New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917.

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transformation of GBS by electroporation, which provided transformation efficiencies of 10(5) CFU/microgram. Variables that influenced the transformation efficiency were the glycine content of the competent cell growth media, the electric field strength during electroporation, the electroporation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. Our transformation protocol provides an efficiency sufficient for cloning from ligation reactions directly into GBS, obviating an intermediate host such as Escherichia coli. Second, temperature-sensitive plasmids of the pWV01 lineage were shown to transform GBS, and their temperature-sensitive replication was confirmed. Lastly, the temperature-sensitive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transposon delivery vector for the construction of genomic Tn917 mutant libraries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, representative clones from a Tn917 library contained single transposon insertions that were randomly located throughout the chromosome. These techniques should provide useful methods for cloning, mutagenesis, and characterization of genes from GBS.     

Use of glnQ as a Counterselectable Marker for Creation of Allelic Exchange Mutations in Group B Streptococci

Efficient allelic exchange mutagenesis in group B streptococci (GBS) has been hampered by the lack of a counterselectable marker system. Growth inhibition of GBS by the glutamine analog gamma-glutamyl hydrazide requires glnQ. We have used this phenomenon to create a counterselectable marker system for efficient selection of allelic exchange mutants in GBS.     

Rapid Microbiological Testing: Monitoring the Development of Bacterial Stress

The ability to respond to adverse environments effectively along with the ability to reproduce are sine qua non conditions for all sustainable cellular forms of life. Given the availability of an appropriate sensing modality, the ubiquity and immediacy of the stress response could form the basis for a new approach for rapid biological testing. We have found that measuring the dielectric permittivity of a cellular suspension, an easily measurable electronic property, is an effective way to monitor the response of bacterial cells to adverse conditions continuously. The dielectric permittivity of susceptible and resistant strains of Escherichia coli and Staphylococcus aureus, treated with gentamicin and vancomycin, were measured directly using differential impedance sensing methods and expressed as the Normalized Impedance Response (NIR). These same strains were also heat-shocked and chemically stressed with Triton X-100 or H₂O₂. The NIR profiles obtained for antibiotic-treated susceptible organisms showed a strong and continuous decrease in value. In addition, the intensity of the NIR value decrease for susceptible cells varied in proportion to the amount of antibiotic added. Qualitatively similar profiles were found for the chemically treated and heat-shocked bacteria. In contrast, antibiotic-resistant cells showed no change in the NIR values in the presence of the drug to which it is resistant. The data presented here show that changes in the dielectric permittivity of a cell suspension are directly correlated with the development of a stress response as well as bacterial recovery from stressful conditions. The availability of a practical sensing modality capable of monitoring changes in the dielectric properties of stressed cells could have wide applications in areas ranging from the detection of bacterial infections in clinical specimens to antibiotic susceptibility testing and drug discovery.     

Characterization of Enterococcus faecalis Alkaline Phosphatase and Use in Identifying Streptococcus agalactiae Secreted Proteins

We have identified and characterized an Enterococcus faecalis alkaline phosphatase (AP, encoded by phoZ). The predicted gene product shows homology with alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases from Bacillus subtilis. Expression of phoZ in Escherichia coli, E. faecalis, Streptococcus agalactiae (group B streptococcus [GBS]), or Streptococcus pyogenes (group A streptococcus [GAS]) produces a blue-colony phenotype on plates containing a chromogenic substrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP). Two tests were made to determine if the activity of the enzyme is dependent upon the enzyme's subcellular location. First, elimination of the signal sequence reduced AP activity to 3% of the wild-type activity (or less) in three species of gram-positive bacteria. Restoration of export, using the signal sequence from C5a peptidase, restored AP activity to at least 50% of that of the wild type. Second, we engineered two chimeric proteins in which AP was fused to either a periplasmic domain or a cytoplasmic domain of lactose permease (a membrane protein). In E. coli, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion. We concluded that AP activity is export dependent. The signal sequence deletion mutant, phoZDeltass, was used to identify random genomic fragments from GBS that encode exported proteins or integral membrane proteins. Included in this set of fragments were genes that exhibited homology with the Rib protein (a cell wall protein from GBS) or with DppB (an integral membrane protein from GAS). AP acts as a reporter enzyme in GBS, GAS, and E. faecalis and is expected to be useful in a variety of gram-positive bacteria.     

Methods for the quantification ofFrankia cell biomass

Six methods for the estimation of microbial biomass were compared for determination ofFrankia cell concentrations. Six strains ofFrankia were cultivated in stationary culture, harvested by centrifugation, washed with saline buffer and diluted to five standardized concentrations. These cell suspensions were then used to assess reliability of each of the biomass determination methods. The destructive total protein determination methods were the most sensitive and reliable. Two non-destructive methods, packed cell volume and turbidity measurement, were also accurate, and because of their simplicity hold advantage for routine growth measurements and inoculum dilutions. Dry weight determinations were inconsistent for the small cell masses used in this study. An ELISA procedure demonstrated reliability but little sensitivity.     

Screening of symbiotic frankiae for host specificity by restriction fragment length polymorphism analysis.

Restriction fragment length polymorphism analysis of numerous Frankia strains, using a nifDH probe, separated the strains into three distinct groups based on hybridization patterns. The groups identified in this study were well correlated with host specificity groups identified in earlier cross-inoculation studies.     

Screening of symbiotic frankiae for host specificity by restriction fragment length polymorphism analysis

Restriction fragment length polymorphism analysis of numerous Frankia strains, using a nifDH probe, separated the strains into three distinct groups based on hybridization patterns. The groups identified in this study were well correlated with host specificity groups identified in earlier cross-inoculation studies.     

Use of glnQ as a counterselectable marker for creation of allelic exchange mutations in group B streptococci

Efficient allelic exchange mutagenesis in group B streptococci (GBS) has been hampered by the lack of a counterselectable marker system. Growth inhibition of GBS by the glutamine analog gamma-glutamyl hydrazide requires glnQ. We have used this phenomenon to create a counterselectable marker system for efficient selection of allelic exchange mutants in GBS.