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Essential gene deletions producing gigantic bacteria.

ABSTRACT: To characterize the consequences of eliminating essential functions needed for peptidoglycan synthesis, we generated deletion mutations of Acinetobacter baylyi by natural transformation and visualized the resulting microcolonies of dead cells. We found that loss of genes required for peptidoglycan precursor synthesis or polymerization led to the formation of polymorphic giant cells with diameters that could exceed ten times normal. Treatment with antibiotics targeting early or late steps of peptidoglycan synthesis also produced giant cells. The giant cells eventually lysed, although they were partially stabilized by osmotic protection. Genome-scale transposon mutant screening (Tn-seq) identified mutations that blocked or accelerated giant cell formation. Among the mutations that blocked the process were those inactivating a function predicted to cleave murein glycan chains (the MltD murein lytic transglycosylase), suggesting that giant cell formation requires MltD hydrolysis of existing peptidoglycan. Among the mutations that accelerated giant cell formation after ß-lactam treatment were those inactivating an enzyme that produces unusual 3->3 peptide cross-links in peptidoglycan (the LdtG L,D-transpeptidase). The mutations may weaken the sacculus and make it more vulnerable to further disruption. Although the study focused on A. baylyi, we found that a pathogenic relative (A. baumannii) also produced giant cells with genetic dependencies overlapping those of A. baylyi. Overall, the analysis defines a genetic pathway for giant cell formation conserved in Acinetobacter species in which independent initiating branches converge to create the unusual cells.

SUBMITTER: Bailey J

PROVIDER: S-EPMC6586353 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Essential gene deletions producing gigantic bacteria.

Bailey Jeannie J Cass Julie J Gasper Joe J Ngo Ngoc-Diep ND Wiggins Paul P Manoil Colin C

PLoS genetics 20190610 6

To characterize the consequences of eliminating essential functions needed for peptidoglycan synthesis, we generated deletion mutations of Acinetobacter baylyi by natural transformation and visualized the resulting microcolonies of dead cells. We found that loss of genes required for peptidoglycan precursor synthesis or polymerization led to the formation of polymorphic giant cells with diameters that could exceed ten times normal. Treatment with antibiotics targeting early or late steps of pept ...[more]

PMID: 31181062

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Project description:Mitochondrial DNA has been the preferential genome biodiversity studies. However, several factors contribute to its inadequacy. Numts constitute one of the main complications that prevent obtaining orthologous mitochondrial sequences. Orthoptera have been a model group in numt studies because of their huge genome size. In this study we aimed to; (i) test efficiency of standard PCR protocol in producing orthologous sequences of cytochrome C oxidase, (ii) study presence/absence of numts in several unstudied Orthoptera species, (iii) test if there is a threshold between the length of mtDNA targeted for amplification and possibility of encountering numts, and (iv) estimate reliability of the sequences in databases in light of these findings. For these aims we studied 38 species of Orthoptera representing different sublineages and genome sizes. DNA extracted from each sample was used to amplify five different fragments of COI region by standard PCR protocol. Sequenced PCR amplicons were checked for numt possibility by several different numt criteria. No sequences without numt signs were obtained for the first fragment. The number of samples with numt signs for the other four fragments differed between the suborders Ensifera and Caelifera. The percentage of samples with numt signs was higher in Caelifera than Ensifera for all fragments. The numt percentage considerably decreased for the longest two fragments. Numts are more prevalent in families with larger genome size. We arrived at the following conclusions: (i) numts are common in all members of Orthoptera, but, their prevalence differs among intra-lineages, especially more prevalent in Caelifera, (ii) there seems a correlation between numt rate and genome size, (iii) there is no threshold to avoid numt co-amplification, but, a 1,000 bp length may be a threshold for Ensifera, (iv) Folmer region of COI doesn't seem an appropriate marker for animal barcoding. Additionally, a phylogenetic tree produced from the numt sequences of fragment four detected in genus Anterastes suggested a paleonumt gained in generic ancestor a 3.5-4 times slower divergence rate for numt sequences.

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Essential gene deletions producing gigantic bacteria.

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Essential gene deletions producing gigantic bacteria.

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