Role and Regulation of Poly-3-Hydroxybutyrate in Nitrogen Fixation in Azorhizobium caulinodans

An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Structures of the ribosome-inactivating protein from barley seeds reveal a unique activation mechanism

Ribosome-inactivating protein (RIP), a defence protein found in various plants, possesses different chain architectures and activation mechanisms. The RIP from barley (bRIP) is a type I RIP and has sequence features that are divergent from those of type I and type II RIPs from dicotyledonous plants and even the type III RIP from maize. This study presents the first crystal structure of an RIP from a cereal crop, barley, in free, AMP-bound and adenine-bound states. For phasing, a codon-optimized syntheticbrip1gene was used and a vector was constructed to overexpress soluble bRIP fusion proteins; such expression has been verified in a number of cases. The overall structure of bRIP shows folding similar to that observed in other RIPs but also shows significant differences in specific regions, particularly in a switch region that undergoes a structural transition between a 310-helix and a loop depending on the liganded state. The switch region is in a position equivalent to that of a proteolytically susceptible and putative ribosome-binding site in type III RIPs. Thus, the bRIP structure confirms the detailed enzymatic mechanism of this N-glycosidase and reveals a novel activation mechanism for type I RIPs from cereal crops.

An Unusual Mutation Results in the Replacement of Diaminopimelate with Lanthionine in the Peptidoglycan of a Mutant Strain of Mycobacterium smegmatis

ABSTRACT Mycobacterial peptidoglycan contains l-alanyl-d-iso-glutaminyl-meso-diaminopimelyl-d-alanyl-d-alanine peptides, with the exception of the peptidoglycan of Mycobacterium leprae, in which glycine replaces the l-alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the meso-diaminopimelate (DAP) moiety of one peptide and the penultimate d-alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of Mycobacterium smegmatis that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to β-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants, M. smegmatis strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the cbs gene, encoding cystathionine β-synthase, an enzyme that is a part of the cysteine biosynthetic pathway.

Genetic and Phenotypic Variations of a Resistant Pseudomonas aeruginosa Epidemic Clone

ABSTRACT From May 1997 to December 2001, a serotype O:6 multidrug-resistant strain of Pseudomonas aeruginosa colonized or infected 201 patients in the University Hospital of Besançon (France). The susceptibility profile of this epidemic clone to fluoroquinolones and aminoglycosides was relatively stable during the outbreak but showed important isolate-to-isolate variations (up to 64-fold) in the MICs of β-lactams. Analysis of 18 genotypically related isolates selected on a quaterly basis demonstrated alterations in the two DNA topoisomerases II and IV (Thr83→Ile in GyrA and Ser87→Leu in ParC) and production of an ANT(2")-I enzyme. Although constitutively overproduced in these bacteria, the MexXY efflux system did not appear to contribute significantly to aminoglycoside resistance. β-Lactam resistance was associated with derepression of intrinsic AmpC β-lactamase (with isolate-to-isolate variations of up to 58-fold) and sporadic deficiency in a 46-kDa protein identified as the carbapenem-selective porin OprD. Of the 18 isolates, 14 were also found to overproduce the efflux system MexAB-OprM as a result of alteration of the repressor protein MexR (His107→Pro). However, complementation experiments with the cloned mexR gene demonstrated that MexAB-OprM contributed only marginally to β-lactam and fluoroquinolone resistance. Of the four isolates exhibiting wild-type MexAB-OprM expression despite the MexR alteration, two appeared to harbor secondary mutations in the mexA-mexR intergenic region and one harbored secondary mutations in the putative ribosome binding site located upstream of the mexAB oprM operon. In conclusion, this study shows that many mechanisms were involved in the multiresistance phenotype of this highly epidemic strain of P. aeruginosa. Our results also demonstrate that the clone sporadically underwent substantial genetic and phenotypic variations during the course of the outbreak, perhaps in relation to local or individual selective drug pressures.

H2O2, Which Causes Macrophage-Related Stress, Triggers Induction of Expression of Virulence-Associated Plasmid Determinants in Rhodococcus equi

ABSTRACT The response of the intracellular pathogen Rhodococcus equi to H2O2 treatment, a situation potentially encountered after the oxidative burst of alveolar macrophages, was analyzed. Compared to other bacteria, including Deinococcus radiodurans, R. equi showed exceptionally high resistance to this stress. A proteomic approach showed that four polypeptides present in the wild-type strain (85F) are missing in the plasmid-cured strain 85F(P-), and by using a DNA macroarray, we identified two plasmid-encoded vap genes, vapA and vapG, whose expression was highly induced by H2O2 treatment. Whereas the transcript size of vapA was compatible with a monocistronic mRNA, the transcript of vapG was considerably longer. Rapid amplification of cDNA ends PCRs showed that the transcriptional start sites of the two operons were 69 and 269 nucleotides (nt) upstream of the start codon, respectively. Analysis of these leader sequences revealed the presence of a small open reading frame named podG, which encodes a sequence of 55 amino acids preceded by a putative ribosome binding site sequence in the vapG transcript. Taking this result into account, the untranslated leader of the podG/vapG operon is 87 nt. Alignment of this sequence with the leader sequences of vapA and vapD, genes previously shown to be induced by acid, revealed significant homologies. Since our results showed that vapA, vapD, and vapG are genes highly induced by macrophage-related stresses, their gene products may, within the Vap protein family, play a dominant role inside these phagocytic cells and may be the most promising candidates for vaccination strategies.

Features of the cellodextrinase gene from Fibrobacter succinogenes S85

The nucleotide sequence of a 2.3-kb DNA fragment containing a cellodextrinase gene (cedA) from the ruminal anaerobe Fibrobacter succinogenes S85 was determined. Activity was expressed from this fragment when it was cloned in both orientations in pBluescript KS+ and SK−, indicating a functional F. succinogenes promoter in Escherichia coli. Promoter sequences (TTGAACA and AATAA) were identified upstream of the ATG initiation codon preceded by a putative ribosome binding site. The cedA open reading frame of 1071 base pairs encoded a protein of 357 amino acid residues with a calculated molecular mass of 41.9 kDa, similar to the40-kDa size of the native protein as determined by gel filtration chromatography. CedA is proposed to belong to family 5 (family A) of the glycosyl hydrolases. The primary structure of the cellodextrinase showed over 40% similarity with endoglucanase 3 from F. succinogenes S85. Short regions of similarity were also demonstrated with endoglucanase C from Clostridium thermocellum, CelA from Ruminococcus flavefaciens, and two exoglucanases from yeast.Key words: Fibrobacter succinogenes, cedA, cellodextrinase, sequence, rumen, gene.