Functional Analysis of the Major Pilin Proteins of Type IV Pili in Streptococcus sanguinis CGMH010.

The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis. Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT, encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion.

Prevalence of Type IV Pili-Mediated Twitching Motility in Streptococcus sanguinis Strains and Its Impact on Biofilm Formation and Host Adherence.

Type IV pili (Tfp) are known to mediate several biological activities, including surface-dependent twitching motility. Although a pil gene cluster for Tfp biosynthesis is found in all sequenced Streptococcus sanguinis strains, Tfp-mediated twitching motility is less commonly detected. Upon examining 81 clinical strains, 39 strains generated twitching zones on blood agar plates (BAP), while 27 strains displayed twitching on Todd-Hewitt (TH) agar. Although BAP appears to be more suitable for the development of twitching zones, 5 strains exhibited twitching motility only on TH agar, indicating that twitching motility is not only strain specific but also sensitive to growth media. Furthermore, different twitching phenotypes were observed in strains expressing comparable levels of pilT, encoding the retraction ATPase, suggesting that the twitching phenotype on agar plates is regulated by multiple factors. By using a PilT-null and a pilin protein-null derivative (CHW02) of twitching-active S. sanguinis CGMH010, we found that Tfp retraction was essential for biofilm stability. Further, biofilm growth was amplified in CHW02 in the absence of shearing force, indicating that S. sanguinis may utilize other ligands for biofilm formation in the absence of Tfp. Similar to SK36, Tfp from CGMH010 were required for colonization of host cells, but PilT only marginally affected adherence and only in the twitching-active strain. Taken together, the results suggest that Tfp participates in host cell adherence and that Tfp retraction facilitates biofilm stability. IMPORTANCE Although the gene clusters encoding Tfp are commonly present in Streptococcus sanguinis, not all strains express surface-dependent twitching motility on agar surfaces. Regardless of whether the Tfp could drive motility, Tfp can serve as a ligand for the colonization of host cells. Though many S. sanguinis strains lack twitching activity, motility can enhance biofilm stability in a twitching-active strain; thus, perhaps motility provides little or no advantage to the survival of bacteria within dental plaque. Rather, Tfp retraction could provide additional advantages for the bacteria to establish infections outside the oral cavity.

Identification of a Putative CodY Regulon in the Gram-Negative Phylum Synergistetes.

CodY is a dominant regulator in low G + C, Gram-positive Firmicutes that governs the regulation of various metabolic pathways and cellular processes. By using various bioinformatics analyses and DNA affinity precipitation assay (DAPA), this study confirmed the presence of CodY orthologues and corresponding regulons in Gram-negative Synergistetes. A novel palindromic sequence consisting of AT-rich arms separated by a spacer region of variable length and sequence was identified in the promoters of the putative codY-containing operons in Synergistetes. The consensus sequence from genera Synergistes and Cloacibacillus (5'-AATTTTCTTAAAATTTCSCTTGATATTTACAATTTT) contained three AT-rich regions, resulting in two palindromic sequences; one of which is identical to Firmicutes CodY box (5'-AATTTTCWGAAAATT). The function of the consensus sequence was tested by using a recombinant CodY protein (His-CodYDSM) of Cloacibacillus evryensis DSM19522 in DAPA. Mutations in the central AT-rich sequence reduced significantly the binding of His-CodYDSM, whereas mutations in the 5' or 3' end AT-rich sequence slightly reduced the binding, indicating that CodYDSM could recognize both palindromic sequences. The proposed binding sequences were found in the promoters of multiple genes involved in amino acids biosynthesis, metabolism, regulation, and stress responses in Synergistetes. Thus, a CodY-like protein from Synergistetes may function similarly to Firmicutes CodY.

PM2.5 impairs macrophage functions to exacerbate pneumococcus-induced pulmonary pathogenesis.

BACKGROUND: Pneumococcus is one of the most common human airway pathogens that causes life-threatening infections. Ambient fine particulate matter (PM) with aerodynamic diameter ≤ 2.5 µm (PM2.5) is known to significantly contribute to respiratory diseases. PM2.5-induced airway inflammation may decrease innate immune defenses against bacterial infection. However, there is currently limited information available regarding the effect of PM2.5 exposure on molecular interactions between pneumococcus and macrophages. RESULTS: PM2.5 exposure hampered macrophage functions, including phagocytosis and proinflammatory cytokine production, in response to pneumococcal infection. In a PM2.5-exposed pneumococcus-infected mouse model, PM2.5 subverted the pulmonary immune response and caused leukocyte infiltration. Further, PM2.5 exposure suppressed the levels of CXCL10 and its receptor, CXCR3, by inhibiting the PI3K/Akt and MAPK pathways. CONCLUSIONS: The effect of PM2.5 exposure on macrophage activity enhances pneumococcal infectivity and aggravates pulmonary pathogenesis.

Functional Analysis of a Fibronectin Binding Protein of Streptococcus parasanguinis FW213.

Streptococcus parasanguinis is a primary colonizer of dental plaque and an opportunistic pathogen for subacute endocarditis. A putative fibronectin binding protein (Spaf_1409) that lacks both an N-terminal signal peptide and a C-terminal cell wall-anchoring motif was identified from the S. parasanguinis FW213 genome. Spaf_1409 was abundantly present in the cytoplasm and also was found in the cell wall preparation and culture supernatant. By using an isogenic mutant strain, MPH4, Spaf_1409 was found to mediate the binding of S. parasanguinis FW213 to fibronectin. Inactivation of Spaf_1409 did not significantly alter the mass of static biofilm, but reduced the resistance of S. parasanguinis against the shearing force in a flow cell biofilm system, resulting in scattered biofilm. The mortality in Galleria mellonella larvae infected with MPH4 was higher than in those infected with wild-type S. parasanguinis. However, fewer viable bacterial cells were recovered from larvae infected with MPH4, compared to those infected with wild-type S. parasanguinis, up to 42 h post infection, suggesting that the infection by MPH4, but not the growth, was responsible for the elevated mortality. The phagocytic analysis using flow cytometry indicated that Spaf_1409 participates in the recognition of S. parasanguinis FW213 by RAW264.7 macrophages, suggesting that inactivation of Spaf_1409 intensified the immune responses in larvae, leading to larval death. Taken together, the data indicate that Spaf_1409 plays different roles in the development of dental biofilm and in systemic infections.

Molecular and Functional Analysis of the Type IV Pilus Gene Cluster in Streptococcus sanguinis SK36.

Streptococcus sanguinis, dominant in the oral microbiome, is the only known streptococcal species possessing a pil gene cluster for the biosynthesis of type IV pili (Tfp). Although this cluster is commonly present in the genome of S. sanguinis, most of the strains do not express Tfp-mediated twitching motility. Thus, this study was designed to investigate the biological functions encoded by the cluster in the twitching-negative strain S. sanguinis SK36. We found that the cluster was transcribed as an operon, with three promoters located 5' to the cluster and one in the intergenic region between SSA_2307 and SSA_2305. Studies using promoter-cat fusion strains revealed that the transcription of the cluster was mainly driven by the distal 5' promoter, which is located more than 800 bases 5' to the first gene of the cluster, SSA_2318. Optimal expression of the cluster occurred at the early stationary growth phase in a CcpA-dependent manner, although a CcpA-binding consensus is absent in the promoter region. Expression of the cluster resulted in a short hairlike surface structure under transmission electron microscopy. Deletion of the putative pilin genes (SSA_2313 to SSA_2315) abolished the biosynthesis of this structure and significantly reduced the adherence of SK36 to HeLa and SCC-4 cells. Mutations in the pil genes downregulated biofilm formation by S. sanguinis SK36. Taken together, the results demonstrate that Tfp of SK36 are important for host cell adherence, but not for motility, and that expression of the pil cluster is subject to complex regulation.IMPORTANCE The proteins and assembly machinery of the type IV pili (Tfp) are conserved throughout bacteria and archaea, and yet the function of this surface structure differs from species to species and even from strain to strain. As seen in Streptococcus sanguinis SK36, the expression of the Tfp gene cluster results in a hairlike surface structure that is much shorter than the typical Tfp. This pilus is essential for the adherence of SK36 but is not involved in motility. Being a member of the highly diverse dental biofilm, perhaps S. sanguinis could more effectively utilize this structure to adhere to host cells and to interact with other microbes within the same niche.

Impact of growth pH and glucose concentrations on the CodY regulatory network in Streptococcus salivarius.

BACKGROUND: Streptococcus salivarius is an abundant isolate of the human oral microbiota. Since both pH and glucose availability fluctuate frequently in the oral cavity, the goal of this study was to investigate regulation by CodY, a conserved pleiotropic regulator of Gram positive bacteria, in response to these two signals. The chemostat culture system was employed to precisely control the growth parameters, and the transcriptomes of wild-type S. salivarius 57.I and its CodY-null derivative (ΔcodY) grown at pH 7 and 5.5, with limited and excessive glucose supply were determined. RESULTS: The transcriptomic analysis revealed that CodY was most active at pH 7 under conditions of glucose limitation. Based on whether a CodY binding consensus could be located in the 5' flanking region of the identified target, the transcriptomic analysis also found that CodY shaped the transcriptome via both direct and indirect regulation. Inactivation of codY reduced the glycolytic capacity and the viability of S. salivarius at pH 5.5 or in the presence of H2O2. Studies using the Galleria mellonella larva model showed that CodY was essential for the toxicity generated from S. salivarius infection, suggesting that CodY regulation was critical for immune evasion and systemic infections. Furthermore, the CodY-null mutant strain exhibited a clumping phenotype and reduced attachment in biofilm assays, suggesting that CodY also modulates cell wall metabolism. Finally, the expression of genes belonging to the CovR regulon was affected by codY inactivation, but CodY and CovR regulated these genes in opposite directions. CONCLUSIONS: Metabolic adaptation in response to nutrient availability and growth pH is tightly linked to stress responses and virulence expression in S. salivarius. The regulation of metabolism by CodY allows for the maximal utilization of available nutrients and ATP production. The counteractive regulation of the CovR regulon could fine tune the transcriptomes in response to environmental changes.

YMC-2011, a Temperate Phage of Streptococcus salivarius 57.I.

Streptococcus salivarius is an abundant isolate of the oral cavity. The genome of S. salivarius 57.I consists of a 2-Mb chromosome and a 40,758-bp circular molecule, designated YMC-2011. Annotation of YMC-2011 revealed 55 open reading frames, most of them associated with phage production, although plaque formation is not observed in S. salivarius 57.I after lytic induction using mitomycin C. Results from Southern hybridization and quantitative real-time PCR confirmed that YMC-2011 exists extrachromosomally, with an estimated copy number of 3 to 4. Phage particles were isolated from the supernatant of mitomycin C-treated S. salivarius 57.I cultures, and transmission electron microscopic examination indicated that YMC-2011 belongs to the Siphoviridae family. Phylogenetic analysis suggests that phage YMC-2011 and the cos-type phages of Streptococcus thermophilus originated from a common ancestor. An extended -10 element (p L ) and a σ70-like promoter (p R ) were mapped 5' to Ssal_phage00013 (encoding a CI-like repressor) and Ssal_phage00014 (encoding a hypothetical protein), respectively, using 5' rapid amplification of cDNA ends, indicating that YMC-2011 transcribes at least two mRNAs in opposite orientations. Studies using promoter-chloramphenicol acetyltransferase reporter gene fusions revealed that p R , but not p L , was sensitive to mitomycin C induction, suggesting that the switch from lysogenic growth to lytic growth was controlled mainly by the activity of these two promoters. In conclusion, a lysogenic state is maintained in S. salivarius 57.I, presumably by the repression of genes encoding proteins for lytic growth.IMPORTANCE The movement of mobile genetic elements such as bacteriophages and the establishment of lysogens may have profound effects on the balance of microbial ecology where lysogenic bacteria reside. The discovery of phage YMC-2011 from Streptococcus salivarius 57.I suggests that YMC-2011 and Streptococcus thermophilus-infecting phages share an ancestor. Although S. salivarius and S. thermophilus are close phylogenetically, S. salivarius is a natural inhabitant of the human mouth, whereas S. thermophilus is commonly found in the mammary mucosa of bovine species. Thus, the identification of YMC-2011 suggests that horizontal gene transfer via phage infection could take place between species from different ecological niches.

Is the whole greater than the sum of its parts? De novo assembly strategies for bacterial genomes based on paired-end sequencing.

BACKGROUND: Whole genome sequence construction is becoming increasingly feasible because of advances in next generation sequencing (NGS), including increasing throughput and read length. By simply overlapping paired-end reads, we can obtain longer reads with higher accuracy, which can facilitate the assembly process. However, the influences of different library sizes and assembly methods on paired-end sequencing-based de novo assembly remain poorly understood. RESULTS: We used 250 bp Illumina Miseq paired-end reads of different library sizes generated from genomic DNA from Escherichia coli DH1 and Streptococcus parasanguinis FW213 to compare the assembly results of different library sizes and assembly approaches. Our data indicate that overlapping paired-end reads can increase read accuracy but sometimes cause insertion or deletions. Regarding genome assembly, merged reads only outcompete original paired-end reads when coverage depth is low, and larger libraries tend to yield better assembly results. These results imply that distance information is the most critical factor during assembly. Our results also indicate that when depth is sufficiently high, assembly from subsets can sometimes produce better results. CONCLUSIONS: In summary, this study provides systematic evaluations of de novo assembly from paired end sequencing data. Among the assembly strategies, we find that overlapping paired-end reads is not always beneficial for bacteria genome assembly and should be avoided or used with caution especially for genomes containing high fraction of repetitive sequences. Because increasing numbers of projects aim at bacteria genome sequencing, our study provides valuable suggestions for the field of genomic sequence construction.

The pH-dependent expression of the urease operon in Streptococcus salivarius is mediated by CodY.

Urease gene expression in Streptococcus salivarius 57.I, a strain of one of the major alkali producers in the mouth, is induced by acidic pH and excess amounts of carbohydrate. Expression is controlled primarily at the transcriptional level from a promoter, pureI. Recent sequencing analysis revealed a CodY box located 2 bases 5' to the -35 element of pureI. Using continuous chemostat culture, transcription from pureI was shown to be repressed by CodY, and at pH 7 the repression was more pronounced than that in cells grown at pH 5.5 under both 20 and 100 mM glucose. The direct binding of CodY to pureI was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP)-quantitative real-time PCR (qPCR). The result of ChIP-qPCR also confirmed that the regulation of CodY is indeed modulated by pH and the binding of CodY at neutral pH is further enhanced by a limited supply of glucose (20 mM). In the absence of CodY, the C-terminal domain of the RNA polymerase (RNAP) α subunit interacted with the AT tracks within the CodY box, indicating that CodY and RNAP compete for the same binding region. Such regulation could ensure optimal urease expression when the enzyme is most required, i.e., at an acidic growth pH with an excess amount of carbon nutrients.

RopB represses the transcription of speB in the absence of SIP in group A Streptococcus.

RopB is a quorum-sensing regulator that binds to the SpeB-inducing peptide (SIP) under acidic conditions. SIP is known to be degraded by the endopeptidase PepO, whose transcription is repressed by the CovR/CovS two-component regulatory system. Both SIP-bound RopB (RopB-SIP) and SIP-free RopB (apo-RopB) can bind to the speB promoter; however, only RopB-SIP activates speB transcription. In this study, we found that the SpeB expression was higher in the ropB mutant than in the SIP-inactivated (SIP*) mutant. Furthermore, the deletion of ropB in the SIP* mutant derepressed speB expression, suggesting that apo-RopB is a transcriptional repressor of speB Up-regulation of PepO in the covS mutant degraded SIP, resulting in the down-regulation of speB We demonstrate that deleting ropB in the covS mutant derepressed the speB expression, suggesting that the speB repression in this mutant was mediated not only by PepO-dependent SIP degradation but also by apo-RopB. These findings reveal a crosstalk between the CovR/CovS and RopB-SIP systems and redefine the role of RopB in regulating speB expression in group A Streptococcus.

Clostridium innocuum, an emerging pathogen that induces lipid raft-mediated cytotoxicity.

Clostridium innocuum is an emerging spore-forming anaerobe that is often observed in Clostridioides difficile-associated inflammatory bowel disease (IBD) exacerbations. Unlike C. difficile, C. innocuum neither produces toxins nor possesses toxin-encoding genetic loci, but is commonly found in both intestinal and extra-intestinal infections. Membrane lipid rafts are composed of dynamic assemblies of cholesterol and sphingolipids, allowing bacteria to gain access to cells. However, the direct interaction between C. innocuum and lipid rafts that confers bacteria the ability to disrupt the intestinal barrier and induce pathogenesis remains unclear. In this study, we investigated the associations among nucleotide-binding oligomerization domain containing 2 (NOD2), lipid rafts, and cytotoxicity in C. innocuum-infected gut epithelial cells. Our results revealed that lipid rafts were involved in C. innocuum-induced NOD2 expression and nuclear factor (NF)-κB activation, triggering an inflammatory response. Reducing cholesterol by simvastatin significantly dampened C. innocuum-induced cell death, indicating that the C. innocuum-induced pathogenicity of cells was lipid raft-dependent. These results demonstrate that NOD2 mobilization into membrane rafts in response to C. innocuum-induced cytotoxicity results in aggravated pathogenicity.

FastAnnotator--an efficient transcript annotation web tool.

BACKGROUND: Recent developments in high-throughput sequencing (HTS) technologies have made it feasible to sequence the complete transcriptomes of non-model organisms or metatranscriptomes from environmental samples. The challenge after generating hundreds of millions of sequences is to annotate these transcripts and classify the transcripts based on their putative functions. Because many biological scientists lack the knowledge to install Linux-based software packages or maintain databases used for transcript annotation, we developed an automatic annotation tool with an easy-to-use interface. METHODS: To elucidate the potential functions of gene transcripts, we integrated well-established annotation tools: Blast2GO, PRIAM and RPS BLAST in a web-based service, FastAnnotator, which can assign Gene Ontology (GO) terms, Enzyme Commission numbers (EC numbers) and functional domains to query sequences. RESULTS: Using six transcriptome sequence datasets as examples, we demonstrated the ability of FastAnnotator to assign functional annotations. FastAnnotator annotated 88.1% and 81.3% of the transcripts from the well-studied organisms Caenorhabditis elegans and Streptococcus parasanguinis, respectively. Furthermore, FastAnnotator annotated 62.9%, 20.4%, 53.1% and 42.0% of the sequences from the transcriptomes of sweet potato, clam, amoeba, and Trichomonas vaginalis, respectively, which lack reference genomes. We demonstrated that FastAnnotator can complete the annotation process in a reasonable amount of time and is suitable for the annotation of transcriptomes from model organisms or organisms for which annotated reference genomes are not avaiable. CONCLUSIONS: The sequencing process no longer represents the bottleneck in the study of genomics, and automatic annotation tools have become invaluable as the annotation procedure has become the limiting step. We present FastAnnotator, which was an automated annotation web tool designed to efficiently annotate sequences with their gene functions, enzyme functions or domains. FastAnnotator is useful in transcriptome studies and especially for those focusing on non-model organisms or metatranscriptomes. FastAnnotator does not require local installation and is freely available at http://fastannotator.cgu.edu.tw.

Structural insight into the role of Streptococcus parasanguinis Fap1 within oral biofilm formation.

The fimbriae-associated protein 1 (Fap1) is a major adhesin of Streptococcus parasanguinis, a primary colonizer of the oral cavity that plays an important role in the formation of dental plaque. Fap1 is an extracellular adhesive surface fibre belonging to the serine-rich repeat protein (SRRP) family, which plays a central role in the pathogenesis of streptococci and staphylococci. The N-terminal adhesive region of Fap1 (Fap1-NR) is composed of two domains (Fap1-NR(α) and Fap1-NR(ß)) and is projected away from the bacterial surface via the extensive serine-rich repeat region, for adhesion to the salivary pellicle. The adhesive properties of Fap1 are modulated through a pH switch in which a reduction in pH results in a rearrangement between the Fap1-NR(α) and Fap1-NR(ß) domains, which assists in the survival of S. parasanguinis in acidic environments. We have solved the structure of Fap1-NR(α) at pH 5.0 at 3.0Ǻ resolution and reveal how subtle rearrangements of the 3-helix bundle combined with a change in electrostatic potential mediates 'opening' and activation of the adhesive region. Further, we show that pH-dependent changes are critical for biofilm formation and present an atomic model for the inter-Fap1-NR interactions which have been assigned an important role in the biofilm formation.

The Bacterial Markers of Identification of Invasive CovR/CovS-Inactivated Group A Streptococcus.

Necrotizing fasciitis is a severe infectious disease that results in significant mortality. Streptococcus pyogenes (group A Streptococcus, GAS) is one of the most common bacterial pathogens of monomicrobial necrotizing fasciitis. The early diagnosis of necrotizing fasciitis is crucial; however, the typical cutaneous manifestations are not always presented in patients with GAS necrotizing fasciitis, which would lead to miss- or delayed diagnosis. GAS with spontaneous inactivating mutations in the CovR/CovS two-component regulatory system is significantly associated with destructive diseases such as necrotizing fasciitis and toxic shock syndrome; however, no specific marker has been used to identify these invasive clinical isolates. This study evaluated the sensitivity and specificity of using CovR/CovS-controlled phenotypes to identify CovR/CovS-inactivated isolates. Results showed that the increase of hyaluronic acid capsule production and streptolysin O expression were not consistently presented in CovS-inactivated clinical isolates. The repression of SpeB is the phenotype with 100% sensitivity of identifying in CovS-inactivated isolates among 61 clinical isolates. Nonetheless, this phenotype failed to distinguish RopB-inactivated isolates from CovS-inactivated isolates and cannot be utilized to identify CovR-inactivated mutant and RocA (Regulator of Cov)-inactivated isolates. In this study, we identified and verified that PepO, the endopeptidase which regulates SpeB expression through degrading SpeB-inducing quorum-sensing peptide, was a bacterial marker to identify isolates with defects in the CovR/CovS pathway. These results also inform the potential strategy of developing rapid detection methods to identify invasive GAS variants during infection. IMPORTANCE Necrotizing fasciitis is rapidly progressive and life-threatening; if the initial diagnosis is delayed, deep soft tissue infection can progress to massive tissue destruction and toxic shock syndrome. Group A Streptococcus (GAS) with inactivated mutations in the CovR/CovS two-component regulatory system are related to necrotizing fasciitis and toxic shock syndrome; however, no bacterial marker is available to identify these invasive clinical isolates. Inactivation of CovR/CovS resulted in the increased expression of endopeptidase PepO. Our study showed that the upregulation of PepO mediates a decrease in SpeB-inducing peptide (SIP) in the covR mutant, indicating that CovR/CovS modulates SIP-dependent quorum-sensing activity through PepO. Importantly, the sensitivity and specificity of utilizing PepO to identify clinical isolates with defects in the CovR/CovS pathway, including its upstream RocA regulator, were 100%. Our results suggest that identification of invasive GAS by PepO may be a strategy for preventing severe manifestation or poor prognosis after GAS infection.

Complete genome sequence of the ureolytic Streptococcus salivarius strain 57.I.

Streptococcus salivarius 57.I is one of the most abundant and highly ureolytic bacteria in the human mouth. It can utilize urea as the sole nitrogen source via the activity of urease. Complete genome sequencing of S. salivarius 57.I revealed a chromosome and a phage which are absent in strain SK126.

New cell surface protein involved in biofilm formation by Streptococcus parasanguinis.

Dental biofilm formation is critical for maintaining the healthy microbial ecology of the oral cavity. Streptococci are predominant bacterial species in the oral cavity and play important roles in the initiation of plaque formation. In this study, we identified a new cell surface protein, BapA1, from Streptococcus parasanguinis FW213 and determined that BapA1 is critical for biofilm formation. Sequence analysis revealed that BapA1 possesses a typical cell wall-sorting signal for cell surface-anchored proteins from Gram-positive bacteria. No functional orthologue was reported in other streptococci. BapA1 possesses nine putative pilin isopeptide linker domains which are crucial for pilus assembly in a number of Gram-positive bacteria. Deletion of the 3' portion of bapA1 generated a mutant that lacks surface-anchored BapA1 and abolishes formation of short fibrils on the cell surface. The mutant failed to form biofilms and exhibited reduced adherence to an in vitro tooth model. The BapA1 deficiency also inhibited bacterial autoaggregation. The N-terminal muramidase-released-protein-like domain mediated BapA1-BapA1 interactions, suggesting that BapA1-mediated cell-cell interactions are important for bacterial autoaggregation and biofilm formation. Furthermore, the BapA1-mediated bacterial adhesion and biofilm formation are independent of a fimbria-associated serine-rich repeat adhesin, Fap1, demonstrating that BapA1 is a new streptococcal adhesin.

Properties and construction of plasmid pFW213, a shuttle vector with the oral Streptococcus origin of replication.

Streptococcus parasanguinis is among the most successful colonizers of the human body. Strain FW213 harbors a 7.0-kb cryptic plasmid, pFW213, with a copy number at 5 to 10 per chromosome. Sequence and functional analyses of pFW213 revealed that the open reading frame (ORF) encoding the replication protein (Rep) is essential for the replication of pFW213, and the putative plasmid addiction system (RelB and RelE) and an ORF (ORF6) with no known function are required for its stability. The minimal replicon of pFW213 contains the rep gene and its 5'-flanking 390-bp region. Within the minimal replicon, an A/T-rich region followed by 5 contiguous 22-bp repeats was located 5' of the ATG of rep. No single-stranded replication intermediates were detected in the derivatives of pFW213, suggesting that pFW213 replicates via the theta replication mechanism. The minimal replicon was unstable in streptococcal hosts without selection, but the stability was greatly enhanced in derivatives containing the intact relBE genes. A Streptococcus-Escherichia coli shuttle vector, pCG1, was constructed with the pFW213 replicon. Plasmid pCG1 features a multiple cloning region and a spectinomycin resistance determinant that is expressed in both Streptococcus spp. and E. coli. Various streptococcal DNA fragments were cloned in pCG1, and the recombinant constructs were stably maintained in the streptococcal hosts. Since pCG1 is compatible with the most widely used streptococcal replicon, pVA380-1, pCG1 will provide a much needed tool allowing the cloning of two genes that work in concert in the same host.

Incidence and Effects of Acquisition of the Phage-Encoded ssa Superantigen Gene in Invasive Group A Streptococcus.

The acquisition of the phage-encoded superantigen ssa by scarlet fever-associated group A Streptococcus (Streptococcus pyogenes, GAS) is found in North Asia. Nonetheless, the impact of acquiring ssa by GAS in invasive infections is unclear. This study initially analyzed the prevalence of ssa+ GAS among isolates from sterile tissues and blood. Among 220 isolates in northern Taiwan, the prevalence of ssa+ isolates increased from 1.5% in 2008-2010 to 40% in 2017-2019. Spontaneous mutations in covR/covS, which result in the functional loss of capacity to phosphorylate CovR, are frequently recovered from GAS invasive infection cases. Consistent with this, Phostag western blot results indicated that among the invasive infection isolates studied, 10% of the ssa+ isolates lacked detectable phosphorylated CovR. Transcription of ssa is upregulated in the covS mutant. Furthermore, in emm1 and emm12 covS mutants, ssa deletion significantly reduced their capacity to grow in human whole blood. Finally, this study showed that the ssa gene could be transferred from emm12-type isolates to the emm1-type wild-type strain and covS mutants through phage infection and lysogenic conversion. As the prevalence of ssa+ isolates increased significantly, the role of streptococcal superantigen in GAS pathogenesis, particularly in invasive covR/covS mutants, should be further analyzed.

Role of GlnR in acid-mediated repression of genes encoding proteins involved in glutamine and glutamate metabolism in Streptococcus mutans.

The acid tolerance response (ATR) is one of the major virulence traits of Streptococcus mutans. In this study, the role of GlnR in acid-mediated gene repression that affects the adaptive ATR in S. mutans was investigated. Using a whole-genome microarray and in silico analyses, we demonstrated that GlnR and the GlnR box (ATGTNAN(7)TNACAT) were involved in the transcriptional repression of clusters of genes encoding proteins involved in glutamine and glutamate metabolism under acidic challenge. Reverse transcription-PCR (RT-PCR) analysis revealed that the coordinated regulation of the GlnR regulon occurred 5 min after acid treatment and that prolonged acid exposure (30 min) resulted in further reduction in expression. A lower level but consistent reduction in response to acidic pH was also observed in chemostat-grown cells, confirming the negative regulation of GlnR. The repression by GlnR through the GlnR box in response to acidic pH was further confirmed in the citBZC operon, containing genes encoding the first three enzymes in the glutamine/glutamate biosynthesis pathway. The survival rate of the GlnR-deficient mutant at pH 2.8 was more than 10-fold lower than that in the wild-type strain 45 min after acid treatment, suggesting that the GlnR regulon participates in S. mutans ATR. It is hypothesized that downregulation of the synthesis of the amino acid precursors in response to acid challenge would promote citrate metabolism to pyruvate, with the consumption of H(+) and potential ATP synthesis. Such regulation will ensure an optimal acid adaption in S. mutans.