Genome sequence of the temperate bacteriophage PH10 from Streptococcus oralis.

Exponential growing cultures of Streptococcus oralis strain OMZ 1038, isolated from human supragingival dental plaque, were found to release a bacteriophage (designated PH10) upon treatment with mitomycin C. The complete genome sequence of phage PH10 was determined. The genome was 31276 bp in size and contained 54 open reading frames. The module encoding structural proteins was highly similar to that of Streptococcus pneumoniae prophage PhiSpn_3. The most abundant phage structural protein was encoded by ORF35 and was likely processed by proteolytic cleavage. The putative endolysin from PH10, which contained a muramidase domain and a choline-binding domain, was purified and shown to have lytic activity with S. oralis, S. pneumoniae and Streptococcus mitis, but not with other streptococcal species.

The Streptococcus mutans serine/threonine kinase, PknB, regulates competence development, bacteriocin production, and cell wall metabolism.

Bacteria can detect, transmit, and react to signals from the outside world by using two-component systems (TCS) and serine-threonine kinases and phosphatases. Streptococcus mutans contains one serine-threonine kinase, encoded by pknB. A gene encoding a serine-threonine phosphatase, pppL, is located upstream of pknB. In this study, the phenotypes of pknB and pppL single mutants and a pknB pppL double mutant were characterized. All mutants exhibited a reduction in genetic transformability and biofilm formation, showed abnormal cell shapes, grew slower than the wild-type strain in several complex media, and exhibited reduced acid tolerance. The mutants had reduced cariogenic capacity but no significant defects in colonization in a rat caries model. Whole-genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes involved in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant, several were likely to be involved in cell wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to also be downregulated in a vicK mutant, which encodes a sensor kinase involved in the response to oxidative stress. Collectively, the results lead us to speculate that PknB may modulate the activity of the two-component signal transduction systems VicKR and ComDE. Real-time reverse transcriptase PCR (RT-PCR) showed that genes downregulated in the pknB mutant were upregulated in the pppL mutant, indicating that PppL serves to counteract PknB.

The serotype-specific glucose side chain of rhamnose-glucose polysaccharides is essential for adsorption of bacteriophage M102 to Streptococcus mutans.

Bacteriophage M102 is a virulent siphophage that propagates in some serotype c Streptococcus mutans strains, but not in S. mutans of serotype e, f or k. The serotype of S. mutans is determined by the glucose side chain of rhamnose-glucose polysaccharide (RGP). Because the first step in the bacteriophage infection process is adsorption of the phage, it was investigated whether the serotype specificity of phage M102 was determined by adsorption. M102 adsorbed to all tested serotype c strains, but not to strains of different serotypes. Streptococcus mutans serotype c mutants defective in the synthesis of the glucose side chain of RGP failed to adsorb phage M102. These results suggest that the glucose side chain of RGP acts as a receptor for phage M102.

Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages.

Clustered regularly interspaced short palindromic repeats (CRISPR) consist of highly conserved direct repeats interspersed with variable spacer sequences. They can protect bacteria against invasion by foreign DNA elements. The genome sequence of Streptococcus mutans strain UA159 contains two CRISPR loci, designated CRISPR1 and CRISPR2. The aims of this study were to analyse the organization of CRISPR in further S. mutans strains and to investigate the importance of CRISPR in acquired immunity to M102-like phages. The sequences of CRISPR1 and CRISPR2 arrays were determined for 29 S. mutans strains from different persons. More than half of the CRISPR1 spacers and about 35 % of the CRISPR2 spacers showed sequence similarity with the genome sequence of M102, a virulent siphophage specific for S. mutans. Although only a few spacers matched the phage sequence completely, most of the mismatches had no effect on the amino acid sequences of the phage-encoded proteins. The results suggest that S. mutans is often attacked by M102-like bacteriophages, and that its acquisition of novel phage-derived CRISPR sequences goes along with the presence of S. mutans phages in the environment. Analysis of CRISPR1 of M102-resistant mutants of S. mutans OMZ 381 showed that some of them had acquired novel spacers, and the sequences of all but one of these matched the phage M102 genome sequence. This suggests that the acquisition of the spacers contributed to the resistance against phage infection. However, since not all resistant mutants had new spacers, and since the removal of the CRISPR1 array in one of the mutants and in wild-type strains did not lead to loss of resistance to infection by M102, the acquisition of resistance must be based on further elements as well.

Kinase activity of the dgk gene product is involved in the virulence of Streptococcus mutans.

C-terminal deletion of the diacylglycerol kinase (Dgk) homologue of the cariogenic oral bacterium Streptococcus mutans resulted in loss of aciduricity. To confirm the role of the C terminus of the Dgk homologue in aciduricity, various mutants of S. mutans UA159 with a C-terminally truncated Dgk homologue were constructed. The deletion of one or two amino acid residues at the C terminus had no effect on the acid-tolerance properties of mutants. When further amino acid residues at the C terminus were removed, mutants became more acid-sensitive. The mutant with deletion of eight amino acid residues at the C terminus did not grow at pH 5.5, suggesting that the C-terminal tail of the Dgk homologue was indispensable for tolerance to acid stress in S. mutans. Kinase activity assays revealed that deletion of the C-terminal amino acids of Dgk led to a reduction of kinase activity for undecaprenol. A truncated mutant that had completely lost kinase activity was unable to grow at pH 5.5. These results suggest that the acid tolerance of S. mutans is closely related to kinase activity of the Dgk homologue. Additionally, the dgk deletion mutant exhibited markedly reduced levels of smooth-surface carious lesions in pathogen-free rats, despite there being no difference between the mutant and the parental organism in the extent of total smooth surface plaque. The results suggest that Dgk activity may play a direct role in the virulence of S. mutans.

Characterization of Streptococcus gordonii prophage PH15: complete genome sequence and functional analysis of phage-encoded integrase and endolysin.

Streptococcus gordonii OMZ1039, isolated from supragingival dental plaque, was found to harbour a prophage, PH15, whose excision could be induced by mitomycin treatment. Phage PH15 belongs to the Siphoviridae. The complete genome sequence of PH15 was determined. The genome was 39 136 bp in size and contained 61 ORFs. The genome of PH15 was most similar in the structural module to the temperate bacteriophages MM1 and phiNIH1.1 from Streptococcus pneumoniae and Streptococcus pyogenes, respectively. In strain OMZ1039, PH15 was found to reside as a prophage in the cysteinyl-tRNA gene. A plasmid, harbouring the attP site and the integrase gene downstream of a constitutive promoter, was capable of site-specific integration into the genomes of different oral streptococcal species. The phage endolysin was purified after expression in Escherichia coli and found to inhibit growth of all S. gordonii strains tested and several different streptococcal species, including the pathogens Streptococcus mutans, S. pyogenes and Streptococcus agalactiae.

Genome sequence of Streptococcus mutans bacteriophage M102.

Bacteriophage M102 is a lytic phage specific for serotype c strains of Streptococcus mutans, a causative agent of dental caries. In this study, the complete genome sequence of M102 was determined. The genome is 31,147 bp in size and contains 41 ORFs. Most of the ORFs encoding putative phage structural proteins show similarity to those from bacteriophages from Streptococcus thermophilus. Bioinformatic analysis indicated that the M102 genome contains an unusual lysis cassette, which encodes a holin and two lytic enzymes.

Quantitation of Guggenheimella bovis and treponemes in bovine tissues related to digital dermatitis.

Digital dermatitis is an inflammation of uncertain aetiology in the skin of the foot of cattle. In 2005, a novel microorganism, Guggenheimella bovis, was isolated from the advancing front of digital dermatitis lesions, suggesting a possible role in pathogenesis. In the present study, tissue samples of 20 affected cows were examined by quantitative PCR for G. bovis, treponemes and the total eubacterial load. High numbers of eubacteria and treponemes were found in most lesions, whereas only a few lesions contained Guggenheimella, and only at low concentrations. The results argue against the relevance of G. bovis in the aetiology of digital dermatitis in cattle, but are consistent with a role for treponemes.

Regulation of bacteriocin production in Streptococcus mutans by the quorum-sensing system required for development of genetic competence.

In Streptococcus mutans, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by comC). Here, it is shown that the same system is also required for expression of the nlmAB genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional nlmAB'-lacZ fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the comDE genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the nlmAB promoter region led to a decrease in CSP-dependent expression of nlmAB'-lacZ. In agreement with these results, a comDE mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci.

Deletion of gtfC of Streptococcus mutans has no influence on the composition of a mixed-species in vitro biofilm model of supragingival plaque.

Glucosyltransferases from Streptococcus mutans are thought to play an important role in bacterial adherence to the tooth surface. The goal of the present study was to determine the effect of the deletion of the gtfC gene, which encodes a glucosyltransferase that catalyses primarily the formation of insoluble glucan (mutan), on colonization of S. mutans in a mixed-species biofilm model of supragingival plaque. A gtfC deletion mutant of S. mutans UA159 grew poorly in biofilms on a polystyrene surface in Todd-Hewitt medium containing sucrose, but biofilm formation in the semi-defined fluid universal medium (FUM) was not affected. The S. mutans gtfC mutant colonized with the same efficiency as the wild-type strain when grown together with five other species in a mixed-species biofilm on hydroxyapatite in a mixture of FUM and saliva with pulses of sucrose and showed the same ability to demineralize enamel in vitro. Colonization of mutant and wild-type strains was also equal in an association experiment in specific-pathogen-free rats. However, the gtfC mutant gave rise to more dentinal fissure lesions and smooth surface caries than the wild-type strain; this could be caused by a change in diffusion properties as a result of to the lack of mutan.

Quantitative detection of Porphyromonas gingivalis fimA genotypes in dental plaque.

We developed quantitative fimA genotype assays and applied them in a pilot study investigating the fimbrial genotype distribution of Porphyromonas gingivalis in European subjects with or without chronic periodontitis. P. gingivalis was found in 71% and 9% of the samples from patients and healthy subjects, respectively. Enumeration of total P. gingivalis cell numbers by polymerase chain reaction and immunofluorescence showed excellent correspondence (r = 0.964). 73% of positive samples contained multiple fimA genotypes, but generally one genotype predominated by one to three orders of magnitude. Genotype II predominated in 60% of the samples. Genotype IV occurred with similar prevalence (73%) as genotype II but predominated in only 20% of the samples. Genotypes I, III and V were of much lower prevalence and cell densities of the latter two remained sparse. Our results suggest marked differences among the fimA genotypes' ability to colonize host sites with high cell numbers.

Transformation of isopropylamine to L-alaninol by Pseudomonas sp. strain KIE171 involves N-glutamylated intermediates.

Pseudomonas sp. strain KIE171 was able to grow with isopropylamine or L-alaninol [S-(+)-2-amino-1-propanol] as the sole carbon source, but not with D-alaninol. To investigate the hypothesis that L-alaninol is an intermediate in the degradation of isopropylamine, two mini-Tn5 mutants unable to utilize both isopropylamine and L-alaninol were isolated. Whereas mutant KIE171-BI transformed isopropylamine to L-alaninol, mutant KIE171-BII failed to do so. The two genes containing a transposon insertion were cloned, and the DNA regions flanking the insertions were sequenced. Two clusters, one comprising eight ipu (isopropylamine utilization) genes (ipuABCDEFGH) and the other encompassing two genes (ipuI and orf259), were identified. Comparisons of sequences of the deduced Ipu proteins and those in the database suggested that isopropylamine is transported into the cytoplasm by a putative permease, IpuG. The next step, the formation of gamma-glutamyl-isopropylamide from isopropylamine, ATP, and L-glutamate, was shown to be catalyzed by IpuC, a gamma-glutamylamide synthetase. gamma-Glutamyl-isopropylamide is then subjected to stereospecific monooxygenation by the hypothetical four-component system IpuABDE, thereby yielding gamma-glutamyl-L-alaninol [gamma(L-glutamyl)-L-hydroxy-isopropylamide]. Enzymatic hydrolysis by a hydrolase, IpuF, was shown to finally liberate L-alaninol and to regenerate L-glutamate. No gene(s) encoding an enzyme for the next step in the degradation of isopropylamine was found in the ipu clusters. Presumably, L-alaninol is oxidized by an alcohol dehydrogenase to yield L-2-aminopropionaldehyde or it is deaminated by an ammonia lyase to propionaldehyde. Genetic evidence indicated that the aldehyde formed is then further oxidized by the hypothetical aldehyde dehydrogenases IpuI and IpuH to either L-alanine or propionic acid, compounds which can be processed by reactions of the intermediary metabolism.

The switch from inorganic to organic sulphur assimilation in Escherichia coli: adenosine 5'-phosphosulphate (APS) as a signalling molecule for sulphate excess.

The utilization of organosulphur compounds as sources of sulphur by Escherichia coli is strongly repressed by sulphate. To search for the signal enabling E. coli to alternate gene expression according to the sulphur source, we investigated the transcriptional control of the ssuEADCB operon, required for the transport and desulphonation of aliphatic sulphonates. We demonstrate that, of the two LysR-type regulators involved in expression from the ssu promoter, Cbl acts as a direct and sufficient activator of transcription in vivo and in vitro, whereas CysB downregulates the promoter efficiency. Most importantly, the Cbl-mediated transcription initiation at the ssu promoter in vitro is abolished in the presence of an early metabolite of the sulphate assimilatory pathway, adenosine 5'-phosphosulphate (APS). This role for APS was confirmed in vivo by measuring the expression of beta-galactosidase from a transcriptional ssu-lacZ fusion in strains containing different mutations blocking the synthesis and consumption of APS. Our data comprise the first evidence that APS may act as the negative cofactor of the transcriptional regulator Cbl, and that APS, and not sulphate itself, serves as the signalling molecule for sulphate excess.

Bacillus subtilis genes for the utilization of sulfur from aliphatic sulfonates.

A 5 kb region upstream of katA at 82 degrees on the Bacillus subtilis chromosome contains five ORFs organized in an operon-like structure. Based on sequence similarity, three of the ORFs are likely to encode an ABC transport system (ssuBAC) and another to encode a monooxygenase (ssuD). The deduced amino acid sequence of the last ORF (ygaN) shows no similarity to any known protein. B. subtilis can utilize a range of aliphatic sulfonates such as alkanesulfonates, taurine, isethionate and sulfoacetate as a source of sulfur, but not when ssuA and ssuC are disrupted by insertion of a neomycin-resistance gene. Utilization of aliphatic sulfonates was not affected in a strain lacking 3'-phosphoadenosine 5'-phosphosulfate (PAPS) sulfotransferase, indicating that sulfate is not an intermediate in the assimilation of sulfonate-sulfur. Sulfate or cysteine prevented expression of beta-galactosidase from a transcriptional ssuD::lacZ fusion. It is proposed that ssuBACD encode a system for ATP-dependent transport of alkanesulfonates and an oxygenase required for their desulfonation.