Early Stationary Growth Phase Research Articles

Extracellular proteins of Clostridium chauvoei are protective in a mouse model

The anaerobic bacillus Clostridium chauvoei is the causative agent of blackleg, a lethal disease that has an important impact on the sheep and cattle industry worldwide. Immunity to C. chauvoei is considered to be mainly anticellular, and for this reason there is scarce information about the immunogenicity of extracellular proteins. In this work variations in protein profiles, immune response by ELISA and protective capacity of culture supernatants of three C. chauvoei strains, collected at different growth phases, are reported. Sera raised against extracellular antigens also recognised cellular antigens of the same molecular masses. Partially purified cell-free supernatants and those concentrated 10 times by ultrafiltration (C-CFS), obtained at the early stationary phase of growth, induced a strong immunoprotective response, even at low doses, that was more marked for C. chauvoei strain ATCC 10092 (p < or = 0.05). With C-CFS formulations, a clear relationship was observed between IgG titres, protective capacity and concentration of the antigen doses, indicating a specific immune response.

Limits of propidium iodide as a cell viability indicator for environmental bacteria

Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.

Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

BackgroundAll organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column.ResultsThe N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period.ConclusionTaken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.

Isolation and characterization of thermophilic bacteria capable of lysing microbial cells in activated sludge

To find an efficient biological method to solubilize waste activated sludge (WAS) from the biological wastewater treatment process, several strains of thermophilic bacteria capable of solubilizing WAS were isolated from sewage sludge compost. The culture supernatants of the isolates were able to lyse vegetable bacterial cells and the lytic activity mainly came from the exoenzyme produced by the isolates. The culture supernatants of the different isolates showed different lysis characteristics. The factors affecting bacterial cell lysis were investigated using E. coli as a model bacterium. The E. coli cells were lysed easily at higher temperature (60 degrees C or 70 degrees C) while little lytic activity by the supernatants of isolates was observed at lower temperature (50 degrees C). The level of pH also had great influence on the lysis of E. coli cells. The E. coli cells in the early stationary growth phase were easier to lyse than those in the late stationary growth phase or death phase.

Involvement of Pyruvate Oxidase Activity and Acetate Production in the Survival of Lactobacillus plantarum during the Stationary Phase of Aerobic Growth

In addition to the previously characterized pyruvate oxidase PoxB, the Lactobacillus plantarum genome encodes four predicted pyruvate oxidases (PoxC, PoxD, PoxE, and PoxF). Each pyruvate oxidase gene was individually inactivated, and only the knockout of poxF resulted in a decrease in pyruvate oxidase activity under the tested conditions. We show here that L. plantarum has two major pyruvate oxidases: PoxB and PoxF. Both are involved in lactate-to-acetate conversion in the early stationary phase of aerobic growth and are regulated by carbon catabolite repression. A strain devoid of pyruvate oxidase activity was constructed by knocking out the poxB and poxF genes. In this mutant, acetate production was strongly affected, with lactate remaining the major end product of either glucose or maltose fermentation. Notably, survival during the stationary phase appeared to be dramatically improved in the poxB poxF double mutant.

The synthesis and role of the mechanosensitive channel of large conductance in growth and differentiation of Bacillus subtilis

A translational lacZ fusion of the Bacillus subtilis mscL gene that encodes the mechanosensitive channel of large conductance (MscL) was expressed at significant levels during log phase growth of B. subtilis, and the level of mscL-lacZ expression was increased 1.5-fold by growth in medium with high salt (1 M NaCl). However, in growth media with either low or high salt, mscL-lacZ expression fell drastically beginning in the late log phase of growth, and fell to even lower levels during sporulation, although a significant amount of beta-galactosidase from mscL to lacZ was accumulated in the developing spore. Deletion of mscL had no effect on B. subtilis growth, sporulation or subsequent spore germination. The DeltamscL strain also grew as well as the wild-type parental strain in medium with 1.2 M NaCl. While log phase wild-type cells grown with 1.2 M NaCl survived a rapid 0.9 M osmotic downshift, log phase DeltamscL cells rapidly lost viability and lysed when subjected to this same osmotic downshift. However, by the early stationary phase of growth, DeltamscL cells had become resistant to a 0.9 M osmotic downshift.

Adsorption Behavior of Rhodamine B onRhizopusoryzaeBiomass

The removal of a carcinogenic dye rhodamine B (C. I. 45170) from wastewater by biomass of different moulds and yeasts is described. Among all of the fungal species tested, the biomass of Rhizopus oryzae MTCC 262 is found to be the most effective. Dye adsorption reaches maximum with the biomass harvested from the early stationary phase of growth. The optimum temperature and pH for adsorption are observed to be 40 degrees C and 7.0, respectively. The adsorption rate is very fast initially and attains equilibrium after 5 h. The adsorption isotherm follows the Langmuir isotherm model satisfactorily within the studied dye concentration range. Of the different metabolic inhibitors tested, 2,4-ditrophenol (DNP) and N,N'-dicyclohexylcarbodiimide (DCCD) decrease dye adsorption by approximately 30% suggesting the role of energy metabolism in the process. Spectrophotometric study indicates that the removal of rhodamine B by R. oryzae biomass involves an adsorption process. Scanning (SEM) and transmission (TEM) electron microscopic investigations have been carried out to understand the probable mechanism of the dye-biomass interaction.

Quantitative detection of gene expression and toxin complex produced by Clostridium botulinum serotype D strain 4947

Botulinum toxin is produced by Clostridium botulinum as a large toxin complex (L-TC) non-covalently assembled with a neurotoxin (NT), a non-toxic non-hemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33, and HA-17). In this study, the gene expressions of five individual L-TC components were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in C. botulinum serotype D strain 4947 (D-4947) during cell growth. Transcripts for the five component genes were successfully detected in the mid-exponential growth phase (6.5 h), reaching a maximum at the early stationary growth phase (12 h). The ratio of the mRNA transcripts of nt and ntnha was approximately 1:1, suggesting that nt and ntnha are bicistronically transcribed. On the other hand, the transcript levels of the ha genes were several-fold higher than those of nt and ntnha, although the mRNA transcript level of ha-33 was less than the other two ha subcomponent genes. The results based on qRT-PCR indicate that a shortage of HA-33 among the proteins associated with botulinum TC could explain the production by D-4947 of other smaller-sized L-TCs (610, 540 and 410 kDa) with fewer HA-33 molecules than the mature 650 kDa L-TC. Western blot analysis demonstrated that TC species in cell lysate were initially observed in the mid-exponential phase, while extracellular TCs were detected subsequently in the early stationary phase.

Identification of erythrobactin, a hydroxamate-type siderophore produced by Saccharopolyspora erythraea

To investigate the production of siderophores by Saccharopolyspora erythraea SGT2 and how this production is affected by the inoculum. When grown in a low-iron, chemically defined medium (CDM), the soil dwelling actinomycete S. erythraea secretes a substance that is reactive in the nonspecific chrome azurol S (CAS) assay. Importantly, the production of CAS-reactive substance is highly reduced upon the addition of 0.925 micromol l(-1) iron to the cultures and has a peak of production in the late-log to early stationary growth phase. In addition, the culture supernatants tested were negative in the Arnow and Rioux assays but positive in the Csáky procedure. Interestingly, we also found evidence that the production of this CAS-reactive substance in CDM was highly reduced, when inoculated with cells that had been previously grown to late-stationary phase. Conversely, inocula derived from late-log to early stationary cultures presented high levels of CAS activity. These results indicate that S. erythraea produces a hydroxamate-type siderophore that we have generically designated as erythrobactin. Additionally, the inocula growth stage plays a key role in siderophore production in S. erythraea. It is the first evidence for siderophore synthesis in S. erythraea and one of the first examples of non-polyketide secondary metabolite production by this organism.

Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations

The RNA polymerase sigma factor, encoded by rpoS gene, controls the expression of a large number of genes in Escherichia coli under stress conditions. The present study investigated the growth characteristics and metabolic pathways of rpoS gene knockout mutant of E. coli growing in LB media under aerobic condition. The analyses were made based on gene expressions obtained by DNA microarray and RT-PCR, enzyme activities and intracellular metabolite concentrations at the exponential and early stationary phases of growth. Although the glucose utilization pattern of the mutant was similar to the parent strain, the mutant failed to utilize acetate throughout the cultivation period. Microarray data indicated that the expression levels of several important genes of acetate metabolism such as acs, aceAB, cysDEK, fadR, etc. were significantly altered in the absence of rpoS gene. Interestingly, there was an increased activity of TCA cycle during the exponential growth phase, which was gradually diminished at the onset of stationary phase. Moreover, rpoS mutation had profound effect on the expression of several other genes of E. coli metabolic pathways that were not described earlier. The changes in the gene expressions, enzyme activities and intracellular metabolite concentrations of the rpoS mutant are discussed in details with reference to the major metabolic pathways of E. coli.

Transformation of suspension cultures of bromegrass (Bromus inermis) by Agrobacterium tumefaciens

Smooth bromegrass (Bromus inermis Leyss) is an extremely cold hardy perennial grass and its cell culture is an excellent system for studying mechanisms of cold hardiness induced by low temperature or abscisic acid (ABA). Agrobacterium tumefaciens-mediated transformation of non-embryogenic bromegrass cultures was attempted. Agrobacterium strain EHA105 carrying a binary vector that contained the neomycin phosphotransferase (NPT II), beta-glucuronidase (GUS) and green fluorescent protein (GFP) genes were co-cultivated for 3 days with bromegrass cells at the late exponential or early stationary growth phase (7–9 days after subculture). These conditions gave optimal transformation efficiency. Putative transformants were identified by selection for geneticin resistance and by examining the calluses using fluorescence microscopy. This allows the elimination of escapes and selection of cells that express the target genes. PCR and Southern blot analyses confirmed the integration of the GUS and GFP genes into the genome of transformed bromegrass cell lines. Transformants with various levels of GUS expression were obtained with a high frequency following Agrobacterium-mediated gene transfer and visual selection by GFP. The successful transformation method described allows reverse genetics approaches for analyzing cold hardiness genes isolated from bromegrass cells.

Transcription Profiling of the mgrA Regulon in Staphylococcus aureus

MgrA has been shown to affect multiple Staphylococcus aureus genes involved in virulence and antibiotic resistance. To comprehensively identify the target genes regulated by mgrA, we employed a microarray method to analyze the transcription profiles of S. aureus Newman, its isogeneic mgrA mutant, and an MgrA-overproducing derivative. We compared genes that were differentially expressed at exponential or early stationary growth phases. Our results showed that MgrA affected an impressive number of genes, 175 of which were positively regulated and 180 of which were negatively regulated in an mgrA-specific manner. The target genes included all functional categories. The microarray results were validated by real-time reverse transcription-PCR quantitation of a set of selected genes from different functional categories. Our data also indicate that mgrA regulates virulence factors in a fashion analogous to that of the accessory gene regulatory locus (agr). Accordingly, exoproteins are upregulated and surface proteins are downregulated by the regulator, suggesting that mgrA may function in concert with agr. The fact that a large number of genes are regulated by mgrA implies that MgrA is a major global regulator in S. aureus.

Anaerobic cells of Bacillus cereus F4430/73 respond to low oxidoreduction potential by metabolic readjustments and activation of enterotoxin expression

In the present study, a food-borne pathogen strain of Bacillus cereus (F4430/73) was anaerobically grown in controlled-batch conditions under low initial oxidoreduction potential (ORP=-148 mV) using hydrogen gas as reducing agent. Its physiological characteristics, including growth, glucose fermentation capacity and enterotoxin production, were compared with anaerobic conditions generated by nitrogen gas (ORP=+ 45 mV). The results showed that low ORP affected growth mainly during the early stages. Maximal specific rates of growth and glucose consumption were reduced, and drastic changes in time profiles of fermentation product concentration were observed. Production of lactate was promoted at the expense of acetate. Nevertheless, low ORP did not affect final biomass yield. Under both ORP conditions, Non-haemolytic enterotoxin (Nhe) was produced early during the exponential growth phase as a first enterotoxin and Haemolysin BL (Hbl) later during the early stationary growth phase as a second enterotoxin. The major effect of low ORP was the strong stimulation of Hbl production and, to a lesser extent, Nhe production. This control was complex, involving different levels of regulation. We discussed the regulation of enterotoxin expression and the involvement of the pleiotropic regulator PlcR.

Evidence for quorum sensing in Clostridium botulinum 56A

Experiments were designed to detect quorum-sensing signals produced by Clostridium botulinum. Clostridium botulinum 56A cell-free supernatants obtained at the end of lag phase, the mid-exponential phase and early stationary phase of growth were assayed for bioluminescence in the Vibrio harveyi quorum-sensing assay system. Twelve and 16-h culture supernatants induced bioluminescence in the auto-inducer 2 (AI-2) but not the auto-inducer 1 (AI-1) assay. Intra-species quorum sensing was also assayed as the ability of the supernatants to promote spore germination and outgrowth in a microtitre plate system. Spore populations exposed to C. botulinum supernatant from the end of lag phase became positive for growth sooner than controls. The influence of cell-free supernatant on ungerminated spores and detection of bioluminescence in the AI-2 assay are evidence for a signalling molecule(s) and provide a first step in characterizing C. botulinum quorum sensing. This study suggests that spores do not behave independently of each other and may explain the inocula size effects observed in challenge studies. Whether AI-2 production in C. botulinum serves as an inter-species signal or as a detoxification mechanism remains to be determined.

Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida

We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.

A comparative study ofBacillus cereus,Bacillus thuringiensis andBacillus anthracis extracellular proteomes

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are closely related species that share a similar genetic background but occupy different ecological niches. Virulence plasmids bearing genes coding for toxins, may explain, at least partly, this specialization. We have compared by 2-DE in the early stationary phase of growth the extracellular proteomes of three strains of these species that have lost their virulence plasmids. Proteins expected to be secreted or to belong to the cell wall or to the cytosol were found in the three proteomes. For the cell wall and cytosolic proteins located in the extracellular space, the three proteomes were similar. Cytosolic proteins included enolase, GroEL, PdhB, PdhD, SodA and others. Cell surface proteins were mainly autolysins, proteases, nucleotidases and OppAs. In contrast, the secreted proteins profiles of B. cereus and B. thuringiensis were quite different from that of B. anthracis. B. cereus and B. thuringiensis extracellular proteomes both contained large amounts of secreted degradative enzymes and toxins, including nine proteases, three phospholipases, two haemolysins and several enterotoxins. Most of the genes encoding these enzymes and toxins are controlled by the transcriptional activator PlcR. The extracellular proteome of the pXO1-, pXO2- B. anthracis 9131 strain contained only one secreted protein: the metalloprotease InhA1, also found in the proteomes of the two other strains and possibly involved in antibacterial peptide degradation.

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Rhodopseudomonas palustris SP5212

Production of poly(3-hydroxybutyric acid) [P(3HB)] by Rhodopseudomonas palustris SP5212 isolated in this laboratory has been optimized under phototrophic microaerophilic conditions. Cells grown in malate medium accumulated 7.7% (w/w) P(3HB) of cellular dry weight at the early stationary phase of growth. The accumulated P(3HB) however, attained 15% (w/w) of cellular dry weight when acetate (1.0%, w/v) was used as the sole carbon source under nitrogen-limiting conditions. Synthesis and accumulation of polymer was favoured by sulphate-free conditions and at a phosphate concentration sub-optimal for growth. The polymer content of cells was increased drastically (34% of cellular dry weight) when the acetate containing medium was supplemented with n-alkanoic acids. Compositional analysis by H1 NMR revealed that these accumulated polymers were composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (3HV). The contents of 3HV in these copolymers ranged from 14 to 38 mol%.

Analysis of the growth phase-associated transcriptome of Streptococcus pyogenes

Streptococcus pyogenes (group A streptococci, GAS) is a human pathogen which probably varies its multiplication rate and thus, growth phases in association with the type of infection caused in its host. To create a basis for future determinations of such associations, the genome-wide growth phase-related GAS transcriptome was assessed in the present study. Therefore, the published serotype M1 S. pyogenes genome sequence as well as the partially sequenced serotype M18 and M49 GAS genomes were used to produce DNA microarrays that carried 2256 oligonucleotide probes matching 3662 open reading frames (ORFs). With these microarrays, the transcriptome of the serotype M49 GAS strain 591 grown to the exponential, transition, and early stationary growth phases was assessed in seven independent experiments. The gained data were compared to real-time RT-PCR assays. Data analysis was refined by a novel approach, i.e. grouping of expressed genes to four classes according to relative transcript abundance and gene functions. At the different growth phases, 86.7%, 79.5% and 55.7% of the at least 1883 ORFs contained in the serotype M49 genome were expressed above the defined detection level. Contrary to the general trend, transcript amounts of genes in the functional groups of transport and membrane proteins as well as stress response factors peaked at the transition phase. The most prominent changes in the transcript abundances were predominantly observed for sugar compound transport and turnover-related ORFs. The majority of known virulence genes had their maximum expression during the transition phase, consistent with the proposed associated change in virulence behavior of the bacteria. With these results, it will now be feasible to assess the in situ growth phase of a given GAS strain during any type of infection by measuring the expression of selected marker genes.

Cell cycle dependent expression of toxicity by the ichthyotoxic prymnesiophyte Chrysochromulina polylepis

The coupling of toxicity expression with cell-cycle phases was studied in the toxic marine prymnesiophyte Chrysochromulina polylepis Manton & Parke, Clone B1511. Cell synchronisation of cul- tures in exponential or early stationary growth phases under nutrient-replete conditions was achieved by manipulation of the photoperiod. Chlorophyll a (chl a) and cell number increased in a stepwise man- ner, but were asynchronous, with chl a increasing during the light period and cell number increasing during the dark period. In the course of the light period, nearly all cells clustered in the G1 (Gap 1) phase, which lasted for about 20 h. DNA synthesis (S phase) occurred mainly in the dark during a discrete period (about 4 h) and G2 (Gap 2) and mitosis (M) were always completed before the end of the dark period. Toxicity expression, measured by the erythrocyte lysis assay (ELA), exhibited a dramatic drop in LC50 values (increase in toxicity) during the light period, although this effect was less pronounced af- ter the first 2 generations of cell division when the cultures had entered the stationary phase. Similarly, haemolytic activity per unit cell volume decreased by a factor of 3 to 4 during the dark period over the first 48 h, but became irregular towards the end of the experiment. In this study, the light-dependent effect on toxicity and relationship to discrete phases of the cell cycle are demonstrated for the first time in a prymnesiophyte.

Growth conditions required for bacteriocin production by strains of Staphylococcus aureus

The influence of growth parameters on the production of bacteriocins (aureocins) by five strains of Staphylococcus aureus was investigated. These aureocins have a broad spectrum of activity and can inhibit important human and animal pathogens. All strains produced large inhibition zones upon the indicator strain when they were grown in rich media such as brain-heart infusion (BHI), N2GT and 2 × YT. Bacteriocin production was not influenced by the initial pH of the medium (6.0–8.0). At lower temperature (28 °C), there was a marked reduction in bacteriocin production. Incubation of the producers under anaerobiosis affected profoundly the production of two related bacteriocins, aureocins MB92 and 146L, and slightly increased the production of aureocins A53 and 215FN. Production of aureocins MB92 and 215FN was apparently abolished in media containing ≥ 2.5 g and ≥ 3.5 g Nacl/100 ml, respectively. Although production of the remaining aureocins was observed in all NaCl concentrations tested (0.5–7.5 g/100 ml), the larger inhibition zones were detected in media containing up to either 1.5 g (for aureocins A70 and 146L) or 2.5 g NaCl/100 ml (for aureocin A53). Aureocin 215FN could not be detected in the culture supernatant. For the remaining aureocins tested, the highest levels of bacteriocin production occurred in either the late-log or early stationary growth phase of cultures grown in BHI medium at 37 °C. Changes in environmental conditions can, therefore, have detrimental effects on the production of active aureocins. Such factors are relevant when considering the potential biotechnological applications of these substances and when testing new S. aureus isolates for bacteriocin production.