Biology subjects

Batista, B. B.

Publications and source records attributed to Batista, B. B..

5 recordsLinked to original sources

bioRxiv2024

Mutations in ampD cause hyperproduction of AmpC and CphA beta-lactamases and high resistance to beta-lactam antibiotics in Chromobacterium violaceum

Bacterial resistance to beta-lactam antibiotics mediated by beta-lactamase enzymes is widespread worldwide. Chromobacterium violaceum, an environmental Gram-negative bacteria pathogen, is intrinsically resistant to some beta-lactam antibiotics. In this work, we found that mutations in an ampD gene, encoding a peptidoglycan-recycling amidase, cause hyperproduction of two chromosomal beta-lactamases (AmpC and CphA), conferring high beta-lactam resistance in C. violaceum. Susceptibility tests using {Delta}ampC, {Delta}cphA, and{Delta} ampC{Delta}cphA mutant strains revealed specific susceptibility profiles to penicillin, cephalosporin, and carbapenem beta-lactams, suggesting that AmpC is a broad-spectrum beta-lactamase (penicillinase and cephalosporinase), while CphA is a narrow-spectrum metallo-carbapenemase. Beta-galactosidase assays indicate that the expression of ampC and cphA increased in response to beta-lactams. We isolated C. violaceum spontaneous mutants resistant to the antibiotic ceftazidime and found that most mutants were also resistant to several other beta-lactams and overexpressed ampC and cphA. DNA sequencing of the three paralog genes encoding the C. violaceum AmpD amidases revealed mutations of different types in AmpD1 (CV_0566) in most of the spontaneous mutants, but no mutation was found in AmpD2 or AmpD3. Analysis of single and combined null amidase mutants revealed overexpression of both beta-lactamases and increased resistance to beta-lactams only in mutants with deleted ampD1. When introduced into ampD1 null or spontaneous mutants, the ampD1 gene rescued the antibiotic-related phenotypes. The AmpD1 amidase from C. violaceum has a unique architecture with an N-terminal acetyltransferase domain. Our work offers new insights into the mechanisms of beta-lactamase-mediated antibiotic resistance and open perspectives to improve the treatment of C. violaceum infections.

microbiology↗

bioRxiv2024

A cytochrome bd repressed by a MarR family regulator confers resistance to metals, nitric oxide, sulfide, and cyanide in Chromobacterium violaceum

Chromobacterium violaceum is a ubiquitous environmental pathogen. Despite its remarkable adaptability, little is known about the mechanisms of stress resistance in this bacterium. Here, in a screen for iron-susceptible transposon mutants, we identified a cytochrome bd that protects C. violaceum against multiple stresses. The two subunits of this cytochrome bd (CioAB) are encoded by the cioRAB operon, which also encodes a GbsR-type MarR family transcription factor (CioR). A {Delta}cioAB mutant strain was sensitive to iron and the iron-requiring antibiotic streptonigrin and showed a decrease in siderophore production. Growth curves and survival assays revealed that the {Delta}cioAB strain was also sensitive to zinc, hydrogen peroxide, nitric oxide, sulfide, and cyanide. Expression analysis showed that the promoter activity of the cioRAB operon and the transcript levels of the cioAB genes were increased in a {Delta}cioR mutant. CioR bound the promoter region of the cio operon in vitro, indicating that CioR is a direct repressor of its own operon. Expression of the cio operon increased at high cell density and was dependent on the quorum-sensing regulator CviR. As cyanide is also a signal for cio expression, and production of endogenous cyanide is known to be a quorum sensing-regulated trait in C. violaceum, we suggest that CioAB is a cyanide-insensitive terminal oxidase that allow respiration under cyanogenic growth conditions. Our findings indicate that the cytochrome bd CioAB protects C. violaceum against multiple stress agents that are potentially produced endogenously or during interactions with a host. IMPORTANCEThe terminal oxidases of bacterial respiratory chains rely on heme-copper (heme-copper oxidases) or heme (cytochrome bd) to catalyze reduction of molecular oxygen to water. Chromobacterium violaceum is a facultative anaerobic bacterium that uses oxygen and other electron acceptors for respiration under conditions of varying oxygen availability. The C. violaceum genome encodes multiple respiratory terminal oxidases, but their role and regulation remain unexplored. Here, we demonstrate that CioAB, the single cytochrome bd from C. violaceum, protects this bacterium against multiple stressors that are inhibitors of heme-copper oxidases, including nitric oxide, sulfide, and cyanide. CioAB also confers C. violaceum resistance to iron, zinc, and hydrogen peroxide. This cytochrome bd is encoded by the cioRAB operon, which is under direct repression by the MarR-type regulator CioR. In addition, the cioRAB operon responds to quorum sensing and to cyanide, suggesting a protective mechanism of increasing CioAB in the setting of high endogenous cyanide production.

microbiology↗

bioRxiv2024

Molecular identification of whole squids and calamari at fairs and markets in regions of Latin America

In recent decades, the commercial importance of cephalopods has increased considerably, being an important fishing resource around the world. However, during the preparation for commercialization of those species, especially those sold in markets, they suffer the process known as "calamari" which includes removing and separating the head, arm, skin or even having the body structure cut into rings, which ends up making it difficult or often prevents the identification of the species, which can lead to replacements. In this sense, the present study aimed to use the large ribosomal region, rrnL (also known as 16S rDNA) to genetically identify cephalopod species sold in markets and fairs in Latin America. Whole and processed samples were collected from supermarkets and directly from local fishers the approximate collection location. Each generated sequence was submitted to the website Genbank for molecular comparison and included in the database for subsequent genetic identification. Comparison of sequences using the Blastn revealed the presence of eight species that are widely traded in the Latin American region. Our results indicate labeling errors in samples from the State of Para that contained the species Dosidicus gigas (d Orbigny, 1823) found only in the Pacific Ocean and were generically labeled as "National Lula". No type of substitution was found among the samples that were being sold at fairs and markets, only labeling errors. Thus, our results demonstrate the effectiveness of the rrnL for identifying species and evaluating labeling errors.

genetics↗

bioRxiv2023

A complex regulatory cascade involving quorum sensing regulates siderophore-mediated iron homeostasis in Chromobacterium violaceum

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos on PSA-CAS plates identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutation of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum. ImportanceThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.

microbiology↗

bioRxiv2022

The regulatory protein ChuP connects heme and siderophore-mediated iron acquisition systems required for Chromobacterium violaceum virulence

Chromobacterium violaceum is an environmental Gram-negative beta-proteobacterium that causes systemic infections in humans. C. violaceum uses siderophore-based iron acquisition systems to overcome the host-imposed iron limitation, but its capacity to use other iron sources is unknown. In this work, we characterized ChuPRSTUV as a heme utilization system employed by C. violaceum to explore an important iron reservoir in mammalian hosts, free heme and hemoproteins. We demonstrate that the chuPRSTUV genes comprise a Fur-repressed operon that is expressed under iron limitation. The chu operon potentially encodes a small regulatory protein (ChuP), an outer membrane TonB-dependent receptor (ChuR), a heme degradation enzyme (ChuS), and an inner membrane ABC transporter (ChuTUV). Our nutrition growth experiments using C. violaceum chu deletion mutants revealed that, with the exception of chuS, all genes of the chu operon are required for heme and hemoglobin utilization in C. violaceum. The mutant strains without chuP displayed increased siderophore halos on CAS plate assays. Significantly, we demonstrate that ChuP connects heme and siderophore utilization by acting as a positive regulator of chuR and vbuA, which encode the TonB-dependent receptors for the uptake of heme (ChuR) and the siderophore viobactin (VbuA). Our data favor a model of ChuP as a heme-binding post-transcriptional regulator. Moreover, our virulence data in a mice model of acute infection demonstrate that C. violaceum uses both heme and siderophore for iron acquisition during infection, with a preference for siderophores over the Chu heme utilization system.

microbiology↗