Insoluble Chitin Research Articles

Purification and Chitinolytic Characteristics of Chitinases A and B from Serratiamarcescens

In Serratiamarcescensthe enzymatic chitinolytic machinery of is one of the best that can convert insoluble polysaccharides.This process includes four chitin-active enzymes: ChiA, ChiBand ChiC, an endo-acting non-processivechitinase, , two processivechitinases moving along chitin chains in opposite directions. Serratiamarcescensas a Gram-negative soil bacterium produces these three chitinases, ChiA, ChiB and ChiC which together enable Serratiamarcescens to perfectly degrade the insoluble chitin polymer. The data collected for the pseudotrisaccharideallosamidinshows a comparative inhibition including the cyclic pentapeptideargadin. ChiA, ChiBand ChiC play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of ChiA, ChiBand ChiC.

A new chitinase-D from a plant growth promoting Serratia marcescens GPS5 for enzymatic conversion of chitin

The current study describes heterologous expression and biochemical characterization of single-modular chitinase-D from Serratia marcescens (SmChiD) with unprecedented catalytic properties which include chitobiase and transglycosylation (TG) activities besides hydrolytic activity. Without accessory domains, SmChiD, hydrolyzed insoluble polymeric chitin substrates like colloidal, α- and β-chitin. Activity studies on CHOS with degree of polymerization (DP) 2–6 as substrate revealed that SmChiD hydrolyzed DP2 with a chitobiase activity and showed TG activity on CHOS with DP3–6, producing longer chain CHOS. But, the TG products were further hydrolyzed to shorter chain CHOS with DP1–2 products. SmChiD with its unique catalytic properties, could be a potential enzyme for the production of long chain CHOS and also for the preparation of efficient enzyme cocktails for chitin degradation.

RpoS is required for natural transformation of Vibrio cholerae through regulation of chitinases.

Vibrio species naturally reside in the aquatic environment and a major metabolite in this habitat is the chitinous exoskeletons of crustacean zooplankton. In addition to serving as a nutrient, chitin-derived oligosaccharides also induce natural genetic competence in many Vibrio spp., a physiological state in which bacteria take up DNA from the extracellular environment and can integrate it into their chromosome by homologous recombination. Another inducing cue required for competence are quorum-sensing autoinducers. The alternative sigma factor RpoS is critical for natural transformation in Vibrio cholerae, and it was previously presumed to exert this effect through regulation of quorum sensing. Here, we show that RpoS does not affect quorum sensing-dependent regulation of competence. Instead, we show that an rpoS mutant has reduced chitinase activity, which is required to liberate the soluble chitin oligosaccharides that serve as an inducing cue for competence. Consistent with this, we demonstrate that RpoS is required for growth of V. cholerae on insoluble chitin. RpoS also regulates the mucosal escape response in pathogenic strains of V. cholerae. Thus, in addition to promoting egress from its human host, RpoS may also prime this pathogen for successful reentry into the aquatic environment.

Phenotypic and Genomic Properties of Chitinispirillum alkaliphilum gen. nov., sp. nov., A Haloalkaliphilic Anaerobic Chitinolytic Bacterium Representing a Novel Class in the Phylum Fibrobacteres.

Anaerobic enrichment from sediments of hypersaline alkaline lakes in Wadi el Natrun (Egypt) with chitin resulted in the isolation of a fermentative haloalkaliphilic bacterium, strain ACht6-1, growing exclusively with insoluble chitin as the substrate in a sodium carbonate-based medium at pH 8.5–10.5 and total Na+ concentrations from 0.4 to 1.75 M. The isolate had a Gram-negative cell wall and formed lipid cysts in old cultures. The chitinolytic activity was associated with cells. Analysis of the 4.4 Mb draft genome identified pathways for chitin utilization, particularly, secreted chitinases linked to the cell surface, as well as genes for the hydrolysis of other polysaccharides and fermentation of sugars, while the genes needed for aerobic and anaerobic respiration were absent. Adaptation to a haloalkaliphilic lifestyle was reflected by the gene repertoire encoding sodium rather than proton-dependent membrane-bound ion pumps, including the Rnf-type complex, oxaloacetate decarboxylase, V-type ATPase, and pyrophosphatase. The phylogenetic analysis using 16S rRNA gene and ribosomal proteins indicated that ACht6-1 forms a novel deep lineage at the class level within the bacterial candidate division TG3. Based on phylogenetic, phenotypic and genomic analyses, the novel chitinolytic bacterium is described as Chitinispirillum alkaliphilum gen. nov., sp. nov., within a novel class Chitinispirillia that could be included into the phylum Fibrobacteres.

A comparative study of transgenic canola (Brassica napus L.) harboring either chimeric or native Chit42 genes against phytopathogenic fungi

Canola (Brassica napus L.), an agro-economically important crop in the world, is sensitive to many fungal pathogens. One strategy to combat fungal diseases is genetic engineering through transferring genes encoding the pathogenesis-related (PR) proteins such as chitinase which cause the chitin degradation of fungal cell wall. Chitinase Chit42 from Trichoderma atroviride (PTCC5220) plays an important role in biocontrol and has high antifungal activity against a wide range of phytopathogenic fungi. This enzyme lacks a chitin binding domain (ChBD) which is involved in binding activity to insoluble chitin. In the present study, we investigated the effect of chitin binding domain fused to Chit42 when compared with native Chit42. These genes were over-expressed under the CaMV35S promoter in B. napus, R line Hyola 308. Transformation of cotyledonary petioles was achieved by pBISM2 and pBIKE1 constructs containing chimeric and native Chit42 genes respectively, via Agrobacterium method. The insertion of transgenes in T0 generation was verified through polymerase chain reaction (PCR) and Southern blot analysis. Antifungal activity of expressed chitinase in transgenic plants was also investigated by bioassays. The transgenic canola expressing chimeric chitinase showed stronger inhibition against phytopathogenic fungi that indicates the role of chitin binding domain.

Effects of C-Terminal Domain Truncation on Enzyme Properties of Serratia marcescens Chitinase C

A chitinase gene (SmChiC) and its two C-terminal truncated mutants, SmChiCG426 and SmChiCG330 of Serratia marcescens, were constructed and cloned by employing specific polymerase chain reaction (PCR) techniques. SmChiCG426 is derived from SmChiC molecule without its C-terminal chitin-binding domain (ChBD) while SmChiCG330 is truncated from SmChiC by its C-terminal deletion of both ChBD and fibronectin type III domain (FnIII). To study the role of the C-terminal domains of SmChiC on the enzyme properties, SmChiC, SmChiCG426, and SmChiCG330 were expressed in Escherichia coli by using the pET-20b(+) expression system. The His-tag affinity-purified SmChiC, SmChiCG426, and SmChiCG330 enzymes had a calculated molecular mass of 51, 46, and 36 kDa, respectively. Certain biochemical characterizations indicated that the enzymes had similar physicochemical properties, such as the optimum pH (5), temperature (37 °C), thermostability (50 °C), and identical hydrolyzing product (chitobiose) from both the soluble and insoluble chitin substrates. The overall catalytic efficiency k cat /K M was higher for both truncated enzymes toward the insoluble α-chitin, whereas the binding abilities toward the insoluble α-chitin substrate were reduced moderately. The fluorescence and circular dichroism (CD) spectroscopy data suggested that both mutants retained a similar folding conformation as that of the full-length SmChiC enzyme. However, a CD-monitored melting study showed that the SmChiCG330 had no obvious transition temperature, unlike the SmChiC and SmChiCG426.

Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains.

Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.

Hydrodynamic characterisation of chitosan and its interaction with two polyanions: DNA and xanthan.

Chitosan, a soluble polycationic derivative of insoluble chitin, has been widely considered for use in the food, cosmetic and pharmaceutical industries. Commercial ("C") and in-house laboratory ("L") prepared chitosan samples extracted from crustaceous shells with different molecular weight and degrees of acetylation (25% and 15%) were compared with regards to (i) weight-average molecular weight (Mw); (ii) sedimentation coefficient (s(o)(20,w)) distribution, and (iii) intrinsic viscosity ([η]). These parameters were estimated using a combination of analytical ultracentrifugation (AUC), size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALS) and differential pressure viscometry. Polydisperse distributions were seen from sedimentation coefficient distributions and elution profiles from SEC-MALS. Mw values obtained for each sample by sedimentation equilibrium measurements were in excellent agreement with those obtained from SEC-MALS. Mark-Houwink-Kuhn-Sakurada (MHKS) and Wales van Holde analyses of the data all suggest a semi-flexible conformation. The principle of co-sedimentation was then used to monitor the interactions of the two different molecular weights of L chitosans with two polyanions, DNA and xanthan (another double helical high molecular weight molecule). Interactions were clearly observed and then quantified from the changes in the sedimentation coefficient distribution of the mixture compared to unmixed controls using sedimentation velocity. The interactions appeared to show a strong dependence on molecular weight. The relevance of this for DNA condensation applications is indicated.

Identification and functional analysis of LysM effectors from Marssonina brunnea

Popular is not only an important tree species economically, but is also a model system for plant biology. Marssonina leaf spot is a serious leaf disease caused by Marssonina brunnea, which can result in significant economic losses. M. brunnea is considered to be a hemibiotrophic pathogen but its pathogenesis remains unclear. Full length cDNA of 24 LysM effector genes were obtained based on the sequencing of the M. brunnea genome. All of these genes belonged to a single gene family for which the characteristics of the amino acid sequences and protein domains were analyzed. Further detailed analysis was carried out on two representative gene family members. These two genes were up-regulated during the infection process and, when transformed into Arabidopsis thaliana, reduced the plant’s sensitivity to chitin thus might compromised its immune system. LysM effectors appear to show a specific affinity for chitin since the expressed proteins co-precipitated with insoluble chitin. We performed a series of studies into the molecular mechanism of LysM effectors from M. brunnea and carried out a preliminary analysis into the interaction between the pathogen and its host. This research lays a foundation for further investigation into forest tree resistance breeding.

2-Acylamido Analogues of N-Acetylglucosamine Prime Formation of Chitin Oligosaccharides by Yeast Chitin Synthase 2

Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N'-diacetylchitobiose (GlcNAc2) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc2 and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc2 as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.

A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection

BackgroundMost of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales’ procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development.ResultsWe report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 μM. In contrast to the other commonly used methods, the Schales’ procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin.ConclusionThe ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional Schales’ procedure and DNS method. The detection limit is lower and there is no requirement for harsh conditions for the development of the signal. The assay also involves fewer and easier handling steps. There is no need for boiling to develop the color and results are available within 15 minutes. These aforementioned features render this newly developed assay method highly suitable for applications in biorefinery-related research.

Flavobacterium johnsoniae chitinase ChiA is required for chitin utilization and is secreted by the type IX secretion system.

Flavobacterium johnsoniae, a member of phylum Bacteriodetes, is a gliding bacterium that digests insoluble chitin and many other polysaccharides. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding motility and for chitin utilization. Five potential chitinases were identified by genome analysis. Fjoh_4555 (ChiA), a 168.9-kDa protein with two glycoside hydrolase family 18 (GH18) domains, was targeted for analysis. Disruption of chiA by insertional mutagenesis resulted in cells that failed to digest chitin, and complementation with wild-type chiA on a plasmid restored chitin utilization. Antiserum raised against recombinant ChiA was used to detect the protein and to characterize its secretion by F. johnsoniae. ChiA was secreted in soluble form by wild-type cells but remained cell associated in strains carrying mutations in any of the T9SS genes, gldK, gldL, gldM, gldNO, sprA, sprE, and sprT. Western blot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses suggested that ChiA was proteolytically processed into two GH18 domain-containing proteins. Proteins secreted by T9SSs typically have conserved carboxy-terminal domains (CTDs) belonging to the TIGRFAM families TIGR04131 and TIGR04183. ChiA does not exhibit strong similarity to these sequences and instead has a novel CTD. Deletion of this CTD resulted in accumulation of ChiA inside cells. Fusion of the ChiA CTD to recombinant mCherry resulted in secretion of mCherry into the medium. The results indicate that ChiA is a soluble extracellular chitinase required for chitin utilization and that it relies on a novel CTD for secretion by the F. johnsoniae T9SS.

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18-10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.

Genome analysis of Chitinivibrio alkaliphilus gen. nov., sp. nov., a novel extremely haloalkaliphilic anaerobic chitinolytic bacterium from the candidate phylum Termite Group 3

Anaerobic enrichments from hypersaline soda lakes with chitin as substrate yielded five closely related anaerobic haloalkaliphilic isolates growing on insoluble chitin by fermentation at pH 10 and salinities up to 3.5 M. The chitinolytic activity was exclusively cell associated. To better understand the biology and evolutionary history of this novel bacterial lineage, the genome of the type strain ACht1 was sequenced. Analysis of the 2.6 Mb draft genome revealed enzymes of chitin-degradation pathways, including secreted cell-bound chitinases. The reconstructed central metabolism revealed pathways enabling the fermentation of polysaccharides, while it lacks the genes needed for aerobic or anaerobic respiration. The Rnf-type complex, oxaloacetate decarboxylase and sodium-transporting V-type adenosine triphosphatase were identified among putative membrane-bound ion pumps. According to 16S ribosomal RNA analysis, the isolates belong to the candidate phylum Termite Group 3, representing its first culturable members. Phylogenetic analysis using ribosomal proteins and taxonomic distribution analysis of the whole proteome supported a class-level classification of ACht1 most probably affiliated to the phylum Fibribacteres. Based on phylogenetic, phenotypic and genomic analyses, the novel bacteria are proposed to be classified as Chitinivibrio alkaliphilus gen. nov., sp. nov., within a novel class Chitinivibrione.

Physicochemical study of a novel chimeric chitinase with enhanced binding ability

Chitinases are slow-reacting but important enzymes as they are anticipated to have diverse applications. The role of a chitin-binding domain (ChBD) in enhancing the quality of binding is essential information for purposeful engineering of chitinases. The idea of making hybrid chitinases by fusing a known ChBD to a chitinase, which naturally lacks ChBD is of interest especially for bio-controlling purposes. Therefore, in the present study, the ChBD of Serratia marcescens chitinase B was selected and fused to the fungal chitinase, Trichoderma atroviride Chit42. Both Chit42 and chemric Chit42 (ChC) showed similar activity towards colloidal chitin with specificity constants of 0.83 and 1.07 min(-1), respectively, same optimum temperatures (40°C), and similar optimum pH (4 and 4.5, respectively). In the presence of insoluble chitin, ChC showed higher activity (70%) and obtained a remarkably higher binding constant (700 times). Spectroscopic studies indicated that chimerization of Chit42 caused some structural changes, which resulted in a reduction of α-helix in ChC structure. Chemical and thermal stability studies suggested that ChC had a more stable structure than Chit42. Hill analysis of the binding data revealed mixed-cooperativity with positive cooperativity governing at ChC concentrations below 0.5 and above 2 µM in the presence of insoluble chitin. It is suggested that the addition of the ChBD to Chit42 affords structural changes which enhance the binding ability of ChC to insoluble chitin, improving its catalytic efficiency and increasing its thermal and chemical stability.

Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

Involvement of Gln679, in addition to Trp687, in chitin-binding activity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12

Chitinase A1 (ChiA1) from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III domains, and a C-terminal chitin-binding domain (ChBD). The ChBD of ChiA1 (ChBDChiA1) belongs to carbohydrate-binding module (CBM) family 12 and specifically binds to insoluble or crystalline chitin. It has been suggested that tryptophan-687 (Trp687) is involved in the chitin-binding activity of this ChBD. Site-directed mutagenesis was used to identify additional amino acid residues required for chitin-binding activity of this domain. Furthermore, a total of 14 amino acid residues in ChBDChiA1 were carefully selected, and it was found that mutation of Gln679, which is not well-conserved in CBM family 12, significantly decreased the binding activity to colloidal chitin. A nuclear magnetic resonance study demonstrated that neither the Q679A nor the W687A mutation altered the overall structure of ChBDChiA1. Therefore, Gln679 was identified as a new residue that is involved in the chitin-binding activity of ChBDChiA1 in addition to Trp687. However, the mechanism of chitin binding by ChBD is still unknown.

Increased virulence of transgenic Trichoderma koningi strains to the Asian corn borer larvae by overexpressing heterologous chit42 gene with chitin-binding domains

The chit42 gene cloned from Metarhizium anisopliae lacks chitin-binding domain (chBD), which plays important roles in binding insoluble chitin. Five kinds of hybrid chitinase Trichoderma transformants were constructed in this study, where the chit42 gene was fused to chBDs derived from plant, bacterial, and insect sources. The transformant Mc4 harboring chBDs from bitter melon (Momordica charantia) displayed the highest chitinase activity among all chBDs. The chitinase activities of Mc4, chit42 Trichoderma transformant Mchit3, and wild-type strain T30 were 44.94, 32.48, and 12.38 U/mL, respectively. The mortality rate of corn borer larvae in Mc4 fermentation liquid treatment increased by 10% and 30% compared with Mchit3 and T30, respectively. The midgut microvilli and goblet cell microvilli of the corn borer larvae exhibited distinct pathological changes after 48 h of feeding in Mc4 treatment. Mc4 also exhibited the strongest antifungal activity against Fusarium verticillioides and Rhizoctonia solani.

Regulation of Chitinase Production by the 5'-Untranslated Region of theybfMinSerratia marcescens2170

Serratia marcescens 2170 produces three chitinases and the chitin-binding protein CBP21, and efficiently degrades insoluble and crystalline chitin. The three chitinases and CBP21 are induced by N,N'-diacetylchitobiose [(GlcNAc)₂], the major product of chitin hydrolysis by S. marcescens chitinases. We have found that uptake of both (GlcNAc)₂ and N-acetylglucosamine (GlcNAc) is important for the efficient utilization of (GlcNAc)₂ because (GlcNAc)₂ is less efficiently fermented by S. marcescens 2170 in the absence of chitobiase. In order to determine the mechanism of utilization of the degradation products of chitin by S. marcescens, chitobiase deficient transposon mutants were screened. A transposon present in chitobiase-deficient mutants was inserted into the ybfMN-ctb cluster. The mutants produced chitinases, except for TT327, in which a transposon was inserted into the 5'-untranslated region (5'-UTR) of ybfM. Ectopic expression of this region in TT327 restored chitinase production. These results indicate that the 5'-UTR of ybfM is important for chitinase induction in S. marcescens.

Bacterial chitin utilisation at extremely haloalkaline conditions

Chitin is produced in large amounts in hypersaline habitats with neutral pH due to the high biomass production of brine shrimp Artemia. Recently, a high abundance of Artemia was also noticed in hypersaline soda lakes in the Kulunda Steppe (Altai, Russia), which prompted us to survey the possibility of microbial chitin utilization at extremely haloalkaline conditions in soda brines. Most active chitin utilisation-supporting microbial growth was found at anaerobic conditions at pH 10 and up to 3.5 M total Na(+). At aerobic conditions, the degradation of chitin was slower, mostly incomplete and active at <2 M total Na(+), although very slow partial degradation was possible up to 4 M Na(+). Anaerobic enrichments at pH 10 yielded two different groups of obligately haloalkaliphilic fermentative anaerobes, exclusively specialized to utilise insoluble chitin as the only growth substrate. One group was represented by a single strain growing at moderate salinity, and another comprised multiple isolates growing up to 3.5 M Na(+). These groups represent two novel bacterial phyla not closely related to any other cultured bacteria. Aerobic enrichments from the lake sediments were dominated by several obligately haloalkaliphilic members of the genus Marinimicrobium in the Gammaproteobacteria. They were less specialised than the anaerobes and grew with chitin and its monomer and oligomers at a pH of 10 up to 2.5 M Na(+). Furthermore, several strains of haloalkaliphilic Gram-positive chitinolytics belonging to bacilli and actinobacteria were isolated from soda lake sediments and surrounding soda soils. In general, the results indicate the presence of an active and diverse haloalkaliphilic chitinolytic microbial community in hypersaline soda habitats.