Gene encoding γ-carbonic anhydrase is cotranscribed with argC and induced in response to stationary phase and high CO2 in Azospirillum brasilense Sp7

<p>Abstract</p> <p>Background</p> <p>Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO₂to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (γ-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only γ-CA of <it>Methanosarcina thermophila </it>(Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium <it>Azospirillum brasilense</it>, revealed occurrence of ORFs encoding one β-CA and two γ-CAs.</p> <p>Results</p> <p>One of the putative γ-CA encoding genes of <it>A. brasilense </it>was cloned and overexpressed in <it>E. coli</it>. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO₂hydration activity. Reverse transcription-PCR analysis indicated that <it>gca1 </it>in <it>A. brasilense </it>is co-transcribed with its upstream gene annotated as <it>argC</it>, which encodes a putative <it>N</it>-acetyl-γ-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between <it>argC </it>and <it>gca1</it>, and the transcription start site located upstream of <it>argC </it>transcribed both the genes (<it>argC-gca1</it>). Using transcriptional fusions of <it>argC</it>-<it>gca1 </it>upstream region with promoterless <it>lacZ</it>, we further demonstrated that <it>gca1 </it>upstream region did not have any promoter and its transcription occurred from a promoter located in the <it>argC </it>upstream region. The transcription of <it>argC-gca1 </it>operon was upregulated in stationary phase and at elevated CO₂atmosphere.</p> <p>Conclusions</p> <p>This study shows lack of CO₂hydration activity in a recombinant protein expressed from a gene predicted to encode a γ-carbonic anhydrase in <it>A. brasilense </it>although it cross reacts with anti-Cam antibody raised against a well characterized γ-CA. The organization and regulation of this gene along with the putative <it>argC </it>gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO₂hydration.</p

Ethanol-induced stress response of Staphylococcus aureus.

Transcriptional profiles of two unrelated clinical methicillin-resistant S. aureus (MRSA) isolates were analyzed following 10 % v/v ethanol challenge (15 min), which arrested growth but did not reduce viability. Ethanol-induced stress (EIS) resulted in differential gene expression of 1091 genes, 600 common to both strains, of which 291 were up-regulated. With the exception of the down-regulation of genes involved with osmotic stress functions, EIS resulted in the up-regulation of genes that contribute to stress response networks, notably those altered by oxidative stress, protein quality control in general, and heat shock in particular. In addition, genes involved with transcription, translation and nucleotide biosynthesis were down-regulated. relP, which encodes a small alarmone synthetase (RelP), was highly up-regulated in both MRSA strains following ethanol challenge and relP inactivation experiments indicated that this gene contributed to EIS growth arrest. A number of persistence-associated genes were also up-regulated during EIS, including those that encode toxin-antitoxin systems. Overall, transcriptional profiling indicated that the MRSA investigated responded to EIS by entering a state of dormancy and altering the expression of elements from cross protective stress response systems in an effort to protect preexisting proteins.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Comparing additionality of tuberculosis cases using GeneXpert or smear-based active TB case-finding strategies among social contacts of index cases in Nepal : tropical medicine and infectious disease

Funding: The study obtained funding from European Union, Horizon 2020—IMPACT TB project (grant number: 733174).This study compares the yield and additionality of community-based active tuberculosis (TB) active case-finding strategies using either smear microscopy or GeneXpert as the TB diagnostic test. Active case-finding strategies screened social contacts of index cases and high-risk groups in four districts of Nepal in July 2017–2019. Two districts (Chitwan and Dhanusha) applied GeneXpert testing and two districts (Makwanpur and Mahotarri) used smear microscopy. Two control districts implemented standard national TB program activities. Districts implementing GeneXpert testing screened 23,657 people for TB, tested 17,114 and diagnosed 764 TB cases, producing a yield of 4.5%. Districts implementing smear microscopy screened 19,961 people for TB, tested 13,285 and diagnosed 437 cases, producing a yield of 3.3%. The screening numbers required were 31 for GeneXpert and 45.7 for smear districts. The test numbers required were 22.4 and 30.4 for GeneXpert and smear. Using the TB REACH additionality method, social contact tracing for TB through GeneXpert testing contributed to a 20% (3958/3322) increase in district-level TB notifications, smear microscopy 12.4% (3146/2798), and −0.5% (2553/2566) for control districts. Therefore, social contact tracing of TB index cases using GeneXpert testing should be implemented throughout Nepal within the TB FREE initiative to close the notification gap and accelerate progress toward END TB strategy targets.Publisher PDFPeer reviewe