Characterization of exopolysaccharides produced by three moderately halophilic bacteria belonging to the family Alteromonadaceae

Aims:  To study the exopolysaccharides (EPSs) produced by three novel moderately halophilic species belonging to the family Alteromonadaceae to optimize EPS yields, characterize their physical and chemical properties and evaluate possible biotechnological applications for these polymers.
Methods and Results:  EPSs synthesized by Idiomarina fontislapidosi F32^T, Idiomarina ramblicola R22^T and Alteromonas hispanica F23^T were collected and analysed under optimum conditions: MY medium supplemented with 7·5% (w/v) salts; 32°C; and 1% (w/v) glucose. Polymers were synthesized mainly during the early stationary growth phase with yields ranging from 1 to 1·5 g l⁻¹. The Idiomarina species each produced an anionic EPS composed mainly of glucose, mannose and galactose. A. hispanica synthesized an anionic EPS composed mainly of glucose, mannose and xylose. Solutions of all the polymers were low in viscosity and pseudoplastic in their behaviour. They showed emulsifying activity and the capacity to bind some metals.
Conclusions:  The Alteromonadaceae species studied in this work produced EPSs with physical and chemical properties different from those produced by other halophilic and nonhalophilic bacteria, suggesting that the wide diversity of micro-organisms being encountered nowadays in hypersaline environments offers enormous potential resources for biotechnological applications.
Significance and Impact of the Study:  We have optimized the EPS production and analysed new biopolymers produced by some recently described, moderately halophilic bacteria. These biopolymers are chemically and physically different from others already in use in biotechnology and offer hopes for new applications, especially in the case of A. hispanica , which may prove to be a viable source of xylo-oligosaccharides.     

Partial characterization of an extracellular polysaccharide produced by the moderately halophilic bacterium Halomonas xianhensis SUR308

A moderately halophilic bacterium, Halomonas xianhensis SUR308 (Genbank Accession No. KJ933394) was isolated from a multi-pond solar saltern at Surala, Ganjam district, Odisha, India. The isolate produced a significant amount (7.87 g l^?1) of extracellular polysaccharides (EPS) when grown in malt extract–yeast extract medium supplemented with 2.5% NaCl, 0.5% casein hydrolysate and 3% glucose. The EPS was isolated and purified following the conventional method of precipitation and dialysis. Chromatographic analysis (paper, GC and GC-MS) of the hydrolyzed EPS confirmed its heteropolymeric nature and showed that it is composed mainly of glucose (45.74 mol%), galactose (33.67 mol %) and mannose (17.83 mol%). Fourier-transform infrared spectroscopy indicated the presence of methylene and carboxyl groups as characteristic functional groups. In addition, its proton nuclear magnetic resonance spectrum revealed functional groups specific for extracellular polysaccharides. X-ray diffraction analysis revealed the amorphous nature (CI_xrd, 0.56) of the EPS. It was thermostable up to 250°C and displayed pseudoplastic rheology and remarkable stability against pH and salts. These unique properties of the EPS produced by H. xianhensis indicate its potential to act as an agent for detoxification, emulsification and diverse biological activities.     

Functionality of exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system

Aims:  To evaluate whether slime-exopolysaccharides (EPS) or capsular-polysaccharide (CPS) production could protect the polymer-producing strains Streptococcus thermophilus CRL 1190 and Lactobacillus casei CRL 87 against the harsh conditions of an in vitro gastric system (GS). EPS stability on the GS was studied.
Methods and Results:  An in vitro GS model containing human saliva and gastric juice was standardized. Polymer functionality on the cell viability and metabolic activity of the EPS-producing strains in the GS acidic conditions was evaluated. Two isogenic EPS/CPS deficient mutants were used for comparison. EPS or CPS conferred no significant protection on the cell viability of the studied strains after passage through the GS conditions. However, the phospho- and β-galactosidase activities of the EPS⁺ strains were higher than those of the EPS⁻. Cytoplasmic alterations in the wild-type and mutant strains and partial degradation of both EPS were detected.
Conclusions:  The presence of EPS/CPS protected the metabolic activity of the assayed LAB strains, but had no effect on survival at low pH.
Significance and Impact of the Study:  The presence of EPS/CPS as well as polymer resistance to the harsh conditions of the human GS could impact positively in probiotic strains to exert their properties in the host.     

Tyrosol degradation via the homogentisic acid pathway in a newly isolated Halomonas strain from olive processing effluents

Aims: To isolate a new Halomonas sp. strain capable of degrading tyrosol, a toxic compound present in olive mill wastewater, through the homogentisic acid (HGA) pathway. Methods and Results: A moderately halophilic Gram‐negative bacterium belonging to the Halomonas genus and designated strain TYRC17 was isolated from olive processing effluents. This strain was able to completely degrade tyrosol (2‐(p‐hydroxyphenyl)‐ethanol), a toxic compound found in such effluent. Tyrosol degradation begins by an oxidation to 4‐hydroxyphenylacetic acid (HPA), which is then converted into HGA by an HPA 1‐monooxygenase, while closest Halomonas species degrade tyrosol through 3,4‐dihydroxyphenylacetic acid (DHPA). In the presence of transition metals, HGA underwent a pH‐dependent abiotic conversion into benzoquinone acetic acid, then into 2,5‐dihydroxybenzaldehyde (gentisaldehyde) and pyomelanin, by oxidative decarboxylation and polymerization, respectively. Conclusions: Tyrosol degradation via HGA by the new Halomonas sp. strain TYRC17 was complete in the absence of trace elements. In their presence, HGA was abiotically converted into gentisaldehyde and pyomelanin. Significance and Impact of the Study: This is the first report on tyrosol degradation via the HGA pathway under hypersaline conditions and on the oxidative decarboxylation of HGA into gentisaldehyde. It underlines the importance of the Halomonas genus in the bioremediation of toxic‐contaminated sites.     

Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley

A number of novel alkaliphilic organotrophic bacteria have been isolated from several saline and alkaline East African soda lakes. The new isolates grow at pH values between 7.0 and 11.0, with pH optima for growth between 9.0 and 10.0. Growth occurs at total salts concentration between 0% and 20% (w/v) with optimum at 0%–7% (w/v). Phylogenetic analyses based on 16S rDNA sequence comparison indicate that these isolates are related (>96% similarity) to members of the Halomonadaceae within the γ-3 subdivision of the Proteobacteria. These analyses indicate that existing species within the Halomonadaceae fell within three main groups, one group comprising the type species of Halomonas, Halomonas elongata, and a number of other known species including one soda lake isolate. A second group constituting most of the remaining known species of Halomonas and related Chromohalobacter spp. includes 3 soda lake isolates with high DNA–DNA homologies. The third group included Halomonas halodenitrificans, Halomonas desiderata, Halomonas cupida, and 13 soda lake isolates. Phenotypic comparisons indicated that the majority of soda lake strains shared similar morphological, phenotypic, and chemotaxonomic properties to known strains of Halomonas but grew under alkaline conditions. The 3 soda lake isolates with high DNA–DNA homologies were, however, significantly different in antibiotic sensitivity pattern and in the utilization of several substrates, were unable to reduce nitrite, and showed low DNA–DNA homologies with known halomonads in the same group. We propose that these isolates comprise a new species of the genus Halomonas that we name Halomonas magadii sp. nov. The type strain is strain 21 MI (NCIMB 13595). Received: July 20, 1999 / Accepted: October 29, 1999     

Use of desalting gel for the rapid separation of simple sugars from exopolysaccharides produced by lactic acid bacteria

Three methods to remove simple carbohydrates prior to the measurement of exopolysaccharide concentration with the phenol/sulphuric acid method were compared. A new method based on size exclusion chromatography on a desalting gel compared favourably with ethanol precipitation (which was simple and rapid, but resulted in underestimation of EPS concentration) and dialysis (which was long and cumbersome). © Rapid Science Ltd. 1998     

小溪自然保护区非盐环境土壤中嗜盐和耐盐菌多样性

【目的】研究湖南小溪国家级自然保护区普通非盐环境(ordinary non-saline environment)土壤样品中可培养嗜盐及耐盐细菌(含放线菌)多样性。【方法】采用纯培养法和基于16S rRNA基因序列的系统发育分析对样品中嗜盐及耐盐细菌多样性进行研究。【结果】用补充5%-20%(w/v)NaCl的MA、ISP2、ISP5、NA和HAA培养基从土壤样品中分离到114株细菌,其中8株为中度嗜盐菌,19株为轻度嗜盐菌,87株为耐盐菌。根据形态观察和部分生理生化实验结果去冗余,选取61个代表性菌株进行基于16S rRNA基因序列的系统发育多样性分析。结果表明,这些菌株属于细菌域(Bacteria)的3个大的系统发育类群(门;phylum)(Actinobacteria,Firmicutes,Proteobacteria)的16个科、18个属,代表了41个物种。多数菌株属于Firmicutes门(38株,62.3%)和Actinobacteria门(18株,29.5%)。大多数菌株与其系统发育关系最密切的已知物种的典型菌株之间存在一定的遗传差异(16S rRNA基因序列相似性为96.9%-99.8%),其中有7个菌株(JSM070026,JSM081004,JSM081006,JSM081008,JSM083058,JSM083085,JSM084035)代表7个潜在新种(potential novel species)。【结论】研究结果表明,湖南小溪国家级自然保护区普通非盐环境土壤中存在较为丰富的可培养嗜盐及耐盐细菌多样性,并且潜藏着较多新的微生物类群(物种)。     

Uptake of zinc and copper by halophilic bacteria isolated from the dead sea shore,Jordan

Ten Gram-positive and gram-negative bacterial cultures were recovered from nine water, mud, and soil samples collected from the Dead Sea shore at Suwaymah. All bacterial cultures were able to grow at 10% NaCl and at 45°C. They were able to grow in nutrient media supplemented with 1250 ppm of Zn. Most of them, except cultures 2 and 8, were able to grow in nutrient medium supplemented with 1000 ppm of Cu. After 2 wk of incubation of these 10 cultures at different concentrations (5, 25, 100, and 500 ppm), stock solutions of both Zn and Cu elements, the maximum absorption using atomic absorption spectrometry for Zn was achieved by culture 7 at 11.2%, 1.0%, 38.4%, and 84.54%, respectively, from the previous stock solutions, whereas the maximum absorption of the same concentration of Cu was achieved by culture 3 at 6.2%, 55.56%, 85.66%, and 90.82%, respectively, of the different concentrations. After 3 wk of incubation, the estimated absorption for Zn was achieved by cultures 2, 9, and 10 at 19.2%, 16.68%, 42.92%, and 76.5%, 18.2%, 21.56%, 32.22%, and 77.43%, and 20.8%, 23.52%, 32.22%, and 82.84% of the previous stocks. The maximum absorption of the same concentration of Cu was achieved by culture 3 at 32.6%, 49.88%, 90.44%, and 91.86%, respectively. The accumulation of the absorbed metals was found to be maximum in the protoplast of all cultures. The accumulation at the cell wall was maximum for cultures 2 and 6 for Zn and Cu, respectively, and between the cell wall and the plasma membrane, it was maximum for cultures 2 and 8 for Zn and Cu, respectively.     

Uptake of Pb2+ by a cyanobacterium belonging to the genus Synechocystis, isolated from East Kolkata Wetlands

East Kolkata Wetlands is a conserved wetland utilizing sewage and garbage, generated by Kolkata Municipal Corporation area for cultivation purpose. Cyanobacteria are the photosynthetic prokaryotes having bioremedial capacity. We have isolated a cyanobacterium from the sewage recycling fish-pond of East Kolkata Wetlands. Partial sequence of 16S rDNA gene of the isolated strain showed 100% similarity with that of genus Synechocystis. Isolated strain and Synechocystis sp. PCC6803 survived up to 300 mug ml(-1) Pb(2+ )and growth was completely inhibited at 400 mug ml(-1) Pb(2+). All experiments were carried out with 100 mug ml(-1) Pb(2+) in which growth was the maximum. 91.67% of the total Pb(2+) got adsorbed to the outer surface of the cell and 1% of the total Pb(2+) entered the cell of the isolated strain as estimated by atomic absorption spectrometry, but in Synechocystis sp. PCC6803 72.72% adsorbed and 0.96% penetrated. Intracellular and periplasmic depositions of Pb(2+) were observed in both the strain. A filamentous structure developed outside the cell wall of the isolated cyanobacterium, but very little change was observed in Synechocystis sp. PCC6803. ZiaR-SmtB like regulator gene was expressed in both the strains after Pb(2+) induction. The cDNA sequence of ZiaR of the isolated cyanobacterium shows 100% homology with that of Synechocystis sp. PCC6803. Upon Pb(2+) induction, expression of SOD gene increased. cDNA sequence of the SOD gene from the isolated strain showed 98% homology with that of Synechocystis sp. PCC6803. Enzymatic activity of catalase and SOD was also increased. No DNA damage was monitored upon induction with Pb(2+).     

Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy)

A total of 41 strains of lactic acid bacteria (LAB) isolated from durum wheat sourdoughs used to produce Cornetto di Matera bread, were identified by SDS-PAGE of whole cell proteins (WCP) and screened for acid production ability, antimicrobial activity and exopolysaccharide (EPS) production. The isolates were identified as Lactobacillus plantarum (49%), Leuconostoc mesenteroides (17%), Lactobacillus curvatus (15%), Lactobacillus paraplantarum (12%), Weissella cibaria (5%) and Lactobacillus pentosus (2%). Several strains of Lb. plantarum and Leuc. mesenteroides showed a high acid production ability. The antagonistic activity was tested using an agar-spot deferred antagonism assay against a set of five indicators. The species had different profiles of inhibition. Lb. plantarum had the largest spectrum of inhibition, while no isolates of W. cibaria and Leuc. mesenteroides showed antimicrobial activity. No strains had antimicrobial activity against Bacillus cereus. The inhibitory activity of five strains was confirmed to be sensitive to proteolytic enzymes and thus potentially due to bacteriocin production. All Leuc. mesenteroides and W. cibaria strains produced EPS from sucrose. Some Lb. plantarum and Lb. paraplantarum strains produced EPS from different sugars in solid media. EPS production in liquid media was different within the species, with the highest production in liquid media containing glucose and maltose. A defined strain starter culture (W. cibaria DBPZ1006, Lb. plantarum DBPZ1015 and S. cerevisiae MTG10) was selected on the basis of technological properties and tested in model sourdough fermentations.     

Antimicrobial activity of lactic acid bacteria isolated from Tenerife cheese: initial characterization of plantaricin TF711, a bacteriocin-like substance produced by Lactobacillus plantarum TF711

AIMS: The screening and initial characterization of bacteriocins produced by lactic acid bacteria (LAB) from raw Tenerife goats' cheese with possible application as biopreservatives or ripening accelerators for Tenerife cheese. METHODS AND RESULTS: One hundred and eighty LAB of the genera Lactobacillus (95), Leuconostoc (64) and Lactococcus (21) isolated from raw Tenerife goats' cheese were screened for the production of antimicrobial substances. Lactobacillus plantarum TF711, which had the broadest spectrum of antimicrobial activity, was selected for further characterization. The antimicrobial compound was determined as a proteinaceous substance, as it was sensitive to proteases. The bacteriocin-like substance, which we called plantaricin TF711, was active against the Gram-positive bacteria Bacillus cereus, Clostridium sporogenes and Staphylococcus aureus; and against the Enterobacteriaceae Shigella sonnei and Klebsiella pneumoniae. It was stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 1 and 9. Plantaricin TF711 exhibited primary metabolite kinetics, a bacteriostatic mode of action and a molecular mass of c. 2.5 kDa as determined by tricine SDS-PAGE. CONCLUSIONS: Lact. plantarum TF711 produces a low molecular mass bacteriocin-like compound with a wide spectrum of activity and interesting technological properties (thermostability, good pH stability and stability against surfactants and organic solvents). SIGNIFICANCE AND IMPACT OF THE STUDY: Plantaricin TF711 was found to have potential for use as a biopreservative in the food industry.     

Histamine, histidine, and growth-phase mediated regulation of the histidine decarboxylase gene in lactic acid bacteria isolated from wine

Fermented foods are frequently contaminated by histamine that is generated by microorganisms with histidine decarboxylase activity. The ingestion of large amounts of histamine can cause serious toxicological problems in humans. A study of the effects of histamine, histidine, and growth phase on histamine production by lactic acid bacteria isolated from wine is reported here. With northern blots and specific activity analysis, we observed that histidine induces the expression of the histidine decarboxylase gene (hdc) and that histamine causes a decrease in the expression of this gene. The expression of hdc is also mediated by the bacterial growth phase. Histidine and histamine do not affect histidine decarboxylase activity, whereas pyridoxal 5'-phosphate does. Data on histamine-producing lactic acid bacteria isolated from wine should contribute to the prevention of histamine formation during winemaking and storage.     

若尔盖高原沼泽土和草甸风沙土细菌16S rDNA PCR-RFLP和系统发育分析

采用稀释平板法从若尔盖高原沼泽土和草甸风沙土分离获得66株细菌,在16S rDNA PCR-RFLP分析的基础上,测定了22株代表菌株的16S rDNA序列,构建了供试细菌的系统发育关系。16S rDNA PCR-RFLP分析中,在78%相似性水平处,除REG14,REG20,REG22和REG55单独成群外,其余菌株分为8个遗传群,其中,群Ⅰ和群Ⅳ最大,均有15个菌株,其次是群Ⅷ,由11个菌株组成,其余21个菌株分布于5个群内。对22个代表菌株16SrDNA全序列系统发育表明,这些菌株分布于不同的系统发育分支。其中,以芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)和分枝杆菌属(Mycobacterium)为主,其余菌株分布于Lysinibacillus属、中华根瘤菌属(Sinorhizobium)、不动杆菌(Acinetobacter)、固氮菌属(Azotobacter)、红球菌属(Rhodococcus)、微球菌属(Micrococcus)和醋酸杆菌属(Acetobacter)等7个属。     

Rhodopirellula aestuarii sp. nov., a novel member of the genus Rhodopirellula isolated from brackish sediments collected in the Tagus River estuary,Portugal

Bacteria within the phylum Planctomycetota are biologically relevant due to unique characteristics among prokaryotes. Members of the genus Rhodopirellula can be abundant in marine habitats, however, only six species are currently validly described. In this study, we expand the explored genus diversity by formally describing a novel species. The pink-coloured strain ICT_H3.1^T was isolated from brackish sediments collected in the Tagus estuary (Portugal) and a 16S rRNA gene sequence-based analysis placed this strain into the genus Rhodopirellula (family Pirellulaceae). The closest type strain is Rhodopirellula rubra LF2^T, suggested by a similarity of 98.4% of the 16S rRNA gene sequence. Strain ICT_H3.1^T is heterotrophic, aerobic and able to grow under microaerobic conditions. The strain grows between 15 and 37 °C, over a range of pH 6.5 to 11.0 and from 1 to 8% (w/v) NaCl. Several nitrogen and carbon sources were utilized by the novel isolate. Cells have an elongated pear-shape with 2.0 ± 0.3 × 0.9 ± 0.2 µm in size. Cells of strain ICT_H3.1^T cluster in rosettes through a holdfast structure and divide by budding. Younger cells are motile. Ultrathin cell sections show cytoplasmic membrane invaginations and polar fimbriae. The genome size is 9,072,081 base pairs with a DNA G + C content of 56.1 mol%. Genomic, physiological and morphological comparison of strain ICT_H3.1^T with its relatives suggest that it belongs to a novel species within the genus Rhodopirellula. Hence, we propose the name Rhodopirellula aestuarii sp. nov., represented by ICT_H3.1^T (=CECT30431^T = LMG32464^T) as the type strain of this novel species.16S rRNA gene accession number: GenBank = OK001858.Genome accession number: The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAMQBK000000000. The version described in this paper is version JAMQBK010000000.     

Three new species of fleas belonging to the genus Macrostylophora from the three‐striped ground squirrel,Lariscus insignis,in Java

Three new species of fleas belonging to the genus Macrostylophora (Siphonaptera, Ceratophyllidae) are described from the three‐striped ground squirrel, Lariscus insignis, from Tjibodas, West Java (Jawa Barat), Indonesia at an elevation of 1500 m. Macrostylophora larisci sp. n. is described from three male specimens, Macrostylophora debilitata sp. n. is described from one male and Macrostylophora wilsoni sp. n. is described from one female. Non‐genital morphological characters of the female specimen, including ctenidial spine shapes and lengths, show that it is not the corresponding female for either M. larisci sp. n. or M. debilitata sp. n. It is unusual for three different species of congeneric fleas to parasitize the same host species in the same geographical location. These three new species represent the first known records of Macrostylophora from Java and they could be enzootic vectors between rodents of flea‐borne zoonotic pathogens such as Rickettsia typhi and Yersinia pestis, both of which are established on Java. A list is provided of the 43 known species and 12 subspecies of Macrostylophora together with their known geographical distributions and hosts. A map depicting the distributions of known Indonesian (and Bornean) species of Macrostylophora is also included.     

Differences between Lactococcus garvieae isolated from the genus Seriola in Japan and those isolated from other animals (trout, terrestrial animals from Europe) with regard to pathogenicity, phage susceptibility and genetic characterization

AIMS: To clarify the epidemiological relationship between Lactococcus garvieae isolates from the Seriola in Japan and isolates from other animals. METHODS AND RESULTS: A total of 32 isolates obtained from aquatic (the genus Seriola and trout) and terrestrial animals (cow, pig, cat, dog and horse) was used to evaluate its pathogenicity to yellowtail and mouse, phenotype (KG+ and KG-), its susceptibility to three bacteriophages and the pattern of pulsed field gel electrophoresis (PFGE). Lactococcus garvieae isolated from Seriola showed strong pathogenicity to yellowtail, while isolates from trout showed weak pathogenicity and those obtained from terrestrial animals showed no distinct pathogenicity. Only, the isolates from the genus Seriola in Japan showed susceptibility to the bacteriophages. The results of PFGE pattern indicate that the isolates obtained from the Seriola predict homogeneity, while there is no similarity among the isolates obtained from different animals. CONCLUSION: This experiment indicates that L. garvieae isolated from Seriola in Japan appears to be very different from the isolates obtained from other animals, and the isolates prevalent among the genus Seriola in Japan might be homogeneous. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that a particular genetic group that has specially adapted and acquired virulence toward yellowtail were prevalent among the genus Seriola in Japan.