Less is known about the MTT1 genes, but because these lager strains contain more than one copy of most chromosomes, it is again expected that they may contain more than one version of each (2.4 and 2.7 kb) MTT1 gene. Therefore, selleck screening library it

should be realized that the genes characterized here probably represent only a part of all maltose transporter genes present in the lager strains. Comparison of the sequences of the long and short versions of the MTT1 isolates makes it likely that the long versions are not transcribed properly because the ORFs of BS07 2.7 kb and BS07 2.4 kb are identical and the WS34/70 2.7 kb-encoded protein differs in only four residues. It is not clear whether reduced transcription might be caused by the 294 bp longer distance between the transcription start site and the Mal63-binding sites in the 2.7-kb versions and whether the Mal63-binding sites are involved in the transcription regulation of these transporter genes. However, the region between 515 and 582 bp upstream of the MAL61 coding region was shown to be required for the induction of MAL61 by maltose

in the S. cerevisiae strain 332-5A (Levine et al., 1992). Our data suggest that the MAL31 genes encode transporters with a lower affinity for maltotriose than those encoded by the MTT1 genes as the Decitabine cloned promoter regions of the MAL31 isolates, with the exception of that from the laboratory strain CENPK113-7D, are identical to those of the MTT1 genes. The differences in the predicted proteins thus must cause the differences in the ability of these genes to restore the growth of A15 on maltotriose in the presence of antimycin A. There are several sequence differences that are common to all MAL31 isolates. Further analyses are necessary to determine which of these is or are Thiamet G responsible for the observed phenotypes. Based on the growth rate on maltotriose in the presence of antimycin A, the four

lager strains used in this study have different maltotriose uptake capacities. Those of BS01 and WS34/70 are efficient, that of A15 is not and BS07 is intermediate in this respect. With the assumption that other maltotriose transporter genes do not play a role and the observation that all four strains contain a short version of the MTT1 gene, it may be concluded that the difference in the maltotriose transport capacity must be caused by either a copy number effect and/or a difference in the transcription rate. In the latter case, this might be caused by strain-specific differences in the activity of transcription factors. Alternatively, sequences further upstream than the cloned parts of the promoters might play a role, because the cloned parts of the promoters are almost identical. It appears unlikely that translation regulation or post-translational modification would explain the differences between the lager strains. This work was funded by a grant from Heineken Supply Chain (to J.D.

In the tripartite

protein complex, MexB is the inner membrane protein and a member of the resistance–nodulation–division (RND) family, MexA is a membrane fusion protein and OprM is an outer membrane protein. Although all three proteins in the complex are necessary for drug efflux from P. aeruginosa, the substrate specificity of the complex is mediated by MexB. MexB recognizes a wide variety of chemically different compounds including antibiotics, Small molecule library cell assay detergents, dyes and molecules involved in quorum sensing (Poole, 2001). MexB bears a close resemblance to its counterpart from Escherichia coli, AcrB (70% identity), and can also functionally substitute for AcrB in the AcrAB-TolC complex (Krishnamoorthy et al., 2008; Welch et al., 2010). Recently, the crystal structure of MexB has 3-MA been solved and it was found to be an asymmetric homotrimer similar to AcrB (Sennhauser et al., 2009). Each monomer of MexB consists of 12 transmembrane α-helices constituting the inner membrane domain and a large periplasmic domain (Sennhauser et al., 2009). The periplasmic domains of the RND family of drug transporter proteins are implicated in drug recognition and transport (Elkins & Nikaido, 2002; Mao et al., 2002; Tikhonova et al., 2002; Middlemiss & Poole,

2004; Murakami et al., 2006; Seeger et al., 2006; Bohnert et al., 2007; Dastidar et al., 2007; Sennhauser & Grutter, 2008; Takatsuka et al., 2010; Nakashima et al., 2011). Based upon the asymmetric structures of the AcrB trimers, a

substrate pathway through the periplasmic domains of the individual subunits has been proposed as an alternative access mechanism with the protomers adopting binding, access and extrusion conformations, respectively (Murakami et al., 2006; Seeger et al., 2006; Sennhauser & Grutter, 2008). Recent biochemical studies have confirmed the peristaltic pump mechanism of transport (Seeger et al., 2008; Takatsuka & Nikaido, 2009), while structural, functional and computational analyses yielded an insight into the entire substrate path through the periplasmic domain of AcrB (Husain & Nikaido, 2010; Schulz et al., 2010, 2011; Yao et al., 2010; Nakashima et al., 2011). Although the drug efflux pathway through the periplasmic MRIP domains of AcrB has now been very well established and characterized, the question still remains if all drugs are effluxed from the periplasm or if substrates could also be removed directly from the cytoplasm/inner cytoplasmic membrane. In MexB and the related RND transporter MexD, mutations affecting resistance against drugs mapped to periplasmic domains affected both periplasmically and cytoplasmically acting antibiotics; therefore, the authors concluded that there are no separate binding sites for antimicrobials with periplasmic vs. cytoplasmic targets (Mao et al., 2002; Middlemiss & Poole, 2004).

Six months after vaccination, fewer than half of the 169 patients had a twofold or greater increase in antibody

titres, suggesting poor immunogenicity of PPV in patients with moderate to severe immunosuppression at HIV diagnosis and at vaccination. The proportions of responders to the three serotypes in the four groups for five consecutive years are shown in Figure 2a, b and c [the proportions of responders are shown in a supplementary table (Supporting Information Table S1) which can be provided upon request]. In each study year, group 1 had a consistently lower proportion of responders to the three serotypes studied compared with the other three groups. For each group, there were decreasing trends of the proportion of responders to all of the three serotypes after vaccination, despite continued increases in CD4 lymphocyte counts for five Cetuximab chemical structure consecutive years of HAART (Table 1). The loss of antibody responses in each follow-up year varied with the serotype selleck studied and it appeared to be faster among patients in group 1 (Fig. 2a, b and c). For example, all of

the subjects in group 1 lost antibody responses to serotype 23F in the first year of follow-up, while none of them lost antibody responses to serotype 19F until year 5; and antibody responses to serotype 14 persisted in two of 22 patients (9.1%) at year 5. At the end of the 5 years of follow-up, approximately one-third of the patients in the other three groups remained responders to serotype 14 while <20% of them were responders to serotype 19F and only 5% of them were responders to serotype 23F. In order to identify risk factors associated with

maintaining significant antibody responses (twofold or greater increase from baseline) from year 1 to year 5, we compared responders and nonresponders with regard to age, sex, risk factor for HIV transmission, nadir CD4 cell count before vaccination, CD4 cell count and plasma HIV RNA load GPX6 at vaccination, proportion of patients with CD4<100 or <200 cells/μL at vaccination, proportion of persons achieving viral suppression and updated absolute CD4 increase at each year of follow-up. The results of univariate analysis for year 5 are shown in Table 2, while those for years 1–4 are shown in supplementary tables (Tables S2–S5, which can be provided upon request). In univariate analysis, we found that patients with CD4<100 cells/μL at vaccination were less likely to achieve twofold or greater antibody responses throughout the 5-year study period. From years 3 to 5, significantly more responders than nonresponders achieved better suppression of HIV replication, as indicated by the proportion of patients with undetectable plasma HIV RNA load (Table 2).

, 2001), gingival fibroblasts and T cells (Belibasakis et al., 2010), which are crucial for the induction of cytokine responses and the establishment of chronic inflammation in periodontitis (Holzhausen et al., 2010; Fagundes et al., 2011). Gingipains can also stimulate IL-6 production by oral

epithelial cells Pritelivir order (Lourbakos et al., 2001) and IL-8 production by gingival fibroblasts (Oido-Mori et al., 2001), enhancing the inflammatory responses. However, they can also proteolytically inactivate both anti-inflammatory (IL-4, IL-5) and pro-inflammatory (IL-12, IFN-γ) cytokines (Yun et al., 1999, 2001, 2002; Tam et al., 2009). A number of particularly interesting effects are exerted by the gingipains on components of the complement system. Arg-X gingipains can cleave the C5 molecule, resulting in release of its C5a component, which is crucial for enhancing C646 cost the recruitment of PMNs (Wingrove et al., 1992; Imamura et al., 2001). On the other hand, Lys-X can inactivate the C5a receptor on PMNs, an action that may actually impair their recruitment (Jagels et al., 1996a, b). Along this line, the Arg-X gingipains can degrade the C3 molecule, potentially contributing to decreased bacterial opsonization (Schenkein et al., 1995). This property could confer increased resistance of P. gingivalis to bactericidal activity. Apart from their effect on immune responses, gingipains may

also be involved in the binding of P. gingivalis to host cells, as Rgp–Kgp complexes have been shown to mediate adherence on gingival epithelial cells and gingival fibroblasts (Chen et al., 2001; Grenier et al., 2003; Andrian et al., 2004). Interestingly, when P. gingivalis intracellularly invades Montelukast Sodium gingival epithelial cells, expression of gingipain is downregulated (Xia et al., 2007). Gingipains may also affect vascular permeability and bleeding at the periodontal site. They can proteolytically activate plasma kallikrein and bradykinin, or alternatively increase the release of thrombin and prothrombin,

which can result in increased vascular permeability and PMN influx (Imamura et al., 1994, 1995a). Moreover, by degrading fibrinogen (Scott et al., 1993), they may contribute to inhibition of blood coagulation and increase bleeding at the site (Imamura et al., 1995a) , thus enhancing the availability of hemin required for P. gingivalis growth. Collectively, studies in various experimental systems indicate that gingipains have seemingly contradicting actions on the innate immune responses, hampering interpretation of their role in the pathogenesis of periodontitis. Nevertheless, such differences may be reconciled by the existence of a concentration gradient of gingipains in the tissue (Pathirana et al., 2010). Closer to the gingival epithelial barrier where the biofilm resides, gingipain concentrations are high, causing degradation or deregulation of various components of the immune response.

Studies were identified by searching the MEDLINE, Embase, Cochrane Clinical Trials Register, and ClinicalTrials.gov databases.

Primary end point was difference in incidence of AMS between acetazolamide and placebo groups. Acetazolamide prophylaxis was associated with a 48% relative-risk Selleck Cabozantinib reduction compared to placebo. There was no evidence of an association between efficacy and dose of acetazolamide. Adverse effects were often not systematically reported but appeared to be common but generally mild. One study found that adverse effects of acetazolamide were dose related. Acetazolamide is effective prophylaxis for the prevention of symptoms of AMS in those going to high altitude. A dose of 250 mg/day has similar efficacy to higher doses and may have a favorable side-effect profile. Acute mountain sickness (AMS), characterized by headache, light-headedness, fatigue, nausea, and insomnia, occurs primarily at altitudes above 2,500 m in those poorly acclimatized to such conditions. If untreated, this symptom complex can progress to the life-threatening conditions of high altitude cerebral edema and high altitude pulmonary edema.[1] It has been suggested that the carbonic anhydrase inhibitor acetazolamide is effective in the prevention of AMS when begun prior to ascent

to altitude. Deforolimus price However, for clinicians prescribing for those ascending to altitude, there has been a lack of clarity regarding the usefulness of acetazolamide, when and for whom it should be recommended, and the optimum dose. While guidelines published by the Wilderness Medical Society recommend acetazolamide for travelers under some circumstances,[2] the Union Internationale des Associations d’Alpinisme does not make a similar suggestion.[3] The side effect profile of acetazolamide includes paraesthesia, urinary frequency, and Tideglusib dysgeusia (taste disorder). As such unpleasant symptoms could affect compliance with treatment, it is desirable to determine the lowest effective dose in order to potentially minimize the harmful effects of acetazolamide. Two systematic reviews of acetazolamide in the prevention of altitude-related symptoms have been

published. The first, published in 1994, included trials measuring a diverse range of outcomes not limited to classic symptoms of AMS.[4] This review found evidence of a benefit associated with acetazolamide but the heterogeneity in measured outcomes limits interpretation in a clinical context. The second systematic review was published in 2000 and had more restrictive inclusion criteria—including only studies reporting the incidence of AMS as an end point.[5] The authors concluded that 750 mg/d of acetazolamide was effective in the prophylaxis of AMS but that there was no evidence of benefit from 500 mg/d. However, this review was limited by the small number of patients in the pooled analysis which significantly limited its power.

Sensitivity to the behavioural effects of the psychotomimetic N-methyl-d-aspartate receptor antagonists MK-801 and phencyclidine (PCP) was examined in mutant mice with heterozygous deletion of NRG1. Social behaviour (sociability, social novelty preference and dyadic interaction), together with exploratory activity, was assessed following acute or subchronic administration of MK-801 (0.1 and 0.2 mg/kg) or PCP (5 mg/kg). In untreated NRG1 mutants, levels of glutamate, N-acetylaspartate VDA chemical and GABA were determined using high-performance liquid chromatography and regional brain volumes were assessed using magnetic resonance imaging at 7T. NRG1 mutants, particularly males, displayed decreased

responsivity to the locomotor-activating effects of acute PCP. Subchronic MK-801 and PCP disrupted Selumetinib purchase sociability and social novelty preference in mutants and wildtypes and reversed the increase in both exploratory activity and social dominance-related behaviours

observed in vehicle-treated mutants. No phenotypic differences were demonstrated in N-acetylaspartate, glutamate or GABA levels. The total ventricular and olfactory bulb volume was decreased in mutants. These data indicate a subtle role for NRG1 in modulating several schizophrenia-relevant processes including the effects of psychotomimetic N-methyl-d-aspartate receptor antagonists. “
“G protein-gated inwardly-rectifying K+ (GIRK/family 3 of inwardly-rectifying K+) channels are coupled to neurotransmitter action and can play

important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult brain of mice and rats using biochemical, immunohistochemical Mephenoxalone and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1-3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1-3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma membrane. During development, GIRK1 and GIRK2 were found primarily at postsynaptic sites, whereas GIRK3 was predominantly detected at presynaptic sites. In addition, GIRK1 and GIRK2 expression on the spine plasma membrane showed identical proximal-to-distal gradients that differed from GIRK3 distribution. Furthermore, although GIRK1 was never found within the postsynaptic density (PSD), the level of GIRK2 in the PSD progressively increased and GIRK3 did not change in the PSD during development.

Jiang and J.-Y. Kim, unpublished data). Past studies have used AAV-GFP virus for in vivo imaging following stereotaxic injection into mice and monkeys (Stettler et al., 2006; Lowery et al., 2009). Local injection has the benefit of eliminating background fluorescence from distant projection neurons, but at the cost of having less control over the density of labeled cells due to a sharp gradient in transduction from the site of injection. Neonatal transduction provides improved

consistency LY294002 in vivo in the expression pattern, and offers a serviceable alternative to Thy1-XFP lines (Feng et al., 2000), particularly when working with models that already require multiple transgenes or modified alleles. Viral transgenesis also Ibrutinib concentration provides access to neurons not labeled in the Thy1-XFP mice, notably Purkinje cells of the cerebellum, which in the past have required acute injection of synthetic dyes for morphological study in vivo (Gobel & Helmchen, 2007). Given the high plasticity of cerebellar circuitry and the progressive but poorly understood degeneration

of Purkinje neurons in many inherited ataxias (Boyden et al., 2004; Carlson et al., 2009), chronic in vivo imaging of these arbors during motor learning and disease will likely grant new insight into cerebellar function and dysfunction. Combined with the potential to genetically manipulate the labeled neurons, neonatal viral transduction opens the possibility for experiments probing the relationship between targeted proteins, dendritic morphology, and neuronal function within single cells of the intact brain (O’Connor et al., 2009). Although this technique has many advantages over past methods, several limitations should be noted. First, as mentioned above, the small packaging size of AAV limits the length and number of transgenes that can be co-expressed. In some situations this can be overcome by trans-splicing of co-injected viruses, but this may not

be possible in every setting (Lai et al., 2005; Ghosh & Duan, 2007). Second, widespread transduction may not be ideal when more restricted expression is needed. Where available, spatial or cell-type specificity could be attained using Cre-dependent flex-signal viruses (Atasoy et al., 2008) with Cre-expressing transgenic Thymidylate synthase lines (e.g. nagy.mshri.on.ca/). In other cases, selectivity might be achieved using an intersectional strategy of complementary elements introduced on co-injected viruses (Dymecki et al., 2010; Haubensak et al., 2010; Fujimoto et al., 2011). Third, the level of viral gene expression varies between cells due to differences in the multiplicity of infection inherent in viral transgenesis. This fluctuation may complicate some studies of neuronal function, but may be lessened at extremes of high and low titers where infection can be maximised or dilution-limited to a single particle.

Jiang and J.-Y. Kim, unpublished data). Past studies have used AAV-GFP virus for in vivo imaging following stereotaxic injection into mice and monkeys (Stettler et al., 2006; Lowery et al., 2009). Local injection has the benefit of eliminating background fluorescence from distant projection neurons, but at the cost of having less control over the density of labeled cells due to a sharp gradient in transduction from the site of injection. Neonatal transduction provides improved

consistency see more in the expression pattern, and offers a serviceable alternative to Thy1-XFP lines (Feng et al., 2000), particularly when working with models that already require multiple transgenes or modified alleles. Viral transgenesis also Sunitinib provides access to neurons not labeled in the Thy1-XFP mice, notably Purkinje cells of the cerebellum, which in the past have required acute injection of synthetic dyes for morphological study in vivo (Gobel & Helmchen, 2007). Given the high plasticity of cerebellar circuitry and the progressive but poorly understood degeneration

of Purkinje neurons in many inherited ataxias (Boyden et al., 2004; Carlson et al., 2009), chronic in vivo imaging of these arbors during motor learning and disease will likely grant new insight into cerebellar function and dysfunction. Combined with the potential to genetically manipulate the labeled neurons, neonatal viral transduction opens the possibility for experiments probing the relationship between targeted proteins, dendritic morphology, and neuronal function within single cells of the intact brain (O’Connor et al., 2009). Although this technique has many advantages over past methods, several limitations should be noted. First, as mentioned above, the small packaging size of AAV limits the length and number of transgenes that can be co-expressed. In some situations this can be overcome by trans-splicing of co-injected viruses, but this may not

be possible in every setting (Lai et al., 2005; Ghosh & Duan, 2007). Second, widespread transduction may not be ideal when more restricted expression is needed. Where available, spatial or cell-type specificity could be attained using Cre-dependent flex-signal viruses (Atasoy et al., 2008) with Cre-expressing transgenic CHIR-99021 mouse lines (e.g. nagy.mshri.on.ca/). In other cases, selectivity might be achieved using an intersectional strategy of complementary elements introduced on co-injected viruses (Dymecki et al., 2010; Haubensak et al., 2010; Fujimoto et al., 2011). Third, the level of viral gene expression varies between cells due to differences in the multiplicity of infection inherent in viral transgenesis. This fluctuation may complicate some studies of neuronal function, but may be lessened at extremes of high and low titers where infection can be maximised or dilution-limited to a single particle.

cereus. Our current findings suggest that the protein is part Selleck SCH 900776 of an outer spore structure, most likely the exosporium or the interspace between the exosporium and the coat. The bacterial strains used in this study were the B. cereus type strain ATCC 14579 (Frankland & Frankland, 1887; Ivanova et al., 2003) and B. subtilis B252 (From et al., 2005). To create a bc1245 deletion mutant in B. cereus ATCC 14579, a shuttle vector modified from pMAD (Arnaud

et al., 2004) with a spectinomycin-resistant cassette in the restriction site SalI (Fagerlund, 2007) was used. Sequence information was obtained from the NCBI bacterial genome database (http://www.ncbi.nlm.nih.gov/guide) or the ergo database (Overbeek et al., 2003). Comparative genomic analyses of bc1245 were performed on selected members of the B. cereus group [B. cereus ATCC 14579 (GenBank: NC004722), B. cereus ATCC 10987 (GenBank: NC003909), Akt inhibitor B. cereus AH187 (GenBank: CP001177), Bacillus thuringiensis YBT-020 (GenBank: CP002508), B. anthracis str. Ames (GenBank: AE016879), Bacillus weihenstephanensis KBAB4 (GenBank: NC010184), B. mycoides DSM 2048 (GenBank: CM000742) and B. pseudomycoidesDSM12442 (GenBank: CM000745)] to investigate whether bc1245 is conserved. Putative σ-binding sites for the bc1245 promotor

were predicted by analyzing the 500-bp upstream region of bc1245 with DBTBS release 5 (Sierro et al., 2008). 4-Aminobutyrate aminotransferase To search for functional motifs, the amino acid sequence of BC1245 was submitted to ScanProSite, (http://www.expasy.ch/prosite; Bairoch et al., 1997). Quantitative PCR experiments were performed as described previously (van der Voort et al., 2010), and primers were designed by use of Primer 3 (Rozen & Skaletsky, 2000) for sigH, sigE, sigF, sigG, sigK, bc1245 and zcDNA (Table 1) using the chromosomal DNA sequence of B. cereus ATCC 14579 as a template.

PCR on genomic DNA was used to check primer efficiency (results not shown). RNA was isolated from two independent cultures withdrawn at different stages of sporulation of B. cereus ATCC 14579 grown in maltose sporulation medium (MSM) as described earlier (van der Voort et al., 2010). cDNA synthesis was performed with ~ 500 ng of total RNA and a mix of relevant reverse primers as described previously (van Schaik et al., 2007). Quantitative PCR was performed with 5 μM of each of the primer pairs listed in Table 1 using an ABI Prism 7700 with SYBR green technology (PE Applied Biosystems, Nieuwekerk a/d Ijssel, the Netherlands) as described previously (van Schaik et al., 2005). By comparing expression of the chosen genes with that of the reference 16S rRNA gene (zcDNA) levels, relative expression values were obtained with the REST-MCS program using the Pair Wise Fixed Reallocation Randomization Test (Pfaffl et al., 2002).

RNA was pelleted at 16 000 g for 20 min at 4 °C, washed once with 1 mL of 70% ethanol, repelleted and briefly air-dried before being resuspended in 100 μL of RNase-free water. The

resuspended RNA was then further purified using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The pure RNA was stored at −80 °C. RNA was DNase treated using the Ambion turbo-free DNA kit according to the manufacturer’s instructions. cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems). A total of ∼1.2−1.5 μg of RNA was used in a 20-μL reaction in all cases. cDNA was synthesized using a PCR cycle of 25 °C for 10 min, SCH772984 37 °C for 120 min and 85 °C for 5 s. qRT-PCR was performed using the custom-made Taqman gene expression assays (Applied Biosystems). A total of 60 ng of cDNA was used in each 20 μL reaction. Reactions were performed in 20 μL containing 10 μL 2 × Taqman gene expression Mastermix (Applied Biosystems),

1 μL Taqman gene expression assay (Applied Biosystems) and 9 μL cDNA (60 ng). The real-time PCR cycle was carried out in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) (50 °C for 2 min, 95 °C for 10 min and then 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min). The fold change in the expression levels of each of the genes was calculated using the ΔΔCt method (Livak & Schmittgen, 2001). RNA was extracted from mid-log cultures of M. smegmatis as described above, and the 5′RACE system for the rapid amplification of cDNA ends see more (Version 2.0, Invitrogen) was used according to the manufacturer’s instructions, using the primers cpn60.1 gsp1, cpn60.1 gsp2 and cpn10 gsp2. cDNA was tailed at the 5′ ends using poly-cytosine and transcriptional start sites were identified by detection

of the junction of this poly-C tail in the sequenced cDNA. The promoterless lacZ E. coli–Mycobacterium shuttle vector pSD5B was used to analyse promoter activity (Jain et al., Chlormezanone 1997). Fragments of varying lengths upstream of the cpn60 or cpn10 genes were amplified with primers containing XbaI and SphI sites, or XbaI sites alone. The products were digested as appropriate and ligated into plasmid pSD5B. The resultant recombinant plasmids contained the various promoter regions just upstream of the lacZ gene (Table 1 and Fig. 1). Each of the pSD5B constructs containing a promoter region was electroporated into M. smegmatis mc2155 cells. The strains were grown in liquid media at 37 °C for 2 days, after which their absorbance at OD600 nm was measured. Each culture (100 μL) was added to 900 μL Z buffer (30 °C). A drop each of 0.1% sodium dodecyl sulphate and chloroform was then added to the tubes, which were vortexed to lyse the cells. The reaction was started by adding 200 μL ONPG (4 mg mL−1) and mixing well. When a significant yellow colour developed, the reaction was stopped by addition of 500 μL 0.