The PII protein, encoded by glnB, has a central role in the control of nitrogen metabolism in nitrogen-fixing prokaryotes. The glnB gene of Rhodospirillum rubrum was isolated and sequenced. The deduced amino acid sequence had very high sequence identity to other PII proteins. The glnA gene, encoding glutamine synthetase, was located 135 bp downstream of glnB and was partially sequenced. glnB is cotranscribed with glnA from a promoter with high similarity to the sigma 54-dependent promoter consensus sequence. A putative sigma 70 promoter was also identified further upstream of glnB. Northern blotting analyses showed that in addition glnA is either transcribed from an unidentified promoter or, more likely, that the glnBA transcript is processed to give the glnA mRNA. The total level of the two transcripts was much higher in nitrogen-fixing cells than in ammonia-grown cells.
In natural environments bacteria and filamentous fungi often compete for the same resources. Consequently, production of antibiotic secondary metabolites and defence mechanisms against these compounds have evolved in these organisms. An experimental model has been developed to study the response in fungi exposed to one such antibiotic. The filamentous fungus Aspergillus nidulans was treated with bafilomycin B-1, a Streptomyces-produced antibiotic which reduces radial growth rate and induces morphological changes in fungi. mRNA differential display was used to study changes in fungal gene expression. For five genes, changes in abundance of the corresponding mRNAs, directly or indirectly caused by bafilomycin, were observed. Of these, three were up-regulated and two repressed. With four of these the change in mRNA abundance measured ranged from 10- to 60-fold. However, for one gene the mRNA was only detected after bafilomycin treatment. One of the downregulated mRNAs encodes ASPND1, a glycoprotein that belongs to a known family of antigens identified in aspergilloma patients. One up-regulated mRNA shows sequence similarities, at the amino acid level, with a cell-wall protein of Saccharomyces cerevisiae. The remaining three genes were also cloned and sequenced; their sequences do not correspond to known genes in A. nidulans, and no similarities with published nucleotide or protein sequences in other organisms were found. These results indicate the feasibility of using mRNA differential display to study interactions between bacteria and filamentous fungi.
The authors have previously reported on molecular responses of Aspergillus nidulans to bacterial antifungal metabolites, e.g. bafilomycins and the related concanamycins. These compounds are known inhibitors of V-ATPases and cause dramatic effects on mycelial growth and morphology. In Neurospora crassa, studies have shown that disruption of the gene encoding subunit A of the V-ATPase results in morphological changes and reduced growth similar to those observed after addition of concanamycin. This phenotype, and the fact that this mutation confers resistance to concanamycin, suggests that V-ATPase is the main (or only) target for the antibiotics. However, growth inhibition and morphology changes in, for example, A. nidulans and Penicillium roqueforti are more severe, and thus other targets are possible. In this study, the vmaA gene of A. nidulans, encoding the subunit A of V-ATPase, was disrupted by homologous recombination. The resulting vmaA 1 mutant strain displayed extremely slow growth and failed to produce asexual spores. Furthermore, an altered morphology similar to that caused by addition of V-ATPase inhibitors, i.e. bafilomycin or concanamycin, was observed, indicating that V-ATPase is the main target for the antibiotics also in A. nidulans. The vmaA 1 mutant was not viable at pH values above 7 and was highly sensitive to high Zn2+ concentrations, in agreement with previous results from studies of Saccharomyces cerevisiae and N. crassa.
There is a growing appreciation that microbiota composition can significantly affect host health and play a role in disease onset and progression. This study assessed the impact of streptozotocin (STZ)-induced type-1-diabetes (T1D) on intestinal microbiota composition and diversity in Sprague-Dawley rats, compared with healthy controls over time. T1D was induced by injection of a single dose (60 mg STZ kg(-1)) of STZ, administered via the intraperitoneal cavity. Total DNA was isolated from faecal pellets at weeks 0 (pre-STZ injection), 1, 2 and 4 and from caecal content at week 5 from both healthy and T1D groups. High-throughput 16S rRNA sequencing was employed to investigate intestinal microbiota composition. The data revealed that although intestinal microbiota composition between the groups was similar at week 0, a dramatic impact of T1D development on the microbiota was apparent post-STZ injection and for up to 5 weeks. Most notably, T1D onset was associated with a shift in the Bacteroidetes : Firmicutes ratio (P<0.05), while at the genus level, increased proportions of lactic acid producing bacteria such as Lactobacillus and Bifidobacterium were associated with the later stages of T1D progression (P<0.05). Coincidently, T1D increased caecal lactate levels (P<0.05). Microbial diversity was also reduced following T1D (P<0.05). Principle co-ordinate analyses demonstrated temporal clustering in T1D and control groups with distinct separation between groups. The results provide a comprehensive account of how T1D is associated with an altered intestinal microbiota composition and reduced microbial diversity over time.
We have previously demonstrated that oral administration of a metabolically active Bifidobacterium breve strain, with ability to form cis-9, trans-11 conjugated linoleic acid (CLA), resulted in modulation of the fatty acid composition of the host, including significantly elevated concentrations of c9, t11 CLA and omega-3 (n-3) fatty acids in liver and adipose tissue. In this study, we investigated whether a recombinant lactobacillus expressing linoleic acid isomerase (responsible for production of t10, c12 CLA) from Propionibacterium acnes (PAI) could influence the fatty acid composition of different tissues in a mouse model. Linoleic-acid-supplemented diets (2 %, w/w) were fed in combination with either a recombinant t10, c12 CLA-producing Lactobacillus paracasei NFBC 338 (Lb338), or an isogenic (vector-containing) control strain, to BALB/c mice for 8 weeks. A third group of mice received linoleic acid alone (2 %, w/w). Tissue fatty acid composition was assessed by GLC at the end of the trial. Ingestion of the strain expressing linoleic acid isomerase was associated with a 4-fold increase (P<0.001) in t10, c12 CLA in adipose tissues of the mice when compared with mice that received the isogenic non-CLA-producing strain. The livers of the mice that received the recombinant CLA-producing Lb338 also contained a 2.5-fold (albeit not significantly) higher concentration of t10, c12 CLA, compared to the control group. These data demonstrate that a single gene (encoding linoleic acid isomerase) expressed in an intestinal microbe can influence the fatty acid composition of host fat.