Peptidoglycan Research Articles

Low Voltage-Enhanced Mechano-Bactericidal Biopatch.

Mechano-bactericidal strategies represent a safe and sustainable method for preventing microbial contamination in the postantibiotic era. However, their effectiveness against Gram-positive bacteria (≤55%) is still limited due to the thick peptidoglycan layer in their cell walls. Herein, an intelligent biomimetic nanopillared biopatch is developed. It is assisted by low-voltage (8 V) electrical stimulation from TENG and significantly enhances antibacterial efficacy (>99%) against three types of stubborn Gram-positive bacteria. These collaborative antibacterial behaviors are solely based on purely physical actions, thus avoiding the risk of triggering bacterial resistance. Moreover, the slight mechanical energy generated by human physiological activities is converted into a power source, exhibiting energy-efficient, eco-friendly, and sustainable features. The conductive hydrogel in the biopatch can also act as an intelligent temperature sensor, monitoring, and real-time assessment of wound conditions. This intelligent biopatch holds immense potential for efficient healing and safe management of both acute and chronic wound infections.

Halobacillus rhizosphaerae sp. nov., a moderately halophilic bacterium with protease activities isolated from the rhizosphere soil of the mangrove Acanthus ebracteatus.

A Gram-positive, moderately halophilic, rod-shaped and facultatively anaerobic strain T66T, was isolated from the rhizosphere soil of mangrove Acanthus ebracteatus in Guangxi, China. Strain T66T was observed to grow at 15-40℃ (optimum 30℃), pH 5-8 (optimum 6-7) and with 2-20% (w/v) NaCl (optimum 6%). The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain T66T to be closely related to Halobacillus litoralis SL-4T (98.72%), Halobacillus dabanensis D-8T (98.70%) and Halobacillus campisalis ASL-17T (98.66%). The phylogenetic analysis based on the 16S rRNA gene sequence showed that strain T66T belonged to the genus Halobacillus and formed a separate branch. The digital DNA-DNA hybridisation and average nucleotide identify values between strain T66T and its related species were 17.80-19.80% and 70.78-73.22%, respectively, which were lower than the threshold recommended for species delineation. The genomic DNA G + C content was 40.9%. The predominant isoprenoid quinone was MK-7, the cell-wall peptidoglycan contained meso-diaminopimelic acid, and the major fatty acids that accounted for more than 10.0% were anteiso-C15:0 (68.4%) and anteiso-C17:0 (13.4%). Phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids and an unidentified glycolipid were found in the polar lipid extraction. Strain T66T could hydrolyse casein and contains several genes (e. g. vanY, vpr, pbpG and elpC) involved in protease. Therefore, strain T66T is salt-tolerant and produces protease, and it may have development and utilisation value in flavouring agents and food curing. Based on phenotypic, chemotaxonomic data and genotypic characteristics, strain T66T represents a novel species of the genus Halobacillus, for which the name Halobacillus rhizosphaerae sp. nov. is proposed. The type strain is T66T (= JCM 36534T = MCCC 1K08701T).

Quinolone-mediated metabolic cross-feeding develops aluminium tolerance in soil microbial consortia

Aluminium (Al)-tolerant beneficial bacteria confer resistance to Al toxicity to crops in widely distributed acidic soils. However, the mechanism by which microbial consortia maintain Al tolerance under acid and Al toxicity stress remains unknown. Here, we demonstrate that a soil bacterial consortium composed of Rhodococcus erythropolis and Pseudomonas aeruginosa exhibit greater Al tolerance than either bacterium alone. P. aeruginosa releases the quorum sensing molecule 2-heptyl-1H-quinolin-4-one (HHQ), which is efficiently degraded by R. erythropolis. This degradation reduces population density limitations and further enhances the metabolic activity of P. aeruginosa under Al stress. Moreover, R. erythropolis converts HHQ into tryptophan, promoting the synthesis of peptidoglycan, a key component for cell wall stability, thereby improving the Al tolerance of R. erythropolis. This study reveals a metabolic cross-feeding mechanism that maintains microbial Al tolerance, offering insights for designing synthetic microbial consortia to sustain food security and sustainable agriculture in acidic soil regions.

Investigating novel Streptomyces bacteriophage endolysins as potential antimicrobial agents.

As antibiotic resistance has become a major global threat, the World Health Organization (WHO) has urgently called for alternative strategies for control of bacterial infections. Endolysin, a phage-encoded protein, can degrade bacterial peptidoglycan in the bacterial cell wall and disrupt bacterial growth. According to the WHO, there are only three endolysin products currently in clinical phase development. In this study we explored novel endolysins from Streptomyces phages as only a few of them have been experimentally characterized. Using several bioinformatics tools, we identified nine different combinations of functional enzymatic domain types from 250 Streptomyces bacteriophages possible endolysins. From these, three potential endolysins were selected for experimental characterization. All three showed positive results in degrading cell wall material and disrupting bacterial growth, indicating their potential as possible antimicrobial agents.

Nanostructure-Mediated Photothermal Effect for Reinforcing Physical Killing Activity of Nanorod Arrays.

The physical killing of bacteria based on surface topography has attracted much attention due to the sustainable and safe prevention of biofilm formation. However, the antibacterial efficiency of biomedical implants derived solely from nanostructures or microstructures is insufficient to combat bacteria against common infections, such as methicillin-resistant Staphylococcus aureus with thick cell walls. Herein, photothermal therapy is carried out in the presence of nanorod arrays to mitigate infection of biomedical implants. Different from traditional photothermal therapy relying on a photosensitizer, the photothermal effect is mediated by light traps rendered by the nanorod arrays, and consequently, the photosensitizer is not needed. Finite element simulations and experiments are performed to elucidate the light-to-thermal conversion mechanism. This photothermal platform, in conjunction with thermosensitive nitric oxide therapy, is applied to treat titanium implant infection. The nanostructure-mediated photothermal effect destroys bacterial cell walls by inhibiting peptidoglycan synthesis and increasing the membrane permeability by affecting fatty acid synthesis. Furthermore, the nanorods synergistically puncture the bacterial membrane easily as demonstrated by experiments and transcriptome analysis. The results provide insights into the development of efficient antibacterial treatment of implants by combining nanostructures and photothermal therapy.

Phage-Derived Endolysins Against Resistant Staphylococcus spp.: A Review of Features, Antibacterial Activities, and Recent Applications.

Antimicrobial resistance is a significant global public health issue, and the dissemination of antibiotic resistance in Gram-positive bacterial pathogens has significantly increased morbidity, mortality rates, and healthcare costs. Among them, Staphylococcus, especially methicillin-resistant Staphylococcus aureus (MRSA), causes a wide range of diseases due to its diverse pathogenic factors and infection strategies. These bacteria also present significant issues in veterinary medicine and food safety. Effectively managing staphylococci-related problems necessitates a concerted effort to implement preventive measures, rapidly detect the pathogen, and develop new and safe antimicrobial therapies. In recent years, there has been growing interest in using endolysins to combat bacterial infections. These enzymes, which are also referred to as lysins, are a unique class of hydrolytic enzymes synthesized by double-stranded DNA bacteriophages. They possess glycosidase, lytic transglycosylase, amidase, and endopeptidase activities, effectively destroying the peptidoglycan layer and resulting in bacterial lysis. This unique property makes endolysins powerful antimicrobial agents, particularly against Gram-positive organisms with more accessible peptidoglycan layers. Therefore, considering the potential benefits of endolysins compared to conventional antibiotics, we have endeavored to gather and review the characteristics and uses of endolysins derived from staphylococcal bacteriophages, as well as their antibacterial effectiveness against Staphylococcus spp. based on conducted experiments and trials.

Determining the rate-limiting processes for cell division in Escherichia coli

A critical cell cycle checkpoint for most bacteria is the onset of constriction when the septal peptidoglycan synthesis starts. According to the current understanding, the arrival of FtsN to midcell triggers this checkpoint in Escherichia coli. Recent structural and in vitro data suggests that recruitment of FtsN to the Z-ring leads to a conformational switch in actin-like FtsA, which links FtsZ protofilaments to the cell membrane and acts as a hub for the late divisome proteins. Here, we investigate this putative pathway using in vivo measurements and stochastic cell cycle modeling at moderately fast growth rates. Quantitatively upregulating protein concentrations and determining the resulting division timings shows that FtsN and FtsA numbers are not rate-limiting for the division in E. coli. However, at higher overexpression levels, they affect divisions: FtsN by accelerating and FtsA by inhibiting them. At the same time, we find that the FtsZ numbers in the cell are one of the rate-limiting factors for cell divisions in E. coli. Altogether, these findings suggest that instead of FtsN, accumulation of FtsZ in the Z-ring is one of the main drivers of the onset of constriction in E. coli at faster growth rates.

Amino acid variability at W194 of Staphylococcus aureus sortase A alters nucleophile specificity.

Bacterial sortases are a family of cysteine transpeptidases in Gram-positive bacteria of which sortase A (SrtA) enzymes are responsible for ligating proteins to the peptidoglycan layer of the cell surface. Engineered versions of sortases are also used in sortase-mediated ligation (SML) strategies for a variety of protein engineering applications. Although a versatile tool, substrate recognition by Staphylococcus aureus SrtA (saSrtA), the most commonly utilized enzyme in SML, is stringent and relies on an LPXTG pentapeptide motif. Previous structural studies revealed that the requirement of a glycine in the binding motif may be due to potential steric hindrance of amino acids possessing a β-carbon by W194, a tryptophan located in the β7-β8 loop of the enzyme. Here, we measured the effect of seven single point mutants of W194 (A, D, F, G, N, S, Y) saSrtA using a FRET-based activity assay. We found that while the LPXTG motif remains a requirement for initial proteolytic cleavage, the nucleophile specificity of our variants is altered. In particular, W194A and W194S saSrtA recognize a D-Ala nucleophile and are able to perform ligation reactions. Notably, an LPXT(D-Ala) peptide was not cleaved by either mutant enzyme. We hypothesize that these variants may potentially be utilized to develop an irreversible sortase-mediated reaction. Taken together, this experiment reveals new insight into sortase specificity and possible future SML strategies.

Molecular Docking of Endolysins for Studying Peptidoglycan Binding Mechanism.

Endolysins of bacteriophages, which degrade the bacterial cell wall peptidoglycan, are applicable in many industries to deal with biofilms and bacterial infections. While multi-domain endolysins have both enzymatically active and cell wall-binding domains, single-domain endolysins consist only of an enzymatically active domain, and their mechanism of peptidoglycan binding remains unexplored, for this is a challenging task experimentally. This research aimed to explore the binding mechanism of endolysins using computational approaches, namely molecular docking and bioinformatical tools, and analyze the performance of these approaches. The docking engine Autodock Vina 1.1.2 and the 3D-RISM module of AmberTools 24 were studied in the current work and used for receptor-ligand affinity and binding energy calculations, respectively. Two possible mechanisms of single-domain endolysin-ligand binding were predicted by Autodock Vina and verified by the 3D-RISM. As a result, the previously obtained experimental results on peptidoglycan binding of the isolated gamma phage endolysin PlyG enzymatically active domain were supported by molecular docking. Both methods predicted that single-domain endolysins are able to bind peptidoglycan, with Autodock Vina being able to give accurate numerical estimates of protein-ligand affinities and 3D-RISM providing comparative values.

Sulfonamide-Based Inhibition of the β-Carbonic Anhydrase from A. baumannii, a Multidrug-Resistant Bacterium.

Acinetobacter baumannii is a Gram-negative opportunistic pathogen responsible for severe hospital-associated infections. Owing to its ability to develop resistance to a wide range of antibiotics, novel therapeutic strategies are urgently needed. One promising approach is to target bacterial carbonic anhydrases (CAs; EC 4.2.1.1), which are enzymes critical for various metabolic processes. The genome of A. baumannii encodes a β-CA (βAbauCA), which is essential for producing bicarbonate ions required in the early stages of uridine triphosphate (UTP) synthesis, a precursor for the synthesis of peptidoglycans, which are vital components of the bacterial cell wall. This study aimed to inhibit βAbauCA in vitro, with the potential to impair the vitality of the pathogen in vivo. We conducted sequence and structural analyses of βAbauCA to explore its differences from those of human CAs. Additionally, kinetic and inhibition studies were performed to investigate the catalytic efficiency of βAbauCAβ and its interactions with sulfonamides and their bioisosteres, classical CA inhibitors. Our results showed that βAbauCA has a turnover rate higher than that of hCA I but lower than that of hCA II and displays distinct inhibition profiles compared to human α-CAs. Based on the obtained data, there are notable differences between the inhibition profiles of the human isoforms CA I and CA II and bacterial βAbauCA. This could open the door to designing inhibitors that selectively target bacterial β-CAs without affecting human α-CAs, as well as offer a novel strategy to weaken A. baumannii and other multidrug-resistant pathogens.

Essential role of proline synthesis and the one-carbon metabolism pathways for systemic virulence of Streptococcus pneumoniae.

Virulence screens have indicated potential roles during Streptococcus pneumoniae infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect S. pneumoniae virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δfhs and ΔproABC mutants. S. pneumoniae capsular serotype 6B BHN418 Δfhs and ΔproABC mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 ΔproABC strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of proABC was strain specific, as the D39 ΔproABC strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δfhs and ΔproABC strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δfhs had an impaired stringent response, and when cultured in sera, BHN418 Δfhs and ΔproABC were under increased oxidative stress and had altered lipid profiles. Loss of proABC also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the S. pneumoniae metabolic functions affected by S. pneumoniae one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae. We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential.

Computational Exploration of Limonin as a Potential Inhibitor of DapB in Klebsiella pneumoniae.

Klebsiella pneumoniae has emerged as a significant multidrug-resistant pathogen, classified as a critical priority by the World Health Organization. The rising rates of antibiotic resistance have led to increased therapeutic failures, diminishing the effectiveness of existing antibiotics. Consequently, there is an urgent need for alternative treatments to effectively inhibit the growth of K. pneumoniae and mitigate associated diseases. Phytochemicals have demonstrated potential advantages over traditional antibiotics, prompting their exploration as innovative therapeutic agents. This study aimed to identify phytochemicals that can inhibit dapB, a vital enzyme in the lysine biosynthesis pathway of K. pneumoniae, which is essential for protein synthesis and the cross-linking of the bacterial peptidoglycan cell wall. We screened 17,934 phytochemicals based on Lipinski's Rule of Five, along with their Absorption, Distribution, Metabolism, Excretion properties and toxicological parameters. Next, we conducted triplicate docking studies against dapB to evaluate the library further. The most promising molecules then underwent 100 ns Molecular Dynamics simulations in triplicate, followed by MM/PBSA based binding free energy calculations to identify potential dapB inhibitors. This in silico analysis highlighted limonin as a promising inhibitor of dapB in K. pneumoniae. Further experimental validation is crucial to enhance limonin's potential as a novel therapeutic agent against K. pneumoniae-associated diseases.

Bifidobacterium favimelis sp. nov., isolated from black comb honey.

A strain of the bacterial genus Bifidobacterium, IMAU50987T, that was isolated from black comb honey from Lianghe County, Dehong Autonomous Prefecture, Yunnan Province, China, could not be accurately identified as a known species using 16S rRNA gene sequencing. In this study, the taxonomic status of strain IMAU50987T was evaluated using whole-genome sequencing combined with phenotypic analyses. Strain IMAU50987T was Gram-positive, non-motile and non-spore-forming. Comparative of 16S rRNA gene sequence similarity analysis indicated that strain IMAU50987T could be attributed to the genus Bifidobacterium and most closely related to the B. mellis Bin7NT (98.5% identity). Comparative sequence analysis of eight housekeeping genes (pyrG, thrS, glnA1, recA, tuf, dnaB, rpoC and xfp) showed that strain IMAU50987T was different from closely related species. Phylogenomic analysis based on the 88 single-copy genes in the phylogenetic tree showed that strain IMAU50987T belonged to the B. asteroides group. The highest average nucleotide identity value for strain IMAU50987T was 77.0% in comparison with B. choladohabitans B14384H11T and B. apis F753-1T. However, the highest digital DNA-DNA hybridization value was 23.1% compared with B. apis F753-1T and B. xylocopae XV2T; the highest average amino acid identity value was 73.2% compared with B. polysaccharolyticum W8117T. These values are below the accepted threshold for novel species boundaries. The draft genome size of strain IMAU50987T was 1.9 Mb, and the DNA G+C content was 63.5%. The major cellular fatty acids of strain IMAU50987T were C16 : 0, C18 : 1 ω9c and Sum in Feature 7 (C19 : 1 ω7c/C19 : 1 ω6c). The cell-wall peptidoglycan was of the A3α l-Lys-Gly type. Based on data from this study, a novel species, Bifidobacterium favimelis sp. nov., is proposed with the type strain IMAU50987T (= GDMCC 1.4189T = JCM 36315T).

Reassessing the substrate specificities of the major Staphylococcus aureus peptidoglycan hydrolases lysostaphin and LytM.

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined. In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase.

Reassessing the substrate specificities of the major Staphylococcus aureus peptidoglycan hydrolases lysostaphin and LytM

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined. In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase.

Type IV pili-associated secretion of a biofilm matrix protein from Clostridium perfringens that forms intermolecular isopeptide bonds.

For bacteria to secrete folded proteins to the environment, they have to overcome the physical barriers of an outer membrane in Gram-negative bacteria and the thick peptidoglycan layer in Gram-positive bacteria. One mechanism to do this is the use of a Type II secretion system in Gram-negative bacteria, which has a similar structure as type IV pili and is modeled to act as a piston that pumps folded proteins through the outer membrane to the environment. Clostridium perfringens , like all or most all of the clostridia, has type IV pili and, in fact, has two sets of pilus-associated genes. Here we present evidence that C. perfringens uses one set of pilus genes to secrete a biofilm associated protein and may be responsible for secreting the main biofilm protein BsaA. We show that BsaA monomers are, unlike most other biofilm matrix proteins, linked by intermolecular isopeptide bonds, enhancing the physical strength of BsaA fibers.

Two codependent routes lead to high-level MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA), in which acquisition of mecA [which encodes the cell wall peptidoglycan biosynthesis component penicillin-binding protein 2a (PBP2a)] confers resistance to β-lactam antibiotics, is of major clinical concern. We show that, in the presence of antibiotics, MRSA adopts an alternative mode of cell division and shows an altered peptidoglycan architecture at the division septum. PBP2a can replace the transpeptidase activity of the endogenous and essential PBP2 but not that of PBP1, which is responsible for the distinctive native septal peptidoglycan architecture. Successful division without PBP1 activity requires the alternative division mode and is enabled by several possible chromosomal potentiator (pot) mutations. MRSA resensitizing agents differentially interfere with the two codependent mechanisms required for high-level antibiotic resistance, which provides opportunities for new interventions.

Synergistic potential of Leu10-teixobactin and cefepime against multidrug-resistant Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a significant Gram-positive opportunistic pathogen behind many debilitating infections. β-lactam antibiotics are conventionally prescribed for treating S. aureus infections. However, the adaptability of S. aureus in evolving resistance to multiple β-lactams contributed to the persistence and spread of infections, exemplified in the emergence of methicillin-resistant S. aureus (MRSA). In the present study, we investigated the efficacies of the synthetic teixobactin analogue, Leu10-teixobactin, combined with the penicillinase-resistant cephalosporin cefepime against MRSA strains. The Leu10-teixobactin and cefepime combination exerted synergism against most strains tested in broth microdilution assay. Time-kill profiles showed that both Leu10-teixobactin and cefepime predominantly exhibited synergistic activity, with > 2.0-log10CFU decrease compared to monotherapy at 24 h. Moreover, biofilm assays revealed a significant inhibition of biofilm production in ATCC™43300 cells treated with sub-MICs of Leu10-teixobactin and cefepime. Subsequent electron microscopy studies showed more extensive damage with the combination therapy compared to monotherapies, including aberrant bacterial morphology, vesicle formation and substantial lysis, indicating combined damage to the cell wall. Quantitative real-time PCR revealed marked perturbation of genes mecA, sarA, atlA, and icaA, substantiating the apparent mode of combined antibacterial action of both antibiotics against peptidoglycan synthesis and initial biofilm production. Hence, the study highlights the prospective utility of the Leu10-teixobactin-cefepime combination in treating MRSA infections via β-lactam potentiation.

Janibacter alittae sp. nov., an actinobacterium isolated from the gut of marine sandworm (Alitta virens).

A novel Gram-stain-positive, facultatively anaerobic, non-motile, catalase-positive, oxidase-negative and ovoid cocci, designated as A1S7T, was isolated from the gut of a marine sandworm (Alitta virens). Strain A1S7T exhibited optimal growth at temperatures of 20-30 ℃, pH 6-8 and in the presence of 2-4% (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain A1S7T belonged to the genus Janibacter, exhibiting a similarity of 99.0% to Janibacter cremeus KCTC 49873T, followed by Janibacter anophelis KCTC 19282T (98.8%), Janibacter hoylei KCTC 49872T (98.4%), Janibacter limosus KACC 20518T (98.2%) and Janibacter corallicola KACC 21120T (97.2%). The complete genome sequence of strain A1S7T revealed a genome size of 3360920 bp with a genomic G+C content of 70.1 mol%. The orthologous average nucleotide identity and the digital DNA-DNA hybridization values between strain A1S7T and Janibacter cremeus KCTC 49873T were determined to be 89.5 and 37.2%, respectively. The major respiratory quinone was MK-8(H4). The predominant fatty acids (>10%) included iso-C16:0, C17:1 ω8c, C18:1 ω9c and C17:0. Polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, one unknown phosphoglycolipid and three unknown polar lipids. The cell-wall peptidoglycan type was A1γ. The major whole-cell sugars were ribose, mannose and glucose. Based on phenotypic, phylogenetic, genotypic and chemotaxonomic properties, strain A1S7T represents a novel species in the genus Janibacter, for which the name Janibacter alittae sp. nov. is proposed. The type strain is A1S7T (=KCTC 49714T = JCM 36706T).

FtsK Initiates the Assembly of a Unique Divisome Complex in the FtsZ-less Chlamydia trachomatis.

Chlamydia trachomatis serovar L2 (Ct), an obligate intracellular bacterium that does not encode FtsZ, divides by a polarized budding process. In the absence of FtsZ, we show that divisome assembly in Ct is initiated by FtsK, a chromosomal translocase. Chlamydial FtsK forms discrete foci at the septum and at the base of the progenitor mother cell, and our data indicate that FtsK foci at the base of the mother cell mark the location of nascent divisome complexes that form at the site where a daughter cell will emerge in the next round of division. The divisome in Ct has a hybrid composition, containing elements of the divisome and elongasome from other bacteria, and FtsK is recruited to nascent divisomes prior to the other chlamydial divisome proteins assayed, including the PBP2 and PBP3 transpeptidases, and MreB and MreC. Knocking down FtsK prevents divisome assembly in Ct and inhibits cell division and septal peptidoglycan synthesis. We further show that MreB does not function like FtsZ and serve as a scaffold for the assembly of the Ct divisome. Rather, MreB is one of the last proteins recruited to the chlamydial divisome, and it is necessary for the formation of septal peptidoglycan rings. Our studies illustrate the critical function of chlamydial FtsK in coordinating divisome assembly and peptidoglycan synthesis in this obligate intracellular bacterial pathogen.