Journal of Microbiology最新文献

Vibrio parahaemolyticus has two flagellar systems, the polar flagellum and lateral flagella, which are both intricately regulated by a multitude of factors. CalR, a LysR-type transcriptional regulator, is sensitive to calcium (Ca) and plays a crucial role in regulating the virulence and swarming motility of V. parahaemolyticus. In this study, we have demonstrated that the deletion of calR significantly enhances the swimming motility of V. parahaemolyticus under low Ca conditions but not under high Ca conditions or in the absence of Ca. CalR binds to the regulatory DNA regions of flgM, flgA, and flgB, which are located within the polar flagellar gene loci, with the purpose of repressing their transcription. Additionally, it exerts an indirect negative control over the transcription of flgK. The overexpression of CalR in Escherichia coli resulted in a reduction in the expression levels of flgM, flgA, and flgB, while having no impact on the expression of flgK. In summary, this research demonstrates that the negative regulation of V. parahaemolyticus swimming motility by CalR under low Ca conditions is achieved through its regulation on the transcription of polar flagellar genes.

Klebsiella pneumoniae, a Gram-negative opportunistic pathogen, is increasingly resistant to carbapenems in clinical settings. This growing problem necessitates the development of alternative antibiotics, with phage therapy being one promising option. In this study, we investigated novel phages targeting carbapenem-resistant Klebsiella pneumoniae (CRKP) and evaluated their lytic capacity against clinical isolates of CRKP. First, 23 CRKP clinical isolates were characterized using Multi-Locus Sequence Typing (MLST), carbapenemase test, string test, and capsule typing. MLST classified the 23 K. pneumoniae isolates into 10 sequence types (STs), with the capsule types divided into nine known and one unknown type. From sewage samples collected from a tertiary hospital, 38 phages were isolated. Phenotypic and genotypic characterization of these phages was performed using Random Amplification of Polymorphic DNA-PCR (RAPD-PCR), transmission electron microscopy (TEM), and whole genome sequencing (WGS) analysis. Host spectrum analysis revealed that each phage selectively lysed strains sharing the same STs as their hosts, indicating ST-specific activity. These phages were subtyped based on their host spectrum and RAPD-PCR, identifying nine and five groups, respectively. Fourteen phages were selected for further analysis using TEM and WGS, revealing 13 Myoviruses and one Podovirus. Genomic analysis grouped the phages into three clusters: one closely related to Alcyoneusvirus, one to Autographiviridae, and others to Straboviridae. Our results showed that the host spectrum of K. pneumoniae-specific phages corresponds to the STs of the host strain. These 14 novel phages also hold promise as valuable resources for phage therapy against CRKP.

The emerging drug resistance and lack of safer and more potent antifungal agents make Candida infections another hot topic in the healthcare system. At the same time, the potential of plant products in developing novel antifungal drugs is also in the limelight. Considering these facts, we have investigated the different extracts of the flowers of Hibiscus rosa-sinensis of the Malvaceae family for their antifungal efficacy against five different pathogenic Candida strains. Among the various extracts, the chloroform extract showed the maximum zone of inhibition (26.6 ± 0.5 mm) against the Candida albicans strain. Furthermore, the chloroform fraction was isolated, and a sterol compound was identified as β-sitosterol. Mechanistic studies were conducted to understand the mechanism of action, and the results showed that β-sitosterol has significant antifungal activity and is capable of interrupting biofilm formation and acts by inhibiting ergosterol biosynthesis in Candida albicans cells. Microscopic and molecular docking studies confirmed these findings. Overall, the study validates the antifungal efficacy of Candida albicans due to the presence of β-sitosterol which can act as an effective constituent for antifungal drug development individually or in combination.

Two novel bacterial strains, 273M-4^T and Sam97^T, were isolated from seawater in the Yellow Sea, Muan-gun, South Korea, and identified as members of the genus Thalassotalea. Both strains were Gram-stain-negative, aerobic, rod-shaped, non-motile, non-flagellated, and oxidase- and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains 273M-4^T and Sam97^T were most closely related to Thalassotalea ponticola KCTC 42155^T, with sequence similarities of 97.5% and 98.3%, respectively. Optimal growth for strain 273M-4^T occurred at 25-30 °C, pH 7.0, and 2% NaCl, while strain Sam97^T grew optimally at 30 °C, pH 8.0, and 2% NaCl. Genome sizes of strains 273M-4^T and Sam97^T were 3.37 and 3.31 Mb, with DNA G + C contents of 41.0 mol% and 42.9 mol%, respectively. The orthologous average nucleotide identity (OrthoANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 71.6% and 24.4%, respectively, indicating that they are distinct species. Further genomic analyses of these two strains revealed OrthoANI values of < 73.5% and dDDH values of < 26.7% within the genus Thalassotalea, suggesting their distinctiveness from other Thalassotalea species. The predominate fatty acids of strains 273M-4^T and Sam97^T were summed feature 3 (consisting of C_16:1 ω7c/C_16:1 ω6c) and C_16:0. All strains contained phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids and ubiquinone-8 (Q-8) as the primary respiratory quinone. Based on phenotypic, phylogenetic, genotypic, and chemotaxonomic data, strains 273M-4^T (= KCTC 8644^T = LMG 33695^T) and Sam97^T (= KCTC 8645^T = LMG 33694^T) represent novel species of the genus Thalassotalea, named Thalassotalea aquiviva sp. nov. and Thalassotalea maritima sp. nov..

The production of recombinant proteins in Escherichia coli is often challenged by cytoplasmic expression due to proteolytic degradation and inclusion body formation. Extracellular expression can overcome these problems by simplifying downstream processing and improving protein yields. This study aims to compare the efficiency of two Bacillus subtilis chitosanase signal peptides in mediating extracellular secretion in E. coli. We identified a naturally occurring mutant signal peptide (mCsn2-SP) from B. subtilis CH2 chitosanase (CH2CSN), which is characterized by a deletion of six amino acids in the N-region relative to the signal peptide (Csn1-SP) from B. subtilis CH1 chitosanase (CH1CSN). The CH1CSN and CH2CSN genes were cloned into the pET-11a vector and protein secretion was evaluated in E. coli BL21(DE3) host cells. Expression was induced with 0.1 mM and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30 °C for one and three days. CH2CSN showed higher secretion levels compared to CH1CSN under all experimental conditions, especially with 0.1 mM IPTG induction for 3 days, which resulted in a 2.37-fold increase in secretion. Furthermore, it was demonstrated that mCsn2-SP is capable of secreting human Cu,Zn-superoxide dismutase (hSOD) in E. coli BL21(DE3) and successfully translocating it to the periplasmic region. This study represents the inaugural investigation into the utilisation of a naturally modified signal peptide, thereby corroborating the assertion that signal peptide deletion variants can influence protein secretion efficiency. Furthermore, the findings substantiate the proposition that such variants can serve as a viable alternative for the secretion of heterologous proteins in E. coli.

Over the past two decades, as the importance of gut microbiota to human health has become widely known, attempts have been made to treat diseases by correcting dysbiosis of gut microbiota through fecal microbiota transplantation (FMT). Apart from current knowledge of gut microbiota, FMT to treat disease has a long history, from the treatment of food poisoning in the fourth century to the treatment of Clostridioides difficile infections in the twentieth century. In 2013, FMT was recognized as a standard treatment for recurrent C. difficile because it consistently showed high efficacy. Though recurrent C. difficile is the only disease internationally recognized for FMT efficacy, FMT has been tested for other diseases and shown some promising preliminary results. Different FMT methods have been developed using various formulations and administration routes. Despite advances in FMT, some issues remain to be resolved, such as donor screening, manufacturing protocols, and unknown components in the fecal microbiota. In this review, we discuss the mechanisms, clinical indications, methods, and challenges of current FMT. We also discuss the development of alternative therapies to overcome the challenges of FMT.

Two Gram-stain-positive, facultatively anaerobic, non-hemolytic, coccoid-shaped bacterial strains, designated MS01^T and MS02, were isolated from cabbage watery kimchi in the Republic of Korea. Cellular growth occurred at 5-25 ℃ (optimum, 20 ℃), pH 5-8 (optimum, pH 7) and in the presence of 0-5% (w/v) NaCl (optimum, 1%). Results of 16S rRNA gene-based phylogenetic analyses showed that strains MS01^T and MS02 shared identical sequences, clustered within the Leuconostoc clade in phylogenetic trees, and were most closely related to Leuconostoc inhae IH003^T and Leuconostoc gasicomitatum LMG 18811^T with sequence similarities of 98.74%. The complete whole-genome sequences of strains MS01^T and MS02 measured 2.04-2.06 Mbp and harbored a 50.6 kb plasmid, with DNA G + C contents of 37.7% for both. Based on average nucleotide identities (ANI) and digital DNA-DNA hybridization (dDDH) values, both strains were confirmed to belong to the same species but showed ≤ 85.9% ANI and ≤ 29.9% dDDH values to other Leuconostoc species, indicating that they represent a novel species. Metabolic pathway reconstruction revealed that both strains perform heterolactic acid fermentation, producing lactate, acetate, and ethanol. Chemotaxonomic analyses, including cellular fatty acids, polar lipids, and peptidoglycan amino acid, confirmed the inclusion of both strains within the genus Leuconostoc. Based on the phylogenetic, genomic, and phenotypic characterization, strains MS01^T and MS02 were considered to represent a novel species within the genus Leuconostoc, for which the name Leuconostoc aquikimchii sp. nov. is proposed with MS01^T (= KACC 23748^T = JCM 37028^T) as the type strain.

The envE gene of Salmonella enterica serovar Typhimurium is encoded within Salmonella Pathogenicity Island-11 (SPI-11) and is located immediately upstream of the virulence gene msgA (macrophage survival gene A) in the same transcriptional orientation. To date, the characteristics and roles of envE remain largely unexplored. In this study, we show that EnvE, a predicted lipoprotein, is localized on the outer membrane using sucrose gradient ultracentrifugation. Under oxidative stress conditions, envE transcription is suppressed, while msgA transcription is induced, indicating an inverse correlation between the mRNA levels of the two neighboring genes. Importantly, inactivation of envE leads to constitutive transcription of msgA regardless of the presence of oxidative stress. Moreover, trans-complementation of the envE mutant with a plasmid-borne envE fails to prevent the induction of msgA transcription, suggesting that envE functions as a cis-regulatory element rather than a trans-acting factor. We further show that both inactivation and complementation of envE confer wild-type levels of resistance to oxidative stress by ensuring the expression of msgA. Our data suggest that the S. enterica envE gene encodes an outer membrane lipoprotein, and its transcription represses msgA expression in a cis-acting manner, probably by transcriptional interference, although the exact molecular details are yet unclear.

Amid rising environmental concerns, attempts have been made to produce glycolic acid (GA) using microbial processes with renewable carbon resources instead of using chemicals. The Dahms pathway for GA production uses xylose as a substrate and consists of relatively simple enzymatic steps. However, employing it leads to a decrease in cell growth and GA productivity. Systematically identifying and addressing metabolic bottlenecks in the Dahms pathway are essential for efficient glycolic acid (GA) production have not yet been performed. Through metabolic flux balance analysis, we found that insufficient aldehyde dehydrogenase (AldA) activity lowers GA production and negatively affects cell growth due to reduced energy production. Thus, we discovered a novel AldA isolated from Buttiauxella agrestis (BaAldA) demonstrated a 1.69-fold lower K_M and a 1.49-fold higher turnover rate (k_cat/K_M) than AldA from Escherichia coli (EcAldA). GA production in E. coli harboring BaAldA was 1.59 times higher than in the original strain. Fed-batch fermentation of E. coli harboring BaAldA produced 22.70 g/L GA with a yield of 0.497 g/g_xylose (98.2% of the theoretical maximum yield in the Dahms pathway), showing a higher final yield for GA than previously reported in E. coli. Our novel BaAldA enzyme shows great potential for the production of GA using microorganisms or enzymes. Furthermore, our approach to identifying metabolic bottlenecks using flux balance analysis could be utilized to enhance the microbial production of various desirable products in future studies.