Genome Analysis Provides Insights into the Osmoadaptation Mechanisms of <em>Halomonas titanicae</em>

Here, we report the osmoadaptation strategies adopted by the halotolerant species Halomonas titanicae BH1(T) inferred from genome sequence analysis. BH strain was isolated in 2010 from a rusticated sample collected in 1991 from the wreck of the Titanic, genome deposited in the database under the accession number (CP059082.1). It showed a high salt tolerance ranging from 0.5 to 25% NaCl (w/v) (optimal growth at 10% NaCl) with no growth in the absence of NaCl. The phylogenomic analysis showed that the BH1 strain is more closely related to the Halomonas sedementi QX-2, a strain isolated from deep-sea sediments. The RAST (Rapid Annotation using Subsystem Technology) annotation revealed divergent mechanisms involved in the primary and secondary response to osmotic stress citing protein implicated in potassium transport, periplasmic glucan synthesis, choline and betaine upake system, biosynthesis of glycine-betaine, ectoine, and proline. These findings provide an overview of the osmoadaptive mechanisms of H. titanicae BH1, and could offer helpful information to future biotechnological applications like osmolyte synthesis and related applications

Patterns and determinants of halophilic Archaea (class Halobacteria) diversity in Tunisian endorheic salt lakes and sebkhet systems

We examined the diversity and community structure of members of the halophilic Archaea (class Halobacteria) in samples from central and southern Tunisian endorheic salt lakes and sebkhet (also known as sebkha) systems using targeted 16S rRNA gene diversity survey and quantitative PCR (qPCR) approaches. Twenty-three different samples from four distinct locations exhibiting a wide range of salinities (2% to 37%) and physical characteristics (water, salt crust, sediment, and biofilm) were examined. A total of 4,759 operational taxonomic units at the 0.03 (species-level) cutoff (OTU0.03s) belonging to 45 currently recognized genera were identified, with 8 to 43 genera (average, 30) identified per sample. In spite of the large number of genera detected per sample, only a limited number (i.e., 2 to 16) usually constituted the majority (>/=80%) of encountered sequences. Halobacteria diversity showed a strong negative correlation to salinity (Pearson correlation coefficient = -0.92), and community structure analysis identified salinity, rather than the location or physical characteristics of the sample, as the most important factor shaping the Halobacteria community structure. The relative abundance of genera capable of biosynthesis of the compatible solute(s) trehalose or 2-sulfotrehalose decreased with increasing salinities (Pearson correlation coefficient = -0.80). Indeed, qPCR analysis demonstrated that the Halobacteria otsB (trehalose-6-phosphatase)/16S rRNA gene ratio decreases with increasing salinities (Pearson correlation coefficient = -0.87). The results highlight patterns and determinants of Halobacteria diversity at a previously unexplored ecosystem and indicate that genera lacking trehalose biosynthetic capabilities are more adapted to growth in and colonization of hypersaline (>25% salt) ecosystems than trehalose producers.Peer reviewedMicrobiology and Molecular Genetic

Halocins, Bacteriocin-Like Antimicrobials Produced by the Archaeal Domain: Occurrence and Phylogenetic Diversity in <em>Halobacteriales</em>

Members of extremely halophilic archaea, currently consisting of more than 56 genera and 216 species, are known to produce their specific bacteriocin-like peptides and proteins called halocins, synthesized by the ribosomal pathway. Halocins are diverse in size, consisting of proteins as large as 35 kDa and peptide “microhalocins” as small as 3.6 kDa. Today, about fifteen halocins have been described and only three genes, halC8, halS8 and halH4, coding C8, S8 and H4 halocins respectively have been identified. In this study, a total of 1858 of complete and nearly complete genome sequences of Halobacteria class members were retrieved from the IMG and Genbank databases and then screened for halocin encoding gene content, based on the BLASTP algorithm. A total of 61 amino acid sequences belonging to three halocins classes (C8, HalH4 and S8) were identified within 15 genera with the abundance of C8 class. Phylogenetic analysis based on amino acids sequences showed a clear segregation of the three halocins classes. Halocin S8 was phylogenetically more close to HalH4. No clear segregation on species and genera levels was observed based on halocin C8 analysiscontrary to HalH4 based analysis. Collectively, these results give an overview on halocins diversity within halophilic archaea which can open new research topics that will shed light on halocins as marker for haloarchaeal phylogentic delineation

Pseudomonas rhizophila S211, a New Plant Growth-Promoting Rhizobacterium with Potential in Pesticide-Bioremediation

A number of Pseudomonas strains function as inoculants for biocontrol, biofertilization, and phytostimulation, avoiding the use of pesticides and chemical fertilizers. Here, we present a new metabolically versatile plant growth-promoting rhizobacterium, Pseudomonas rhizophila S211, isolated from a pesticide contaminated artichoke field that shows biofertilization, biocontrol and bioremediation potentialities. The S211 genome was sequenced, annotated and key genomic elements related to plant growth promotion and biosurfactant (BS) synthesis were elucidated. S211 genome comprises 5,948,515 bp with 60.4% G+C content, 5306 coding genes and 215 RNA genes. The genome sequence analysis confirmed the presence of genes involved in plant-growth promoting and remediation activities such as the synthesis of ACC deaminase, putative dioxygenases, auxin, pyroverdin, exopolysaccharide levan and rhamnolipid BS. BS production by P. rhizophila S211 grown on olive mill wastewater based media was effectively optimized using a central-composite experimental design and response surface methodology (RSM). The optimum conditions for maximum BS production yield (720.80 ± 55.90 mg/L) were: 0.5% (v/v) inoculum size, 15% (v/v) olive oil mill wastewater (OMWW) and 40◦C incubation temperature at pH 6.0 for 8 days incubation period. Biochemical and structural characterization of S211 BS by chromatography and spectroscopy studies suggested the glycolipid nature of the BS. P. rhizophila rhamnolipid was stable over a wide range of temperature (40–90◦C), pH (6–10), and salt concentration (up to 300mM NaCl). Due to its low-cost production, emulsification activities and high performance in solubilization enhancement of chemical pesticides, the indigenous BS-producing PGPR S211 could be used as a promising agent for environmental bioremediation of pesticide-contaminated agricultural soils

Different Modes of Regulation of the Expression of Dextransucrase in Leuconostoc lactis AV1n and Lactobacillus sakei MN1

Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter PdsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the PdsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20°C than at 37°C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc, AV1n was transformed with plasmids carrying either the PdsrLL-mrfp fusion or the PdsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from PdsrLL upon growth at low temperature (20°C versus 30°C and 37°C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from PdsrLL. A different behavior was detected, when expression from PdsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30°C and 20°C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications

Archaea - New Biocatalysts, Novel Pharmaceuticals and Various Biotechnological Applications

Besides the Introductory Chapter that gives a brief overview of archaeal applications, the present book contains four chapters. The first chapter, by Castro-Fernandez et al., provides an interesting depiction of the phylum Euryarchaeota and its biotechnological applications. The second chapter, by Ben Hania and coauthors, focuses on the promotion of the idea that some specific Archaea are potential next-generation probiotics. The third chapter, by Torregrosa-Crespo et al., emphasizes the main characteristics of biocompounds from haloarchaea and their potential uses in biomedicine, pharmacy, and industry. The concluding chapter, by Mizuno et al., proposes a plasmid curing approach for improving the potential of thermophiles in various biotechnological applications and opens new perspectives on industrial valorization

MADFORWATER. WP2 Adaptation of wastewater treatment technologies for agricultural reuse. Task2.2 Municipal wastewater and drainage canal water treatment. UMA-Tunisia

This dataset contains the data produced by UMA team in the framework of task 2.2 of MADFORWATER project. This task deal with a set of data generated regarding the detection of antibiotic resistance genes and enterovirus, particularly Hepatitis A virus from Treated Municipal Wastewater. To monitor the prevalence of antibiotic resistant bacteria in MWW before and after treatment, the qualitative and quantitative PCR methods, allowing the detection of resistance genes in all microorganisms including the non-culturable species, were performed. PCR products of target antibiotic genes (tet(O), tet(Q), tet(W), tet(G), amp(C) and bla TEM) were loaded and visualized on agarose gels. For qPCR, the analysis was performed in order to detect and quantify the antibiotic resistance genes copy number. The investigation of the Hepatitis A virus prevalence in MTWW was performed using two different extraction methods (virus extraction, concentration and concentrate decontamination method according to US Environmental Protection Agency (1992) from Mud (M) and Supernatant (S) of the influent and from the effluent and virus concentration from water with adsorption/elution on glass wool (XP T 90-451. March 1996) (Rodier et al., 2009)) followed by qPCR. A specific attention was dedicated to the detection of SARS-CoV-2 in wastewaters using the same protocols developed and optimized under MADFORWATER (RNA extraction/concentration, cDNA synthesis, Q-RT-PCR) by using specific DNA primers approved in the RT-PCR test. This could contribute to better understanding and studying the emerged SARS-CoV-2 and its propagation routes and epidemiology in a given population (i.e. Tunis city). Regarding Microarray, the generated data were produced and elaborated basing on a list of targeted genes downloaded from accessible databases (NCBI, IMG, KEEG). The effectiveness of MWWTP cannot be approved without accepted microbiological quality. In the literature, there is a huge number of publications and standards related fecal or pathogen bacteria removal in wastewater treatment plant (WWTP), many publications show inefficiency of current MWWTP in removing virus but no standards dealing with virus detection from MWW in Tunisia. Generated data allowed the design of the WWchip, able to monitor catabolic genes markers of fecal indicators, pathogens indicators, virus and antibiotic resistant bacteria. A total of 3744 genes and 12832 probes were designated. In silico validation and verification of all probes was performed using the BLASTN algorithm and custom-made databases. Two set of data are proposed. The first one summarizes the gene type, number of genes and number of probes considered in the design of the WWchip. The list and sequences of all the probes of target genes is presented a second set of data. The data format produced by the microarray consists of a list of genes and corresponding values that represent relative DNA levels of each targeted gene. The developed WWChip will constitute a new rapid tool for pathogen monitoring of different types of treated wastewaters

Population genetics of Lactobacillus sakei reveals three lineages with distinct evolutionary histories

Lactobacillus sakei plays a major role in meat fermentation and in the preservation of fresh meat. The large diversity of L. sakei strains represents a valuable and exploitable asset in the development of a variety of industrial applications; however, an efficient method to identify and classify these strains has yet to be developed. In this study, we used multilocus sequence typing (MLST) to analyze the polymorphism and allelic distribution of eight loci within an L. sakei population of 232 strains collected worldwide. Within this population, we identified 116 unique sequence types with an average pairwise nucleotide diversity per site (pi) of 0.13%. Results from STRUCTURE, goeBURST, and CLONALFRAME software analyses demonstrated that the L. sakei population analyzed here is derived from three ancestral lineages, each of which shows evidence of a unique evolutionary history influenced by independent selection scenarios. However, the signature of selective events in the contemporary population of isolates was somewhat masked by the pervasive phenomenon of homologous recombination. Our results demonstrate that lineage 1 is a completely panmictic subpopulation in which alleles have been continually redistributed through the process of intra-lineage recombination. In contrast, lineage 2 was characterized by a high degree of clonality. Lineage 3, the earliest-diverging branch in the genealogy, showed evidence of both clonality and recombination. These evolutionary histories strongly indicate that the three lineages may correspond to distinct ecotypes, likely linked or specialized to different environmental reservoirs. The MLST scheme developed in this study represents an easy and straightforward tool that can be used to further analyze the population dynamics of L. sakei strains in food products