SummaryIn preparation for transfer conjugative type IV secretion systems (T4SS) produce a nucleoprotein adduct containing a relaxase enzyme covalently linked to the 5Ј end of single-stranded plasmid DNA. The bound relaxase is expected to present features necessary for selective recognition by the type IV coupling protein (T4CP), which controls substrate entry to the envelope spanning secretion machinery. We prove that the IncF plasmid R1 relaxase TraI is translocated to the recipient cells. Using a Cre recombinase assay (CRAfT) we mapped two internally positioned translocation signals (TS) on F-like TraI proteins that independently mediate efficient recognition and secretion. Tertiary structure predictions for the TS matched best helicase RecD2 from Deinococcus radiodurans. The TS is widely conserved in MOB F and MOBQ families of relaxases. Structure/function relationships within the TS were identified by mutation. A key residue in specific recognition by T4CP TraD was revealed by a fidelity switch phenotype for an F to plasmid R1 exchange L626H mutation. Finally, we show that physical linkage of the relaxase catalytic domain to a TraI TS is necessary for efficient conjugative transfer.

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membranespanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7,8,10,30,41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site-and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5Ј end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3Ј-OH generated at nic and the subsequent DNA unwind...

Enterococci have controversial status due to their emerging role in nosocomial infections and transmission of antibiotic resistance genes, while some enterococci strains are used as probiotics for humans and animals and starter cultures in dairy industry. In order to improve our understanding of factors involved in the safe use of enterococci as potential probiotics, the antibiotic susceptibility, virulence and probiotic traits of 75 dairy enterococci isolates belonging to Enterococcus durans (50), En. faecium (15), En. faecalis (6), En. italicus (3), and En. hirae (1) were evaluated. The results revealed that ciprofloxacin resistance and biofilm formation are correlated with isolates originated from Golija mountain (Serbia), while gelatinase activity was more common in isolates from Prigorje region (Croatia), pointing to uncontrolled use of antibiotics and anthropogenic impact on dairy products' microbiota in these regions. The virulence genes were sporadically present in 13 selected dairy enterococci isolates. Interestingly, biofilm formation was correlated with higher ability of strains to reduce the adhesion of E. coli and Salmonella Enteritidis to HT29-MTX cells. To our knowledge this is the first study reporting the presence of the esp gene (previously correlated with pathogenesis) in dairy enterococci isolates, mostly associated with the genes involved in adhesion property. Hence, the results of this study revealed that the virulence genes are sporadically present in dairy isolates and more correlated to adhesion properties and biofilm formation, implicating their role in gut colonization rather than to the virulence traits.

Controlled duplex DNA unwinding is a crucial prerequisite for the expression and maintenance of genomes. Genomemanipulating and -regulating proteins are central to that biological function in recognizing appropriate DNA targets at initiation sequences and unwinding the complementary strands to provide single-stranded DNA (ssDNA) templates for nucleic acid synthesis and other processing reactions. The protein machineries involved include nucleic acid helicases. DNA helicases are powerful enzymes that convert the energy of nucleoside triphosphate hydrolysis to directional DNA strand translocation and separation of the double helix into its constituent single strands (for reviews, see references 13, 14, 16, 38, 55, and 64). By necessity, these enzymes interact with DNA strands via mechanisms independent of sequence recognition. At replication initiation helicases gain controlled access to the doublestranded genome at positions determined by the DNA binding properties of initiator proteins that comprise an origin recognition complex (1,9,17,31,45,66). The mechanisms supporting localized unwinding within the complex include initiatorinduced DNA looping, wrapping, and bending and feature regions of low thermodynamic stability. The exposed ssDNA mediates helicase binding followed by directional translocation along that strand until the enzyme engages the duplex for unwinding.In the MOB F family of conjugation systems, the plasmid DNA strand destined for transfer (T strand) is unwound from its complement by a dedicated conjugative helicase, TraI of F-like plasmids or TrwC of the IncW paradigm. These enzymes are remarkable in that the same polypeptides additionally harbor in a distinct domain a DNA transesterase activity. That function is required to recognize and cleave the precise phosphodiester bond, nic, in the T strand where unwinding of the secretion substrate begins. In current models the conjugative helicases are thus targeted to the transfer origin (oriT) of their cognate plasmid by the high-affinity DNA sequence interactions of their N-terminal DNA transesterase domains. In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10,12,29,33,51,52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2,15,49). In current models, the T4CP forms a hexameric translocation pore at the cytoplasmic membrane that not only governs substrate entry to the envelope spanning type IV secretion machinery but also provides energy for macromolecular transport via ATP hydrolysis (36,50). These models propose that T4CPs provide not only a physical bridge between the plasmid and the type IV ...

Probiotic bacteria are recognized for their health-promoting properties, including maintenance of gut epithelial integrity and host immune system homeostasis. Taking into account the beneficial health-promoting effects of GABA, the presence of the gadB gene, encoding glutamate decarboxylase that converts L-glutamate to GABA, was analyzed in Lactic Acid Bacteria (LAB) natural isolates from Zlatar cheese. The results revealed that 52% of tested Lactobacillus spp. and 8% of Lactococcus spp. isolates harbor the gadB gene. Qualitative and quantitative analysis of GABA production performed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed the highest GABA production by Lactobacillus brevis BGZLS10-17. Since high GABA-producing LAB natural isolates are the most valuable source of naturally produced GABA, the probiotic properties of BGZLS10-17 were characterized. This study demonstrated high adhesion of BGZLS10-17 strain to Caco-2 cells and the ability to decrease the adhesion of Escherichia coli ATCC25922 and Salmonella enterica C29039. Treatment of differentiated Caco-2 cells monolayer with BGZLS10-17 supernatant containing GABA alleviated inflammation (production of IL-8) caused by IL-1β and significantly stimulated the expression of tight junction proteins (zonulin, occludin, and claudin 4), as well as the expression of TGF-β cytokine leading to the conclusion that immunosuppression and strengthening the tight junctions can have significant role in the maintenance of intestinal epithelial barrier integrity. Taken together the results obtained in this study support the idea that using of GABA producing BGZLS10-17 probiotic strain could be a good strategy to modulate immunological response in various inflammatory diseases, and at the same time, it could be a good candidate for adjunct starter culture for production of GABA-enriched dairy foods and beverages offering new perspectives in designing the novel functional foods.

Black soldier fly larvae (BSFL) are fast-growing, resilient insects that can break down a variety of organic substrates and convert them into valuable proteins and lipids for applications in the feed industry. Decomposition is mediated by an abundant and versatile gut microbiome, which has been studied for more than a decade. However, little is known about the phylogeny, properties and functions of bacterial isolates from the BSFL gut. We therefore characterized the BSFL gut microbiome in detail, evaluating bacterial diversity by culture-dependent methods and amplicon sequencing of the 16S rRNA gene. Redundant strains were identified by genomic fingerprinting and 105 non-redundant isolates were then tested for their ability to inhibit pathogens. We cultivated representatives of 26 genera, covering 47% of the families and 33% of the genera detected by amplicon sequencing. Among these isolates, we found several representatives of the most abundant genera: Morganella, Enterococcus, Proteus and Providencia. We also isolated diverse members of the less-abundant phylum Actinobacteria, and a novel genus of the order Clostridiales. We found that 15 of the isolates inhibited at least one of the tested pathogens, suggesting a role in helping to prevent colonization by pathogens in the gut. The resulting culture collection of unique BSFL gut bacteria provides a promising resource for multiple industrial applications.

Carbapenem-resistant A. baumannii present a significant therapeutic challenge for the treatment of nosocomial infections in many European countries. Although it is known that the gradient of A. baumannii prevalence increases from northern to southern Europe, this study provides the first data from Serbia. Twenty-eight carbapenem-resistant A. baumannii clinical isolates were collected at a Serbian pediatric hospital during a 2-year period. The majority of isolates (67.68%) belonged to the sequence type Group 1, European clonal complex II. All isolates harbored intrinsic OXA-51 and AmpC cephalosporinase. OXA-23 was detected in 16 isolates (57.14%), OXA-24 in 23 isolates (82.14%) and OXA-58 in 11 isolates (39.29%). Six of the isolates (21.43%) harbored all of the analyzed oxacillinases, except OXA-143 and OXA-235 that were not detected in this study. Production of oxacillinases was detected in different pulsotypes indicating the presence of horizontal gene transfer. NDM-1, VIM and IMP were not detected in analyzed clinical A. baumannii isolates. ISAba1 insertion sequence was present upstream of OXA-51 in one isolate, upstream of AmpC in 13 isolates and upstream of OXA-23 in 10 isolates. In silico analysis of carO sequences from analyzed A. baumannii isolates revealed the existence of two out of six highly polymorphic CarO variants. The phylogenetic analysis of CarO protein among Acinetobacter species revised the previous classification CarO variants into three groups based on strong bootstraps scores in the tree analysis. Group I comprises four variants (I-IV) while Groups II and III contain only one variant each. One half of the Serbian clinical isolates belong to Group I variant I, while the other half belongs to Group I variant III.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system (CNS). It is widely accepted that autoimmune response against the antigens of the CNS is the essential pathogenic force in the disease. It has recently become increasingly appreciated that activated encephalitogenic cells tend to migrate toward gut associated lymphoid tissues (GALTs) and that interrupted balance between regulatory and inflammatory immunity within the GALT might have decisive role in the initiation and propagation of the CNS autoimmunity. Gut microbiota composition and function has the major impact on the balance in the GALT. Thus, our aim was to perform analyses of gut microbiota in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Albino Oxford (AO) rats that are highly resistant to EAE induction and Dark Agouti (DA) rats that develop EAE after mild immunization were compared for gut microbiota composition in different phases after EAE induction. Microbial analyses of the genus Lactobacillus and related lactic acid bacteria showed higher diversity of Lactobacillus spp. in EAE-resistant AO rats, while some members of Firmicutes and Proteobacteria (Undibacterium oligocarboniphilum) were detected only in feces of DA rats at the peak of the disease (between 13 and 16 days after induction). Interestingly, in contrast to our previous study where Turicibacter sp. was found exclusively in non-immunized AO, but not in DA rats, in this study it was detected in DA rats that remained healthy 16 days after induction, as well as in four of 12 DA rats at the peak of the disease. Similar observation was obtained for the members of Lachnospiraceae. Further, production of a typical regulatory cytokine interleukin-10 was compared in GALT cells of AO and DA rats, and higher production was observed in DA rats. Our data contribute to the idea that gut microbiota and GALT considerably influence multiple sclerosis pathogenesis.