Characteristics and living patterns of marine myxobacterial isolates

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.     

海水盐离子对耐盐粘球菌生长和发育的影响

黄褐粘球菌 (Myxococcusfulvus)HW 1菌株是分离自海水样品的耐盐粘细菌 ,能够在海水 (盐浓度为3.8% )或稀释海水条件下生长。随着生长环境中海水浓度的提高 ,HW 1在生长与分化发育上表现出不同于陆地粘细菌的特性。对海水中主要盐离子的进一步分析表明 ,Na+ 对HW 1生长有抑制作用 ,当Ca2 + 超过海水中浓度时对HW 1生长抑制最为明显 ,而Mg2 + 对HW 1生长几乎没有抑制作用 ,但能促进子实体的形成。海水盐成分中SO42 -,K+ 和不含K+ 的其它盐组合都能导致孢子抗热能力的提高。     

Resistance of the oil-oxidizing microorganism <Emphasis Type="Italic">Dietzia</Emphasis> sp. to hyperosmotic shock in reconstituted biofilms

A number of halotolerant and halophilic bacterial strains were isolated from the Romashkinskoe oil field (Tatarstan) stratal waters having a salinity of up to 100 g/l. The isolation of pure cultures involved biofilm reconstitution on M9 medium with paraffins. The associations obtained were dispersed and reinoculated onto solid media that contained either peptone and yeast extract (PY medium) or paraffins. It was shown that such associations included both oil-oxidizing bacteria and accompanying chemoheterotrophic bacteria incapable of oil oxidation. The pure cultures that were isolated were used for creating binary biofilms. In these biofilms, interactions between halophilic and nonhalophilic bacteria under hypo-and hyperosmotic shocks were investigated. We conducted a detailed study of a biofilm obtained from an oil-oxidizing halotolerant species (with an upper growth limit of 10–12% NaCl) identified as Dietzia sp. and an extremely halophilic gram-negative bacterium (growing within the 5–20% NaCl concentration range) of the genus Chromohalobacter that did not oxidize paraffins. If these microorganisms were grown in a mixed suspension (planktonic) culture that was not supplemented with an additional amount of NaCl, no viable cells of the halophilic microorganism were detected after reinoculation. In contrast, only halophilic cells were detected at a NaCl concentration of 15%. Thus, no mutual protective influence of the microorganisms manifested itself in suspension culture, either under hypoor under hyperosmotic shock. Neither could halophile cells be detected after reinoculating a biofilm obtained on a peptone medium without the addition of NaCl. However, biofilms produced at a NaCl concentration of 15% contained approximately equal numbers of cells of the halophilic and halotolerant organisms. Thus, the halophile in biofilms sustaining a hyperosmotic shock exerts a protective influence on the halotolerant microorganism. Preliminary data suggest that this effect is due to release by the halophile of osmoprotective substances (ectoine and glutamate), which are taken up by the halotolerant species. Such substances are diluted by a large medium volume in suspension cultures, whereas, in biofilms, their diffusion into the medium is apparently hampered by their interaction with the intercellular polymer matrix.     

Enhygromyxa salina gen. nov., sp. nov., a slightly halophilic myxobacterium isolated from the coastal areas of Japan

Six isolates of novel marine myxobacteria, designated strains SHK-1T, SMK-1-1, SMK-1-3, SMK-10, SKK-2, and SMP-6, were obtained from various coastal samples (mud, sands and algae) collected around Japan. All of the isolates had Gram-negative rod-shaped cells, motile by gliding and grew aerobically. They showed bacteriolytic action, fruiting body formation, and NaCl requirement for growth with an optimum concentration of 1.0-2.0% (w/v). In addition, divalent cationic components of seawater, such as Mg2+ or Ca2+, were also needed for growth. The major respiratory quinone was MK-7. The G+C content of genomic DNA ranged from 65.6 to 67.4 mol% (by HPLC). The isolates shared almost identical 16S rDNA sequences, and clustered with a recently described marine myxobacterium, Plesiocystis pacifica, as their closest relative on a phylogenetic tree (95.9-96.0% similarity). Physiological and chemotaxonomic differences between the new strains and strains of the genus Plesiocystis justify the proposal of a new genus. Therefore, we propose to classify the six isolates into a new taxon of marine myxobacteria with the name, Enhygromyxa salina gen. nov., sp. nov. The type strain is SHK-1(T) (JCM 11769(T) = DSM 15217(T) = AJ 110011(T)).     

Haliangium ochraceum gen. nov., sp. nov. and Haliangium tepidum sp. nov.: novel moderately halophilic myxobacteria isolated from coastal saline environments

Phenotypic and phylogenetic studies were performed on two myxobacterial strains, SMP-2 and SMP-10, isolated from coastal regions. The two strains are morphologically similar, in that both produce yellow fruiting bodies, comprising several sessile sporangioles in dense packs. They are differentiated from known terrestrial myxobacteria on the basis of salt requirements (2-3% NaCl) and the presence of anteiso-branched fatty acids. Comparative 16S rRNA gene sequencing studies revealed that SMP-2 and SMP-10 are genetically related, and constitute a new cluster within the myxobacteria group, together with the Polyangium vitellinum Pl vt1 strain as the closest neighbor. The sequence similarity between the two strains is 95.6%. Based on phenotypic and phylogenetic evidence, it is proposed that these two strains be assigned to a new genus, Haliangium gen. nov., with SMP-2 designated as Haliangium ochraceum sp. nov. (= JCM 11303(T) = DSM 14365(T)), and SMP-10 as Haliangium tepidum sp. nov. (= JCM 11304(T)= DSM 14436(T)).     

Improved methods of isolation and purification of myxobacteria and development of fruiting body formation of two strains

By using baiting techniques and different purification methods, a high number of myxobacterial strains have been isolated as pure cultures from soil of different regions of China. Because myxobacterial cells do not disperse easily in liquid media, a medium containing an enzymatic hydrolysate of casein (CEH) medium have been used for purification and purity tests combined in a single step. The key method, in which isolates are reintroduced to sterile rabbit dung to induce fruiting bodies formation, facilitates purification of myxobacteria. Sterile rabbit dung pellets are used to mimic the natural growth substance of these organisms which has the advantage that characteristic fruiting bodies emerge, which is a key characteristics in the taxonomy of myxobacteria. In this study, the optimum program of isolation and purification of some myxobacteria strains has been established which will facilitate screening programs. Moreover, the development of fruiting body formation of strain BD20 (Chondromyces) and strain BD54 (Cystobacter) have been recorded in this study.     

Improving Cellular Properties for Genetic Manipulation by Dispersed Growing Mutagenesis in Myxococcus fulvus HW-1

One of the key limitations to genetic manipulation in myxobacteria is that the cells grow in clumps in liquid. A salt-tolerant strain HW-1 of Myxococcus fulvus was treated with UV irradiation and produced a completely dispersedly growing mutant UV684. There were no significant differences between the parent HW-1 and the mutant UV684 in terms of salt-tolerant growth. The mutant UV684 and the parent strain had the similar abilities of the fruiting body formation and S motility. Interestingly, the mutant exhibited high transformation/transposition efficiency with 10⁵–10⁶ colony-forming units per μg DNA, which was about 10³–10⁵ fold higher than HW-1. The results indicate that the mutation that led to dispersed growth in the UV684 mutant strain had a few impacts on social behavior, but greatly facilitated molecular genetic manipulation.     

Isolation of myxobacteria from the marine environment

In an attempt to isolate indigenous marine myxobacteria from coastal samples, we obtained two swarm forming bacteria. Both isolates formed cell aggregates which, at least in one isolate, developed to fruiting body-like structures consisting of a mass of myxospore-like cells. The optimum NaCl concentrations for their growth were between 2 and 3%, comparable to the NaCl concentration of seawater. This growth characteristic strongly suggests that the two isolates are specific marine bacteria. The 16S rDNA sequence studies indicated that the two isolates were related to the genus Nannocystis. Based on the phylogenetic distances between branches, we concluded that the isolates should be assigned to two new myxobacterial genera.     

Genome sequence of the halotolerant marine bacterium Myxococcus fulvus HW-1

Myxococcus fulvus HW-1 (ATCC BAA-855) is a halotolerant marine myxobacterium. This strain exhibits complex social behaviors in the presence of low concentrations of seawater but adopts an asocial living pattern under oceanic conditions. The whole genome of M. fulvus HW-1 will enable us to further investigate the details of its evolution.     

Evolution of bacterial flora from a subterranean saline well by graduated salinity changes in enrichment media

The bacterial flora of samples taken from a subterranean saline well was enriched (1) by periodic salinity increase and (2) by periodic salinity decrease, both at 25° and 35°C. During the enrichment process, bacterial flora, including halotolerant, marine, moderately and extremely halophilic bacteria, were enumerated. Results were similar at both temperatures. Marine bacteria and moderately halophilic microorganisms were the most favoured groups, predominating between 3 and 30% (w/v) salt content; extremely halophilic bacteria also appeared when salinity reached 30% (w/v) and halotolerant bacteria were poorly represented. The taxonomic distribution of 125 selected strains, chosen at random from counting media, was essentially similar to those from other hypersaline environments.     

The effect of gradual increase in salinity on the biomass productivity and biochemical composition of several marine,halotolerant<Emphasis Type="Bold">,</Emphasis> and halophilic microalgae

Open ponds are the preferred cultivation system for large-scale microalgal biomass production. To be more sustainable, commercial scale biomass production should rely on seawater, as freshwater is a limiting resource, especially in places with high irradiance. If seawater is used for both pond fill and evaporative volume makeup, salinity of the growth media will rise over time. It is not possible for any species to achieve optimum growth over the whole saline spectrum (from seawater salinity level up to salt saturation state). In this study, we investigated the effects of gradual salinity increase (between 35 and 233 ppt) on biomass productivity and biochemical composition (lipid and carbohydrate) of six marine, two halotolerant, and a halophilic microalgae. A gradual and slow stepped salinity increase was found to expand the salinity tolerance range of tested species. A gradual reduction in biomass productivity and maximum photochemical efficiency was observed as a consequence of increased salinity in all tested species. Among the marine microalgae, Tetraselmis showed highest biomass productivity (32 mg L^?1 day^?1) with widest salinity tolerance range (35 to 109 ppt). Halotolerant Amphora and Navicula were able to grow from 35 ppt to 129 ppt salinity. Halophilic Dunaliella was the only species capable of growing between 35 and 233 ppt and showed highest lipid content (56.2%) among all tested species. This study showed that it should be possible to maintain high biomass in open outdoor cultivation utilizing seawater by growing Tetraselmis, Amphora, and Dunaliella one after another as salinity increases in the cultivation system.     

微生物高盐渗透适应策略及其耐盐强化研究进展

高盐废水因具有硬度高、可生化性差、水质成分复杂等特点,是较难处理的工业废水之一。现有物化处理技术存在运行成本高、处理效率低、二次污染重等诸多瓶颈。耐盐/嗜盐微生物可在高盐环境下进行正常生理代谢,因此,开发经济、高效、可靠的高盐废水生物处理技术有望成为高盐废水处理的主流方向之一。本文系统综述了耐盐/嗜盐微生物盐溶、胞内小分子相容溶质积累、蛋白质稳定和细胞表面稳定等高渗透压适应策略。由于嗜盐微生物存在生长条件苛刻、功能微生物种类稀缺等问题,因此,耐盐微生物在高盐废水处理的未来应用空间更大。最新研究发现强化调控技术(电、光、磁)可提升微生物的高渗透压适应能力,其中电调控技术或是未来高盐废水生物处理的重点研究方向。     

Adaptation of Salt-tolerant <Emphasis Type="Italic">Myxococcus</Emphasis> Strains and their Motility Systems to the Ocean Conditions

More and more studies have indicated that myxobacteria are able to live in seawater conditions, which, however, can decrease the fruiting body formation ability and also the adventurous (A) and social (S) motility systems of the myxobacteria. To learn the adaptation mechanism of the salt-tolerant myxobacteria to marine conditions, we analyzed 10 salt-tolerant Myxococcus strains of their fruiting body formation and motility. The isolates were from marine samples and possessed different levels of salt tolerance. They had the dual motility system and formed fruiting bodies in the presence of suitable seawater concentrations. Some high salt-tolerant strains even lost their fruiting abilities in the absence of seawater. In response to the presence of seawater, the S-motility was found to be increased in the high salt-tolerants but decreased in the low salt-tolerants. The A-motility, on the other hand, was observed in all the salt-tolerant Myxococcus strains, but increased or decreased in response to the presence of seawater. Perceived shifts of fruiting body formation abilities and motilities discovered in the salt-tolerant Myxococcus strains suggested an ecological adaptation of myxobacterial social behaviors to the marine environments.     

Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations

Solar salterns, based on a multi-pond system, give a discontinuous gradient of salt concentrations. The heterotrophic bacterial populations of ponds containing from 10% salt to saturation have been studied. Saltern samples were spread on agar plates containing different media for halophilic bacteria and one medium made with water of the pond plus nutrients. Replica plating was done to determine the salt range for growth of the colonies. We studied 150 strains to determine the salt spectra of growth, the morphology, and nutrient requirements. The following conclusions were reached: (a) In salt concentrations above 10% (total salts), most bacteria are halophilic and few are halotolerant; (b) the two types of halophilic bacteria, moderate and extreme, show different distributions; in these ponds a narrow overlap exists between 25% and 32% salts with moderate halophiles predominating below this interval and extreme halophiles above it; (c) the populations of moderate halophiles are highly heterogeneous, and the salt concentration of their habitat affects their taxonomic composition, salt range for growth, and nutrient requirements. The population composition of extreme halophiles is less affected by the salt concentrations at which these bacteria are found.     

Synthesis of osmoprotectors by halophilic and alkalophilic methanotrophs

The 1H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5-1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium of Methylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-containing osmoprotectants, ectoine and 5-oxo-1-proline. M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH4+ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.     

CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO₂ fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.     

Halophily and halotolerance in cyanophytes

The survival, growth and distribution of organisms in hypersaline environments is discussed using cyanophytes (cyanobacteria) as examples. The distinction between halophilic (Na⁺-requiring) and halotolerant organisms is not adequate to describe the entire spectrum of adaptations to salt. The classical division into stenohaline (narrow) and euryhaline (wide) adaptational types, with optima identified as oligo-, meso- and polyhaline, better reflects both organismal adaptations and the environmental conditions to which these are adjusted and is therefore recommended as a conceptual model.Two independent properties of organisms are growth and survival. Organisms requiring narrow ranges of salt concentration are considered specialists and are restricted to environments with relatively constant salinities at any particular concentration. Organisms which tolerate wide ranges of fluctuation in salinity are considered generalists. The existence of separate and distinct microbial assemblages in these two types of environments is demonstrated in marine intertidal zones and seasonal salt works, representative of fluctuating salinity, and in the open ocean. The hypersaline ponds of Yallahs, Jamaica, and Solar Lake, Sinai represent different but relatively constant salinities. It is concluded that cyanophytes speciate along the salinity gradient, and that separate halophilic taxa occupy environments with relatively constant salinities.Proceedings of the fourth College Park Colloquium on Chemical EvolutionLimits of Life, University of Maryland, College Park, 18–20 October 1978.     

Synthesis of osmoprotectants by halophilic and alkaliphilic methanotrophs

The¹H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5–1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium ofMethylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-contaihing osmoprotectants, ectoine and 5-oxo-1-proline.M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH ₄ ⁺ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.     

A simple method to isolate salt-tolerant myxobacteria from marine samples

This paper describes a simple method for the isolation of salt-tolerant myxobacteria from marine conditions. As the results show in this paper, salt-tolerant myxobacteria are found to be able to grow, but unable to form fruiting bodies at high salt concentrations. The fruiting body structures of the salt-tolerant strains were all formed in conditions with lower seawater content, i.e. lower than 60% seawater (about 2.0% salt content) or distilled water supplemented with MgCl(2). The method picked up the fruiting bodies for isolation.     

Intracellular Changes in Ions and Organic Solutes in Halotolerant Brevibacterium sp. Strain JCM 6894 after Exposure to Hyperosmotic Shock

In the present study we aimed to observe the intracellular responses when there was a hyperosmotic shock with a large shift in ionic strength in nutrient-rich and nutrient-poor external environments in order to clarify the availability of substrates. To do this, we used the halotolerant organism Brevibacterium sp. strain JCM 6894, which is able to grow in the presence of a wide range of salt concentrations. Hyperosmotic shock was induced by transferring cells in the late exponential phase of growth in a complex medium containing 0.5 M NaCl into either old or fresh culture medium containing 2 M NaCl. Changes in the growth rate, in the pH of the medium, and in the internal cation or organic solute concentrations in the cytosol after an upshock were analyzed as a function of incubation time. The cells exhibited very different responses to upshocks in fresh culture medium and in old culture medium; in fresh culture medium, growth was stimulated and the medium became more acidic, whereas the old culture medium repressed growth and the medium became more alkaline. The intracellular free Na⁺ concentrations remained low (80 nmol mg of protein⁻¹) after an upshock in fresh culture medium, although they quickly increased twofold in the old culture medium. In contrast, K⁺ ions immediately accumulated in the cells in fresh culture medium, whereas K⁺ ions were taken up quite slowly in old culture medium. Furthermore, the cells placed in fresh culture medium transiently accumulated alanine and glutamine in response to the upshock, but the cells placed in old culture medium did not. Growth of the Brevibacterium strain at higher levels of salinity was supported by ectoine synthesis but was not observed after the shift to high-osmolarity conditions in the old culture. In the fresh culture, however, ectoine was vigorously synthesized in cells for more than 5 h after the upshock; the concentration of ectoine in cells was more than 3,500 nmol mg of protein⁻¹ at 10 h, which corresponded to a ninefold increase compared to the concentration before the shock. These findings are consistent with the results of an analysis of the extracellular medium composition before and after the upshock.