Structural studies of the deacylated glycolipids and lipoteichoic acid of Lactococcus cremoris 3107.

Lactococcus cremoris and Lactococcus lactis are among the most extensively exploited species of lactic acid bacteria in dairy fermentations. The cell wall of lactococci, like other Gram-positive bacteria, possesses a thick peptidoglycan layer, which may incorporate cell wall polysaccharides (CWPS), wall teichoic acids (WTA), and/or lipoteichoic acids (LTA). In this study, we report the isolation, purification and structural analysis of the carbohydrate moieties of glycolipids (GL) and LTA of the L. cremoris model strain 3107. Chemical structures of these compounds were studied by chemical methods, NMR spectroscopy and positive and negative mode ESI MS. We found that the LTA of strain 3107 is composed of short chains of 1,3-polyglycerol phosphate (PGP), attached to O-6 of the non-reducing glucose of the kojibiose-Gro backbone of the glycolipid anchor. Extraction of cells with cold TCA afforded the detection of 1,3-glycerol phosphate chains randomly substituted at O-2 of glycerol by D-Ala. Unlike the LTA of L. lactis strains studied to date, the PGP backbone of the LTA of L. cremoris 3107 did not carry any glycosyl substitution. The deacylated glycolipid fraction contained the free kojibiose-Gro oligosaccharide, identical to the backbone of the GL anchor of LTA, and its shorter fragment α-Glc-1-Gro. These OS may have originated from the GL precursors of LTA biosynthesis.

Promoter DNA recognition by the Enterococcus faecalis global regulator MafR.

When Enterococcus faecalis is exposed to changing environmental conditions, the expression of many genes is regulated at the transcriptional level. We reported previously that the enterococcal MafR protein causes genome-wide changes in the transcriptome. Here we show that MafR activates directly the transcription of the OG1RF_10478 gene, which encodes a hypothetical protein of 111 amino acid residues. We have identified the P10478 promoter and demonstrated that MafR enhances the efficiency of this promoter by binding to a DNA site that contains the -35 element. Moreover, our analysis of the OG1RF_10478 protein AlphaFold model indicates high similarity to 1) structures of EIIB components of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system, and 2) structures of receiver domains that are found in response regulators of two-component signal transduction systems. However, unlike typical EIIB components, OG1RF_10478 lacks a Cys or His residue at the conserved phosphorylation site, and, unlike typical receiver domains, OG1RF_10478 lacks a conserved Asp residue at the position usually required for phosphorylation. Different from EIIB components and receiver domains, OG1RF_10478 contains an insertion between residues 10 and 30 that, according to ColabFold prediction, may serve as a dimerization interface. We propose that OG1RF_10478 could participate in regulatory functions by protein-protein interactions.

Host genetic requirements for DNA release of lactococcal phage TP901-1.

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.

PclR is a transcriptional activator of the gene that encodes the pneumococcal collagen-like protein PclA.

The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that shows high levels of genetic variability. The pneumococcal R6 genome harbours several gene clusters that are not present in all strains of the species. One of these clusters contains two divergent genes, pclA, which encodes a putative surface-exposed protein that contains large regions of collagen-like repeats, and spr1404 (here named pclR). PclA was shown to mediate pneumococcal adherence to host cells in vitro. In this work, we demonstrate that PclR (494 amino acids) is a transcriptional activator. It stimulates transcription of the pclA gene by binding to a specific DNA site upstream of the core promoter. In addition, we show that PclR has common features with the MgaSpn transcriptional regulator (493 amino acids), which is also encoded by the R6 genome. These proteins have high sequence similarity (60.3%), share the same organization of predicted functional domains, and generate multimeric complexes on linear double-stranded DNAs. However, on the PpclA promoter region, MgaSpn binds to a site different from the one recognized by PclR. Our results indicate that PclR and MgaSpn have similar DNA-binding properties but different DNA-binding specificities, pointing to a different regulatory role of both proteins.

Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein.

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

Lysogenization of a Lactococcal Host with Three Distinct Temperate Phages Provides Homologous and Heterologous Phage Resistance.

Lactococcus lactis is the most widely exploited microorganism in global dairy fermentations. Lactococcal strains are described as typically harboring a number of prophages in their chromosomes. The presence of such prophages may provide both advantages and disadvantages to the carrying host. Here, we describe the deliberate generation of three distinct lysogens of the model lactococcal strain 3107 and the impact of additional prophage carriage on phage-resistance and anti-microbial susceptibility. Lysogen-specific responses were observed, highlighting the unique relationship and impact of each lysogenic phage on its host. Both homologous and heterologous phage-resistance profiles were observed, highlighting the presence of possible prophage-encoded phage-resistance factors. Superinfection exclusion was among the most notable causes of heterologous phage-resistance profiles with resistance observed against members of the Skunavirus, P335, P087, and 949 lactococcal phage groups. Through these analyses, it is now possible to identify phages that may pursue similar DNA injection pathways. The generated lysogenic strains exhibited increased sensitivity to the antimicrobial compounds, nisin and lysozyme, relative to the parent strain, although it is noteworthy that the degree of sensitivity was specific to the individual (pro)phages. Overall, the findings highlight the unique impact of each prophage on a given strain and the requirement for strain-level analysis when considering the implications of lysogeny.

In vitro DNA Inversions Mediated by the PsrA Site-Specific Tyrosine Recombinase of Streptococcus pneumoniae.

Site-specific recombination is a DNA breaking and reconstructing process that plays important roles in various cellular pathways for both prokaryotes and eukaryotes. This process requires a site-specific recombinase and direct or inverted repeats. Some tyrosine site-specific recombinases catalyze DNA inversions and regulate subpopulation diversity and phase variation in many bacterial species. In Streptococcus pneumoniae, the PsrA tyrosine recombinase was shown to control DNA inversions in the three DNA methyltransferase hsdS genes of the type I restriction-modification cod locus. Such DNA inversions are mediated by three inverted repeats (IR1, IR2, and IR3). In this work, we purified an untagged form of the PsrA protein and studied its DNA-binding and catalytic features. Gel retardation assays showed that PsrA binds to linear and supercoiled DNAs, containing or not inverted repeats. Nevertheless, DNase I footprinting assays showed that, on linear DNAs, PsrA has a preference for sites that include an IR1 sequence (IR1.1 or IR1.2) and its boundary sequences. Furthermore, on supercoiled DNAs, PsrA was able to generate DNA inversions between specific inverted repeats (IR1, IR2, and IR3), which supports its ability to locate specific target sites. Unlike other site-specific recombinases, PsrA showed reliance on magnesium ions for efficient catalysis of IR1-mediated DNA inversions. We discuss that PsrA might find its specific binding sites on the bacterial genome by a mechanism that involves transitory non-specific interactions between protein and DNA.

Transcriptional activation by MafR, a global regulator of Enterococcus faecalis.

Proteins that act as global transcriptional regulators play key roles in bacterial adaptation to new niches. These proteins recognize multiple DNA sites across the bacterial genome by different mechanisms. Enterococcus faecalis is able to survive in various niches of the human host, either as a commensal or as a leading cause of serious infections. Nonetheless, the regulatory pathways involved in its adaptive responses remain poorly understood. We reported previously that the MafR protein of E. faecalis causes genome-wide changes in the transcriptome. Here we demonstrate that MafR functions as a transcription activator. In vivo, MafR increased the activity of the P12294 and P11486 promoters and also the transcription levels of the two genes controlled by those promoters. These genes are predicted to encode a calcium-transporting P-type ATPase and a QueT transporter family protein, respectively. Thus, MafR could have a regulatory role in calcium homeostasis and queuosine synthesis. Furthermore, MafR recognized in vitro specific DNA sites that overlap the -35 element of each target promoter. The MafR binding sites exhibit a low sequence identity, suggesting that MafR uses a shape readout mechanism to achieve DNA-binding specificity.

DNA-binding properties of MafR, a global regulator of Enterococcus faecalis.

Global transcriptional regulators play key roles during bacterial adaptation to environmental fluctuations. Protein MafR from Enterococcus faecalis was shown to activate the transcription of many genes on a genome-wide scale. We proposed that MafR is a global regulator of the Mga/AtxA family. Here, we purified an untagged form of the MafR protein and found that it binds to linear double-stranded DNAs in a nonsequence-specific manner. Moreover, multiple MafR units (likely dimers) bind sequentially to the DNA molecule generating multimeric complexes. On DNAs that contain the promoter of the mafR gene, MafR recognizes a potentially curved DNA region. We discuss that a characteristic of the Mga/AtxA regulators might be their ability to recognize particular DNA shapes across the bacterial genomes.

When Humans Met Superbugs: Strategies to Tackle Bacterial Resistances to Antibiotics.

Bacterial resistance to antibiotics poses enormous health and economic burdens to our society, and it is of the essence to explore old and new ways to deal with these problems. Here we review the current status of multi-resistance genes and how they spread among bacteria. We discuss strategies to deal with resistant bacteria, namely the search for new targets and the use of inhibitors of protein-protein interactions, fragment-based methods, or modified antisense RNAs. Finally, we discuss integrated approaches that consider bacterial populations and their niches, as well as the role of global regulators that activate and/or repress the expression of multiple genes in fluctuating environments and, therefore, enable resistant bacteria to colonize new niches. Understanding how the global regulatory circuits work is, probably, the best way to tackle bacterial resistance.

Global Regulation of Gene Expression by the MafR Protein of Enterococcus faecalis.

Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. However, as an opportunistic pathogen, it is able to colonize other host niches and cause life-threatening infections. Its adaptation to new environments involves global changes in gene expression. The EF3013 gene (here named mafR) of E. faecalis strain V583 encodes a protein (MafR, 482 residues) that has sequence similarity to global response regulators of the Mga/AtxA family. The enterococcal OG1RF genome also encodes the MafR protein (gene OG1RF_12293). In this work, we have identified the promoter of the mafR gene using several in vivo approaches. Moreover, we show that MafR influences positively the transcription of many genes on a genome-wide scale. The most significant target genes encode components of PTS-type membrane transporters, components of ABC-type membrane transporters, and proteins involved in the metabolism of carbon sources. Some of these genes were previously reported to be up-regulated during the growth of E. faecalis in blood and/or in human urine. Furthermore, we show that a mafR deletion mutant strain induces a significant lower degree of inflammation in the peritoneal cavity of mice, suggesting that enterococcal cells deficient in MafR are less virulent. Our work indicates that MafR is a global transcriptional regulator. It might facilitate the adaptation of E. faecalis to particular host niches and, therefore, contribute to its potential virulence.

Mobilizable Rolling-Circle Replicating Plasmids from Gram-Positive Bacteria: A Low-Cost Conjugative Transfer.

Conjugation is a key mechanism for horizontal gene transfer in bacteria. Some plasmids are not self-transmissible but can be mobilized by functions encoded in trans provided by other auxiliary conjugative elements. Although the transfer efficiency of mobilizable plasmids is usually lower than that of conjugative elements, mobilizable plasmids are more frequently found in nature. In this sense, replication and mobilization can be considered important mechanisms influencing plasmid promiscuity. Here we review the currently available information on two families of small mobilizable plasmids from Gram-positive bacteria that replicate via the rolling-circle mechanism. One of these families, represented by the streptococcal plasmid pMV158, is an interesting model since it contains a specific mobilization module (MOBV) that is widely distributed among mobilizable plasmids. We discuss a mechanism in which the promiscuity of the pMV158 replicon is based on the presence of two origins of lagging strand synthesis. The current strategies to assess plasmid transfer efficiency as well as to inhibit conjugative plasmid transfer are presented. Some applications of these plasmids as biotechnological tools are also reviewed.

Mobilizable Rolling-Circle Replicating Plasmids from Gram-Positive Bacteria: A Low-Cost Conjugative Transfer.

Conjugation is a key mechanism for horizontal gene transfer in bacteria. Some plasmids are not self-transmissible but can be mobilized by functions encoded in trans provided by other auxiliary conjugative elements. Although the transfer efficiency of mobilizable plasmids is usually lower than that of conjugative elements, mobilizable plasmidsare more frequently found in nature. In this sense, replication and mobilization can be considered as important mechanisms influencing plasmid promiscuity. Here we review the present available information on two families of small mobilizable plasmids from Gram-positive bacteria that replicate via the rolling-circle mechanism. One of these families, represented by the streptococcal plasmid pMV158, is an interesting model since it contains a specific mobilization module (MOBV) that is widely distributed among mobilizable plasmids. We discuss a mechanism in which the promiscuity of the pMV158 replicon is based on the presence of two origins of lagging strand synthesis. The current strategies to assess plasmid transfer efficiency as well as to inhibit conjugative plasmid transfer are presented. Some applications of these plasmids as biotechnological tools are also reviewed.

Novel plasmid-based genetic tools for the study of promoters and terminators in Streptococcus pneumoniae and Enterococcus faecalis.

Promoter-probe and terminator-probe plasmid vectors make possible to rapidly examine whether particular sequences function as promoter or terminator signals in various genetic backgrounds and under diverse environmental stimuli. At present, such plasmid-based genetic tools are very scarce in the Gram-positive pathogenic bacteria Streptococcus pneumoniae and Enterococcus faecalis. Hence, we developed novel promoter-probe and terminator-probe vectors based on the Streptococcus agalactiae pMV158 plasmid, which replicates autonomously in numerous Gram-positive bacteria. As reporter gene, a gfp allele encoding a variant of the green fluorescent protein was used. These genetic tools were shown to be suitable to assess the activity of promoters and terminators (both homologous and heterologous) in S. pneumoniae and E. faecalis. In addition, the promoter-probe vector was shown to be a valuable tool for the analysis of regulated promoters in vivo, such as the promoter of the pneumococcal fuculose kinase gene. These new plasmid vectors will be very useful for the experimental verification of predicted promoter and terminator sequences, as well as for the construction of new inducible-expression vectors. Given the promiscuity exhibited by the pMV158 replicon, these vectors could be used in a variety of Gram-positive bacteria.