- Volume 168, Issue 6, 2022
Volume 168, Issue 6, 2022
- Reviews
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Lactobacilli and human dental caries: more than mechanical retention
More LessLactobacilli have been considered as major contributors to human dental caries for over a century. Recent in vitro model studies have shown that when compared to Streptococcus mutans, a keystone pathogen of human dental caries, the ability of lactobacilli to form biofilms is poor, although differences exist between the different major species. Further studies using molecular and bioinformatics approaches provide evidence that multiple mechanisms, including adhesin-receptor mediated physical contact with S. mutans , facilitate the adherence and establishment of lactobacilli on the tooth surface. There is also evidence that under conditions like continuous sugar consumption, weak acids and other antimicrobials such as bacteriocins from lactobacilli can become detrimental to the microbial community, especially those in the proximity. Details on the underlying mechanisms of how different Lactobacillus sp. establish and persist in the highly complex microbiota on the tooth surface await further investigation.
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- Microbe Profiles
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Microbe Profile: Gigaspora margarita, a multifaceted arbuscular mycorrhizal fungus
More LessGigaspora margarita is a cosmopolitan arbuscular mycorrhizal fungus, which - as an obligate symbiont- requires being associated to a host plant to accomplish its life cycle. It is characterized by huge white spores, the development of extraradical auxiliary cells, and the lack of intraradical vesicles. Its genome is dominated by transposable elements and is one of the largest fungal genomes so far sequenced. G. margarita has the peculiar feature to host taxonomically different endobacteria in its cytoplasm. The development of a cured line has allowed us to demonstrate how the endobacteria have a positive impact on the fungal physiology and -with a cascade effect- on the mycorrhizal plant.
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- Antimicrobials and AMR
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Deciphering the role of residues in the loops nearing the active site of OXA-58 in imparting beta-lactamase activity
More LessThe existence of OXA-58 carbapenemase alone or in combination with other beta-lactam resistance factors poses significant beta-lactam resistance. The exact mechanism of action of OXA type beta-lactamases is debatable due to the involvement of multiple residues within or outside the active site. In the present work, we have elucidated the relative role of residues present in the putative omega (W169, L170, K171) and β6-β7 (A226 and D228) loops on the activity of OXA-58 by substituting into alanine (and aspartate for A226) through site-directed mutagenesis. E. coli cells harbouring OXA-58, substituted at the putative omega loop, manifest a significant decrease in the beta-lactam resistance profile than that of the cells expressing OXA-58. Further, a reduction in the catalytic efficiency is observed for the purified variants of OXA-58 carrying individual substitutions in the putative omega loop than that of OXA-58. However, the addition of NaHCO3 (for carbamylation of K86) increases catalytic efficiency of the individual protein as revealed by nitrocefin hydrolysis assay and steady state kinetics. Moreover, W169A and K171A substitutions show significant effects on the thermal stability of OXA-58. Therefore, we conclude that the putative omega loop residues W169, L170 and K171, individually, have significant role in the activity and stability of OXA-58, mostly by stabilising carbamylated lysine of active site.
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Genomic characterization of antimicrobial resistance in mcr-carrying ESBL-producing Escherichia coli from pigs and humans
More LessWhole-genome sequencing (WGS) was conducted to characterize mcr-carrying extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (n=7). These E. coli isolates originated from two pigs (TH2 and TH3) and two humans (TH8 and TH9) from Thailand, and three pigs from Lao PDR (LA1, LA2 and LA3). Four E. coli sequence types/serotypes – ST6833/H20 (TH2 and TH3), ST48/O160:H40 (TH8 and TH9), ST5708/H45 (LA1) and ST10562/O148:H30 (LA2 and LA3) – were identified. The plasmid replicon type IncF was identified in all isolates. The point mutations Ser31Thr in PmrA and His2Arg in PmrB were found concurrently in all isolates (colistin MIC=4–8 µg ml−1). LA1 contained up to five point mutations in PmrB, and the colistin MIC was not significantly different from that for the other isolates. All mcr-1.1 was located in the ISApl1-mcr-1-pap2 element, while all mcr-3.1 was located in the TnAs2-mcr-3.1-dgkA-ISKpn40 element. The mcr-3.1 and bla CTX-M-55 genes were co-localized on the same plasmid, which concurrently contained cml, qnrS1 and tmrB. The bla CTX-M-55 and mcr-3.1 genes were located on conjugative plasmids and could be transferred horizontally under selective pressure from ampicillin or colistin. In conclusion, comprehensive insights into the genomic information of ESBL-producing E. coli harbouring mcr were obtained. As mcr-carrying ESBL-producing E. coli were detected in pigs and humans, a holistic and multisectoral One Health approach is required to contain antimicrobial resistance (AMR).
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- Cell and Developmental Microbiology (formerly Cell Biology)
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DNA damage-induced block of sporulation in Streptomyces venezuelae involves downregulation of ssgB
DNA damage often causes an arrest of the cell cycle that provides time for genome integrity to be restored. In bacteria, the classical SOS DNA damage response leads to an inhibition of cell division resulting in temporarily filamentous growth. This raises the question as to whether such a response mechanism might similarly function in naturally filamentous bacteria such as Streptomyces. Streptomyces exhibit two functionally distinct forms of cell division: cross-wall formation in vegetative hyphae and sporulation septation in aerial hyphae. Here, we show that the genotoxic agent mitomycin C confers a block in sporulation septation in Streptomyces venezuelae in a mechanism that involves, at least in part, the downregulation of ssgB. Notably, this DNA damage response does not appear to block cross-wall formation and may be independent of canonical SOS and developmental regulators. We also show that the mitomycin C-induced block in sporulation can be partially bypassed by the constitutive expression of ssgB, though this appears to be largely limited to mitomycin C treatment and the resultant spore-like cells have reduced viability.
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- Microbial Physiology, Biochemistry and Metabolism
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FtsZ at mid-cell is essential in Escherichia coli until the late stage of constriction
More LessThere has been recent debate as to the source of constriction force during cell division. FtsZ can generate a constriction force on tubular membranes in vitro, suggesting it may generate the constriction force in vivo. However, another study showed that mutants of FtsZ did not affect the rate of constriction, whereas mutants of the PG assembly did, suggesting that PG assembly may push the constriction from the outside. Supporting this model, two groups found that cells that have initiated constriction can complete septation while the Z ring is poisoned with the FtsZ targeting antibiotic PC190723. PC19 arrests treadmilling but leaves FtsZ in place. We sought to determine if a fully assembled Z ring is necessary during constriction. To do this, we used a temperature-sensitive FtsZ mutant, FtsZ84. FtsZ84 supports cell division at 30 °C, but it disassembles from the Z ring within 1 min upon a temperature jump to 42 °C. Following the temperature jump we found that cells in early constriction stop constricting. Cells that had progressed to the later stage of division finished constriction without a Z ring. These results show that in Escherichia coli , an assembled Z ring is essential for constriction except in the final stage, contradicting the simplest interpretation of previous studies using PC19.
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- Microbial Physiology, Biochemistry and Metabolism (formerly Physiology and Metabolism)
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Markerless gene editing in Neisseria gonorrhoeae
More LessNeisseria gonorrhoeae , the gonococcus, is a pathogen of major public health concern, but sophisticated approaches to gene manipulation are limited for this species. For example, there are few methods for generating markerless mutations, which allow the generation of precise point mutations and deletions without introducing additional DNA sequence. Markerless mutations are central to studying pathogenesis, the spread of antimicrobial resistance (AMR) and for vaccine development. Here we describe the use of galK as a counter-selectable marker that can be used for markerless mutagenesis in N. gonorrhoeae . galK encodes galactokinase, an enzyme that metabolizes galactose in bacteria that can utilize it as a sole carbon source. GalK can also phosphorylate a galactose analogue, 2-deoxy-galactose (2-DOG), into a toxic, non-metabolisable intermediate, 2-deoxy-galactose-1-phosphate. We utilized this property of GalK to develop a markerless approach for mutagenesis in N. gonorrhoeae . We successfully deleted both chromosomally and plasmid-encoded genes, that are important for gonococcal vaccine development and studies of AMR spread. We designed a positive-negative selection cassette, based on an antibiotic resistance marker and galK, that efficiently rendered N. gonorrhoeae susceptible to growth on 2-DOG. We then adapted the galK-based counter-selection and the use of 2-DOG for markerless mutagenesis, and applied biochemical and phenotypic analyses to confirm the absence of target genes. We show that our markerless mutagenesis method for N. gonorrhoeae has a high success rate, and should be a valuable gene editing tool in the future.
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- Regulation, Sensing and Signalling (formerly Regulation)
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Sulphate repression of ssuD-dependent alkanesulphonate-sulphur assimilation in Escherichia coli
More LessEscherichia coli cells utilize alkanesulphonates including taurine as the sulphur source. We previously reported that when E. coli cells carrying a double deletion in tauD and cysN were inoculated into a taurine-containing minimal medium, they started to grow only after long-term incubation (Nishikawa et al. 2018, Microbiology 164: 1446–1456). We show here that cells that can induce ssuD-dependent alkanesulphonate–sulphur assimilation (SASSA) are essentially rare, but suppressors that can induce SASSA appear during long-term incubation. Mutant cells carrying ΔtauD and ΔcysN, ΔcysC or ΔcysH generated suppressor cells that can induce SASSA at a frequency of about 10−6 in a population. Whereas ΔtauD ΔcysN cells without prior SASSA did not express ssuD even when necessary, the cells with prior SASSA properly expressed ssuD. Whole-genome DNA sequencing of a clone isolated from ΔtauD ΔcysN cells with prior SASSA revealed that the influx of sulphate or thiosulphate may be related to the regulation of SASSA. To clarify whether sulphate or thiosulphate affects the induction of SASSA, the effect of mutations in sbp and cysP, which are responsible for sulphate and thiosulphate uptake with different preferences for substrates, was examined. Only the ΔtauD ΔcysN Δsbp mutant did not show repression of SASSA when no sulphate was added to the medium. When the concentration of the sulphate added was over 10 μM, the Δsbp mutant showed repression of SASSA. Therefore, it was considered that the influx of extracellular sulphate resulted in repression of SASSA.
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Volumes and issues
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