Jak-STAT regulation of cyst stem cell development in the Drosophila testis.

Establishment and maintenance of functional stem cells is critical for organ development and tissue homeostasis. Little is known about the mechanisms underlying stem establishment during organogenesis. Drosophila testes are among the most thoroughly characterized systems for studying stem cell behavior, with germline stem cells (GSCs) and somatic cyst stem cells (CySCs) cohabiting a discrete stem cell niche at the testis apex. GSCs and CySCs are arrayed around hub cells that also comprise the niche and communication between hub cells, GSCs, and CySCs regulates the balance between stem cell maintenance and differentiation. Recent data has shown that functional, asymmetrically dividing GSCs are first established at ∼23 h after egg laying during Drosophila testis morphogenesis (Sheng et al., 2009). This process correlates with coalescence of the hub, but development of CySCs from somatic gonadal precursors (SGPs) was not examined. Here, we show that functional CySCs are present at the time of GSC establishment, and that Jak-STAT signaling is necessary and sufficient for CySC maintenance shortly thereafter. Furthermore, hyper-activation of Jak in CySCs promotes expansion of the GSC population, while ectopic Jak activation in the germline induces GSC gene expression in GSC daughter cells but does not prevent spermatogenic differentiation. Together, these observations indicate that, similar to adult testes, Jak-STAT signaling from the hub acts on both GSCs and CySC to regulate their development and differentiation, and that additional signaling from CySCs to the GSCs play a dominant role in controlling GSC maintenance during niche formation.

Bioenergetic aspects of the translocation of macromolecules across bacterial membranes.

Bacteria are extremely versatile in the sense that they have gained the ability to transport all three major classes of biopolymers through their cell envelope: proteins, nucleic acids, and polysaccharides. These macromolecules are translocated across membranes in a large number of cellular processes by specific translocation systems. Members of the ABC (ATP binding cassette) superfamily of transport ATPases are involved in the translocation of all three classes of macromolecules, in addition to unique transport ATPases. An intriguing aspect of these transport processes is that the barrier function of the membrane is preserved despite the fact the dimensions of the translocated molecules by far surpasses the thickness of the membrane. This raises questions like: How are these polar compounds translocated across the hydrophobic interior of the membrane, through a proteinaceous pore or through the lipid phase; what drives these macromolecules across the membrane; which energy sources are used and how is unidirectionality achieved? It is generally believed that macromolecules are translocated in a more or less extended, most likely linear form. A recurring theme in the bioenergetics of these translocation reactions in bacteria is the joint involvement of free energy input in the form of ATP hydrolysis and via proton sym- or antiport, driven by a proton gradient. Important similarities in the bioenergetic mechanisms of the translocation of these biopolymers therefore may exist.

Uptake and processing of DNA by Acinetobacter calcoaceticus--a review.

In natural transformation, DNA in the form of macromolecular fragments can be translocated across the cell envelope of prokaryotic microorganisms. During the past two decades, several, largely mutually contradictory, hypotheses have been forwarded to explain the molecular mechanism and bioenergetics of this translocation process. Other biomacromolecules are translocated across the bacterial cell envelope as well, such as polysaccharides and proteins, the latter for instance in the process of the assembly of type-IV pili. This brings up the question whether or not common components are involved. Here, we review analyses of DNA translocation in Acinetobacter calcoaceticus, a Gram-negative eubacterium that is able to migrate through twitching motility, and also shows a high frequency of natural transformation. DNA uptake in this organism is an energy-dependent process. Upon entry into the cells, the DNA fragments are integrated into the resident chromosome when a sufficiently large region of mutual homology is available (200 to 400 bp). However, this process is rather inefficient, and on the average 500 bp of each incoming fragment is degraded through exonuclease activity. Upon covalent attachment of a bulky protein molecule to the transforming DNA, the DNA-translocation machinery becomes blocked in further translocation activity. Since A. calcoaceticus is not well suited for transposon mutagenesis, a random mutagenesis procedure has been developed, based on the ligation of an antibiotic-resistance marker to random fragments of chromosomal DNA. This method was used to generate several mutants impaired in the natural transformation process. Three of these have been characterized in detail. No components, common to the translocation of macromolecules through the cell envelope of Acinetobacter, have been detected in this screen.

Acinetobacter calcoaceticus liberates chromosomal DNA during induction of competence by cell lysis.

A transformation assay was used to assay the amount of DNA present in the extracellular medium of a growing culture of Acinetobacter calcoaceticus. It was observed that small amounts of DNA were liberated during the entire exponential growth phase in a batch culture. Release of DNA could be fully accounted for by lysis of cells. Lysis was quantified via simultaneous measurement of beta-galactosidase activity of cells and supernatant, with a strain that contained a plasmid (pAPA100) with lacZ under control of a constitutive beta-lactamase promoter. In conclusion, no evidence could be obtained indicating that Acinetobacter calcoaceticus actively excretes DNA, to be used for DNA exchange.

Characterization of transformation-deficient mutants of Acinetobacter calcoaceticus.

Three Acinetobacter calcoaceticus transformation-deficient mutants, obtained by insertional mutagenesis with the nptll gene, have been characterized physiologically. One mutant (AAC211) was found to be completely transformation deficient, while two others, AAC213 and AAC214, were severely impaired in transformation efficiency (100-1000 times lower than the wild type). The latter applied to both chromosomal as well as plasmid DNA. Analysis of the chromosomal DNA fragments flanking the nptll gene in the mutants showed that mutants AAC213 and AAC214 had an insertion of the nptll gene in the same chromosomal region, but that they were the result of two independent mutational events, whereas the insertion in mutant AAC211 was at a different position. None of the three mutants showed phenotypic or genotypic characteristics typical of a RecA-deficient strain.

Osmoregulation in Rhodobacter sphaeroides.

Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light. The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths. The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions. Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized. The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 microM to 2.0 mM). Only one kinetically distinguishable choline transport system could be detected. Kt values of 2.4 and 3.0 microM and maximal rates of choline uptake (Vmax) of 5.4 and 4.2 nmol of choline/min.mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively. Choline transport was not inhibited by a 25-fold excess of L-proline or betaine. Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl. In cells grown and assayed in the absence of NaCl, betaine transport occurred with a Kt of 15.1 microM and a Vmax of 3.2 nmol/min . mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 microM and 9.2 nmol of betaine/min . mg of protein. This system was also able to transport L-proline, but with a lower affinity than that for betaine. The addition of choline of betaine to the growth medium did not result in the induction of additional transport systems.

Physiological characterization of natural transformation in Acinetobacter calcoaceticus.

Acinetobacter calcoaceticus BD413 develops competence for natural transformation immediately after the start of the exponential growth-phase and remains competent up to e few hours into the stationary phase, after which competence gradually declines. The transformation frequencies obtained strongly depend on the kind of transforming DNA and the incubation time with DNA. Up to 25% of the cells in a culture can be transformed. DNA uptake in Acinetobacter does not display sequence specificity, is Mg(2+)-, Mn(2+)- or Ca(2+)-dependent and is uncoupler sensitive. The transforming DNA enters the cells in single-stranded form. These properties constitute a unique combination, not previously observed in other bacteria, and make A. caloaceticus ideally suited for detailed studies of the bioenergetics of DNA translocation.

Insertional mutation of orfD of the DCW cluster of Streptococcus pneumoniae attenuates virulence.

Mutational analysis of a 5.5 kb fragment of the genome Streptococcus pneumoniae led to the identification of a putative new virulence gene, designated orfD. Insertion mutagenesis of flanking genes on the fragment suggested that the corresponding gene products were required for in vitro growth. In contrast, insertion mutation of orfD did not alter in vitro growth or the transformability pattern of the mutated strain. However, it did reduce bacterial growth in mice and attenuated virulence in an intraperitoneal model of infection. orfD is flanked by orfC (63 codons) and ftsL (105 codons) and all three genes are upstream of pbpx. orfC showed no similarity with other known proteins. ftsL of S. pneumoniae exhibits minimal sequence similarity with ftsL of E. coli, but shares 16% identical residues with the ftsL homologue encoded by ylld of B. subtilis. Also, ftsL of S. pneumoniae has a predicted topology similar to that described for ftsL of E. coli. Putative promoters with an extended -10 box could be identified upstream of both orfC or orfD. The four open reading frames (including pbpx) are orientated in the same direction, and polycistronic transcription could theoretically start at either promoter. Interestingly, this region shows organizational and sequence homologies with genes controlling division and cell wall biosynthesis (DCW) in other bacteria. The attenuation of virulence in the orfD insertion mutant might be due to the loss of function of the orfD gene product or to an altered level of expression of downstream genes.