Lactococcus lactis ZitR is a zinc-responsive repressor active in the presence of low, nontoxic zinc concentrations in vivo.

In the family Streptococcaceae, the genes encoding zinc ABC uptake systems (called zit or adc) are regulated by a coencoded MarR family member (i.e., ZitR or AdcR), whereas in the great majority of bacteria, these genes are regulated by Zur, the Fur-like zinc-responsive repressor. We studied the zit operon from Lactococcus lactis and its regulation in response to Zn(II) in vivo. zit transcription is repressed by Zn(II) in a wide concentration range starting from nontoxic micromolar levels and is derepressed at nanomolar concentrations. The level of zit promoter downregulation by environmental Zn(II) is correlated with the intracellular zinc content. The helix-turn-helix domain of ZitR is required for downregulation. In vitro, the purified protein is a dimer that complexes up to two zinc ligands per monomer and specifically binds two intact palindromic operator sites overlapping the -35 and -10 boxes of the zit promoter. DNA binding is abolished by the chelator EDTA or TPEN and fully restored by Zn(II) addition, indicating that the active repressor complexes Zn(II) with high affinity. These results suggest that derepression under starvation conditions could be an essential emergency mechanism for preserving Zn(II) homeostasis by uptake; under Zn(II)-replete conditions, the function of ZitR repression could be to help save energy rather than to avoid Zn(II) toxicity. The characterization of a MarR family zinc-responsive repressor in this report gives insight into the way Streptococcaceae efficiently adapt to Zn(II) fluctuations in their diverse ecological niches.

Cloning, expression, and characterization of a peculiar choline-binding beta-galactosidase from Streptococcus mitis.

A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative beta-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric beta-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40 degrees C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In S. mitis, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first beta-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.

Mutations in the tacF gene of clinical strains and laboratory transformants of Streptococcus pneumoniae: impact on choline auxotrophy and growth rate.

The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.

Skl, a novel choline-binding N-acetylmuramoyl-L-alanine amidase of Streptococcus mitis SK137 containing a CHAP domain.

The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been purified and biochemically characterized. Analysis of the degradation products obtained by digestion of pneumococcal cell walls with Skl revealed that this enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), showing optimum activity at 30 degrees C and at a pH of 6.5. Skl is a unique member of the choline-binding family of proteins since it contains a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The CHAP domain of Skl showed homology to lysins of unknown especificity from a variety of streptococcal prophages. Skl represents the first characterized member of a new subfamily of CHAP-containing choline-binding proteins.

PpiA, a surface PPIase of the cyclophilin family in Lactococcus lactis.

BACKGROUND: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. RESULTS: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H(2)O(2)) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H(2)O(2). Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. CONCLUSIONS: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.

In vitro bactericidal activity of the antiprotozoal drug miltefosine against Streptococcus pneumoniae and other pathogenic streptococci.

Miltefosine (hexadecylphosphocholine), the first oral drug against visceral leishmaniasis, triggered pneumococcal autolysis at concentrations higher than 2.5 microM. Bactericidal activity was also observed in cultures of other streptococci, although these failed to undergo lysis. The autolysis elicited by miltefosine can be attributed to triggering of the pneumococcal autolysin LytA.

Isolation and characterization of a new plasmid pSpnP1 from a multidrug-resistant clone of Streptococcus pneumoniae.

A novel Streptococcus pneumoniae plasmid (pSpnP1; 5413bp) has been isolated from the multidrug-resistant clone Poland(23F)-16, and its complete nucleotide sequence has been determined. Sequence analysis predicted seven co-directional open reading frames and comparative analyses revealed that plasmid pSpnP1 is different to pDP1, the only previously described pneumococcal plasmid, whereas it is highly similar to pSt08, a plasmid from Streptococcus thermophilus. A double-stranded origin for replication similar to the replication origin of the pC194/pUB110 family was located upstream of the putative rep gene (orf2). It also contained a 144-bp region with over 60% identity to the single-stranded origin type A of the Streptococcus agalactiae plasmid pMV158/pLS1. Detection of single-stranded DNA by Southern blot analysis indicated that pSpnP1 replicates via a rolling circle mechanism. Interestingly, the product of orf1 has a putative Zonular occludens toxin conserved domain present in toxigenic strains of Vibrio cholerae. Real-time PCR assays revealed that this ORF was expressed. Hybridization experiments showed that the pSpnP1 replicon was unusual among other examined antibiotic-resistant pneumococcal clones, although the recombinant plasmids based on pSpnP1 were able to replicate in Bacillus subtilis and Lactococcus lactis.

Characteristic signatures of the lytA gene provide a basis for rapid and reliable diagnosis of Streptococcus pneumoniae infections.

The nucleotide sequences of the lytA gene from 29 pneumococcal isolates of various serotypes and 22 additional streptococci of the mitis group (including two Streptococcus pseudopneumoniae strains) have been compared and found to correspond to 19 typical (927-bp-long) and 20 atypical (921-bp-long) alleles. All the Streptococcus pneumoniae strains harbored typical lytA alleles, whereas nonpneumococcal isolates belonging to the mitis group always carried atypical alleles. A sequence alignment showed that the main difference between typical and atypical lytA alleles resided in 102 nucleotide positions (including the 6 bp absent from atypical alleles). These nucleotides were perfectly conserved in all the typical alleles studied, and the corresponding nucleotides of the atypical alleles were also perfectly conserved. The presence in these signatures of distinctive restriction sites (namely, SnaBI, XmnI, and BsaAI) allowed the development of a simple, reliable, and fast method that combines PCR amplification of the lytA gene, digestion with BsaAI, and separation of the products by agarose gel electrophoresis. This assay allows the rapid and consistent identification of true S. pneumoniae strains and represents an improved diagnostic tool for the study of pneumococcal carriage.

Molecular characterization of disease-associated streptococci of the mitis group that are optochin susceptible.

Eight optochin-susceptible (Opt(s)) alpha-hemolytic (viridans) streptococcus isolates were characterized at the molecular level. These isolates showed phenotypic characteristics typical of both viridans streptococci and Streptococcus pneumoniae. Comparison of the sequence of housekeeping genes from these isolates with those of S. pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae suggested that the Opt(s) isolates corresponded to streptococci of the mitis group. Besides, the Opt(s) streptococci were negative by a Gen-Probe AccuProbe pneumococcus test and hybridized with specific pneumococcal probes (lytA and ply) but also with ant, a gene not present in most S. pneumoniae strains. Moreover, the isolates were insoluble in 1% sodium deoxycholate but completely dissolved in 0.1% deoxycholate. Sequence analysis of the lytA gene revealed that the Opt(s) streptococci carried lytA alleles characteristic of those present in nonpneumococcal streptococci of the mitis group. The determination of the partial nucleotide sequence embracing the atp operon encoding the F(o)F(1) H(+)-ATPase indicated that the optochin susceptibility of the isolates was due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae by horizontal gene transfer.

Immobilization-based isolation of capsule-negative mutants of Streptococcus pneumoniae.

The capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococcus pneumoniae, a human pathogen of the upper respiratory tract. One limitation in studies of S. pneumoniae surface virulence factors is the lack of a reliable procedure for isolation of capsule-negative mutants of clinical strains. This paper presents an approach, based on the immobilization of pneumococci in semi-liquid (0.04 % agar) medium, to easily distinguish and select for non-capsulated mutants. A clinical S. pneumoniae type 37 strain was used as a model to show that CPS production results in bacterial immobilization in semi-liquid agar medium and restricts cell sedimentation. Descendants of CPS(-) mutants sedimented faster under these conditions and therefore could be separated from immobilized parental cells. The CPS(-) phenotype of the obtained mutants was confirmed by both immunoagglutination and immunostaining experiments using specific type 37 capsular antibodies. Complementation of immobilization with the cloned tts gene, encoding type 37 CPS synthase, confirmed that faster sedimentation of mutants was specifically due to loss of the capsule. DNA sequence determination of three independent mutants revealed a point mutation, a 46 nt deletion and a heptanucleotide duplication in the tts gene. Immobilization of strains producing other CPSs (type 2, 3 and 6) also resulted in the appearance of CPS(-) mutants, thus showing that immobilization-based isolation is not restricted to type 37 pneumococci. Bacterial growth in semi-liquid medium proved to be a useful model system to identify the genetic consequences of immobilization. The results indicate that immobilization due to CPS may impose selective pressure against capsule production and thus contribute to capsule plasticity.