- Volume 140, Issue 12, 1994

Francisella novicidais a facultative intracellular pathogen that can survive and grow in macrophages by preventing phagolysosomal fusion. In this study in vitrocassette mutagenesis was used to generate a library of insertion mutants of F. novicida.Two related mutants, KM14 and KM14S, initially identified as defective for growth in macrophages, were found to be sensitive to serum. These mutants were also found to grow approximately 1000-fold less well in the livers and spleens of infected mice. We cloned a genetic locus that was presumably mutagenized in these mutants and found that it included genes that had high similarity in their deduced amino acid sequence to those of msbAand orfEof Escherichia coli.The former is a member of the superfamily of ABC transporter proteins. We named the corresponding genes in F. novicida, valAB.Integration of a cloned valABlocus into the chromosome of KM14S partially restored the serum resistance phenotype found in wild-type F. novicida.

Porphyromonas gingivalishas been implicated in human periodontal diseases. It expresses a number of exoglycosidase enzymes capable of hydrolysing host proteoglycan residues. As a first stage to explore the role of these enzymes in periodontal tissue damage, the nahAgene of P. gingivalisW83, which encodes β-N-acetylhexosaminidase (β-Nahase), was cloned. The gene was expressed poorly in Escherichia coli, but increased expression was achieved by cloning the nahAgene downstream of the tac promoter. Southern blot analysis revealed that nahAwas present as a single copy, and it was found in all the other P. gingivalisstrains tested. In contrast, sequences homologous to nahAwere not detected in either P. endodontalisor P. asaccharolytica.The nahAgene was 2331 bp long and encoded a β-Nahase enzyme of 777 amino acids with a predicted molecular mass of 87 kDa. A characteristic signal peptide for an acylated lipoprotein was present at the amino-terminus, suggesting that the mature β-Nahase is a lipoprotein. The predicted amino acid sequence of the P. gingivalisβ-Nahase shared homology with the catalytic domains of the human β-Nahase enzyme and the chitinase of Vibrio harveyi, suggesting a common catalytic mechanism.

Hog gastric mucin has been used as a model glycoprotein to determine the role of particular glycosidases produced by different oral bacteria in the development of stable, diverse microbial communities. The patterns of glycosidase and protease activity were determined in pure cultures of ten representative species of oral bacteria using synthetic substrates. A five member mixed culture was established in a chemostat, comprising species with minimal glycosidase and protease activity, in which hog gastric mucin was the major carbon and energy source. Introduction of additional species with novel enzyme activities (e.g. sialidase, α-fucosidase and endopeptidase) led to their establishment within the community to make communities with seven, eight, nine and ten members and resulted in an increase in the total viable counts of the microbial consortium. This increase in viable count was made up of the numbers of the newly added species as well as from a rise in the numbers of the existing community members. This result suggested that glycoprotein catabolism involved the synergistic and concerted action of several species with overlapping patterns of enzyme activity. Such metabolic cooperation results in the liberation of additional nutrients, and this may help to maintain the characteristic diversity of resident microbial communities found in many natural habitats.

Exact knowledge of microbial diffusion coefficients is a prerequisite for the application of mass transport theories to microbial deposition data. Microbial diffusion coefficients can be calculated on the basis of cell radii using the Einstein equation. This approach, however, does not take into account the additional effects of structural cell surface features such as fibrils or a fuzzy coat. Microbial diffusion coefficients of micro-organisms in suspension can be experimentally measured using dynamic light scattering. In this paper we compare experimental microbial diffusion coefficients with those calculated from radii for a variety of microbial strains suspended in 10 mM or 40 mM potassium phosphate solutions of different pH. Furthermore, the pH dependence of the microbial diffusion coefficients is related to the pH dependence of the microbial zeta potentials in similar ionic strength solutions. Microbial diffusion coefficients ranged from 2 × 1013to 5 × 1013 m2s−1and were generally higher at low pH (pH 2) than at high pH (pH 7) for strains with structural surface features. Experimental diffusion coefficients at pH 2 were approximately similar to those based on microscopic radii. This indicates that at pH 2 structural surface features are in a collapsed state, presumably due to the lack of stabilizing electrostatic repulsion between the cell surface structures as shown by the zeta potentials measured. Alternatively, a hydrodynamic radius of a micro-organism can be calculated from the experimental diffusion coefficients and the Einstein equation. Assuming that the maximal difference in hydrodynamic radius, observed over the pH range employed, represents the maximal length of structural cell surface features present, then the calculated fibrillar lengths for Streptococcus mitisBA and S. salivariusHB are in reasonable agreement with electron microscopical estimates.

Lactococcus lactistakes up glucose and the nonmetabolizable glucose analogue 2-deoxyglucose (2DG) via the phosphotransferase system and extrudes the accumulated sugar phosphates in a process apparently dependent on a cytoplasmic sugar-phosphate phosphatase. Uptake of 2DG into L. lactisvesicles was shown to be dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate (PEP). 2DG phosphate (2DG-P) accumulation in these vesicles was not inhibited, and preaccumulated 2DG-P was not released from them, upon electroporation of fructose 1,6-diphosphate (FDP), gluconate 6-phosphate or 2-phosphoglycerate into the vesicles. Intravesicular but not extravesicular wild-type HPr of Bacillus subtilisalone stimulated uptake, but in the presence of any one of these metabolites, it prevented accumulation of 2DG-P. Intravesicular H15A mutant HPr inhibited uptake and allowed further inhibition of 2DG-P accumulation in the presence of the intravesicular metabolites. Intravesicular S46A mutant HPr stimulated uptake but could not promote inhibition in the presence of the phosphorylated metabolites. The S46D mutant HPr protein promoted regulation, even in the absence of a metabolite. The V maxbut not the K mvalue for 2DG uptake was affected. Accumulation of the natural, metabolizable substrates of the lactose, glucose, mannose and ribose permeases was inhibited by wild-type HPr in the presence of FDP or by S46D mutant HPr. The results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase selectively determines the levels of sugar accumulation via the glucose and lactose permeases in L. lactis.They suggest that two primary functions of HPr(Ser) phosphorylation are: (1) to feedback-inhibit the activities of carbohydrate permeases and not merely to create a hierarchy of preferred carbon sources, and (2) to regulate the cytoplasmic concentrations of carbohydrate inducers by exclusion and expulsion mechanisms.

Campylobacter-like organisms were isolated from the liver, duodenum and caecum of broiler and layer chickens, and from humans with gastroenteritis. They formed a unique DNA homology group and a polyphasic taxonomic analysis was made of 16 strains. Analysis of the nucleotide sequence of the 16S rRNA gene from seven of the strains identified them as belonging to a single species, within the genus Helicobacter.This conclusion was supported by the studies of relative DNA homology and of total protein electrophoretic patterns. The new species could be biochemically differentiated from other helicobacters and its ultrastructure in the electron microscope was typical of the genus except that the flagellum was not sheathed. We propose the name Helicobacter pullorumsp. nov. for this group. Like H. fennelliaeor H. cinaediit represents another non-gastric urease-negative Helicobacterspecies colonizing the lower bowel. Its isolation from the livers of chickens with vibrionic hepatitis is significant. We describe a species-specific PCR assay for H. pullorumsp. nov. which will facilitate its identification and further studies of its epidemiology.

Two isolates of a novel, moderately thermophilic Thiobacillusspecies have been studied. The isolates, KU and BC13, are Gram-negative, motile bacteria having a pH optimum for growth of 2-2.5 and an optimum growth temperature of 45 °C. Both isolates are capable of chemolithotrophic growth on reduced sulfur substrates. They can also use molecular hydrogen as an electron donor. These two isolates can grow mixotrophically with sulfur or tetrathionate and yeast extract or glucose. The G + C content is 63.1-63.9 mol% and the isolates exhibit no significant DNA homology to any other Thiobacillusspecies. Strains KU and BC13 both contain ubiquinone Q.8. 16S rRNA analysis indicates that these strains belong to a group of bacteria which includes other chemolithotrophic sulfur oxidizers such as T. ferrooxidansand T. thiooxidans. These characteristics distinguish KU and BC13 from any other species described previously and they thus represent the first acidophilic, thermophilic Thiobacillusspecies, named T. caldussp. nov., to be described. The type strain, referred to as strain KU in this paper, has been deposited in the Deutsche Sammlung von Mikroorganismen, Braunschweig, FRG, with the accession number DSM 8584.