- Volume 159, Issue Pt_11, 2013

The initiation of chromosome replication is tightly regulated in bacteria to ensure that it takes place only once per cell cycle. In many proteobacteria, this process requires the ATP-bound form of the DnaA protein. The regulatory inactivation of DnaA (RIDA) facilitates the conversion of DnaA-ATP into replication-inactive DnaA-ADP, thereby preventing overinitiation. Homologues of the HdaA protein, together with the β-clamp of the DNA polymerase (DnaN), are required for this process. Here, we used fluorescence resonance energy transfer experiments to demonstrate that HdaA interacts with DnaN in live Caulobacter crescentus cells. We show that a QFKLPL motif in the N-terminal region of HdaA is required for this interaction and that this motif is also needed to recruit HdaA to the subcellular location occupied by the replisome during DNA replication. An HdaA mutant protein that cannot colocalize or interact with DnaN can also not support the essential function of HdaA. These results suggest that the recruitment of HdaA to the replisome is needed during RIDA in C. crescentus, probably as a means to sense whether chromosome replication has initiated before DnaA becomes inactivated. In addition, we show that a conserved R145 residue located in the AAA+ domain of HdaA is also needed for the function of HdaA, although it does not affect the interaction of HdaA with DnaN in vivo. The AAA+ domain of HdaA may therefore be required during RIDA after the initial recruitment of HdaA to the replisome by DnaN.

Motility driven by rotational movement of flagella allows bacteria and archaea to seek favourable conditions and escape toxic ones. However, archaeal flagella share structural similarities with bacterial type IV pili rather than bacterial flagella. The Haloferax volcanii genome contains two flagellin genes, flgA1 and flgA2. While FlgA1 has been shown to be a major flagellin, the function of FlgA2 is elusive. In this study, it was determined that although FlgA2 by itself does not confer motility to non-motile ΔflgA1 Hfx. volcanii, a subset of these mutant cells contains a flagellum. Consistent with FlgA2 being assembled into functional flagella, FlgA1 expressed from a plasmid can only complement a ΔflgA1 strain when co-expressed with chromosomal or plasmid-encoded FlgA2. Surprisingly, a mutant strain lacking FlgA2, but expressing chromosomally encoded FlgA1, is hypermotile, a phenotype that is accompanied by an increased number of flagella per cell, as well as an increased flagellum length. Site-directed mutagenesis resulting in early translational termination of flgA2 suggests that the hypermotility of the ΔflgA2 strain is not due to transcriptional regulation. This, and the fact that plasmid-encoded FlgA2 expression in a ΔflgA2 strain does not reduce its hypermotility, suggests a possible regulatory role for FlgA2 that depends on the relative abundance of FlgA1. Taken together, our results indicate that FlgA2 plays both structural and regulatory roles in Hfx. volcanii flagella-dependent motility. Future studies will build upon the data presented here to elucidate the significance of the hypermotility of this ΔflgA2 mutant, and will illuminate the regulation and function of archaeal flagella.

MADS-box transcription factors (TFs) regulate functionally diverse gene targets in eukaryotes. In select ascomycetes, MADS-box TFs have been shown to play a role in virulence, and vegetative and sexual development. Here, we characterized Fusarium verticillioides MADS-box TFs, Mads1 and Mads2, in terms of their roles in secondary metabolism and sexual mating. Sequence analyses showed that MADS1 and MADS2 encode TFs with a SRF-type dimerization domain and a MEF2-type dimerization domain, respectively. The MADS1 and MADS2 knockout mutants (Fmt1 and Fmt2 strains, respectively) exhibited decreased vegetative growth and FB1 production when compared to the wild-type. Fmt1 showed reduced expression of 14 polyketide synthase (PKS) genes present in the organism, whereas Fmt2 did not display a change in PKS gene expression. Significantly, the deletion of MADS1 and MADS2 in the MAT1-2 genotype (Fmt4 and Fmt5 strains, respectively) led to strains that failed to produce perithecia and ascospores when crossed with the MAT1-1 wild-type strain. Notably, deletion of either gene did not have an effect on the ability of the fungus to colonize maize stalk or kernels. FB1 production and PKS expression data suggest that Mads1 is a broad regulator of secondary metabolism in F. verticillioides, and may target regulons upstream of Mads2 to influence FB1 production. In addition, MADS-box TFs in F. verticillioides play a critical role in the perithecia development.

Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers.

The Bacillus cereus type strain ATCC 14579 harbours pBClin15, a linear plasmid with similar genome organization to tectiviruses. Since phage morphogenesis is not known to occur it has been suggested that pBClin15 may be a defect relic of a tectiviral prophage without relevance for the bacterial physiology. However, in this paper, we demonstrate that a pBClin15-cured strain is more tolerant to antibiotics interfering with DNA integrity than the WT strain. Growth in the presence of crystal violet or the quinolones nalidixic acid, norfloxacin or ciprofloxacin resulted in aggregation and lysis of the WT strain, whereas the pBClin15-cured strain was unaffected. Microarray analysis comparing the gene expression in the WT and pBClin15-cured strains showed that pBClin15 gene expression was strongly upregulated in response to norfloxacin stress, and coincided with lysis and aggregation of the WT strain. The aggregating bacteria experienced a significant survival benefit compared with the planktonic counterparts in the presence of norfloxacin. There was no difference between the WT and pBClin15-cured strains during growth in the absence of norfloxacin, the pBClin15 genes were moderately expressed, and no effect was observed on chromosomal gene expression. These data demonstrate for the first time that although pBClin15 may be a remnant of a temperate phage, it negatively affects the DNA stress tolerance of B. cereus ATCC 14579. Furthermore, our results warrant a recommendation to always verify the presence of pBClin15 following genetic manipulation of B. cereus ATCC 14579.

O antigens are polysaccharides consisting of repeat units of three to eight sugars, generally assembled by genes in a discrete O antigen gene cluster. Salmonella enterica produces 46 forms of O antigen, and most of the variation is determined by genes in the gene cluster. However in some cases the structures are modified by enzymes encoded outside of the gene cluster, and several such modifications have been reported for Salmonella enterica group E, some with the genes on bacteriophages and one gene at a distant chromosomal site. We identified the enzyme, WbaK, that is responsible for O-acetylating the subgroup E1 O antigen, and found that the gene is located just downstream of the gene cluster as currently known. The wbaK gene appears to have been imported by a recombination event that also replaced the last 37 bp of the wbaP gene, indicating that homologous recombination was involved. Some of the group E strains we studied must have the original gene cluster, as they lack wbaK and the sequence downstream of wbaP is very similar to that in several other S. enterica O antigen gene clusters. In effect the gene cluster was extended by one gene in subgroup E1. It appears that a function that is usually encoded by a gene outside of the gene cluster has been added to the gene cluster, in this case giving an example of how such gene clusters can evolve.

Nearly half of US clinical isolates of the emerging pathogen Mycobacterium abscessus were reported to exhibit smeared DNA during PFGE. This DNA degradation (Dnd) phenotype results from DNA phosphorothioation, a sulfur modification found in other bacteria and conferred by dnd genes located on mobile elements. Putative dnd genes are located on a 19.6 kbp genomic island (GI) in the M. abscessus type strain ATCC 19977. We confirmed that ATCC 19977T is Dnd-positive by PFGE and we developed a PCR assay to predict Dnd phenotype. Dnd-positive strains generated an amplicon from dndC whereas Dnd-negative strains generated a bridge amplicon that spanned the GI insertion site, indicating they lacked the entire ‘Dnd-GI’. Comparative analyses of sequences from the bridge amplicon with ATCC 19977T revealed the Dnd-GI is flanked by 22 bp repeats in M. abscessus sensu stricto and inserted downstream of a tRNA-Ala gene and between inverted repeats. Regions flanking the Dnd-GI were highly conserved within the M. abscessus complex. Bioinformatics studies suggest the Dnd-GI inserted independently into a strain of Mycobacterium massiliense and that other species of mycobacteria also have dnd genes, supporting reports that the Dnd phenotype is common among actinomycetes. Within the M. abscessus complex, Dnd-positive clinical isolates were primarily M. abscessus sensu stricto, and tandem repeat typing indicated these isolates were highly related, confirming previous PFGE studies and revealing a widespread family of strains with significance in human disease.

The 2006 US spinach outbreak of Escherichia coli O157 : H7, characterized by unusually severe disease, has been attributed to a strain (TW14359) with enhanced pathogenic potential, including elevated virulence gene expression, robust adherence and the presence of novel virulence factors. This study proposes a mechanism for the unique virulence expression and adherence phenotype of this strain, and further expands the role for regulator RcsB in control of the E. coli locus of enterocyte effacement (LEE) pathogenicity island. Proteomic analysis of TW14359 revealed a virulence proteome consistent with previous transcriptome studies that included elevated levels of the LEE regulatory protein Ler and type III secretion system (T3SS) proteins, secreted T3SS effectors and Shiga toxin 2. Basal levels of the LEE activator and Rcs phosphorelay response regulator, RcsB, were increased in strain TW14359 relative to O157 : H7 strain Sakai. Deletion of rcsB eliminated inherent differences between these strains in ler expression, and in T3SS-dependent adherence. A reciprocating regulatory pathway involving RcsB and LEE-encoded activator GrlA was identified and predicted to co-ordinate LEE activation with repression of the flhDC flagellar regulator and motility. Overexpression of grlA was shown to increase RcsB levels, but did not alter expression from promoters driving rcsB transcription. Expression of rcsDB and RcsB was determined to increase in response to physiological levels of bicarbonate, and bicarbonate-dependent stimulation of the LEE was shown to be dependent on an intact Rcs system and ler activator grvA. The results of this study significantly broaden the role for RcsB in enterohaemorrhagic E. coli virulence regulation.

Chronic Pseudomonas aeruginosa infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa isolates undergo significant transcriptomic and proteomic modulation as they adapt to the niche environment of the CF lung and the host defences. This study characterized the in vitro virulence of isogenic strain pairs of P. aeruginosa epidemic or frequent clonal complexes (FCCs) and non-epidemic or infrequent clonal complexes (IFCCs) that were collected 5–8 years apart from five chronically infected adult CF patients. Strains showed a significant decrease in virulence over the course of chronic infection using a Caenorhabditis elegans slow-killing assay and in phenotypic tests for important virulence factors. This decrease in virulence correlated with numerous differentially expressed genes such as oprG, lasB, rsaL and lecB. Microarray analysis identified a large genomic island deletion in the IFCC strain pair that included type three secretion system effector and fimbrial subunit genes. This study presents novel in vitro data to examine the transcriptomic profiles of sequentially collected P. aeruginosa from CF adults. The genes with virulence-related functions identified here present potential targets for new therapies and vaccines against FCCs and IFCCs.

Francisella tularensis is a highly infectious facultative intracellular bacterium and aetiological agent of tularaemia. The conserved hypothetical lipoprotein with homology to thiol/disulphide oxidoreductase proteins (FtDsbA) is an essential virulence factor in F. tularensis. Its protein sequence has two different domains: the DsbA_Com1_like domain (DSBA), with the highly conserved catalytically active site CXXC and cis-proline residue; and the domain amino-terminal to FKBP-type peptidyl-prolyl isomerases (FKBP_N). To establish the role of both domains in tularaemia infection models, site-directed and deletion mutagenesis affecting the active site (AXXA), the cis-proline (P286T) and the FKBP_N domain (ΔFKBP_N) were performed. The generated mutations led to high attenuation with the ability to induce full or partial host protective immunity. Recombinant protein analysis revealed that the active site CXXC as well as the cis-proline residue and the FKBP_N domain are necessary for correct thiol/disulphide oxidoreductase activity. By contrast, only the DSBA domain (and not the FKBP_N domain) seems to be responsible for the in vitro chaperone activity of the FtDsbA protein.

We describe a previously cryptic phenotype associated with the opportunistic phytopathogen Pectobacterium atrosepticum (Pca): surface swarming. We found that when Pca was spotted onto plates containing <0.5 % (w/v) agar, the culture produced copious amounts of extracellular matrix material containing highly motile cells. Once produced, this ‘slime layer’ spread rapidly across the plate either as an advancing front or as tendrils. Transposon mutagenesis was used to identify mutants that were affected in swarming. Hypo-swarmer mutants mostly carried insertions in a horizontally acquired island (HAI5), which encodes a cluster of genes involved in O antigen biosynthesis. Hyper-swarmer mutants mostly carried insertions in hexY, a known antagonist of the class I flagellar master regulator, FlhD4C2. In addition, we found that the nucleoid protein, histone-like nuclear structuring protein 2 (H-NS2), also regulated swarming behaviour. A mutant in which hns2 was overexpressed displayed a hyper-swarming phenotype, whereas a mutant in which the hns2 ORF was inactivated had a hypo-swarming phenotype. Swarming was also regulated by quorum sensing (QS) and by the carbon source being utilized. We show, using a range of epistasis experiments, that optimal swarming requires both motility and O antigen biosynthesis, and that H-NS2 and QS both promote swarming through their effects on motility.

Dickeya dadantii is a phytopathogenic bacterium secreting a large array of plant-cell-wall-degrading enzymes that participate in the infection and maceration of the host plant tissue. Sequencing of the D. dadantii 3937 genome predicted several genes encoding potential glycosidases. One of these genes, bgxA, encodes a protein classified in family 3 of glycosyl hydrolases. Inactivation of bgxA and the use of a gene fusion revealed that this gene is not essential for D. dadantii pathogenicity but that it is expressed during plant infection. The bgxA expression is induced in the presence of glycosidic or non-glycosidic aromatic compounds, notably ferulic acid, cinnamic acid, vanillic acid and salicin. The BgxA enzyme has a principal β-d-glucopyranosidase activity and a secondary β-d-xylopyranosidase activity (ratio 70 : 1). This enzyme activity is inhibited by different aromatic glycosides or phenolic compounds, in particular salicin, arbutin, ferulic acid and vanillic acid. Together, the induction effects and the enzyme inhibition suggest that BgxA is mostly involved in the cleavage of aromatic β-glucosides. There is evidence of functional redundancy in the D. dadantii β-glucoside assimilation pathway. In contrast to other β-glucoside assimilation systems, involving cytoplasmic phospho-β-glucosidases, the cleavage of aromatic glucosides in the periplasmic space by BgxA may avoid the release of a toxic phenolic aglycone into the cytoplasm while still allowing for catabolism of the glucose moiety.

The oxygenated long-chain mycolic acids from many mycobacteria are characterized by the presence of mid-chain cyclopropane groups, which can have either cis-configuration or trans-configuration with an adjacent methyl branch. To determine the effect of these functional groups on mycolic acid conformation, surface pressure (π) versus mean molecular area isotherms of methoxy- (MeO-) mycolic acids (MAs) from Mycobacterium kansasii, Mycobacterium tuberculosis (Mtb) Canetti and Mtb H37Ra, and of keto-MAs from Mycobacterium avium-intracellulare complex (MAC) and Mtb H37Ra were recorded and analysed. The MeO- and keto-MAs from Mtb H37Ra, containing scarcely any trans-cyclopropyl groups, apparently took no fully folded ‘W-form’ conformations. Keto-MA from MAC, whose trans-cyclopropyl group content is nearly 90 %, showed a very solid W-form conformation. MeO-MAs from M. kansasii and Mtb Canetti gave stable W-form conformations at lower temperatures and surface pressures and extended conformations at higher temperatures and surface pressures; their W-form conformation was not as stable as expected from their cis-cyclopropyl group content, probably because they had a wide range of constituent homologues. Energy level calculations of cis- or α-methyl trans-cyclopropane-containing model molecules and computer simulation studies confirmed the superior folding properties of the latter functional unit. The present results were compared with those of MeO- and keto-MAs from Mtb and from M. bovis Bacillus Calmette–Guérin (BCG) reported previously. Among the oxygenated MAs, those having higher trans-cyclopropane content tended to take W-form conformations more firmly, implying that the meromycolate proximal intra-chain α-methyl trans-cyclopropane groups facilitated MA folding more than cis-cyclopropane groups.

The probiotic Gram-positive bacterium Lactobacillus casei BL23 is naturally confronted with salt-stress habitats. It has been previously reported that growth in high-salt medium, containing 0.8 M NaCl, leads to modifications in the cell envelope of this bacterium. In this study, we report that L. casei BL23 has an increased ability to form biofilms and to bind cations in high-salt conditions. This behaviour correlated with modifications of surface properties involving teichoic acids, which are important cell wall components. We also showed that, in these high-salt conditions, L. casei BL23 produces less of the cell wall polymer lipoteichoic acid (LTA), and that this anionic polymer has a shorter mean chain length and a lower level of d-alanyl-substitution. Analysis of the transcript levels of the dltABCD operon, encoding the enzymes required for the incorporation of d-alanine into anionic polymers, showed a 16-fold reduction in mRNA levels, which is consistent with a decrease in d-alanine substitutions on LTA. Furthermore, a 13-fold reduction in the transcript levels was observed for the gene LCABL_09330 coding for a putative LTA synthase. To provide further experimental evidence that LCABL_09330 is a true LTA synthase (LtaS) in L. casei BL23, the enzymic domain was cloned and expressed in E. coli. The purified protein was able to hydrolyse the membrane lipid phosphatidylglycerol as expected for an LTA synthase enzyme, and hence LCABL_09330 was renamed LtaS. The purified enzyme showed Mn2+-ion dependent activity, and its activity was modulated by differences in NaCl concentration. The decrease in both ltaS transcript levels and enzyme activity observed in high-salt conditions might influence the length of the LTA backbone chain. A putative function of the modified LTA structure is discussed that is compatible with the growth under salt-stress conditions and with the overall envelope modifications taking place during this stress condition.