Current Microbiology

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease gene (Cas), originating from the bacteria acquired immune system, have revolutionized gene editing technology. In this regard, type II (Cas9) been extensively studied and widely applied CRISPR system so far. The mechanism for precise manipulation of genomic sequences is guided by small RNA called CRISPR RNA (crRNA). In this study we devised and optimized CRISPR-Cas9 screening system based on Cas9 gene detection, targeting a conserved part of recognition domain (REC) consisting of arginine rich bridge helix (BH). We used hemi-nested PCR approach for screening sensitivity and reproducibility. The recombinant E. coli DH5 alpha containing the pRGEB32 vector (DH5 alpha/pRGEB32) with the Cas9 gene was used for system optimization. Subsequently, the screening system was applied and validated on different environmental bacterial strains including Alcaligenes faecalis and Pseudomonas stutzeri, isolated from sewerage samples. The optimized hemi-nested PCR resulted in amplification of targeted region in environmental bacterial strains and results were reproduced successfully. Furthermore, nucleotides and amino acid sequence, motif and domain analysis of PCR products, confirmed the targeted Cas9 REC-BH domain. Presently, no rapid and cost effective CRISPR-Cas screening system is available except expensive whole genome sequencing approach. Our investigation aimed to device rapid and cost effective screening system for identification of new variants of Cas9 proteins in environmental bacterial species. In this context, the developed Cas9 gene-based CRISPR-Cas screening system (C9CSS) may be a potential rapid screening tool to identify new Cas9 orthologs in different bacterial genomes with improved functions.

Antimicrobial resistance in Pseudomonas aeruginosa has emerged as a major clinical concern. Ciprofloxacin has potent intrinsic activity against P. aeruginosa, but resistance to this antibiotic is increasingly reported in clinical settings. In this study, P. aeruginosa ATCC 27853 strain was exposed to gradually increasing ciprofloxacin concentrations for 50 passages ([~]100 days). The final selected resistant strain (CIP-R) with 256-fold of minimum inhibitory concentration was studied further. Whole-genome sequencing revealed 12 genomic alterations, including known mutations in the quinolone resistance-determining genes gyrA (Thr83Ile), parC (Thr177Asn), parE (Glu459Lys). A duplication mutation in nfxB (Tyr153_Gly154dup) was also observed. In addition to these genes, we observed mutations in pilA, tadB, psdR, NP446_RS32055 (TRAP transporter permease), multiple dppA3 variants, and a prophage-associated hypothetical gene (NP446_RS24255), which have not been reported previously. The gyrA Thr83Ile substitution was conserved in 86.38% of ciprofloxacin-resistant clinical isolates retrieved from the NCBI database. Resistance acquisition was accompanied by slower growth, impaired swimming and swarming motility, diminished surface attachment, reduced biofilm formation. The resistant strain has enhanced {beta}-lactamase activity, and resistance to levofloxacin, cefepime, and meropenem together with sensitivity to piperacillin-tazobactam and aztreonam. This study highlights the gyrA Thr83Ile mutation as a key genomic marker for molecular screening of ciprofloxacin resistance and reveals secondary adaptive trade-offs that can be targeted for clinical diagnostic and therapeutic decision-making. In conclusion, achieving high-level ciprofloxacin resistance in P. aeruginosa involves non-target-site genomic adaptations and physiological trade-offs beyond classical target mutations.

Background Bryozoans are ancient colonial animals that have existed for over 470 million years and are widely distributed across aquatic environments. Recently, many spherical colloids (average diameter{approx}1 meter in diameter), containing bryozoans and diverse microorganisms, were found in the Hanjiang river in China. In this study, we investigated these bryozoan-dominated microbial complexes using metagenomic sequencing, metabolomic profiling, and complementary approaches. Results The results revealed that the microbial structure dominated by bryophytes included eukaryotes such as fungi, microalgae, and small parasites etc., as well as prokaryotes such as bacteria and viruses. All members form a highly integrated consortium that collaboratively maintains community function and stability, which displayed the shape and structure of a regular microbial complex and increased in size. Additionally, the results suggested the predominately presence of Proteobacteria were particularly abundant and are known to produce bryostatins in response to bryozoan signals. Interesting, it was implied that a decrease or even a loss in virulence occurs when pathogenic microorganisms become members of the complex. This is the first study focusing on the composition and metabolites of the unusual microorganism complex. The results discovered the close and intriguing connections within the microbial communities dominated by the ancient organism bryozoan through multi-omics technologies. It showed that both eukaryotes and prokaryotes can coordinate or compete, thereby forming a stable composite structure.

The Enterobacteriaceae pathogens like E. coli, Shigella, Klebsiella and Salmonella cause a wide range of gastrointestinal and other mucosal infections when the contaminated food and water are consumed. Conventional microbiological methods are time-consuming and laborious process to detect these pathogens. Molecular assays are fast and sensitive to detect the pathogens. But a potential target for the detection is very essential to develop a molecular assay for the rapid detection. In this study, we explored the cpn60 UT region as a potential target for the detection of significant Enterobacteriaceae members like E. coli, Shigella sp, Salmonella, Klebsiella etc. We did the phylogenetic analysis of cpn60 UT region among the Enterobacteriaceae pathogenic groups. This analysis has revealed the presence of 13 clusters and two oligonucleotide regions with highest nucleotide conservation. Targeting these regions we have designed a set of oligonucleotide primer pairs and developed a SYBR green real time PCR assay to detect the Enterobacteriaceae pathogenic members. The degenerate oligonucleotide primers designed amplified a 132 bp UT region of cpn60 gene present in Enterobacteriaceae pathogens. The sensitivity of the assay was initially investigated by 10-fold dilution series of overnight broth culture of E. coli ATCC 10536 and had a detection limit of 101 CFU/mL. The specificity was assessed by comparing the amplification plot of a set of Enterobacteriaceae pathogens and non-Enterobacteriaceae members like Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes) and found to detect all the Enterobacteriaceae cultures included in the study whereas amplification was not seen in S. aureus and L. monocytogenes.

The emergence of antimicrobial resistant pathogens has led to increase in mortality rate, longer hospital stays, and need for complex and expensive treatment regimens. However, it is not very clear from where does the bacteria get the building blocks to develop resistance to these synthetic antimicrobial agents which have been integral part of modern medicine. One hypothesis is that non-coding cryptic genes without any vital function or positive contribution to fitness act as versatile endogenous genetic repertoires that help in bacterial adaptation and evolution of new phenotypes when conditions change. This study reports the presence of putative cryptic metallo beta lactamase gene in the marine bacterium Cytobacillus oceanisediminis isolated from the Gulf of Mannar, India. Using in silico, in vitro and in vivo assay, we demonstrate that putative cryptic metallo beta lactamase encodes an enzyme that can cleave only imipenem, has two characteristic domains: HXHXDH and TPGH, but does not share homology with the other reported Metallo-beta-Lactamase genes. The findings underscore the importance for comprehensive screening of putative cryptic antimicrobial resistance genes to fully address and mitigate the challenges posed by antimicrobial resistance.

Microorganisms, found in every environment, play an important role in recycling matter and providing energy to the ecosystem. Here, we used 18S and 16S metabarcoding to study bacteria, cyanobacteria and protist community diversity and functional ecology among different environments of typical lake, river, marine and soil from the Yangtze Delta of China. The results showed that the similarity of cyanobacteria and protozoa communities in soil and aquatic environments was both higher than that of bacteria and microalgae communities. While the diversity of cyanobacteria and bacteria in lake was higher than that in river, marine and soil, the diversity of microalgae and protozoa in river was higher than that in lake, marine and soil. The {beta} diversity of cyanobacteria and bacteria marked differences in lake from other environments. But The distribution of dominant families of cyanobacteria, bacteria, microalgae and protozoa is similar to diversity. While significant positive correlations were found among dominant species of cyanobacteria, protozoa, bacteria and microalgae in lake the dominant species among bacteria, microalgae and protozoa in river, marine and soil all showed more negative correlations. Our study provides the basis for understanding the functional ecology of microbes in the micro-food webs of different environments.

Bacterial cellulose (BC) is a biopolymer that comes from natural sources. It has high purity, crystallinity, mechanical strength, and biocompatibility, which makes it suitable for various biomedical and industrial uses. Unlike plant cellulose, BC does not contain lignin or hemicellulose. This results in better material quality and simpler processing. However, producing BC on a large scale is limited by high production costs, expensive culture media, and dependence on a few bacterial strains. It is essential to find cost-effective production methods and alternative microbial sources to expand its commercial use. This study looked at the cellulose-producing ability of Acetobacter diazotrophicus, a safe and relatively unexplored bacterium, under different growth conditions. We compared bacterial growth and cellulose production using Hestrin-Schramm (HS) medium, the standard for BC production, and LB supplemented with glucose (LB+Glucose), which we explored as a more affordable option. We analyzed growth rates, inoculum age, and pH levels to find the best conditions for cellulose production. We observed faster bacterial growth in HS medium, with a doubling time of 3.906 hours, compared to 6.241 hours in LB+Glucose medium. Cellulose production was greatly affected by inoculum age, with successful synthesis from cultures that were agitated for 36 to 40 hours. The highest cellulose yield was at pH 6.0 in HS medium (4.6 mg/mL) and at pH 5.5 in LB+Glucose medium (3.6 mg/mL). FTIR analysis confirmed the presence of characteristic functional groups of bacterial cellulose. These results suggest that Acetobacter diazotrophicus is a promising and cost-effective option for producing bacterial cellulose and highlight the importance of medium composition, inoculum age, and pH for optimizing production.

Hypervirulent Klebsiella pneumoniae represents a growing clinical threat, particularly where multidrug resistance limits treatment options. Here, we report the isolation and characterization of three novel lytic phages from Benin (Kp1Bj_HH11_M23, Kp2Bj_LN294_M23, and Kp10Bj_LN54_14). These Myoviruses, belonging to the genera Marfavirus and Jiaodavirus, display broad host range activity against multidrug-resistant and hypervirulent K. pneumoniae strains. Genomic analysis confirmed the absence of virulence and antimicrobial resistance genes. The phages exhibit rapid adsorption, short latency periods, and high burst sizes (119-2208 PFU/cell). All three phages significantly inhibited biofilm formation and reduced established biofilms in vitro. Their stability across a wide range of temperatures and pH further supports their potential for therapeutic development. Together, these data highlight the value of locally sourced phages as candidates for tackling region-specific antimicrobial resistance challenges.

Bacterial melanin are macromolecules found in nature that provide a wide range of biological functions, including pigmentation, resistance to radiation, scavenging of free radicals, thermoregulation and protected from oxidative stress and harmful heavy metals. The melanin is crucial for pathogenesis and bacterial survival in a variety of circumstances, and they can also influence how bacteria interact with other organisms. Usually, bacteria produce the melanin is either black or brown colour. The produced melanin has excellent properties like antimicrobial, antioxidant, photoprotective and antibiofilm. This is a report on Corynebacterium amycolatum melanin-producing bacteria isolated from the marine sediment of Thiruvanmiyur beach in Tamil Nadu, India. Corynebacterium amycolatum was screened using tyrosine basal broth (TBB), and UV-visible spectroscopy, FTIR, and SEM were used to analyse the extracted melanin. The non-pathogenic nature of the Cornynebacterium amycolatum strain was verified through antibiotic sensitivity profiling. The cosmetic potential was evaluated using antioxidant and SPF assays. Corynebacterium amycolatum predominantly uses the DOPA pathway for melanin production, was confirmed using kojic acid inhibitor study. The in vitro studies on mouse fibroblast cell line (L929) and in vivo studies on zebra fish embryos shows non-cytotoxicity using this melanin, even in lower concentration confirms its potential to use in cosmetic formulation. This research aims to demonstrate that bacterial melanin is safe for the environment and has qualities that make it safer and more effective in cosmetics.

Vibrio species are ubiquitous in shrimp hatchery systems, where their opportunistic pathogenic characteristics threaten the vulnerable larval stage, and antibiotics are routinely applied for disease control and prevention. Therefore, studying Vibrio composition and its antibiotic resistance burden is critical for hatchery environments and public health. The present study characterized Vibrio composition and its antimicrobial resistance patterns in water-flow systems and postlarvae from shrimp hatcheries in Coxs Bazar, Bangladesh. The findings revealed that Vibrio composition in these hatchery systems comprised nineteen different species. The Harveyi clade was the most dominant group, including V. alginolyticus, V. rotiferianus, and V. harveyi. The Vibrio isolates were highly resistant to erythromycin, ampicillin, and streptomycin, while remaining susceptible to chloramphenicol, nitrofurantoin, and gentamicin. Seven antibiotics, including trimethoprim-sulfamethoxazole, ampicillin, and erythromycin, were categorized as poorly active, suggesting their inefficacy against vibriosis outbreaks. More than 50% of the isolates had multiple antibiotic resistance index greater than 0.2 and 67.8% were categorized as multidrug-resistant. Both indices progressively increased along the water-flow systems and postlarvae had the highest values, pointing to antibiotic contamination within hatchery systems. Resistance gene screening identified sul2 and tetC as most prevalent, while association analysis showed cross-resistance patterns among antibiotics, concordance between trimethoprim-sulfamethoxazole and streptomycin resistance with the presence of sul2 and strA-strB genes, and co-selection of sul2, ermB, and strA-strB genes. The findings provide baseline data on Vibrio diversity and resistance burden, and inform antibiotic stewardship policies and monitoring strategies that support a One Health approach in shrimp hatchery systems.

ObjectiveTo investigate the combined effects of multiple drugs and provide more therapeutic options for invasive fungal infections, this study evaluated the in vitro susceptibility of Aspergillus spp., Candida auris, Cryptococcus neoformans, and Exophiala dermatitidis to pibrentasvir (PIB) in combination with itraconazole (ITR), voriconazole (VOR), posaconazole (POS), or fluconazole (FLU). MethodsAccording to the M27-A3 and M38-A2 guidelines established by the Clinical and Laboratory Standards Institute (CLSI), the in vitro antifungal activities of PIB combined with ITR, VOR, POS, or FLU against 78 clinical isolates, including Aspergillus spp., E. dermatitidis, C. auris, and C. neoformans, were determined. The minimum inhibitory concentrations (MICs) and fractional inhibitory concentration indices (FICIs) were calculated to evaluate the synergistic effects. ResultsPIB alone exhibited no antifungal activity. Significant synergistic effects were observed when PIB was combined with azole antifungal agents. The PIB-POS combination showed synergistic effects against Aspergillus spp. (27/41, 65.90%), C. auris (9/10, 90.00%), C. neoformans (2/9, 22.20%), and E. dermatitidis (11/18, 61.11%). The PIB-ITR combination also showed synergistic effects against Aspergillus spp. (18/41, 43.9%), C. auris (9/10, 90.0%), C. neoformans (2/9, 22.2%), and E. dermatitidis (10/18, 55.5%). Synergistic effects were less frequently observed with the PIB-VOR or PIB-FLU combinations, and no antagonistic effects were observed. ConclusionThis study demonstrates that PIB acts as an azole sensitizer, with the strongest synergistic effects observed when combined with POS or ITR, providing a new research direction for combination therapy against invasive fungal infections.

A Gram-stain-negative, aerobic, moderately halophilic, non-motile, rod-shaped bacterium, designated MC10-B4T, was isolated from the coral Oulastrea crispata collected from Hong Kong waters. The strain showed the ability to persist in a coral holobiont during an eight-month monitoring period in a field trial, indicating a long-term association with the coral host. Growth occurred at 10-37 {degrees}C (optimum, 28-30 {degrees}C), at NaCl concentrations up to 4.0% (w/v; optimum, 0.5-2.0%), and at pH 7.0-10.0 (optimum, pH 7.0-8.0) under standard laboratory conditions. Colonies on Marine Agar 2216 were round, smooth, convex, white, opaque, and had entire margins. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MC10-B4T belongs to the genus Ruegeria and shares the highest sequence similarity with Ruegeria conchae TW15T (99.64%), followed by Ruegeria profundi ZGT108T (98.92%), Ruegeria lacuscaerulensis ITI-1157T (98.46%), Ruegeria denitrificans CECT 5091T (98.31%), Ruegeria arenilitoris G-M8T (97.69%), Ruegeria atlantica NBRC 15792T (97.55%), Ruegeria halocynthiae MA1-6 (97.11%), Ruegeria atlantica IAM 14463T (96.98%), Ruegeria meonggei MA-E2-3 (96.83%), and Ruegeria pomeroyi DSS-3T (96.65%). Genome sequencing revealed a genome size of 4.55 Mbp and a DNA G+C content of 57.5 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain MC10-B4T and Ruegeria conchae TW15T were 77.6% and 18.2-22.8%, respectively. These values are below the recommended thresholds for species delineation (95% and 70%). Strain MC10-B4T shares the same predominant fatty acid, summed feature 8 (consisting of C18:1 {omega}7c and/or C18:1 {omega}6c), with other Ruegeria strains; however, it also contains C19:0 cyclo {omega}8c, which was not detected in Ruegeria conchae TW15T and Ruegeria pomeroyi DSS-3T. The type strain is MC10-B4T (= GDMCC 1.6889T = KCTC 18785T).

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.

Comamonas testosteroni TA441 is a model aerobic steroid-degrading bacterium whose sterane degradation pathway has been elucidated in greater detail than that of any other bacterium to date. Similar pathways have been identified in many bacterial genera, including members of Proteobacteria and Actinomycetota, such as Mycobacterium tuberculosis. Steroid degradation genes in TA441 form an approximately 120-kb mega-cluster, and 40 genes involved mainly in the degradation of the sterane structure and the C20?C22 portion of the side chain at C17 have been identified. Here, we present the complete genome sequence of TA441 together with our findings on its steroid degradation genes. We also compare the genes in the mega-cluster with corresponding genes in other Comamonas strains, ?-Proteobacteria other than Comamonas, ?-Proteobacteria, ?-Proteobacteria, and Actinomycetota using CAGECAT to analyze similarities and differences in steroid degradation genes among these bacteria.

The vancomycin-resistant Enterococcus faecium (VREfm) has been declared as a high priority pathogen by World Health Organisation (WHO). It presents a major therapeutic challenge to healthcare, remaining with limited antibiotic options. Bacteriophage therapy has emerged as a promising alternative for combating antimicrobial-resistant pathogens. This study reports the isolation and characterization of bacteriophages active against VREfm and MDR E. faecium clinical isolates from India. Two bacteriophages, NIMS_EF375_N69_P9 ({Phi}1) and NIMS_EF375_N74_P12 ({Phi}2), were isolated from sewage by enrichment using MDR E. faecium strain EF375 as the propagation host. Lytic activity was confirmed by spot assay and double-layer soft agar plaque assay; both phages produced clear plaques of 1.5-2.0 mm. Transmission electron microscopy showed icosahedral heads with long non-contractile tails, presenting siphovirus-like morphotype within the class Caudoviricetes. Host range was assessed against 10 MDR E. faecium isolates (including the propagation host), 2 of which were VREfm. {Phi}1 lysed 5 of 10 isolates, including the VanA-type VREfm, whereas {Phi}2 lysed 7 of 10, including both VanA- and VanB-type VREfm. In time-kill assays against EF375 at an MOI of 1, {Phi}1 produced effective decline in optical density sustained through 24 h (~80% reduction relative to the untreated control), whereas {Phi}2 suppressed bacterial growth with only ~50% reduction. To the best of our knowledge, this study represents the first report from India on the isolation and characterization of bacteriophages active against clinical VREfm isolates. The broader host range of {Phi}2 and the stronger killing kinetics of {Phi}1 suggest complementary roles in a phage cocktail, warranting genomic characterization and in vivo evaluation.

Mesozooplankton is a vital component of pelagic food webs, serving as a key trophic link between primary producers and higher marine organisms, and driving coastal and oceanic biogeochemical cycles. This study investigated the abundance, biovolume, community structure, and spatial distribution of mesozooplankton in relation to hydrographic variables in the Northeastern Arabian Sea (NEAS 15{whitebullet}N - 21{whitebullet}N) during the Spring Intermonsoon (March 2019) on board FORV Sagar Sampada. Sampling across 14 stations comparing coastal (> 50 m) and oceanic (< 1000 m) waters revealed distinct environmental gradients. Both Sea Surface Temperature (SST: 24.33{whitebullet}C - 28.68{whitebullet}C) and Sea Surface Salinity (SSS: 35.25 - 36.65) exhibited a declining trend toward northern latitudes, whereas Dissolved Oxygen (DO: 4.55 - 5.24 ml * L-1) increased northward. Chlorophyll concentrations were significantly higher in coastal regions (mean 1.45 mg * m-3) than in oceanic waters (mean 0.36 mg * m-3). Correspondingly, mesozooplankton biovolume (excluding large gelatinous taxa) was threefold higher in coastal waters (mean 1.39 mL * m-3) compared to oceanic waters (mean 0.38 mL * m-3). Mean numerical abundance was also higher coastally (605 ind. * m-3) than oceanically (524 ind. * m-3). A total of 21 mesozooplankton taxa were identified. Copepods dominated the community structure across both regimes (coastal: 61.07%; oceanic: 56.69%). In coastal waters, Chaetognatha (14.76%), Cladocera (9.39%), and Decapod larvae (5.69%) were the secondary drivers, with localised salp and siphonophore swarms observed at 17{whitebullet} - 19{whitebullet}N. In oceanic waters, Ostracoda (22.93%) and Salpidae (12.51%) were major secondary contributors. These results indicate that during the Spring Intermonsoon, the NEAS exhibits moderate productivity representative of a receding winter cooling regime.

The marine bacterium Vibrio alginolyticus and the food poisoning bacterium V. parahaemolyticus possess two types of flagella in one cell: proton-driven lateral flagella (Laf) extending from the periphery of the cell body, and sodium ion-driven polar flagella (Pof) extending from a cell pole. For swimming in seawater they use polar flagella, suppressing the expression of lateral flagella. When they attach to the body surface of fish or intestinal tract, lateral flagella are induced, allowing it to crawl along the surface or through mucus. The dynamometer hypothesis, which proposes that polar flagella sense rotation and control the expression of lateral flagellar genes, has been widely accepted. However, how rotation is sensed and how expression is controlled remains unclear. Although swarming has recently been analyzed by physical, biological, or biochemical perspectives, it remains unclear how this motility is controlled, or which substances and conditions are necessary for swarming ability. In this study, we discovered that adding gelatin to agar medium promotes swarming on the agar surface by the lateral flagella of Vibrio. Our data suggested that surfactants or viscous polysaccharides secreted extracellularly are important for promoting swarming on the agar surface and we identified that swarming is likely to be driven by S (social)-motility, in which bacteria move by interacting with each other, and A (adventure)-motility, in which bacteria move by interacting with the agar surface. Our study provides clues that help clarify the mechanism of bacterial swarming IMPORTANCEWe discovered that adding gelatin to hard agar medium promoted swarming on agar surfaces by the lateral flagella of Vibrio cells. The surfactants or viscous polysaccharides secreted extracellularly seem to be important for swarming ability on agar surfaces. We proposed that the swarming is thought to occur through S(social)-motility, where cells move by interacting with cell bodies each other, and A(adventure)-motility, where cells move by interacting with the agar surface and cell body. The present study should provide the clues to clarify the mechanism of bacterial swarming and how to move in a viscous environment.

Pectobacterium comprises Gram-negative, pectinolytic plant pathogens that cause soft rot and blackleg diseases on numerous economically important crops. During sampling in 2018 in taro (Colocasia esculenta) fields in Hawaii, two bacterial strains PL152T and PL155 were isolated from taro. Peliminary molecular characterization placed both strains in the genus Pectobacterium. However, their taxanomic postions could not be resoved at the species level. A polyphasic taxonomic approach incorporating phenotypic characterization, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), whole genome based phylogeny, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) was therefore employed to determine their taxonomic status. Phylogenetic and phylogenomic analyses consistently placed PL152 and PL155 together in a distinct and well supported lineage within the genus Pectobacterium, separate from currently recognized species. The two strains exhibited high genomic relatedness to each other, whereas the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, were lower than the accepted species delineation thresholds of 95% and 70%, respectively, supported the delineation of a novel bacterial species. The genomic characteristic of PL152 included a genome size of 5.02 Mb and a DNA G+C content of 52%. Collectively, based on a polyphasic approach including phylogenetic, phlyogenomic, biochemical, and phenotypic evidences supports the classification of strains PL152 and PL155 as representatives of a novel species within the genus Pectobacterium, for which the name Pectobacterium pacificum sp. nov. is proposed. Strain PL152 (ICMP 2436274, LMG 32536) is designated as the type strain.

The obscure life histories of many kickxellalean genera remain a bottleneck for comprehensive taxon sampling and phylogenetic reconstruction of the order. While Kickxellales has long been regarded as saprobes inhabiting soil or dung, the discovery of "amphibious fungi" such as Unguispora, which exhibits dimorphic growth between the animal gut and feces, suggests a cryptic gut-dwelling stage within these genera. Given its phylogenetic proximity to Unguispora, ecophysiological traits of Linderina were investigated to evaluate its potential association with the animal gut. Two isolates of L. macrospora were obtained from Japanese soil, representing the first record of this species in Japan. Physiological assays revealed that the optimal temperature for both vegetative growth and sporulation was 25-30 {degrees}C. Furthermore, comparative growth assays on different media demonstrated that sporocladium abundance per sporangiophore is sensitive to nutrient availability, and nutrient-poor media were determined to be the most suitable for evaluating morphological characterization. Under anaerobic, nutrient-rich conditions which are known to induce yeast-like growth in Unguispora, sporangiospores of L. macrospora produce arthrospores. Although marked morphological plasticity was observed during the arthrospore formation, the occurrence of yeast-like unicellular proliferation suggests a potential relationship with the animal gut. Additionally, vegetative growth and sporulation were markedly inhibited by white light exposure; notably, a lethal effect on growth was observed during incubation at 20 {degrees}C, indicating that the natural niche of the species is restricted to light-shielded environments. Our findings will help to elucidate the cryptic life cycles and evolutionary trajectories within Kickxellales.

The human gut is home to more than 100 trillion bacteria, where various environmental factors, such as nutrients and gases, exist and influence them. The levels of fermentation gases in the gut, including CO2, fluctuate depending on the host's diet. However, how CO2, an environmental factor, affects the physiological states of gut anaerobes remains poorly understood, but CO2 is often utilized in their cultivation. Akkermansia muciniphila (A. muciniphila) is widely studied as a beneficial human gut microbe. However, the effects of CO2 on the physiological state of A. muciniphila are not fully understood. In this study, we focused on CO2 as an environmental factor and aimed to clarify how CO2 affects the physiological state of A. muciniphila. Culture experiments showed that compared with the absence of CO2, the presence of CO2 significantly increased the growth of A. muciniphila. The results of the metabolomic analysis revealed distinct metabolic profiles in the presence or absence of CO2. The tracing analysis using stable 13C-labelled CO2 demonstrated that CO2 was incorporated into various metabolites in the TCA cycle, the de novo purine/pyrimidine synthesis pathways, and other pathways. In addition, the activity of pyruvate carboxylase, which catalyses an anaplerotic reaction involving CO2 fixation to supply oxaloacetate (OAA) to the TCA cycle, was significantly higher in A. muciniphila cells grown in the presence of CO2. Moreover, supplementation with OAA increased the growth of A. muciniphila in a dose-dependent manner, mirroring the effects of CO2 supplementation. These findings suggest that A. muciniphila utilizes CO2 for an anaplerotic reaction in the TCA cycle and other pathways involving CO2 incorporation, facilitating metabolic flux to promote its robust growth.