Early Exponential Growth Phase Research Articles

Cancer growth: Predictions of a realistic model

Simulations of avascular cancer growth are performed using experimental values of the relevant parameters. This permits a realistic assessment of the influence of these parameters on cancer growth dynamics. In general, an early exponential growth phase is followed by a linear regime (as observed in recent experiments), while the thickness of the viable cell layer remains approximately constant. Contrary to some predictions, a transition to latency is not observed.

Physiologic mechanisms of sequential products synthesis in 1,3‐propanediol fed‐batch fermentation by Klebsiella pneumoniae

The glycerol fed-batch fermentation by Klebsiella pneumoniae CGMCC 1.6366 exhibited the sequential synthesis of products, including acetate, 1,3-propanediol (1,3-PD), 2,3-butanediol, ethanol, succinate, and lactate. The dominant flux distribution was shifted from acetate formation to 1,3-PD formation in early- exponential growth phase and then to lactate synthesis in late-exponential growth phase. The underlying physiological mechanism of the above observations has been investigated via the related enzymes, nucleotide, and intermediary metabolites analysis. The carbon flow shift is dictated by the intrinsic physiological state and enzymatic activity regulation. Especially, the internal redox state could serve as a rate-controlling factor for 1,3-PD production. The q(1,3-PD) formation was the combined outcomes of regulations of glycerol dehydratase activity and internal redox balancing. The q(ethanol)/q(acetate) ratios demonstrated the flexible adaptation mechanism of K. pneumoniae preferring ATP generation in early-exponential growth phase. A low PEP to pyruvate ratio corresponded LDH activity increase, leading to lactate accumulation in stationary phase.

Concerted Action of Lactate Oxidase and Pyruvate Oxidase in Aerobic Growth of Streptococcus pneumoniae : Role of Lactate as an Energy Source

Streptococcus pneumoniae was shown to possess lactate oxidase in addition to well-documented pyruvate oxidase. The activities of both H(2)O(2)-forming oxidases in wild-type cultures were detectable even in the early exponential phase of growth and attained the highest levels in the early stationary phase. For each of these oxidases, a defective mutant was constructed and compared to the parent regarding the dynamics of pyruvate and lactate in aerobic cultures. The results obtained indicated that the energy-yielding metabolism in the wild type could be best described by the following scheme. (i) As long as glucose is available, approximately one-fourth of the pyruvate formed is converted to acetate by the sequential action of pyruvate oxidase and acetate kinase with acquisition of additional ATP; (ii) the rest of the pyruvate is reduced by lactate dehydrogenase to form lactate, with partial achievement of redox balance; (iii) the lactate is oxidized by lactate oxidase back to pyruvate, which is converted to acetate as described above; and (iv) the sequential reactions mentioned above continue to occur as long as lactate is present. As predicted by this model, exogenously added lactate was shown to increase the final growth yield in the presence of both oxidases.

Post-translational regulation of the RpoS and PsrA genes in pseudomonas putida WCS358: The role of ClpXP protease

The RpoS and PsrA proteins are key transcriptional regulators that are activated in response to the stationary phase of growth in pseudomonads. This study was designed to establish whether ClpXP (ATP-dependent serine protease) regulates levels of RpoS and PsrA in Pseudomonas putida WCS358. Western blot analysis of P. putida WCS358 protein extracts from the early exponentianl, late exponential, and stationary phases of growth with antibodies against RpoS and PsrA revealed that these proteins are degraded by ClpXP in the early exponential phase of growth. The obtained results demonstrate a role for ClpXP protease in post-translational regulation of proteins encoded by the rpoS and psrA genes in Pseudomonas spp.

Massetolide A Biosynthesis inPseudomonas fluorescens

Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.

The glgX gene product of Corynebacterium glutamicum is required for glycogen degradation and for fast adaptation to hyperosmotic stress

Corynebacterium glutamicum cells growing in medium containing sugars accumulate glycogen in the early exponential-growth phase, and start to degrade this polymer at entry into the stationary phase. In a first attempt to investigate glycogen degradation, the C. glutamicum glgX gene, which encodes a protein with 46 % identity to the isoamylase-type debranching enzyme of Escherichia coli, was analysed, expressed and inactivated. The purified C. glutamicum gene product showed debranching activity towards glycogen, amylopectin and starch. Chromosomal inactivation of glgX in C. glutamicum wild-type led to slower growth and to a higher intracellular glycogen pool throughout growth, when compared to those in the parental strain. This result suggests that glycogen synthesis and degradation occur simultaneously in C. glutamicum. When exposed to hyperosmotic shock, C. glutamicum rapidly degrades glycogen, and at the same time, synthesizes the osmoprotectant trehalose. The glgX mutant, however, synthesized only minor amounts of trehalose throughout cultivation, and its growth ceased after hyperosmotic shock. Taken together, the results indicate that the glgX gene product is essential for glycogen degradation in C. glutamicum, that glycogen is constantly recycled in C. glutamicum, and that it serves as a carbon store for trehalose synthesis via the TreYZ pathway after hyperosmotic shock.

Glycogen formation in Corynebacterium glutamicum and role of ADP-glucose pyrophosphorylase

Glycogen is generally assumed to serve as a major reserve polysaccharide in bacteria. In this work, glycogen accumulation in the amino acid producer Corynebacterium glutamicum was characterized, expression of the C. glutamicum glgC gene, encoding the key enzyme in glycogen synthesis, ADP-glucose (ADP-Glc) pyrophosphorylase, was analysed, and the relevance of this enzyme for growth, survival, amino acid production and osmoprotection was investigated. C. glutamicum cells grown in medium containing the glycolytic substrates glucose, sucrose or fructose showed rapid glycogen accumulation (up to 90 mg per g dry weight) in the early exponential growth phase and degradation of the polymer when the sugar became limiting. In contrast, no glycogen was detected in cells grown on the gluconeogenic substrates acetate or lactate. In accordance with these results, the specific activity of ADP-Glc pyrophosphorylase was 20-fold higher in glucose-grown than in acetate- or lactate-grown cells. Expression analysis suggested that this carbon-source-dependent regulation might be only partly due to transcriptional control of the glgC gene. Inactivation of the chromosomal glgC gene led to the absence of ADP-Glc pyrophosphorylase activity, to a complete loss of intracellular glycogen in all media tested and to a distinct lag phase when the cells were inoculated in minimal medium containing 750 mM sodium chloride. However, the growth of C. glutamicum, its survival in the stationary phase and its glutamate and lysine production were not affected by glgC inactivation under either condition tested. These results indicate that intracellular glycogen formation is not essential for growth and survival of and amino acid production by C. glutamicum and that ADP-Glc pyrophosphorylase activity might be advantageous for fast adaptation of C. glutamicum to hyperosmotic stress.

Hawaii as a Model System for Human Ecodynamics

The human ecodynamics approach in archaeology privileges landscape as a core concept, asserting that there can be no environment or ecosystem detached from humans and their behavior. Drawing on recent research of a multidisciplinary biocomplexity project, I explore in this article the Hawaiian archipelago as a model system for studying human ecodynamics. Natural patterns of biogeochemical and climate gradients constrained the development of intensive agroecosystems over 1,000 years. An early phase of exponential population growth was linked with agricultural intensification of terraced irrigation systems, primarily on the older islands. After C.E. 1400, expansion of population onto the leeward slopes of the young islands of Maui and Hawai'i was accompanied by intensification of dryland agricultural field systems. These changes were in turn linked to significant transformations of social and political formations, including restructuring of the system of land tenure and descent group organization, and the imposition of a system of surplus extraction organized around a ritual hierarchy of temples.

Growth and reproductive performance in cultured nearshore rockfish ( Sebastes spp.)

The present study evaluates the extent to which the viviparous reproductive strategy that characterizes the genus Sebastes (rockfish) can be expressed in culture. In addition, the growth patterns of rockfish were examined during the early phases of growth pertinent to the culture of the species. Using grass rockfish ( Sebastes rastrelliger) as our model, it was demonstrated that all phases of ovarian seasonal cyclicity could proceed under controlled environmental conditions. Rockfish successfully spawned in our facility and subsequently passed through successive phases of oocyte development during the normal reproductive season before follicular atresia was observed in the absence of fertilization. Fish introduced into culture in an advanced stage of vitellogenesis became pregnant in the absence of males 14–55 days later. This observation is consistent with the view that delayed fertilization occurs in rockfish and establishes that wild caught specimens for spawning purposes are not confined to pregnant females. By monitoring growth parameters in two groups of brown rockfish ( S. auriculatus) introduced into culture during their first and second year of development a comprehensive picture of early growth was established. Overall changes in both weight and length followed a sigmoid curve comprising an early phase of exponential growth transitioning to a phase of exponential decay. The constituent phases of this curve, as expressed in the two age groups, and the combined growth data over a period of 1083 culture-days were precisely described by the Gompertz equation. This equation also described the growth of young-of-the-year copper rockfish ( S. caurinus) with similar accuracy. Validation of the applicability of our Gompertz equation was achieved by reference to growth parameters of brown rockfish of known age raised from birth at UCSB. Our mathematical model precisely depicted growth with age from birth to the period of sexual maturity. These data on growth in relation to age may find broad application in fisheries management plans allowing for evaluation of population changes over time and be useful in determining and improving the culture potential of nearshore rockfish species.

Production profile of the soluble lytic transglycosylase homologue inStaphylococcus aureusduring bacterial proliferation

The immunodominant antigen A, IsaA, of Staphylococcus aureus is considered to be a sequence homologue of gram-negative soluble lytic transglycosylase. Recently, it was reported that IsaA production is probably regulated by the essential two-component regulatory system consisting of YycG and YycF. Therefore, we investigated the isaA expression profile by quantifying its gene products at different stages of bacterial growth. In the culture supernatant, IsaA was detectable in the early exponential growth phase and its concentration constantly increased until the early stationary phase. Cell surface IsaA was mainly found on growing bacteria, and disappeared proportionately with a decline in cell proliferation. On the other hand, the isaA transcript rapidly increased at the beginning of the culture, and then stayed at a constant level until the late exponential growth phase. These findings indicated that isaA gene expression was stimulated during the exponential growth phase and repressed in the stationary phase. Thus, IsaA production was associated with active bacterial growth. Our results support the previous report that isaA expression is regulated by YycF, and also suggest that IsaA is probably involved in bacterial proliferation by residing on the cell wall. We also discuss the possibility that isaA transcription may be controlled by SarA.

Leaf Growth and Photosynthetic Performance of Two Co-existing Oak Species in Contrasting Growing Seasons

Ecophysiological investigations of Quercus petraea and Quercus cerris were performed at the Sikfőkút research site in the dry and humid growing seasons of 2003 and 2004. The results suggested that leaf growth and the photosynthetic apparatus of Q. petraea exhibited higher sensitivity to drought in 2003 than that of Q. cerris. In leaves of Q. petraea, chlorophyll content showed larger inter-annual and within-canopy variability than in those of Q. cerris. Fully developed leaves of Q petraea showed lower SLM which indicated higher leaf cell wall elasticity allowing them to maintain a water spending strategy, while high specific leaf mass (SLM) values reflected a water saving strategy for Q. cerris. Water use efficiency of Q. cerris was higher than in the case of Q. petraea, which may provide an advantage for this species in dry periods. In the contrasting years the final leaf area and leaf mass of both species were determined by the amount of rainfall and temperature conditions during the period of early exponential phase of leaf growth. As indicated by the low values of the Fv/Fm chlorophyll fluorescence parameter the photosynthetic apparatus of both species exhibited high susceptibility to abiotic stress factors in early spring. A large VAZ cycle pool indicated that zeaxanthin dependent heat dissipation was the main contributor to photoprotection of photosynthetic apparatus in young leaves but in fully developed leaves the relatively high light saturated ETR and low Pmax as well as the maintenance of high Fv/Fm even in severe dry periods reflected the potential involvement of photorespiratory electron transport in photoprotection of both species in summer. Drought in 2003 may have resulted in serious depletion of dry matter reserves influencing the vitality of trees in following year. Q. petraea showed lower photochemical activity in the successive vegetation period after the dry year than Q. cerris which suggested lower tolerance to drought in the long term.

Cephalopod hatchling growth: the effects of initial size and seasonal temperatures

Temperature is known to have a strong influence on cephalopod growth during the early exponential growth phase. Most captive growth studies have used constant temperature regimes and assumed that populations are composed of identically sized individuals at hatching, overlooking the effects of seasonal temperature variation and individual hatchling size heterogeneity. This study investigated the relative roles of initial hatchling size and simulated natural seasonal temperature regimes on the growth of 64 captive Octopus pallidus over a 4-month period. Initial weights were recorded, and daily food consumption and fortnightly growth monitored. Two temperature treatments were applied replicating local seasonal water temperatures: spring/summer (14–18°C) and summer/autumn (18–14°C). Overall octopuses in the spring/summer treatment grew at a rate of 1.42% bwd−1 (% body weight per day) compared to 1.72% bwd−1 in the summer/autumn treatment. Initial size influenced growth rate in the summer/autumn treatment with smaller octopuses (<0.25 g) growing faster at 1.82% bwd−1 compared to larger octopuses at 1.68% bwd−1. This was opposite to individuals in the spring/summer treatment where smaller octopuses grew slower at 1.29% bwd−1 compared to larger octopuses at 1.60% bwd−1. Initial size influenced subsequent growth, however, this was dependent on feeding rate and appears to be secondary to the effects of temperature.

Identification of IspC, an 86-Kilodalton Protein Target of Humoral Immune Response to Infection with Listeria monocytogenes Serotype 4b, as a Novel Surface Autolysin

We identified and biochemically characterized a novel surface-localized autolysin from Listeria monocytogenes serotype 4b, an 86-kDa protein consisting of 774 amino acids and known from our previous studies as the target (designated IspC) of the humoral immune response to listerial infection. Recombinant IspC, expressed in Escherichia coli, was purified and used to raise specific rabbit polyclonal antibodies for protein characterization. The native IspC was detected in all growth phases at a relatively stable low level during a 22-h in vitro culture, although its gene was transiently transcribed only in the early exponential growth phase. This and our previous findings suggest that IspC is upregulated in vivo during infection. The protein was unevenly distributed in clusters on the cell surface, as shown by immunofluorescence and immunogold electron microscopy. The recombinant IspC was capable of hydrolyzing not only the cell walls of the gram-positive bacterium Micrococcus lysodeikticus and the gram-negative bacterium E. coli but also that of the IspC-producing strain of L. monocytogenes serotype 4b, indicating that it was an autolysin. The IspC autolysin exhibited peptidoglycan hydrolase activity over a broad pH range of between 3 and 9, with a pH optimum of 7.5 to 9. Analysis of various truncated forms of IspC for cell wall-hydrolyzing or -binding activity has defined two separate functional domains: the N-terminal catalytic domain (amino acids [aa] 1 to 197) responsible for the hydrolytic activity and the C-terminal domain (aa 198 to 774) made up of seven GW modules responsible for anchoring the protein to the cell wall. In contrast to the full-length IspC, the N-terminal catalytic domain showed hydrolytic activity at acidic pHs, with a pH optimum of between 4 and 6 and negligible activity at alkaline pHs. This suggests that the cell wall binding domain may be of importance in modulating the activity of the N-terminal hydrolase domain. Elucidation of the biochemical properties of IspC may have provided new insights into its biological function(s) and its role in pathogenesis.

Determination of soluble and granular inorganic polyphosphate in Corynebacterium glutamicum

Corynebacterium glutamicum forms inorganic polyphosphate (poly P) that may occur as soluble (cytosolic) poly P and/or as volutin granules. A suitable method for monitoring soluble and granular poly P in C. glutamicum was developed and applied to C. glutamicum cells cultivated under different growth conditions. Under phosphate-limiting conditions, C. glutamicum did not accumulate poly P, but it rebuilt its poly P storages when phosphate became available. The poly P content of C. glutamicum growing on glucose minimal medium with sufficient phosphate varied considerably during growth. While the poly P content was minimal in the midexponential growth phase, two maxima were observed in the early exponential growth phase and at entry into the stationary growth phase. Cells in the early exponential growth phase primarily contained granular poly P, while cells entering the stationary growth phase contained soluble, cytosolic poly P. These results and those obtained for C. glutamicum cells cultivated under hypo- or hyperosmotic conditions or during glutamate production revealed that the poly P content of C. glutamicum and the partitioning between cytosolic and granular forms of poly P are dynamics and depend on the growth conditions.

Global gene expression during stringent response in Corynebacterium glutamicum in presence and absence of the rel gene encoding (p)ppGpp synthase

BackgroundThe stringent response is the initial reaction of microorganisms to nutritional stress. During stringent response the small nucleotides (p)ppGpp act as global regulators and reprogram bacterial transcription. In this work, the genetic network controlled by the stringent response was characterized in the amino acid-producing Corynebacterium glutamicum.ResultsThe transcriptome of a C. glutamicum rel gene deletion mutant, unable to synthesize (p)ppGpp and to induce the stringent response, was compared with that of its rel-proficient parent strain by microarray analysis. A total of 357 genes were found to be transcribed differentially in the rel-deficient mutant strain. In a second experiment, the stringent response was induced by addition of DL-serine hydroxamate (SHX) in early exponential growth phase. The time point of the maximal effect on transcription was determined by real-time RT-PCR using the histidine and serine biosynthetic genes. Transcription of all of these genes reached a maximum at 10 minutes after SHX addition. Microarray experiments were performed comparing the transcriptomes of SHX-induced cultures of the rel-proficient strain and the rel mutant. The differentially expressed genes were grouped into three classes. Class A comprises genes which are differentially regulated only in the presence of an intact rel gene. This class includes the non-essential sigma factor gene sigB which was upregulated and a large number of genes involved in nitrogen metabolism which were downregulated. Class B comprises genes which were differentially regulated in response to SHX in both strains, independent of the rel gene. A large number of genes encoding ribosomal proteins fall into this class, all being downregulated. Class C comprises genes which were differentially regulated in response to SHX only in the rel mutant. This class includes genes encoding putative stress proteins and global transcriptional regulators that might be responsible for the complex transcriptional patterns detected in the rel mutant when compared directly with its rel-proficient parent strain.ConclusionIn C. glutamicum the stringent response enfolds a fast answer to an induced amino acid starvation on the transcriptome level. It also showed some significant differences to the transcriptional reactions occuring in Escherichia coli and Bacillus subtilis. Notable are the rel-dependent regulation of the nitrogen metabolism genes and the rel-independent regulation of the genes encoding ribosomal proteins.

LuxS-mediated signalling in Streptococcus anginosus and its role in biofilm formation

The autoinducer-2 signal (AI-2) produced by several Gram-positive and Gram-negative bacteria mediates interspecies communication. In this study we were able to identify an orthologue of luxS, required for the synthesis of AI-2 signals, in Streptococcus anginosus. Comparative analyses revealed conserved sequences in the predicted S. anginosus LuxS. Expression of luxS was highest during early exponential growth phase. Compared to other oral streptococci, conditioned media from growth of members of the anginosus group were the most efficient in inducing bioluminescence in Vibrio harveyi, indicative of AI-2 signalling. Disruption of luxS in S. anginosus resulted in a mutant deficient in biofilm formation, whereas no effect on planktonic growth rate was observed under various growth conditions. S. anginosus is part of the human flora found in biofilms of the oral cavity, as well as of the upper respiratory, gastrointestinal and urogenital tracts. Such habitats harbour large varieties of bacterial species, among which cell-cell communication may play an important role. S. anginosus has also been associated with purulent infections and cancer in the upper digestive tract. Knowledge about the molecular mechanisms involved in S. anginosus communication is important for understanding its commensalism and its pathogenic transition.

Differential expression profiles of Streptococcus mutans ftf, gtf and vicR genes in the presence of dietary carbohydrates at early and late exponential growth phases

Dental caries is one of the most common infectious diseases that affects humans. Streptococcus mutans, the main pathogenic bacterium associated with dental caries, produces a number of extracellular sucrose-metabolizing enzymes, such as glucosyltransferases (GTFB, GTFC and GTFD) and fructosyltransferase (FTF). The cooperative action of these enzymes is essential for sucrose-dependent cellular adhesion and biofilm formation. A global response regulator (vicR) plays important roles in S. mutans ftf and gtf expression in response to a variety of stimuli. A real-time reverse-transcription polymerase chain-reaction was used to quantify the relative levels of ftf, gtfB, gtfC, gtfD and vicR transcription of S. mutans in the presence of various dietary carbohydrates: sucrose, d-glucose, d-fructose, d-glucitol (d-sorbitol), d-mannitol and xylitol. Ftf was highly expressed at late exponential phase in the presence of sorbitol and mannitol. GtfB was highly expressed in the presence of all the above carbohydrates except for xylitol at early exponential growth phase and glucose and fructose at late exponential growth phase. Similar to gtfB, the expression of gtfC was also induced with the presence of all the tested carbohydrates except for xylitol at early growth and glucose and fructose at late exponential phase. In addition, no effect of mannitol on gtfC expression at early exponential phase was observed. GtfD was less influenced compared to the gtfB and gtfC, demonstrating enhanced expression especially in the presence of sorbitol, glucose, mannitol and xylitol at early exponential phase and mannitol at late exponential phase. VicR expression was induced only at the presence of xylitol at late exponential phase, and a decrease in expression was recorded at early exponential phase. Our findings show that dietary carbohydrates have a major influence on the transcription of ftf, gtfB, gtfC and gtfD, but less on vicR. Sorbitol and mannitol, which are considered as noncariogenic sugar substitutes, may indirectly affect caries by promoting biofilm formation via enhanced expression of gtfs and ftf. These results suggest regulatory circuits for exopolysaccharide gene expression in S. mutans.

Characterization of a flocculation-like phenotype in Cryptococcus neoformans and its effects on pathogenesis

We investigated the phenomenon of cell-cell aggregation (flocculation) in a serotype D strain of Cryptococcus neoformans (ATCC 24067, isolate RC-2). Cell aggregation into clumps of 5-40 cells (clump+ cells) occurred during the early log phase and disappeared in the beginning of the stationary phase (clump- cells). The cell aggregation phenomenon was medium dependent. Clump+ cells could be dispersed by either vortexing or proteinase K digestion. Most importantly, the transient change in cellular phenotype changed several important host-pathogen interactions. Adherence of clump+ cells to murine macrophage-like cells J774.16 was significantly (P < 0.001) enhanced compared with adherence of clump- cells. Furthermore, complement-mediated phagocytosis efficacy of dispersed clump+ cells was significantly higher (P < 0.001) compared with clump- cells. Similar findings were documented with an in vivo phagocytosis assay. Infection of mice with a low inoculum (10(4)) of clump+ cells resulted in lower fungal burden when compared with mice infected with clump- cells. Accordingly, mice infected with clump+ cells survived significantly longer than mice infected with clump- cells. These results indicate that the cellular phenotype undergoes significant changes that result in a transient flocculation-like phenotype. We hypothesize that this cell-cell aggregation is the result of changes in protein content in the polysaccharide capsule. We conclude from our data that the change in cellular phenotype has a dramatic effect on cell adherence, and on complement-mediated phagocytosis, both of which can affect the pathogenesis of the disease in the host. Our results underscore the complexity of studies that investigate host pathogen interactions and may explain differences and inconsistencies observed in in vitro and in vivo assays.

The WD-40 repeat protein PkwA ofThermomonospora curvatais associated with rapid growth and is localized in the tips of growing hyphae

The PkwA protein of the thermophilic actinomycete Thermomonospora curvata has already been reported as the first instance of a WD-40 module-containing protein of prokaryotic origin. This protein is composed of an N-terminal eukaryotic-type protein kinase domain and of seven C-terminal WD-40 repeats. PkwA is a peripheral membrane protein that is linked to the early exponential growth phase of the bacterium. Its intracellular concentrations are extremely low. We have shown that the protein forms high molecular weight complexes and is localized mainly in the tips of the young Thermomonospora vegetative hyphae.

Paenibacillus curdlanolyticusStrain B-6 Xylanolytic-Cellulolytic Enzyme System That Degrades Insoluble Polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, beta-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, beta-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.