- Volume 157, Issue 7, 2011

The coupling between chromosome replication and cell division includes temporal and spatial elements. In bacteria, these have globally been resolved during the last 40 years, but their full details and action mechanisms are still under intensive study. The physiology of growth and the cell cycle are reviewed in the light of an established dogma that has formed a framework for development of new ideas, as exemplified here, using the Cell Cycle Simulation (CCSim) program. CCSim, described here in detail for the first time, employs four parameters related to time (replication, division and inter-division) and size (cell mass at replication initiation) that together are sufficient to describe bacterial cells under various conditions and states, which can be manipulated environmentally and genetically. Testing the predictions of CCSim by analysis of time-lapse micrographs of Escherichia coli during designed manipulations of the rate of DNA replication identified aspects of both coupling elements. Enhanced frequencies of cell division were observed following an interval of reduced DNA replication rate, consistent with the prediction of a minimum possible distance between successive replisomes (an eclipse). As a corollary, the notion that cell poles are not always inert was confirmed by observed placement of division planes at perpendicular planes in monstrous and cuboidal cells containing multiple, segregating nucleoids.

The streptococcal protein required for cell separation B (PcsB) is predicted to play an important role in peptidoglycan metabolism, based on sequence motifs and altered phenotypes of gene deletion mutant cells exhibiting defects in cell separation. However, no enzymic activity has been demonstrated for PcsB so far. By generating gene deletion mutant strains in four different genetic backgrounds we could demonstrate that pcsB is not essential for cell survival in Streptococcus pneumoniae, but is essential for proper cell division. Deletion mutant cells displayed cluster formation due to aberrant cell division, reduced growth and antibiotic sensitivity that were fully reverted by transformation with a plasmid carrying pcsB. Immunofluorescence staining revealed that PcsB was localized to the cell poles, similarly to PBP3 and LytB, enzymes with demonstrated peptidoglycan-degrading activity required for daughter cell separation. Similarly to other studies with PcsB homologues, we could not detect peptidoglycan-lytic activity with recombinant or native pneumococcal PcsB in vitro. In addition to defects in septum placement and separation, the absence of PcsB induced an increased release of several proteins, such as enolase, MalX and the SP0107 LysM domain protein. Interestingly, genes encoding both LysM domain-containing proteins that are present in the pneumococcal genome (SP0107 and SP2063) and predicted to be involved in cell wall metabolism were found to be highly overexpressed (14–33-fold increase) in ΔpcsB cells in two different genetic backgrounds. Otherwise, we detected very few changes in the global gene expression profile of cells lacking PcsB. Thus our data suggest that LysM domain proteins partially compensate for the lack of PcsB function and allow the survival and slow growth of the pneumococcus.

Anabaena sp. strain PCC 7120 (Anabaena PCC 7120) is a filamentous, nitrogen-fixing cyanobacterium. Upon deprivation of combined nitrogen, about 5–10 % of the cells become heterocysts, i.e. cells devoted to N2 fixation. Heterocysts are intercalated among vegetative cells and distributed in a semi-regular pattern, and adjacent heterocysts are rarely observed. Previously, we showed that the cell cycle could play a regulatory function during heterocyst development, although the mechanism involved remains unknown. As a further step to understand this phenomenon, we identified the oriC region for chromosomal DNA replication, located between dnaA and dnaN. The oriC region of Anabaena PCC 7120 was able to support the self-replication of a plasmid in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Surprisingly, integration of the oriC region into the chromosome of Anabaena PCC 7120 through homologous recombination led to much slower cell growth in the absence of a combined-nitrogen source and to multiple contiguous proheterocysts after prolonged incubation. Real-time RT-PCR showed that expression of two heterocyst-related genes, hetR and hetN, was altered in these strains: hetR expression remained high 48 h after induction, and hetN increased to high levels after induction for 12 h. These results suggest that the balance between oriC and DnaA could be important for heterocyst development.

Type IV pili (TFP) are membrane-anchored filaments with a number of important biological functions. In the model organism Myxococcus xanthus, TFP act as molecular engines that power social (S) motility through cycles of extension and retraction. TFP filaments consist of several thousand copies of a protein called PilA or pilin. PilA contains an N-terminal α-helix essential for TFP assembly and a C-terminal globular domain important for its activity. The role of the PilA sequence and its structure–function relationship in TFP-dependent S motility remain active areas of research. In this study, we identified an M. xanthus PilA mutant carrying an alanine to valine substitution at position 32 in the α-helix, which produced structurally intact but retraction-defective TFP. Characterization of this mutant and additional single-residue variants at this position in PilA demonstrated the critical role of alanine 32 in PilA stability, TFP assembly and retraction.

Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during infection natriuretic peptides, particularly CNP, could enhance the virulence of PAO1. This activity is relayed by Vfr and PtxR activation, and a general diagram of the virulence activation cascade involving AHL, HCN and exotoxin A is proposed.

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of deoxyribonucleotides (dNTPs). Surprisingly, Escherichia coli cells carrying the nrdA101 allele, which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 °C under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at 42 °C and that its degradation under restrictive conditions is prevented by the presence of rifampicin. Nevertheless, the mere stability of the RNR protein at 42 °C cannot explain the completion of chromosomal DNA replication in the nrdA101 mutant. We found that inactivation of the DnaA protein by using several dnaAts alleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in the nrdA101 mutant at 42 °C. As both inactivation of the DnaA protein and inhibition of RNA synthesis block the occurrence of new DNA initiations, the consequent decrease in the number of forks per chromosome could be related to those effects. In support of this notion, we found that avoiding multifork replication rounds by the presence of moderate extra copies of datA sequence increases the relative amount of DNA synthesis of the nrdA101 mutant at 42 °C. We propose that a lower replication fork density results in an improvement of the progression of DNA replication, allowing replication of the entire chromosome at the restrictive temperature. The mechanism related to this effect is also discussed.

Aspergillus fumigatus is the most common airborne fungal pathogen, causing fatal invasive aspergillosis in immunocompromised patients. The crude mortality is 60–90 % and remains around 29–42 % even with treatment. The main reason for patient death is the low efficiency of the drug therapies. As protein N-glycosylation is involved in cell wall biogenesis in A. fumigatus, a deeper understanding of its role in cell wall biogenesis will help to develop new drug targets. The Afstt3 gene encodes the essential catalytic subunit of oligosaccharyltransferase, an enzyme complex responsible for the transfer of the N-glycan to nascent polypeptides. To evaluate the role of N-glycosylation in cell wall biosynthesis, we constructed the conditional mutant strain CPR-stt3 by replacing the endogenous promoter of Afstt3 with the nitrogen-dependent niiA promoter. Repression of the Afstt3 gene in the CPR-stt3 strain led to a severe retardation of growth and a slight defect in cell wall integrity (CWI). One of the most interesting findings was that upregulation of the cell wall-related genes was not accompanied by an activation of the MpkA kinase, which has been shown to be a central element in the CWI signalling pathway in both Saccharomyces cerevisiae and A. fumigatus. Considering that the unfolded protein response (UPR) was found to be activated, which might upregulate the expression of cell wall protein and chitin, our data suggest that the UPR, instead of the MpkA-dependent CWI signalling pathway, is the major compensatory mechanism induced by repression but not abolition of N-glycosylation in A. fumigatus. Our finding is a key to understanding the complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development.

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64 % of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Gemmata obscuriglobus has a highly condensed nucleoid which is implicated in its resistance to radiation. However, the mechanisms by which such compaction is achieved, and the proteins responsible, are still unknown. Here we have examined the genome of G. obscuriglobus for the presence of proteins homologous to those that have been associated with nucleoid condensation. We found two different proteins homologous to the bacterial nucleoid-associated protein HU, one with an N-terminal and one with a C-terminal extension relative to the amino acid sequence of the HU found in Escherichia coli. Sequence analysis revealed that one of these HU homologues represents a novel type with a high number of prolines in its C-terminal extension, whereas the other one has motifs similar to the N terminus of the HU homologue from the radio-resistant bacterium Deinococcus radiodurans. The occurrence of two such HU homologue proteins with these two different terminal extensions in one organism appears to be unique among the Bacteria.

In our previous study, extensive genomic rearrangements were found in two strains of the Gram-negative anaerobic bacterium Porphyromonas (Por.) gingivalis, and most of these rearrangements were associated with mobile genetic elements such as insertion sequences and conjugative transposons (CTns). CTnPg1, identified in Por. gingivalis strain ATCC 33277, was the first complete CTn reported for the genus Porphyromonas. In the present study, we found that CTnPg1 can be transferred from strain ATCC 33277 to another Por. gingivalis strain, W83, at a frequency of 10−7 to 10−6. The excision of CTnPg1 from the chromosome in a donor cell depends on an integrase (Int; PGN_0094) encoded in CTnPg1, whereas CTnPg1 excision is independent of PGN_0084 (a DNA topoisomerase I homologue; Exc) encoded within CTnPg1 and recA (PGN_1057) on the donor chromosome. Intriguingly, however, the transfer of CTnPg1 between Por. gingivalis strains requires RecA function in the recipient. Sequencing analysis of CTnPg1-integrated sites on the chromosomes of transconjugants revealed that the consensus attachment (att) sequence is a 13 bp sequence, TTTTCNNNNAAAA. We further report that CTnPg1 is able to transfer to two other bacterial species, Bacteroides thetaiotaomicron and Prevotella oralis. In addition, CTnPg1-like CTns are located in the genomes of other oral anaerobic bacteria, Porphyromonas endodontalis, Prevotella buccae and Prevotella intermedia, with the same consensus att sequence. These results suggest that CTns in the CTnPg1 family are widely distributed among oral anaerobic Gram-negative bacteria found in humans and play important roles in horizontal gene transfer among these bacteria.

The genome sequence of Bacillus subtilis subsp. spizizenii W23 has been determined. The sequence strongly suggests that W23 is a direct descendant of B. subtilis ATCC 6633. W23 shares a 3.6 Mb core genome with the intensively studied model organism B. subtilis subsp. subtilis 168, and gene order within this core has been strongly conserved. Additionally, the W23 genome has 157 accessory (that is, non-core) genome segments that are not found in 168, while the 168 genome has 141 segments not found in W23. The distribution of sequences similar to these accessory segments among other genomes of the B. subtilis species complex shows that those sequences having entered into the phylogeny of the complex more recently tend to be larger and more AT-rich than those having entered earlier. A simple model can account for these observations, in which parasitic or symbiotic DNAs are transferred into the genome and then are reduced in size and modified in base composition during speciation.

The protein MsRbpA from Mycobacterium smegmatis rescues RNA polymerase (RNAP) from the inhibitory effect of rifampicin (Rif). We have reported previously that MsRbpA interacts with the β-subunit of RNAP and that the effect of MsRbpA on Rif-resistant (RifR) RNAP is minimal. Here we attempted to gain molecular insights into the mechanism of action of this protein with respect to its role in rescuing RNAP from Rif-mediated transcription inhibition. Our experimental approach comprised multiple-round transcription assays, fluorescence spectroscopy, MS and surface plasmon resonance in order to meet the above objective. Based on our molecular studies we propose here that Rif is released from its binding site in the RNAP–Rif complex in the presence of MsRbpA. Biophysical studies reveal that the location of MsRbpA on RNAP is at the junction of the β- and β′-subunits, close to the Rif-binding site and the (i+1) site on RNAP.

Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE+ and SopE− S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH–gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA–gfp reporter mirrored that of PprgH–gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection.

The ability of serovars of Salmonella enterica to cause systemic disease is dependent upon their survival and replication within macrophages. To do this, bacteria must withstand or surmount bacteriostatic and bactericidal responses by the host cell, including the delivery of hydrolytic enzymes from lysosomes to the phagosome. The bacterial two-component regulatory system PhoP/Q has been implicated in avoidance of phagolysosomal fusion by S. enterica serovar Typhimurium (S. Typhimurium) in murine macrophages. In this study, the involvement of PhoP/Q-activated genes in avoidance of phagolysosomal fusion was analysed: of all the S. Typhimurium mutant strains tested, only an mgtC mutant strain partially reproduced the phenotype of the phoP mutant strain. As this gene is required for bacterial growth in magnesium-depleted conditions in vitro, the contributions of PhoP/Q to intramacrophage replication and survival were reappraised. Although PhoP/Q was required for both replication and survival of S. Typhimurium within murine macrophages, subsequent analysis of the kinetics of phagolysosomal fusion, taking account of differences in the replication rates of wild-type and phoP mutant strains, provided no evidence for a PhoP/Q-dependent role in this process. PhoP/Q appeared to act subsequent to the process of phagolysosomal avoidance and to promote replication of those bacteria that had already escaped a phagolysosomal fate. Therefore, we conclude that the PhoP/Q regulon enables S. Typhimurium to adapt to intramacrophage stresses other than phagolysosomal fusion.