This review presents the data on structural diversity of rhamnans in the cell walls and S-layers of Gram-positive (monoderm) bacteria – representatives of the phyla Bacillota (“Firmicutes”) and Actinomycetota (“Actinobacteria”). Diversity of the rhamnan structures is determined by configuration and alternation of the (1→2)- and (1→3)-glycosidic bonds in the polymer core, as well as by the presence and composition of side glycosyl and other substituents. Structural features of rhamnans in several bacterial groups are noted: presence of single glycerol phosphate units in the side chains of polymers in Streptococcus, Lactococcus, and Ruminococcus (Mediterraneibacter); presence of rhamnans with l- and d-isomers of rhamnose in actinobacteria; and complexes of rhamnans and teichoic acids or glycosyl 1-phosphate polymers (two-component heteropolysaccharides) in Enterococcus and Lactobacillus. The review provides information on the structural features of rhamnans underlying serological classification of streptococci. The role of rhamnose and rhamnans in bacterial cell physiology (cell wall biogenesis and cell division, viability, adaptation to adverse conditions, antibiotic resistance), phage reception, adhesion, biofilm formation, and in the “bacterial–host” interaction (induction of inflammatory processes in tissues, immune system activation, and virulence manifestation) is discussed.

Using chemical methods and methods of NMR spectroscopy, the structures of three cell wall glycopolymers of Rathayibacter sp. VKM Ac-2856 (family Microbacteriaceae, class Actinomycetes), isolated from Brachypodium distachyon (family Poaceae), were determined. The first polymer, an unsubstituted mannan with a repeating unit, →2)-α-d-Manp-(1→, was discovered in the cell walls of Rathayibacter for the first time. The second, a rhamnomannan, consists of regularly alternating residues of α-d-rhamnose and α-d-mannose and has non-stoichiometric substitution with β-d-Xylp residues in a 3 : 1 ratio, →2)-α-d-Rhap-(1→3)-[β-d-Xylp-(1→2)]-α-d-Manp-(1→. The third, a minor, teichuronic acid-like polymer with a trisaccharide repeating unit and R-pyruvic acid, bears side residues of β-d-glucuronic acid, →3)-α-d-[4,6-R-Pyr]-Galp-(1→3)-β-d-Glcp-(1→6)-[β-d-GlcpA-(1→3)]-α-d-Manp-(1→. The latter distinguishes this polymer from teichuronic acids, which contain uronic acid residues in the main chain. Structure of the teichuronic acid-like polymer is novel for both Rathayibacter and prokaryotes in general. The results of this study demonstrate structural diversity of microbial glycopolymers and are consistent with the previously reported data on the species-specific composition of glycopolymers in the representatives of the genus Rathayibacter.

Antimicrobial resistance is a major threat to modern society and healthcare, as it severely compromises the efficacy of standard antibiotic treatments. To meet the ever-increasing demand for novel antimicrobial drugs, it is crucial to develop new strategies for screening antimicrobial compounds and improve existing high-throughput techniques. Reporter systems that employ specific genetic markers are powerful tools not only for detecting antimicrobial activity of the substance being studied, but also for identifying the potential mechanism of its action. Among other metabolic pathways, RNA biosynthesis machinery is considered a promising molecular target as it remains underutilized in current antimicrobial therapy and therefore is rarely exposed to drug pressure. However, there is no suitable biomarker for identifying compounds that inhibit the transcription in Gram-negative bacteria. Combining bioinformatic search and RT-qPCR experimental validation, we have established the overexpression of the spermidine synthase gene (speE) as a biomarker associated with impaired transcription in Escherichia coli. Monitoring the expression level of speE in antibiotic-treated cells enables reliable detection of compounds that inhibit bacterial RNA-polymerase, such as rifampicin and fidaxomicin. Moreover, our screening system was successfully applied in practice to analyze chromatography fractions from fermentation broth of antibiotic producers, with compounds of the rifamycin family being identified as hits and isolated. The proposed method has the potential to be used in sequential screening procedures to reveal active antimicrobial compounds that inhibit bacterial transcription process, giving the world novel antimicrobials with minimal risk of resistance development.

In recent decades, most studies of microbial rhodopsins have focused on their identification and characterization in aquatic bacteria. In 2021, actinomycetes of the family Geodermatophilaceae, commonly inhabiting terrestrial ecosystems in hot and arid regions, have been reported to contain rhodopsins with DTEW, DTEF and NDQ amino acid motifs. An advanced bioinformatics analysis performed in this work additionally revealed NTQ rhodopsin and heliorhodopsins. The absorption maxima identified for rhodopsins from the above five groups ranged from 513 nm (NTQ rhodopsin) to 559 nm (heliorhodopsin). An assessment of pumping specificity showed that DTEW and DTEF rhodopsins possessed outward H+-transport activities. Ca2+ ions were required for pumping if E. coli C43(DE3) was used as an expression strain, but were unnecessary in the case of E. coli BL21(DE3). For NDQ rhodopsin, outward H+-transport was detected in NaCl and KCl solutions at pH 5 and 6, but not at neutral pH. A weak Na+-efflux was observed for this rhodopsin at pH 6 and 7 in a NaCl solution only in the presence of proton ionophore. NTQ rhodopsin acted as an inward Cl--, Br--, and I-- pump, with a much weaker activity towards NO3-.No pumping activity was detected for the heliorhodopsin tested. The finding of rhodopsins with novel properties further expands the rhodopsin landscape.

The structures of two cell wall glycopolymers from the strain Rathayibacter sp. VKM Ac-2927 (family Microbacteriaceae, class Actinomycetes), isolated from the leaves of a linden affected by leaf miner, were established using chemical and NMR spectroscopy methods. The first polymer, rhamnomannan, is a heterogeneous chain polymer composed of regularly alternating α-D-rhamnose and α-D-mannose residues linked by (1→3)- and (1→2)-glycosidic bonds and contains a minor amount of lateral β-D-xylopyranose residues (structure is presented in the text). The second polymer, acetalized with R- and S-pyruvic acid, consists of branched tetrasaccharide units, →3)-α-D-Galp4,6-(R-Pyr)-(1→3)-β-D-Glcp-(1→6)-[β-D-Galp3,4-(S-Pyr)-(1→4)]-α-D-Manp-(1→. Structure of this polysaccharide is new for the representatives of the genus Rathayibacter and prokaryotes in general. The results of present study indicate structural diversity of microbial glycopolymers and are consistent with the previously obtained data on specificity of their composition for the species of the Rathayibacter genus.

Rathayibacter festucae VKM Ac-1390T (family Microbacteriaceae, class Actinomycetes) contains three rhamnose-containing glycopolymers in the cell wall, the structures of which were established by chemical and NMR spectroscopy methods. The first polymer, a rhamnomannan, consists of repeating tetrasaccharide units with xylopyranose side residues, →2)-α-[β-D-Xylp-(1→3)]-D-Rhap-(1→3)-α-D-Manp-(1→2)-α-D-Rhap-(1→3)-α-D-Manp-(1→. The second polymer found in minor amounts, is a rhamnan, →2)-α-D-Rhap-(1→3)-α-D-Rhap-(1→. The third polymer is a teichuronic acid acetalated with pyruvic acid, →2)-α-[4,6-S-Pyr]-D-Manp-(1→4)-α-L-Rhap-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→4)-β-D-GlcpA-(1→. The structures of rhamnomannan and teichuronic acid are new for Ratayibacter and prokaryotes in a whole. The results of this study expand our understanding of the structural diversity of microbial glycopolymers and are consistent with previous reports on the species specificity of glycopolymer structures and composition in the genus Rathayibacter.

American beech (Fagus grandifolia) trees exhibiting symptoms of beech leaf disease (BLD) induced by the foliar nematode, Litylenchus crenatae, were found growing in Delmar, New York, USA, in 2019. Foliar symptoms included leaf drop, galling, and darkening associated with leaf curl. Bacterial isolates were cultured from infected leaf tissue and identified to genus by 16S rRNA gene sequencing and MALDI-TOF-MS profiling. In addition to many taxa with putatively environmental roles, strains of the genera Agrobacterium, Curtobacterium, Pantoea, Xanthomonas, as well as Erwinia and Pseudomonas were detected. Strains of the latter two genera have been previously associated with BLD, but isolates have not yet been identified at the species level. Phylogenetic trees generated from sequences of 16S rRNA and housekeeping genes (gyrB, rpoB, and rpoD) combined with MALDI-TOF-MS, showed that the isolates from nematode-infected leaves of American beech belonged to the known plant pathogenic species C. flaccumfaciens, E. billingiae, E. rhapontici, Pa. agglomerans, and X. arboricola. The species affiliation of strains showing close relatedness to A. skierniewicense, A. vaccinii, Pa. alfalfae, Pa. ananatis, Ps. amygdali, Ps. cerasi, and Ps. syringae requires further support. The identities of other bacteria isolated from the same samples are also provided.

Two novel yellow-pigmented, rod-shaped and non-motile coryneform actinobacteria, strains VKM Ac-2596T and VKM Ac-2761, were isolated from a plant Tanacetum vulgare (Asteraceae) infested by foliar nematode Aphelenchoides sp. The strains exhibited the highest 16S rRNA gene sequence similarities to Rathayibacter agropyri CA4T (99.71

The cell wall of endophytic strain Rathayibacter oskolensis VKM Ac-2121T (family Microbacteriaceae, class Actinomycetes) was found to contain neutral and acidic glycopolymers. The neutral polymer is a block-type rhamnomannan partially should be substitutied by xylose residues, [→2)-α-[β-D-Xylp-(1→3)]-D-Manp-(1→3)-α-D-Rhap-(1→]∼30 [→2)-α-D-Manp-(1→3)-α-D-Rhap-(1→]∼45. The acidic polymer has branched chain, bearing lactate and pyruvate residues, →4)-α-D-[S-Lac-(2―3)-α-L-Rhap-(1→3)]-D-Manp-(1→3)-α-D-[4,6-R-Pyr]-D-Galp-(1→3)-β-D-Glcp-(1→. The structures of both glycopolymers were not described in the Gram-positive bacteria to date. The glycopolymers were studied by chemical and NMR spectroscopic methods. The results of this study provide new data on diversity of bacterial glycopolymers and may prove useful in the taxonomy of the genus Rathayibacter and for understanding the molecular mechanisms of interaction between plants and plant endophytes.

Mycelium-forming aerobic actinomycete, strain VKM Ас-2527T, was isolated from a soil sample collected from the North Caucasus mountains. The 16S rRNA gene sequence similarities of this strain to the type strains of the known Kribbella species ranged from 95.5 to 98.5

Four salt-tolerant and aromatics degrading strains used in this study were isolated from polluted technogenic soil on the territory of the Verkhnekamsk potash deposit (Russia). The strains were aerobic, Gram-stain-positive, non-motile, non-endospore-forming irregular rods, exhibiting a marked rod-coccus growth cycle. They contained lysine-based peptidoglycan, teichulosonic acid and poly(glycosyl phosphate) polymers in the cell walls. The major menaquinone was MK-9(H2), the predominant fatty acids were saturated, anteiso- and iso-branched, and the major compounds of polar lipid profiles included phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and two glycolipids (monogalactosyldiacylglycerol and dimannosylglyceride). The strains showed the highest 16S rRNA gene sequence similarity to Arthrobacter crystallopoietes (99.6–99.9

Puromycin (Puro) is a natural aminonucleoside antibiotic that inhibits protein synthesis by its incorporation into elongating peptide chains. The unique mechanism of Puro finds diverse applications in molecular biology, including the selection of genetically engineered cell lines, in situ protein synthesis monitoring, and studying ribosome functions. However, the key step of Puro biosynthesis remains enigmatic. In this work, pur6-guided genome mining is carried out to explore the natural diversity of Puro-like antibiotics. The diversity of biosynthetic gene cluster (BGC) architectures suggests the existence of distinct structural analogs of puromycin encoded by pur-like clusters. Moreover, the presence of tRNACys in some BGCs, i.e., cst-like clusters, leads us to the hypothesis that Pur6 utilizes aminoacylated tRNA as an activated peptidyl precursor, resulting in cysteine-based analogs. Detailed metabolomic analysis of Streptomyces sp. VKM Ac-502 containing cst-like BGC revealed the production of a cysteinyl-based analog of Puro—cystocin (Cst). Similar to puromycin, cystocin inhibits both prokaryotic and eukaryotic translation by the same mechanism. Aminonucleoside N-acetyltransferase CstC inactivated Cst, mediating antibiotic resistance in genetically modified bacteria and human cells. The substrate specificity of CstC originated from the steric hindrance of its active site. We believe that novel aminonucleosides and their inactivating enzymes can be developed through the directed evolution of the discovered biosynthetic machinery.

Two types of cell wall glycopolymers of different structure were found in the type strains of two species of phytopathogenic bacteria, Clavibacter insidiosus VKM Ac-1402T and Clavibacter nebraskensis VKM Ac-1404T (family Microbacteriaceae, class Actinomycetes). The first type was represented by new, previously undescribed (1→6)-linked β-D-galactofuranans, which in the studied strains differed in the structure of side oligosaccharide chains (the structures are given in the text). The structure of the second glycopolymer, a pyruvate-containing galactomannan, was identical in both strains. The results obtained in this work, together with those published previously, indicate that the presence of the pyruvate-containing galactomannan and galactofuranans (with an identical core structure and different side oligosaccharide substituents) can be considered as a chemotaxonomic trait of the genus Clavibacter, and galactofuranans with di-, tri-, or tetrasaccharide substituents of different composition and structures can serve as chemotaxonomic markers of the species. The data obtained expand our understanding of the structural diversity of natural glycopolymers and structural features of the bacterial cell walls in various taxa and may be of interest for taxonomic studies and the studies aimed at elucidating the molecular mechanisms of interaction between bacteria and plant cells.

An aerobic, rod-shaped, non-motile, endospore-forming bacterium, strain V-9T, was found in the frozen volcanic ash, Kamchatka peninsula, Russia. Phylogenies based on 16S rRNA gene and genome sequences demonstrated that strain V-9T was affiliated with the family Paenibacillaceae and clustered outside the clade that includes the type species of this genus, Paenibacillus polymyxa, and closely related species. V-9T showed the highest 16S rRNA gene sequences similarity to Paenibacillus chartarius CCUG 55240T (96.7

— Information is provided on the cell wall glycopolymers of members of eight species of the genus Rathayibacter (family Microbacteriaceae , class Actinomycetes ). The composition, structure, and structural components of polymers that are common to the genus and characteristic of species are noted, which can be useful as chemotaxonomic characters. The differences between Rathayibacter and other genera of the family Microbacteriaceae ( Clavibacter, Curtobacterium ) in the type and composition of glycopolymers are shown.

A glycopolymer of novel structure was found in the cell wall of plant pathogen Clavibacter phaseoli VKM Ac-2641T (family Microbacteriaceae, class Actinomycetes). The glycopolymer was (1 → 6)-linked β-d-galactofuranan with side branched trisaccharide, α-D-Manp-(1 → 2)-[α-D-Manp-(1 → 3)]-α-D-Ribf-(1→ at O-2 on every second galactofuranose residue. The galactofuranan structure was established by chemical and NMR spectroscopic methods using one- and two-dimensional techniques 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC. The results of this study provide new data on diversity of bacterial glycopolymers, may prove useful for bacterial taxonomy and contribute to the understanding of the host plant-microbiota interaction mechanisms.

Seven actinomycete strains assigned to seven new species of the genus Kribbella were isolated from soils of different regions in Russia. The strains exhibited 98.2–99.3

The structures of two cell wall glycopolymers were studied in the plant pathogenic bacterium Clavibacter tesselarius VKM Ac-1406T (family Microbacteriaceae, order Micrococcales, class Actinomycetes). The predominant polymer was a novel (1 → 6)-linked β-d-galactofuranan with a highly branched repeating unit, α-L-Rhap-(1 → 3)-α-D-Galp-(1 → 2)-[α-L-Rhap-(1 → 3)]-α-D-Fucp-(1 →, at O-2 on every second galactofuranose residue. The second polymer present in small amounts was acidic with the repeating unit, →3)-α-D-Galp-(1 → 3)-α-D-[4,6-S-Pyr]-Manp-(1 → 3)-α-D-Manp-[2OAc]0.2-(1→, and was reported in all Clavibacter species investigated to date. The presented results expand our knowledges of structural diversity of phosphate-free cell wall glycopolymers and provide evidence in support of their taxonomic specificity for bacterial species and genera.

Bacteria of the genus “Candidatus Cardinium” and related organisms composing the Cardinium clade are intracellular endosymbionts frequently occurring in several arthropod groups, freshwater mussels and plant-parasitic nematodes. Phylogenetic analyses based on two gene sequences (16S rRNA and gyrB) showed that the Cardinium clade comprised at least five groups: A, B, C, D and E. In this study, a screening of 142 samples of plant-parasitic nematodes belonging to 93 species from 12 families and two orders using PCR with specific primers and sequencing, revealed bacteria of Cardinium clade in 14 nematode samples belonging to 12 species of cyst nematodes of the family Heteroderidae. Furthermore, in this study, the genome of the Cardinium cHhum from the hop cyst nematode, Heterodera humuli, was also amplified, sequenced and analyzed. The comparisons of the average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values for the strain Cardinium cHhum with regard to related organisms with available genomes, combined with the data on 16S rRNA and gyrB gene sequence identities, showed that this strain represents a new candidate species within the genus “Candidatus Paenicardinium”. The phylogenetic position of endosymbionts of the Cardinium clade detected in nematode hosts was also compared to known representatives of this clade from other metazoans. Phylogenetic reconstructions based on analysis of 16S rRNA, gyrB, sufB, gloEL, fusA, infB genes and genomes and estimates of genetic distances both indicate that the endosymbiont of the root-lesion nematode Pratylenchus penetrans represented a separate lineage and is designated herein as a new group F. The phylogenetic analysis also confirmed that endosymbionts of ostracods represent the novel group G. Evolutionary relationships of bacterial endosymbionts of the Cardinium clade within invertebrates are presented and discussed.

Draft genome sequences of 11 strains of putative new species of Geodermatophilaceae were generated using Illumina technology. The genome sizes ranged from 4.19 to 4.99 Mb, with G+C contents of 73.5% to 74.6%, and contained genes for microbial rhodopsins. This study will contribute to our knowledge of the ecology and diversity of members of the family Geodermatophilaceae.