Diversity and functional analysis of luxS genes in Vibrios from marine sponges Mycale laxissima and Ircinia strobilina

Sponges harbor highly diverse and dense microbial communities, providing an environment in which bacterial signaling may be important. Quorum sensing (QS) is a cell density-dependent signaling process that bacteria employ to coordinate and regulate their gene expression. Previous studies have found that bacteria isolated from sponges are able to produce acyl-homoserine lactones (AHLs), an important class of QS molecules found in proteobacteria. Autoinducer-2 (AI-2) is a second class of QS molecule, and is considered to be an interspecies signal. However, AI-2 signaling has not been reported in sponge bacterial symbionts. In this study, degenerate primers were designed based on known Vibrio luxS sequences to amplify the luxS genes encoding AI-2 synthases of several Vibrio isolates from marine sponges Mycale laxissima and Ircinia strobilina. All the vibrios isolated from these two sponges had luxS genes and were able to produce signals with AI-2 activity as detected using a biological reporter. A novel group of luxS sequences was found, thus extending the known diversity of luxS genes. One isolate was chosen for further analysis of its luxS gene by expression of the gene in Escherichia coli DH5α and by characterization of the profile of AI-2 activity. This work provides the first information about luxS genes and AI-2 activity in sponge-associated bacterial communities.     

Influence of Turning and Environmental Contamination on the Dynamics of Populations of Lactic Acid and Acetic Acid Bacteria Involved in Spontaneous Cocoa Bean Heap Fermentation in Ghana

The influence of turning and environmental contamination on six spontaneous cocoa bean heap fermentations performed in Ghana was studied through a multiphasic approach, encompassing both microbiological (culture-dependent and culture-independent techniques) and metabolite target analyses. A sensory analysis of chocolate made from the fermented, dried beans was performed as well. Only four clusters were found among the isolates of acetic acid bacteria (AAB) identified: Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter senegalensis, and a potential new Acetobacter lovaniensis-like species. Two main clusters were identified among the lactic acid bacteria (LAB) isolated, namely, Lactobacillus plantarum and Lactobacillus fermentum. No differences in biodiversity of LAB and AAB were seen for fermentations carried out at the farm and factory sites, indicating the cocoa pod surfaces and not the general environment as the main inoculum for spontaneous cocoa bean heap fermentation. Turning of the heaps enhanced aeration and increased the relative population size of AAB and the production of acetic acid. This in turn gave a more sour taste to chocolate made from these beans. Bitterness was reduced through losses of polyphenols and alkaloids upon fermentation and cocoa bean processing.     

Diversity of Predominant Lactic Acid Bacteria Associated with Cocoa Fermentation in Nigeria

The fermentation of cocoa relies on a complex succession of bacteria and filamentous fungi, all of which can have an impact on cocoa flavor. So far, few investigations have focused on the diversity of lactic acid bacteria involved in cocoa fermentation, and many earlier investigations did not rely on polyphasic taxonomical approaches, which take both phenotypic and genotypic characterization techniques into account. In our study, we characterized predominant lactic acid bacteria from cocoa fermentations in Nigeria, using a combination of phenotypic tests, repetitive extragenic palindromic PCR, and sequencing of the 16S rRNA gene of representative strains for accurate species identification. Thus, of a total of 193 lactic acid bacteria (LAB) strains isolated from common media used to cultivate LAB, 40 (20.7%) were heterofermentative and consisted of either L. brevis or L. fermentum strains. The majority of the isolates were homofermentative rods (110 strains; 57% of isolates) which were characterized as L. plantarum strains. The homofermentative cocci consisted predominantly of 35 (18.1% of isolates) Pediococcus acidilactici strains. Thus, the LAB populations derived from these media in this study were accurately described. This can contribute to the further assessment of the effect of common LAB strains on the flavor characteristics of fermenting cocoa in further studies.     

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as “Weissella ghanaensis,” was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named “Acetobacter senegalensis” (A. tropicalis-like) and “Acetobacter ghanaensis” (A. syzygii-like).     

Quorum Sensing Circuit and Reactive Oxygen Species Resistance in Deinococcus sp.

Genus Deinococcus is characterized by an increased resistance toward reactive oxygen species (ROS). The chromosome of five strains belonging to this genus has been sequenced and the presence of a luxS-like gene was deduced from their genome sequences. The aim of this study was to assess if a complete QS circuit is present in Deinococcus sp. and if this QS is associated with ROS. Primers for searching luxS-like gene and the putative receptor gene, namely ai2R, were designed. AI-2 signal production was evaluated by luminescence analysis using Vibrio harveyi BB170 as reporter strain. AI-2 signal was also evaluated by competitive assays using cinnamaldehyde, ascorbic acid, and 3-mercaptopropionic acid as interfering molecules. Potassium tellurite and metronidazole were used as oxidative stressors. A luxS-like gene as well as an ai2R gene was detected in strain UDEC-P1 by PCR. Cell-free supernatant of strain UDEC-P1 culture induced luminescence in V. harveyi BB170, and this property was inhibited with the three interfering molecules. The oxidative stressors metronidazole and potassium tellurite decreased Deinococcus sp. viability, but increased luminescence of the reporter strain. The results demonstrate that both a functional luxS-like gene and a putative receptor for AI-2 signal are present in Deinococcus sp. strain UDEC-P1. This finding also suggests that a complete QS circuit is present in this genus, which could be related to oxidative stress.     

Type 2 Quorum Sensing Monitoring,Inhibition and Biofilm Formation in Marine Microrganisms

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 μM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 μM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that QSI compounds must be selected in the specific system in which they are to function, and they cannot easily be transferred from one QS system to another.     

The clam Meretrix lamarckii (Bivalvia: Veneridae) is a rich repository of marine lactic acid bacterial strains

Lactic acid bacteria (LAB) were isolated from the intestinal tract of the wild clam Meretrix lamarckii caught from the coastal waters of Kashima, Ibaraki, Japan. As many as 415 isolates were obtained using the culture method, of which 70 were considered presumptive LAB strains based on phenotypic tests. Phylogenetic analysis of these presumptive isolates of LAB based on the sequence of the 16S rRNA gene demonstrated that the species belonged to several genera of Lactobacillus, Lactococcus and Pediococcus. Interestingly, however, the species composition was different between the samples in July and October 2010. Further analyses based on the fermentation profiles revealed that the LAB from the clam caught in July 2010 were identified to be Lactobacillus curvatus, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris and Pediococcus pentosaceus, whereas those in October 2010 were identified to be Lactobacillus plantarum, Lactococcus lactis subsp. lactis and P. pentosaceus. The diversity of LAB in the intestinal tract of the clam suggests that the filter feeder bivalves such as M. lamarckii are a rich repository of marine isolates of LAB.     

产Ⅱ类细菌素乳酸菌群体感应及其应用

群体感应(quorum sensing,QS)是微生物通过感知与细胞密度相关的信号分子的浓度来调控基因表达的一种行为。许多产Ⅱ类细菌素乳酸菌通过自诱导肽介导的QS系统调控其细菌素的合成。本文综述了乳酸菌Ⅱ类细菌素合成的QS调控现象、调控机制、QS系统组分以及QS的应用。产Ⅱ类细菌素乳酸菌QS的研究,必将为揭示发酵调控机理、调控发酵过程提供新的研究平台,为食品级基因表达系统的开发提供新的选择。     

Diversity of lactic acid bacteria from Miang,a traditional fermented tea leaf in northern Thailand and their tannin-tolerant ability in tea extract

The microbiota of lactic acid bacteria (LAB) in thirty-five samples of Miang, a traditional fermented tea leaf product, collected from twenty-two different regions of eight provinces in upper northern Thailand was revealed through the culture-dependent technique. A total of 311 presumptive LAB strains were isolated and subjected to clustering analysis based on repetitive genomic element-PCR (rep-PCR) fingerprinting profiles. The majority of the strains belonged to the Lactobacillus genera with an overwhelming predominance of the Lb. plantarum group. Further studies of species-specific PCR showed that 201 of 252 isolates in the Lb. plantarum group were Lb. plantarum which were thus considered as the predominant LAB in Miang, while the other 51 isolates belonged to Lb. pentosus. In contrast to Lb. plantarum, there is a lack of information on the tannase gene and the tea tannin-tolerant ability of Lb. pentosus. Of the 51 Lb. pentosus isolates, 33 were found to harbor the genes encoding tannase and shared 93-99% amino acid identity with tannase obtained from Lb. pentosus ATCC 8041^T. Among 33 tannase gene-positive isolates, 23 isolates exhibited high tannin- tolerant capabilities when cultivated on de Man Rogosa and Sharpe agar-containing bromocresol purple (0.02 g/L, MRS-BCP) supplemented with 20% (v/v) crude tea extract, which corresponded to 2.5% (w/v) tannins. These Lb. pentosus isolates with high tannin-tolerant capacity are expected to be the high potential strains for functional tannase production involved in Miang fermentation as they will bring about certain benefits and could be used to improve the fermentation of tea products.     

Screening and molecular identification of potential probiotic lactic acid bacteria in effluents generated during ogi production

Screening and molecular identification of probiotic lactic acid bacteria (LAB) in effluents generated during the production of ogi, a fermented cereal (maize, millet, and sorghum) were done. LAB were isolated from effluents generated during the first and second fermentation stages in ogi production. Bacterial strains isolated were identified microscopically and phenotypically using standard methods. Probiotic potential properties of the isolated LAB were investigated in terms of their resistance to pH 1.5 and 0.3% bile salt concentration for 4 h. The potential LAB isolates ability to inhibit the growth of pathogenic organisms (Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium) was evaluated in vitro. The pH and LAB count in the effluents ranged from 3.31 to 4.49 and 3.67 to 4.72 log cfu/ml, respectively. A total of 88 LAB isolates were obtained from the effluents and only 10 LAB isolates remained viable at pH 1.5 and 0.3% bile salt. The zones of inhibition of the LAB isolates with probiotic potential ranged from 7.00 to 24.70 mm against test organsisms. Probiotic potential LAB isolates were molecularly identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecium, Pediococcus acidilactici, Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus brevis. Survival and proliferation of LAB isolates at low pH, 0.3% bile salt condition, and their inhibition against some test pathogens showed that these LAB isolates could be a potential probiotics for research and commercial purposes.     

Population Diversity of Yeasts and Lactic Acid Bacteria in Pig Feed Fermented with Whey, Wet Wheat Distillers' Grains, or Water at Different Temperatures

The diversity of populations of yeast and lactic acid bacteria (LAB) in pig feeds fermented at 10, 15, or 20°C was characterized by rRNA gene sequencing of isolates. The feeds consisted of a cereal grain mix blended with wet wheat distillers' grains (WWDG feed), whey (W feed), or tap water (WAT feed). Fermentation proceeded for 5 days without disturbance, followed by 14 days of daily simulated feed outtakes, in which 80% of the contents were replaced with fresh feed mixtures. In WWDG feed, Pichia galeiformis became the dominant yeast species, independent of the fermentation temperature and feed change. The LAB population was dominated by Pediococcus pentosaceus at the start of the fermentation period. After 3 days, the Lactobacillus plantarum population started to increase in feeds at all temperatures. The diversity of LAB increased after the addition of fresh feed components. In W feed, Kluyveromyces marxianus dominated, but after the feed change, the population diversity increased. With increasing fermentation temperatures, there was a shift toward Pichia membranifaciens as the dominant species. L. plantarum was the most prevalent LAB in W feed. The WAT feed had a diverse microbial flora, and the yeast population changed throughout the whole fermentation period. Pichia anomala was the most prevalent yeast species, with increasing occurrence at higher fermentation temperatures. Pediococcus pentosaceus was the most prevalent LAB, but after the feed change, L. plantarum started to proliferate. The present study demonstrates that the species composition in fermented pig feed may vary considerably, even if viable cell counts indicate stable microbial populations.     

Isolation and characterization of lactic acid bacteria from pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan

Lactobacillus pobuzihii is a novel species which has been previously found in pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan. However, the lactic acid bacteria (LAB) microflora in pobuzihi has not been studied in detail. In this study, LAB from pobuzihi were isolated, identified, and characterized. A total of 196 LAB were isolated; 79 cultures were isolated from the sample collected from a manufacturing factory, 38 from pobuzihi samples collected from 4 different markets, and 79 from 2 fresh cummingcordia samples. These isolates were characterized phenotypically and then divided into eight groups (A to H) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Lactobacillus plantarum was the most abundant LAB found in most samples during the fermentation of pobuzihi. On the other hand, Enterococcus casseliflavus and Weissella cibaria were, respectively, the major species found in the two fresh cummingcordia samples. A potential novel species or subspecies of lactococcal strain was found. In addition, seven L. plantarum and five W. cibaria strains showed inhibitory activity against the indicator strain Lactobacillus sakei JCM 1157^T. This is the first report describing the distribution and varieties of LAB existing in the pobuzihi during its fermentation process and the final product on the market.     

Vibrio vulnificus quorum-sensing system operates in cirrhotic ascites, a human ex vivo experimental system

This study was conducted to experimentally determine whether a Vibrio vulnificus quorum sensing (QS) system actually operates ex vivo. The expressions of luxS encoding autoinducer-2 synthase, of smcR encoding the master regulator of QS system, and of vvpE encoding a metalloprotease, the best known target directly regulated by QS, were increased with increasing bacterial density in cirrhotic ascites used as a human ex vivo experimental system. A luxS or smcR mutation significantly decreased vvpE expression under the same conditions. Accordingly, V. vulnificus QSS is likely to operate ex vivo.     

Oxidation of Metabolites Highlights the Microbial Interactions and Role of Acetobacter pasteurianus during Cocoa Bean Fermentation

Four cocoa-specific acetic acid bacterium (AAB) strains, namely, Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848^T, Acetobacter fabarum LMG 24244^T, and Acetobacter senegalensis 108B, were analyzed kinetically and metabolically during monoculture laboratory fermentations. A cocoa pulp simulation medium (CPSM) for AAB, containing ethanol, lactic acid, and mannitol, was used. All AAB strains differed in their ethanol and lactic acid oxidation kinetics, whereby only A. pasteurianus 386B performed a fast oxidation of ethanol and lactic acid into acetic acid and acetoin, respectively. Only A. pasteurianus 386B and A. ghanensis LMG 23848^T oxidized mannitol into fructose. Coculture fermentations with A. pasteurianus 386B or A. ghanensis LMG 23848^T and Lactobacillus fermentum 222 in CPSM for lactic acid bacteria (LAB) containing glucose, fructose, and citric acid revealed oxidation of lactic acid produced by the LAB strain into acetic acid and acetoin that was faster in the case of A. pasteurianus 386B. A triculture fermentation with Saccharomyces cerevisiae H5S5K23, L. fermentum 222, and A. pasteurianus 386B, using CPSM for LAB, showed oxidation of ethanol and lactic acid produced by the yeast and LAB strain, respectively, into acetic acid and acetoin. Hence, acetic acid and acetoin are the major end metabolites of cocoa bean fermentation. All data highlight that A. pasteurianus 386B displayed beneficial functional roles to be used as a starter culture, namely, a fast oxidation of ethanol and lactic acid, and that these metabolites play a key role as substrates for A. pasteurianus in its indispensable cross-feeding interactions with yeast and LAB during cocoa bean fermentation.     

Genomic Characterization of Burkholderia pseudomallei Isolates Selected for Medical Countermeasures Testing: Comparative Genomics Associated with Differential Virulence

Burkholderia pseudomallei is the causative agent of melioidosis and a potential bioterrorism agent. In the development of medical countermeasures against B. pseudomallei infection, the US Food and Drug Administration (FDA) animal Rule recommends using well-characterized strains in animal challenge studies. In this study, whole genome sequence data were generated for 6 B. pseudomallei isolates previously identified as candidates for animal challenge studies; an additional 5 isolates were sequenced that were associated with human inhalational melioidosis. A core genome single nucleotide polymorphism (SNP) phylogeny inferred from a concatenated SNP alignment from the 11 isolates sequenced in this study and a diverse global collection of isolates demonstrated the diversity of the proposed Animal Rule isolates. To understand the genomic composition of each isolate, a large-scale blast score ratio (LS-BSR) analysis was performed on the entire pan-genome; this demonstrated the variable composition of genes across the panel and also helped to identify genes unique to individual isolates. In addition, a set of ~550 genes associated with pathogenesis in B. pseudomallei were screened against the 11 sequenced genomes with LS-BSR. Differential gene distribution for 54 virulence-associated genes was observed between genomes and three of these genes were correlated with differential virulence observed in animal challenge studies using BALB/c mice. Differentially conserved genes and SNPs associated with disease severity were identified and could be the basis for future studies investigating the pathogenesis of B. pseudomallei. Overall, the genetic characterization of the 11 proposed Animal Rule isolates provides context for future studies involving B. pseudomallei pathogenesis, differential virulence, and efficacy to therapeutics.     

The Presence of Quorum-Sensing Genes in Pseudomonas isolates Infecting Cystic Fibrosis and Non-cystic Fibrosis Patients

Ninety-one Pseudomonas aeruginosa isolates recovered from cystic fibrosis and non-cystic fibrosis patients were evaluated regarding the ability to form biofilm and acyl-homoserine lactones production and for the presence of five quorum-sensing (QS) regulatory genes (lasI, lasR, rhlI, rhlR, and vfr). Most isolates (90.1 %) presented all five QS genes. Five isolates shown to be lasI/lasR-deficient were not able to produce biofilm in vitro. Moreover, one isolate harboring all five QS genes was also not able to form a biofilm. The function of rhlR gene may be compensated by the las QS system. However, in our study, all isolates which were deficient for the rhlR gene were also deficient for the lasI/lasR system. This may point to some hierarchy in QS regulation which may pose a potential for controlling biofilm infections due to P. aeruginosa.     

A sensitive fluorescence reporter for monitoring quorum sensing regulated protease production in Vibrio harveyi

Many bacteria produce and secrete proteases during host invasion and pathogenesis. Vibrio harveyi, an opportunistic pathogen of shrimp, is known to use a two-component quorum sensing (QS) mechanism for coordination of gene expression including protease secretion at high population densities. We examined the role of V. harveyi's QS signaling molecules, N-(3-hydroxybutanoyl)-l-homoserine lactone (AI-1) and the boron derivative of autoinducer-2 (BAI-2) in extracellular protease production. A fusion protein, M3CLPY (Rajamani et al., 2007), consisting of a large protease sensitive BAI-2 mutant receptor LuxP (~ 38 kDa) flanked by two protease insensitive cyan and yellow variants of GFP (~ 28 kDa each) was utilized as a substrate to detect secreted protease activity. The M3CLPY fusion, with the addition of wild-type V. harveyi (BB120) cell-free culture filtrate showed a time-dependent loss in fluorescence resonance energy transfer (FRET) associated with the cleavage of the LuxP linker protein and hence separation of the two flurophores. This cleavage of LuxP linker protein leading to decreased FRET efficiency was further confirmed by immunoblotting using anti-GFP antibody. The addition of cell-free filtrates from strains defective in one or both of the two-component QS pathways: luxN⁻ (defective in AI-1), luxS⁻ (defective in BAI-2), and luxN⁻/luxS⁻ (defective in both AI-1/BAI-2) showed differential levels of protease production. The observed protease activities were most pronounced in wild-type, followed by the AI-1 defective mutant (BB170) and the least for luxS⁻ mutant (MM30) and luxN⁻/luxS⁻ double mutant (MM32) strains. Incidentally, the lowest protease producing strains MM30 and MM32 were both defective in BAI-2 production. This observation was validated by addition of synthetic BAI-2 to MM30 and MM32 strains to restore protease production. Our results indicate that BAI-2 signaling in the two-component QS pathway plays the key role in regulating extracellular protease production in V. harveyi.