Xianglong Liu

The vertical distribution of bacterial and archaeal communities in the water and sediment of Lake Taihu.

This study was conducted to characterize the vertical distribution of bacterial and archaeal communities in the water and sediment of Lake Taihu, which underwent a change in trophic status from oligotrophic to hypertrophic in last half of the 20th century. The results revealed that the bacterial communities in different layers of sediment sample were very similar, and were related to Alpha-, Beta-, Gamma- and Deltaproteobacteria, Nitrospira, Bacteroidetes, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Chlorobi, Actinobacteria and Acidobacteria. In contrast, the archaeal communities varied greatly with depth. The archaeal communities were primarily related to Euryarchaeota and Crenarchaeota, with methanogenic Archaea accounting for approximately 2-35% of the total Archaea. Additionally, sequences related to putative ammonia-oxidizing Archaea and ammonia-oxidizing Bacteria were detected in different layers of sediment samples. The abundance of Archaea, Bacteria, methanogenic Archaea and Nitrospira was further characterized by real-time PCR.

Isolation, identification and characterization of an algicidal bacterium from Lake Taihu and preliminary studies on its algicidal compounds

In an effort to identify a bio-agent capable of controlling cyanobacterial blooms, we isolated a bacterial strain, A27, which exhibited strong algicidal activity against the dominant bloom-forming species of Microcystis aeruginosa in Lake Taihu. Based on 16S rRNA gene sequence analysis, this strain belongs to the genus Exiguobacterium. This is the first report of an algicidal bacterial strain belonging to the genus Exiguobacterium. Strain A27 exhibited algicidal activity against a broad range of cyanobacteria, but elicited little or no algicidal activity against the two green algal strains tested. The algicidal activity of strain A27 was shown to be dependent on the density of the bacteria and to have a threshold density of 1.5x10(6) CFU/mL. Our data also showed that the algicidal activity of strain A27 depended on different growth stages of Microcystis aeruginosa (exponential approximately lag phase > early stationary) rather than that of the bacterium itself. Our results also suggested the algicidal activity of strain A27 occurred via the production of extracellular algicidal compounds. Investigation of the algicidal compounds revealed that there were at least two different algicidal compounds produced by strain A27. These results indicated that strain A27 has great potential for use in the control of outbreaks of cyanobacterial blooms in Lake Taihu.

Diversity of bile salt hydrolase activities in different lactobacilli toward human bile salts

This study was conducted to evaluate the diversity of bile salt hydrolase (BSH) activities in eight species of lactobacilli. BSH activities were quantified based on the amount of taurine or glycine liberated from six main human bile sodium salts [glycocholic, glycodeoxycholic, glycochenodeoxycholic, taurocholic (TC), taurochenodeoxycholic, taurodeoxycholic] and a mixture of bile salts that resembled human bile. The eight species differed in their BSH activities. Specifically, Lactobacillus helveticus, Lactobacillus fermentum and Lactobacillus gallinarum had the ability to deconjugate taurine-conjugated bile salts, but not glycine-conjugated bile salts, which suggested that microbial BSHs recognize bile salts on both the cholate steroid nucleus and the amino acid moiety. Of the eight species evaluated, Lactobacillus acidophilus strains exhibited the highest specific BSH activity toward human bile salts, with the exception of TC. In addition, the L. acidophilus specific BSH activity toward glycine-conjugated bile salts was ten times higher than that toward taurine-conjugated bile salts. Moreover, the specific BSH activity of Lactobacillus plantarum did not vary significantly toward different bile salts, and Lactobacillus gasseri Am1 exhibited higher specific BSH activity toward TC than other lactobacilli. A comparison of bsh genes indicated that the LA-bshA, LA-bshB, LG-bsh and LP-bsh1 genes that encode the BSH enzymes are highly homologous (higher than 45%), while the LP-bsh2, LP-bsh3 and LP-bsh4 genes, which might not encode BSH enzymes, had lower similarity (lower than 26.3%).

Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa

A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms.

Cloning and heterologous expression of the manganese superoxide dismutase gene from Lactobacillus casei Lc18

Many of the species of Lactobacillus can be considered to be “probiotics” with a variety of benefits, including imparting antioxidative effects to the host. Lactobacillus species evolve different mechanisms to defend themselves against oxygen toxicity, such as superoxide dismutases (SODs), hydroperoxidases and high intracellular levels of metal ions. The SODs provide a cellular defense mechanism against oxidative stress by scavenging O2−. Most Lactobacillus species appear to lack a sod gene. To date, only two species of Lactobacillus (Lactobacillus casei and L. sakei) may have sod genes, as evidenced by sequence analysis of the genome, but no experimental verification, including cloning and heterologous expression of the Lactobacillus sod gene, has been reported. It is therefore unknown whether these sod genes can express SOD or are functional. We have PCR-amplified the gene from L. casei Lc18 that encodes the SOD using primers designed according to the genome of L. casei ATCC 334 and ligated it into the vector pET-28a(+) for heterologous expression in Escherichia coli BL21(DE3). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The superoxide radical scavenging activity of the recombinant strain was found to be increased relative to that of the control strain. The SOD was also purified by nickel ion affinity chromatography and found to consist of a single band, as determined by SDS-PAGE analysis, with an activity of 39.97 U/mg. N-terminal amino acid sequence analysis indicated that it may be a manganese-containing SOD. This is the first report of a sod gene from Lactobacillus spp. being expressed in other prokaryotes.

The influence of pH on heat stress response by probiotic Lactobacillus plantarum LP-Onlly

The influence of pH on the heat stress response of a human-derived probiotic strain of Lactobacillus plantarum, LP-Onlly, was investigated. The heat resistance of mid-log- and stationary-phase cells that were heat-adapted (45°C) in cultures that were or were not pH adjusted (pH 4.3–7.0) was evaluated at a lethal temperature of 55°C. The results demonstrated that the heat resistance of heat-adapted stationary-phase cells increased sharply as the pH for heat adaptation in cultures was adjusted from pH 4.3 (non-adjusted pH) to pH 7.0 step by step (pH 5.0, 5.5, 6.0, 6.5, and 7.0); however, the mid-log-phase cells did not show the similar pH-related trend. The optimal adjusted pH for heat adaptation in cultures was found to be 6.5, with stationary-phase cells that were heat-adapted at pH 6.5 exhibiting a 3.4-log-cycle increase in heat resistance and 6-fold greater storage stability than controls. Real-time reverse transcription PCR analysis of five major heat shock protein genes (dnak, dnaJ, grpE, groES, and groEL) revealed that the heat-inductive expression of these genes after heat adaptation was up-regulated in stationary-phase cells when the pH in cultures for heat adaptation was adjusted from 4.3 (non-adjusted pH) to 6.5. Nevertheless, the transcription levels of these genes after heat adaptation was down-regulated in mid-log-phase cells when the pH in cultures for heat adaptation was shifted from 6.0 (non-adjusted pH) to 6.5.

The algicidal activity of Aeromonas sp. strain GLY-2107 against bloom-forming Microcystis aeruginosa is regulated by N-acyl homoserine lactone-mediated quorum sensing.

Cyanobacterial blooms have disrupted the efficient utilization of freshwater worldwide. A new freshwater bacterial strain with strong algicidal activity, GLY-2107, was isolated from Lake Taihu and identified as Aeromonas sp. It produced two algicidal compounds: 2107-A (3-benzyl-piperazine-2,5-dione) and 2107-B (3-methylindole). Both compounds exhibited potent algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. The EC50 values (concentration for 50% maximal effect) of 3-benzyl-piperazine-2,5-dione and 3-methylindole were 4.72 and 1.10 μg ml-1 respectively. Based on a thin-layer chromatography biosensor assay and ultra-performance liquid chromatography-coupled high resolution-tandem mass spectrometry (UPLC-HRMS/MS), the N-acyl homoserine lactone (AHL) profile of strain GLY-2107 was identified as two short side-chain AHLs: N-butyryl-homoserine lactone (C4-HSL) and N-hexanoyl-homoserine lactone (C6-HSL). The production of the two algicidal compounds was controlled by AHL-mediated quorum sensing (QS), and C4-HSL was the key QS signal for the algicidal activity of the strain GLY-2107. Moreover, 3-methylindole was found to be positively regulated by C4-HSL-mediated QS, whereas 3-benzyl-piperazine-2,5-dione might be negatively controlled by C4-HSL-mediated QS. This study suggests that a QS-regulated algicidal system may have potential use for the development of a novel control strategy for harmful cyanobacterial blooms.

Analysis of newly detected tetracycline resistance genes and their flanking sequences in human intestinal bifidobacteria

Due to tetracycline abuse, the safe bifidobacteria in the human gastrointestinal intestinal tract (GIT) may serve as a reservoir of tetracycline resistance genes. In the present investigation of 92 bifidobacterial strains originating from the human GIT, tetracycline resistance in 29 strains was mediated by the tet(W), tet(O) or tet(S) gene, and this is the first report of tet(O)- and tet(S)-mediated tetracycline resistance in bifidobacteria. Antibiotic resistance genes harbored by bifidobacteria are transferred from other bacteria. However, the characteristics of the spread and integration of tetracycline resistance genes into the human intestinal bifidobacteria chromosome are poorly understood. Here, conserved sequences were identified in bifidobacterial strains positive for tet(W), tet(O), or tet(S), including the tet(W), tet(O), or tet(S) and their partial flanking sequences, which exhibited identity with the sequences in multiple human intestinal pathogens, and genes encoding 23 S rRNA, an ATP transporter, a Cpp protein, and a membrane-spanning protein were flanking by the 1920-bp tet(W), 1920-bp tet(O), 1800-bp tet(O) and 252-bp tet(S) in bifidobacteria, respectively. These findings suggest that tetracycline resistance genes harbored by human intestinal bifidobacteria might initially be transferred from pathogens and that each kind of tetracycline resistance gene might tend to insert in the vicinity of specific bifidobacteria genes.

Relationship between acid tolerance and cell membrane in Bifidobacterium, revealed by comparative analysis of acid-resistant derivatives and their parental strains grown in medium with and without Tween 80

The acid tolerance is particularly important for bifidobacteria to function as probiotics because they usually encounter acidic environments in food products and gastrointestinal tract passage. In this study, two acid-resistant derivatives Bifidobacterium longum JDY1017dpH and Bifidobacterium breve BB8dpH, which displayed a stable acid-resistant phenotype, were generated. The relationship between acid tolerance and cell membrane was investigated by comparing the two acid-resistant derivatives and their parental strains grown in medium with and without Tween 80. The fold increase in acid tolerance of the two acid-resistant derivatives relative to their parental strains was much higher when cells were grown in medium with Tween 80 (104 ~ 105-fold) than without Tween 80 (181- and 245-fold). Moreover, when cells were grown in medium with Tween 80, the two acid-resistant derivatives exhibited more C18:1 and cycC19:0, higher mean fatty acid chain length, lower membrane fluidity, and higher expression of cfa gene encoding cyclopropane fatty acid synthase than their parental strains. No significant differences in cell membrane were observed between the two acid-resistant derivatives and their parental strains when cells were grown in medium without Tween 80. The present study revealed that, when cells were grown in medium with Tween 80, the significant fold increase in acid tolerance of the two acid-resistant derivatives was mainly ascribed to the pronounced changes in cell membrane compared with their parental strains. Results presented here could provide a basis for developing new strategies of cell membrane modification to enhance acid tolerance in bifidobacteria.

A BSH volumetric activity dependent method for determination of coprecipitated cholesterol and the assimilation/coprecipitation proportion of cholesterol removal byLactobacillus plantarum

Thein vitro removal of cholesterol by probiotics has been categorized into two phases: assimilation by bacterial cells, and coprecipitation with deconjugated bile salts. Bile salt hydrolase (BSH) produced by probiotics catalyzes the deconjugation reaction of bile salts. A novel method that depends on the BSH volumetric activity (μg min−1 ml−1 bacteria culture) was developed to evaluate the cholesterol coprecipitation in MRS culture supplemented with synthetic human bile and cholesterol. Six probioticLactobacillus plantarum strains from the healthy youth intestinal tract were screened. The amount of cholesterol coprecipitation that occurred in eachL. Plantarum culture was then determined according to the method developed here and verified by a modified redissolution method. Next, the assimilation/coprecipitation proportion of cholesterol removal for each strain was deduced. The results revealed that after 12 h of incubation, all six strains ofL. plantarum removed a larger amount of cholesterol by assimilation (63.45–81.62%) than by coprecipitation (18.38–36.55%). Finally, we investigated the effects of synthetic human bile on BSH specific activity (μg min−1 mg−1 protein) and found that the BSH specific activity of all strains showed a rapid, limited and reversible decrease in response to synthetic human bile stress. We also found that variations in the BSH specific activity were related to the growth phases and that the maximum emerged after approximately 8 h (middle exponential phase) of growth.