based assays showed the SCF cells to be significantly more susceptible than NCF cells to a range of concentrations of rifampicin and isoniazid (antibiotic stress), H2O2 (oxidative stress),and acidified NaNO2 (nitrite stress). Live cell imaging showed significantly higher susceptibility of the SCs of SC-NC sister daughter cell pairs, formed from highly-deviated ACD of normal/long-sized mother cells, to rifampicin and H2O2, as compared to the sister daughter NCs, irrespective of their comparable growth rates. The SC-SC sister daughter cell pairs, formed from the SCDs of short-sized mother cells and having comparable growth rates, always showed comparable stress-susceptibility. These observations and the presence of M. tuberculosis SCs and NCs in pulmonary tuberculosis patients' sputum earlier reported by us imply a physiological role for the SCs and the NCs under the stress conditions. The plausible reasons for the higher stress susceptibility of SCs and lower stress susceptibility of NCs are discussed.

Evaluating the potential of cytokinins for biomass and lipid enhancement in microalga Acutodesmus obliquus under nitrogen stress

Despite the significant reduction in biomass production, nitrogen stress is the most feasible strategy used for lipid enhancement in microalgae. The present study evaluates the potential for application of exogenous cytokinins to enhance the lipid accumulation and overcome the biomass production constraint of microalgae under nitrogen stressed conditions. This study was undertaken to investigate the effect of exogenously supplied cytokinins viz. kinetin and zeatin at various stages of growth on biomass and lipid productivities of Acutodesmus obliquus under nitrogen stress (ON). The effect of cytokinins on photosynthetic performance and biochemical composition of biomass was also evaluated. Enhanced biomass and lipid productivities were observed upon supplementation of the microalgae with selected cytokinins. Supplementation with kinetin (1mgL−1) and zeatin (0.1mgL−1) resulted in increased biomass productivity up to 50% and 60.7% respectively as compared to ON control. The highest biomass productivity of 176.79mgL−1 d−1 was obtained with the addition of 0.1mgL−1 zeatin (ON-Zi) at the initial log phase. Cytokinin supplementation resulted in improvement in photosynthetic performance and exhibited higher rETR (relative electron transport rate) values as compared to ON control. The addition of kinetin (ON-Km) at mid log phase and Zeatin (ON-Zi) at initial log phase resulted in 64.95% and 63.06% increase in lipid productivity respectively, compared to ON control. Carbohydrate productivity was also found to be increased with zeatin (ON-Zi) supplementation. The supplementation of cytokinins is an easy and scalable strategy for biomass and lipid enhancement in Acutodesmus obliquus under nitrogen stress.

Metabolic Interference of sod gene mutations on catalase activity in Escherichia coli exposed to Gramoxone® (paraquat) herbicide

Herbicides are continuously used to minimize the loss of crop productivity in agricultural environments. They can, however, cause damage by inhibiting the growth of microbiota via oxidative stress, due to the increased production of reactive oxygen species (ROS). Cellular responses to ROS involve the action of enzymes, including superoxide dismutase (SOD) and catalase (CAT). The objective of this study was to evaluate adaptive responses in Escherichia coli K-12 to paraquat, the active ingredient in the herbicide Gramoxone®. Mutant bacterial strains carrying deletions in genes encoding Mn-SOD (sodA) and Fe-SOD (sodB) were used and resulted in distinct levels of hydrogen peroxide production, interference in malondialdehyde, and viability. Mutations also resulted in different levels of interference with the activity of CAT isoenzymes and in the inactivation of Cu/Zn-SOD activity. These mutations may be responsible for metabolic differences among the evaluated strains, resulting in different patterns of antioxidative responses, depending on mutation background. While damage to the ΔsodB strain was minor at late log phase, the reverse was true at mid log phase for the ΔsodA strain. These results demonstrate the important role of these genes in defense against oxidative stress in different periods of growth. Furthermore, the lack of Cu/Zn-SOD activity in both mutant strains indicated that common metal cofactors likely interfere in SOD activity regulation. These results also indicate that E. coli K-12, a classical non-environmental strain, constitutes a model of phenotypic plasticity for adaptation to a redox-cycling herbicide through redundancy of different isoforms of SOD and CAT enzymes.

Recombinant Human REG I<i>α</i> Aggregates <i>Staphylococcus aureus</i>—Exhibits a Lectin-Like Function

Staphylococcus aureus is pathogenic to humans with worldwide health care concern due to its ability to evade the immune system and develop resistance to multiple drugs. Reg family proteins are C-type lectins with antimicrobial properties. Bacterial aggregation through binding to microbial cell surface sugar and/or lipid moieties is key mechanism employed in the process. In the present study we have analysed the antimicrobial effect of human REG Iα on S. aureus. Aggregation of mid-log phase culture of S. aureus was observed in presence of purified recombinant REG Iα. Therefore REG Iα can be applied in eliminating S. aureus infections in humans.

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling.

Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development.

SigCH, an extracytoplasmic function sigma factor of Porphyromonas gingivalis regulates the expression of cdhR and hmuYR

Extracytoplasmic function (ECF) sigma factors play an important role in the bacterial response to various environmental stresses. Porphyromonas gingivalis, a prominent etiological agent in human periodontitis, possesses six putative ECF sigma factors. So far, information is limited on the ECF sigma factor, PGN_0319. The aim of this study was to investigate the role of PGN_0319 (SigCH) of P. gingivalis, focusing on the regulation of hmuY and hmuR, which encode outer-membrane proteins involved in hemin utilization, and cdhR, a transcriptional regulator of hmuYR. First, we evaluated the gene expression profile of the sigCH mutant by DNA microarray. Among the genes with altered expression levels, those involved in hemin utilization were downregulated in the sigCH mutant. To verify the microarray data, quantitative reverse transcription PCR analysis was performed. The RNA samples used were obtained from bacterial cells grown to early-log phase, in which sigCH expression in the wild type was significantly higher than that in mid-log and late-log phases. The expression levels of hmuY, hmuR, and cdhR were significantly decreased in the sigCH mutant compared to wild type. Transcription of these genes was restored in a sigCH complemented strain. Compared to the wild type, the sigCH mutant showed reduced growth in log phase under hemin-limiting conditions. Electrophoretic mobility shift assays showed that recombinant SigCH protein bound to the promoter region of hmuY and cdhR. These results suggest that SigCH plays an important role in the early growth of P. gingivalis, and directly regulates cdhR and hmuYR, thereby playing a potential role in the mechanisms of hemin utilization by P. gingivalis.

Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact.

Staphylococcus aureus (Sau) strains are a main cause of disease, including nosocomial infections which have been linked to the production of biofilms and the propagation of antibiotic resistance strains such as methicillin-resistant Staphylococcus aureus (MRSA). A previous study found that Streptococcus pneumoniae (Spn) strains kill planktonic cultures of Sau strains. In this work, we have further evaluated in detail the eradication of Sau biofilms and investigated ultrastructural interactions of the biofilmicidal effect. Spn strain D39, which produces the competence stimulating peptide 1 (CSP1), reduced Sau biofilms within 8 h of inoculation, while TIGR4, producing CSP2, eradicated Sau biofilms and planktonic cells within 4 h. Differences were not attributed to pherotypes as other Spn strains producing different pheromones eradicated Sau within 4 h. Experiments using Transwell devices, which physically separated both species growing in the same well, demonstrated that direct contact between Spn and Sau was required to efficiently eradicate Sau biofilms and biofilm-released planktonic cells. Physical contact-mediated killing of Sau was not related to production of hydrogen peroxide as an isogenic TIGR4ΔspxB mutant eradicated Sau bacteria within 4 h. Confocal micrographs confirmed eradication of Sau biofilms by TIGR4 and allowed us to visualize ultrastructural point of contacts between Sau and Spn. A time-course study further demonstrated spatial colocalization of Spn chains and Sau tetrads as early as 30 min post-inoculation (Pearson's coefficient >0.72). Finally, precolonized biofilms produced by Sau strain Newman, or MRSA strain USA300, were eradicated by mid-log phase cultures of washed TIGR4 bacteria within 2 h post-inoculation. In conclusion, Spn strains rapidly eradicate pre-colonized Sau aureus biofilms, including those formed by MRSA strains, by a mechanism(s) requiring bacterium-bacterium contact, but independent from the production of hydrogen peroxide.

An assessment of early colonisation of implant-abutment metal surfaces by single species and co-cultured bacterial periodontal pathogens

ObjectiveNumerous studies have proposed that smooth metal surfaces reduce initial bacterial attachment in the establishment of an early biofilm formation. However, these studies have largely examined single bacterial species, which are not always relevant as pathogens identified as initiators of inflammatory peri-implantitis. This study investigated the adherence of four periodontally-relevant bacterial species to implant and abutment surfaces in current clinical use. MethodsDiscs of polished cobalt chromium (CoCr-polished) and milled titanium (Ti-milled), representing two clinically relevant surfaces, were prepared and surfaces were characterised. Bacterial species Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were cultured to mid-log or stationary growth phase. Co-cultures of P. gingivalis, F. nucleatum and P. gingivalis, F. nucleatum, Pr. intermedia were similarly prepared. Bacteria were inoculated onto discs for 2h, stained with a live/dead fluorescent stain and percentage bacterial coverage was calculated by confocal microscopy and image analysis. ResultsCoCr-polished discs had smooth surfaces with gentle valley structures, whilst Ti-milled discs had sharp edged peaks. Both discs demonstrated a partial wetting ability capable of initiating bacterial adhesion. P. gingivalis, F. nucleatum and co-cultures, at both mid-log and stationary concentrations, demonstrated equally high coverage of both the smooth CoCr-polished and the rougher Ti-milled metal surfaces. Pr. intermedia and A. actinomycetemcomitans demonstrated lower surface coverage which was slightly higher for Ti-milled. ConclusionVariability was noted in the adherence potential for the respective periodontal pathogens examined. Particularly high adherence was noted for P. gingivalis and F. nucleatum, despite the manufacture of a smooth surface. Clinical significanceBoth surfaces studied may be used at implant-abutment junctions and both possess an ability to establish a bacterial biofilm containing a periodontally-relevant species. These surfaces are thus able to facilitate the apical migration of bacteria associated with peri-implantitis.