Trigalacturonate-producing pectate lyase PelQ1 from Saccharobesus litoralis with unique exolytic activity.

PelQ1 from Saccharobesus litoralis is a Ca2+-dependent pectate lyase belonging to the polysaccharide lyase family 1 (PL1). Although being an endolytic enzyme, it degraded polygalacturonate into predominantly unsaturated trimer in an exolytic manner with delayed production of dimer, tetramer and pentamer. The enzyme harbours a C-terminal domain from the carbohydrate-binding module family 13 (CBM13), whose presence facilitated the production of dimer. PelQ1's homology model showed that it possessed a well-conserved catalytic cleft, with R232 acting as the general base and R203 as the general acid. Structural comparison with DcPelC, a similar trimer-generating pectate lyase from Dickeya chrysanthemi EC16, implied that both enzymes' catalytic clefts encompassed at least eight subsites, i.e. -5 to +3. The unequal distribution of the subsites between the reducing and non-reducing ends of the cleavage site might be responsible for the exolytic generation of the trimer. As all but the -1, +1 and + 2 subsites could accommodate methylated galacturonate, this subclass of PL1 pectate lyases may function to help break up methylated pectin.

Polysaccharide degradation in Cellvibrionaceae: Genomic insights of the novel chitin-degrading marine bacterium, strain KSP-S5-2, and its chitinolytic activity.

In this study, we present the genome characterization of a novel chitin-degrading strain, KSP-S5-2, and comparative genomics of 33 strains of Cellvibrionaceae. Strain KSP-S5-2 was isolated from mangrove sediment collected in Balik Pulau, Penang, Malaysia, and its 16S rRNA gene sequence showed the highest similarity (95.09%) to Teredinibacter franksiae. Genome-wide analyses including 16S rRNA gene sequence similarity, average nucleotide identity, digital DNA-DNA hybridization, and phylogenomics, suggested that KSP-S5-2 represents a novel species in the family Cellvibrionaceae. The Cellvibrionaceae pan-genome exhibited high genomic variability, with only 1.7% representing the core genome, while the flexible genome showed a notable enrichment of genes related to carbohydrate metabolism and transport pathway. This observation sheds light on the genetic plasticity of the Cellvibrionaceae family and the gene pools that form the basis for the evolution of polysaccharide-degrading capabilities. Comparative analysis of the carbohydrate-active enzymes across Cellvibrionaceae strains revealed that the chitinolytic system is not universally present within the family, as only 18 of the 33 genomes encoded chitinases. Strain KSP-S5-2 displayed an expanded repertoire of chitinolytic enzymes (25 GH18, two GH19 chitinases, and five GH20 ß-N-acetylhexosaminidases) but lacked genes for agar, xylan, and pectin degradation, indicating specialized enzymatic machinery focused primarily on chitin degradation. Further, the strain degraded 90% of chitin after 10 days of incubation. In summary, our findings provided insights into strain KSP-S5-2's genomic potential, the genetics of its chitinolytic system, genomic diversity within the Cellvibrionaceae family in terms of polysaccharide degradation, and its application for chitin degradation.

Complete genome sequence of marine filamentous bacterium, Saprospira grandis strain WHT.

Here, we report the complete genome sequence of a type strain of the genus Saprospira, Saprospira grandis strain WHT. The genome consists of one circular chromosome and plasmid comprising 4,250,550 bp and 53,161 bp with GC content of 46.6% and 46.8%, respectively.

Characterisation and substrate binding modes of exopolygalacturonase PGQ1 from Saccharobesus litoralis.

Among the enzymes required for the efficient utilisation of pectin is polygalacturonase. Saccharobesus litoralis harbours two polygalacturonases belonging to glycoside hydrolase family 28 (GH28). One of them, PGQ1, cleaved polygalacturonate exolytically at the non-reducing end into monomeric units. It was most active at 60 °C and pH 8, with Km and kcat values of 2.3 mg/ml and 6.4 s-1 respectively. Its homology model of a right-handed parallel ß-helix core consisted of Asp297 as the general acid and either Asp276 or Asp298 as the general base. By inferring the substrate binding modes at the -1 and +1 subsites from known crystal structures, a hexagalacturonate could be docked into the highly electropositive binding cleft. Interestingly, while no residues were present in the vicinity to make up the +2 and +4 subsites, Arg361 and Arg430 could readily bind to the carboxyl groups of the galacturonates at the +3 and +5 subsites respectively. Structural comparison suggested that this binding pattern with missing subsites might be unique to closely related exopolygalacturonases. As S. litoralis grew much more slowly on extracellular galacturonate due to the lack of a transporter for the monosaccharide, PGQ1 probably functioned in the periplasm to help degrade oligopectates completely.Communicated by Ramaswamy H. Sarma.

Genome Sequence of Vibrio sp. Strain CCB-PB317, Isolated from a Malaysian Mangrove, and Its Arsenic Resistance Mechanisms.

Vibrio sp. strain CCB-PB317 with potential arsenic detoxification was isolated from a mangrove in Pulau Betong, Malaysia. Here, we report a draft genome sequence of strain CCB-PB317, which comprised 5,157,574 bp with a G+C content of 44.9%. The genome contains genes related to an arsenic resistance system coupled with glycolytic metabolism.

Application of Bacterial Endophytes to Control Bacterial Leaf Blight Disease and Promote Rice Growth.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) disease in rice (Oryza sativa L.) and it is among the most destructive pathogen responsible for severe yield losses. Potential bacterial biocontrol agents (BCAs) with plant growth promotion (PGP) abilities can be applied to better manage the BLB disease and increase crop yield, compared to current conventional practices. Thus, this study aimed to isolate, screen, and identify potential BCAs with PGP abilities. Isolation of the BCAs was performed from internal plant tissues and rhizosphere soil of healthy and Xoo-infected rice. A total of 18 bacterial strains were successfully screened for in vitro antagonistic ability against Xoo, siderophore production and PGP potentials. Among the bacterial strains, 3 endophytes, Bacillus sp. strain USML8, Bacillus sp. strain USML9, and Bacillus sp. strain USMR1 which were isolated from diseased plants harbored the BCA traits and significantly reduced leaf blight severity of rice. Simultaneously, the endophytic BCAs also possessed plant growth promoting traits and were able to enhance rice growth. Application of the selected endophytes (BCAs-PGP) at the early growth stage of rice exhibited potential in suppressing BLB disease and promoting rice growth.

Genome sequence data of Bacillus sp. CCB-MMP212 isolated from Malaysian mangrove: A potential strain in arsenic resistance with ArsI, C•As lyase.

Bacillus sp. CCB-MMP212 is a Gram-positive bacterium isolated from mangrove sediment in Matang Perak, Malaysia (4.85496°E, 100.73495°N). Genome sequencing was performed using the Oxford Nanopore and Illumina platforms. The assembled genome was annotated using the rapid annotation subsystem technology server (RAST) (rast.nmpdr.org). The genome size of the Bacillus sp. CCB-MMP212 was 6,151,644 base pairs (bp) with a G+C content of 34.75%. The genome includes 6,311 coding sequences and 58 RNAs. The sequence has been deposited at Genbank with the accession number of JALDQE000000000. Interestingly, an arsenic resistance (ars) operon consisted of arsenic resistance operon repressor (arsR), ACR3 family arsenite efflux transporter (arsB), and arsenate reductase (arsC) genes were found in the genome. In addition, the arsenic inducible gene (arsI), which encoded a dioxygenase with C•As lyase activity, was also found in the ars operon. The enzyme is crucial for the methylation of methylarsonous acid [MAs(III)] and trivalent roxarsone [Rox(III)]. This dataset reveals the genetic ability of this strain in arsenic resistance. To the best of our knowledge, the arsI encoding C•As lyase is rarely reported within the genus Bacillus. Therefore, the dataset presented in this manuscript provides further insight into the arsenic resistance mechanisms of the genus Bacillus.

Comparative Genomic Analyses of the Genus Photobacterium Illuminate Biosynthetic Gene Clusters Associated with Antagonism.

The genus Photobacterium is known for its ecophysiological versatility encompassing free-living, symbiotic, and pathogenic lifestyles. Photobacterium sp. CCB-ST2H9 was isolated from estuarine sediment collected at Matang Mangrove, Malaysia. In this study, the genome of CCB-ST2H9 was sequenced, and the pan-genome of 37 Photobacterium strains was analysed. Phylogeny based on core genes showed that CCB-ST2H9 clustered with P. galatheae, forming a distinct clade with P. halotolerans, P. salinisoli, and P. arenosum. The core genome of Photobacterium was conserved in housekeeping functions, while the flexible genome was well represented by environmental genes related to energy production and carbohydrate metabolism. Genomic metrics including 16S rRNA sequence similarity, average nucleotide identity, and digital DNA-DNA hybridization values were below the cut-off for species delineation, implying that CCB-ST2H9 potentially represents a new species. Genome mining revealed that biosynthetic gene clusters (BGCs) involved in producing antimicrobial compounds such as holomycin in CCB-ST2H9 could contribute to the antagonistic potential. Furthermore, the EtOAc extract from the culture broth of CCB-ST2H9 exhibited antagonistic activity against Vibrio spp. Intriguingly, clustering based on BGCs profiles grouped P. galatheae, P. halotolerans, P. salinisoli, P. arenosum, and CCB-ST2H9 together in the heatmap by the presence of a large number of BGCs. These BGCs-rich Photobacterium strains represent great potential for bioactive secondary metabolites production and sources for novel compounds.

SARS-CoV-2 viral shedding and susceptibility: perspectives on gender and asymptomatic patients.

Despite efforts to contain and manage the SARS-CoV-2 outbreak which was declared a public health emergency of international concern in January 2020 by the World Health Organization (WHO), the COVID-19 pandemic still remains a major global challenge. Patients who display the classical symptoms of the infection are easily identified, tested, isolated and monitored. However, many cases of infected asymptomatic patients have been documented. These patients are not easily identified even though many evidences suggest that they can spread the virus to others. How and why these COVID-19 asymptomatic presentations occur remain unclear. The many theories and views are conjectural, and supporting evidences are still needed. In this review, we described the trend in SARS-CoV-2 viral shedding and susceptibility, providing perspectives on gender differences and asymptomatic patients. We further discussed how genetics, gender, viral inoculum, and pre-existing immunity may influence asymptomatic presentations in COVID-19 infections. We hope that this article improves our understanding of asymptomatic SAR-CoV-2 infection and it sheds light on some salient areas that should be considered as the search for a potent vaccine continues.

Removal of Microcystis aeruginosa cells using the dead cells of a marine filamentous bacterium, Aureispira sp. CCB-QB1.

Inorganic and synthetic flocculants are widely investigated for removing harmful microalgae, such as Microcystis aeruginosa. However, their toxicity and non-biodegradability are shortcomings. Bioflocculants based on extracellular polysaccharides have attracted much attention as alternative flocculants. However, its high production cost is a limiting factor for applying bioflocculants. Here, we investigate the potential of the dead cells of a marine filamentous bacterium, Aureispira sp. CCB-QB1, as a novel flocculant on M. aeruginosa cells. The removal efficiency of M. aeruginosa cells by the dead cells was measured by mixing and shaking both components in a buffer with 5 mM CaCl2 in different incubation times and concentrations of the dead cells. After that, the minimum effective concentration of CaCl2 was determined. The combination effect of FeCl3 and the dead cells on the removal efficiency was tested. The structure of cell aggregates consisted of the dead cells and M. aeruginosa cells were also observed using a scanning electron microscope. The maximum removal efficiency (75.39%) was reached within 3 min in the presence of CaCl2 when 5 mg/ml of the dead cells (wet cells) were added. The optimal concentration of CaCl2 was 5 mM. The combination of the dead cells and a low concentration of FeCl3 (10 mg/L) with 5 mM of CaCl2 significantly improved the removal efficiency by about 1.2 times (P < 0.05). This result indicates that the combination usage of the dead cells can reduce the use of FeCl3. These results indicated that the dead cells could potentially be a novel biolfocculant to remove M. aeruginosa cells.

Metabolic strategies of dormancy of a marine bacterium Microbulbifer aggregans CCB-MM1: Its alternative electron transfer chain and sulfate-reducing pathway.

Bacterial dormancy plays a crucial role in maintaining the functioning and diversity of microbial communities in natural environments. However, the metabolic regulations of the dormancy of bacteria in natural habitats, especially marine habitats, have remained largely unknown. A marine bacterium, Microbulbifer aggregans CCB-MM1 exhibits rod-to-coccus cell shape change during the dormant state. Therefore, to clarify the metabolic regulation of the dormancy, differential gene expression analysis based on RNA-Seq was performed between rod- (vegetative), intermediate, and coccus-shaped cells (dormancy). The RNA-Seq data revealed that one of two distinct electron transfer chains was upregulated in the dormancy. Dissimilatory sulfite reductase and soluble hydrogenase were also highly upregulated in the dormancy. In addition, induction of the dormancy of MM1 in the absence of MgSO4 was slower than that in the presence of MgSO4. These results indicate that the sulfate-reducing pathway plays an important role in entering the dormancy of MM1.

Saccharobesus litoralis gen. nov., sp. nov., a novel alginate-degrading bacterium isolated from the surface of intertidal algal turf.

A novel rod-shaped, Gram-stain-negative, strictly aerobic and alginate-degrading marine bacterium, designated CCB-QB4T, was isolated from a surface of algal turf collected from a coastal area of Penang, Malaysia. The cells showed motility by a lateral flagellum. The rod-shaped cells formed long chains end-to-end. Phylogenetic analysis based on the 16S rRNA gene sequence of strain CCB-QB4T showed 94.07, 92.69, 91.52 and 90.90 % sequence similarity to Algibacillus agarilyticus RQJ05T, Catenovulum maritimum Q1T, Catenovulum agarivorans YM01T and Catenovulum sediminis D2T, respectively. Strain CCB-QB4T formed a cluster with A. agarilyticus RQJ05T. Strain CCB-QB4T was catalase-negative, oxidase-positive, and degraded agar, alginate, and starch. Cell growth was observed at 15-40 °C, at pH 7.0-10.0 and in the presence of 1-6 % (w/v) NaCl and glucose. The major fatty acids were summed feature 3 (C16 : 1 ω7c/iso-C15 : 0 2-OH), C16 : 0 and C18 : 1 ω7c. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, two unidentified glycolipids, an unidentified phospholipid and unidentified lipid. The major respiratory quinone was ubiquinone-8. The genomic DNA G+C content was 46.7 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain CCB-BQ4T represents a novel species in a new genus, for which the name Saccharobesus litoralis gen. nov., sp. nov. is proposed. The type strain is CCB-QB4T (=JCM 33513T=CCB-MBL 5008T).

An assimilatory sulfite reductase, CysI, negatively regulates the dormancy of Microbulbifer aggregans CCB-MM1T.

Sulfur is one of the common and essential elements of all life. Sulfate, which is a major source of sulfur, plays an important role in synthesizing sulfur-containing amino acids, such as cysteine and methionine, organic compounds essential to all living organisms. Some investigations reported that the assimilatory sulfate reduction pathway (ASRP) involved in cysteine synthesis is crucial to entering bacterial dormancy in pathogens. Our previous investigation reported that the halophilic marine bacterium, Microbulbifer aggregans CCB-MM1T , possesses an ASRP and the dissimilatory sulfate reduction pathway (DSRP). The bacterium might use DSRP to generate energy required for entering its dormant. However, the role of the ASRP in the dormancy of M. aggregans CCB-MM1T was so far unknown. In this study, we found that genes involved in ASRP were downregulated in the dormancy. The disruption of the gene encoding an assimilatory sulfite reductase, cysI, suppressed a completely dormant state under low nutrient conditions. In addition, the cysI mutant showed cell aggregation at the middle-exponential phase under high nutrient conditions, indicating that the mutation might be stimulated to enter the dormancy. The wild-type phenotype of the bacterium was recovered by the addition of cysteine. These results suggested that cysteine concentration may play an important role in inducing the dormancy of M. aggregans.

Genes for degradation and utilization of uronic acid-containing polysaccharides of a marine bacterium Catenovulum sp. CCB-QB4.

BACKGROUND: Oligosaccharides from polysaccharides containing uronic acids are known to have many useful bioactivities. Thus, polysaccharide lyases (PLs) and glycoside hydrolases (GHs) involved in producing the oligosaccharides have attracted interest in both medical and industrial settings. The numerous polysaccharide lyases and glycoside hydrolases involved in producing the oligosaccharides were isolated from soil and marine microorganisms. Our previous report demonstrated that an agar-degrading bacterium, Catenovulum sp. CCB-QB4, isolated from a coastal area of Penang, Malaysia, possessed 183 glycoside hydrolases and 43 polysaccharide lyases in the genome. We expected that the strain might degrade and use uronic acid-containing polysaccharides as a carbon source, indicating that the strain has a potential for a source of novel genes for degrading the polysaccharides. METHODS: To confirm the expectation, the QB4 cells were cultured in artificial seawater media with uronic acid-containing polysaccharides, namely alginate, pectin (and saturated galacturonate), ulvan, and gellan gum, and the growth was observed. The genes involved in degradation and utilization of uronic acid-containing polysaccharides were explored in the QB4 genome using CAZy analysis and BlastP analysis. RESULTS: The QB4 cells were capable of using these polysaccharides as a carbon source, and especially, the cells exhibited a robust growth in the presence of alginate. 28 PLs and 22 GHs related to the degradation of these polysaccharides were found in the QB4 genome based on the CAZy database. Eleven polysaccharide lyases and 16 glycoside hydrolases contained lipobox motif, indicating that these enzymes play an important role in degrading the polysaccharides. Fourteen of 28 polysaccharide lyases were classified into ulvan lyase, and the QB4 genome possessed the most abundant ulvan lyase genes in the CAZy database. Besides, genes involved in uronic acid metabolisms were also present in the genome. These results were consistent with the cell growth. In the pectin metabolic pathway, the strain had genes for three different pathways. However, the growth experiment using saturated galacturonate exhibited that the strain can only use the pathway related to unsaturated galacturonate.

Multifaceted interactions between the pseudomonads and insects: mechanisms and prospects.

Insects and bacteria are the most widespread groups of organisms found in nearly all habitats on earth, establishing diverse interactions that encompass the entire range of possible symbiotic associations from strict parasitism to obligate mutualism. The complexity of their interactions is instrumental in shaping the roles of insects in the environment, meanwhile ensuring the survival and persistence of the associated bacteria. This review aims to provide detailed insight on the multifaceted symbiosis between one of the most versatile bacterial genera, Pseudomonas (Gammaproteobacteria: Pseudomonadaceae) and a diverse group of insect species. The Pseudomonas engages with varied interactions with insects, being either a pathogen or beneficial endosymbiont, as well as using insects as vectors. In addition, this review also provides updates on existing and potential applications of Pseudomonas and their numerous insecticidal metabolites as biocontrol agents against pest insects for the improvement of integrated pest management strategies. Here, we have summarized several known modes of action and the virulence factors of entomopathogenic Pseudomonas strains essential for their pathogenicity against insects. Meanwhile, the beneficial interactions between pseudomonads and insects are currently limited to a few known insect taxa, despite numerous studies reporting identification of pseudomonads in the guts and haemocoel of various insect species. The vector-symbiont association between pseudomonads and insects can be diverse from strict phoresy to a role switch from commensalism to parasitism following a dose-dependent response. Overall, the pseudomonads appeared to have evolved independently to be either exclusively pathogenic or beneficial towards insects.

Crystal structure of a neoagarobiose-producing GH16 family ß-agarase from Persicobacter sp. CCB-QB2.

PdAgaC from the marine bacterium Persicobacter sp. CCB-QB2 is a ß-agarase belonging to the glycoside hydrolase family 16 (GH16). It is one of only a handful of endo-acting GH16 ß-agarases able to degrade agar completely to produce neoagarobiose (NA2). The crystal structure of PdAgaC's catalytic domain, which has one of the highest Vmax value at 2.9 × 103 U/mg, was determined in order to understand its unique mechanism. The catalytic domain is made up of a typical ß-jelly roll fold with two additional insertions, and a well-conserved but wider substrate-binding cleft with some minor changes. Among the unique differences, two unconserved residues, Asn226 and Arg286, may potentially contribute additional hydrogen bonds to subsites -1 and +2, respectively, while a third, His185 from one of the additional insertions, may further contribute another bond to subsite +2. These additional hydrogen bonds may probably have enhanced PdAgaC's affinity for short agaro-oligosaccharides such as neoagarotetraose (NA4), rendering it capable of binding NA4 strongly enough for rapid degradation into NA2.

Draft Genome Sequence of the Halophilic Pararhodobacter-Like Strain CCB-MM2, Which Has Polyhydroxyalkanoate-Synthesizing Potential.

Pararhodobacter-like strain CCB-MM2 is a halophilic alphaproteobacterium isolated from estuarine sediment collected from Matang Mangrove Forest in Malaysia. Here, we present the draft genome sequence of CCB-MM2 and provide insights into its physiological roles and metabolic potential.

Biochemical Characterization of Thermostable and Detergent-Tolerant ß-Agarase, PdAgaC, from Persicobacter sp. CCB-QB2.

Persicobacter sp. CCB-QB2 belonging to the family Flammeovirga is an agarolytic bacterium and exhibits a diauxic growth in the presence of tryptone and agarose. A glycoside hydrolase (GH) 16 ß-agarase, PdAgaC, was identified in the genome of the bacterium and was highly expressed during the second growth phase, indicating the agarase may play an important role in the diauxic growth. In this study, the catalytic domain of PdAgaC (PdAgaCgh) was cloned and characterized. PdAgaCgh showed thermostability at 50 °C and tolerance towards several detergents. In addition, the activity of PdAgaCgh after incubation with 0.1% of SDS and Triton X-100 increased approximately 1.2-fold. On the other hand, PdAgaCgh was sensitive to Fe2+, Ni2+, and Cu2+. The Km and Vmax of PdAgaCgh were 5.15 mg/ml and 2.9 × 103 U/mg, respectively. Interestingly, although the major hydrolytic product was neoagarobiose (NA2), monomeric sugar was also detected by thin-layer chromatographic analysis.

Modelling of polyhydroxyalkanoate synthase from Aquitalea sp. USM4 suggests a novel mechanism for polymer elongation.

Polyhydroxyalkanoate (PHA) synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace petroleum-based plastics. While two structures have been determined, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. A model of the class I PhaC from Aquitalea sp. USM4, characterised with Km of 394 µM and kcat of 476 s-1 on 3-(R)-hydroxybutyryl-CoA, revealed a three-branched channel at the dimeric interface. Two of them are opened to the solvent and are expected to serve as the putative routes for substrate entrance and product exit, while the third is elongated in the class II PhaC1 model from Pseudomonas aeruginosa, indicating a role in accommodating the hydroxyalkanoate (HA) moiety of a HA-CoA substrate. Docking of the two tetrahedral intermediates, formed during the transfer of the growing PHA chain from the catalytic Cys to a new molecule of substrate and back to Cys, suggests a common elongation mechanism requiring the HA moiety of the ligand to rotate ~180°. Substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates.

Functional and Structural Studies of a Multidomain Alginate Lyase from Persicobacter sp. CCB-QB2.

AlyQ from Persicobacter sp. CCB-QB2 is an alginate lyase with three domains - a carbohydrate-binding domain modestly resembling family 16 carbohydrate-binding module (CBM16), a family 32 CBM (CBM32) domain, and an alginate lyase domain belonging to polysaccharide lyase family 7 (PL7). Although AlyQ can also act on polyguluronate (poly-G) and polymannuronate (poly-M), it is most active on alginate. Studies with truncated AlyQ showed that the CBM32 domain did not contribute to enhancing AlyQ's activity under the assayed conditions. Nevertheless, it could bind to cleaved but not intact alginate, indicating that the CBM32 domain recognises alginate termini. The crystal structure containing both CBM32 and catalytic domains show that they do not interact with one another. The CBM32 domain contains a conserved Arg that may bind to the carboxyl group of alginate. The catalytic domain, meanwhile, shares a conserved substrate-binding groove, and the presence of two negatively charged Asp residues may dictate substrate specificity especially at subsite +1. As Persicobacter sp. CCB-QB2 was unable to utilise alginate, AlyQ may function to help the bacterium degrade cell walls more efficiently.