growth-curves-generating-growth-curves-using-colony-forming-units

Growth Curves: Generating Growth Curves Using Colony Forming Units and Optical Density Measurements

Source: Andrew J. Van Alst1, Rhiannon M. LeVeque1, Natalia Martin1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

microscopy-and-staining-gram-capsule-and-endospore-staining

Microscopy and Staining: Gram, Capsule, and Endospore Staining

Source: Rhiannon M. LeVeque1, Natalia Martin1, Andrew J. Van Alst1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

transformation-e-coli-cells-using-an-adapted-calcium-chloride

Transformation of <em>E. coli</em> Cells Using an Adapted Calcium Chloride Procedure

Source: Natalia Martin1, Andrew J. Van Alst1, Rhiannon M. LeVeque1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

large-scale-sars-cov-2-testing-utilizing-saliva-transposition-sample

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. ...

Michigan State University

Actinobacillus pleuropneumoniae is the causative agent of severe necrotizing pneumonia in swine. Previously, we identified the ohr gene encoding organic hydroperoxide reductase as specifically induced during infection of pigs, induced in vitro by organic peroxides but not other oxygen radicals, and present in A. pleuropneumoniae serotypes 1, 9 and 11 but not in other serotypes (Shea & Mulks, 2002). Through analysis of flanking genomic sequence, we identify a homologue of gst, which encodes glutathione-S-transferase, immediately downstream of ohr and demonstrate that ohr-gst confers low but uninducible Ohr activity to serotype 5. We further identify a genomic island of 9.3 kb, flanked by lysR and araC homologues, in serotypes 1, 9 and 11, which contains ohr and gst. In serotypes 2-8, 10 and 12, this region of the genome contains a 1.1-kb islet with a putative transposase flanked by lysR and araC.

Ohr, an in vivo-induced gene in Actinobacillus pleuropneumoniae, is located on a genomic island and requires glutathione-S-transferase for activity.

In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and that A. pleuropneumoniae mutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 micromol/liter, which is 10 to 17% of the concentration in plasma. The expression of both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew in CDM+BCAA but not in CDM-BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis that A. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens.

Branched-chain amino acids are required for the survival and virulence of Actinobacillus pleuropneumoniae in swine.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, an economically important disease of pigs. The hfq gene in A. pleuropneumoniae, encoding the RNA chaperone and posttranscriptional regulator Hfq, is upregulated during infection of porcine lungs. To investigate the role of this in vivo-induced gene in A. pleuropneumoniae, an hfq mutant strain was constructed. The hfq mutant was defective in biofilm formation on abiotic surfaces. The level of pgaC transcript, encoding the biosynthesis of poly-β-1,6-N-acetylglucosamine (PNAG), a major biofilm matrix component, was lower and PNAG content was 10-fold lower in the hfq mutant than in the wild-type strain. When outer membrane proteins were examined, cysteine synthase, implicated in resistance to oxidative stress and tellurite, was not found at detectable levels in the absence of Hfq. The hfq mutant displayed enhanced sensitivity to superoxide generated by methyl viologen and tellurite. These phenotypes were readily reversed by complementation with the hfq gene expressed from its native promoter. The role of Hfq in the fitness of A. pleuropneumoniae was assessed in a natural host infection model. The hfq mutant failed to colonize porcine lungs and was outcompeted by the wild-type strain (median competitive index of 2 × 10(-5)). Our data demonstrate that the in vivo-induced gene hfq is involved in the regulation of PNAG-dependent biofilm formation, resistance to superoxide stress, and the fitness and virulence of A. pleuropneumoniae in pigs and begin to elucidate the role of an in vivo-induced gene in the pathogenesis of pleuropneumonia. 

The RNA chaperone Hfq promotes fitness of Actinobacillus pleuropneumoniae during porcine pleuropneumonia.

, a leading cause of bacterial gastroenteritis, is naturally competent. Like many competent organisms,  restricts the DNA that can be used for transformation to minimize undesirable changes in the chromosome. Although  can be transformed by -derived DNA, it is poorly transformed by the same DNA propagated in  or produced with PCR. Our work indicates that methylation plays an important role in marking DNA for transformation. We have identified a highly conserved DNA methyltransferase, which we term  transformation system methyltransferase (), which methylates an overrepresented 6-bp sequence in the chromosome. DNA derived from a  mutant transforms  significantly less well than DNA derived from  (parental) cells. The  mutation itself does not affect transformation efficiency when parental DNA is used, suggesting that CtsM is important for marking transforming DNA, but not for transformation itself. The mutant has no growth defect, arguing against ongoing restriction of its own DNA. We further show that  plasmid and PCR-derived DNA can efficiently transform  when only a subset of the CtsM sites are methylated in vitro. A single methylation event 1 kb upstream of the DNA involved in homologous recombination is sufficient to transform , whereas otherwise identical unmethylated DNA is not. Methylation influences DNA uptake, with a slight effect also seen on DNA binding. This mechanism of DNA discrimination in  is distinct from the DNA discrimination described in other competent bacteria.

Methylation-dependent DNA discrimination in natural transformation of .

Porcine tonsils are lympho-epithelial tissues, colonized by numerous bacteria and viruses, that act as a reservoir for both host-specific pathogens and zoonotic pathogens with a high potential of transmission to humans. There are no existing studies describing the development of the tonsillar microbiome. We sequenced 16S rRNA genes from tonsillar samples of pigs to follow the development of the microbial communities from birth through weaning. Samples derived from sows were also analyzed to determine potential sources for the tonsil microbiome in piglets.

Development of the tonsillar microbiome in pigs from newborn through weaning.

Tonsils, lympho-epithelial tissues located at the junction of the oropharynx and nasopharynx, play a key role in surveillance, colonization, and persistence of inhaled and ingested pathogens. In pigs, the tonsils are a reservoir for numerous bacteria and viruses, including host-specific pathogens and potential zoonotic pathogens as well as commensal organisms. However, there are no in depth studies of the development of the tonsillar microbiome in pigs, or any mammal, over time. The goal of this study was to follow the development of the tonsil microbiome in healthy pigs from birth to market weight. Samples were collected using tonsil brushes from 16 piglets (4 each from 4 sows) at newborn, 1, 2, 3, and 4 weeks of age, and from 8 of those piglets at 6, 8, 10, 12, 16, and 19 weeks of age. Bacterial DNA was isolated from each sample and 16S rDNA genes were amplified and sequenced. Sequence analysis showed that members of the , and  were present at all time points and represent the three most abundant families identified. Other community members appeared transiently or increased or decreased significantly with disruption events or stress. We observed four significant shifts in the tonsil community that coincided with well-defined disruption events: weaning plus addition of Carbadox plus movement to the nursery at week 3, removal of Carbadox and addition of Tylan at week 5, removal of Tylan and habitat change at week 9, and habitat change at week 16. Weaning triggered a bloom of  and decrease of . The shift from Carbadox to Tylan led to reduction in a and  but an increase in other , accompanied by a dramatic increase in community richness. Cessation of Tylan coincided with a return to a less rich community, and a bloom in . The final shift in habitat was accompanied by a decrease in  and increase in . The tonsillar microbiome of older pigs resembled the previously described mature core tonsillar microbiome. This study demonstrates a temporal succession in the development of the pig tonsillar microbiome, and significant community shifts that correlate with disruption events.

Development of the Tonsil Microbiome in Pigs and Effects of Stress on the Microbiome.

 causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of  were among the most abundant when the bacterium was grown in chickens. Elevated levels of the  mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of  Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the :: mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source  Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of  physiology and  fitness and survival.  causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in , there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by  in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other  phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.

ArticleTotal : 4	Year
Publication title	2023
Publication title	2023
Publication title	2023
Publication title Cited by 4	2022

Article	Year
Ohr, an in vivo-induced gene in Actinobacillus pleuropneumoniae, is located on a genomic island and requires glutathione-S-transferase for activity. FEMS immunology and medical microbiology\| PubMed ID: 19656190	2009
Branched-chain amino acids are required for the survival and virulence of Actinobacillus pleuropneumoniae in swine. Infection and immunity\| PubMed ID: 19703979	2009
The RNA chaperone Hfq promotes fitness of Actinobacillus pleuropneumoniae during porcine pleuropneumonia. Infection and immunity\| PubMed ID: 23732171	2013
Methylation-dependent DNA discrimination in natural transformation of . Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 28855338	2017
Development of the tonsillar microbiome in pigs from newborn through weaning. BMC microbiology\| PubMed ID: 29661149	2018
Development of the Tonsil Microbiome in Pigs and Effects of Stress on the Microbiome. Frontiers in veterinary science\| PubMed ID: 30283792	2018
Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens. Journal of bacteriology\| PubMed ID: 31932316	2020

Rhiannon M. LeVeque

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