Initiation of chromosome replication in bacteria is precisely timed in the cell cycle. Bacteria that harbor multiple chromosomes face the additional challenge of orchestrating replication initiation ...of different chromosomes. In Vibrio cholerae, the smaller of its two chromosomes, Chr2, initiates replication after Chr1 such that both chromosomes terminate replication synchronously. The delay is due to the dependence of Chr2 initiation on the replication of a site, crtS, on Chr1. The mechanism by which replication of crtS allows Chr2 replication remains unclear. Here, we show that blocking Chr1 replication indeed blocks Chr2 replication, but providing an extra crtS copy in replication-blocked Chr1 permitted Chr2 replication. This demonstrates that unreplicated crtS copies have significant activity, and suggests that a role of replication is to double the copy number of the site that sufficiently increases its activity for licensing Chr2 replication. We further show that crtS activity promotes the Chr2-specific initiator function and that this activity is required in every cell cycle, as would be expected of a cell-cycle regulator. This study reveals how increase of gene dosage through replication can be utilized in a critical regulatory switch.
The antitumor efficacy of cancer immunotherapy can correlate with the presence of certain bacterial species within the gut microbiome. However, many of the molecular mechanisms that influence host ...response to immunotherapy remain elusive. In this study, we show that members of the bacterial genus
improve checkpoint inhibitor immunotherapy in mouse tumor models. Active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in nonprotective
was sufficient to promote immunotherapy response, and its activity required the peptidoglycan sensor NOD2. Notably, SagA-engineered probiotics or synthetic muropeptides also augmented anti-PD-L1 antitumor efficacy. Taken together, our data suggest that microbiota species with specialized peptidoglycan remodeling activity and muropeptide-based therapeutics may enhance cancer immunotherapy and could be leveraged as next-generation adjuvants.
The Escherichia coli origin of replication, oriC, comprises mostly binding sites of two proteins: DnaA, a positive regulator, and SeqA, a negative regulator. SeqA, although not essential, is required ...for timely initiation, and during rapid growth, synchronous initiation from multiple origins. Unlike DnaA, details of SeqA binding to oriC are limited. Here we have determined that SeqA binds to all its sites tested (9/11) and with variable efficiency. Titration of DnaA alters SeqA binding to two sites, both of which have overlapping DnaA sites. The altered SeqA binding, however, does not affect initiation synchrony. Synchrony is also unaffected when individual SeqA sites are mutated. An apparent exception was one mutant where the mutation also changed an overlapping DnaA site. In this mutant, the observed asynchrony could be from altered DnaA binding, as selectively mutating this SeqA site did not cause asynchrony. These results reveal robust initiation synchrony against alterations of individual SeqA binding sites. The redundancy apparently ensures SeqA function in controlling replication in E. coli.
Abstract
Studies of bacterial chromosomes and plasmids indicate that their replication initiator proteins bind to origins of replication at many double-stranded sites and also at AT-rich regions ...where single-stranded DNA is exposed during origin opening. Single-strand binding apparently promotes origin opening by stabilizing an open structure, but how the initiator participates in this process and the contributions of the several binding sites remain unclear. Here, we show that the initiator protein of Vibrio cholerae specific to chromosome 2 (Chr2) also has single-strand binding activity in the AT-rich region of its origin. Binding is strand specific, depends on repeats of the sequence 5′ATCA and is greatly stabilized in vitro by specific double-stranded sites of the origin. The stability derives from the formation of ternary complexes of the initiator with the single- and double-stranded sites. An IHF site lies between these two kinds of sites in the Chr2 origin and an IHF-induced looping out of the intervening DNA mediates their interaction. Simultaneous binding to two kinds of sites in the origin appears to be a common mechanism by which bacterial replication initiators stabilize an open origin.
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA
Correspondence Indranil Biswas ...ibiswas{at}kumc.edu
A set of shuttle plasmids containing four different constitutive promoters was generated to facilitate overexpression of foreign and native genes in streptococci, such as Streptococcus mutans . The four promoters that were chosen were: P ami , P spac , P 23 and P veg . These promoters are active in many Gram-positive bacteria, and allow various levels of gene expression depending on the host bacterium. Shuttle plasmids were constructed based on two types of broad-host-range replication origins: a rolling-circle replicon (pSH71) and a theta replicon (pAM β 1). Shuttle plasmids derived from the pAM β 1 replicon were generated to avoid the structural and segregational stability problems associated with rolling-circle replication, since these problems may be encountered during large gene cloning. In a complementation assay, we used one such plasmid to express a gene in trans to show the utility of these plasmids. In addition, a series of plasmids was generated for the expression of recombinant proteins with an N-terminal 6 x His tag or a C-terminal Strep-tag fusion, and, using a gene derived from S. mutans , we showed a high level of recombinant protein expression in S. mutans and Streptococcus pyogenes . Since these plasmids contain broad-host-range replication origins, and because the selected promoters are functional in many bacteria, they can be used for gene expression studies, such as complementation and recombinant protein expression.
Abbreviations: CSP, competence-stimulating peptide; GUS, β -glucuronidase; MCS, multiple-cloning site; MU, Miller units; RCR, rolling-circle replicon
Abstract
Replication of Vibrio cholerae chromosome 2 (Chr2) initiates when the Chr1 locus, crtS (Chr2 replication triggering site) duplicates. The site binds the Chr2 initiator, RctB, and the binding ...increases when crtS is complexed with the transcription factor, Lrp. How Lrp increases the RctB binding and how RctB is subsequently activated for initiation by the crtS-Lrp complex remain unclear. Here we show that Lrp bends crtS DNA and possibly contacts RctB, acts that commonly promote DNA-protein interactions. To understand how the crtS-Lrp complex enhances replication, we isolated Tn-insertion and point mutants of RctB, selecting for retention of initiator activity without crtS. Nearly all mutants (42/44) still responded to crtS for enhancing replication, exclusively in an Lrp-dependent manner. The results suggest that the Lrp-crtS controls either an essential function or more than one function of RctB. Indeed, crtS modulates two kinds of RctB binding to the origin of Chr2, ori2, both of which we find to be Lrp-dependent. Some point mutants of RctB that are optimally modulated for ori2 binding without crtS still remained responsive to crtS and Lrp for replication enhancement. We infer that crtS-Lrp functions as a unit, which has an overarching role, beyond controlling initiator binding to ori2.
Graphical Abstract
Graphical Abstract
RctB, the initiator of replication of Vibrio cholerae chromosome 2 (chr2), binds to the origin of replication to specific 12-mer sites both as a monomer and a dimer. Binding to 12-mers is essential ...for initiation. The monomers also bind to a second kind of site, 39-mers, which inhibits initiation. Mutations in rctB that reduce dimer binding increase monomer binding to 12-mers but decrease monomer binding to 39-mers. The mechanism of this paradoxical binding behavior has been unclear. Using deletion and alanine substitution mutants of RctB, we have now localized to a 71 amino acid region residues important for binding to the two kinds of DNA sites and for RctB dimerization. We find that the dimerization domain overlaps with both the DNA binding domains, explaining how changes in the dimerization domain can alter both kinds of DNA binding. Moreover, dimerization-defective mutants could be initiation-defective without apparent DNA binding defect. These results suggest that dimerization might be important for initiation beyond its role in controlling DNA binding. The finding that determinants of crucial initiator functions reside in a small region makes the region an attractive target for anti-V. cholerae drugs.
The origin region of Vibrio cholerae chromosome II (chrII) resembles plasmid origins that have repeated initiator-binding sites (iterons). Iterons are essential for initiation as well as preventing ...over-initiation of plasmid replication. In chrII, iterons are also essential for initiation but over-initiation is prevented by sites called 39-mers. Both iterons and 39-mers are binding sites of the chrII specific initiator, RctB. Here, we have isolated RctB mutants that permit over-initiation in the presence of 39-mers. Characterization of two of the mutants showed that both are defective in 39-mer binding, which helps to explain their over-initiation phenotype. In vitro, RctB bound to 39-mers as monomers, and to iterons as both monomers and dimers. Monomer binding to iterons increased in both the mutants, suggesting that monomers are likely to be the initiators. We suggest that dimers might be competitive inhibitors of monomer binding to iterons and thus help control replication negatively. ChrII replication was found to be dependent on chaperones DnaJ and DnaK in vivo. The chaperones preferentially improved dimer binding in vitro, further suggesting the importance of dimer binding in the control of chrII replication.
The present investigation evaluated the effect of continuous application (>43 years) of organic and inorganic fertilisers on soil aggregate stability, aggregate size distribution, ...aggregate-associated carbon and its fractions, and total macro-nutrient content under the soybean–wheat cropping system in vertisols of the semi-arid region. Seven contrasting treatments consisted of T1 (50% NPK), T2 (100% NPK), T3 (150% NPK), T4 (100% NP), T5 (100% N), T6 (100% NPK + FYM) and T7 Control (crop raised without addition of any nutrient). The highest and lowest percentage of large macroaggregates (11.3%) was found in T6 and T7 treatments. The NPK + FYM (T6) treatments substantially increased the proportion of the macroaggregate fractions (>2 mm and 2–0.25 mm) than other treatments. However, different manure and fertilisation treatments did not affect the proportion of silt + clay aggregates. Long-term application of 100% NPK + FYM increased mean weight diameter (MWD) and stable water aggregates (WSA) by 35.7 and 6.01% over control. The aggregate-associated SOC followed the trend of large macroaggregates > microaggregates > small macroaggregates > silt + clay fractions. Application of long-term manure plus inorganic fertiliser (T6) has also increased Walkley Black soil organic carbon (WBSC), permanganate oxidisable carbon (KMnO4-C), soil microbial biomass carbon (SMBC), carbon mineralisation (CM), total soil carbon (TSC), total soil N (TSN), total soil phosphorus (TSP) and total soil potassium (STK) by 82.1, 71.6, 182, 42.4, 23.9, 41.6, 117 and 18.4%, respectively, over control (T7). The lowest metabolic quotient (MetQ) value of 5.13 mg CO2–C mg−1 MBC h−1 was obtained in the control treatment (T7). The lowest MetQ was recorded in the integrated application of manure + inorganic fertiliser, i.e., 100% NPK + FYM (T6). Similarly, microbial quotient (MiQ) was also higher in treatment T6 (100% NPK + FYM) and lower in T7 (control). It is concluded that the application of inorganic fertiliser alone is insufficient to maintain soil health and sustainability so, combined application of manure plus inorganic fertilisation is the most important nutrient management practice for long-term soil sustainability because it maintains SOC levels in soils for long periods and ultimately ensures the soil health of soybean–wheat cropping systems in the vertisols of semi-arid regions.
Chromosome dynamics in multichromosome bacteria Jha, Jyoti K.; Baek, Jong Hwan; Venkova-Canova, Tatiana ...
Biochimica et biophysica acta,
07/2012, Letnik:
1819, Številka:
7
Journal Article
Recenzirano
Odprti dostop
On the basis of limited information, bacteria were once assumed to have no more than one chromosome. In the era of genomics, it has become clear that some, like eukaryotes, have more than one ...chromosome. Multichromosome bacteria provide opportunities to investigate how split genomes emerged, whether the individual chromosomes communicate to coordinate their replication and segregation, and what selective advantages split genomes might provide. Our current knowledge of these topics comes mostly from studies in Vibrio cholerae, which has two chromosomes, chr1 and chr2. Chr1 carries out most of the house-keeping functions and is considered the main chromosome, whereas chr2 appears to have originated from a plasmid and has acquired genes of mostly unknown origin and function. Nevertheless, unlike plasmids, chr2 replicates once and only once per cell cycle, like a bona fide chromosome. The two chromosomes replicate and segregate using separate programs, unlike eukaryotic chromosomes. They terminate replication synchronously, suggesting that there might be communication between them. Replication of the chromosomes is affected by segregation genes but in a chromosome specific fashion, a new development in the field of DNA replication control. The split genome allows genome duplication to complete in less time and with fewer replication forks, which could be beneficial for genome maintenance during rapid growth, which is the norm for V. cholerae in broth cultures and in the human host. In the latter, the expression of chr2 genes increases preferentially. Studies of chromosome maintenance in multichromosomal bacteria, although in their infancy, are already broadening our view of chromosome biology. This article is part of a Special Issue entitled: Chromatin in time and space.
► Some bacteria, like eukaryotes, have multiple chromosomes ► Some of these chromosomes appear to be of plasmid provenance ► Maintenance mechanisms of individual chromosomes are distinct, not as in eukaryotes
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