Conjugal transfer of plasmid pAM beta 1 in Lactobacillus reuteri and between lactobacilli and Enterococcus faecalis.

The broad-host-range plasmid pAM beta 1 (erythromycin resistance) was transferred conjugally from Streptococcus lactis to Lactobacillus reuteri, L. murinus, and L. fermentum. Transfer of pAM beta 1 between two L. reuteri strains occurred, and lactobacillus transconjugants could act as donors of pAM beta 1 in crosses with Enterococcus faecalis JH2-2.     

Biotin-labeled plasmid DNA probes for detection of epithelium-associated strains of lactobacilli

Biotin-labeled DNA probes prepared from whole plasmids (5.5 and 4.8 kilobases) of two lactobacillus strains (Lactobacillus delbrueckii 21 and Lactobacillus reuteri 100-23) were used to detect the homologous bacteria in microtome-cryostat-prepared sections of murine forestomach. The forestomach sections were incubated on nylon filter membranes placed on agar plates and, after lysis of the lactobacillus cells and denaturation of their DNA, were used in hybridization experiments with the strain-specific DNA probes. Hybridization of the probes to membranes containing sections from lactobacillus-free mice did not occur. The probes detected the presence of homologous strains of lactobacilli in sections cut from the forestomach of mice harboring one or both of the strains.     

Lactobacilli and bile salt hydrolase in the murine intestinal tract.

Mice that have a complex intestinal microflora but that do not harbor lactobacilli were used to determine the contribution of lactobacilli to the total bile salt hydrolase activity in the murine intestinal tract. Bile salt hydrolase activity in the ileal contents of these mice was reduced 86% in the absence of lactobacilli and by greater than 98% in the absence of lactobacilli and enterococci compared with samples from conventional mice. Bile salt hydrolase activities were lower in ileal and cecal contents from lactobacillus-free mice colonized with enterococci than in samples from lactobacillus-free mice colonized with lactobacilli. Bile salt hydrolase activity in the duodena, jejuna, ilea, and ceca of reconstituted lactobacillus-free mice colonized by lactobacilli was similar to that in samples from the intestinal tracts of conventional mice. We conclude from these studies that lactobacilli are the main contributors to total bile salt hydrolase activity in the murine intestinal tract.     

A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators.

In a screen of mouse erythroleukemia cDNA expression libraries with anti-phosphotyrosine antibodies, designed to isolate tyrosine kinase coding sequences, we identified several cDNAs encoding proteins identical or very similar to known protein-tyrosine kinases. However, two frequently isolated cDNAs, clk and nek, encode proteins which are most closely related to protein kinases involved in regulating progression through the cell cycle, and contain motifs generally considered diagnostic of protein-serine/threonine kinases. The clk gene product contains a C-terminal cdc2-like kinase domain, most similar to the FUS3 catalytic domain. The Clk protein, expressed in bacteria, becomes efficiently phosphorylated in vitro on tyrosine as well as serine/threonine, and phosphorylates the exogenous substrate poly(glu, tyr) on tyrosine. Direct biochemical evidence indicates that both protein-tyrosine and protein-serine/threonine kinase activities are intrinsic to the Clk catalytic domain. These results suggest the existence of a novel class of protein kinases, with an unusual substrate specificity, which may be involved in cell cycle control.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides.

Cells of the oral bacterium Streptococcus gordonii express three cytoplasmic membrane-bound lipoproteins with apparent molecular masses of 76 to 78 kDa that are the products of three genes (designated hppA, hppG, and hppH). The lipoproteins are immunologically cross-reactive, contain 60% or more identical amino acid residues, and are highly similar to the AmiA, AliA (PlpA), and AliB substrate-binding protein components of an oligopeptide permease in Streptococcus pneumoniae. Insertional inactivation of the hppA or hppH gene resulted in loss of the ability of S. gordonii cells to utilize specific peptides of five to seven amino acid residues for growth. An insertion within the COOH-terminal coding region of hppG that caused apparent truncation of the HppG polypeptide had a similar effect; however, S. gordonii mutants in which HppG polypeptide production was abolished were still able to grow on all oligopeptides tested. Inactivation of hppA gene (but not inactivation of the hppG or hppH gene) caused reduced growth rate of cells in complex medium, slowed the rate of development of competence for transformation, reduced the efficiency of transformation, and increased the resistance of cells to aminopterin. These results suggest that the formation of a solute-binding-protein complex consisting of at least the HppA and the HppH lipopolypeptides is necessary for binding and subsequent uptake of primarily hexa- or heptapeptides by a Hpp (Hexa-heptapeptide permease) system in S. gordonii. In addition, Hpp may play a role in the control of metabolic functions associated with the growth of streptococcal cells on complex nitrogen sources and with the development of competence.     

Molecular analysis of the composition of the bifidobacterial and lactobacillus microflora of humans.

The bifidobacterial and lactobacillus populations of fecal samples collected from two human subjects during a 12-month period were studied. The total numbers of bifidobacteria were stable throughout the study period in both subjects, but lactobacillus numbers were less constant. Analysis of the composition of the bifidobacterial populations by using ribotyping or pulsed-field gel electrophoresis to differentiate between bacterial strains demonstrated major differences between the subjects. Subject 1 harbored five strains of bifidobacteria throughout the 12-month period, and one strain was numerically predominant. In contrast, subject 2 harbored a more complex bifidobacterial population (five to six strains per sample) whose composition fluctuated throughout the 12 months. One lactobacillus strain was numerically predominant throughout the study in both subjects. Strains of bifidobacteria and lactobacilli common to both subjects were not detected.     

Differentiation of Lactobacillus strains by ribotyping.

Fifty-four lactobacillus strains were differentiated by ribotyping. The stability of ribotypes characteristic of four strains of lactobacilli inhabiting the digestive tract of mice was investigated. One of four isolates of Lactobacillus delbrueckii GT21, which had been associated with mice for 22 months, had an altered ribotype.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.